CROSS SECTIONAL STUDY
Dissertation submitted to
THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY In partial fulfillment for the degree of
MASTER OF DENTAL SURGERY
BRANCH – II PERIODONTOLOGY
THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY CHENNAI – 600032
2016 – 2019
ACKNOWLEDGEMENT
Firstly, I thank the LORD ALMIGHTY for providing me this opportunity and granting me the capability to proceed successfully.
I owe a debt of gratitude to my guide, Dr. H. Esther Nalini, M.D.S, Professor & Head, Department of Periodontics, K.S.R. Institute of Dental Science & Research. She is an inspiration to all and her guidance and mentorship has been the navigation for my dissertation, without her help completion of this dissertation wouldn’t have been possible. Her unending belief in me was the key element which helped me to bring this work to a successful conclusion.
I would like to express my sincere gratitude to Dr. Arun Kumar Prasad M.D.S, Professor, who has provided me with constant support and guidance whenever necessary.
I must offer my profoundest gratitude to Dr. R. Renuka Devi M.D.S, Professor, Department of Periodontics, for her unreserved help and guidance in times of need.
I am deeply grateful to Dr. G.S. Kumar, Principal, K.S.R. Institute of Dental Science and Research for his kind permission, encouragement and for providing me with all the facilities needed to complete this work.
It gives me great pleasure to thank the staff members, Dr. Thirumalai, Dr. Kokila Priya, Dr. Tamilselvi and Dr. Nithya, Department of Periodontology, K.S.R. Institute of Dental Science & Research, for their valuable insights during my study.
I feel so blessed to have such a wonderful batchmate Dr. Y. Abirami Sri for her moral support, encouragement and help which were the sustaining factors for carrying out this work successfully.
I would also like to thank my juniors and my seniors for their continuous encouragement and support.
I am indebted to my parents and my sister. This work would not have been possible without their understanding, love and care. They supported me in each and every way, believed in me permanently and inspired me in all dimensions of life.
I would like to express my heartfelt thanks to Dr. J. Philip Robinson Ph.D, Professor and H.O.D, Department of Biotechnology, K.S.R. College of Technology for giving me the opportunity to complete my thesis work. I would also like to thank G. Ayyappadasan Ph.D, Assistant Professor, Dr. S. Rubavathi, Assistant Professor, staff and students of Department of Biotechnology for supporting me in my thesis work by carrying out the biochemical analysis.
A special thanks to all the patients who participated in the study. This dissertation would not have been possible without their support and co-operation.
Finally, I take this opportunity to express my thanks to the non-teaching staff of the department, who have supported me to complete the research work directly or indirectly.
CONTENTS
S. No TITLE PAGE. NO
1 INTRODUCTION 1-2
2 AIMS AND OBJECTIVES 3
3 REVIEW OF LITERATURE 4-16
4 MATERIALS AND METHODS 17-35
5 STATISTICAL ANALYSIS 36
6 RESULTS 37-47
7 DISCUSSION 48-52
8 SUMMARY AND CONCLUSION 53-54
9 BIBLIOGRAPHY 55-59
10 ANNEXURES 60-67
LIST OF FIGURES
FIGURE NO
CONTENTS PAGE NO
1 Two way relationship between Diabetes Mellitus and Chronic periodontitis
4
2 Chemerin, its receptors and functions
5
3 Actions of chemerin and its release 7
4 Armamentarium for saliva collection 29
5 Collection of salivary sample (Figure 5A & 5B) 29
6 Intra oral photograph of a patient of Group-I (GCG) (Fig. 6A-6E) 30
7 Intra oral photograph of a patient of Group-II (GCP) (Fig. 7A-7E) 31
8 Intra oral photograph of a patient of Group-III (GCP with Type-II DM) (Fig. 8A-8E)
32
9 Salivary chemerin analysis using ELISA (Fig. 9A-9H) 33-35
LIST OF TABLES
TABLE
NO CONTENT PAGE NO
1 Distribution of age (in years) between the groups 37
2 Distribution of gender between the groups 37
3 Comparison of Body mass index (BMI) between the groups 38
4 Comparison of Mean Random blood glucose (RBG), glycated haemoglobin (HbA1c) between the groups
39
5 Comparison of PI, GI, PPD and CAL between the groups 41 6 Comparison of mean chemerin values between the groups 44
7 Correlation between chemerin and HbA1c, CAL 45
LIST OF GRAPHS
GRAPH NO
CONTENT PAGE NO
Graph 3a Comparison of Body mass index (BMI) between the groups 38
Graph 4a Comparison of Mean Random blood glucose (RBG) levels between the groups
40
Graph 4b Comparison of Mean glycated haemoglobin (HbA1c) levels between the groups
40
Graph 5a Comparison of Mean Plaque Index (PI) between the groups 42
Graph 5b Comparison of Mean Gingival Index (GI) between the groups 42
Graph 5c Comparison of Mean Probing pocket depth (PPD) between the groups 43
Graph 5d Comparison of Mean Clinical Attachment level (CAL) between the groups
43
Graph 6a Comparison of Mean Chemerin levels between the groups 44
Graph 7a Correlation between chemerin and HbA1c 45
Graph 7b Correlation between chemerin and CAL 45
ABBREVIATIONS
AGEs Advanced Glycation End Products (AGEs)
BMD Bone Mineral Density
BMI Body Mass Index
BOP Bleeding On Probing
C-terminal Carboxy Terminal
CAL Clinical Attachment Level
CEJ Cemento Enamel Junction
ChemR23 Chemerin R23
CMKLR1 Chemokine Receptor-Like 1
CCRL2 C-C Chemokine Receptor Like-2
CRP C-Reactive Protein
DGAT-2 Diacyl Glycerol Acyl Transferase 2
DC Dendritic Cells
DM Diabetes Mellitus
ELISA Enzyme Linked Immunosorbent Assay
GCF Gingival Crevicular Fluid
GCG Generalized Chronic Gingivitis
GCP Generalized Chronic Periodontitis
GI Gingival Index
GM Gingival Margin
GPR-1 G-Protein Coupled Receptor-1
GLUT-4 Glucose Transporter-4
hsCRP High Sensitivity C-Reactive Protein
HbA1c Glycated Hemoglobin
HDL High Density Lipoproteins
HRP Horse Radish Peroxide
IL Interleukin
IL-8 Interleukin-8
IL-1α Interleukin 1 Alpha
IL-1β Interleukin 1 Beta
LPS Lipopolysaccharides
mRNA Messenger Ribonucleic Acid
MIP Macrophage Inflammatory Protein
Ml Milliliter
MMP Matrix Metallo Proteinase
MPO Myeloperoxidase
NDD Newly Diagnosed Diabetes
NK cells Natural Killer Cells
ng Nanogram
nm Nanometer
OD Optical Density
OGTT Oral Glucose Tolerance Test
OHI Oral Hygiene Instructions
PPD Probing Pocket Depth
PI Periodontal Index
PlI Plaque Index
PMN Polymorphonuclear Neutrophils
POC Point Of Care
PPD Probing Pocket Depth
PG Prostaglandins
qPCR Quantitative Polymerase Chain Reaction
RBG Random Blood Glucose
RARRES2 Retinoic Acid Receptor Responder 2
RT-PCR Real Time Polymerase Chain Reaction
SD Standard Deviation
SOD Superoxide Dismutase
SRP Scaling And Root Planing
TIMPs Tissue Inhibitors Of Metalloproteinases
TNF-α Tumour Necrosis Factor Alpha
TG Triglycerides
µl Microliter
WAT White Adipose Tissue
WHR Waist Hip Ratio
Introduction
Periodontitis is a chronic inflammatory disease characterised by destruction of hard and soft tissues supporting the teeth.1 An imbalance between a localized infection and an exaggerated host inflammatory response plays a pivotal role in determining the tissue damage.
Evidence suggests that the effect of periodontal disease might not be limited just to the oral cavity but it might have systemic consequences as well due to an associated systemic inflammatory response.
Diabetes Mellitus (DM) comprises a clinically and genetically heterogenous group of metabolic disorders manifested by abnormally elevated glucose levels in the blood.2 In DM, neutrophil adherence, chemotaxis and phagocytosis are often impaired which may significantly increase periodontal destruction. This could explain the increased risk of impaired metabolic control in diabetes related complications and adverse effects of Diabetes Mellitus on periodontal health. Periodontitis is described as the sixth complication of Diabetes Mellitus.
Diabetes is now viewed as an inflammatory condition and its development is preceded by a low grade systemic inflammation with elevated plasma concentrations of pro-inflammatory markers such as CRP, cytokines (IL-1β, IL-6 and TNF- ) and prostanoids (PGE2). Periodontal disease and Diabetes Mellitus show a bidirectional relationship.
Adipose tissue is a complex and metabolically active endocrine organ. The adipocytes secrete bioactive molecules called adipokines which act as signalling molecules to liver, muscle and endothelium.3 They are soluble proteins that initiate intercellular signalling cascades. Some adipokines exhibit both pro-inflammatory and anti-inflammatory properties. The relationship between pro-inflammatory and anti-inflammatory adipokines can lead to a low grade inflammatory condition, as occurs with periodontitis and Diabetes Mellitus.
Chemerin is an adipose tissue specific and pro-inflammatory adipocytokine that is produced by adipose tissue, liver, epithelial cells, endothelium, fibroblasts and keratinocytes.4
Under normal physiological conditions, it remains in an inactive form as prochemerin which gets activated during inflammation. The inflammatory effect of chemerin occurs both through the induction of pro-inflammatory cytokines and through the stimulation of chemotaxis of the inflammatory cells to the site of inflammation. Fluctuating chemerin volumes have been seen to correlate positively with recognized markers of inflammation such as TNF- , IL-6, CRP and it can be identified within inflamed tissues and inflammatory fluids.5
Chemerin levels were found to be higher within serum of patients with Type-II DM than in those with normal glucose tolerance. Chemerin may also be detected within the periodontium and might represent chronic inflammation in subjects with chronic periodontitis with / without Type-2 DM. This suggests the active role of chemerin in the pathogenesis of periodontitis and its association with Diabetes Mellitus.6
In view of the above, chemerin levels may be considered a potential pro-inflammatory marker for Diabetes Mellitus and periodontal disease.
Saliva is a clinically informative, biological fluid that contains specific soluble biological markers (biomarkers).7 It is useful for laboratory and clinical diagnosis, determining the prognosis and monitoring and management of patients with both oral and systemic diseases.
However, till date, salivary chemerin concentrations in subjects with chronic periodontitis with Type-II DM has not been explored. Hence, based on the above findings, we found it of interest to investigate and compare the salivary levels of chemerin in healthy subjects and in subjects with chronic periodontitis with/without Type-II Diabetes Mellitus.
Aims and Objectives
To evaluate the salivary levels of chemerin in three groups i. systemically and periodontally healthy subjects ii. systemically healthy subjects with chronic periodontitis iii. Type II Diabetes Mellitus subjects with chronic periodontitis.
To compare the salivary levels of chemerin among systemically and periodontally healthy subjects, subjects with CP with / without Type II DM.
To correlate the salivary levels of chemerin with Glycated Hemoglobin (HbA1c) and Clinical Attachment level (CAL) in systemically and periodontally healthy subjects, subjects with chronic periodontitis with / without Type II DM.
Review of literature
Periodontitis is an inflammatory disease of the supporting tissues of the teeth caused by specific microorganisms or groups of specific microorganisms, resulting in progressive destruction of the periodontal ligament and alveolar bone with increased probing depth formation, recession or both.1 Diabetes Mellitus comprises a clinically and genetically heterogeneous group of metabolic disorders manifested by abnormally elevated levels of glucose in the blood.2 Periodontal disease and Diabetes Mellitus show a bidirectional relationship.
Figure. 1
Adipose tissue is a complex and metabolically active endocrine organ. The adipocytes secrete bioactive molecules called adipokines which act locally or systemically as signalling molecules to liver, muscle and endothelium. They are soluble proteins that bind receptors on target cells and initiate intercellular signalling cascades resulting in phenotype changes to the cell through altered gene expression and regulation.
Some adipokines play a protective role in tissue protection, while some others have a pro inflammatory effect and certain types exhibit both pro inflammatory & anti-inflammatory
properties. The relationship between pro-inflammatory and anti-inflammatory adipokines can lead to a low-grade inflammatory condition, as occurs with periodontitis and Diabetes Mellitus.
These adipocytokines activate monocytes which increases the production of pro-inflammatory cytokines thus playing an important role in the initiation of periodontal disease. In 2007 chemerin was described as a novel adipokine that is expressed in mouse and human adipocytes and associated with obesity and factors contributing to the development of the metabolic syndrome (e.g., blood pressure and circulating triglycerides).
CHEMERIN:
Chemerin is an adipose tissue specific and pro-inflammatory adipokine which is also known as tazarotene induced gene 2 and retinoic acid receptor responder 2 (RARRES2).
Chemerin functions via three receptors: Chemokine receptor like 1 (CMKLR1), C-C chemokine receptor-like 2 (CCRL2) and G-protein coupled receptor 1 (GPR1). These receptors are involved in adipogenesis, osteoclastogenesis, angiogenesis and inflammatory processes in skin and adipose tissue. Chemerin and its receptors are released from adipose tissue, epithelial cells, endothelium, fibroblasts and keratinocytes.4
Figure. 2
The above picture depicts the three chemerin receptors, ChemR23, GPR1 and CCRL2 which are represented at the surface of cells, together with the main events triggered by ligand binding.4 ChemR23 leads to strong signalling (promoting leukocyte chemotaxis) and internalization of the chemerin–receptor complex. GPR1 internalizes in response to chemerin, but signals inefficiently through classical cascades. CCRL2 does not signal nor internalizes, but might present chemerin to nearby cells displaying functional receptors.
Chemerin is expressed as prochemerin that consists of 163 amino acids. Under normal physiological conditions, it remains in an inactive form as prochemerin. However, under inflammatory conditions, it is activated by the removal of C-terminal amino acid by proteolytic enzymes such as cathepsin-G. The inflammatory effect of Chemerin occurs both through the induction of the pro-inflammatory cytokines such as IL-1β, IL-8 and through the stimulation of chemotaxis of the inflammatory cells to the site of inflammation. It plays an important role in adipocyte differentiation and development and also influences glucose, lipid metabolism and inflammation levels. Fluctuating Chemerin volumes have been seen to correlate positively with recognised markers of inflammation such as TNF- , IL-6, CRP and it can be identified within inflamed tissues and inflammatory fluids.5 Chemerin is involved in pathogenesis of several diseases like Ulcerative colitis, Crohn’s disease, Osteoarthritits, Psoriasis and Multiple sclerosis.8 Chemerin plays an important role in the regulation of multi-organ physiopathological processes.
ACTIONS OF CHEMERIN AND ITS RELEASE:
The adipocytes, pre-adipocytes and macrophages have metabolic and inflammatory functions, which make it to release several mediators. These exert different biological effects
in the white adipose tissue (WAT) itself or other tissues, acting in paracrine or endocrine way.
Adipose tissue macrophages are responsible for the circulating levels of specific inflammatory molecules that determines “low grade” chronic obesity-related inflammation. Adipocyte hypertrophy induces the macrophage activation due to hypoxic condition resulting in adiposopathy, the process that converts healthy adipose tissue to inflamed adipose tissue.
Adipocytokines, particularly chemerin release, causes the harmful effects linked with a major or minor risk to develop metabolic and inflammatory diseases.8
Figure. 3 CHEMERIN AS AN ADIPOKINE:
Chemerin regulates mature adipocyte functions, by controlling the expression of key effectors of glucose and lipid metabolism, the glucose transporter GLUT4, diacyl glycerol acyl transferase (DGAT2), mediating the synthesis of triglycerides, and the adipokines leptin and adiponectin. Obesity is frequently associated with a chronic systemic inflammatory state.
Adipocyte enlargement is associated with the secretion of cytokines such as TNF- and IL-6,
which increase in the bloodstream, and adipose tissue becomes infiltrated by macrophages, T cells, NK cells and immature dendritic cells. Pro-inflammatory cytokines secreted by adipocytes and macrophages interfere with insulin signalling and promote insulin resistance.
Chemerin promotes chemotaxis of leukocytes found in adipose tissue from obese individuals hence regarded as a potential link between obesity and inflammation. It helps the cells such as macrophages and polymorphonuclear leukocytes that contain ChemR23 receptors move to the site of inflammation. Serum chemerin levels are elevated in obese and Type 2 Diabetic patients and correlated positively with body mass index (BMI), fasting glucose, fasting insulin, triglycerides and total cholesterol, but negatively with HDL cholesterol. Chemerin levels also correlate with serum concentrations of TNF- , IL-6 and C reactive protein (CRP) and reflect the inflammatory status associated with obesity.
CHEMERIN IN PERIODONTITIS:
Chemerin promotes chemotaxis of monocytes and macrophages to the site of inflammation. As chemerin binds to its receptor ChemR23, it leads to increased expression of pro-inflammatory cytokines such as TNF- and IL-1β and release of proteases such as MMP- 9 and MMP-13 causing matrix degradation.9 It also causes irreversible tissue damage by increasing the MMP levels and thus may lead to periodontal tissue destruction.10
In the present study, we found it of interest to investigate and compare the salivary levels of chemerin in healthy subjects and in subjects with chronic periodontitis with / without Type-II Diabetes Mellitus.
CHEMERIN IN SALIVA:
Human saliva is an easily accessible biological fluid and contains a variety of disease- related biomarkers, which makes it a potential diagnostic tool. Saliva could be an alternative to plasma / serum analysis for screening, diagnostic and prognostic purposes as well as evaluation of treatment outcome. The biggest advantage of using saliva is that the collection is non-invasive and a plausible method.
The inflammatory ‘biomarker’ refers to biologic substances that can be measured and evaluated to serve as indicators of biological health, pathogenic processes, environmental exposure and pharmacologic responses to a therapeutic intervention.11 The study of salivary proteins and the complete human salivary proteome provides an insight into the intricate cellular functional interactions that maintain oral and general health. The biomarkers analyzed in saliva samples are interleukins, tumour necrosis factors, lysozymes, Matrix metalloproteinases (MMPs), Tissue Inhibitors of metalloproteinases (TIMPs), Myeloperoxidase (MPO) and other proteins.
Chemerin is an adipose tissue specific cytokine which was discovered recently.
Chemerin levels were found to be higher within serum of patients with Type-II DM than in those with normal glucose tolerance. It may also be detected within the periodontium and might represent chronic inflammation in subjects with chronic periodontitis with / without Type-2 DM.12
Jentsch et al (2017)13 assessed the levels of acylated and total ghrelin chemerin and IL- 1β in saliva, GCF and serum of periodontally healthy, diseased individuals with respect to different body mass categories. The authors categorized the subjects into chronic periodontitis, periodontally healthy / gingivitis, which were further subdivided into normal and overweight group according to BMI. GCF, stimulated saliva, serum were collected and assessed for the
levels of acylated and total ghrelin, chemerin, IL-1β using ELISA kits. The results showed an increase in GCF chemerin, IL-1β levels and a decrease in GCF ghrelin levels in subjects with chronic periodontitis with BMI < 25 and a positive correlation was observed between BMI and serum levels of chemerin. The authors concluded that low ghrelin and increased GCF chemerin levels are linked to periodontal disease and obesity and unlike IL-1β, GCF chemerin and ghrelin are not potential markers of periodontal destruction.
Özcan et al (2017)14 evaluated mRNA and protein expression of chemerin and its receptors, chemR23 and CCRL2 in healthy and chronic periodontitis subjects and investigated their relationships with clinical parameters. The authors categorised the subjects into chronic periodontitis, healthy subjects and tissue biopsy samples were obtained from chronic periodontitis subjects and from gingiva of healthy subjects undergoing a crown lengthening procedure. mRNA expression of chemerin, chemR23 and CCRL2 was examined using qPCR.
The results showed that both qPCR and immunohistochemistry revealed higher expression of chemerin, chemR23 in tissues with periodontitis than in healthy tissues and there was a positive correlation between mRNA expression levels and clinical parameters. The authors concluded that chemerin plays an important role in the pathogenesis of periodontitis by causing chemo attraction of immune cells that direct chemR23 receptors to the site of inflammation.
Jose et al (2016)15 compared and evaluated the serum chemerin levels in healthy subjects and chronic generalised periodontitis patients. The authors categorised the subjects into Group-I (periodontally healthy); Group-II (chronic generalised periodontitis). Blood samples were collected and serum was obtained and assayed for the levels of chemerin using ELISA kit. The results showed a significantly greater serum levels of chemerin in chronic
periodontitis group compared to healthy subjects and positively correlated with periodontal disease severity. The authors concluded that chemerin is a potential biomarker of periodontal tissue destruction.
Balli et al (2016)16 investigated the changes in the levels of GCF chemerin and IL-6 in both obese / non-obese subjects with periodontitis following non-surgical periodontal therapy.
The authors categorised the subjects into (I) periodontally healthy without obesity (II) chronic periodontitis without obesity (III) periodontally healthy with obesity (IV) chronic periodontitis with obesity and subjects underwent non-surgical periodontal therapy. GCF samples were collected prior to therapy and 6 weeks thereafter and assessed for the levels of chemerin and IL-6 using ELISA kits. The results showed an increase in levels of chemerin and IL-6 in obese subjects compared to the non-obese and in subjects with chronic periodontitis compared to healthy subjects and decrease in levels following therapy and there was a positive correlation between chemerin and IL-6 in obese groups. The authors summarized that chemerin levels act as both diagnostic / prognostic indicators and play an important role in pathologic mechanisms that relate adipokines to both periodontal disease and obesity.
Özcan et al (2016)17 assessed the levels of some adipokines, pro-inflammatory cytokines and MMPs in periodontitis and healthy subjects and evaluated the changes after non- surgical therapy. The authors categorized the subjects into two groups: Chronic periodontitis and healthy. Unstimulated whole saliva was collected and the supernatant samples were obtained and assessed for the levels of visfatin, chemerin, progranulin, IL-1β, IL-8, MMP-8 and MMP-13 using ELISA kits. Salivary samples were collected at 3, 6 months after non- surgical therapy. The results showed increased levels of visfatin, chemerin, progranulin, IL-1β,
IL-8, MMP-8 and MMP-13 in periodontitis group compared to healthy subjects and their levels decreased following nonsurgical periodontal therapy. The authors concluded that the adipokines cause the periodontal tissue breakdown in periodontitis by increasing the expression of pro inflammatory cytokines and MMPs and added that visfatin, chemerin and MMP-8 in saliva can be considered as reliable biomarkers for monitoring the progress of the treatment of periodontal disease.
Seyma et al (2016)6 determined whether the GCF chemerin is a novel marker in chronic periodontitis patients with and without Type-II Diabetes Mellitus and the relationship between chemerin and IL-6 in healthy and chronic periodontitis patients with and without Type-II DM and evaluated the effects of non-surgical periodontal therapy on GCF chemerin levels. This study included 80 subjects categorised into healthy, Type-II DM, chronic periodontitis and chronic periodontitis with Type-II DM. Chronic periodontitis subjects underwent SRP and clinical parameters were recorded before and 6 weeks after SRP. GCF samples were collected and assessed for the levels of chemerin and IL-6 using ELISA kits. The results showed an increase in the level of GCF chemerin and IL-6 in chronic periodontitis subjects and chronic periodontitis with Type-II DM. GCF levels of chemerin and IL-6 decreased following non- surgical therapy. The authors concluded that GCF chemerin can be considered as an inflammatory marker for periodontal disease and Diabetes mellitus.
Patnaik et al (2015)12 evaluated the levels and correlated human chemerin in GCF and tear fluid in chronic periodontitis subjects with and without type-II Diabetes Mellitus. The authors categorised the subjects into three groups: Group-I (periodontally healthy), Group-II (Chronic periodontitis); Group-III (Type-II Diabetes Mellitus with chronic periodontitis). The
GCF and tear fluid samples were collected and assessed for the levels of human chemerin using ELISA kits. The results showed a significant increase in the human chemerin levels from group 1 to group 3 and a positive correlation was observed between the GCF, tear fluid values of inflammatory mediators with evaluated periodontal parameters.
Jing et al (2015)18 evaluated the serum chemerin levels and investigated the relationship between serum chemerin levels and bone mineral density in subjects with osteoporosis and healthy subjects. The authors categorised the subjects into two groups:
osteoporosis and healthy subjects based on the T-score. Fasting blood samples were taken for the measurement of serum chemerin levels by using ELISA. The results showed increased serum chemerin levels in subjects with osteoporosis than in healthy subjects and a negative correlation was observed between femoral and lumbar bone mineral density and chemerin in both the groups. The authors concluded that subjects with osteoporosis presented elevated levels of serum chemerin and BMD showed an inverse correlation with serum chemerin levels.
Bobbert et al (2015)19 determined whether chemerin is an independent risk factor for T2DM or related subtraits of glucose metabolism. In this study, a total of 440 subjects with Metabolic syndrome Berlin-Potsdam without Diabetes Mellitus were included. An oral glucose tolerance test (OGTT) was performed and fasting blood samples were taken prior to OGTT at baseline and follow-up observation. Plasma and serum were obtained for all the subjects and supernatant was obtained and the levels of chemerin were assayed using ELISA kit. The results showed chemerin significantly correlated with age, HOMA-IR, HbA1c, 2hr glucose, TG, waist-hip ratio and BMI. The chemerin levels was significantly elevated in persons with IFG and or IGT at baseline. Chemerin also predicted worsening of fasting glucose and HbA1c
(adjusted for age, sex, BMI, time of follow-up, WHR, cholesterol, TGL). The authors concluded that chemerin is a weak predictor of Type-II DM.
Fatima et al (2015)20 determined whether chemerin along with other inflammatory markers (TNF- and hs-CRP) can discriminate subjects with sub-clinical Diabetes. The authors categorised the subjects into healthy and subjects with Type-II DM. They were subjected to a 75g oral glucose tolerance test and were further classified as either newly diagnosed Diabetes (NDM) or healthy subjects. BMI were calculated and blood samples were collected and plasma was obtained and cytokine / adipokine levels were determined using ELISA kits. The results showed that the mean levels of chemerin and TNF- were highest in NDM group, followed by T2DM and control group. The hs-CRP levels were highest in T2DM compared to NDM group.
The cut off of 13.7 ng/ml of chemerin helps to discriminate 73% of NDM subjects with impaired glucose level with 91% and 96% of sensitivity and specificity respectively. The authors concluded that elevated serum chemerin in NDM group is a surrogate of impaired glucose metabolism in obese individuals.
Özcan et al (2014)10 investigated the salivary levels of visfatin, chemerin and progranulin and their relationship with periodontal health and disease. The authors categorised the subjects into healthy, gingivitis and periodontitis groups. Unstimulated whole saliva was collected and the supernatant samples were obtained and assessed for the levels of visfatin, chemerin, progranulin using ELISA kits. The concentrations of TNF- , SOD, Malonialdehyde (MDA), were also determined using ELISA kits. The results showed an increase in the levels of visfatin, chemerin and progranulin in subjects with periodontitis and gingivitis. There was a positive correction between visfatin and TNF- , PI, GI; progranulin and TNF- , SOD;
chemerin and GI, PD, CAL. The authors concluded that chemerin is a newly emerged candidate biomarker involved in the pathogenesis of several chronic inflammatory diseases and is used for screening purpose in large groups of people to evaluate the risk of destructive periodontal disease.
Zabel et al (2014)21 reviewed about the mechanism underlying chemerin regulation and its function in host defense. The authors discussed about the role of chemerin in immune cell migration, adipogenesis, osteoblastogenesis, angiogenesis, myogenesis glucose homeostasis, protective barrier and the regulation of chemerin expression by nuclear receptors / nuclear receptor ligands, metabolic signalling proteins and intermediates and pro- inflammatory cytokines. The authors also mentioned about the post-translational regulation of chemerin activity and summarized that chemerin acts as an antimicrobial protein and a leukocyte attractant for macrophages, dendritic cells (DC) and Natural Killer (NK) cells.
Roh et al (2007)22 examined the effects and mechanism of action of chemerin and its receptor in adipocytes. The authors obtained white adipose and non-adipose tissues from mice who were fed with standard diet and high fed diet. 3T3-L1 cells and total mRNA extraction and semi-quantitative RT-PCR was performed to measure levels of chemerin, chemerin R, PPAR-γ2, β-actin mRNA. The results showed that chemerin and chemR mRNA expression were highly expressed in adipose tissues and that their expression levels were up-regulated in mice fed with high fat diet. Both chemerin and chemR mRNA expression significantly increased during the differentiation of 3T3-L1 cells and human preadipocytes into adipocytes.
The authors concluded that chemerin regulates both adipocyte differentiation and lipid
metabolism and that adipokine chemerin regulates adipocyte function by autocrine / paracrine mechanism.
Materials and methods
PATIENT SELECTION
The study sample included 75 patients selected from the outpatient ward, Department of Periodontology, K.S.R. Institute of Dental Science and Research, Tiruchengode, Namakkal district, Tamil Nadu. Study protocol was explained to the patients and written informed consent and institutional ethical clearance was obtained.
INCLUSION CRITERIA
Patients diagnosed with generalized chronic periodontitis (male and female)
Patients with at least four teeth with a probing depth of ≥ 4mm
Lack of furcation involvement and mobility
Patients with Type II Diabetes Mellitus diagnosed and under medication for the same with no other Diabetic complications
EXCLUSION CRITERIA
Pregnant and lactating women
Patients with systemic diseases other than Type-II Diabetes Mellitus
Periodontal treatment within the past 6 months
Use of systemic antibiotics within the last 6 months
Smokers and pan chewers STUDY DESIGN
The study sample included 75 individuals. They were divided into three groups
Group 1 - Generalized chronic gingivitis
Group 2 - Generalized chronic periodontitis
Group 3 - Generalized chronic periodontitis with Type-II Diabetes Mellitus
The first group included 25 individuals who were systemically and periodontally healthy. The second group included 25 individuals who were systemically healthy with generalised chronic periodontitis and third group included 25 individuals with generalised chronic periodontitis with Type-II DM. The clinical parameters including Plaque index (PI), Gingival index (GI), Probing depth (PD), Clinical attachment level (CAL) and Body mass index (BMI) and haematological parameters like Random blood glucose (RBG), Glycated haemoglobin (HbA1c) levels were recorded and saliva samples were collected from all the subjects prior to non-surgical periodontal therapy. The levels of chemerin in saliva samples were analysed by using a sandwich enzyme-linked immunosorbent assay.
Armamentarium for in vivo examination
Mouth mirror
William’s graduated periodontal probe
Dental explorer
Cotton pliers
Cotton roll
Stainless steel tray
Gloves & Face mask
Armamentarium for unstimulated saliva collection
Plastic vials
Thermocol box
Dry ice
CLINICAL PARAMETERS:
1. Plaque Index - Loe’s modification (1967)23 2. Gingival Index - Loe’s modification (1967)23 3. Probing pocket depth (PPD)
4. Body Mass Index (BMI)
HAEMATOLOGICAL PARAMETER:
1. Random Blood glucose (RBG) 2. Glycated Hemoglobin (HbA1c)
PLAQUE INDEX (PlI):
The Plaque Index was described by Silness J and Loe H. in 1964 and modified by Loe H. in 1967. The plaque was assessed using a mouth mirror and dental explorer after air drying the teeth to assess plaque on the different areas namely mesiofacial, facial, distofacial and lingual surfaces.
Instruments used:
Mouth mirror and the dental explorer
The teeth were air dried and examined visually. When no plaque was visible an explorer was used on the surface. The explorer was passed across the surface in the cervical third and near the entrance to the gingival sulcus. The following scores were given.
Scores for Plaque Index
Score Criteria
0 No plaque.
1 A film of plaque adhering to the free gingival margin and adjacent area of the tooth. The plaque may be seen only by running a probe, across the tooth surface.
2 Moderate accumulation of soft deposits within the gingival pocket, on the gingival margin and / or adjacent tooth surface, which can be seen by the naked eye.
3 Abundance of soft matter within the gingival pocket and /or on the gingival margin and adjacent tooth surface.
Calculation of Pl I:
Pl I score for the area:
Each area (disto-facial, facial, mesio-facial, lingual) is assigned a score from 0 to 3.
Pl I score for a tooth:
The scores from the four areas of the tooth are added and then divided by four.
Pl I score for the individual:
The indices for each of the teeth are added and then divided by the total number of teeth examined. The scores range from 0 to 3.
Interpretation:
Excellent 0
Good 0.1-0.9
Fair 1-1.9
Poor 2-3
GINGIVAL INDEX (GI)
The Gingival Index (GI) was developed by Loe H. and Silness J. in 1963, solely for the purpose of assessing the severity of gingivitis and its location in four possible areas by examining only the qualitative changes (i.e., severity of the lesion) of the gingival soft tissue.
In 1967, Loe detailed the sequence of examination to include entire teeth instead of index teeth.
Instruments used:
Mouth mirror and the Periodontal probe.
The tissues surrounding each tooth were divided into four gingival scoring units:
mesiofacial papilla, facial margin, disto-facial papilla and the entire lingual gingival margin.
The teeth and gingiva should be dried lightly with a blast of air and /or cotton rolls. Each of the 4 gingival units were assessed and following scores were given.
Scores for Gingival Index
Score Criteria
0 Absence of inflammation / normal gingiva
1 Mild inflammation, slight change in color, slight edema; no bleeding on probing.
2 Moderate inflammation; moderate glazing, redness, edema and hypertrophy, bleeding on probing.
3 Severe inflammation; marked redness and hypertrophy, ulceration, tendency to spontaneous bleeding.
Calculation of GI:
GI Score for the area:
Each area (disto-facial, facial, mesio-facial, lingual) is assigned a score from 0 to 3.
GI Score for a tooth:
The scores from the four areas of the tooth are added and then divided by four.
GI score for the individual:
The indices for each of the teeth are added and then divided by the total number of teeth examined. The scores range from 0 to 3.
Interpretation:
Gingival scores Conditions
0.1-1.0 Mild gingivitis
1.1-2.0 Moderate gingivitis
2.1-3.0 Severe gingivitis
PROBING POCKET DEPTH (PPD):
Probing pocket depth was measured as the distance between the free gingival margin and the base of the pocket.
Instruments used:
Mouth mirror and William’s Periodontal probe.
CLINICAL ATTACHMENT LEVEL (CAL):
Clinical attachment level was measured as the distance from the CEJ to the base of the pocket.
BODY MASS INDEX (BMI):
BMI was measured using the following formula.
Weight (in kgs) (Height (in metres))2
HEMATOLOGICAL PARAMETER:
5 ml of blood was drawn from antecubital fossa under aseptic conditions and random blood glucose and glycated haemoglobin (HbA1c) were evaluated for all the subjects.
SALIVA SAMPLING:
Saliva samples were collected according to the unstimulated saliva collection procedure. All of the saliva samples were collected between 9 am and 12 pm to minimize diurnal variations associated with saliva sampling. Participants were asked to sit comfortably with head tilted forward and were asked to swallow their saliva first, and then to allow the saliva to drain passively for almost 10-15 minutes over the lower lip into the polypropylene tube before clinical measurements and approximately 2 ml saliva was collected. The samples were immediately frozen and stored at -70ºC until the biochemical analysis was performed.
ASSAY PROCEDURE:
The chemerin levels in the collected saliva samples were measured using Enzyme Linked Immunosorbent Assay (ELISA) kit (Human chemerin ELISA kit; Sincere Biotech co.
ltd., China). The assay was a quantitative sandwich ELISA technique.
ELISA is one of the immunoassay methods using antibodies to capture an antigen and an enzyme labelled antibody to estimate the amount of antigen. A Sandwich ELISA quantifies the amount of antigen between two layers of antibody. The desired antigen is captured by one antibody and bound to the plate. A second antibody binds the immobilized antigen for detection and quantification. The antigen must contain at least two non-overlapping epitope sites capable of binding different antibodies.
Human chemerin ELISA kit from bioassay technology laboratory was employed to analyse the chemerin levels in saliva.
MATERIALS USED TO ASSESS CHEMERIN LEVEL BY ELISA:
1. Precoated ELISA plate (Microtiter 96 wells) 2. 96-Well cover sheet
3. Standard solution 4. Standard diluent
5. HRP Conjugate reagent
6. Wash buffer concentrate (30X) 7. Chromogen solution A
8. Chromogen solution B
9. Stop Solution (6ml)
10. An incubator which can provide stable incubation conditions up to 37°C±0.5°C 11. Microplate reader capable of measuring absorbance at 450 nm
12. Precision pipettes to deliver 2 μl to 1 ml volumes 13. Adjustable 1-25 ml pipettes for reagent preparation 14. 100 ml and 1 litre graduated cylinders
15. Absorbent paper, Distilled / deionized water
16. Log-log graph paper or computer and software for ELISA data analysis 17. Tubes to prepare standard or sample dilutions
18. Container for wash solution ASSAY PROCEDURE
The stored frozen saliva samples were thawed before assay and they were refined by centrifugation using 3000 rpm for 5 minutes.
1. Samples were duplicated and the procedure was performed in two different kits.
2. All reagents and samples were brought to room temperature (18 - 25ºC) before use.
REAGENTS PREPARATION:
Preparation of standard: The 10 standard wells on the ELISA plates were coated and labelled from 1 to 10. First, standard 100µl & then standard diluent 50µl was added to first and second well and mixed and 100µl from the above wells were taken out and added to the third and fourth well separately and then standard diluent 50µl was added to third and fourth well and mixed and 50µl taken out from third and fourth well and discarded and 50µl was taken out
from third and fourth well and added to fifth and sixth well. Standard diluent 50µl was added to the fifth and sixth well and mixed. 50µl was taken out from fifth and sixth well and added to seventh and eighth well and standard diluent 50µl was added to seventh and eighth well and mix and again take out 50µl from the seventh and eighth well and added to ninth and tenth well. Standard diluent 50µl was added to ninth and tenth well and mixed and 50µl was taken out and discarded. After dilution, the total volume in each well was 50µl and the concentration was 9 ng/ml, 6 ng/ml, 3 ng/ml, 1.5 ng/ml, 0.75 ng/ml respectively.
3. Sample diluent 40µl was added to the testing sample well and later testing sample 10µl was added. Precaution was taken to add sample to the bottom of the pre-coated well and not to touch the well wall as far as possible and mix gently.
4. Samples were incubated for 30 min at 37°C after closing the plate with closure plate membrane.
5. Washing buffer was prepared: 30 fold wash solution diluted with Distilled water until 600 ml.
6. Closure plate membrane was uncovered, liquid was discarded and dried by swing and washing buffer was added to each well for 30 seconds and then drained and procedure was repeated 5 times and the dried.
7. HRP conjugate reagent 50µl was added to each well except the blank well.
8. Incubation was done for 30 minutes at 37°C after closing the plate with closure plate membrane.
9. After incubation, closure plate membrane was uncovered, liquid was discarded and dried by swing and washing buffer was added to each well, still for 30 seconds and then drained and procedure was repeated for 5 times and dried.
10.TMB chromogen solution A 50µl and TMB chromogen solution B 50µl was added to each well and gently mixed and care was taken to evade the light and continued to be for 15 minutes at 37°C.
11.Stop solution 50µl was added to each well and the reaction was stopped. (Blue colour changes to yellow colour immediately).
12.Optical density (OD) was measured under 450 nm wavelength using ELISA reader and was carried out within 15 minutes after adding the stop solution.
13.The standard curve was plotted on log-log graph paper and using Sigma plot software, with standard concentration on the x-axis and absorbance on the y axis.
14.The best-fit straight line was drawn through the standard points. The equation was derived to convert the chemerin values from nm to ng/ml.
FORMULA: y = 0.17x + 0.075 x = chemerin values in nm
y = 0.0179x + 0.0759 R² = 0.8264
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2
0.75 1.5 3 6 9
Series1 Linear (Series1)
Absorbance at 450 nm
Concentration (ng/ml)
FORMULA: y = 0.044x + 0.071 x = chemerin values in nm
y = 0.0441x + 0.071 R² = 0.9827
0 0.05 0.1 0.15 0.2 0.25 0.3
1 2 3 4
Series1 Linear (Series1)
Absorbance at 450 nm
Concentration (ng/ml)
Fig. 4 Armamentarium for saliva collection
Fig. 5 Collection of salivary sample
5A. Collection of saliva by passive drooling method 5B. Collected saliva
Fig. 6 GROUP-I (GCG) 6A. Front view
6C. Right lateral view 6B. Left lateral view
6D. Lingual view 6E. Palatal view
Fig. 7 GROUP-II (GCP) 7A. Front view
7B. Left lateral view 7C. Right lateral view
7D. Lingual view 7E. Palatal view
Fig. 8 GROUP-III (GCP with T2DM) 8A. Front view
8B. Right lateral view 8C. Left lateral view
8D. Palatal view 8E. Lingual view
9A. ELISA Kit
9B. ELISA 96 well plate
Fig. 9 Salivary Chemerin analysis using ELISA
9C. Samples for processing
9D. Centrifugation of saliva samples
9H. Reading of ELISA plate 9E. Preparation of Standards
9F. Addition of samples to the wells coated with primary antibody
9G. ELISA plates for processing
Statistical analysis
Salivary chemerin levels in healthy subjects (25) and in subjects with chronic periodontitis with (25) / without Type-II DM (25) were evaluated. Data were analysed using the statistical package for the Social Sciences (SPSS, ver.18.0; SPSS Inc., Chicago, IL, USA).
Shapiro Wilks test was used to assess normality of the obtained data. Data were found to be in non-normal distribution hence descriptive statistics were obtained. Comparison of variables between the study groups were performed using Kruskal Wallis H test. Kendall's tau correlation coefficient was employed to find correlation between overall volumes of chemerin with HbA1c levels and CAL.
P VALUE:
The P value or calculated probability is the estimated probability of rejecting the null hypothesis of a study question when that hypothesis is true. Differences between the populations were considered significant when p < 0.05.
Results
Table No. 1 Distribution of age (in years) between the groups
Table No. 2 Distribution of gender between the groups
Gender
Total
Male Female
Total Count 39 36 75
% within gr 52.0% 48.0% 100.0%
% of Total 52.0% 48.0% 100.0%
Group Mean (in years)
Standard deviation
Median Interquartile range
Kruskal Wallis test p value
Group-I 23.44 3.852 22.0 1 0.000*
Group-II 52.36 9.652 52 14
Group-III 57.08 10.428 56 12
Table No. 3 Comparison of Body mass index (BMI) between the groups
Graph 3a. Comparison of Mean Body Mass Index (BMI) between the groups
22.862
24.958
26.134
21 22 23 24 25 26 27
grp 1 grp 2 grp 3
Body Mass Index (BMI)
grp 1 grp 2 grp 3
Groups N Mean Std. Deviation Mean rank Median IQR P value Group-I 25 22.8620 3.95075 29.30 21.92 6.80 0.000*
Group-II 25 24.9580 5.29104 38.84 24.37 9.05 Group-III 25 26.1340 3.23064 45.86 26.22 5.23
Total 75 24.6513 4.40259
Table No.4 Comparison of Mean Random blood glucose (RBG), Glycated haemoglobin (HbA1c) between the groups
N Mean Std. Deviation
Mean
rank Median IQR
Kruskal wallis test Clinical
Parameters
p value
RBG Grp 1 25 90.52 8.357 32.18 91 12
Grp 2 25 85.20 10.251 23.10 85 16 0.000*
Grp 3 25 158.76 62.224 58.72 145 68
Total 75 111.49 49.491
HbA1c Grp 1 25 5.3440 .40423 20.58 5.3 0.5 0.000*
Grp 2 25 5.6920 .39887 32.10 5.7 0.6
Grp 3 25 9.1920 2.27887 61.32 9.1 4.0
Total 75 6.7427 2.20216
Graph 4a. Comparison of Mean Random blood glucose (RBG) levels between the groups
Graph 4b. Comparison of Mean Glycated Hemoglobin (HbA1c) levels between the groups
90.52 85.2
158.76
0 20 40 60 80 100 120 140 160 180
grp 1 grp 2 grp 3
RBG
grp 1 grp 2 grp 3
5.344 5.692
9.192
0 1 2 3 4 5 6 7 8 9 10
grp 1 grp 2 grp 3
HbA1c
grp 1 grp 2 grp 3
Table No. 5 Comparison of PI, GI, PPD and CAL between the groups
N Mean
Std.
Deviation
Mean
rank Median IQR
Kruskal wallis test Clinical
parameters
p value PI Grp 1 25 1.6416 .13111 13.88 1.62 0.26
Grp 2 25 2.1348 .26544 49.96 2.14 0.20 0.000*
Grp 3 25 2.1032 .11968 50.16 2.12 0.19 Total 75 1.9599 .29080
GI Grp 1 25 1.6980 .15626 13.50 1.74 0.28
Grp 2 25 2.0448 .17633 52.28 2.0 0.0 0.000*
Grp 3 25 1.9940 .05553 48.22 2.0 0.0
Total 75 1.9123 .20665
PPD Grp 1 25 2.1032 .23203 13.16 2.14 0.12
Grp 2 25 3.3112 .61711 53.88 3.09 0.99 0.000*
Grp 3 25 3.0966 .82200 46.96 2.89 0.42 Total 75 2.8370 .80048
CAL Grp 1 25 .2079 .03676 13.02 0.21 0.06
Grp 2 25 4.4464 1.01286 48.48 4.35 1.41 0.000*
Grp 3 25 4.6803 1.30962 52.50 4.78 1.06 Total 75 3.1115 2.27402
Graph 5a. Comparison of Mean Plaque Index (PI) between the groups
Graph 5b. Comparison of Mean Gingival Index (GI) between the groups
1.6416
2.1348 2.1032
0 0.5 1 1.5 2 2.5
grp 1 grp 2 grp 3
PI
grp 1 grp 2 grp 3
1.698
2.0448 1.994
0 0.5 1 1.5 2 2.5
grp 1 grp 2 grp 3
GI
grp 1 grp 2 grp 3
Graph 5c. Comparison of Mean Probing Pocket Depth (PPD) between the groups
Graph 5d. Comparison of Mean Clinical Attachment level (CAL) between the groups
2.1032
3.3112
3.0966
0 0.5 1 1.5 2 2.5 3 3.5
grp 1 grp 2 grp 3
PPD
grp 1 grp 2 grp 3
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
grp 1 grp 2 grp 3
CAL
grp 1 grp 2 grp 3
Table No. 6 Comparison of mean chemerin values between the groups
Graph 6a. Comparison of Mean chemerin levels between the groups
43.8068
47.8733
71.4742
0 10 20 30 40 50 60 70 80
grp 1 grp 2 grp 3
Chemerin levels
grp 1 grp 2 grp 3
N Mean Std. Deviation
Mean
rank Median IQR
Kruskal wallis test
p value Chemerin
values
Grp 1 25 43.8068 22.89437 31.72 35.88 35.30
Grp 2 25 47.8733 18.01104 39.18 45.0 22.19 0.172 Grp 3 25 71.4742 72.45908 43.10 44.09 32.27
Total 75 54.3848 46.13895
Table No. 7 Correlation between chemerin and HbA1c, CAL Variables Correlation Coefficient
(r value)
P value
Chemerin and HbA1C 0.143 0.077(NS)
Chemerin and CAL 0.125 0.115 (NS)
Kendall's tau’s correlation test NS-Not significant
Graph 7a. Correlation between chemerin and HbA1c
Graph 7b. Correlation between chemerin and CAL
0 10 20 30 40 50 60 70 80
grp 1 grp 2 grp 3
Correlation between chemerin and HbA1c
chemerin values HbA1C
0 10 20 30 40 50 60 70 80
grp 1 grp 2 grp 3
Correlation between chemerin and CAL
chemerin values CAL
Table No. 1 & No. 2 shows the mean and standard deviation of age and gender between the three groups. Kruskal Wallis test has been used to compare the mean values. The mean age of subjects in group-I, II and III was 23.44 ± 3.852, 52.36 ± 9.652 and 57.08 ± 10.428 respectively. Among the population studied, 52.0% of the subjects were males and 48% of subjects were females.
Table No. 3 & graphs 3a show the mean and standard deviation of the Body Mass Index (BMI) of healthy subjects and in subjects with generalised chronic periodontitis with / without Type-II DM. The mean BMI were 22.8620 ± 3.95075, 24.9580 ± 5.29104 and 26.1340
± 3.23064 in group-I, group-II and group-III respectively and showed a statistical significant higher BMI scores in group-III and group-II compared to group-I (p < 0.05).
Table No. 4 & graphs 4a, 4b show the mean and standard deviation of the Random Blood Glucose (RBG) and glycated haemoglobin (HbA1c) levels of healthy subjects, subjects with generalised chronic periodontitis with / without Type-II DM. The mean RBG values were 90.52 ± 8.357, 85.20 ± 10.251 and 158.76 ± 62.224 in group-I, group-II and group-III respectively and showed significant elevated values in group-III compared to group-II and group-I (p < 0.05). The mean HbA1c values were 5.3440 ± .40423, 5.6920 ± .39887 and 9.1920
± 2.27887 in group-I, group-II and group-III respectively and showed a statistical significant higher HbA1c values in group-III compared to group-II and group-I (p < 0.05).
Table No. 5 & graphs 5a, 5b, 5c & 5d show the mean and standard deviation of PI, GI, PPD, CAL in three groups. Kruskal Wallis test has been used to compare the mean values between the groups and the mean PI, GI, PPD and CAL were elevated in group-III and group- II and least in group-I and showed statistical significance. (p < 0.05).
Table No. 6 and graph 6a show the mean salivary chemerin levels in group-I, group- II and group-III. Higher levels were observed in group-III (71.4742 ng/ml) compared to group-II (47.8733 ng/ml) and group-I (43.8068 ng/ml). The differences were not statistically significant (p < 0.172).
Table No. 7 and graphs 7a & 7b show the correlation coefficient (r value) and p-value correlating salivary chemerin levels with glycated haemoglobin (HbA1c) and Clinical attachment level (CAL). Kendall tau’s correlation test has been used and there is no statistically significant correlation between chemerin and HbA1c (p - 0.077); chemerin and CAL (p - 0.115).
Discussion
Periodontitis is a chronic inflammatory disease, which if left untreated may lead to irreversible destruction of periodontal tissues. Type-II DM is a pathological condition with multiple disorders like dyslipidaemia, visceral obesity and hyperglycemia. Dyslipidemia is one of the most common cause of DM. Some active molecules involved in the pathogenesis of periodontitis are known to disrupt the systemic lipid metabolism. Unhealthy adipose tissue metabolism in Type-II DM might affect other organs due to the creation of adipokines, TNF-
, IL-6 and other pro-inflammatory cytokines.
Adipose tissue was thought to be an inert organ for many years and currently adipose tissue is considered as a complex and metabolically active endocrine organ that secretes numerous immunomodulatory factors that control lipid metabolism and inflammation.
Chemerin is a recently identified adipokine that participates in both metabolic and immune dysfunction. Evidence shows that inflammatory cytokines play a role in chemerin regulation within adipose tissue. The abnormal production of chemerin may elicit or intensify the progression of Type-II DM and periodontitis. Moreover, periodontitis contributes to the undue expression of chemerin which in turn hastens disproportionate lipid metabolism in DM.
The aim and objective of my study were as follows: (1) To evaluate the salivary levels of chemerin in three groups i. systemically and periodontally healthy subjects (Group-I) ii.
systemically healthy with chronic periodontitis (Group-II) iii. Type II Diabetes Mellitus with chronic periodontitis (Group-III) (2) To compare the salivary levels of chemerin among systemically and periodontally healthy subjects, subjects with CP with / without Type II DM.
(3) To correlate the salivary levels of chemerin with Glycated Hemoglobin (HbA1c) and Clinical Attachment level (CAL) in systemically and periodontally healthy subjects, subjects with chronic periodontitis with / without Type II DM. 75 subjects were chosen and categorised into three groups based on their periodontal status and a positive / negative diabetic history.
The mean age of subjects in Group - I, II and III was 23.44 ± 3.852, 52.36 ± 9.652 and 57.08 ± 10.428 respectively. Among the population studied, 52.0% of the subjects were males and 48% of subjects were females. Clinical parameters including plaque index (PI), gingival index (GI), probing pocket depth (PPD), Body Mass Index (BMI) and haematological parameters like Random Blood Glucose (RBG) and glycated haemoglobin (HbA1c) and salivary chemerin levels were evaluated. The mean PI, GI, PPD and CAL were elevated in group-III and group-II and least in group-I and showed statistical significance (p < 0.05). The mean BMI were 22.8620 ± 3.95075, 24.9580 ± 5.29104 and 26.1340 ± 3.23064 in group-I, group-II and group-III respectively and showed a statistical significant higher BMI scores in group-III and group-II compared to group-I (p < 0.05). HbA1c and RBG values were significantly higher in chronic periodontitis subjects with Type-II DM compared to the other subjects.
Clinical parameters assess the patient’s gingival and periodontal status. PI provides an assessment of local factors as well patient’s oral hygiene maintenance. GI determines the inflammatory changes in the gingiva. Probing depth (PD) is useful in the assessment of the depths of the periodontal pockets which are the principal habitats of pathogens. CAL reveals the extent of tissue destruction. Clinical parameters can only assess the past disease activity and not the current disease status. Hence biomarkers help to determine the presence of current disease activity, predict the sites vulnerable for future breakdown and evaluate the response to periodontal therapy, thereby improving the clinical management of periodontal patients.
The present study aims at assessing the role of chemerin as a biomarker by evaluating and comparing its levels in healthy subjects and in subjects with chronic periodontitis with / without Type-II DM.
Chemerin is a recently identified adipokine expressed in the liver, adipose tissue, epithelial cells, endothelium, fibroblasts and keratinocytes that plays an important role in adipocyte differentiation and development. The role of chemerin in inflammation and metabolic syndrome links its pathophysiological mechanism with insulin resistance and body fat accumulation. Hence, chemerin may be a link between obesity and development of Type- II DM.5,24 There has been a clear link manifesting the influence of chemerin in the pathogenesis of chronic periodontitis25 and Type-II DM24, providing a two-way relationship between them.
The production of pro-inflammatory cytokines like IL-1β, IL-6 and TNF- play a role in the chemerin release from adipose tissue. Both IL-1β and TNF- induced chemerin mRNA expression and secretion from adipocytes attributes to the increase in the serum chemerin levels in DM and periodontitis where there is an increase in inflammatory mediators.
Unstimulated whole saliva has been used as adiagnostic fluid as it is easy to collect, non-invasive, and reliable. The whole saliva represents a sample with contribution from all periodontal sites and also includes some molecules originating from GCF and is produced locally in the oral cavity. Analysis of biomarkers in saliva provides overall assessment of disease status, in contrast to the site specific GCF analysis. Majority of the constituents present in the saliva, enter saliva from blood by passing through spaces between cells by transcellular, paracellular routes. Hence, most compounds found in blood are also present in saliva; hence saliva is functionally equivalent to serum in reflecting the physiological state of body.
In the present study, the mean salivary chemerin levels were remarkably elevated in group-III (71.4742 ng/ml) compared to group-II (47.8733 ng/ml) and group-I (43.8068 ng/ml).
The difference observed was not statistically significant (p < 0.172). On the contrary, the study carried out by Dogan et al (2016)6 reported elevated levels of chemerin in subjects with chronic periodontitis with / without Type-II DM compared to healthy subjects.
