Inflamm. Res. (2014) 63:317–323 DOI 10.1007/s00011-013-0703-3
Inflammation Research
ORIGINAL RESEARCH PAPER
Correlation of human S100A12 (EN-RAGE) and high-sensitivity C-reactive protein as gingival crevicular fluid and serum markers of inflammation in chronic periodontitis and type 2 diabetes A. R. Pradeep • Santosh S. Martande • Sonender Pal Singh Deepak Kumar Suke • Arjun P. Raju • Savitha B. Naik
•
Received: 30 July 2013 / Revised: 21 November 2013 / Accepted: 18 December 2013 / Published online: 31 December 2013 Ó Springer Basel 2013
Abstract Objective The aim of the present study was to evaluate the levels and correlation of human S100A12 and highsensitivity C-reactive protein (hs-CRP) in gingival crevicular fluid (GCF) and serum in chronic periodontitis (CP) subjects with and without type 2 diabetes mellitus (DM). Materials and methods A total of 44 subjects were divided into three groups: group 1 had 10 periodontally healthy subjects, group 2 consisted of 17 CP subjects and group 3 had 17 type 2 DM subjects with CP. GCF and serum levels of human S100A12 and hs-CRP were quantified using enzyme-linked immunosorbent assay and immunoturbidimetric analysis, respectively. The clinical outcomes evaluated were gingival index, probing depth and clinical attachment level and the correlations of the two inflammatory mediators with clinical parameters were evaluated. Results Both human S100A12 and hs-CRP levels increased from group 1 to group 2 to group 3. The GCF and serum values of both these inflammatory mediators corre-
Responsible Editor: Ji Zhang. A. R. Pradeep (&) S. S. Martande S. P. Singh D. K. Suke Department of Periodontics, Government Dental College and Research Institute, Bangalore 560002, Karnataka, India e-mail: [email protected] A. P. Raju Bangalore Medical College and Research Institute, Bangalore 560002, Karnataka, India S. B. Naik Department of Conservative Dentistry and Endodontics, Government Dental College and Research Institute, Bangalore 560002, Karnataka, India
lated positively with each other and with the periodontal parameters evaluated (p \ 0.05). Conclusion Human S100A12 and hs-CRP can be considered as possible GCF and serum markers of inflammatory activity in CP and DM. Keywords Chronic periodontitis Inflammation Diabetes mellitus Gingival crevicular fluid Biomarkers
Introduction Chronic periodontitis (CP) is a multi-factorial infection elicited by a complex group of bacterial species that interact with host tissues and cells leading to the release of a broad array of inflammatory mediators, some of which lead to destruction of the periodontal structures, including the tooth supporting tissues, alveolar bone, and periodontal ligament [1]. The inflammatory host response is a fundamental feature of periodontal disorder and is regarded as the primary cause for periodontal breakdown [2]. Polymorphonuclear neutrophils (PMNs) perform an innate cellular host defense role within the gingival crevice and overall oral cavity. These neutrophils make up half of the leukocytes infiltrating the junctional epithelium and 90 % of the leukocytes isolated from crevicular fluid, thus suggesting a key role in periodontal inflammation [3]. Diabetes mellitus (DM) is a metabolic disorder which, due to disturbances in insulin production, leads to abnormal fat, sugar and protein metabolism and resultant hyperglycaemia which may lead to various systemic complications [4]. Diabetic subjects have a two- to fourfold higher risk of developing severe periodontitis and accelerated periodontal disease progression [5]. Periodontitis, in fact, has been referred as the sixth complication of
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DM [6]. Diabetic subjects with periodontal infection have a much higher risk of worsening glycaemic control over time compared with diabetic subjects without periodontitis, thus suggesting a two-way street between these diseases [7]. Human S100A12, also known as calgranulin C, MRP6 or extracellular newly identified receptor for advanced glycation endproducts (EN-RAGE) binding protein, is a calcium-binding proinflammatory protein predominantly secreted by PMNs [8]. S100A12 was first described in humans by Guignard and colleagues in 1995 [9]. S100A12 is a member of the S100 multigene family of calciumbinding proteins which play a major role in the Ca21dependent regulation of a variety of intracellular activities, including protein phosphorylation, enzyme activities, cell proliferation and differentiation, intracellular Ca21 homeostasis, inflammation and protection from oxidative cell damage [10]. It exhibits several proinflammatory properties, and has a chemotactic activity comparable to that of other strongly chemotactic agents [11]. When secreted extracellularly from neutrophils, S100A12 contributes to innate immune responses. The proinflammatory activities of S100A12 include chemotactic activity and activation of intracellular signalling cascades which leads to production of various cytokines and induction of oxidative stress [12, 13]. Intracellular signalling via protein kinases induces nuclear factor (NF)-jB-dependent secretion of different cytokines [14]. Bovine S100A12 is a ligand for the receptor for advanced glycation end products (RAGE) expressed on macrophages, endothelium and lymphocytes [15]. It is hypothesized that binding of S100A12 to RAGE mediates the proinflammatory properties of this protein [15]. S100A12 is thought to play an important role in local inflammatory responses and serum S100A12 levels are elevated in chronic inflammatory diseases like rheumatoid arthritis and psoriatic arthritis [16], inflammatory bowel disease [17] and cystic fibrosis [18]. The other members of the S100 family have been implicated in the inflammatory periodontal diseases. S100A2, S100A8 and S100A9 were found to be significantly elevated in gingival tissues of gingivitis and moderate and severe chronic periodontitis [19]. Furthermore, S100A2 protein levels were found to be increased in gingival crevicular fluid (GCF) of gingivitis and chronic periodontitis analysed by enzyme-linked immunosorbent assay (ELISA), thus suggesting S100A2 as a potential biomarker of inflammatory periodontal disease [19]. C-reactive protein (CRP) is an acute-phase reactant released in response to inflammatory cytokines, interleukin (IL-6), IL-1, and tumor necrosis factor-alpha (TNF-a). CRP has several proinflammatory actions and circulating CRP levels are a marker of systemic inflammation and are associated with periodontal disease [20]. Fasting plasma glucose, a parameter of diabetic status, is a significant
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determinant of CRP and IL-6 plasma concentrations [21]. Plasma S100A12 and hs-CRP levels are elevated in subjects with type 2 diabetes, suggesting that S100A12 and hsCRP levels are regulated by factors related to subclinical inflammation and glucose control in type 2 diabetes [22]. To date, no study has reported human S100A12 levels in GCF and serum and its correlation with hs-CRP levels in CP subjects with and without type 2 DM. In this context, this clinico-biochemical study was designed to assess the levels of human S100A12 in GCF and serum and its correlation with hs-CRP levels in CP subjects with and without type 2 DM.
Materials and methods This was a 3-month-long cross-sectional study performed from November 2012 to February 2013. The study group consisted of 44 age- and sex-balanced individuals (22 males and 22 females, aged 25–45 years) attending the outpatient section, Department of Periodontics, Government Dental College and Research Institute (GDCRI), Bangalore, India. Written informed consent was obtained from all the subjects participating in the study. The ethical clearance was approved by the Institutional Ethical Committee and Review Board, GDCRI. Inclusion and exclusion criteria The inclusion criteria were: subjects aged 25–45 years; presence of at least 20 natural teeth, subjects with a diagnosis of CP based on clinical parameters like probing depth (PD), clinical attachment level (CAL) [23], gingival index (GI) [24], body mass index (BMI) in the range of 18.5–22.9 kg/m2 and waist circumference\90 cm (men) or \80 cm (women) (WHO, 2004) [25]; subjects should not have received periodontal therapy within preceding 6 months; well-controlled type 2 diabetic patients classified based on criteria given by American Diabetic Association (ADA) in 2011 and glycated haemoglobin levels [26]. Patients with: (1) aggressive periodontitis, (2) hypertension and other cardiovascular diseases, (3) smoking tobacco in any form, (4) gross oral pathology, (5) rheumatoid arthritis, (6) tumors, (7) any other systemic disease that can alter the course of periodontal disease, (8) any course of medication affecting periodontal status, or (9) received periodontal therapy in the preceding 6 months were excluded from the study. Subject grouping Participants were categorized into three groups based on the GI, pocket PD, CAL and radiographic evidence of bone
S100A12 and hs-CRP in chronic periodontitis and diabetes
loss. Group 1 (healthy) consisted of ten individuals with clinically healthy periodontium, GI = 0 (absence of clinical inflammation), PD B 3 mm and CAL = 0, with no evidence of bone loss on radiographs. Group 2 (CP without type 2 DM) consisted of 17 individuals who had signs of clinical inflammation, GI [ 1, PD C 5 mm and CAL C 3 mm, with radiographic evidence of bone loss. Group 3 (type 2 DM among individuals with CP) consisted of 17 individuals who had signs of clinical inflammation, GI [ 1, PD C 5 mm, CAL C 3 mm and haemoglobin A1c (HbA1c) B7 % with radiographic evidence of bone loss. Only well-controlled (HbA1c B 7 %) type 2 DM individuals were selected based on American Diabetes Association’s criteria for diagnosis of diabetes [26]. Clinical evaluation of subjects Group allocation and sample site selection in each subject was performed by the chief coordinator (A.R.P.). A thorough case history was taken for each subject. Each subject then went through a full-mouth periodontal probing and charting and BMI charting procedure according to the World Health Organization guidelines [25]. A calibrated examiner (S.S.M.) performed the clinical evaluation, measuring the clinical parameters including PD, CAL and GI using a periodontal probe (University of North Carolina-15 periodontal probe, Hu-Friedy, Chicago, IL, USA). 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 (S.S.M.) performed the radiographic evaluation and collected the GCF samples. Site selection and GCF collection GCF sample collection was done from most inflammed sites. Two test sites for GCF sample collection were selected based on the highest scored sites. In group 2 and group 3 subjects, the two sites showing the greatest CAL and signs of inflammation, along with radiographic confirmation of bone loss, were selected for sampling. One of the two sites selected per subject was used for hs-CRP and the other for S100A12 analysis. In the healthy group, to standardize site selection and obtain adequate fluid volume, sampling was predetermined to be from the mesio-buccal region of the maxillary right first molar, in the absence of which the left first molar was sampled. Initially the selected site was cleaned, isolated and airdried using sterile cotton rolls and the supragingival
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plaque was removed gently using a Gracey curette (Universal Gracey Curette #4R/4L, Hu-Friedy) so as to avoid contamination of the paper strips (Periopaper, Ora Flow Inc., Amityville, NY, USA). The paper strips were placed gently at the entrance of the gingival sulcus/crevice until light resistance was felt [27], care being taken to avoid mechanical injury, and left in place for 60 s. The absorbed GCF volume of each strip was determined by electronic impedance (Periotron 8000, ProFlow Inc., Amityville, NY, USA). 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 per site that absorbed GCF from each subject were pooled and were immediately transferred to microcentrifuge tubes (premarked with the biomarker name) containing 400 ll of phosphate buffered 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 (S.P.S.). Blood collection Blood collection was done at the same appointment for the purpose of serum sample collection. Two milliliters of blood were collected from the antecubital fossa by venipuncture using a 20-gauge needle with a 2-ml syringe and immediately transferred to the laboratory. The blood sample was allowed to clot at room temperature and after 1 h the serum was separated from the blood by centrifuging at 3,000g for 5 min. The serum was immediately transferred to a plastic vial and stored at -70 °C until the time of assay. Human S100A12 analysis The samples were assayed for S100A12 using ELISA according to the manufacturer’s instructions. The GCF sample tubes were first homogenized for 30 s and then centrifuged for 5 min at 1,500g to elute. The elute was used as sample for ELISA estimation from GCF samples. Each sample was assayed using a commercially available ELISA kit (Human S100A12 Elisa kit, USCN Life Sciences, USA) in accordance with the manufacturer’s instructions. Colour development was monitored using a microplate reader until an optimum optical density was reached, a stop solution was added and the optical density was read at 450 nm. The total S100A12 was determined in nanograms (ng), and the calculation of the concentration in each sample was performed by dividing the amount of S00A12 by the volume of sample (ng/ml).
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Table 1 Descriptive statistics of study population (mean ± SD) Study group
Group 1 (n = 10)
Group 2 (n = 17)
Group 3 (n = 17)
Age (years)
39.30 ± 3.52
38.35 ± 3.20
40.88 ± 4.44
Sex (M/F)
5/5
9/8
8/9
GI
–
1.84 ± 0.23
2.16 ± 0.31
PPD (mm)
2.10 ± 0.73
6.12 ± 0.92
6.88 ± 0.99
CAL (mm)
0
5.71 ± 0.68
6.35 ± 0.93
Serum S100A12 (ng/ml)
32.10 ± 10.02 113.29 ± 8.93 127.65 ± 10.4
GCF S100A12 (ng/ml)
38.10 ± 11.37 124.71 ± 8.16 143.35 ± 10.6
Serum hs-CRP (mg/l)
2.05 ± 0.78
3.44 ± 0.38
4.47 ± 0.77
GCF hs-CRP (mg/l) 0.49 ± 0.25
0.85 ± 0.17
0.98 ± 0.20
hs-CRP analysis The samples for CRP were measured immunoturbidimetrically. The microcentrifuge tubes containing the Periopaper strips and plastic vials containing serum were transferred to the lab for immunoturbidimetric analysis. Serum was used undiluted. The measuring range of CRP was 0–220 mg/l, the normal value of CRP being 0–5 mg/l.
Statistical analysis Power calculations were performed before the study was initiated and the sample size was selected based on the previous study [28]. Sample size and grouping was based on the power of the study and the 95 % confidence interval (p \ 0.05). Statistical analysis was done using SPSS statistical software (SPSS version 16.0, Chicago, IL, USA). Analysis of variance (ANOVA) was carried out for a comparison of GCF and serum hs-CRP and S100A12 levels between the groups. Using Pearson’s correlation coefficient, the relationships between hs-CRP and S100A12 concentration and the clinical parameters were analyzed. The intra-group correlation of serum and GCF concentrations of hs-CRP and S100A12 was also performed using Pearson’s correlation coefficient. Pair-wise comparisons between groups for S100A12 and hs-CRP concentrations in GCF and serum were carried out by Scheff’s test. p values \0.05 were considered statistically significant. The mean intraexaminer standard deviation of differences in repeated PD measurements and CAL measurements was obtained using single passes of measurements with a UNC-15 probe (correlation coefficients between duplicate measurements; r = 0.95).
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Results Table 1 gives the descriptive statistics (mean ± SD) of the study population. The mean S100A12 and hs-CRP concentrations in both serum and GCF were highest for group 3, followed by group 2, and least in group 1. ANOVA was carried out to find out the equality of means between the three groups (Table 2). A significant difference in the serum and GCF levels of S100A12 and hs-CRP was found between the three groups. The Pearson correlation coefficient test was applied to evaluate the correlation, and statistically significant correlations existed between the serum levels of S100A12 and the serum levels of hs-CRP, and also the GCF values between the two. Table 3 shows the correlation coefficients and p values. Table 4 demonstrates the correlation between GCF and serum values of S100A12 and hs-CRP and the clinical parameters. The serum and GCF levels of S100A12 and that of hs-CRP were found to be 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 5 demonstrates pair-wise comparisons between groups for S100A12 and hs-CRP concentrations in GCF and serum. The comparisons were statistically significant for GCF and serum S100A12 and serum hs-CRP levels between all groups. While GCF hs-CRP levels showed statistical significance between groups 1 and 2 and groups 1 and 3, the difference was not significant between groups 2 and 3.
Discussion This study aimed at evaluating the GCF and serum levels of S100A12 and hs-CRP in CP with and without association of type 2 DM. The results of the study indicated an increase in the GCF and serum levels of both S100A12 and hs-CRP from healthy 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 both S100A12 and hs-CRP can be considered as potential biomarkers for inflammatory periodontal disease and type 2 diabetes. Human S100A12 or EN-RAGE, a calcium-binding proinflammatory protein predominantly secreted by PMNs, is implicated in the innate immune response during early inflammation [9]. The interaction of S100A12 with its multi-ligand receptor RAGE and soluble receptor sRAGE plays a central role in proinflammatory properties of this protein [15]. The S100 group of proteins are overall
S100A12 and hs-CRP in chronic periodontitis and diabetes
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Table 2 Results of ANOVA comparing the mean serum and GCF S100A12 and hs-CRP concentrations between the three groups Groups
S100A12
Hs-CRP
Serum
GCF
Serum
GCF
F value
p value
F value
p value
F value
p value
F value
p value
324.71
\0.001*
376.92
\0.001*
43.43
\0.001*
17.73
\0.001*
Group 1 Group 2 Group 3 * Significant at p \ 0.001
Table 3 Correlation of serum and GCF S100A12 and hs-CRP in each group using Spearman’s rank correlation coefficient test Group
Serum Correlation coefficient
Group 1 Group 2 Group 3
0.970 0.869
Table 5 Pair-wise comparisons using Scheff’s test for S100A12 (ng/ ml) and hs-CRP (mg/l) concentrations in GCF and serum
GCF p value \0.001* \0.001*
0.680
0.006*
Correlation coefficient 0.641 0.794 0.822
Study groups p value 0.046*
S100A12 (GCF)
0.012* \0.001*
S100A12 (serum)
* Significant at p \ 0.05 hs-CRP (GCF) Table 4 Relationship of S100A12 and hs-CRP to clinical parameters Parameters
Group 1
Group 2
Group 3
S100A12 Serum
GCF
GI
–
PD
0.007*
CAL
–
GI
–
PD
0.006*
CAL
–
0.004*
0.002*
\0.001*
\0.001*
0.012*
\0.001*
0.024*
0.006*
\0.001*
\0.001*
0.003*
\0.001*
hs-CRP Serum
GCF
GI
–
0.004*
0.002*
PD
0.002*
\0.001*
\0.001*
CAL GI
– –
\0.001* 0.016*
0.002* 0.062
PD
0.004*
\0.001*
0.004*
CAL
–
\0.001*
\0.001*
* Significant at p \ 0.05
involved in various inflammatory conditions; S100A12 is thought to play an important role in local inflammatory responses and serum S100A12 levels are elevated in chronic inflammatory diseases [16–18]. S100A12 and CRP levels were found to be increased in various conditions like rheumatoid arthritis and psoriatic arthritis [16], inflammatory bowel disease [17] and cystic fibrosis [18]. Thus, the increase in GCF and serum concentrations of both S100A12 and hs-CRP in CP and type 2 DM in the present study can be attributed to the proinflammatory properties of these
hs-CRP (serum)
Mean Standard p value difference error 3.95
\0.0001*
Group 1 and group 3 109.35
3.95
\0.0001*
Group 2 and group 3
18.64
3.40
\0.0001*
Group 1 and group 2
81.19
3.19
\0.0001*
Group 1 and group 3
95.54
3.29
\0.0001*
Group 2 and group 3
14.35
3.35
\0.0001*
Group 1 and group 2
86.600
Group 1 and group 2
0.363
0.083
\0.008*
Group 1 and group 3
0.486
0.083
\0.0001*
Group 2 and group 3
0.124
0.071
0.210
Group 1 and group 2
1.391
0.260
\0.0001*
Group 1 and group 3 Group 2 and group 3
2.421 1.029
0.260 0.244
\0.0001* \0.003*
* Significant at p \ 0.05
proteins. Moreover, the other members of the S100 family have been implicated in the inflammatory periodontal diseases. S100A2, S100A8 and S100A9 levels were found to be significantly elevated in gingival tissues of gingivitis and moderate and severe chronic periodontitis analysed by realtime PCR, implicating the role of these proteins in inflammatory conditions [19]. Furthermore, S100A2 protein levels were found to be elevated in GCF of gingivitis and chronic periodontitis subjects as compared to healthy subjects when analysed by ELISA, thus suggesting the potential role of S100A2 in the immune response in inflammatory periodontal disease [19]. The concentration of S100A12 in our study was slightly higher in GCF than in serum, which could be explained by local production of S100A12 in diseased periodontal tissues, suggesting that S100A12 levels might serve as a marker for local disease activity. It has been widely accepted that periodontitis and diabetes share a common link and that impaired metabolic control in diabetes can worsen periodontal condition through the influence of advanced glycation endproducts (AGEs) [5, 29]. Thus, the hyperglycaemic condition can increase the intensity of inflammation by accumulation of
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AGEs and via interactions with the cellular receptors for AGEs (RAGE) [29]. This can explain the highest values of GCF and serum S100A12 in CP subjects with type 2 DM in our study. Moreover, these results are in accordance with a study by Kosaki et al. [22] in which plasma S100A12 and hs-CRP levels were elevated in subjects with type 2 diabetes. In the same study, plasma S100A12 values were found to be correlated with glycated haemoglobin (HbA1c) levels, thus suggesting that S100A12 levels are regulated by glycaemic control [22]. Various other factors can be postulated to explain the correlation of glycaemic control with the levels of S100A12 and hs-CRP. Numerous studies have established that increased circulating levels of proinflammatory cytokines (IL-6, TNF, IL-1) and cytokineresponsive acute phase proteins such as CRP, plasminogen activator inhibitor-1 and fibrinogen are associated with the hyperglycaemic condition in diabetes and these increased levels of cytokines may in turn upregulate the production of S100A12 in type 2 DM [22, 30–33]. The increase in hs-CRP, an acute phase reactant protein and one of the most important markers of inflammation from healthy to CP and CP with type 2 DM, is in accordance with a previous study where CRP levels in GCF and serum were found to correlate with CP and type 2 DM [34], while in another study CRP levels in gingiva and GCF were found to increase in periodontitis sites, thus suggesting that periodontal infection may influence systemic inflammation [35]. The mean GCF and serum concentration of CRP in the periodontitis group with diabetes was greater than that of the periodontitis group, which was, in turn, greater than that of the healthy group. Thus, the mean concentration of CRP was elevated in GCF and serum with the simultaneous progression of the periodontal disease and worsening of glycaemic control. The results of the present study showed that serum levels of hs-CRP in GCF and serum were significantly correlated with S100A12 in CP. This might indicate some element of systemic inflammation in addition to local periodontal inflammation in some subjects. To our knowledge this is the first study evaluating and correlating S100A12 and hs-CRP in CP with and without type 2 DM. One limitation of this cross-sectional study is the small sample size evaluated. Further longitudinal studies with larger sample size should be carried out to confirm the findings of the study and better understand the role of these inflammatory biomarkers in CP and type 2 DM.
Conclusion Within the limitations of the study, S100A12 and hs-CRP can be considered as potential biomarkers of periodontal
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disease. The highest levels of the two inflammatory mediators in CP with type 2 DM may indicate active inflammatory process both locally in periodontal tissues and systemically, thus suggesting a correlation between glycaemic control and periodontitis. These biomarkers can therefore be valuable in detecting high-risk individuals with periodontitis and systemic diseases like diabetes. Further longitudinal prospective studies must be carried out to confirm these findings and understand the possible roles of S100A12 and hs-CRP in the pathogenesis of periodontitis and diabetes. Acknowledgments The authors are grateful to Mr. Manjunath Sharma, Institute of Statistical Services, Bangalore for providing the statistical analysis. The authors report no conflict of interests.
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Inflammation Research
ORIGINAL RESEARCH PAPER
Correlation of human S100A12 (EN-RAGE) and high-sensitivity C-reactive protein as gingival crevicular fluid and serum markers of inflammation in chronic periodontitis and type 2 diabetes A. R. Pradeep • Santosh S. Martande • Sonender Pal Singh Deepak Kumar Suke • Arjun P. Raju • Savitha B. Naik
•
Received: 30 July 2013 / Revised: 21 November 2013 / Accepted: 18 December 2013 / Published online: 31 December 2013 Ó Springer Basel 2013
Abstract Objective The aim of the present study was to evaluate the levels and correlation of human S100A12 and highsensitivity C-reactive protein (hs-CRP) in gingival crevicular fluid (GCF) and serum in chronic periodontitis (CP) subjects with and without type 2 diabetes mellitus (DM). Materials and methods A total of 44 subjects were divided into three groups: group 1 had 10 periodontally healthy subjects, group 2 consisted of 17 CP subjects and group 3 had 17 type 2 DM subjects with CP. GCF and serum levels of human S100A12 and hs-CRP were quantified using enzyme-linked immunosorbent assay and immunoturbidimetric analysis, respectively. The clinical outcomes evaluated were gingival index, probing depth and clinical attachment level and the correlations of the two inflammatory mediators with clinical parameters were evaluated. Results Both human S100A12 and hs-CRP levels increased from group 1 to group 2 to group 3. The GCF and serum values of both these inflammatory mediators corre-
Responsible Editor: Ji Zhang. A. R. Pradeep (&) S. S. Martande S. P. Singh D. K. Suke Department of Periodontics, Government Dental College and Research Institute, Bangalore 560002, Karnataka, India e-mail: [email protected] A. P. Raju Bangalore Medical College and Research Institute, Bangalore 560002, Karnataka, India S. B. Naik Department of Conservative Dentistry and Endodontics, Government Dental College and Research Institute, Bangalore 560002, Karnataka, India
lated positively with each other and with the periodontal parameters evaluated (p \ 0.05). Conclusion Human S100A12 and hs-CRP can be considered as possible GCF and serum markers of inflammatory activity in CP and DM. Keywords Chronic periodontitis Inflammation Diabetes mellitus Gingival crevicular fluid Biomarkers
Introduction Chronic periodontitis (CP) is a multi-factorial infection elicited by a complex group of bacterial species that interact with host tissues and cells leading to the release of a broad array of inflammatory mediators, some of which lead to destruction of the periodontal structures, including the tooth supporting tissues, alveolar bone, and periodontal ligament [1]. The inflammatory host response is a fundamental feature of periodontal disorder and is regarded as the primary cause for periodontal breakdown [2]. Polymorphonuclear neutrophils (PMNs) perform an innate cellular host defense role within the gingival crevice and overall oral cavity. These neutrophils make up half of the leukocytes infiltrating the junctional epithelium and 90 % of the leukocytes isolated from crevicular fluid, thus suggesting a key role in periodontal inflammation [3]. Diabetes mellitus (DM) is a metabolic disorder which, due to disturbances in insulin production, leads to abnormal fat, sugar and protein metabolism and resultant hyperglycaemia which may lead to various systemic complications [4]. Diabetic subjects have a two- to fourfold higher risk of developing severe periodontitis and accelerated periodontal disease progression [5]. Periodontitis, in fact, has been referred as the sixth complication of
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DM [6]. Diabetic subjects with periodontal infection have a much higher risk of worsening glycaemic control over time compared with diabetic subjects without periodontitis, thus suggesting a two-way street between these diseases [7]. Human S100A12, also known as calgranulin C, MRP6 or extracellular newly identified receptor for advanced glycation endproducts (EN-RAGE) binding protein, is a calcium-binding proinflammatory protein predominantly secreted by PMNs [8]. S100A12 was first described in humans by Guignard and colleagues in 1995 [9]. S100A12 is a member of the S100 multigene family of calciumbinding proteins which play a major role in the Ca21dependent regulation of a variety of intracellular activities, including protein phosphorylation, enzyme activities, cell proliferation and differentiation, intracellular Ca21 homeostasis, inflammation and protection from oxidative cell damage [10]. It exhibits several proinflammatory properties, and has a chemotactic activity comparable to that of other strongly chemotactic agents [11]. When secreted extracellularly from neutrophils, S100A12 contributes to innate immune responses. The proinflammatory activities of S100A12 include chemotactic activity and activation of intracellular signalling cascades which leads to production of various cytokines and induction of oxidative stress [12, 13]. Intracellular signalling via protein kinases induces nuclear factor (NF)-jB-dependent secretion of different cytokines [14]. Bovine S100A12 is a ligand for the receptor for advanced glycation end products (RAGE) expressed on macrophages, endothelium and lymphocytes [15]. It is hypothesized that binding of S100A12 to RAGE mediates the proinflammatory properties of this protein [15]. S100A12 is thought to play an important role in local inflammatory responses and serum S100A12 levels are elevated in chronic inflammatory diseases like rheumatoid arthritis and psoriatic arthritis [16], inflammatory bowel disease [17] and cystic fibrosis [18]. The other members of the S100 family have been implicated in the inflammatory periodontal diseases. S100A2, S100A8 and S100A9 were found to be significantly elevated in gingival tissues of gingivitis and moderate and severe chronic periodontitis [19]. Furthermore, S100A2 protein levels were found to be increased in gingival crevicular fluid (GCF) of gingivitis and chronic periodontitis analysed by enzyme-linked immunosorbent assay (ELISA), thus suggesting S100A2 as a potential biomarker of inflammatory periodontal disease [19]. C-reactive protein (CRP) is an acute-phase reactant released in response to inflammatory cytokines, interleukin (IL-6), IL-1, and tumor necrosis factor-alpha (TNF-a). CRP has several proinflammatory actions and circulating CRP levels are a marker of systemic inflammation and are associated with periodontal disease [20]. Fasting plasma glucose, a parameter of diabetic status, is a significant
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determinant of CRP and IL-6 plasma concentrations [21]. Plasma S100A12 and hs-CRP levels are elevated in subjects with type 2 diabetes, suggesting that S100A12 and hsCRP levels are regulated by factors related to subclinical inflammation and glucose control in type 2 diabetes [22]. To date, no study has reported human S100A12 levels in GCF and serum and its correlation with hs-CRP levels in CP subjects with and without type 2 DM. In this context, this clinico-biochemical study was designed to assess the levels of human S100A12 in GCF and serum and its correlation with hs-CRP levels in CP subjects with and without type 2 DM.
Materials and methods This was a 3-month-long cross-sectional study performed from November 2012 to February 2013. The study group consisted of 44 age- and sex-balanced individuals (22 males and 22 females, aged 25–45 years) attending the outpatient section, Department of Periodontics, Government Dental College and Research Institute (GDCRI), Bangalore, India. Written informed consent was obtained from all the subjects participating in the study. The ethical clearance was approved by the Institutional Ethical Committee and Review Board, GDCRI. Inclusion and exclusion criteria The inclusion criteria were: subjects aged 25–45 years; presence of at least 20 natural teeth, subjects with a diagnosis of CP based on clinical parameters like probing depth (PD), clinical attachment level (CAL) [23], gingival index (GI) [24], body mass index (BMI) in the range of 18.5–22.9 kg/m2 and waist circumference\90 cm (men) or \80 cm (women) (WHO, 2004) [25]; subjects should not have received periodontal therapy within preceding 6 months; well-controlled type 2 diabetic patients classified based on criteria given by American Diabetic Association (ADA) in 2011 and glycated haemoglobin levels [26]. Patients with: (1) aggressive periodontitis, (2) hypertension and other cardiovascular diseases, (3) smoking tobacco in any form, (4) gross oral pathology, (5) rheumatoid arthritis, (6) tumors, (7) any other systemic disease that can alter the course of periodontal disease, (8) any course of medication affecting periodontal status, or (9) received periodontal therapy in the preceding 6 months were excluded from the study. Subject grouping Participants were categorized into three groups based on the GI, pocket PD, CAL and radiographic evidence of bone
S100A12 and hs-CRP in chronic periodontitis and diabetes
loss. Group 1 (healthy) consisted of ten individuals with clinically healthy periodontium, GI = 0 (absence of clinical inflammation), PD B 3 mm and CAL = 0, with no evidence of bone loss on radiographs. Group 2 (CP without type 2 DM) consisted of 17 individuals who had signs of clinical inflammation, GI [ 1, PD C 5 mm and CAL C 3 mm, with radiographic evidence of bone loss. Group 3 (type 2 DM among individuals with CP) consisted of 17 individuals who had signs of clinical inflammation, GI [ 1, PD C 5 mm, CAL C 3 mm and haemoglobin A1c (HbA1c) B7 % with radiographic evidence of bone loss. Only well-controlled (HbA1c B 7 %) type 2 DM individuals were selected based on American Diabetes Association’s criteria for diagnosis of diabetes [26]. Clinical evaluation of subjects Group allocation and sample site selection in each subject was performed by the chief coordinator (A.R.P.). A thorough case history was taken for each subject. Each subject then went through a full-mouth periodontal probing and charting and BMI charting procedure according to the World Health Organization guidelines [25]. A calibrated examiner (S.S.M.) performed the clinical evaluation, measuring the clinical parameters including PD, CAL and GI using a periodontal probe (University of North Carolina-15 periodontal probe, Hu-Friedy, Chicago, IL, USA). 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 (S.S.M.) performed the radiographic evaluation and collected the GCF samples. Site selection and GCF collection GCF sample collection was done from most inflammed sites. Two test sites for GCF sample collection were selected based on the highest scored sites. In group 2 and group 3 subjects, the two sites showing the greatest CAL and signs of inflammation, along with radiographic confirmation of bone loss, were selected for sampling. One of the two sites selected per subject was used for hs-CRP and the other for S100A12 analysis. In the healthy group, to standardize site selection and obtain adequate fluid volume, sampling was predetermined to be from the mesio-buccal region of the maxillary right first molar, in the absence of which the left first molar was sampled. Initially the selected site was cleaned, isolated and airdried using sterile cotton rolls and the supragingival
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plaque was removed gently using a Gracey curette (Universal Gracey Curette #4R/4L, Hu-Friedy) so as to avoid contamination of the paper strips (Periopaper, Ora Flow Inc., Amityville, NY, USA). The paper strips were placed gently at the entrance of the gingival sulcus/crevice until light resistance was felt [27], care being taken to avoid mechanical injury, and left in place for 60 s. The absorbed GCF volume of each strip was determined by electronic impedance (Periotron 8000, ProFlow Inc., Amityville, NY, USA). 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 per site that absorbed GCF from each subject were pooled and were immediately transferred to microcentrifuge tubes (premarked with the biomarker name) containing 400 ll of phosphate buffered 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 (S.P.S.). Blood collection Blood collection was done at the same appointment for the purpose of serum sample collection. Two milliliters of blood were collected from the antecubital fossa by venipuncture using a 20-gauge needle with a 2-ml syringe and immediately transferred to the laboratory. The blood sample was allowed to clot at room temperature and after 1 h the serum was separated from the blood by centrifuging at 3,000g for 5 min. The serum was immediately transferred to a plastic vial and stored at -70 °C until the time of assay. Human S100A12 analysis The samples were assayed for S100A12 using ELISA according to the manufacturer’s instructions. The GCF sample tubes were first homogenized for 30 s and then centrifuged for 5 min at 1,500g to elute. The elute was used as sample for ELISA estimation from GCF samples. Each sample was assayed using a commercially available ELISA kit (Human S100A12 Elisa kit, USCN Life Sciences, USA) in accordance with the manufacturer’s instructions. Colour development was monitored using a microplate reader until an optimum optical density was reached, a stop solution was added and the optical density was read at 450 nm. The total S100A12 was determined in nanograms (ng), and the calculation of the concentration in each sample was performed by dividing the amount of S00A12 by the volume of sample (ng/ml).
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Table 1 Descriptive statistics of study population (mean ± SD) Study group
Group 1 (n = 10)
Group 2 (n = 17)
Group 3 (n = 17)
Age (years)
39.30 ± 3.52
38.35 ± 3.20
40.88 ± 4.44
Sex (M/F)
5/5
9/8
8/9
GI
–
1.84 ± 0.23
2.16 ± 0.31
PPD (mm)
2.10 ± 0.73
6.12 ± 0.92
6.88 ± 0.99
CAL (mm)
0
5.71 ± 0.68
6.35 ± 0.93
Serum S100A12 (ng/ml)
32.10 ± 10.02 113.29 ± 8.93 127.65 ± 10.4
GCF S100A12 (ng/ml)
38.10 ± 11.37 124.71 ± 8.16 143.35 ± 10.6
Serum hs-CRP (mg/l)
2.05 ± 0.78
3.44 ± 0.38
4.47 ± 0.77
GCF hs-CRP (mg/l) 0.49 ± 0.25
0.85 ± 0.17
0.98 ± 0.20
hs-CRP analysis The samples for CRP were measured immunoturbidimetrically. The microcentrifuge tubes containing the Periopaper strips and plastic vials containing serum were transferred to the lab for immunoturbidimetric analysis. Serum was used undiluted. The measuring range of CRP was 0–220 mg/l, the normal value of CRP being 0–5 mg/l.
Statistical analysis Power calculations were performed before the study was initiated and the sample size was selected based on the previous study [28]. Sample size and grouping was based on the power of the study and the 95 % confidence interval (p \ 0.05). Statistical analysis was done using SPSS statistical software (SPSS version 16.0, Chicago, IL, USA). Analysis of variance (ANOVA) was carried out for a comparison of GCF and serum hs-CRP and S100A12 levels between the groups. Using Pearson’s correlation coefficient, the relationships between hs-CRP and S100A12 concentration and the clinical parameters were analyzed. The intra-group correlation of serum and GCF concentrations of hs-CRP and S100A12 was also performed using Pearson’s correlation coefficient. Pair-wise comparisons between groups for S100A12 and hs-CRP concentrations in GCF and serum were carried out by Scheff’s test. p values \0.05 were considered statistically significant. The mean intraexaminer standard deviation of differences in repeated PD measurements and CAL measurements was obtained using single passes of measurements with a UNC-15 probe (correlation coefficients between duplicate measurements; r = 0.95).
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Results Table 1 gives the descriptive statistics (mean ± SD) of the study population. The mean S100A12 and hs-CRP concentrations in both serum and GCF were highest for group 3, followed by group 2, and least in group 1. ANOVA was carried out to find out the equality of means between the three groups (Table 2). A significant difference in the serum and GCF levels of S100A12 and hs-CRP was found between the three groups. The Pearson correlation coefficient test was applied to evaluate the correlation, and statistically significant correlations existed between the serum levels of S100A12 and the serum levels of hs-CRP, and also the GCF values between the two. Table 3 shows the correlation coefficients and p values. Table 4 demonstrates the correlation between GCF and serum values of S100A12 and hs-CRP and the clinical parameters. The serum and GCF levels of S100A12 and that of hs-CRP were found to be 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 5 demonstrates pair-wise comparisons between groups for S100A12 and hs-CRP concentrations in GCF and serum. The comparisons were statistically significant for GCF and serum S100A12 and serum hs-CRP levels between all groups. While GCF hs-CRP levels showed statistical significance between groups 1 and 2 and groups 1 and 3, the difference was not significant between groups 2 and 3.
Discussion This study aimed at evaluating the GCF and serum levels of S100A12 and hs-CRP in CP with and without association of type 2 DM. The results of the study indicated an increase in the GCF and serum levels of both S100A12 and hs-CRP from healthy 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 both S100A12 and hs-CRP can be considered as potential biomarkers for inflammatory periodontal disease and type 2 diabetes. Human S100A12 or EN-RAGE, a calcium-binding proinflammatory protein predominantly secreted by PMNs, is implicated in the innate immune response during early inflammation [9]. The interaction of S100A12 with its multi-ligand receptor RAGE and soluble receptor sRAGE plays a central role in proinflammatory properties of this protein [15]. The S100 group of proteins are overall
S100A12 and hs-CRP in chronic periodontitis and diabetes
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Table 2 Results of ANOVA comparing the mean serum and GCF S100A12 and hs-CRP concentrations between the three groups Groups
S100A12
Hs-CRP
Serum
GCF
Serum
GCF
F value
p value
F value
p value
F value
p value
F value
p value
324.71
\0.001*
376.92
\0.001*
43.43
\0.001*
17.73
\0.001*
Group 1 Group 2 Group 3 * Significant at p \ 0.001
Table 3 Correlation of serum and GCF S100A12 and hs-CRP in each group using Spearman’s rank correlation coefficient test Group
Serum Correlation coefficient
Group 1 Group 2 Group 3
0.970 0.869
Table 5 Pair-wise comparisons using Scheff’s test for S100A12 (ng/ ml) and hs-CRP (mg/l) concentrations in GCF and serum
GCF p value \0.001* \0.001*
0.680
0.006*
Correlation coefficient 0.641 0.794 0.822
Study groups p value 0.046*
S100A12 (GCF)
0.012* \0.001*
S100A12 (serum)
* Significant at p \ 0.05 hs-CRP (GCF) Table 4 Relationship of S100A12 and hs-CRP to clinical parameters Parameters
Group 1
Group 2
Group 3
S100A12 Serum
GCF
GI
–
PD
0.007*
CAL
–
GI
–
PD
0.006*
CAL
–
0.004*
0.002*
\0.001*
\0.001*
0.012*
\0.001*
0.024*
0.006*
\0.001*
\0.001*
0.003*
\0.001*
hs-CRP Serum
GCF
GI
–
0.004*
0.002*
PD
0.002*
\0.001*
\0.001*
CAL GI
– –
\0.001* 0.016*
0.002* 0.062
PD
0.004*
\0.001*
0.004*
CAL
–
\0.001*
\0.001*
* Significant at p \ 0.05
involved in various inflammatory conditions; S100A12 is thought to play an important role in local inflammatory responses and serum S100A12 levels are elevated in chronic inflammatory diseases [16–18]. S100A12 and CRP levels were found to be increased in various conditions like rheumatoid arthritis and psoriatic arthritis [16], inflammatory bowel disease [17] and cystic fibrosis [18]. Thus, the increase in GCF and serum concentrations of both S100A12 and hs-CRP in CP and type 2 DM in the present study can be attributed to the proinflammatory properties of these
hs-CRP (serum)
Mean Standard p value difference error 3.95
\0.0001*
Group 1 and group 3 109.35
3.95
\0.0001*
Group 2 and group 3
18.64
3.40
\0.0001*
Group 1 and group 2
81.19
3.19
\0.0001*
Group 1 and group 3
95.54
3.29
\0.0001*
Group 2 and group 3
14.35
3.35
\0.0001*
Group 1 and group 2
86.600
Group 1 and group 2
0.363
0.083
\0.008*
Group 1 and group 3
0.486
0.083
\0.0001*
Group 2 and group 3
0.124
0.071
0.210
Group 1 and group 2
1.391
0.260
\0.0001*
Group 1 and group 3 Group 2 and group 3
2.421 1.029
0.260 0.244
\0.0001* \0.003*
* Significant at p \ 0.05
proteins. Moreover, the other members of the S100 family have been implicated in the inflammatory periodontal diseases. S100A2, S100A8 and S100A9 levels were found to be significantly elevated in gingival tissues of gingivitis and moderate and severe chronic periodontitis analysed by realtime PCR, implicating the role of these proteins in inflammatory conditions [19]. Furthermore, S100A2 protein levels were found to be elevated in GCF of gingivitis and chronic periodontitis subjects as compared to healthy subjects when analysed by ELISA, thus suggesting the potential role of S100A2 in the immune response in inflammatory periodontal disease [19]. The concentration of S100A12 in our study was slightly higher in GCF than in serum, which could be explained by local production of S100A12 in diseased periodontal tissues, suggesting that S100A12 levels might serve as a marker for local disease activity. It has been widely accepted that periodontitis and diabetes share a common link and that impaired metabolic control in diabetes can worsen periodontal condition through the influence of advanced glycation endproducts (AGEs) [5, 29]. Thus, the hyperglycaemic condition can increase the intensity of inflammation by accumulation of
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AGEs and via interactions with the cellular receptors for AGEs (RAGE) [29]. This can explain the highest values of GCF and serum S100A12 in CP subjects with type 2 DM in our study. Moreover, these results are in accordance with a study by Kosaki et al. [22] in which plasma S100A12 and hs-CRP levels were elevated in subjects with type 2 diabetes. In the same study, plasma S100A12 values were found to be correlated with glycated haemoglobin (HbA1c) levels, thus suggesting that S100A12 levels are regulated by glycaemic control [22]. Various other factors can be postulated to explain the correlation of glycaemic control with the levels of S100A12 and hs-CRP. Numerous studies have established that increased circulating levels of proinflammatory cytokines (IL-6, TNF, IL-1) and cytokineresponsive acute phase proteins such as CRP, plasminogen activator inhibitor-1 and fibrinogen are associated with the hyperglycaemic condition in diabetes and these increased levels of cytokines may in turn upregulate the production of S100A12 in type 2 DM [22, 30–33]. The increase in hs-CRP, an acute phase reactant protein and one of the most important markers of inflammation from healthy to CP and CP with type 2 DM, is in accordance with a previous study where CRP levels in GCF and serum were found to correlate with CP and type 2 DM [34], while in another study CRP levels in gingiva and GCF were found to increase in periodontitis sites, thus suggesting that periodontal infection may influence systemic inflammation [35]. The mean GCF and serum concentration of CRP in the periodontitis group with diabetes was greater than that of the periodontitis group, which was, in turn, greater than that of the healthy group. Thus, the mean concentration of CRP was elevated in GCF and serum with the simultaneous progression of the periodontal disease and worsening of glycaemic control. The results of the present study showed that serum levels of hs-CRP in GCF and serum were significantly correlated with S100A12 in CP. This might indicate some element of systemic inflammation in addition to local periodontal inflammation in some subjects. To our knowledge this is the first study evaluating and correlating S100A12 and hs-CRP in CP with and without type 2 DM. One limitation of this cross-sectional study is the small sample size evaluated. Further longitudinal studies with larger sample size should be carried out to confirm the findings of the study and better understand the role of these inflammatory biomarkers in CP and type 2 DM.
Conclusion Within the limitations of the study, S100A12 and hs-CRP can be considered as potential biomarkers of periodontal
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disease. The highest levels of the two inflammatory mediators in CP with type 2 DM may indicate active inflammatory process both locally in periodontal tissues and systemically, thus suggesting a correlation between glycaemic control and periodontitis. These biomarkers can therefore be valuable in detecting high-risk individuals with periodontitis and systemic diseases like diabetes. Further longitudinal prospective studies must be carried out to confirm these findings and understand the possible roles of S100A12 and hs-CRP in the pathogenesis of periodontitis and diabetes. Acknowledgments The authors are grateful to Mr. Manjunath Sharma, Institute of Statistical Services, Bangalore for providing the statistical analysis. The authors report no conflict of interests.
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