Inflammation, Vol. 34, No. 3, June 2011 ( # 2010) DOI: 10.1007/s10753-010-9225-0 Plasma Levels of C-Telopeptide Pyridinoline Cross-Links of Type I Collagen and Osteocalcin in Chronic Periodontitis Özgün Özçaka,1 Ayşe Nalbantsoy,2 Nurgün Bıçakçı,1 Timur Köse,3 and Nurcan Buduneli1,4 Abstract—This study was planned to investigate whether chronic periodontitis patients exhibit different plasma concentrations of C-telopeptide pyridinoline cross-links of type I collagen (ICTP) and osteocalcin (OC) compared to the clinically healthy controls. Before initiation of any periodontal intervention, plasma samples and full-mouth clinical periodontal recordings were obtained from 58 otherwise healthy patients with chronic periodontitis and also from 47 systemically and periodontally healthy control subjects. Plasma ICTP levels were determined by radioimmunoassay and OC levels by enzyme-linked immunosorbent assay. Data were tested statistically using t test and ANOVA. The healthy control group exhibited significantly lower values in all clinical periodontal measurements (P<0.001). There were no significant differences between the study groups in plasma ICTP and OC levels (P>0.05). Within the limits of this study, it may be suggested that plasma levels of ICTP and OC may not provide distinguishing data between chronic periodontitis patients and clinically healthy subjects. KEY WORDS: ICTP; inflammation; osteocalcin; periodontal disease; plasma. C-telopeptide pyridinoline cross-links of type I collagen (ICTP) are released into the circulation as a consequence of collagen degradation and alveolar bone resorption [4]. Type I collagen composes 90% of the organic matrix of bone and is the most abundant collagen in osseous tissue [5]. Studies assessing the role of ICTP levels in gingival crevicular fluid (GCF) or peri-implant crevicular fluid as a diagnostic marker of periodontal disease activity have reported promising results so far [6, 7]. ICTP has been suggested to predict future bone loss, to correlate with clinical parameters, and also to reduce following periodontal therapy [8]. Increased circulating amounts of ICTP indicate an increase in osteoclast activity. Osteocalcin (OC) produced by osteoblasts is a calcium-binding protein of bone and the most abundant non-collagenous protein of the mineralized tissue [9]. Serum level of OC is considered as a marker of bone formation [10]. Serum levels of OC were reported to be lower in periodontitis patients compared with healthy subjects suggesting lower osteoblastic activity and bone formation ability [11]. On the contrary, higher serum OC levels were detected in an experimental periodontitis study in rats possibly indicating higher bone formation [12]. As yet, the relationship between plasma ICTP, osteocalcin concentrations and chronic periodontitis has INTRODUCTION Chronic periodontitis is an inflammatory disease of the supporting tissues of the teeth leading to attachment loss, bone loss, and possibly tooth loss if left untreated. Pathogen bacteria in the microbial dental plaque trigger host response mechanisms resulting in progressive destruction of the periodontal ligament and alveolar bone [1]. Complex interactions between the host’s immune, inflammatory responses and periodontopathogens appear to underlie the tissue destruction observed in chronic periodontitis. Bone formation and resorption are coupled events that are necessary to maintain the bone homeostasis. Changes in circulating levels of bone turnover markers have been reported in regards with periodontitis patients [2, 3]. 1 Department of Periodontology, School of Dentistry, Ege University, Bornova, 35100 İzmir, Turkey 2 Department of Bioengineering, Faculty of Engineering, Ege University, Bornova, 35100 İzmir, Turkey 3 Department of Biostatistics and Medical Informatics, School of Medicine, Ege University, Bornova, 35100 İzmir, Turkey 4 To whom correspondence should be addressed at Department of Periodontology, School of Dentistry, Ege University, Bornova, 35100 İzmir, Turkey. E-mail: nurcanbuduneli@yahoo.com; nurcan.budune li@ege.edu.tr 203 0360-3997/11/0300-0203/0 # 2010 Springer Science+Business Media, LLC 204 not been fully clarified. Our hypothesis was that altered plasma concentrations of ICTP and OC may be one systemic manifestation of periodontal tissue destruction in chronic periodontitis. Thus, the aim of this study was to investigate whether chronic periodontitis patients exhibit different plasma concentrations of ICTP and OC than those of the periodontally healthy control subjects. Özçaka, Nalbantsoy, Bıçakçı, Köse, and Buduneli Plasma Sampling The venous blood samples were collected by a standard venipuncture method into tubes containing an anticoagulant, and the plasma was separated from the blood by centrifugation at 1,500 g for 10 min. The plasma samples were then stored at −40°C until subsequent biochemical analyses and thawed immediately before assays. MATERIALS AND METHODS Clinical Measurements Study Population Plasma samples were obtained before initiation of any periodontal intervention. Subsequent to plasma sampling, clinical periodontal recordings, including dichotomous plaque index (present or absent), PD, CAL, and presence of bleeding on probing (BOP; present or absent) were performed at six sites (mesiobuccal, mid-buccal, disto-buccal, mesio-lingual, midlingual, and disto-lingual locations) on each tooth present, except the third molars, using a Williams probe. CAL was assessed from the cemento-enamel junction to the base of the probable pocket. BOP (deemed positive if it occurred within 15 s after periodontal probing) and visible plaque accumulation were recorded dichotomously by visual examination. All measurements were performed by a single calibrated examiner (ÖÖ). A total of 105 subjects seeking dental treatment at the School of Dentistry, Ege University were included in the present study. Otherwise healthy 58 chronic periodontitis (CP) patients and 47 periodontally healthy subjects were recruited between February 2005 and December 2008. The study protocol was approved by the ethics committee of Ege University, School of Medicine. The study was conducted in full accordance with ethical principles, including the World Medical Association Declaration of Helsinki. The study protocol was explained, and written informed consent was received from each individual before clinical examination and blood sampling. Medical and dental histories were obtained. All of the CP patients had at least 20 teeth present. Patients with medical disorders such as diabetes mellitus, immunological disorders, hepatitis, and those who had antibiotic or periodontal treatment in the previous 6 months were excluded from the study. Smoking history was recorded basing on self-reports on a standardized questionnaire, but smokers were not excluded from the study. CP patients were diagnosed in accordance with the clinical criteria stated in the consensus report of the World Workshop in Periodontitis [1]. The CP group had at least four teeth in each jaw with a probing depth (PD) of ≥5 mm, clinical attachment level (CAL) of ≥4 mm, and ≥50% alveolar bone loss in at least two quadrants. Assessment of the extent and severity of alveolar bone loss was done radiographically. Bitewing radiographs were evaluated for interproximal bone loss from the cemento-enamel junction (CEJ) of the tooth to the bone crest. These patients also had bleeding on probing (BOP) at >80% of the proximal sites. Moreover, the CAL was commensurate with the amount of supragingival plaque. The healthy control group had at least 20 teeth, ≥90% of the measured sites exhibited PD <3 mm and CAL ≤1 mm, and no BOP and no radiographic sign of alveolar bone loss (i.e., a distance of <3 mm between the CEJ and bone crest at >95% of the proximal tooth sites). Measurement of ICTP and OC in Plasma Samples The level of ICTP and OC in plasma samples were determined using commercially available radioimmunoassay (RIA) kit (Orion Diagnostica Oy Espoo, Finland) and solid phase enzyme-amplified sensitivity immunoassay kit (BioSource Europe S.A., Belgium), respectively, in accordance with the manufacturers’ directions. The lower detection thresholds for the ICTP and OC assays were 0.4 and 0.08 ng/ml, respectively. Plasma samples were diluted 1/20 times for ICTP analysis. The ICTP kit was based on the competitive RIA technique. A known amount of labeled ICTP and unknown amount of unlabelled ICTP in the sample compete for the limited numbers of high affinity binding sites of the antibody within incubate for 2 h at 37°C. After separating the free antigen, the amount of labeled ICTP in the sample tube was inversely proportional to the amount of ICTP in the sample. The concentrations in unknown samples were obtained from a calibration curve. Plasma samples were diluted 1/10 times for OC analysis. The assay uses monoclonal antibodies (MAbs) directed against distinct epitopes of human OC. Calibrators and samples were 205 Plasma ICTP, Osteocalcin, and Periodontitis reacting with the capture MAb 1 coated on microtiter well and with a MAb 2 labeled with horseradish peroxidase. Following 2 h incubations at room temperature with MAb 1 and MAb 2, the microtiter plate was washed to remove unbound enzyme-labeled antibody. Bound enzyme-labeled antibody was measured through a chromogenic reaction following 30 min incubation at room temperature. The reaction was stopped with the addition of stop solution and the microtiter plates were then read at 450 nm wavelength. A calibration curve was plotted using curve fitting and OC concentrations in the samples were determined by interpolation from the calibration curve. Statistical Analysis A pilot experiment, where osteocalcin levels were measured and a 10% difference was obtained, was utilized in statistical power calculations. With a power of 80% and α=0.05 the minimum number of patients required for the comparisons was 29 for each group. Chi-square test was performed for comparisons of sex and smoking status between study groups. Numerical demographic variables were compared between the chronic periodontitis and healthy controls using the t test. Plasma osteocalcin and ICTP concentrations were analyzed between chronic periodontitis and healthy controls by ANOVA (Randomized Block Design, to control the smoking effect). Bleeding on probing and plaque index measurements were obtained in terms of scores at six sites of each tooth present and then the full-mouth percentages were calculated for bleeding on probing as well as plaque accumulation. Therefore, the possible correlations between the biochemical variables and clinical periodontal measurements were computed by the Pearson correlation coefficient. All tests were done at α=0.05 significance level. All analyses were performed using the SPSS version 17.0 statistical software package. RESULTS Clinical Analyses Demographic variables and mean values of clinical measurements are outlined in Table 1. The age range of the healthy control group was 35–67 years, whereas that of the CP group was 37–69 years. The female to male and the smoker to non-smoker ratios were similar in the chronic periodontitis and healthy control groups (P> 0.05). The healthy control group exhibited significantly lower clinical periodontal measurement values than the chronic periodontitis patients (P<0.001). Plasma ICTP and OC Measurements Plasma ICTP and OC concentrations were similar in the chronic periodontitis and the healthy control groups (P>0.05; Table 2). The overall correlation analyses between clinical parameters and biochemical data revealed a significant positive correlation between plasma ICTP and osteocalcin concentrations and BOP (r=0.313, P=0.03; r= 0.448, P=0.002, respectively). There were no significant correlation between plasma ICTP and osteocalcin concentrations (r=0.024, P=0.86). DISCUSSION In the present exploratory study, we analyzed the plasma levels of ICTP and osteocalcin in otherwise healthy chronic periodontitis patients in comparison to periodontally healthy counterparts. Circulating levels of these bone turnover markers could help to clarify the systemic manifestations of periodontal tissue destruction. Accordingly, we hypothesized that chronic periodontitis patients may have altered plasma levels of ICTP and OC compared to the clinically healthy subjects. However, our findings revealed similar plasma levels of these bone turnover markers in the healthy and diseased individuals. One possible explanation for this finding may be the unknown nature of the disease activity in the present chronic periodontitis group as this is a crosssectional study and no attempt was made to determine whether periodontal destruction is active or not. ICTP and osteocalcin levels in peri-implant crevicular fluid samples of dental implants with or without peri-implant bone destruction have been investigated in a recent study [13]. Significant increases in all clinical periodontal measurements as well as OC levels in periimplantitis sites compared with the clinically healthy sites were reported. Although a slight increase was detected in ICTP levels, the difference between the diseased and healthy sites was not statistically significant. Our present findings are in line with this report and those by Shi et al. [11] and Lappin et al. [2] as no significant differences were found between the chronic periodontitis and healthy control groups in plasma ICTP concentrations. 206 Özçaka, Nalbantsoy, Bıçakçı, Köse, and Buduneli Table 1. Demographic Variables and Clinical Periodontal Measurements (Mean±SD) in The Study Groups Age (years) Female/male (n) Smoker/Non-smoker (n) PD (mm) CAL (mm) PI (%) BOP (%) Chronic periodontitis (n=58) Healthy control (n=47) P 48.38±8.9 25/33 15/43 4.0±0.8 5.4±1.5 80.67±26.5 66.28±26.9 40.42±7.4 26/21 14/33 1.6±0.2 0.1±0.2 12.12±10.9 9.10±9.3 <0.001 0.213 0.655 <0.001 <0.001 <0.001 <0.001 Healthy control group exhibited significantly lower values than the chronic periodontitis group in all clinical periodontal parameters (P<0.001) PD probing depth, CAL clinical attachment level, PI plaque index, BOP bleeding on probing Increased levels of ICTP have been observed in the GCF of periodontitis patients [8, 14]. Lappin et al. [2] reported higher median plasma concentrations of ICTP in periodontitis groups than the healthy subjects where the differences failed to reach statistical significance. In a recent study by Gürlek et al. [15], similar salivary ICTP levels were detected in smoker, non-smoker, and ex-smoker patient groups with similar clinical periodontal findings. There was no clinically healthy control group in that study and the number of teeth present, average probing depths, and attachment levels were all similar in the three study groups. It may be suggested that the similarity in clinical parameters of periodontal disease may explain the similar salivary ICTP levels obtained in these studies. On the other hand, our failure to detect a statistically significant difference may be due to measuring ICTP in plasma rather than gingival crevicular fluid as changes in bone turnover markers may be limited to local level not being reflected in circulation in periodontitis. Thus, one possible limitation of our study may be the lack of analyzing local levels of these proteins. Serum OC is presently considered to be a valid marker of bone turnover when resorption and formation are coupled and a specific marker of bone formation when formation and resorption are uncoupled [14]. In an experimental periodontitis study in rats, significant increases in serum OC levels was suggested as a sign of an increase in bone remodeling and thus inhibition of progression of alveolar bone resorption [12]. Lappin et al. [2] reported reduced plasma OC levels in type 1 diabetics compared to systemically healthy counterparts suggesting that these patients have a reduction in their intrinsic ability to replace bone, such as that which has been destroyed during “acute bursts” of periodontitis. They speculated that this lower OC may increase their susceptibility to progression of this disease. Reduced circulating levels of OC in the presence of periodontitis have been reported before by other researchers [16]. Shi et al. [11] demonstrated lower osteocalcin levels in serum of periodontitis patients. Recently, Lappin et al. [2] confirmed this finding who reported lower osteocalcin levels in the plasma of systemically healthy patients with periodontitis compared to individuals without periodontitis, where the periodontitis patients had to have ≥2 sites with PD and CAL >4 mm, whereas the non-periodontitis group had no sites with CAL or PD >4 mm. Furthermore, OC concentrations were reported to correlate negatively with the extent of periodontitis [2, 3]. However, the present findings indicated no significant difference in plasma OC concentrations between the chronic periodontitis and clinically healthy control groups. The discrepancy between the findings of our study and those of the previous ones may at least partly explained by the differences in the definitions of the study groups as the present control group consisted systemically healthy subjects with no sign of gingivitis or periodontitis whereas, Lappin et al. [2] defined Table 2. Plasma Concentrations (Mean±SD) of ICTP and OC in the Study Groups ICTP (ng/ml) OC (ng/ml) Chronic periodontitis (n=58) Healthy control (n=47) P 4.1±1.9 30.56±5.9 4.2±1.8 29.8±3.6 0.761 0.430 No significant differences were found between the chronic periodontitis patients and healthy controls (P>0.05) 207 Plasma ICTP, Osteocalcin, and Periodontitis the control group as non-periodontitis subjects who might possibly have chronic gingivitis. Another likely explanation may be the differences in patient numbers and/or possible differences in the state of disease activity in periodontitis groups of various studies as it is unknown. Bleeding on probing continues to be the most reliable clinical marker of disease activity, and our present finding of the overall positive correlations of BOP with plasma ICTP, OC concentrations may suggest a relation with circulating levels of these proteins and periodontal disease activity. OC, predominantly produced by osteoblasts is considered as a marker of osteoblast activity and bone formation as well as bone resorption. Because of its tissue-specific expression, circulating level of OC could be regarded as a specific indicator of the overall activities of cells responsible for bone metabolism. Abnormal concentrations of OC are also known to reflect the occurrence of bone diseases [9]. OC levels in GCF of periodontitis patients have been investigated in cross-sectional studies [17–20], suggesting that GCF level of OC reflects the present degree of periodontal tissue inflammation. Nakashima et al. [18] have reported significantly increased concentrations of OC in GCF of both gingivitis and periodontitis patients compared with the healthy subjects. The total amounts (pg/sample) but not concentrations (ng/ml) of OC were found to be significantly higher in the periodontitis group than the gingivitis group. The authors speculated that significant amount of OC might be produced locally by periodontal tissues, since they have found GCF concentrations at least ten times higher than the serum values reported previously by Koyama et al. [21]. Furthermore, Murata et al. [20] have analyzed the levels of OC and IL-1β as markers of bone metabolism in peri-implant crevicular fluid and serum from peri-implantitis patients as well as healthy subjects. They concluded that OC and IL-1β are generated locally and that the elevation of the local levels of these markers in diseased sites did not necessarily contribute to the systemic level. Moreover, Nakashima et al. 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