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. [18] have suggested that the significant
positive correlations between GCF levels of OC,
prostaglandin E2, and alkaline phosphatase might be
due to their partially common origins.
In conclusion, within the limits of the present study,
it may be suggested that systemically healthy chronic
periodontitis patients do not exhibit altered plasma
concentrations of ICTP and OC compared to the
clinically healthy subjects.
Source of Funding. This study has been funded solely
by the institutions of the authors.
Conflict of Interest. The authors declare that they have
no conflict of interest.
REFERENCES
1. Armitage, G.C. 1999. Development of a classification system for
periodontal diseases and conditions. Annals of Periodontology 4:
1–6.
2. Lappin, D.F., B. Eapen, D. Robertson, J. Young, and P.J. Hodge.
2009. Markers of bone destruction and formation and periodontitis
in type 1 diabetes mellitus. Journal of Clinical Periodontology 36:
634–641.
3. Yoshihara, A., T. Deguchi, N. Hanada, and H. Miyazaki. 2009.
Relation of bone turnover markers to periodontal disease and jaw
bone morphology in elderly Japanese subjects. Oral Diseases 15:
176–181.
4. Seibel, M.J. 2003. Biochemical markers of bone metabolism in the
assessment of osteoporosis: useful or not? Journal of Endocrinological Investigation 26: 464–471.
5. Narayanan, A.S., and R.C. Page. 1983. Connective tissues of the
periodontium: a summary of current work. Collagen and Related
Research 3: 33–64.
6. Oringer, R.J., M.D. Palys, A. Iranmanesh, J.P. Fiorellini, A.D.
Haffajee, S.S. Socransky, and W.V. Giannobile. 1998. C-telopeptide pyridinoline cross-links (ICTP) and periodontal pathogens
associated with endosseous oral implants. Clinical Oral Implants
Research 9: 365–373.
7. Oringer, R.J., K.F. Al-Shammari, W.A. Aldredge, V.J. Iacono,
R.M. Eber, H.L. Wang, B. Berwald, R. Nejat, and W.V.
Giannobile. 2002. Effect of locally delivered minocycline
microspheres on markers of bone resorption. Journal of
Periodontology 73: 835–842.
8. Giannobile, W.V. 1999. C-telopeptide pyridinoline cross-links.
Sensitive indicators of periodontal tissue destruction. Annals of the
New York Academy of Sciences 878: 404–412.
9. Lian, J.B., and C.M. Gundberg. 1988. Osteocalcin: biochemical
considerations and clinical applications. Clinical Orthopaedics and
Related Research 226: 267–291.
10. Christerson, R.H. 1997. Biochemical markers of bone metabolism:
an overview. Clinical Biochemistry 30: 573–593.
11. Shi, F., S. Yu, and L. Xu. 1996. Analysis of serum osteocalcin of
patients with periodontitis. Zhonghua Kou Qiang Yi Xue Za Zhi 31:
300–302. Article in Chinese.
12. Buduneli, E., N. Buduneli, S. Vardar-Şengül, L. Kardeşler, G.
Atilla, D. Lappin, and D.F. Kinane. 2005. Systemic low-dose
doxycycline and alendronate administration and serum interleukin1beta, osteocalcin, and C-reactive protein levels in rats. Journal of
Periodontology 76: 1927–1933.
13. Tümer, C., Y. Aksoy, G.N. Güncü, R.M. Nohutçu, K. Kılınç, and
T.F. Tözüm. 2008. Possible impact of inflammatory status on Ctelopeptide pyridinoline cross-links of type I collagen and
osteocalcin levels around oral implants with peri-implantitis: a
controlled clinical trial. Journal of Oral Rehabilitation 35: 934–
939.
208
14. Oringer, R.J., K.F. Al-Shammari, and D.P. Sarment. 2003. Matrix
molecules and growth factors as indicators of periodontal disease
activity. Periodontology 2000 31: 125–134.
15. Gürlek, Ö., D.F. Lappin, and N. Buduneli. 2009. Effects of
smoking on salivary C-telopeptide pyridinoline cross-links of type
I collagen and osteocalcin levels. Archives of Oral Biology 54:
1099–1104.
16. Bullon, P., B. Goberna, J.M. Guerro, J.J. Segura, R. Parez-Cano,
and A. Martinez-Sahuguillo. 2005. Serum, saliva and gingival
crevicular fluid osteocalcin: their relation to periodontal status and
bone mineral density in postmenopausal women. Journal of
Periodontology 76: 513–519.
17. Kunimatsu, K., S. Mataki, H. Tanaki, N. Mine, M. Kiyoki, K.
Hosoda, Y. Kato, and I. Kato. 1993. A cross-sectional study on
osteocalcin levels in gingival crevicular fluid from periodontal
patients. Journal of Periodontology 64: 865–869.
Özçaka, Nalbantsoy, Bıçakçı, Köse, and Buduneli
18. Nakashima, K., N. Roehrich, and G. Cimasoni. 1994. Osteocalcin,
prostaglandin E2 and alkaline phosphatase in gingival crevicular
fluid: their relations to periodontal status. Journal of Clinical
Periodontology 21: 327–333.
19. Lee, A.J., T.F. Walsh, S.J. Hodges, and A. Rawlinson. 1999.
Gingival crevicular fluid osteocalcin in adult periodontitis. Journal
of Clinical Periodontology 26: 252–256.
20. Murata, M., J. Tatsumi, Y. Kato, S. Suda, Y. Nunokawa, Y.
Kobayashi, H. Takeda, H. Araki, K. Shin, K. Okuda, T. Miyata,
and H. Yoshie. 2002. Osteocalcin, deoxypyridinoline and interleukin-1beta in peri-implant crevicular fluid of patients with periimplantitis. Clinical Oral Implants Research 13: 637–643.
21. Koyama, K., K. Ohara, H. Yokota, T. Kurome, M. Katayama, and
F. Hino. 1991. A one step sandwich enzyme immunoassay for γcarboxylated osteocalcin using monoclonal antibodies. Journal of
Immunological Methods 139: 17–23.