Journal of Dental Research

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Salivary Procalcitonin and Periodontitis in Diabetes

C.W. Bassim, R.S. Redman, D.J. DeNucci, K.L. Becker and E.S. Nylen
J DENT RES 2008 87: 630
DOI: 10.1177/154405910808700707

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 RESEARCH REPORTS
Clinical
C.W. Bassim1,2*, R.S. Redman1,
D.J. DeNucci1,2, K.L. Becker1,3,
and E.S. Nylen1,3
1 Washington, DC, Veterans Affairs Medical Center;
2NIDCR, CRC, NIH, Bldg. 10, Rm 1N-118, MSC 1191, 10
Center Drive, Bethesda, MD 20892, USA; and 3George
Washington University, Washington, DC, USA;
*corresponding author, bassimc@mail.nih.gov
J Dent Res 87(7):630-634, 2008
ABSTRACT
Periodontitis and type 2 diabetes are co-morbid
conditions, both characterized by infectious
susceptibility. We investigated procalcitonin
(ProCT) levels in the serum and saliva of persons
with periodontitis and type 2 diabetes (n = 20), to
determine if these levels are altered by
periodontitis activity or by hyperglycemia.
Persons with severe periodontitis showed higher
levels of salivary-ProCT than did those with
moderate periodontitis (241 ± 71 vs. 77 ± 516
pg/mL, p = 0.02) and higher levels than did
healthy control individuals (118 ± 26 pg/mL, p =
0.05). Salivary-ProCT levels were correlated with
bleeding-on-probing (r = 0.45, p = 0.05), as well
as with HgbA 1c (r = 0.49, p = 0.03). Salivary
levels of ProCT were higher than serum levels for
the periodontitis/diabetes group (152 ± 37 vs. 78 ±
17 pg/mL, p = 0.02) and the control group (118 ±
146 vs. 48 ± 17 pg/mL, p = 0.01). Persons with
periodontitis and type 2 diabetes have salivary-
ProCT levels that reflect their degree of
periodontitis activity and hyperglycemia. This
study demonstrates, for the first time, the presence
of procalcitonin (ProCT), an established serum
marker of infection, in saliva.
KEY WORDS: Saliva, procalcitonin, periodontitis,
diabetes.
Received June 22, 2007; Last revision February 28, 2008;
Accepted March 17, 2008
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International and American Associations for Dental Research
Salivary Procalcitonin and
Periodontitis in Diabetes
INTRODUCTION
Tsalivary
he simple and non-invasive nature of saliva collection and high-
sensitivity assay development has led to an emphasis on the promise of
biomarkers (Tabak, 2001). Saliva may reflect levels of therapeutic,
hormonal, and immunologic molecules and can yield markers for infectious
and neoplastic diseases (Kaufman and Lamster, 2002). For the local
inflammatory process of periodontitis, salivary diagnostics may promote
early diagnosis and aid in the monitoring of treatment (Miller et al., 2006).
For some diagnostic purposes, salivary biomarkers may prove more useful
than serum analysis (Streckfus and Bigler, 2005).
Diabetes has long been theorized to influence the course of
periodontitis, and it has been well-established that persons with diabetes
have an increased prevalence and severity of periodontitis (Löe, 1993).
Although the pathogenesis of periodontitis in diabetes remains unclear, the
co-morbidity of type 2 diabetes and periodontitis suggests that the infectious
up-regulation of the local periodontal inflammatory processes and the low-
level systemic inflammation involved with diabetic complications may
interact (Shoelson et al., 2006). Furthermore, the effect of periodontitis on
other disease processes has become a focus of study, with evidence
supporting the connection between periodontitis and poor outcomes in type
2 diabetes (Janket et al., 2005; Saremi et al., 2005; Shultis et al., 2007),
cardiovascular disease (Scannapieco et al., 2003a), and other systemic
pathologies (Slavkin and Baum, 2000; Scannapieco et al., 2003b;
Quagliarello et al., 2005).
Procalcitonin (ProCT) circulates at very low levels normally, but rises
dramatically in persons with systemic infection. It is used as a biomarker for
the presence and severity of severe infection, often with a sensitivity and
persistence unmatched by traditional clinical or routine laboratory tests
(Snider et al., 1997; Whang et al., 1998; Becker et al., 2004). In the
individual with sepsis, ProCT, usually produced only in the thyroid to be
processed into calcitonin, assumes tissue-wide expression and secretion
(Müller et al., 2001). Although ProCT is a significant serum marker, to our
knowledge there is no comparable study in saliva. The present investigation
was initiated to study levels of serum and salivary-ProCT and to analyze
how these might be related to periodontal disease indicators and/or to
hyperglycemia. We hypothesized that periodontitis and type 2 diabetes may
act as a stimulus for local ProCT production.
INDIVIDUALS & METHODS
Saliva and serum were collected from persons with periodontitis and poorly
controlled type 2 diabetes (HgbA1c > 7.0%, n = 20). Serum was analyzed for
HgbA1c, fasting glucose levels, and white blood counts (WBC), according to
standard clinical chemistry laboratory techniques. Periodontitis was classified as
severe (n = 9) or moderate (n = 11) chronic, generalized periodontitis based on a
full periodontal examination, with severe periodontitis showing probing depths
J Dent Res 87(7) 2008 Salivary Procalcitonin in Periodontitis 631

(PD) of > 6 mm and moderate periodontitis showing PD of 4-6
mm on more than 30% of available sites (Wiebe and Putnins,
2000). The examination recorded a full-mouth charting, including
bleeding-on-probing (BOP), and PD percentages, measured as the
ratio of either BOP or a PD at a periodontal site and the total
number of periodontal sites per person (6 sites per tooth). The
individual’s number of teeth, age, and smoking status were
recorded. One-stage, full-mouth periodontal scaling and root-
planing therapy, an efficient and effective treatment for chronic
periodontitis, was performed on each person (Wennström et al.,
2005). Follow-up visits at 3 mos after the initial visit were
scheduled for all persons; 13 persons reported for this secondary
visit and had saliva and serum samples taken. The individuals who
returned for the second visit were similar in initial HgbA1c (9.7 ±
2.3 vs. 10.6 ± 2.3%, p = 0.3) and BOP (31 ± 20 vs. 23 ± 25%, p =
0.4) to the group lost to follow-up; average time to follow-up was
84 ± 27 days.
The serum of non-diabetic control individuals was analyzed to
provide a control level for serum-ProCT (n = 34, 45 ± 12 yrs old).
A separate group of control individuals without periodontal disease
provided salivary samples for salivary-ProCT analysis (n = 8, aged
42 ± 5 yrs old). These control groups were comprised of both
males and females.
Unstimulated whole saliva was collected from the individuals
described above and was considered an admixture from salivary
gland secretion, crevicular fluid, mucosal seepage, resident
microflora, and debris. Saliva collection was performed by
individuals expectorating into 50-mL polyethylene centrifuge tubes
containing one drop of Tween® 20 (Fisher Scientific, Fairlawn, NJ,
USA) until 5 mL had been collected (range of 4-5 min,
approximately 1 mL/min). Saliva samples were then agitated by
vortex for 5 sec, centrifuged at 220 x g for 5 min, and divided into
1-mL aliquots stored in microcentifuge tubes at -27°C, then
thawed for assay.
Written informed consent for participating individuals was
included in the study protocols, approved by the Institutional
Review governing the Washington, DC, Veterans Affairs Medical
Center.
Analysis for ProCT
The salivary-ProCT samples were analyzed by an
aminoprocalcitonin assay (molecular weight 6222 Da). This
ELISA assay (EIA) was performed with a R2B7 antiserum
(developed in rabbits against synthetic human aminoprocalcitonin
(Bachem, Torrance, CA, USA) at a final dilution of 1:80,000. The
antiserum (25 ␮L of 1:20,000) was pre-incubated with standards or
unknowns (20-50 ␮L) in 0.1 mL 0.13 M borate buffer (containing
0.2% gelatin and 0.05% Tween-20; pH = 7.5) at 4°C for 24-72 hrs
(depending on desired sensitivity) in a Reacti-BindGoat antirabbit
coated, 96-well microtiter plate (Pierce, Rockford, IL, USA). After
the plate was rinsed 5 times with PBS containing Tween-20 in an
ELP-40 plate washer (Bio-Tek Instruments, Winooski, VT, USA),
a 2-ng quantity of biotin-human calcitonin (AnaSpec, San Jose,
CA, USA) in 0.1 mL borate buffer was added and incubated at
room temperature in an orbital shaker (150 rpm) for 1 hr. After the
plate was rinsed 5 times, a 100- ␮ L quantity of 1:10,000
strepavidin peroxidase (Ultrasensitive, Sigma-Aldrich, St. Louis,
MO, USA) was added to the plate, and the plate was incubated in
an orbital shaker (150 rpm) for 60 min. The plate was again rinsed
5 times, and a 0.1-mL quantity of a solution of o-
phenylenediamine (0.75 mg/mL) in phosphate-citrate buffer
containing perborate (Sigma-Aldrich, St. Louis, MO, USA) was

Table. Characteristics of Persons with Periodontitis/Diabetes before and after Periodontal Treatment

Periodontitis/Diabetes
Initial vs.
Periodontitis/Diabetes Periodontitis/Diabetes
Follow up Initial vs.
(matched data) Control

Periodontitis/Diabetes Periodontitis/Diabetes Control—No

Parameter Initial Follow up Periodontitis

n 20 13
Age (yrs) 56 ± 2* 55 ± 2
Sex
(male/female) 20/0 13/0
Smoking status
(yes/no) 7/13 4/9
Number of teeth 23 ± 1 22 ± 1

P value P value

HgbA1c% 10.1 ± 0.5 9.9 ± 0.8 0.62

Fasting glucose (mg/dL) 158.2 ± 16.2 169.7 ± 22.1 0.53
WBC (K/cmm) 7.5 ± 0.6 7.9 ± 1.2 0.91
Salivary ProCT 152 ± 37 146 ± 45 118 ± 26 0.41 0.51
(pg/mL) (n = 8)
Serum ProCT 78 ± 17 103.5 ± 40 48 ± 3 0.37 0.04
(pg/mL) (n = 34)
BOP% 27 ± 5 27 ± 5 0.31
PD% 30 ± 5 31 ± 6 0.09

* Data are means ± SEM.

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632
Figure 1. Severe periodontitis is associated with increased
procalcitonin. (A) Persons with severe periodontitis (■, n = 9) had
significantly higher levels of salivary-ProCT than did those with
■, n=11, 241 ± 71 vs. 77 ± 516 pg/mL, p =
moderate periodontitis (■
0.02), and also significantly higher levels than in healthy control
individuals (■, n = 8, 241 ± 71 vs. 118 ± 26 pg/mL, p = 0.05). The
difference in serum-ProCT between persons with severe periodontitis (■,
n = 9) and those with moderate periodontitis (■■ , n = 11) did not reach
significance (107 ± 31 vs. 57 ± 6 pg/mL, p = 0.16), although the
difference between those with severe periodontitis and healthy control
individuals (■, n = 38) was significant (102 ± 20 vs. 48 ± 3, p = 0.01).
(B) Clinical radiographs of the periodontium (from left to right) of an
individual with severe periodontitis, one with moderate periodontitis,
and a healthy control individual.
added to each well. Following an additional 30 min of incubation
in the orbital shaker (150 rpm), the results were read at 450 nm on
an Elx808 ELISA reader (Bio-Tek Instruments, Winooski, VT,
USA). After the enzyme reaction was stopped by the addition of 50
␮L 3 M H2SO4 to each of the wells, the plate was read again at
490 nm. Usually, the results obtained at 490 nm were used in
calculating the assay curves and results. For this assay, the
functional sensitivity was 20 pg/mL and the 50% B/Bo (initial
binding of the labeled peptide to the antibody in the absence of
standards) was 240 pg/mL. Intra-assay reliability for this ELISA
was < 10%, and inter-assay reliability was < 18%. The serum
levels of ProCT were analyzed for ProCT (molecular weight,
12,741 Da) by means of the KRYPTOR-TRACE technology
(BRAHMS, Henningsdorf, Germany). Intra-assay reliability for
serum-ProCT was < 7%, and inter-assay reliability was < 10%
(data provided by the manufacturer and tested by the authors). The
functional sensitivity for this assay was 60 pg/mL. Assays were
run in duplicate.
Statistical Analysis
Statistical analysis was performed with the R statistical analysis
package (R Development Core Team, 2006). Normally distributed
continuous variables were expressed as means ± SEM. We used
Student’s t tests to analyze differences in groups and performed
Pearson’s correlation on data to indicate a linear relationship
between variables. P values < 0.05 were considered statistically
significant.
RESULTS
Salivary-ProCT levels for the diabetes/periodontitis group were
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International and American Associations for Dental Research
Bassim et al. J Dent Res 87(7) 2008
Figure 2. Salivary-procalcitonin is correlated with periodontitis and
diabetes status. (A) A positive correlation is shown between salivary-
ProCT and bleeding-on-¬probing (BOP) (white triangle, n = 20, r =
0.45, p = 0.05). (B) Positive correlation between salivary-ProCT and
HgbA1c (black squares, n = 20, r = 0.49, p = 0.03).
not significantly higher than those for the healthy control
individuals (152 ± 37 vs. 118 ± 3, p = 0.51); serum-ProCT
values for the periodontitis/diabetes group were significantly
higher than those found in control serum (78 ± 17 vs. 48 ± 3
pg/mL, p = 0.04; Table). Individuals with severe periodontitis
had significantly higher levels of salivary-ProCT than did those
with moderate periodontitis (241 ± 71 vs. 77 ± 516 pg/mL, p =
0.02), and also significantly higher levels than did healthy
control individuals (118 ± 26 pg/mL, p = 0.05). The difference
in serum-ProCT between persons with severe periodontitis and
those with moderate periodontitis did not reach significance
(107 ± 31 vs. 57 ± 6 pg/mL, p = 0.16), though that between
those with severe periodontitis and healthy control individuals
did (102 ± 20 vs. 48 ± 3, p = 0.01; Fig. 1). The HgbA1c values
were not different between these groups separated by
periodontal severity. Salivary-ProCT levels were not correlated
with matched serum ProCT levels.
Positive correlations exist between salivary-ProCT and BOP
(r = 0.45, p = 0.05, Fig. 2A), and between salivary-ProCT and
HgbA1c values (r = 0.50, p = 0.03, Fig. 2B). Neither of these
parameters was correlated with PD. Possible confounding
parameters were analyzed for data from this initial visit, with no
further correlations being seen between salivary-ProCT or
serum-ProCT and teeth number, age, smoking, WBC, or
glucose levels. Further, periodontal indicators were not
correlated with HgbA1c values. Serum-ProCT was not correlated
with periodontal indicators, or with glycemic control.
Levels of salivary-ProCT were higher than matched serum-
ProCT levels for the periodontitis/diabetes group (152 ± 37 vs.
78 ± 17 pg/mL, p = 0.02). Salivary-ProCT in the control group
J Dent Res 87(7) 2008 Salivary Procalcitonin in Periodontitis
was also higher than the serum-ProCT of the control
individuals (118 ± 146 vs. 48 ± 17 pg/mL, p = 0.01; Fig. 3).
After periodontal therapy, 13 people returned for a follow-
up visit. All showed improvements in periodontal parameters
on the follow-up visit (6% improvement in PD and 4%
improvement in BOP), but there was no change observed in
salivary-ProCT values (182 ± 55 vs. 146 ± 45 pg/mL, p = 0.41),
serum-ProCT values (92 ± 27 vs. 103 ± 40 pg/mL, p = 0.37), or
HgbA1c (9.8 ± 0.6 vs. 9.9 ± 0.8 %, p = 0.62) (Table). The
correlation found between salivary-ProCT and HgbA1c can be
seen in this group, both at the initial visit (r = 0.65, p = 0.02)
and at the follow-up visit (r = 0.66, p = 0.01).
DISCUSSION
This study showed the heretofore-unreported presence of
ProCT in saliva and its correlation with the degree of
periodontitis in type 2 diabetes. We hypothesized that
periodontitis in diabetes may act as a stimulus for ProCT
production, since endotoxin is a potent stimulator for the
production of ProCT and can promote the systemic release of
calcitonin precursors from nearly all tissues of the body
(Dandona et al., 1994; Preas et al., 2001). Periodontitis is
initiated and promoted by pathogenic Gram-negative,
endotoxin-producing bacterial infections (Mealey and Oates,
2006), and may promote a local up-regulation of ProCT in
saliva. We found significantly higher mean levels of salivary-
ProCT for persons with severe periodontitis when compared
with those with moderate periodontitis. As a confirmation of
this effect, salivary-ProCT was found to be significantly
correlated with the periodontal indicator BOP, a measure of
active periodontal inflammation. No correlation, however, was
found between salivary and serum levels of ProCT, which
argues against a simple reflection of serum components in the
salivary fluid. Future work may determine the extent to which
variations in stimulated and unstimulated salivary-ProCT might
be related to salivary circadian rhythms, the fasting state, and
flow rate. Larger population samples, with age- and gender-
matched controls, will be needed to compensate for the
limitations of this present study.
The correlation between salivary-ProCT and HgbA1c values
may be another supportive indication of the underlying
connection between the local and systemic inflammatory states
of periodontitis and type 2 diabetes (Temelkova-Kurktschiev et
al., 2002; Syrenicz et al., 2006). Since serum-ProCT was not
linked with hyperglycemia in this study, an increasing proximal
expression of ProCT with worsening diabetic status or
periodontitis activity may be postulated. In this regard,
procalcitonin levels have recently been measured in extremity
wound effluent and have been correlated with dehiscence in
wartime extremity injuries, suggesting a local production of
ProCT at the extremity wound site (Forsberg et al., 2008). Our
findings show the comparable analogy of a local up-regulation
of ProCT found in saliva and its correlation with the diabetic
complication of periodontitis.
Our results show that serum-ProCT was higher in our
periodontitis/diabetes group than in our control group, and that
it was further influenced by periodontitis severity, but not by
glycemic control. This may form the basis for further research
on the systemic influence of periodontitis on systemic disease.
The impact of the local chronic infection of periodontitis on
glycemic control and systemic inflammation may add to the
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International and American Associations for Dental Research
633
Figure 3. Levels of salivary-ProCT (■) were higher than matched serum-
ProCT levels (■) for the periodontitis/diabetes group (n = 20, 152 ± 37
vs. 78 ± 17 pg/mL, p = 0.02). Salivary-ProCT in the control group (■,
n = 8) was also higher than the serum-ProCT of the healthy control
individuals (■, n = 38, 118 ± 146 vs. 48 ± 17 pg/mL, p = 0.01).
rationale for the evidence-based aggressive treatment of
periodontitis in the uncontrolled diabetic population. Mounting
evidence that systemic inflammation, particularly in
cardiovascular disease, is modulated with periodontitis severity
and improves with periodontal therapy supports this
interventional approach (Tonetti et al., 2007). The use of
ProCT as a local as well as a systemic biomarker for
inflammation and infection may prove useful for future
research in this field.
Periodontal therapy did not reduce BOP% or improve PD
of the 13 individuals who returned for a follow-up visit. Poorly
controlled diabetes is a risk factor for aggressive periodontitis
and contributes to treatment resistance. This may explain the
lack of a reduction in salivary-ProCT values for persons with
treatment. Matrix metalloproteinase (MMP)-8, a putative
salivary biomarker of periodontitis (Miller et al., 2006), is
reduced with periodontal therapy (Choi et al., 2004; Emingil et
al., 2004) and does exist in higher levels in the saliva of an
individual with diabetes (Collin et al., 2000; Kumar et al.,
2006), but has not clearly been shown to be reduced in a
diabetic population with periodontal treatment. If salivary-
ProCT can be postulated as a salivary biomarker for
periodontal disease activity, its reduction with treatment could
be used as an objective end-point and therapeutic goal for
guided intervention, especially for the individual with diabetes.
Another member of the procalcitonin gene family,
adrenomedullin, has wide tissue distribution in epithelial
surfaces, and both it and calcitonin gene-related peptide
(CGRP) have antimicrobial activities, implicating this family of
gene proteins in a role in mucosal defense (Marutsuka et al.,
2001; Lopez and Martinez, 2002). Adrenomedullin has been
found in saliva at higher concentrations than serum and is
expressed in salivary tissue, and CGRP is found in saliva
(Kapas et al., 2004; Lundy and Linden, 2004; Bellamy et al.,
2006). We have shown that salivary-ProCT levels were higher
than serum-ProCT levels for both our periodontitis/diabetes
group and for the healthy control individuals. It is therefore
possible that our suggestion of the local production of ProCT in
the oral environment may have yet to determine the local
functions in the gingival tissues’ response to be bacterial
634
infection.
In summary, procalcitonin has previously been shown to be
a marker and potential mediator of systemic inflammation
(Nylen et al., 1998), and our results suggest additional
pathogenic links between the co-morbid conditions of
periodontitis and diabetes. Persons with periodontitis and type
2 diabetes have salivary-ProCT levels that, independently from
and better than serum levels, reflect their degree of
periodontitis activity and hyperglycemia. The local and
systemic role uncovered in periodontitis and hyperglycemia
vis-à-vis the expression of ProCT needs further exploration.
ACKNOWLEDGMENTS
This study was conducted through a Veterans Affairs Research
Fellowship at the Washington, DC, VA Medical Center.
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