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Journal of Dental Research

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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International and American Associations for Dental Research

International and American Associations for Dental Research

RESEARCH REPORTS

Clinical

C.W. Bassim 1,2 *, R.S. Redman 1 , D.J. DeNucci 1,2 , K.L. Becker 1,3 , and E.S. Nylen 1,3

1 Washington, DC, Veterans Affairs Medical Center; 2 NIDCR, CRC, NIH, Bldg. 10, Rm 1N-118, MSC 1191, 10 Center Drive, Bethesda, MD 20892, USA; and 3 George 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

630

Salivary Procalcitonin and Periodontitis in Diabetes

INTRODUCTION

T he simple and non-invasive nature of saliva collection and high- sensitivity assay development has led to an emphasis on the promise of

salivary 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 (HgbA 1c > 7.0%, n = 20). Serum was analyzed for HgbA 1c , 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

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International and American Associations for Dental Research

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 HgbA 1c (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) Sex (male/female) Smoking status (yes/no) Number of teeth

56 ± 2*

55 ± 2

20/0

13/0

7/13

4/9

23 ± 1

22 ± 1

 

P value

P value

HgbA 1c % Fasting glucose (mg/dL) WBC (K/cmm) Salivary ProCT (pg/mL) Serum ProCT (pg/mL) BOP% PD%

10.1 ± 0.5 158.2 ± 16.2 7.5 ± 0.6 152 ± 37

9.9 ± 0.8 169.7 ± 22.1 7.9 ± 1.2 146 ± 45

 

0.62

0.53

0.91

118 ± 26 (n = 8) 48 ± 3 (n = 34)

0.41

0.51

78 ± 17

103.5 ± 40

0.37

0.04

27 ± 5 30 ± 5

27 ± 5 31 ± 6

0.31

 

0.09

* Data are means ± SEM.

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International and American Associations for Dental Research

632

Bassim et al.

J Dent Res 87(7) 2008

632 Bassim et al. J Dent Res 87(7) 2008 Figure 1. Severe periodontitis is associated with

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

vs. 77 ± 516 pg/mL, p =

moderate periodontitis (, n=11, 241 ± 71

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.

moderate periodontitis, and a healthy control individual. Figure 2. Salivary-procalcitonin is correlated with

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 HgbA 1c (black squares, n = 20, r = 0.49, p = 0.03).

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 H 2 SO 4 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

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 HgbA 1c 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 HgbA 1c 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 HgbA 1c 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

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J Dent Res 87(7) 2008

Salivary Procalcitonin in Periodontitis

633

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 HgbA 1c (9.8 ± 0.6 vs. 9.9 ± 0.8 %, p = 0.62) (Table). The correlation found between salivary-ProCT and HgbA 1c 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 HgbA 1c 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

on glycemic control and systemic inflammation may add to the Figure 3. Levels of salivary-ProCT (

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

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Bassim et al.

J Dent Res 87(7) 2008

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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