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Salivary biomarkers of existing periodontal disease: A cross-sectional study Craig S. Miller, Charles P. King, Jr., M.

Chris Langub, Richard J. Kryscio and Mark V. Thomas J Am Dent Assoc 2006;137;322-329

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Salivary biomarkers of existing periodontal disease


A cross-sectional study
Craig S. Miller, DMD, MS; Charles P. King Jr., DMD, MS; M. Chris Langub, PhD; Richard J. Kryscio, PhD; Mark V. Thomas, DMD

hole saliva is an important physiologic fluid that contains a highly complex mixture of substances. It is secreted primarily by three paired major salivary glands and secondarily by hundreds of minor salivary glands located below the mucosal surfaces of the mouth. Salivary gland secretions contain locally produced proteins, as well as other molecules from the systemic circulation. Variable amounts of blood, serum, serum products, gingival crevicular fluid (GCF), electrolytes, epithelial and immune cells, microorganisms, bronchial products and other foreign substances also are present in whole saliva.1,2 It is this rich mixture of substances that makes saliva a likely source for identifying unique biomarkers that reflect oral and systemic health changes. Periodontal disease is a chronic bacterial infection characterized by persistent inflammation, connective tissue breakdown and alveolar bone destruction. Contributing inflammatory mediators and tissuedestructive molecules have been detected in the gingival tissues, GCF and saliva of patients affected by periodontitis.3-9 Thus, saliva should contain biomarkers specific for the unique physiological aspects of periodontitis, and qualitative changes in the composition of these biomarkers could have diagnostic

Dr. Miller is a professor, Oral Medicine Section, Department of Oral Health Practice, College of Dentistry, and Department of Microbiology, Immunology & Molecular Genetics, University of Kentucky College of Medicine, MN 324, University of Kentucky College of Dentistry, 800 Rose Street, Lexington, Ky. 40536-0297, e-mail cmiller@uky.edu. Address reprint requests to Dr. Miller. Dr. King maintains a private practice in periodontics, Easley, S.C. Dr. Langub is scientific review administrator, Office of Public Health Research, Centers for Disease Control and Prevention, Atlanta. Dr. Kryscio is a professor, Department of Statistics, College of Arts and Sciences, and Department of Biostatistics, College of Public Health, University of Kentucky, Lexington. Dr. Thomas is an associate professor and the chairman, Department of Oral Health Practice, College of Dentistry, University of Kentucky, Lexington.

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ABSTRACT

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Background. The authors conducted a study to determine if salivary biomarkers specific for three N C U aspects of periodontitisinflammation, collagen A ING EDU 1 N RT C U U degradation and bone turnovercorrelate with clinical A I N GI CDL E E 1 RT features of periodontal disease. ICLE Methods. The relationship between periodontal disease and the levels of interleukin-1 beta (IL-1), matrix metalloproteinase (MMP)-8, and osteoprotegerin (OPG) in whole saliva of 57 adults (28 case subjects with moderate-to-severe periodontal disease and 29 healthy control subjects) was examined in a case-control trial. Results. Mean levels of IL-1 and MMP-8 in saliva were significantly higher in case subjects than in controls. Both analytes correlated with periodontal indexes, whereas, after adjustment for confounders, OPG did not. Elevated salivary levels of MMP-8 or IL-1 (more than two standard deviations above the mean of the controls) significantly increased the risk of periodontal disease (odds ratios in the 11.3-15.4 range ). Combined elevated salivary levels of MMP-8 and IL-1 increased the risk of experiencing periodontal disease 45-fold, and elevations in all three biomarkers correlated with individual clinical parameters indicative of periodontal disease. Conclusion. Salivary levels of MMP-8 and IL-1 appear to serve as biomarkers of periodontitis. Clinical Implications. Qualitative changes in the composition of salivary biomarkers could have significance in the diagnosis and treatment of periodontal disease. Key Words. Periodontal disease; saliva; biomarkers; interleukin1 beta; matrix metalloproteinase. JADA 2006;137:322-9.
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and therapeutic significance. We conducted a study to test the hypothesis that levels of salivary biomarkers specific for three aspects of periodontitisinflammation, collagen degradation and bone turnovercorrelate with the clinical features of the disease. The biomarkers we tested were the following: dinterleukin 1 beta (IL-1), a proinflammatory cytokine that stimulates the induction of adhesion molecules and other mediators that facilitate and amplify the inflammatory response that occurs in periodontal disease10-16; dmatrix metalloproteinase (MMP)-8, a key enzyme in extracellular collagen matrix degradation17,18 derived predominantly from polymorphonuclear leukocytes during acute stages of periodontal disease19; dOPG, a glycoprotein that acts as an osteoblastsecreted decoy receptor and competitively inhibits osteoclast differentiation and activity by preventing osteoclast differentiation factor or the receptor activator of NF-B ligand (RANKL) from binding to osteoclast precursors and promoting the formation of bone-resorbing osteoclasts.20,21 We used enzyme immunosorbent assays to analyze IL-1 for the inflammatory phase, MMP-8 for the collagen degradation phase and OPG for bone turnover in a case-control study to determine their potential as biomarkers of periodontal disease.
SUBJECTS, METHODS AND MATERIALS

Subjects. Subjects 18 years and older who were in good general health and had at least 20 erupted teeth were eligible to participate. We sought subjects by means of advertisements. The case subjects12 men and 16 womenhad existing generalized moderate-to-severe periodontitis.22,23 We included subjects for study if at least 30 percent of their periodontal sites demonstrated bleeding on probing, at least 20 percent of periodontal sites had probing depths (PD) of 4 millimeters or greater, at least 5 percent of periodontal sites had interproximal clinical attachment loss (CAL) greater than 2 mm and radiographic bone loss was evident in posterior vertical bitewing films. We enrolled as control subjects, 29 healthy adults of similar age, race and sex who had BOP in less than 10 percent of their periodontal sites, had PD of 5 mm or greater in less than 2 percent of their periodontal sites, had no PD greater than or equal to 6 mm, had CAL of greater than 2 mm in less than 1 percent of their periodontal sites, and had no radiographic

bone loss evident in posterior vertical bitewing films. We excluded patients if they had a history of alcoholism, liver or kidney dysfunction, inflammatory bowel disease, granulomatous disease or immunosuppression, or if they were undergoing or had undergone organ transplantation or cancer therapy. Additional exclusion criteria were pregnancy, use of antibiotics or immunosuppressant medication within the last six months, need for antibiotics for infective endocarditis prophylaxis during dental procedures, symptoms of acute illness (such as fever, sore throat, body aches and diarrhea) or detection of an oral mucosal inflammatory condition (for example, aphthae, lichen planus, leukoplakia or oral cancer). The study was approved by the University of Kentuckys institutional review board. All subjects provided written informed consent and received incentives (monetary compensation as well as a clinical examination) as part of the study protocol. Methods and materials. Saliva collection, clinical examination and biomarker analysis. We collected unstimulated whole expectorated saliva from each subject according to a modified version of the method described by Navazesh.24 Subjects rinsed their mouths with tap water, then expectorated whole saliva into sterile tubes. We placed collected samples on ice immediately and put them into aliquots before freezing them at 80 C. We thawed and analyzed samples within six months of collection. One examiner (C.P.K.) recorded scores on clinical periodontal indexes (PD, BOP and CAL) for each subject after the collection of saliva. He meas ured probing depths at six locations per tooth (mesial-buccal, midbuccal, distal-buccal, mesiallingual, midlingual and distal-lingual) using a PUNC 15 probe (Hu-Friedy, Chicago). CALs were obtained by measuring interproximal sites only. Technicians determined concentrations of salivary biomarkers IL-1, MMP-8 and OPG in duplicate for each subject using enzyme immunosorbent assays (ELISAs or EIAs). Technologists in the Clinical Laboratory Improvements Amendments (CLIA)-certified General Clinical Research Center laboratory core at the University of Kentucky Medical Center used human IL-1 and MMP-8 assay kits (Human IL-1 Quantikine kit and Human Quantikine MMP-8 ELISA kit, R&D Systems, Minneapolis) and an OPG kit (Osteoprotegerin EIA kit, ALPCO Diagnostics, Salem, N.H.) according to each manufacturers directions. The IL-1 assay recognizes natural and
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TABLE 1

Comparison of case subjects* and control


CHARACTERISTIC CONTROL SUBJECTS (n = 29)

Demographics Range in age in years Mean age in years SD Mean no. teeth SD Percentage male Percentage white Percentage smoking Percentage using alcohol Dental Index Scores Percentage of sites with probing depth 5 millimeters (mean SD) Percentage of sites with probing depth 4 mm (mean SD) Percentage of sites with clinical attachment loss > 2 mm (mean SD) Percentage of sites with bleeding on probing (mean SD) * Case subjects: Subjects with periodontal disease. SD: Standard deviation. 0.16 0.38 1.69 1.71 1.01 1.57 4.93 3.77 28-61 43.1 7.2 27.2 1.8 41.4 62.1 27.6 31.0

recombinant human IL-1. Significant cross-reactivity or interference is not observed with IL-1. Statistical analysis. We compared demographic variables between the case subjects and the control subjects using the Fisher exact test, two sample t tests or both. We compared mean periodontal index scores and concentrations of the salivary analytes between the two groups using two sample t tests with unequal variances, and we used a linear mixed model to analyze the covariates age, race, tobacco use and alcohol use. We measured correlations between an analyte and a dental index by computing Pearsons correlation coefficient and then by computing the odds ratio between the events: high expression of the analyte (more than two standard deviations above the mean of the controls) and high levels of the dental indexes (large percentage of sites with the index of interest). Statistical significance was determined at the .05 level.
RESULTS

This study evaluated 57 dental outpatients ranging in age from 28 to 61 years. We enrolled
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two groups of subjects on the basis of measubjects. sures of periodontal health (case subjects CASE SUBJECTS P VALUE and control subjects). (n = 28) The 28 case subjects had periodontitis and were distributed 31-60 evenly by age among 45.4 8.5 .26 the decades (data not shown). The 29 control 26.4 3.1 .19 subjects were demo42.9 .88 graphically similar to 50.0 .14 the case subjects (Table 1) but were 33.3 .20 clearly distinct on the 57.1 .17 basis of the clinical periodontal indexes measured 30.24 20.8 < .0001 (P < .0001). One hundred percent of the 45.2 21.9 < .0001 case subjects had chronic, generalized 40.57 26.2 < .0001 moderate-to-severe periodontal disease.22,23 45.9 15.6 < .0001 Elevated salivary levels of IL-1 , MMP-8 and OPG in periodontitis. We determined concentrations and mean values of IL-1, MMP-8 and OPG in whole expectorated saliva using EIA. For each analyte examined, the mean coefficient of variance was less than 10 percent. Table 2 shows that the mean salivary levels of IL-1 and MMP-8 were significantly higher in the case subjects (3.5 times and 4.3 times, respectively) than in the control subjects. Salivary OPG levels also were elevated (1.4 times) in the case subjects. However, after adjustments were made for covariates, the difference in concentration in OPG between groups was not significant. Salivary analyte levels correlate with indexes of periodontitis. BOP, CAL and percentage of sites with PD of 4 mm or greater were significantly correlated with elevated levels of MMP-8 and IL-1 in saliva (Table 3). Elevated OPG levels also correlated with BOP and percentage of PD sites greater than or equal to 4 mm (P < .05). However, the correlation of OPG level with CAL greater than or equal to 2 mm was not significant. Table 4 (page 326) shows the odds ratios for salivary analytes predicting the periodontal index

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TABLE 2 scores (BOP, CAL and PD) consistent with periComparison of mean salivary analytes between control odontal disease. Our calsubjects and case subjects.* culations were based on elevated salivary levels ANALYTE CONTROL SUBJECTS CASE SUBJECTS UNADJUSTED ADJUSTED (MEAN SD) P VALUE P VALUE (MEAN SD) that were more than two standard deviations 753.7 1022.4 212.8 167.4 .009 .0096 Interleukin-1 Beta above the mean of the (Picograms/Milliliter) controls. This is similar 408.6 423.3 95.1 80.1 .0005 .0006 Matrix Metalloproteinase-8 to studies in which (Nanograms/mL) researchers have used 3.6 2.58 2.6 1.37 .052 .08 Osteoprotegerin cutoffs for defining other (Picomoles/Liter) metabolic bone diseases * Case subjects: Subjects with periodontitis. such as osteoporosis.25-27 SD: Standard deviation. P value based on two-sample t test with unequal variances. On the basis of these P value based on a linear mixed model with the covariates age, race, tobacco smoking, and alcohol use and analyses, the odds of a unequal variances. high percentage of sites TABLE 3 with BOP (> 30 percent) and CAL ( 2 mm) with Correlations between dental index scores and salivary elevated salivary MMP-8 analyte levels. and IL-1 (that is, more DENTAL INDEX SALIVARY ANALYTE than two standard deviations above the mean of Osteoprotegerin Matrix Interleukin-1 Beta Metalloproteinase-8 the control subjects) were more than 11 times the Correlation P Value P Value Correlation P Value Correlation odds of a low percentage 0.41 0.58 .0001 0.27 .04 Bleeding on Probing .001 of BOP and CAL sites, Clinical Attachment Loss respectively. Similarly, 0.34 0.39 .003 0.07 .59 2 millimeters .009 the odds ratios for Sites With Probing increased number of PD 0.53 0.62 .0001 0.35 .009 Depth 4 mm .0001 sites (greater than or Sites With Probing equal to 4 mm) with ele0.58 0.62 .0001 0.38 .003 Depth 5 mm .0001 vated salivary MMP-8 and IL-1 were 12.8 (confidence interval [CI] 3.04 to 53.9) and 14.6 (CI smoking and alcohol use were not significantly 2.85 to 74.7), respectively. All of the odds ratios correlated with elevated MMP-8, IL-1 and OPG for MMP-8 and IL-1 were highly significant. In salivary levels (data not shown). Racedefined contrast, the odds ratios for elevated salivary here as white and nonwhitewas not signifiOPG with BOP, CAL and PD were between 2.3 cantly correlated with elevated salivary bioand 3.0, and the CI indicated a nonsignificant difmarker levels (data not shown). ference between the groups. DISCUSSION Table 5 shows that the probability of having existing periodontitis was 11.3 times more likely Salivary analysis. Although the diagnostic when salivary MMP-8 was elevated, 15.4 times value of oral fluid has been recognized for some more likely when salivary IL-1 was elevated and time28-30 and potential biomarkers of periodontal 45.5 times more likely when both salivary MMP-8 disease have been identified in saliva,4-6,9,31 this, to and IL-1 were elevated. When OPG was elevated our knowledge, is one of the first studies to examine levels of salivary analytes critical for or when all three salivary analytes were elevated, three distinct events that occur during periodonthe odds ratio ranged between 2 and 10. However, titis (inflammation, collagen degradation and there was not a significant difference between the bone turnover). We found that salivary levels of case and control groups in terms of the likelihood IL-1 and MMP-8 were significantly higher in of having periodontitis. Age (younger than 35 case subjects than in healthy control subjects. years versus older than 54 years), sex, tobacco

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TABLE 4

Odds ratios for elevations of salivary protein analytes correlating with clinical signs of periodontal disease.
ELEVATED* ANALYTE ODDS RATIOS (95% CONFIDENCE INTERVALS [CI]) Bleeding on Probing (> 30% versus < 10% sites) Interleukin-1 Beta Matrix Metalloproteinase-8 Osteoprotegerin 14.6 (CI 2.85-74.7) 12.8 (CI 3.04-53.9) 3.0 (CI 0.52-17.0) % Clinical Attachment Loss 2 Millimeters (> 10% versus < 10% sites) 15.4 (CI 3.1-77.4) 11.3 (CI 2.8-46.4) 2.6 (CI 0.46-14.7) Probing Depths 4 mm (> 25% versus < 5% sites) 14.6 (CI 2.85-74.7) 12.8 ( CI 3.04-53.9) 2.3 (CI 0.37-13.6)

* Elevated: More than two standard deviations above the mean of the controls.

TABLE 5

in bone.35 Although both isoforms of IL-1 (IL-1 and IL-1) have similar biological activiN (%) OF PATIENTS ELEVATED* ODDS RATIO 95% CL ties and appear to be WITH PERIODONTITIS ANALYTE Upper Lower contributory, IL-1 is 15.4 16 (57.1) 3.1 76.8 Interleukin-1 Beta (IL-1 ) more potent in stimulating bone resorption 11.3 17 (60.7) 2.8 45.8 Matrix Metalloproteinase-8 (MMP-8) and is the form more frequently occurring in 2.7 5 (17.9) 0.48 15.2 Osteoprotegerin (OPG) periodontitis.36-38 In 45.5 13 (46.4) 2.54 814.0 IL-1 and MMP-8 clinical studies, 2.08 4 (14.3) 0.35 12.3 MMP-8 and OPG increased levels of IL1 in GCF have been 10.02 4 (14.3) 0.51 195.0 IL-1, MMP-8 and OPG associated with gin* Elevated: More than two standard deviations above the mean of the controls. CL: Confidence limit. gival inflammation, severe periodontitis Both analytes correlated positively with periand periodontal disease progression,39-43 and levels odontal index scores (that is, BOP, CAL and perdecrease after therapy.34,44 Investigators also have centage of sites with PD greater than or equal to detected IL-1 at elevated levels in tissues of 4 mm) (Table 3). Elevated levels of MMP-8 or patients who have progressive periodontitis36,38,45,46 IL-1 in saliva (that is, more than two standard and observed the levels to increase in GCF before deviations above the mean of the controls) significlinical changes were observed.47 Thus, our deteccantly increased the risk of higher BOP, CAL and tion of elevated levels of IL-1 in the whole saliva PD (odds ratio 11 to 15.4). Furthermore, comof subjects with chronic periodontitis was consisbined elevated levels of MMP-8 and IL-1 in tent with the cytokines role in inflammation and saliva increased the risk of periodontal disease suggests that salivary IL-1 may be a sensitive 45-fold. In contrast, elevated salivary levels of marker of periodontal inflammation. OPG did not distinguish case subjects from conMMPs. MMPs are zinc-dependent endopeptitrol subjects but nevertheless were associated dases and key enzymes in extracellular collagen with elevated BOP and PDs. matrix degradation17,18 derived predominantly IL-1. IL-1, a proinflammatory cytokine, is from polymorphonuclear leukocytes during acute important in the pathogenesis of periodontal disstages of periodontal disease.19 Investigators have 10-15 ease. It induces widespread gene expression of detected several of the 25 types of MMPs at elecyclo-oxygenase-2, inducible nitric oxide synvated levels in inflamed human gingivae48 and thetase and metalloproteinases, which results in GCF49 in subjects with adult periodontitis.19,50,51 32-34 activation of osteoclasts and bone resorption However, researchers have detected MMPs less and downregulation of type I collagen expression frequently in saliva.52 Since MMP-8 has the

Odds ratio for elevations of salivary protein analytes correlating with periodontitis.

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unique ability to break down collagen of types I and III, which are critical for periodontal destruction, and subantimicrobial doses of doxycycline inhibit MMP-8 and reduce periodontal disease activity,53-56 we investigated the role of MMP-8 as a putative marker of periodontitis. We observed mean elevated levels of MMP-8 (that is, more than four times that in the saliva of healthy control subjects) in the saliva of subjects with periodontal disease. Elevated MMP-8 levels were highly correlated with BP, CAL and PD measures in a manner consistent with the features of periodontal disease. This indicates that analytes reflective of the collagen degradation phase of periodontitis are measurable in saliva and may be useful for monitoring disease activity. OPG. OPG is a glycoprotein that inhibits osteoclast differentiation and activity competitively by preventing osteoclast differentiation factor RANKL from binding to osteoclast precursors and promoting the formation of bone-resorbing osteoclasts.20,21 Levels in saliva are not well-defined in health or disease. OPGs role in bone metabolism leads one to hypothesize that its levels would be increased in circumstances that require inhibition of osteoclastogenesis. In fact, we observed elevated levels in the case subjects compared with the levels in the control subjects (P = .086). Although our results did not show a significant difference in the level of OPG between the two groups, the trend toward elevation in the case subjects offers insight into the possible biological mechanisms at work. One possibility is that an increase in osteoclast precursors might lead to the upregulation of OPG. In turn, OPG would inhibit an increased number of osteoclast precursors. However, during periodontal destruction, there may be a threshold whereby OPG is operating at full capacity, yet cannot overcome the overwhelming number of osteoclast precursors; this would result in bone turnover. This might explain why, in this study, the mean level of OPG was elevated slightly in case subjects compared with control subjects. Additional contributing factors could be the sample size and demographic characteristics, as well as the fact that periodontitis is episodic. That is, bone turnover may not have been occurring in each subject at the time of sampling in this cross-sectional study. Alternatively, OPG may not have been the most appropriate marker for assessment of active bone resorption, and future studies should address this possibility. GCF sampling versus analysis of salivary

biomarkers. To date, the majority of investigations regarding biomarkers and periodontal status have used GCF collected on filter paper strips. Much valuable information about the inflammatory response in periodontal disease has been determined from the biochemical analysis of this fluid.39-44,47 However, there are a few problems inherent in GCF sampling and analysis. For example, such sampling involves miniscule amounts of fluid, which can affect laboratory analysis.57 Contamination of the sample with blood, saliva or plaque also is a potential problem. Samples contaminated with blood often are discarded, but as these may represent more inflamed sites, this may introduce a differential bias into results of such studies.57 Sampling time also is a problem, as is the manner in which the strip is inserted. In one study, researchers found that GCF volume was correlated positively with clinical indicators of inflammation only after a fifth sequential strip had been inserted.58 Given some of the problems inherent in sampling GCF, the analysis of salivary biomarkers offers some advantages. One of these is that whole saliva represents a pooled sample from all periodontal sites, thereby giving an overall assessment of disease status. A second advantage is that levels of salivary analytes may reflect current disease activity as well as severity. Third, salivary analytes may offer a way of assessing subject-level (as opposed to site-level) risk or status. Finally, collection of whole saliva is easy, noninvasive and rapid and requires no special equipment or expertise. There are, however, some disadvantages to using whole saliva for diagnostic purposes. Whole or mixed saliva is a complex fluid mixture derived from the major and minor salivary glands, and it contains contributions from the GCF, oral bacteria, cells and other sources that make identification of the exact site of disease activity difficult. In addition, the flow rate of saliva varies within and between subjects. Various medications affect salivary flow rate, and there is evidence of sex and biorhythm differences.59-61 Changes in flow rate could have a significant impact on the concentration of analytes in the saliva, particularly those at low concentrations. Also, the composition of stimulated and unstimulated saliva differs, and this could affect the analytes of interest.62,63 We have yet to address whether different results would be obtained if stimulated saliva was analyzed, or
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whether results might be affected if the data were reported as concentrations per flow rate. Some investigators have proposed methods of estimating resting salivary flow rate, methods that may be useful in determining the concentration of analytes.64,65 Future studies could address these issues. Also, it remains to be determined whether the salivary biomarkers analyzed are capable of distinguishing health from disease when the nature of disease is less generalized in subjects.
CONCLUSION

In summary, these data support our hypothesis that levels of salivary biomarkers specific for three aspects of periodontitisinflammation, collagen degradation and bone turnovercorrelate with the clinical features of periodontal disease and suggest that elevated salivary levels of MMP8 and IL-1 are candidate biomarkers of periodontal disease. These findings are consistent with our previous demonstration that C-reactive protein, a marker of inflammation, is elevated in saliva of patients with periodontal disease compared with that of healthy and edentulous control subjects.66 While considerable work needs to be done to confirm the usefulness of these salivary biomarkers, analytes characteristic of the active phases of periodontitis could prove valuable in identifying patients with enhanced disease susceptibility, identifying sites with active disease, predicting sites that will have active disease in the near future and/or serving as surrogate endpoints for monitoring of therapy. Rigorous studies that result in improvements in our diagnostic capacity regarding these areas would greatly facilitate the clinicians treatment of periodontal diseases and the assessment of periodontal status in medical and research settings. s
The authors thank Ken Westberry and Jason Stevens for providing technical assistance. This study was supported by grants from the National Institutes of Health (UO1 DE15017 and M01-RR02602) and the University of Kentucky General Clinical Research Center. This article was written in a personal capacity and does not necessarily represent the opinions or reflect the views of the National Institutes of Health, the U.S. Department of Health and Human Services or the federal government (M.C.L.). 1. Edgar WM. Saliva: its secretion, composition and functions. Br Dent J 1992;172(8):305-12. 2. Schenkels LC, Veerman EC, Nieuw Amerongen AV. Biochemical composition of human saliva in relation to other mucosal fluids. Crit Rev Oral Biol Med 1995;6(2):161-75. 3. Armitage GC; Research, Science and Therapy Committee of the American Academy of Periodontology. Diagnosis of periodontal diseases [published erratum appears in J Periodontol 2004;75:779]. J Periodontol 2003;74(8):1237-47.

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