Studies have indicated that host inflammatory proteins, enzymes and indicators of bone metabolism present in saliva differ in different types of periodontal disease. However, the number of markers analyzed was limited and the effect of edentulousness was not examined.We measured the concentration of host inflammatory proteins: C-reactive protein (CRP), C3 and C4 complement components, alpha-2-macroglobulin (a -2M) and tumor-necrosis factor (TNF) in unstimulated saliva of 14 periodontally healthy (PH), 9 edentulous persons (EP), 10 patients with chronic periodontitis (CP) and 18 with aggressive periodontitis (AgP). TNF was below the level of detection in all samples except one. Edentulous persons and patients with CP had significantly reduced concentrations of CRP, C3 and a -2M. Edentulous persons and AgP patients had lower C4 concentrations. We can conclude that edentulous persons and CP patients have reduced salivary concentrations of host inflammatory proteins. These findings suggest that a reduction in host responsiveness might play a role in the pathogenesis of CP

Ana Stavljenić-Rukavina

Andrej Aurer

Darije Plančak

Jelena Aurer-Koželj

Ksenija Jorgić-Srdjak

English

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Coll. Antropol. 29 (2005) 2: 435–439UDC 616.314-085Original scientific paperProinflammatory Factors in Saliva as PossibleMarkers for Periodontal DiseaseAndrej Aurer1, Ksenija Jorgi}-Srdjak1, Darije Plan~ak1, Ana Stavljeni}-Rukavina2 andJelena Aurer-Ko`elj11 Department of Periodontology, School of Dental Medicine, University of Zagreb, Zagreb, Croatia2 Department of Laboratory Diagnostics, University Hospital Center »Zagreb«, Zagreb, CroatiaA B S T R A C TStudies have indicated that host inflammatory proteins, enzymes and indicators of bone metabolism present in sa-liva differ in different types of periodontal disease. However, the number of markers analyzed was limited and the ef-fect of edentulousness was not examined. We measured the concentration of host inflammatory proteins: C-reactive pro-tein (CRP), C3 and C4 complement components, alpha-2-macroglobulin (-2M) and tumor-necrosis factor (TNF) inunstimulated saliva of 14 periodontally healthy (PH), 9 edentulous persons (EP), 10 patients with chronic period-ontitis (CP) and 18 with aggressive periodontitis (AgP). TNF was below the level of detection in all samples except one.Edentulous persons and patients with CP had significantly reduced concentrations of CRP, C3 and -2M. Edentulouspersons and AgP patients had lower C4 concentrations. We can conclude that edentulous persons and CP patients havereduced salivary concentrations of host inflammatory proteins. These findings suggest that a reduction in host respon-siveness might play a role in the pathogenesis of CP.Key words: saliva, markers, proinflammatory factors, periodontitisIntroductionThe diagnostics of periodontal disease relies primar-ily on clinical and radiographic parameters. These mea-sures are useful in detecting evidence for past disease,or verifying periodontal health, but provide limited in-formation about patients at risk for future periodontalbreakdown1,2.The gingival crevicular fluid (GCF) is derived fromgingival capillary beds (serum components) and resi-dent and migrating inflammatory cells. Factors derivedfrom GCF and the subgingival plaque is found in wholesaliva, rather than gland-specific saliva3.Saliva can be easily collected, contains locally de-rived and systemically-derived markers of periodontaldisease, and hence may offer the basis for a patient spe-cific diagnostic test for periodontitis4. Furthermore, theanalysis of saliva may offer a cost-effective approach toassess periodontal disease incidence in large popula-tions1,5.The use of saliva in periodontal diagnostics has beenthe subject of considerable research activity, and pro-posed markers for disease include proteins of host ori-gin, phenotypic markers, host cells, hormones, bacteriaand bacterial products, volatile compounds and ions6–8.Studies have examined enzyme activity in saliva inrelation to periodontal status and in response to perio-dontal treatment1,4,5,8. Their results indicate that salivamight contain markers useful for diagnosis of perio-dontal disease. However, their exact value or the opti-mal markers combination has not been defined. Also,the influence of other factors, such as edentulousness,on saliva constituents has not been analyzed.We therefore measured following markers of inflam-mation: C-reactive protein (CRP), C3 and C4 compo-nents of complement (C3, C4), -2 macroglobulin (-2M)and tumor necrosis factor (TNF) in whole saliva of pa-tients with chronic periodontitis (CP), aggressive perio-dontitis (AgP), periodontal healthy (PH) and edentulouspersons (EP) to identify potential markers useful for di-agnosis of periodontal disease.435Received for publication February 15, 2005Material and MethodsSubjectsThe study included 51 persons, 25 males and 26 fe-males. Two study groups consisted of 18 untreated AgPpatients, 9 males and 9 females and 10 untreated CPpatients, 5 males and 5 females, refered to our depart-ment at the Dental School University of Zagreb, fortreatment of periodontal disease. The mean age in theAgP group was 33 years, ranging from 19–52 years, andthe mean age in the CP group was 38 years, rangingfrom 29–49 years. Control group consisted of 14 PH per-sons, 7 males and 7 females with mean age 24 years,ranging from 23–25 years, and 9 EP persons, 4 malesand 4 females with mean age of 73 years, ranging from65–81 years.Criteria for AgP were: familial history of periodontaldisease, generalized interproximal attachment loss af-fecting at least 4 permanent teeth other than first mo-lars and incisors, and radiographically-proven verticalbone resorption9. Criteria for CP were: gingival inflam-mation, pocketing with clinical attachment loss of atleast 10 sites with more than 5 mm of clinical attach-ment loss spread over several teeth and radiographicalevidence of alveolar bone loss of more than 1/3 of theroot length on at least 1 tooth per quadrant10. Perio-dontal screening was part of initial periodontal exami-nation, using following clinical measurements: plaqueindex (PI)11, sulcus bleeding index (SBI)12, probing depthand clinical attachment loss (CAL)13. Periodontal tissuedestruction was determined by measuring the attach-ment level loss calculated from the periodontal probingdepth and gingival retraction. Probing depth was mea-sured on mesial, distal, oral and vestibular sides of allteeth while gingival retraction was measured on oraland vestibular sides of all teeth using a calibrated man-ual probe. Diseased groups did not differ regarding thenumber of teeth.PH persons were without clinical signs of destructionof the periodontal ligament, had low plaque and gingivalinflammation levels, and showed no signs of pocketing.According to anamnestic data reasons for teeth lossin the EP group were caries and its complications orperiodontal disease.A dietary and medical history was obtained from allsubjects and all participants received a complete dentalcheck-up. Individuals were encouraged to follow theirnormal daily nutritional regimen; no specific instruc-tions regarding food or fluid intake were given.Exclusion criteria were: individuals who had serioussystemic diseases, such as diabetes or cancer, smoking(including persons who stopped smoking less than 2 yrs.ago) extensive carious lesions, medication in the 6months preceding the study (e.g. immunosuppressivedrugs, antibiotics, antiphlogistics) or pregnancy.After the purpose and scope of the study was ex-plained to them, all subjects gave an written informedconsent for participation in this study.Approval for this investigation was given by the Eth-ical Committee.Collection of salivaAll saliva samples were collected in the morning, atleast 2 hours after any food intake. Unstimulated salivawas collected after oral cavity irrigation with water. Af-ter the irrigation, the subjects took 5 ml of water andheld it for 5 minutes in oral cavity, after which the sam-ple was taken. Unstimulated saliva was obtained bypassive drooling during 10 min with the subjects sittingin a relaxed position. About 4–5 ml of saliva was placedin an ice chilled test tube and transported to the Depart-ment of Laboratory Diagnostics, University HospitalCenter Zagreb. All samples were homogenized, stored at–70°C and tested simultaneously.To determine the temporal variability of tested sali-vary substances in 5 patients with AgP and 5 with CP,samples were obtained 3 times during a period of 1 h.Furthermore, the concentration of tested substanceswas determined in the same subjects on a standard dietfor 3 consecutive days.Laboratory methodsCRP concentrations were determined by simple ra-dial immunodiffusion, using specific anti-human CRPantibodies forming insoluble aggregates after binding(intra assay CV%=1.02, inter-assay CV%=1.29)14. C3and C4 were determined photometrically after radial im-munodiffusion on LC-Partingen plates (Behring, Frank-furt, Germany), based on formation of immune com-plexes with specific antibodies (intra-assay CV% = 1.9,1.8; inter-assay CV%=5.9, 2.9 for C3 and C4 respec-tively)15. -2M concentrations were determined usingthe same method (intra-assay CV%=1.6, inter-assayCV%=3.0)15. TNF was determined employing linkedimmunosorbent assay ELISA with specific monoclonalantibodies (Endogen, Cambridge, Ma, USA) (inter-as-say CV%=1.6, intra-assay CV%=3.3)14.Statistical analysesDifferences between groups for tested variables wereassessed using variance analysis, and for clinical at-tachment loss using t-test. Normality of distributionwas tested with Kolmogorov-Smirnov test. The correla-tion of variables was tested with Pearson correlation co-efficient.ResultsSex did not influence the finding. The mean CALlevel in CP patients was 3.4 mm, range 1.8–4.7 mm, andin AgP patients 4.2 mm, range 3.0–6.4 mm. This differ-ence was not significant (p=0.07). Maximal attachmentloss (CALMAX) is different in CP and AgP groups. Inthe CP group CALMAX was 5.4 mm, range 2.8–9.0 mm.In the AgP group CALMAX was 6.7 mm, range 4.8–9.5mm (Table 1).A. Aurer et al.: Salivary Markers of Periodontal Inflammation, Coll. Antropol. 29 (2005) 2: 435–439436The mean PI for the CP group was 0.8, and 1.9 forthe AgP group. Mean SBI scores were 1.26 in CP pa-tients and 3.02 in AgP patients. In the PH group meanPI was 0.6, mean SBI score was 0.5 and mean CAL 0.3mm, range 0–0.8 mm.TNF was below the level of detection in all samplesexcept one from a patient with AgP.Table 1 shows mean values and standard deviations(SD) of variables: CRP, C3, C4 and -2M. The groupsdiffer in proinflammatory marker concentrations observ-ed in unstimulated saliva. CRP values in saliva werehighest in the AgP group (102.11±79.02), then in the PHgroup (90.20±79.67), while CP and EP groups show sig-nificantly lower values (27.45±29.59) and (20.72±29.51).Observing the paired-differences significant differenceexists in CRP concentrations between EP and AgP, andCP and AgP groups.Tested groups show similar relationship when C3concentrations are analysed. Similarly high values areobserved in PH and AgP groups (10.74±8.03 and 10.39±5.08), while EP and CP groups show almost three timeslower C3 concentrations in saliva (3.79±2.25 and 3.62±2.94). The differences between pairwise PH and EP, PHand CP, AgP and EP, and AgP and CP groups are statis-tically significant.The highest C4 concentration was observed in thePH group (35.36±11.74), somewhat lower in CP group(24.10±10.67). For AgP group C4 concentration was20.55±7.82, and EP group showed lowest concentrationsC4 in saliva (17.50±12.84). Statistically significant dif-ferences in C4 concentrations were found between fol-lowing pairs: PH and EP, PH and CP, EP and AgP, andCP and AgP.When differences in -2M concentrations in the sa-liva are analysed between the groups, they show rela-tion similar to differences of CRP and C3 values be-tween the groups. Mean concentration of -2M in salivaof PH group is 401.14±484.58, and 384.57±383.48 in theAgP group. In the EP and CP group -2M concentra-tions were approximately seven times lower (57.00±A. Aurer et al.: Salivary Markers of Periodontal Inflammation, Coll. Antropol. 29 (2005) 2: 435–439437TABLE 1VARIABLES FOR MARKERS OF PERIODONTAL DISEASE IN SALIVA AND ATTACHMENT LOSS FOR CP AND AGP GROUPSVariables Groups N X SD Standard error Test statistics df pCRP PH 14 90.20 79.67 21.294.95a 3, 47 0.005EP 18 20.72 29.51 9.84CP 10 27.45 29.59 9.36AgP 9 102.11 79.02 18.62Total 51 69.84 73.18 10.25C3 PH 14 10.74 8.03 2.156.15a 3, 47 0.001EP 18 3.79 2.95 0.98CP 10 3.62 2.94 0.93AgP 9 10.39 5.08 1.20Total 51 8.00 6.30 0.88C4 PH 14 35.36 11.74 3.147.18a 3, 47 <0.001EP 18 17.50 12.84 4.28CP 10 24.10 10.67 3.37AgP 9 20.55 7.82 1.84Total 51 24.77 12.29 1.72-2M PH 14 401.14 484.58 129.513.74a 3, 47 0.017EP 18 57.00 42.25 14.08CP 10 58.70 42.97 13.59AgP 9 384.57 383.48 90.39Total 51 267.42 371.89 52.07CAL CP 10 3.40 0.89 0.28–1.89b 26 0.070AgP 18 4.18 1.12 0.26Total 28 3.91 1.09 0.21CALMAX CP 10 5.40 1.80 0.57–2.03b 26 0.050AgP 18 6.69 1.49 0.35Total 28 6.23 1.69 0.32aF-test, bt-test, CP – chronic periodontitis, AgP – aggressive periodontitis, PH – periodontally healthy, EP – edentulous persons, CAL– mean clinical attachment loss,CALMAX – maximal clinical attachment loss, CRP – C-reactive protein, -2M – -2 macroglobulin,C3 – C3 complement component, C4 – C4 complement component42.25, and 58.70±42.97). Statistically significant differ-ences in the -2M concentrations were found pairwisebetween PH and EP, CP and AgP, and EP and CP.Correlation between the tested variables (CRP, C3,C4 and -2M) was tested using Pearson correlation coef-ficient. In the PH group significant positive correlationwas observed between -2M and CRP (r=0.703, p=0.005).Significant positive correlation was also observed be-tween -2M and CRP (r=0.922, p<0.001).Between immunological and clinical variables, therewas no significant correlation in the CP group (Table 2).Similarly, no correlation was observed in the AgP groupbetween immunological and clinical variables (Table 3).DiscussionOur data show that patients with different types ofperiodontitis differ between themselves and from he-althy persons in their salivary content of inflammatoryor bone destruction markers. This supports the hypoth-esis that biochemical analyses of saliva might be an im-portant non-invasive way of studying disease processesin periodontitis16. However, since teeth loss exerts avery strong influence on these variables, future studiesmust take this factor into account. TNF concentration insaliva is below the level of detection of commerciallyavailable assays and investigators who wish to measureit should use alternative methods.CRP, C3 and -2M are humoral inflammatory mark-ers and acute phase reactants. Reduced levels of theseproteins in saliva of patients with CP, indicates that inthis chronic disorder host inflammatory response isreduced17–20. C4 is also a humoral inflammatory markerand acute phase reactant. In contrast to C4, C3 and-2M is higher in AgP in comparison to CP patients orPH group. Reduced concentrations of these proteins andC4 in EP group can be explained by the lack of gingivalfluid component in their saliva.In summary, our data show that the measurement ofhost inflammatory markers in the saliva might be avaluable path to follow in studying periodontal healthand disease, taking into account that teeth loss pro-foundly affects these findings. CP is characterized by areduction in concentrations of different inflammatorymarkers, indicating that a reduced host response mightbe important in the pathogenesis of this disorder21.A. Aurer et al.: Salivary Markers of Periodontal Inflammation, Coll. Antropol. 29 (2005) 2: 435–439438TABLE 2CORRELATION OF VARIABLES FOR CP GROUP (N=10)Variables CRP C3 C4 -2M CAL CALMAXCRP – 0.903* –0.031 0.326 –0.327 –0.229C3 0.903* – 0.118 0.092 –0.205 –0.224C4 –0.031 0.118 – 0.021 0.010 –0.333-2M 0.326 0.092 0.021 – 0.082 –0.010CAL –0.327 –0.205 0.010 0.082 – 0.890*CALMAX –0.229 –0.224 –0.333 –0.010 0.890* –*correlation is significant at the 0.01 level, CP – chronic periodontitis, CAL – mean clinical attachment loss, CALMAX – maximalclinical attachment loss, CRP – C-reactive protein, -2M – -2 macroglobulin, C3 – C3 complement component, C4 – C4 complementcomponentTABLE 3CORRELATION OF VARIABLES FOR AGP GROUP (N=18)Variables CRP C3 C4 -2M CAL CALMAXCRP – 0.387 –0.132 0.384 0.123 0.245C3 0.387 – –0.090 0.000 –0.257 –0.297C4 –0.132 –0.090 – 0.222 –0.035 0.063-2M 0.384 0.000 0.222 – 0.023 0.183CAL 0.123 –0.257 –0.035 0.023 – 0.917*CALMAX 0.245 –0.297 0.063 0.183 0.917* –*correlation is significant at the 0.01 level, AgP – aggressive periodontitis, CAL – mean clinical attachment loss, CALMAX – maxi-mal clinical attachment loss, CRP – C-reactive protein, -2M – -2 macroglobulin, C3 – C3 complement component, C4 – C4 comple-ment componentR E F E R E N C E S1. OHSHIMA, M., K. FUJIKAWA, H. AKUTAGAWA, K. ITO, K. J.OTSUKA, Oral. Sci., 44 (2002) 35. — 2. LAMSTER, I. B., Ann. Perio-dontol., 2 (1997) 123. — 3. EBERSOLE, J. L., Periodontology 2000, 31(2003) 135. — 4. PEZELJ-RIBARIC, S., I. B. PRSO, M. ABRAM, I. GLA-ZAR, G. BRUMINI, M. [IMUNOVI]-[O[KI], Mediators Inflamm., 13(2004) 131. — 5. KAUFMAN, E., I. B. LAMSTER, J. Clin. Periodontol.,27 (2000) 453. — 6. MANDEL, I. D., J. Oral Pathol. Med., 19 (1990) 119.— 7. FDI WORKING GROUP 10, CORE, Int. Dent. J., 42 (1992) 287. —8. LAMSTER, I. B., E. KAUFMAN, J. T. GRBIC, L. J. WINSTON, R. E.J. SINGER, J. Periodontol., 74 (2003) 353. — 9. TONETTI, M. S., A.MOMBELLI, Aggressive periodontitis. In: LINDHE, J., T. KARRING,N. P. LANG (Eds.): Clinical Periodontology and Implant Dentistry. (Black-well, Munksgaard, 2003). — 10. KINANE, D. F., J. LINDHE, Chronicperiodontitis. In: LINDHE, J., T. KARRING, N. P. LANG (Eds.): ClinicalPeriodontology and Implant Dentistry. (Blackwell, Munksgaard, 2003).— 11. SILLNESS, J., H. LÖE, Acta Odontol. Scand., 22 (1964) 121. —12. MÜHLEMAN, H., S. SON, Helvet. Odontol. Acta, 15 (1971) 107. —13. PIHLSTROM, B. L., J. Periodontol., 63 (1992) 1072. — 14. YOUNG,D. S.: Effects of drugs on clinical laboratory tests. (Washington, AACCPress, 2003). — 15. THOMAS, L.: Clinical Laboratory Diagnostics Useand Assessment of Clinical Laboratory Results. (TH-Books Verlagsge-sellschaft, Frankfurt/Main, 1998). — 16. JENTSCH, H., Y. SIEVERT, R.J. GOCKE, Clin. Periodontol., 31 (2004) 511. — 17. BROCK, G. R., C. J.BUTTERWORTH, J. B. MATTHEWS, I. L. J. CHAPPLE, J. Clin Perio-dontol., 31 (2004) 515. — 18. SCULLEY, D. V., S. C. LANGLEY-EVANS,Proc. Nutr. Soc., 61 (2002) 137. — 19. ADONOGIANAKI, E., J. MOO-NEY, D. F. J. KINANE, J. Clin. Periodontol., 19 (1992) 98. — 20. PE-DERSON, E. D., S. R. STANKE, P. T. WHITENER, B. L. SEBASTIANI,D. W. TURNER, Arch. Oral Biol. 40 (1995) 1151. — 21. ZUABI, O., E. E.MACHTEI, H. BEN-ARYEH, L. ARDEKIAN, M. PELED, D. J. LAU-FER, J. Periodontol., 70 (1999) 1240.A. Aurer et al.: Salivary Markers of Periodontal Inflammation, Coll. Antropol. 29 (2005) 2: 435–439439A. AurerDepartment of Periodontology, School of Dental Medicine, University of Zagreb, Gunduli}eva 5, 10000 Zagreb, Croatiae-mail: aurer@sfzg.hrFAKTORI UPALE U SLINI KAO MOGU]I BILJEZI PARODONTNE BOLESTIS A @ E T A KSlina ~ini pogodan medij za otkrivanje proupalnih biljega u usnoj {upljini. Brojni serumski i tkivni sastojci kojiprolaze kroz stijenku spojnog epitela gingivnog sulkusa i ulaze u slinu, mogu pomo}i u odre|ivanju aktivnosti paro-dontitisa. Stoga smo istra`ivali me|uzavisnost medijatora lokalne imunosti u slini doma}ina i klini~ke parametredestrukcije u oboljelih od kroni~nog (CP) i agresivnog (AgP) parodontitisa. Koncentraciju upalnih proteina doma}inaodre|ivali smo enzim-imunokemijskim testovima i u slini bezubih (E), {to do sada nije istra`ivano, te u parodontzdravih osoba (PH). Stupanj destrukcije parodonta mjeren je gubitkom vezivnog pri~vrstka (CAL). Istra`ivanje jeobuhvatilo 51 osobu. Struktura navedenih uzoraka i njihova me|uzavisnost testirana je neparametrijskim meto-dama. Dobiveni rezultati pokazuju kako se razina C-reaktivnog proteina (CRP), C3-komponente komplementa (C3),-2 makroglobulina zna~ajno razlikuju u nativnoj slini oboljelih od CP i AgP. Bezube osobe, sli~no CP skupini, poka-zivale su zna~ajnu redukciju koncentracije CRP, C3 i -2M. C4 komponenta komplementa ne mjeri razliku me|ubolesnima. Zaklju~iti se mo`e kako se razina medijatora obrane doma}ina razlikuje za pojedine vrste parodontitisa unativnoj slini.

Proinflammatory Factors in Saliva as Possible Markers for Periodontal Disease

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