Volume 71 • Number 4

Analysis of Human Gingival Tissue andGingival Crevicular Fluid �-GlucuronidaseActivity in Specific Periodontal DiseasesMeral Layik,* Nermin Yamalik,* Feriha Çaglayan,* Kamer Kilinç,† Ilker Etikan,‡ and Kenan Eratalay*

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Background: β-glucuronidase (βG) is one of the enzymesinvolved in the destruction of non-collagenous components ofthe extracellular matrix. It is also considered an indicator or pre-dictor of periodontal disease activity. The present study wasconducted to determine the presence and the levels of βG activ-ity in gingival tissue and gingival crevicular fluid (GCF) in peri-odontal disease and health status. The validity of 2 expressionsof data, total βG activity versus βG concentration, and the cor-relations between clinical periodontal status and βG profile wasalso evaluated.

Methods: βG activities in gingival tissues and GCF samplesfrom 57 individuals, divided into 3 equal groups of adult peri-odontitis (AP), early-onset periodontitis (EOP), and periodon-tally healthy subjects were spectrophotometrically examined.

Results: Both patient groups had higher βG levels in bothgingiva and GCF than controls. Significant differences wereobserved among all groups when total GCF βG activities wereexamined (P <0.05). However, the difference between AP andcontrols was not significant when concentration values werecompared (P >0.05). The highest GCF βG activity, with bothexpressions, was detected in EOP group. No absolute correla-tions between clinical parameters and βG activity were observed,except for random correlations in the patient groups with meantotal βG activities. Also GCF/gingiva βG levels and the 2 expres-sions did not show absolute correlations.

Conclusions: The findings of the present study confirm therelationship between βG activity and periodontal diseases. Thedifferences in data concerning GCF total βG activity and βGconcentration may suggest that they are not matching mea-sures. Data presentation seems to be an important factor inGCF/enzyme profile studies. J Periodontol 2000;71:618-624.

KEY WORDSGingiva/chemistry; gingival crevicular fluid/chemistry;periodontitis/pathophysiology; periodontitis, early-onset/pathophysiology.

* Department of Periodontology, Faculty of Dentistry, University of Hacettepe, Ankara,Turkey.

† Faculty of Medicine, Department of Biochemistry.‡ Faculty of Medicine, Department of Biostatistics.

Tissue destruction as a consequenceof host-bacteria interaction is a well-described process in the pathogene-

sis of periodontal diseases. During peri-odontal destruction, host cells (mainlypolymorphonuclear leukocytes [PMNs]),release their granular enzymes that arecapable of attacking all extracellular matrixcomponents. Thus, extracellular presence ofenzymes seems to play an important rolein connective tissue damage.1-3

β-glucuronidase (βG) is an importantcomponent of the primary granules of PMN(also found in other cells and bacteria).3-5

βG, together with hyaluronidase, is involvedin the catabolism of proteoglycans. Theendoglycosidase hyaluronidase cleaves hex-osaminidic linkages, producing tetrasac-charides with the structure of (GlcUA-β1,3-GlcNAc β1,4). This tetrasaccharide is fur-ther degraded by βG and β-N-acetylhex-osaminidase. βG is an exoglycosidase thatremoves both GlcUA and IdUA from nonre-ducing ends of tetrasaccharides or largerpolysaccharides. Its substrates include der-matan sulfate, heparan sulfate, chondroitinsulfate, and hyaluronic acid.6 Therefore, βGmost likely contributes to non-collagenousmatrix degradation in periodontal dis-eases.5,7-10 In fact, the relationship betweenβG activity and periodontal disease has beenclearly shown.5,9-15 Furthermore, gingivalcrevicular fluid (GCF) βG activity might bea good indicator or predictor of periodontaldisease activity10-13 and its potential for indi-cating primary granular release from PMNshas been observed.3

Periodontal diseases present significanthetereogenity in prevelance, severity, clin-

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ical course, and mode of treatment. While adult peri-odontitis (AP) is still the most frequently seen form,early-onset periodontitis (EOP), which exhibits differ-ent clinical patterns and microbial/host features hasbeen described.16-19 Studies based on biochemicalaspects of periodontal pathogenesis demonstrated sig-nificant differences among various periodontal diseasegroups.20-25 Increased βG activity levels have beenshown in EOP patients in some previous studies.15,24,25

The validity of GCF as a diagnostic body fluid iswidely accepted.1,4,9,26 Therefore, a description of itsenzymatic profile is likely to provide important datafor the pathogenesis of periodontal diseases and alsofor development of reliable diagnostic tests. However,several aspects of studies on GCF and its enzymaticcontent, including sampling method, time, sample usedfor analysis (rest or flow GCF), and data presentationshould be standardized.9,10,27-30

The aim of the present study is to determine thepresence/activity of βG in GCF and gingiva in peri-odontal disease and health; analyze the potential dif-ferences between AP and EOP patients regarding βGactivity; evaluate the correlations between clinical peri-odontal status and βG activity; and evaluate the valid-ity of 2 models of data presentation, total enzyme activ-ity versus enzyme concentration.

MATERIALS AND METHODSStudy PopulationA total of 57 subjects (32 women and 25 men; meanage 32.90 ± 1.42 years; range, 15 to 53 years) wereincluded in the study and were divided into 3 equalgroups. Two groups were patients with periodontal dis-ease fulfilling the following criteria: no history of a sys-temic disorder; no history of antibiotics and/or antiin-flammatory drugs within the past 6 months; no historyof periodontal treatment (including scaling) within thepast 6 months; presenting >20 teeth; maxillary siteswith ≥5 mm probing depth (PD) and ≥50% alveolarbone loss (preferentially incisors-canines); requiringsurgical pocket elimination; and a clinical and radi-ographical diagnosis of either AP or a form of EOP(juvenile periodontitis or rapidly progressive peri-odontitis).

There were 9 women and 10 men in the AP group(mean age 44.73 ± 1.18 years; range, 37 to 53 years)and 12 women and 7 men in the EOP group (meanage 30.47 ± 1.41; range, 18 to 33 years).

Eleven women and 8 men systemically and peri-odontally healthy (clinical and radiographical) (meanage 23.52 ± 1.67 years, range 15 to 36 years) servedas controls. Third molars in all control subjects werescheduled for extraction. Special care was taken toensure that neither subjects or sites had signs of inflam-mation. The procedures were explained to all subjectsand their consent obtained.

Clinical StudiesDetermination of periodontal status. To determine theclinical periodontal status, probing depths (PD) weremeasured and gingival index (GI),31 plaque index(PI),32 and gingival bleeding index (GBI)33 scores wererecorded. These data were taken from whole mouthand GCF/gingiva sampling area.

Sampling of GCF. Teeth requiring surgical pocketelimination (≥5 mm PD, ≥50% alveolar bone loss) wereselected from the maxillary (preferentially incisors/canines) arch to eliminate the risk of salivary conta-mination. To avoid irritation, samples were obtainedbefore clinical recordings and sampling was madebetween 8.00 and 10.00 a.m. The selected area wasisolated with cotton rolls and gently air-dried. Stan-dardized strips§ were used for GCF sampling, accord-ing to the method described by Rüdin et al.34 In brief,the paper strips with a 1 mm safeguard notch wereinserted into the pocket or sulcus and left for 30 sec-onds. To eliminate the risk of evaporation, paper stripsdamped with rest GCF were immediately transferredto the measuring apparatus� which was previously cal-ibrated by distilled water according to the manufac-turer’s instructions. GCF volume was converted to µLby a special computer program. Sites with no appar-ent signs of clinical inflammation or any periodontalpathology were selected from maxillary teeth in thecontrol group and GCF samples were obtained withthe same procedure. For GCF sampling, the sameanatomical sites were selected whenever possible.Paper strips were placed in sterile tubes and were firmlysealed after GCF collection. Samples were stored inEppendorf tubes at −20°C until analysis.

Sampling of gingival tissue. In the patient groups,gingival tissue was obtained from the same site wherethe GCF sampling was performed. Following initial treat-ment (scaling and polishing), the patients underwentsurgical pocket elimination with modified Widman flapprocedure and gingival tissue was obtained duringsurgery. In the control group, tissues were harvestedduring the surgical removal of completely impacted thirdmolars, or during tooth extraction for orthodontic rea-sons. Special care was taken to ensure that selectedsites presented no clinical signs of infection and/orinflammation. Tissues were stored in firmly wrappedsterile Eppendorf tubes at −20°C until βG analysis.

Laboratory StudiesDetermination of �-glucuronidase activity. β-glu-curonidase activity in GCF and gingival tissuehomogenates was measured using the synthetic sub-strate phenolphthalein glucuronic acid.35 GCF was ex-tracted from the paper strips into phosphate bufferedsaline (300 µL, pH = 7.5) and the tubes were vortexed

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§ Periopaper, Ora Flow, Inc. Plainview, NY.� Periotron 8000, Ora Flow, Inc.

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to maintain elution of GCF from the strips. βG activitywas determined in 25 mM phosphate buffer (pH = 4.9)containing 5 mM substrate. The reaction was initiatedby the addition of 100 µL of GCF extract, and tubes wereincubated at 65°C for 1 hour and the reaction was ter-minated by the addition 100 µL glycine buffer (200 mM,

pH = 11). The intensity of red color of phenolphthaleinwas measured at 550 nm. Commercial phenolphthaleinwas used as the standard. βG activity was calculatedand expressed by 2 different models. The total βGenzyme activity refers to the amount of the enzyme col-lected in 30 seconds, as units (U); and βG concentra-

tion refers to the enzyme activity as unitsper microliter of GCF fluid (U/µL).

Tissue samples were homogenized in50 mM phosphate buffer (pH = 7.5) toobtain 10% (w/v) homogenates. Formeasurement of βG activity, the abovemethod was applied, except that 50 µLof tissue homogenate was used insteadof 100 µL GCF extract. Tissue βG activ-ity was calculated as units per miligramwet gingival tissue (U/mg wet tissue).

Statistical AnalysisDifferences among groups were ana-lyzed by 1-way analysis of variance(ANOVA).36 When the difference wassignificant, groups were analyzed byTukey’s HSD test bilaterally. ANOVA wasperformed on patient averages for wholemouth clinical measures. In order to ana-lyze the correlations between total βGactivity and βG concentration, GCF andgingival tissue βG activity and the clin-ical status of sampling area, simple cor-relation analysis-Pearson correlationcoefficient was used.37 For bilateral cor-relations Bonferroni correction was usedin order to determine the P value.38

RESULTSClinical FindingsMean clinical parameters for both wholemouth and the sampling areas are givenin Table 1. All of the clinical parameterswere significantly higher in the patientgroups when compared to controls (P =0.0001). Bilateral comparisons withTukey’s HSD test revealed that, exceptfor the higher mean sampling area GIscores in the EOP group, there was nosignificant difference between the clinicalparameters of 2 patient groups (P >0.05).

Laboratory FindingsMean βG levels in GCF and gingiva aregiven in Table 2.

�-glucuronidase activity in gingiva.The patient groups had significantlyhigher βG activity than the control group(P = 0.0001). Bilateral comparisonsshowed that the difference between the

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Table 1.

Descriptive Statistical Data on Clinical Parameters of Whole Mouth (WM) and Sampling Area (SA)

Clinical Group/Mean SD SE Minimum Maximum PParameter

PD EOP4.64 (WM) 1.10 0.25 3.02 6.855.60 (SA) 1.03 0.24 4.05 8.16

AP4.28 (WM) 0.61 0.14 3.26 5.425.57 (SA) 0.97 0.22 4.11 7.50

Control1.49 (WM) 0.32 0.07 1.12 2.03 0.0001 (WM)1.35 (SA) 0.31 0.07 1.00 1.94 0.0001 (SA)

PI EOP0.72 (WM) 0.54 0.12 0.19 2.130.75 (SA) 0.64 0.15 0.08 2.33

AP0.59 (WM) 0.35 0.08 0.08 1.310.68 (SA) 0.45 0.10 0.16 1.66

Control0.12 (WM) 0.04 0.01 0.05 0.19 0.0001 (WM)0.05 (SA) 0.06 0.01 0.00 0.16 0.0001 (SA)

GI EOP1.20 (WM) 0.64 0.15 0.21 2.731.32 (SA) 0.64 0.15 0.50 2.77

AP0.93 (WM) 0.43 0.10 0.38 1.770.88 (SA) 0.42 0.10 0.16 1.66

Control0.06 (WM) 0.04 0.01 0.00 0.15 0.0001 (WM)0.01 (SA) 0.03 0.01 0.00 0.08 0.0001 (SA)

GBI EOP64.51 (WM) 28.07 6.44 9.09 100.071.03 (SA) 34.20 7.85 0.00 100.0

AP49.81 (WM) 32.06 7.35 0.00 100.057.89 (SA) 40.96 9.40 0.00 100.0

Control2.14 (WM) 3.37 0.77 0.00 11.11 0.0001 (WM)0.00 (SA) 0.00 0.00 0.00 0.00 0.0001 (SA)

GCF volume (µl) EOP1.07 0.47 0.11 0.35 1.88

AP 0.90 0.42 0.10 0.30 1.60

Control0.19 0.05 0.01 0.11 0.27 0.0001

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patient groups was not significant(P >0.05).

�-glucuronidase activity in GCF.When the mean βG levels are ex-pressed as total activity, the differ-ences among the 3 groups was statis-tically significant (P = 0.0001). Tukey’sHSD test revealed that bilateral dif-ferences were all significant (P <0.05).The EOP group had the highest βGactivity.

As far as the βG enzyme concen-tration is concerned, the differencebetweenEOPand theothergroups wassignificant (P = 0.0018). In bilateralcomparison with Tukey’s HSD test, thedifference between AP and the controlgroup was not significant (P >0.05).The EOP group again presentedthe highest βG activity.

Correlations between clinicalparameters/�G activity/data ex-pressions. Correlations betweenenzyme levels and clinical para-meters, gingival and GCF βG lev-els, and between total βG activityand βG concentration expressionsare shown in Table 3. When alldata were taken as one group,correlations were more frequentthan when the 3 groups were ana-lyzed separately. When the groupswere considered, GCF βG con-centration did not show any sig-nificant correlations with the clin-ical parameters in either patientgroup. Total GCF βG activitypresented significant correlationswith GCF volume (EOP r =0.9372, P = 0.0001; AP r =0.8771, P = 0.001) and the meanGI scores in EOP group (r =0.7309, P = 0.0001). Gingival tis-sue βG activity showed no signif-icant correlations with any of theclinical parameters. In the controlgroup, neither gingiva nor GCFβG levels showed any significantcorrelations with the clinical para-meters. Significant correlationsbetween total βG activity and βGconcentration were not observedin any of the groups. It was seenthat all the groups lacked signifi-cant correlations regarding GCF/gingiva βG activity.

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Table 2.

Descriptive Statistical Data on Gingiva and GCF �G Levels

βG Activity Group Mean SD SE Minimum Maximum P

GCF βG (U) EOP 2.81 1.59 0.36 0.71 5.64AP 1.81 0.86 0.20 0.44 3.47Control 0.36 0.10 0.02 0.18 0.55 0.0001

GCF βG (U/µl) EOP 2.53 0.61 0.14 1.71 3.51 (EOP-AP)AP 2.03 0.50 0.12 1.43 3.16 0.0018Control 1.94 0.45 0.10 0.68 2.42

Gingiva βG EOP 134.76 31.57 7.24 91.59 223.75 (EOP-control)(U/gr wet tissue) AP 159.25 93.02 21.34 87.32 422.04 (AP-control)

Control 54.59 7.67 1.76 33.09 66.70 0.001

Table 3.

Correlations Between Clinical Parameters/�G Activity, Total �GActivity/�G Concentration, and Gingiva/GCF �G Activity

All EOP AP Control

Parameter r P r P r P r P

PI-βG U 0.637 0.0001* 0.4528 0.052 0.3868 0.102 0.2127 0.382

PI-βG U/µL 0.227 0.090 0.2759 0.253 –0.2728 0.259 –0.0365 0.882

PI-βG tissue 0.394 0.002* –0.3774 0.111 0.3121 0.193 0.0015 0.995

GI-βG U 0.852 0.0001* 0.7309 0.0001† 0.5688 0.011 –0.0198 0.936

GI-βG U/µL 0.377 0.004 0.2806 0.245 –0.0837 0.733 0.1350 0.581

GI-βG tissue 0.448 0.0001* –0.1913 0.433 0.2255 0.353 0.0238 0.923

GBI-βG U 0.695 0.0001* 0.5135 0.025 0.3434 0.150

GBI-βG U/µL 0.303 0.022 0.1840 0.451 0.0383 0.876

GBI-βG tissue 0.594 0.0001* 0.0117 0.962 0.4518 0.052

PD- βG U 0.648 0.0001* 0.1138 0.643 0.3511 0.141 0.4018 0.088

PD-βG U/µL 0.233 0.081 0.0410 0.868 –0.3332 0.163 0.1184 0.629

PD-βG tissue 0.598 0.0001* –0.2447 0.313 0.2633 0.276 0.0325 0.895

GCFµL- βG U 0.932 0.0001* 0.9372 0.0001† 0.8771 0.0001† 0.4195 0.074

GCFµL- βG U/µL 0.344 0.009 0.4153 0.077 –0.1013 0.680 –0.4358 0.062

GCFµL- βG tissue 0.437 0.001 –0.3428 0.151 0.1793 0.463 –0.2129 0.381

βG U-βG U/µL 0.607 0.0001* 0.6858 0.001 0.3386 0.156 0.6170 0.005

βG U-βG tissue 0.318 0.016 –0.3506 0.141 0.0709 0.773 0.4640 0.045

βG U/µL-βG tissue 0.035 0.795 –0.1900 0.436 –0.2098 0.389 0.6442 0.003

Simple correlation analysis (Bonferroni correction [α = 0.05/18 = 0.00277778; α = 0.05/54 = 0.000925]).* Significant, P <0.002777.† Significant, P <0.000925.

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These findings indicated that, although random cor-relations were present, there were no absolute corre-lations between clinical parameters and βG activities,between total activity and concentration expressionsand gingival and GCF βG levels in any of the groups.

DISCUSSIONThe findings of the present study confirm the rela-tionship between βG and periodontal disease, sinceincreased enzyme activity in gingiva and in GCF wasobserved when clinical periodontal destruction waspresent. When gingival tissue was analyzed for thepresence and levels of βG activity, all subjects, includ-ing the control group, showed the presence ofdetectable levels of βG. However, the diseased groupspresented higher enzyme activity than periodontallyhealthy subjects. Similar results were observed withtotal βG activity levels in GCF samples as well. There-fore, these findings support the previous studies sug-gesting a role for increased host-originated βG in thepathogenesis of periodontal disease.9,10,14,15,39-41

As indicated in some previous studies conducted onvarious GCF components, including enzymes,15,22-25,28

the highest GCF βG activity (with both expressions)was found in patients with EOP. This increase in EOPforms can be attributed to the hyperactive state andpronounced response of PMN as a consequence ofseverity of microbial virulence factors1,42,43 and alsoto the lytic effect of more pathogenic subgingival bac-teria on host cells leading to an intense host enzymerelease.44-46 Lysosomal release from host cells inresponse to plaque in the absence of phagocytosis47

and the relatively increased production of oxygenmetabolites acting as inactivators of inhibitors and acti-vator of enzymes43,48 and PMN priming in the gingivalcrevice1 can also contribute to the enzymatic profile ofGCF. Lamster et al.12,13 have suggested that elevatedGCF βG activity could be valuable in the identificationof patients at risk for active periodontal disease. Fur-thermore, it has been shown that patients with rapidlyprogressive periodontitis, a form of EOP, presentedhigher intracellular βG activity in peripheral PMN, whichresulted in increased extracellular βG release upon stim-ulation.25,26 Since no single effector system can be heldresponsible for the complicated pathology of periodontaldestruction,49 it seems that all of these pathways, in acoordinated manner, may contribute to the highest GCFβG activity seen in these patients and may suggest thatGCF might have a different biochemical profile in EOPpatients than the healthy subjects and AP patients.

GCF has a widely accepted diagnostic poten-tial.1,4,9,29,30 Although GCF analysis performed onpaper strips is a frequently used method for evaluation,future studies should be standardized and consensusreached on methodological details, including data pre-sentation, as suggested by Lamster et al.9,50,51

Concentration expression has been the preferredmode of data presentation for biochemical profile ofGCF. However, at least for some components, this is notlikely to be the best expression,9,41,50-52 since GCF vol-ume and composition do not have an absolute corre-lation. Therefore, the preference of total activity expres-sion, which considers a time-based standardization, hasbeen recommended for various GCF components,including βG.9-11,30,41,50-52 One of the purposes of thepresent study was to compare the validity of total βGactivity and βG concentration. Thus, enzyme activitywas calculated and analyzed both as U and U/µL.

The results of the present study support the reason-ing of Lamster et al.9,50,51 for reporting total βG activ-ity, due to several factors. Data presentation should beable to reflect even the slightest differences in cross-sec-tional studies. Our findings showed that total βG activ-ity was precise enough to demonstrate the differencesamong all the groups. However, βG concentration,which is a volume-based measurement,9,51,52 was notable to show a difference between the AP and controlgroups. It has been reported that the difficulty of mea-suring the true volume of extremely small quantities ofGCF and/or small losses of GCF results in erroneouslyhigh concentrations of GCF constituents.41,51,52 Theerror in the calculation of concentration is suggested toreach more than 50% if fluid volume is small, but adetectable amount of enzyme is evident.51 This poten-tial source of error in calculating concentration expres-sion seems to be of utmost importance in periodontalhealth status where GCF volume is invariably low.41,51,52

According to the results of the present study, the sig-nificant difference between AP and control groupsobserved in total βG activity turned out to be non-significant with βG concentration. Although we used asensitive device for volume determination and mea-sured GCF volume in a chair side manner, we still con-sider this possibility of erroneously high GCF βG con-centration in periodontally-healthy subjects. Thecorrelations between βG activity and clinical parame-ters were also better with total βG than for βG concen-trations. For these reasons, we support the standard-ization on collection-time basis, rather than GCF-volumebasis, for analysis of GCF βG activity.

In recent years the question of which GCF sample toanalyze has also been debated.27,28,30,52,53 Studies areavailable for a preference of either rest GCF or flowGCF sampling.27,28,52 Rest GCF contains more enzymeactivity than the flow GCF, which makes it an appro-priate choice for cross-sectional models aiming to detecteven minor differences among individual sites/patients/groups.28,29,52,53 Also rest GCF is likely to reflect anundisturbed situation within the sulcus or pocket, wherebacteria-host interaction takes place. This can be inter-preted as the actual situation, rather than being a stat-ic status as suggested.27 In contrast, flow GCF sam-

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ples seem to lose the existing reservoir activity anddilute rest GCF composition as a consequence of vol-umetric effect of the first sampling.28,30 Based on thesefacts, in the present study, analyses of rest GCF sam-ples were preferred. Therefore, the high GCF βG activ-ity found in the GCF samples could be interpreted asthe actual situation with undisturbed reservoir activity.Furthermore, the continous PMN influx to the sulcus(target), the ongoing frustrated phagocytosis process,1

and the effect of inhibitors and/or activators54,55 couldall play a role in the detectable GCF enzyme activity.

The relationship between clinical parameters andGCF composition is another concern. Several GCFcomponents have been shown to correlate with someclinical parameters; however, there are also studieswhich lack such correlations.15,29,30,50,52 In the pres-ent study, GCF total βG activity mostly presented pos-itive correlations with GCF volume and gingival inflam-mation in the patient groups. However, there was nota steady correlation which could be applied to allgroups. The lack of an absolute correlation betweenseverity of periodontal breakdown and GCF enzymeactivity was interpreted as GCF profile preceding theclinical course of disease.15,50,51 It is also suggestedthat the method of data presentation may affect theclinical and laboratory correlations.50 The lack of cor-relations between GCF/gingiva βG levels and the 2models of data presentation observed in the presentstudy may support the previous suggestions12,15,50

that these are not exactly matching measures. We sug-gest consideration of both total activity and concen-tration expressions, if the appropriate mode of datapresentation for a certain GCF component has notbeen defined. Furthermore, for GCF βG activity, wesupport the previous suggestions9-11,41,50,51 regardingthe preference of total βG activity.

The findings of the present study confirm the evi-dence that GCF enzymatic profile alters with peri-odontal disease, and also indicate the importance andnecessity of appropriate data presentation in GCF andenzyme profile studies.

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Send reprint requests to: Dr. Nermin Yamalik, Departmentof Periodontology, Faculty of Dentistry, University ofHacettepe, 06100 Ankara, Turkey. Fax: 90 (312) 418 1121; e-mail: eratalay@ato.org.tr

Accepted for publication October 4, 1999.

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