Archs oral Bid. Vol. 35, No. 5, pp. 329-335, Printed in Great Britain. All rights reserved
1990 Copyright
0
0003~9969/90 $3.00 + 0.00 1990 Pergamon Press plc
PROTEASE ACTIVITY IN GINGIVAL CREVICULAR FLLJID FROM DISCRETE PERIODONTAL SITES IN HUMANS WITH PERIODONTITIS OR GINGIVITIS D. BEIGHTON,’ J. R. RADFORD’ and M. N. NAYLOR’ ‘Hunterian Dental Research Unit, London Hospital Medical College, Turner Street, Whitechapel, London El 2AD, *Department of Conservative Dental Surgery and Department of Periodontology and Preventive Dentistry, United Medical and Dental Schools of Guy’s and St Thomas’s Hospitals, London Bridge, London SE1 9RT, England (Accepted 24 November 1989) Summary-Sensitive fluorogenic assays were used to compare the protease activities of fluid collected from eight such sites in each of 21 adult patients with gingivitis and 22 with periodontitis. The degradation of N-carbobenzoxy-gly-gly-arginine-AMC, L-arginine-AMC, glyproline-AMC, L-leucine-AMC, N-u-benzoyl-L-arginine-AMC, N-[p-toluenesulphonyl]~gly-pro-arginine-AMC, N-tert-butoxycarbonylN-tert-butoxycarbonyl-ileu-glut-gly-arginine-AMC and N-tert-butoxycarleu-ser-thr-arginine-AMC, bonyl-val-leu-lysine-AMC was significantly greater by fluid from the periodontitis group. The specific rates of degradation of L-arginine-AMC, gly-proline-AMC, N-a-benzoyl-L-arginine-AMC and N-[p-toluenesulphonyllglypro-arginine-AMC were significantly greater in that group, indicating that the composition of their gingival crevicular fluid was different from that of the gingivitis group. Discriminant analysis of the substrate hydrolysis data alone correctly identified 77.6% of sites with sensitivity and specificity values of 73.3 and &2.1%, respectively. The predictive value of these assays requires further investigation, but it is possible that they will prove useful for monitoring the success of periodontal treatment. Key words: gingival crevicular fluid, gingivitis, periodontitis, protease activity.
INTRODUCIION The study of components
of gingival crevicular
fluid
is being pursued by many investigators, with the aim of developing an accurate method of diagnosing active periodontal disease (Cimasoni and Giannopoulou, 1988; Fine and Mandel, 1986). One approach is to characterize the biochemical profile (Lamster, Hartley and Vogel., 1985a) of fluid collected from discrete periodontal sites of subjects with a history of destructive periodontal disease and from subjects with gingivitis. Many different host- and bacteria-derived enzymes have been measured in gingival crevicular fluid, including phosphata:se (acid and alkaline), /?-glucuronidase, aryl sulphatase, myeloperoxidase and lactate dehydrogenase (Lamster et af., 1985a, 1985b; Smith, Hinrichs and Melynk, 1986; Wolff et al., 1988). Protease activity has been measured by a variety of methods, including the degradation of native proteins, such as collagen and gelatin, and the hydrolysis of synthetic protease substrates for the detection of dipeptidase and endopeptidase activities (Ando, 1980; Cimasoni, 1983; Paunio, Makinen and Scheinin, 1971; Villela et al., 1987). In general, sites with a history of periodontal destruction have greater proteolytic activity l:han those that are predominantly healthy. However, comparison between studies is Abbreviations : AMC, 7-amido-4-methylcoumarin; TES, N-tris[hydroxymethyl]methyl-2-aminoethanesulphonic acid.
often difficult as gingival crevicular fluid has not always been collected in a standardized protocol (Lamster, Oshrain and Gordon, 1986). Loesche, Syed and Stoll (1987) found increased trypsin-like activity, as determined by measuring the hydrolysis of N-u benzoyl-L-arginine-/? -naphthylamide hydrochloride, in subgingival plaque sampled from sites with periodontitis; the extent of this activity was significantly correlated with the number of spirochaetes in the plaque. Suido et al. (1988) measured the hydrolysis of three chromogenic N-u benzoyl-p,L-arginine-/I synthetic substrates, naphthylamide hydrochloride, N-carbenzoxy-glycylglycyl-L-arginine- P-naphthylamide hydrochloride and glycyl-L-proline-4-methoxy# naphthylamide hydrochloride, by extracts of gingival crevicular fluid from sites with periodontitis. They found the rates of hydrolysis of all three substrates were correlated significantly with the numbers of Bucteroides gingivalis and spirochaetes in subgingival plaque from the same site, and with the degree of gingival inflammation. Beighton and Life (1989) found that in subjects with different degrees of gingivitis, but no loss of periodontal attachment, the rates of hydrolysis of the fluorogenic substrates, N-u benzoyl-L-arginineAMC and glycyl proline-AMC, by gingival crevicular fluid from discrete periodontal sites increased significantly with an increase in gingival inflammation. The examination of gingival crevicular fluid for potential markers of destructive periodontal disease and its use in simple, non-invasive tests that could monitor the success of periodontal therapy warrants
D.
330
BEIGHTQNet
further investigation. We have now developed sensitive assays, using synthetic fluorogenic substrates, to compare the protease activity of such fluid sampled from discrete periodontal sites in patients with gingivitis or periodontitis. MATERIALS AND METHODS
al.
Co. Ltd, West By&et, Surrey, England) and by the same examiner. The probe tip was of a modified WHO 621 type, with a 0.5 mm ball-end to aid calculus detection, and with graduations marked at 1 mm intervals. The probing force was 30 g. Collection of gingival crevicular &id
Subjects
Forty-three patients, referred to Guy’s Dental Hospital, London for periodontal or conservative dental treatment, were selected for this study; 22 were adults with chronic periodontitis [13 males and 10 females; mean age (f SD) 37.0 &-10.7 yr] and 21 were adults with gingivitis or healthy periodontal tissues [ll and 10 females; mean age (*SD) males 32.2 f 13.1 yr]. To be assigned to the periodontitis group, patients had to have at least 4 of 8 designated sites with greater than 5 mm loss of periodontal attachment, together with bleeding on probing at those same sites. Patients allocated to the gingivitis group had no loss of attachment greater than 3 mm at all designated sites, with bleeding on probing at no more than 2 of the designated sites. None of the subjects had received periodontal treatment or antibiotic therapy during the previous 3 months. Each gave consent to participate in the study, the protocol of which had been approved by the hospital ethical committee. Clinical measurements
Gingival crevicular fluid and clinical measurements were made at these, 8 designated mesio-buccal tooth sites: the upper right and lower left second molar, the lower right and upper left second premolar, the upper right and lower left canine, and the lower right and upper left second incisor. At each site, the degree of gingival inflammation (Lee and Silness, 1963), the amount of plaque (Silness and Lee, 1964), loss of attachment and probing depth measurements (to the nearest millimetre), and the presence (score = 1) or absence (score = 0) of subgingival calculus were recorded. Bleeding on probing was measured by twice inserting the tip of a probe to the full depth of the pocket and, after 30 s recording as 0 = no bleeding on probing, 1 = pin-prick, when a small bead of blood appeared and 2 = copious, when blood filled the gingival sulcus and flowed away from the gingival margin. All measurements were made with a Brodontic Constant Pressure Probe (The Prima Instrument
Each tooth was isolated and dried with cottonwool rolls and a 2 x 13 mm filter paper strip (Whatman No. 4 filter paper) was held just within the gingival crevice for 30 s. The volume of fluid collected was quantified in a Periotron 6000 (Harco Electronics) and the strip placed into 500 ~1 of ice-cold, 50 mM TES (Sigma Chemical Co. Ltd, Poole, Dorset, England) buffer, pH 7.5. The fluid eluates were stored on ice and analysed within 6 h of collection. A total of 344 individual fluid samples was examined in this study. Enzyme assays
The gingival crevicular fluid eluates were extracted by the addition of 600 ~1 of TES buffer containing 20 mM dithiothreitol (Sigma), giving a total volume of 1100 ~1, and incubation at 4°C for 30 min. The dithiothreitol was added to maintain reducing conditions. The extracts were centrifuged at 13,000 g for 2 min and supernatants were assayed for their ability to hydrolyse each of the fluorogenic protease substrates (Sigma) listed in Table 1. All substrates were dissolved in a minimum volume of dimethylsulphoxide and made up to a working concentration of lOOpg/ml in TES buffer. Enzyme assays (Zimmerman et al., 1977; Kato et al., 1978) were performed in opaque microtitre trays (Perkin-Elmer) by incubating 100 p 1of gingival crevicular fluid extract with 20 ~1 of substrate solution at 37°C for 20 h. Substrate hydrolysis was measured by determining the amount of AMC released, using a Perkin-Elmer LS-3B fluorimeter fitted with a microplate reading attachment, at an excitation wavelength of 380 nm and an emission wavelength of 460 nm. In order to account for the non-enzymic hydrolysis of substrates, control assays were set up for each substrate without the inclusion of crevicular fluid; control fluorescence values were subtracted from the appropriate test assays. The number of moles of substrate hydrolysed was determined by reference to a standard curve relating fluorescence to moles of AMC (Sigma). Enzyme activity was
Table 1. List of fluorescent substrates and code used to identify substrates in the text Substrate code El E2 E3 FYI E5 E6 E7 E8 E9 El0
Substrate N-carbobenzoxy-gly-gly-arginine-AMC L-arginine-AMC gly-proline-AMC L-leucine-AMC N-a benzoyl-L-a&tine-AMC N-succinyl-ala-phe-lysine-AMC N-[p-toluenesulphonyl]~gly-pro-arginine-AMC N-tert-butoxycarbonyl-leu-ser-thr-arginine-AMC N-tert-butoxycarbonyl-ileu-glut-gly-arginine-AMC N-tert-butoxycarbonyl-val-leu-lysine-AMC
Protease activity in gingival crevicular fluid
331
Table 2. Comparison of mean (*SD) clinical scores for sites in the gingivitis and periodontitis groups Periodontitis (n = 176) Gingivitis (n = 168) Plaque Index Gingival Index Bleeding Index Probing depth (mm) Loss of attachment (mm) Calculus, score Volume gingival crevicular fluid @I)
0.31 f 0.58 0.76 f 0.85 0.25 * 0.49 2.67 f 0.79 2.72 &-0.98 0.49 * 0.50 0.09 * 0.17
1.025 1.82f 0.91 * 3.99 * 5.27 f 0.58 f 0.17 *
0.82** 0.77** 0.74** 1.22** 1.53** 0.50 0.19**
**Signif:cantly different; p < 0.001. expressed in units with one unit defined as one micromole of substrate hydrolysed per minute at 37°C. The extent of hydrolysis of each substrate by each fluid sample was determined without replication as preliminary studies had shown that the correlation coefficients between duplicate determinations for each substrate were greater than 0.92 (p < 0.0001) and that the mean values were not significantly different (p > 0.01). Statistical analysis
Protease activity was calculated as units of enzyme activity per 30 s sample. This represents total activity towards each substrate but does not take into account the concentration of the enzyme activity in the fluid sample. We therefore also calculated the number of units of enzyme activity per microlitre of crevicular fluid. Means and standard deviations of all measurements were calculated; means were compared by the appropriate Student’s t-test or Analysis of Variance and Duncan’s Mu1 tiple Range Test; Pearson correlation coefficients were calculated. To normalize the distribution of the (data for substrate hydrolysis, the values were, for some analyses, transformed to log,,,. To investigate if those data could identify the clinical status of the subject from whom the fluid samples originated, we used Wilk’s stepwise method of discriminant analysis (Norusis, 1988). The extent of hydrolysis of each of the substrates as transformed to log,, and entered as the discriminating variable, with the actual clinical status (gingivitis or periodontitis) of the individual as the predictor variable. The predictive reliability of the enzyme data for each periodontal site was determined by calculating the
sensitivity and specificity of the predicted clinical status versus the actual clinical status of the subject from whom the fluid sample had been taken. RESULTS
Clinical measurements
The mean scores for the clinical measurements are shown in Table 2. The designated sites in the periodontitis group had significantly higher plaque scores, a greater amount of gingivitis and higher bleeding indices. The mean probing depth of the periodontitis group was greater than that of the gingivitis group and, in accordance with the original criteria for inclusion in one or other group, the periodontitis group had a significantly greater mean loss of attachment. The volume of gingival crevicular fluid collected from the periodontitis group was significantly greater than from the gingivitis group, but there was no difference in group mean scores for the presence of subgingival calculus. The bleeding indices were strongly correlated with the degree of gingival inflammation (r = 0.841 and r = 0.874 for gingivitis and periodontitis groups respectively). Regardless of group, the volume of gingival crevicular fluid correlated only weakly with all of the other clinical measurements (range of r values, 0.05-0.25). Protease activity of individual sites
The results for the total activity are shown in Table 3. For all substrates, except E6, the extent of hydrolysis was significantly (p < 0.001) greater in the periodontitis group than in the gingivitis group; a
Table 3. Comparison of the mean (+ SD) number of units ( x 106)of enzyme activity per 30 s sample (total activity) and of the number of units ( x 106)per microlitre of gingival crevicular fluid (concentration) in gingival crevicular fluid from sites in the -
gingivitis group (n = 168 sites) and periodontitis group (n = 176 sites) Total activity ljubstrate
Gingivitis
El E2 E3 E4 E5 E6 E7 E8 E9 El0
0.12 * 0.15 0.09 * 0.09 0.08 &-0.15 0.06 + 0.08 0.03 &-0.05 0.03 + 0.04 0.05 + 0.07 0.04 * 0.03 0.01 * 0.02 0.02 * 0.01
Concentration
Periodontitis
Gingivitis
Periodontitis
0.41 + 0.46 k 0.43 k 0.30 + 0.28 + 0.05 * 0.26 + 0.14 k 0.05 * 0.06 f
2.62 5 4.19 1.70 k 1.38 1.80 k 4,53 1.26 i 1.64 0.76 + 0.98 0.68 * 0.73 1.05 & 1.66 0.86 k 1.16 0.52 &-0.83 0.46 + 0.42
3.53 + 4.19 3.25 k 3.32” 3.02 k 3.51* 2.00 & 4.03 2.10 + 4.20’: 0.47 f 1.00 1.85 + 3.07: 0.97 * 1.41 0.47 &-0.94 0.62 f 1.70
*Significantly different; p < 0.01. **!Egnificantly different; p < 0.001.
0.55** 0.65** 0.58** 0.74** 0.62” 0.14 0.38” 0.26** 0.10** 0.17+*
D. BEIGHTON et al.
332
Table 4. Correlation coefficients relating clinical scores to log,, (units of activity per 30 s sample) for sites in the gingivitis and neriodontitis arouns Gingivitis group (n = 168)
Plaque Index
Gingival Index
Bleeding Index
El E2 E3 E4 E5 E6 E7 E8 E9
0.128 0.416** 0.392** 0.294** 0.268** 0.259** 0.106 -0.055 0.010 0.200* 0.016 0.186’ 0.107 0.334** 0.097 0.248** -0.118 0.053
0.342’; 0.186* 0.253’; -0.055 0.242** 0.136 0.333** 0.174 0.131
El0
- 0.028
Substrate
0.213’
0.181*
Periodontitis group (n = 176) Probing depth measurement (mm) 0.274** 0.204* 0.159 -0.044 0.179* 0.001 0.108 0.183* -0.005 0.128
Plaque Index
Gingival Index
Bleeding Index
Probing depth measurement (mm) 0.162 0.231* 0.097 0.210* 0.101 0.116 0.070 0.181* -0.012
0.139 0.178* 0.084 0.065 0.102 0.266** 0.058 0.149 0.061
0.185* 0.199* 0.167 0.087 0.178* 0.004 0.078 0.238** 0.158
0.224; 0.243** 0.1998 0.120 0.265** 0.003 0.111 0.259** 0.130
0.157
0.108
0.184*
0.030
*p < 0.01; **p < 0.001.
similar trend was found for the concentration of activities (Table 3). The concentration of enzyme activities that could degrade substrates E2, E3, E5 and E7 was significantly greater in the periodontitis group. Relationships between protease activity
clinical measurements
and
Associations between clinical indices and proteolytic activities (transformed to log,,,) were determined for both clinical groups (Table 4). The pattern of such associations was similar for the two groups:
for example, the hydrolysis of substrate E5 was significantly associated with the bleeding index in the gingivitis group (r = 0.242; p < 0.001) and in the periodontitis group (r = 0.265; p < 0.001). Although there were similar relationships between clinical indices and the extent of hydrolysis of many of the substrates, the levels of substrate hydrolysis were lower in the gingivitis group than in the periodontitis group, even for sites having the same clinical score (Fig. 1). The correlation coefficients between the concentration of enzyme activity and the clinical scores tended to be lower that those given above (data not shown). In the gingivitis group, 138 (82.1%) out of 168 individual sites examined were correctly identified by the discriminant analysis, on the basis of their protease activity. Similarly, 129 (73.3%) of the 176 individual sites in the periodontitis group were correctly assigned to that group. Of the remaining sites, 17.9% of those in the gingivitis group were assigned to the periodontitis group, and 26.7% in the periodontitis group were assigned to the gingivitis group. The sensitivity and specificity of these assignments were 73.3 and 82.1% respectively. The significant discriminating variables were the extent of hydrolysis of substrates E2, E4, ES, E6, E8 and ElO. In 19 of the subjects in the gingivitis group, 5 or more of the 8 sites examined were correctly classified, but in one subject all sites were classified as belonging to the periodontitis group. In the periodontitis group, 18 of 22 subjects were determined as having 5 or more sites classified correctly, but in 2 subjects only one or no sites were correctly classified.
Consideration of these incorrectly classified sites showed that those in the periodontitis group assigned to the gingivitis group were, on the basis of the proteolytic activities in the crevicular fluid, indistinguishable from the correctly classified gingivitis group sites. However, their clinical measurements were not significantly different from those of the correctly classified sites in the periodontitis group, except for a smaller volume of fluid collected. The fluid from the 30 sites in the gingivitis group incorrectly assigned to the periodontitis group hydrolysed substrates El and E3 to a significantly greater extent than did the fluid from the correctly assigned gingivitis group sites. The mean values of the clinical measurements of the correctly and incorrectly identified sites from the gingivitis group were not significantly different, except for the calculus score of the incorrectly classified sites, which was significantly less than that of all the other 3 groups (0.30 versus 0.53 to 0.62 for the other groups; p < 0.05). DISCUSSION
We have examined the ability of gingival crevicular fluid samples, taken from discrete periodontal sites in subjects with either gingivitis or with a history of destructive periodontal disease, to hydrolyse 10 synthetic fluorogenic protease substrates. This was a cross-sectional study and the hydrolysis of these substrates was investigated to ascertain their ability, individually and collectively, to distinguish between sites in the two groups. The substrates included two for arylamidase activities @arginine-AMC and L-leucine-AMC), one for glycylprolyl dipeptidase activity (gly-prolineAMC) and several for endopeptidase activity (Ncarbobenzoxy-gly-gly-arginine- AMC, N-u benzoyl-L arginine-AMC, N-succinyl-ala-phe-lysine-AMC, N[p -toluenesulphonyl]-gly-pro-arginine-AMC, N-tertN-tertbutoxycarbonyl-leu-ser-thr-arginine-AMC, butoxycarbonyl-ileu-glut-gly-arginine-AMC and Ntert-butoxycarbonyl-val-leu-lysine-AMC). These substrates were selected because a preliminary survey had shown that strains of the putative periodontopathic bacteria, B. gingivalis, T. denticola and
Proteasc activity in gingival crevicular fluid
1
/
/’
333
r
A.(BLEEDING
INDEX)
t N I
:
0.8
P $ I
0.6
:
0.4
V I
0.2 ERIODONTITIS
0 8”BSTR’ATE
2 E2
rj
1
SUBSTRATE
2 E5
B.(GINGIVAL
INDEX)
PERIODONTITIS
2 z 0 SUB&RATE E2
3 2 Ci S”B;TRATE E5
Fig. 1. (A) R.elationship between the units of activity towards E2 (L-arginine-AMC) and E5 (N-a-bcnzoylr-arginine-AMC) per 30-s sample by gingival crevicular fluid from sites in the gingivitis and periodontitis groups, cate,gorized according to bleeding indices. (B) Relationship between the units of activity towards E2 (L-arginine-AMC) and E5 (N-a Jxnzoyl-L-arginine-AMC) per 30-s sample by gingival crevicular fluid from sites in the gingivitis and periodontitis groups, categorized according to gingival indices. For each clinical scorl: the number of units of enzyme activity towards each substrate was significantly greater (p < 0.05) in fluid from sites in the periodontitis group. Capnocyfophugu spp., each could hydrolyse most of them at demonstrably high rates in our assay. However, we did find differences both within and between bacterial species in the rates at which the substrates
the compromise assay conditions now reported; only if the composition of the subgingival plaque flora at each site had been known would it have been possible to attempt an assay for the hydrolysis of each sub-
had been hydrolysed (data not shown) and the pH optima for these activities were not identical but generally varied between pH 7 and 8. We thus chose
strate under optimal conditions. However, even knowledge of the flora at each site might not have made substantial difference to our
334
D. BEIGHTON et al.
ability to assay optimally because the proteolytic activities in gingival crevicular fluid could originate from host cells, including macrophages, polymorphs and fibroblasts (Uitto, 1987; Hino et al., 1975) or from serum, all of which have similar enzyme activities. Many of our substrates had been developed for the study of blood clotting: El is used to measure trypsin-like activity in serum, E7 for thrombin, E8 for protein C, E9 for factor Xa and El0 for plasmin (Lottenberg et al., 1981). In addition, several host proteases than can degrade synthetic peptide substrates similar to those used in our study have been identified in gingival crevicular fluid, including arginine aminopeptidase (Makinen and Hyyppa, 1975), cathepsin B (Eisenhauer et al., 1983) and mast cell tryptase (Cox and Eley, 1989). Thus it is unlikely that in our assays any of the substrates were selective for individual bacterial or host enzymes; most were probably cleaved by a combination of the two types of activities. The assay conditions may have permitted the hydrolysis of substrates by the participation of both endopeptidases and exopeptidases. In our method, unlike that of Suido et al. (1988) intact bacteria and host cells were removed by centrifugation before assay. Therefore, either the enzymes were already in a soluble form when the crevicular fluid samples were taken or they were extracted by suspension in the buffer. In agreement with the findings of others (Cimasoni, 1983; Fine and Mandel, 1986), the total proteolytic activity was greater in fluid collected from sites in subjects with a history of destructive periodontitis. the increased levels of glycylprolyl dipeptidase activity were associated with increased gingival inflammation; this confirms an earlier finding in subjects with gingivitis only (Beighton and Life, 1989) and supports the observations of Suido et al. (1988) and Ando (1980). The relationship between the extent of hydrolysis of ES and gingival inflammation in our gingivitis group confirms the findings of Beighton and Life (1989) while that for E5 and El, for our periodontitis group, agrees with the findings of Suido et al. (1988). We also identified a number of other endopeptidase substrates (E7-ElO), the hydrolysis of which was associated with increased gingival inflammation. That the rates of hydrolysis of the arylamidase substrates, E2 and E4, were also significantly greater with increased inflammation suggests that these may be candidates for further study, either as potential markers of destructive periodontal disease or in monitoring the response to periodontal treatment. The concentration of enzyme activity towards substrates E2, E3, E5 and E7 was greater in gingival crevicular fluid collected from the periodontitis than from the gingivitis group. This suggests that the composition of the fluid from the 2 groups was different and that the increased total activities of fluid from the periodontitis group were not simply due to the larger volume collected. Whether these increased specific activities originated from changes in the bacterial populations or from an increased number of host cells contributing to the overall proteolytic activity of the crevicular fluid is not known. Only a study that simultaneously examines protease activities derived from the bacterial flora
and the host cells at discrete sites will clarify this problem. Account should be taken of the fact that the levels of measurable protease activity may be modified by the cc,-antitrypsin and qmacroglobulin that are present in gingival crevicular fluid as a derivative of serum (Cimasoni, 1983). If these serum protease inhibitors are degraded by the subgingival flora (ter Steeg et al., 1988) it is probable that they would increase the host contribution to the protease activities of the fluid (Condacci, Cimasoni and Ahmed-Zadeh, 1982). Our discriminant analysis clearly showed that these tests correctly assigned the majority of sites in both groups according to the predetermined clinical status of the subject. In 3 subjects, 7 or more sites were incorrectly assigned; in a cross-sectional study it is not possible to ascertain the significance of these observations in regard of the future clinical status of these sites. Current clinical measurements are certainly poor predictors of destructive periodontal disease activity (Haffajee, Socransky and Goodson, 1983) but any predictive value for our tests in identifying individual subjects or individual periodontal sites at risk of a destructive episode could only be determined in a suitable longitudinal study. It is possible, though, that such tests might have considerable application in monitoring the success of periodontal treatment. Acknowledgements -We
should like to thank the subjects who consented to take part in this study which was funded, in part, by a grant from the Dental Funds, Committee, United Medical and Dental School of Guy’s and St Thomas’ Hospital.
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Paunio K. U., Makinen K. K. and Scheinin A. (1971) Aminopeptidase B in human gingival exudate as affected by reduced oral hygiene and sugar diet. Acra odont. stand. 29, 583-590.
Silness J. and Liie H. (1964) Periodontal disease in pregnancy. II. Correlation between oral hygiene and periodontal condition. Acra odont. stand. 2i, 121-135. _ Smith 0. T.. Hinrichs J. E. and Melnvk R. S. (1986) Ging&l cr&icular fluid myeloperoxida& at periodontitis sites. J. Periodont. Res. 21, 45-55. Steeg P. F. ter, Hoeven J. S. van der, Jong M. H. de, Munster P. J. J. van and Jansen M. J. H. (1988) Modelling the gingival pocket by enrichment of subgingival microflora in human serum in chemostats. Microbial Ecol. Hlih Db. 1, 7344.
Suido H., Nakamura M., Mashimo P., Zambon J. and Genco R. J. (1986) Arylaminopeptidase activities of oral bacteria. J. dent. Res. 65, 1335-1340. Suido H., Zambon J. J., Mashimo P. A., Dunford R. and Genco R. J. (1988) Correlations between crevicular fluid enzymes and the subgingival microflora. J. dent. Res. 67, 107&1074.
Uitto V-J. (1987) Human gingival proteases. 1: Extraction and preliminary characterization of trypsin-like and elastase-like enzymes. J. Periodonr. Res. 22, 5843. Villela B., Cogen R. B., Bartolucci A. A. and BirkedalHansen H. (1987) Collagenolytic activity in crevicular fluid from patients with chronic adult periodontitis, localized juvenile periodontitis and gingivitis, and from healthy control subjects. J. Periodont. Res. 22, 381-389. Wolff L. F., Smith Q. T., Snyder W. K., Bedrick J. A., Liljemark W. F., Aeppli D. M. and Bandt C. L. (1988) Relationship between lactate dehydrogenase and myeloperoxidase levels in human gingival crevicular fluid and clinical and microbial measurements. J. clin. Periodonr. 15, 11&115. Zimmerman M., Ashe B., Yurewicz E. C. and Pate1 G. (1977) Sensitive assays for trypsin, elastase, and chymotrypsin using new fluorogenic substrates. Analyt. Biochem. 78, 47-51.
1990 Copyright
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0003~9969/90 $3.00 + 0.00 1990 Pergamon Press plc
PROTEASE ACTIVITY IN GINGIVAL CREVICULAR FLLJID FROM DISCRETE PERIODONTAL SITES IN HUMANS WITH PERIODONTITIS OR GINGIVITIS D. BEIGHTON,’ J. R. RADFORD’ and M. N. NAYLOR’ ‘Hunterian Dental Research Unit, London Hospital Medical College, Turner Street, Whitechapel, London El 2AD, *Department of Conservative Dental Surgery and Department of Periodontology and Preventive Dentistry, United Medical and Dental Schools of Guy’s and St Thomas’s Hospitals, London Bridge, London SE1 9RT, England (Accepted 24 November 1989) Summary-Sensitive fluorogenic assays were used to compare the protease activities of fluid collected from eight such sites in each of 21 adult patients with gingivitis and 22 with periodontitis. The degradation of N-carbobenzoxy-gly-gly-arginine-AMC, L-arginine-AMC, glyproline-AMC, L-leucine-AMC, N-u-benzoyl-L-arginine-AMC, N-[p-toluenesulphonyl]~gly-pro-arginine-AMC, N-tert-butoxycarbonylN-tert-butoxycarbonyl-ileu-glut-gly-arginine-AMC and N-tert-butoxycarleu-ser-thr-arginine-AMC, bonyl-val-leu-lysine-AMC was significantly greater by fluid from the periodontitis group. The specific rates of degradation of L-arginine-AMC, gly-proline-AMC, N-a-benzoyl-L-arginine-AMC and N-[p-toluenesulphonyllglypro-arginine-AMC were significantly greater in that group, indicating that the composition of their gingival crevicular fluid was different from that of the gingivitis group. Discriminant analysis of the substrate hydrolysis data alone correctly identified 77.6% of sites with sensitivity and specificity values of 73.3 and &2.1%, respectively. The predictive value of these assays requires further investigation, but it is possible that they will prove useful for monitoring the success of periodontal treatment. Key words: gingival crevicular fluid, gingivitis, periodontitis, protease activity.
INTRODUCIION The study of components
of gingival crevicular
fluid
is being pursued by many investigators, with the aim of developing an accurate method of diagnosing active periodontal disease (Cimasoni and Giannopoulou, 1988; Fine and Mandel, 1986). One approach is to characterize the biochemical profile (Lamster, Hartley and Vogel., 1985a) of fluid collected from discrete periodontal sites of subjects with a history of destructive periodontal disease and from subjects with gingivitis. Many different host- and bacteria-derived enzymes have been measured in gingival crevicular fluid, including phosphata:se (acid and alkaline), /?-glucuronidase, aryl sulphatase, myeloperoxidase and lactate dehydrogenase (Lamster et af., 1985a, 1985b; Smith, Hinrichs and Melynk, 1986; Wolff et al., 1988). Protease activity has been measured by a variety of methods, including the degradation of native proteins, such as collagen and gelatin, and the hydrolysis of synthetic protease substrates for the detection of dipeptidase and endopeptidase activities (Ando, 1980; Cimasoni, 1983; Paunio, Makinen and Scheinin, 1971; Villela et al., 1987). In general, sites with a history of periodontal destruction have greater proteolytic activity l:han those that are predominantly healthy. However, comparison between studies is Abbreviations : AMC, 7-amido-4-methylcoumarin; TES, N-tris[hydroxymethyl]methyl-2-aminoethanesulphonic acid.
often difficult as gingival crevicular fluid has not always been collected in a standardized protocol (Lamster, Oshrain and Gordon, 1986). Loesche, Syed and Stoll (1987) found increased trypsin-like activity, as determined by measuring the hydrolysis of N-u benzoyl-L-arginine-/? -naphthylamide hydrochloride, in subgingival plaque sampled from sites with periodontitis; the extent of this activity was significantly correlated with the number of spirochaetes in the plaque. Suido et al. (1988) measured the hydrolysis of three chromogenic N-u benzoyl-p,L-arginine-/I synthetic substrates, naphthylamide hydrochloride, N-carbenzoxy-glycylglycyl-L-arginine- P-naphthylamide hydrochloride and glycyl-L-proline-4-methoxy# naphthylamide hydrochloride, by extracts of gingival crevicular fluid from sites with periodontitis. They found the rates of hydrolysis of all three substrates were correlated significantly with the numbers of Bucteroides gingivalis and spirochaetes in subgingival plaque from the same site, and with the degree of gingival inflammation. Beighton and Life (1989) found that in subjects with different degrees of gingivitis, but no loss of periodontal attachment, the rates of hydrolysis of the fluorogenic substrates, N-u benzoyl-L-arginineAMC and glycyl proline-AMC, by gingival crevicular fluid from discrete periodontal sites increased significantly with an increase in gingival inflammation. The examination of gingival crevicular fluid for potential markers of destructive periodontal disease and its use in simple, non-invasive tests that could monitor the success of periodontal therapy warrants
D.
330
BEIGHTQNet
further investigation. We have now developed sensitive assays, using synthetic fluorogenic substrates, to compare the protease activity of such fluid sampled from discrete periodontal sites in patients with gingivitis or periodontitis. MATERIALS AND METHODS
al.
Co. Ltd, West By&et, Surrey, England) and by the same examiner. The probe tip was of a modified WHO 621 type, with a 0.5 mm ball-end to aid calculus detection, and with graduations marked at 1 mm intervals. The probing force was 30 g. Collection of gingival crevicular &id
Subjects
Forty-three patients, referred to Guy’s Dental Hospital, London for periodontal or conservative dental treatment, were selected for this study; 22 were adults with chronic periodontitis [13 males and 10 females; mean age (f SD) 37.0 &-10.7 yr] and 21 were adults with gingivitis or healthy periodontal tissues [ll and 10 females; mean age (*SD) males 32.2 f 13.1 yr]. To be assigned to the periodontitis group, patients had to have at least 4 of 8 designated sites with greater than 5 mm loss of periodontal attachment, together with bleeding on probing at those same sites. Patients allocated to the gingivitis group had no loss of attachment greater than 3 mm at all designated sites, with bleeding on probing at no more than 2 of the designated sites. None of the subjects had received periodontal treatment or antibiotic therapy during the previous 3 months. Each gave consent to participate in the study, the protocol of which had been approved by the hospital ethical committee. Clinical measurements
Gingival crevicular fluid and clinical measurements were made at these, 8 designated mesio-buccal tooth sites: the upper right and lower left second molar, the lower right and upper left second premolar, the upper right and lower left canine, and the lower right and upper left second incisor. At each site, the degree of gingival inflammation (Lee and Silness, 1963), the amount of plaque (Silness and Lee, 1964), loss of attachment and probing depth measurements (to the nearest millimetre), and the presence (score = 1) or absence (score = 0) of subgingival calculus were recorded. Bleeding on probing was measured by twice inserting the tip of a probe to the full depth of the pocket and, after 30 s recording as 0 = no bleeding on probing, 1 = pin-prick, when a small bead of blood appeared and 2 = copious, when blood filled the gingival sulcus and flowed away from the gingival margin. All measurements were made with a Brodontic Constant Pressure Probe (The Prima Instrument
Each tooth was isolated and dried with cottonwool rolls and a 2 x 13 mm filter paper strip (Whatman No. 4 filter paper) was held just within the gingival crevice for 30 s. The volume of fluid collected was quantified in a Periotron 6000 (Harco Electronics) and the strip placed into 500 ~1 of ice-cold, 50 mM TES (Sigma Chemical Co. Ltd, Poole, Dorset, England) buffer, pH 7.5. The fluid eluates were stored on ice and analysed within 6 h of collection. A total of 344 individual fluid samples was examined in this study. Enzyme assays
The gingival crevicular fluid eluates were extracted by the addition of 600 ~1 of TES buffer containing 20 mM dithiothreitol (Sigma), giving a total volume of 1100 ~1, and incubation at 4°C for 30 min. The dithiothreitol was added to maintain reducing conditions. The extracts were centrifuged at 13,000 g for 2 min and supernatants were assayed for their ability to hydrolyse each of the fluorogenic protease substrates (Sigma) listed in Table 1. All substrates were dissolved in a minimum volume of dimethylsulphoxide and made up to a working concentration of lOOpg/ml in TES buffer. Enzyme assays (Zimmerman et al., 1977; Kato et al., 1978) were performed in opaque microtitre trays (Perkin-Elmer) by incubating 100 p 1of gingival crevicular fluid extract with 20 ~1 of substrate solution at 37°C for 20 h. Substrate hydrolysis was measured by determining the amount of AMC released, using a Perkin-Elmer LS-3B fluorimeter fitted with a microplate reading attachment, at an excitation wavelength of 380 nm and an emission wavelength of 460 nm. In order to account for the non-enzymic hydrolysis of substrates, control assays were set up for each substrate without the inclusion of crevicular fluid; control fluorescence values were subtracted from the appropriate test assays. The number of moles of substrate hydrolysed was determined by reference to a standard curve relating fluorescence to moles of AMC (Sigma). Enzyme activity was
Table 1. List of fluorescent substrates and code used to identify substrates in the text Substrate code El E2 E3 FYI E5 E6 E7 E8 E9 El0
Substrate N-carbobenzoxy-gly-gly-arginine-AMC L-arginine-AMC gly-proline-AMC L-leucine-AMC N-a benzoyl-L-a&tine-AMC N-succinyl-ala-phe-lysine-AMC N-[p-toluenesulphonyl]~gly-pro-arginine-AMC N-tert-butoxycarbonyl-leu-ser-thr-arginine-AMC N-tert-butoxycarbonyl-ileu-glut-gly-arginine-AMC N-tert-butoxycarbonyl-val-leu-lysine-AMC
Protease activity in gingival crevicular fluid
331
Table 2. Comparison of mean (*SD) clinical scores for sites in the gingivitis and periodontitis groups Periodontitis (n = 176) Gingivitis (n = 168) Plaque Index Gingival Index Bleeding Index Probing depth (mm) Loss of attachment (mm) Calculus, score Volume gingival crevicular fluid @I)
0.31 f 0.58 0.76 f 0.85 0.25 * 0.49 2.67 f 0.79 2.72 &-0.98 0.49 * 0.50 0.09 * 0.17
1.025 1.82f 0.91 * 3.99 * 5.27 f 0.58 f 0.17 *
0.82** 0.77** 0.74** 1.22** 1.53** 0.50 0.19**
**Signif:cantly different; p < 0.001. expressed in units with one unit defined as one micromole of substrate hydrolysed per minute at 37°C. The extent of hydrolysis of each substrate by each fluid sample was determined without replication as preliminary studies had shown that the correlation coefficients between duplicate determinations for each substrate were greater than 0.92 (p < 0.0001) and that the mean values were not significantly different (p > 0.01). Statistical analysis
Protease activity was calculated as units of enzyme activity per 30 s sample. This represents total activity towards each substrate but does not take into account the concentration of the enzyme activity in the fluid sample. We therefore also calculated the number of units of enzyme activity per microlitre of crevicular fluid. Means and standard deviations of all measurements were calculated; means were compared by the appropriate Student’s t-test or Analysis of Variance and Duncan’s Mu1 tiple Range Test; Pearson correlation coefficients were calculated. To normalize the distribution of the (data for substrate hydrolysis, the values were, for some analyses, transformed to log,,,. To investigate if those data could identify the clinical status of the subject from whom the fluid samples originated, we used Wilk’s stepwise method of discriminant analysis (Norusis, 1988). The extent of hydrolysis of each of the substrates as transformed to log,, and entered as the discriminating variable, with the actual clinical status (gingivitis or periodontitis) of the individual as the predictor variable. The predictive reliability of the enzyme data for each periodontal site was determined by calculating the
sensitivity and specificity of the predicted clinical status versus the actual clinical status of the subject from whom the fluid sample had been taken. RESULTS
Clinical measurements
The mean scores for the clinical measurements are shown in Table 2. The designated sites in the periodontitis group had significantly higher plaque scores, a greater amount of gingivitis and higher bleeding indices. The mean probing depth of the periodontitis group was greater than that of the gingivitis group and, in accordance with the original criteria for inclusion in one or other group, the periodontitis group had a significantly greater mean loss of attachment. The volume of gingival crevicular fluid collected from the periodontitis group was significantly greater than from the gingivitis group, but there was no difference in group mean scores for the presence of subgingival calculus. The bleeding indices were strongly correlated with the degree of gingival inflammation (r = 0.841 and r = 0.874 for gingivitis and periodontitis groups respectively). Regardless of group, the volume of gingival crevicular fluid correlated only weakly with all of the other clinical measurements (range of r values, 0.05-0.25). Protease activity of individual sites
The results for the total activity are shown in Table 3. For all substrates, except E6, the extent of hydrolysis was significantly (p < 0.001) greater in the periodontitis group than in the gingivitis group; a
Table 3. Comparison of the mean (+ SD) number of units ( x 106)of enzyme activity per 30 s sample (total activity) and of the number of units ( x 106)per microlitre of gingival crevicular fluid (concentration) in gingival crevicular fluid from sites in the -
gingivitis group (n = 168 sites) and periodontitis group (n = 176 sites) Total activity ljubstrate
Gingivitis
El E2 E3 E4 E5 E6 E7 E8 E9 El0
0.12 * 0.15 0.09 * 0.09 0.08 &-0.15 0.06 + 0.08 0.03 &-0.05 0.03 + 0.04 0.05 + 0.07 0.04 * 0.03 0.01 * 0.02 0.02 * 0.01
Concentration
Periodontitis
Gingivitis
Periodontitis
0.41 + 0.46 k 0.43 k 0.30 + 0.28 + 0.05 * 0.26 + 0.14 k 0.05 * 0.06 f
2.62 5 4.19 1.70 k 1.38 1.80 k 4,53 1.26 i 1.64 0.76 + 0.98 0.68 * 0.73 1.05 & 1.66 0.86 k 1.16 0.52 &-0.83 0.46 + 0.42
3.53 + 4.19 3.25 k 3.32” 3.02 k 3.51* 2.00 & 4.03 2.10 + 4.20’: 0.47 f 1.00 1.85 + 3.07: 0.97 * 1.41 0.47 &-0.94 0.62 f 1.70
*Significantly different; p < 0.01. **!Egnificantly different; p < 0.001.
0.55** 0.65** 0.58** 0.74** 0.62” 0.14 0.38” 0.26** 0.10** 0.17+*
D. BEIGHTON et al.
332
Table 4. Correlation coefficients relating clinical scores to log,, (units of activity per 30 s sample) for sites in the gingivitis and neriodontitis arouns Gingivitis group (n = 168)
Plaque Index
Gingival Index
Bleeding Index
El E2 E3 E4 E5 E6 E7 E8 E9
0.128 0.416** 0.392** 0.294** 0.268** 0.259** 0.106 -0.055 0.010 0.200* 0.016 0.186’ 0.107 0.334** 0.097 0.248** -0.118 0.053
0.342’; 0.186* 0.253’; -0.055 0.242** 0.136 0.333** 0.174 0.131
El0
- 0.028
Substrate
0.213’
0.181*
Periodontitis group (n = 176) Probing depth measurement (mm) 0.274** 0.204* 0.159 -0.044 0.179* 0.001 0.108 0.183* -0.005 0.128
Plaque Index
Gingival Index
Bleeding Index
Probing depth measurement (mm) 0.162 0.231* 0.097 0.210* 0.101 0.116 0.070 0.181* -0.012
0.139 0.178* 0.084 0.065 0.102 0.266** 0.058 0.149 0.061
0.185* 0.199* 0.167 0.087 0.178* 0.004 0.078 0.238** 0.158
0.224; 0.243** 0.1998 0.120 0.265** 0.003 0.111 0.259** 0.130
0.157
0.108
0.184*
0.030
*p < 0.01; **p < 0.001.
similar trend was found for the concentration of activities (Table 3). The concentration of enzyme activities that could degrade substrates E2, E3, E5 and E7 was significantly greater in the periodontitis group. Relationships between protease activity
clinical measurements
and
Associations between clinical indices and proteolytic activities (transformed to log,,,) were determined for both clinical groups (Table 4). The pattern of such associations was similar for the two groups:
for example, the hydrolysis of substrate E5 was significantly associated with the bleeding index in the gingivitis group (r = 0.242; p < 0.001) and in the periodontitis group (r = 0.265; p < 0.001). Although there were similar relationships between clinical indices and the extent of hydrolysis of many of the substrates, the levels of substrate hydrolysis were lower in the gingivitis group than in the periodontitis group, even for sites having the same clinical score (Fig. 1). The correlation coefficients between the concentration of enzyme activity and the clinical scores tended to be lower that those given above (data not shown). In the gingivitis group, 138 (82.1%) out of 168 individual sites examined were correctly identified by the discriminant analysis, on the basis of their protease activity. Similarly, 129 (73.3%) of the 176 individual sites in the periodontitis group were correctly assigned to that group. Of the remaining sites, 17.9% of those in the gingivitis group were assigned to the periodontitis group, and 26.7% in the periodontitis group were assigned to the gingivitis group. The sensitivity and specificity of these assignments were 73.3 and 82.1% respectively. The significant discriminating variables were the extent of hydrolysis of substrates E2, E4, ES, E6, E8 and ElO. In 19 of the subjects in the gingivitis group, 5 or more of the 8 sites examined were correctly classified, but in one subject all sites were classified as belonging to the periodontitis group. In the periodontitis group, 18 of 22 subjects were determined as having 5 or more sites classified correctly, but in 2 subjects only one or no sites were correctly classified.
Consideration of these incorrectly classified sites showed that those in the periodontitis group assigned to the gingivitis group were, on the basis of the proteolytic activities in the crevicular fluid, indistinguishable from the correctly classified gingivitis group sites. However, their clinical measurements were not significantly different from those of the correctly classified sites in the periodontitis group, except for a smaller volume of fluid collected. The fluid from the 30 sites in the gingivitis group incorrectly assigned to the periodontitis group hydrolysed substrates El and E3 to a significantly greater extent than did the fluid from the correctly assigned gingivitis group sites. The mean values of the clinical measurements of the correctly and incorrectly identified sites from the gingivitis group were not significantly different, except for the calculus score of the incorrectly classified sites, which was significantly less than that of all the other 3 groups (0.30 versus 0.53 to 0.62 for the other groups; p < 0.05). DISCUSSION
We have examined the ability of gingival crevicular fluid samples, taken from discrete periodontal sites in subjects with either gingivitis or with a history of destructive periodontal disease, to hydrolyse 10 synthetic fluorogenic protease substrates. This was a cross-sectional study and the hydrolysis of these substrates was investigated to ascertain their ability, individually and collectively, to distinguish between sites in the two groups. The substrates included two for arylamidase activities @arginine-AMC and L-leucine-AMC), one for glycylprolyl dipeptidase activity (gly-prolineAMC) and several for endopeptidase activity (Ncarbobenzoxy-gly-gly-arginine- AMC, N-u benzoyl-L arginine-AMC, N-succinyl-ala-phe-lysine-AMC, N[p -toluenesulphonyl]-gly-pro-arginine-AMC, N-tertN-tertbutoxycarbonyl-leu-ser-thr-arginine-AMC, butoxycarbonyl-ileu-glut-gly-arginine-AMC and Ntert-butoxycarbonyl-val-leu-lysine-AMC). These substrates were selected because a preliminary survey had shown that strains of the putative periodontopathic bacteria, B. gingivalis, T. denticola and
Proteasc activity in gingival crevicular fluid
1
/
/’
333
r
A.(BLEEDING
INDEX)
t N I
:
0.8
P $ I
0.6
:
0.4
V I
0.2 ERIODONTITIS
0 8”BSTR’ATE
2 E2
rj
1
SUBSTRATE
2 E5
B.(GINGIVAL
INDEX)
PERIODONTITIS
2 z 0 SUB&RATE E2
3 2 Ci S”B;TRATE E5
Fig. 1. (A) R.elationship between the units of activity towards E2 (L-arginine-AMC) and E5 (N-a-bcnzoylr-arginine-AMC) per 30-s sample by gingival crevicular fluid from sites in the gingivitis and periodontitis groups, cate,gorized according to bleeding indices. (B) Relationship between the units of activity towards E2 (L-arginine-AMC) and E5 (N-a Jxnzoyl-L-arginine-AMC) per 30-s sample by gingival crevicular fluid from sites in the gingivitis and periodontitis groups, categorized according to gingival indices. For each clinical scorl: the number of units of enzyme activity towards each substrate was significantly greater (p < 0.05) in fluid from sites in the periodontitis group. Capnocyfophugu spp., each could hydrolyse most of them at demonstrably high rates in our assay. However, we did find differences both within and between bacterial species in the rates at which the substrates
the compromise assay conditions now reported; only if the composition of the subgingival plaque flora at each site had been known would it have been possible to attempt an assay for the hydrolysis of each sub-
had been hydrolysed (data not shown) and the pH optima for these activities were not identical but generally varied between pH 7 and 8. We thus chose
strate under optimal conditions. However, even knowledge of the flora at each site might not have made substantial difference to our
334
D. BEIGHTON et al.
ability to assay optimally because the proteolytic activities in gingival crevicular fluid could originate from host cells, including macrophages, polymorphs and fibroblasts (Uitto, 1987; Hino et al., 1975) or from serum, all of which have similar enzyme activities. Many of our substrates had been developed for the study of blood clotting: El is used to measure trypsin-like activity in serum, E7 for thrombin, E8 for protein C, E9 for factor Xa and El0 for plasmin (Lottenberg et al., 1981). In addition, several host proteases than can degrade synthetic peptide substrates similar to those used in our study have been identified in gingival crevicular fluid, including arginine aminopeptidase (Makinen and Hyyppa, 1975), cathepsin B (Eisenhauer et al., 1983) and mast cell tryptase (Cox and Eley, 1989). Thus it is unlikely that in our assays any of the substrates were selective for individual bacterial or host enzymes; most were probably cleaved by a combination of the two types of activities. The assay conditions may have permitted the hydrolysis of substrates by the participation of both endopeptidases and exopeptidases. In our method, unlike that of Suido et al. (1988) intact bacteria and host cells were removed by centrifugation before assay. Therefore, either the enzymes were already in a soluble form when the crevicular fluid samples were taken or they were extracted by suspension in the buffer. In agreement with the findings of others (Cimasoni, 1983; Fine and Mandel, 1986), the total proteolytic activity was greater in fluid collected from sites in subjects with a history of destructive periodontitis. the increased levels of glycylprolyl dipeptidase activity were associated with increased gingival inflammation; this confirms an earlier finding in subjects with gingivitis only (Beighton and Life, 1989) and supports the observations of Suido et al. (1988) and Ando (1980). The relationship between the extent of hydrolysis of ES and gingival inflammation in our gingivitis group confirms the findings of Beighton and Life (1989) while that for E5 and El, for our periodontitis group, agrees with the findings of Suido et al. (1988). We also identified a number of other endopeptidase substrates (E7-ElO), the hydrolysis of which was associated with increased gingival inflammation. That the rates of hydrolysis of the arylamidase substrates, E2 and E4, were also significantly greater with increased inflammation suggests that these may be candidates for further study, either as potential markers of destructive periodontal disease or in monitoring the response to periodontal treatment. The concentration of enzyme activity towards substrates E2, E3, E5 and E7 was greater in gingival crevicular fluid collected from the periodontitis than from the gingivitis group. This suggests that the composition of the fluid from the 2 groups was different and that the increased total activities of fluid from the periodontitis group were not simply due to the larger volume collected. Whether these increased specific activities originated from changes in the bacterial populations or from an increased number of host cells contributing to the overall proteolytic activity of the crevicular fluid is not known. Only a study that simultaneously examines protease activities derived from the bacterial flora
and the host cells at discrete sites will clarify this problem. Account should be taken of the fact that the levels of measurable protease activity may be modified by the cc,-antitrypsin and qmacroglobulin that are present in gingival crevicular fluid as a derivative of serum (Cimasoni, 1983). If these serum protease inhibitors are degraded by the subgingival flora (ter Steeg et al., 1988) it is probable that they would increase the host contribution to the protease activities of the fluid (Condacci, Cimasoni and Ahmed-Zadeh, 1982). Our discriminant analysis clearly showed that these tests correctly assigned the majority of sites in both groups according to the predetermined clinical status of the subject. In 3 subjects, 7 or more sites were incorrectly assigned; in a cross-sectional study it is not possible to ascertain the significance of these observations in regard of the future clinical status of these sites. Current clinical measurements are certainly poor predictors of destructive periodontal disease activity (Haffajee, Socransky and Goodson, 1983) but any predictive value for our tests in identifying individual subjects or individual periodontal sites at risk of a destructive episode could only be determined in a suitable longitudinal study. It is possible, though, that such tests might have considerable application in monitoring the success of periodontal treatment. Acknowledgements -We
should like to thank the subjects who consented to take part in this study which was funded, in part, by a grant from the Dental Funds, Committee, United Medical and Dental School of Guy’s and St Thomas’ Hospital.
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