Assessment of a novel screening test for neutrophil collagenase activity in the diagnosis of periodontal diseases.
BACKGROUND
Increased levels of active neutrophil collagenase (MMP-8) in the gingival crevicular fluid (GCF) are associated with progressive periodontitis. The measurement of this enzyme in GCF could facilitate diagnosis. However, assays with sufficient sensitivity to detect collagenase in whole-mouth GCF currently use radiolabeled substrates and require several days to complete. To provide more rapid analyses of collagenase activity that are better adapted to clinical studies, we developed and validated a novel assay (soluble biotinylated-collagen assay: SBA) based on chemiluminescent detection of biotinylated collagen digestion products.


METHODS
The concordance of the novel SBA assay with a radioactive collagen substrate assay was assessed by parallel analyses of enzyme from 35 neutrophil preparations and from 41 samples of GCF from periodontitis patients, followed by Pearson correlation analysis. To test whether the assay appropriately measured MMP-8 activity, enzyme activity was assessed after incubation with specific collagenase blockers. We examined the diagnostic utility of the SBA in cross-sectional and longitudinal analyses of 125 patients with adult periodontitis, 5 patients with early-onset periodontitis, 1 edentulous patient, and in 32 control patients without periodontitis.


RESULTS
The assay detected <56 pg collagen degraded/hour/microl sample, which is comparable to the most sensitive radioactive assay. The total assay time was 22 hours and reproducibility on replicate measurements was high (r = 0.96). In direct comparisons of MMP-8 activity in GCF with enzyme from peripheral blood neutrophils using the SBA and radioactive assays, there was a high correlation (r = 0.97). As expected, EDTA and TIMP-1 and -2, known inhibitors of MMP-8, completely blocked enzyme activity with this assay. Cross-sectional and longitudinal analyses of GCF showed that MMP-8 activity was >18-fold higher in severe periodontitis than in stable periodontitis and decreased to <25% of pretreatment levels following therapy. Based on measurements of collagenase activity in different disease groups, we estimated a value of 80 nano units as a threshold for severe periodontitis.


CONCLUSIONS
These results indicate that active MMP-8 is detected in GCF by a novel assay that is specific, simple, rapid, and reproducible and which may facilitate diagnostic discrimination between stable and progressive lesions.
Mancini
S
S
Medical Research Council Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, ON.
Romanelli
R
R
Laschinger
C A
CA
Overall
C M
CM
Sodek
J
J
McCulloch
C A
CA
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D016022:Case-Control Studies; D004796:Clinical Enzyme Tests; D018450:Disease Progression; D005260:Female; D005883:Gingival Crevicular Fluid / Q000201:enzymology*; D006801:Humans; D016014:Linear Models; D008137:Longitudinal Studies; D008297:Male; D020784:Matrix Metalloproteinase 8 / Q000032:analysis*; D061965:Matrix Metalloproteinase Inhibitors; D008875:Middle Aged; D010518:Periodontitis / Q000175:diagnosis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D018709:Statistics, Nonparametric; D013552:Swine
D061965:Matrix Metalloproteinase Inhibitors; D020784:Matrix Metalloproteinase 8