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 th e gingival crevicular fluid (GCF) are associated with progressive periodont itis. The measurement of this enzyme in GCF could facilitate diagnosis. How ever, assays with sufficient sensitivity to detect collagenase in whole-mou th GCF currently use radiolabeled substrates and require several days to co mplete. To provide more rapid analyses of collagenase activity that are bet ter adapted to clinical studies, we developed and validated a novel assay ( soluble biotinylated-collagen assay: SBA) based on chemiluminescent detecti on 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 neutro phil preparations and from 41 samples of GCF from periodontitis patients, f ollowed by Pearson correlation analysis. To test whether the assay appropri ately measured MMP-8 activity, enzyme activity was assessed after incubatio n 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 edentulo us patient, and in 32 control patients without periodontitis. Results: The assay detected <56 pg collagen degraded/hour/mu l sample, whic h is comparable to the most sensitive radioactive assay. The total assay ti me 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 per ipheral blood neutrophils using the SEA and radioactive assays, there was a high correlation (r = 0.97). As expected, EDTA and TIMP-1 and -2, known in hibitors of MMP-8, completely blocked enzyme activity with this assay. Cros s-sectional and longitudinal analyses of GCF showed that MMP-8 activity was >18-fold higher in severe periodontitis than in stable periodontitis and d ecreased to <25% of pretreatment levels following therapy Based on measurem ents of collagenase activity in different disease groups, we estimated a va lue 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 les ions.