Elastase Activity in Gingival Crevicular Fluid of Healthy Volunteers

The role of elastase in connective tissue disintegration and collagen breakdown in periodontitis has been demonstrated. Not only the concentration of elastase, but its proteolytic activity seems to be more significant for gingival inflammation and periodontal destruction. Alone or in a complex witha-1-proteinase-inhibitor or a-2-macroglobulin this enzyme might cause the progression of the disease. The aim of this study was to show a possible detection of elastolytic activity even in healthy volunteers. Previous investigations of our laboratory (1997) found high elastolytic activities in GCF samples from patients with marginal periodontitis. We analysed the samples with a synthetic polipeptide substrateMeO-Succ-Ala-Ala-Pro-Val-7-amino-4-methylcoumarin, according to investigations of Castillo et al. (1978) and Turner et al. (1994). The elastolytic activity was measured in a micro plate reader (Fluostar®). Recovery rates were performed at 3200, 1600 and 200 µU/µl of elastolytic activity. According to our results the reliability of this method is above 90%.In order to assess a possible loss of activity during storage, pooled samples were tested at various temperatures and measured after 6, 12, 24, 48 and 72 hours. We observed that when the samples were kept at room temperature for two days, their activity decreased to 85%.Maximum excitation of the substrate was found at 390 nm and fluorogenic emission at 460 nm. Assay pH was adjusted to 8.1 and samples were incubated at 25 °C for 6, 12 and 18 hours.27 male, healthy volunteers were treated in a period of two weeks with professional tooth cleaning and oral hygiene instructions. To avoid effects of a possible gingival irritation GCF was collected from the teeth 13 and 25 one week after the last professional tooth cleaning. Therefore two Periopapers® were placed in mesio- and distobuccal site of the gingival sulcus. The GCF-volume was measured with a Periotron 8000®. The samples from one tooth were immediately pooled in a buffer and stored at -80 °C and then analysed in our laboratory.The volunteers received fully oral and special periodontal examination. Persons, with no response to hygiene instruction, and those who received medication, as well as smokers, volunteers with subgingival prostheodontic restaurations, signs of gingivitis, and marginal periodontitis were excluded.Our microassay explored elastase in the GCF of all volunteers. The median elastolytic activity was 452 µU/µl. In comparison with other workgroups this activity can be explained, because the buffers used in this test are able to liberate elastase from neutrophile granulocytes contained in the GCF-sample. Keywords: elastase, experimental gingivitis, fluorometric assay, Periotron 8000, gingival crevicular fluid