Exploration of whole saliva proteolysis as a diagnostic marker for periodontal disease
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Human whole saliva (WS)is an oral fluid derived mainly from salivary glands. It also contains non-exocrine components, such as gingival crevicular fluid, oral microorganisms, desquamated oral epithelial cells and host and bacteria-derived components. WS is a wealthy source of information regarding biological processes in oral health and disease and may contain markers that can be exploited diagnostically. One disease that could potentially be diagnosed in saliva is Periodontal disease, as it involves proteolytic breakdown of matrix proteins by either host- or bacteria-derived proteases. While whole saliva is a unique fluid source for diagnosing oral diseases, it is also a challenging fluid due to its mixed composition of fluids and particulate components and high proteolytic activity. An important question relates to how saliva should be treated in order to prevent 'destruction' of the diagnostic markers. Alternatively, perhaps the enzymatic activities themselves can be used for the diagnosis of diseases that involve proteolytic activity, such as periodontal disease. In the first series of experiments, the focus was on finding ways to minimize the proteolytic activity of whole saliva in order to preserve diagnostic markers. This is important when the diagnostic marker(S) of interest are sensitive to proteolytic degradation. The results indicated that a) there were inter-individual differences in protease activities between saliva donors, and the t1/2 values, representing the time needed to degrade half of the added amount of histatin 5, ranged from 8 to 22 minutes, b) acidification of whole saliva to pH 4.0 effectively prevented histatin 5 degradation, c) tl/2 values at 37 degrees C, 22 degrees C, 4 degrees C and 0 degrees C were 22 min, 35 min, 3 h 10 min and 4 h 20 min, indicating it is possible to retard but not to abolish proteolysis on ice, d) boiling of saliva for 10 min delayed histatin degradation but, surprisingly, was not able to completely prevent its degradation. The second part of the study focused on the diagnostic potential of the enzymes themselves. Whole saliva was collected from 48 individuals of which 23 were periodontally healthy and 25 were affected by moderate to severe periodontitis. To study endogenous proteolytic activities in WSS, three substrates were used: 1) human salivary histatin 5; 2) a histatin-derived synthetic analog (Z-RGYR-MCA); and 3) gelatin. Degradation of histatin 5 (0.2 mg/ml) was monitored by cationic PAGE and densitometric analysis of the histatin 5 band. Enzymatic cleavage of ZRGYR-MCA (0.5 M), was measured fluorimetrically. Degradation of gelatin (denatured collagen) was studied by gelatin zymography and densitometric analysis of a selected region of the zymogram gel. In addition, histatin 5 and Collagen type IV zymography was performed for four representative periodontally healthy and four diseased subjects. The rates of degradation of histatin 5 and hydrolysis of Z-RGYRMCA did not differ between healthy and periodontal patients (P[more than]0.05; NS). Histatin 5 zymography confirmed these results. In contrast, WSS activities directed at gelatin did show statistically significant differences between the healthy and the periodontally diseased groups (289.1[plus or minus]138.6 AU and 470.1[plus or minus]144.9 AU, respectively; P[less than]0.01). Collagen type IV zymography results were consistent with gelatin zymography results, showing that there is advanced proteolytic activity in the periodontally diseased group and indicating that these activities have a clear diagnostic potential.
PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact firstname.lastname@example.org.Thesis (MSD) --Boston University, Henry M. Goldman School of Dental Medicine, 2012 (Department of Periodontology and Oral Biology).Includes bibliographic references: leaves 59-65.
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