Biochemical and immunological studies of periodontal disease in humans with emphasis on the analyses of breakdown products emanating from the gingival crevice

Active periodontal disease is characterized by marked destruction of collagen in periodontally-involved tissues. Biochemical characterization of the disease is complicated as it is an episodic disorder that is believed to cycle through periods of high activity and quiescence. The first part of the study focused on the measurements and analysis of volatile sulphur compounds (VSC) collected at gingival sulci. Their concentrations in mouth air correlate with the severity of the disease. The second part was devoted to the evaluation of breakdown products of collagen metabolism found in the gingival crevicular fluid (GCF) in relation to periodontal disease. A novel method and device was developed for collection and analysis of volatile compounds from specific gingival crevice sites. It demonstrated that volatile sulphur compound profiles of crevicular air differ from that of mouth air. It showed that total sulphur content of either inflamed or deep (PD z4 mm) periodontal sites was significantly higher than the non inflamed or shallow (PD<4 mm) sites (p<.05). The ratio of CH3SH to H2S was significantly higher in inflamed than noninflamed sites (p<.05) and in deep versus shallow sites (p<.1). This is the first known study to quantitate VSC directly from individual crevicular sites. Furthermore, in the course of the study, methodology was developed for high performance liquid chromatography (HPLC) analysis of hydroxyproline (Hyp) from GCF using precolumn derivatization with phenylisothiocynate. The method was applied to a study of crevicular fluid collected from 30 periodontally-involved subjects that participated in a clinical study which evaluated spiramycin as an adjunct in treatment to scaling and root planing. The study compared Hyp values to several recorded clinical periodontal parameters. Analysis of hydrolyzed and unhydrolyzed samples indicated that the dominant source of Hyp was present in a peptide or bound form. Hyp did not correlate with pocket depth or crevicular fluid volume. Hyp levels measured at inflamed and noninflamed sites fluctuated at given examination points (0, 2, 8, 12, 24 weeks) and between these time points during the study. The most complete data were obtained for time points0 and 12 weeks which were used to make the best longitudinal comparison between these two time points. Accordingly, the Hyp levels in both treated groups at week twelve in periodontal sites that were inflamed at week zero and remained inflamed at week 12 were higher than in sites that remained noninflamed. This relationship was statistically significant in the spiramycin treated group (p<.05). In both treatment groups Hyp levels at week twelve were higher in healing sites that experienced a mm gain of attachment between weeks 0 and 12, than sites that remained unresolved. Again in the spiramycin treated group this difference was found to be statistically significant (p<.05). Hence the study indicated an increase in Hyp levels in GCF during episodes of increased collagen metabolism. This metabolism occurred during both healing and active phases of periodontal disease. A cross sectional study of GCF from inflamed and noninflamed periodontal sites was performed to confirm that Hyp content was higher in inflamed sites and to investigate the Hyp contribution from type I collagen and Clq sources. Again Hyp content was found to be higher ininflamed than noninflamed sites (p<.001). Using ELISA, type I collagen was found to contribute approximately 6-fold more Hyp than Clq to total Hyp content of fluid from inflamed and noninflamed sites. SDS/PAGE gels of GCF from inflamed sites exhibited more intense protein banding pattern than GCF from noninflamed sites. Western blot analyses showed markedly more intense staining for type I collagen peptides than for Clq reactive peptides. This finding, together with the ELISA assay results which indicated that Clq contributed less than 10% of total Hyp to both inflamed or noninflamed GCF, strongly implies that collagen is the dominant source of the total Hyp content in the processsed GCF samples. The presence of higher VSC content and CH3SH to H2S ratio in crevicular air and higher Hyp content in GCF, can distinguish inflamed from noninflamed periodontal sites. These indicators can distinguish the presence of disease at specific sites at the time of examination. Since Hyp levels were found to be increased at periodontal sites that exhibited healing during a 3 month period, Hyp can also ascertain the effectiveness of periodontal therapy.Dentistry, Faculty ofGraduat