Proteomic Investigation of Salivary Biomarkers in Periodontal Diseases
Introduction: The aim of the study was to investigate novel biomarkers of periodontal disease present in GCF and saliva. -- Methods: The identities of specific putative biomarkers identified by SELDI-TOF MS were confirmed using 1-D PAGE coupled with LC-MS/MS. These biomarkers were then tested using ELISA in the samples that had been initially screened using SELDI-TOF MS as well as in saliva samples in an independent cohort selected according to diabetic status rather than periodontal status. The effect of non-surgical periodontal therapy, diabetes, saliva sample collection method, eating, time of day, day of the week and storage conditions on the salivary concentration of human neutrophil peptide 1-3 (HNP 1-3), cathelicidin LL-37 and protein S100A8 were tested. Copy number variation for HNP 1-3 gene, DEFA1A3 was tested in an independent cohort and the data analysed for association with presence and degree of periodontal disease. -- Results: Antimicrobial peptides, HNP 1-3, LL-37 and S100A8 were identified. GCF and salivary HNP 1-3 and LL-37 concentrations were significantly elevated in periodontitis than in gingivitis or health. LL-37 was significantly elevated in aggressive periodontitis (AgP) compared with chronic periodontitis (CP) and gingivitis/health. Periodontal therapy reduced the salivary levels of HNP 1-3. Diabetes, eating and day of week had no significant effect on AMP levels. Saliva collection using spitting method yielded significantly more AMPs than using Salivettes®. Sample collection at 16:00 hours yielded significantly higher amounts of AMPs than samples collected at other times. Sample storage at room temperature (RT) for up to 48 hours had no significant impact on the salivary HNP 1-3 and LL-37 concentration while a proteinase inhibitor did not did improve the recovery of the AMPs after 72 hours.<= 6 copies of DEFA1A3 copy numbers were partly associated with periodontal disease.
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