ABSTRACT: Aim: This study aimed to analyse the salivary composition and proteomic profile of saliva and the salivary pellicle in smokers compared to non-smokers, and to examine potential differences of the erosion-protective capacity of the salivary pellicle. Materials and Methods: Twenty-five smokers and 25 non-smokers were included. Unstimulated and stimulated saliva samples were analysed regarding flow rate, pH, buffer capacity, calcium, phosphate, fluoride, and protein content. Salivary pellicles were formed intraorally on resin specimens (n=8). Saliva and salivary pellicle samples were used for data-independent acquisition mass spectrometry. Proteins were identified using the UniProtKB Homo sapiens reference proteome. For the saliva samples, proteomes of 15 commonly observed bacterial species from the oral microbiome were also included. In an in situ experiment, the participants used intraoral splints loaded with bovine enamel and dentin specimens for 120 min (each n=4). Pellicle-covered specimens were extraorally eroded (1mL HCl, pH 2.3, 60 s). Calcium release into the acid was determined photometrically and compared to pellicle-free specimens (control, each n=30). Differentially abundant proteins between smokers and non-smokers were identified using t tests (≥1.5-fold differences and padj.≤0.05). In addition, a pathway enrichment analysis of the associated genes was performed (KEGG, Reactome). Statistical analysis of the salivary samples was performed by one-way ANOVAs followed by pairwise t tests, t tests, Pearson correlations, and multiple linear regression analysis (p<0.05). Results: Except for phosphate in stimulated saliva (smokers: 5.5±2.1 mmol/L, non-smokers: 3.6±1.0 mmol/L; padj.=0.003), salivary parameters were not significantly different between smokers and non-smokers. For both saliva and the salivary pellicle, gene set enrichment analysis indicated common biological themes of genes enriched in either the smokers or the non-smokers. Despite the different proteomic profiles, the erosion-protective capacity of the salivary pellicle of smokers and non-smokers did not differ, and calcium release was not correlated with or predicted by the assessed salivary parameters. The salivary pellicle had a significant erosion-protective effect in enamel compared to the control (41.4±6.3 nmol/mm2), but no differences between smokers (33.2±10.6 nmol/mm2, padj.=0.001 ) and non-smokers (32.7±8.6 nmol/mm2, padj.=0.001) were found. Calcium release did not correlate with or was predicted by the assessed salivary parameters. Conclusion: The proteomic profiles of both unstimulated and stimulated saliva and the salivary pellicle differ between smokers and non-smokers. Evidence for different activity of different biological metabolic pathways such as carcinogesis exists. However, there was no detectable influence on the erosion-protective capacity of the pellicle.
Christof Lenz