Characterization of Saliva Biochemical Profile

Samples of saliva were collected in the morning in fasted subjects subjects. Resting and stimulated saliva were collected in pre weighed cups for 5 min. Stimulated saliva was produced by chewing a piece of Parafilm^{59 (link)}. Samples were centrifuged (30 min, 15,000 g, 4 °C) and the supernatants were stored at −80 °C until biochemical assays. Analyses (flow, protein concentration, enzymatic activities, total antioxidant capacity (TAC) and CA-VI levels were performed as described previously^{38 (link),40} . In brief, protein concentration was measured by using the Bradford protein assay (Bio-Rad, France). Proteolytic activity was determined using a Pierce Fluorescent Assay Kit (Pierce Biotechnology, Rockford, IL). Amylolytic activity was assayed using CPNG3 Assay Kit (Biolabo, Maizy, France). Lipolytic activity was explored according a method fully detailed in^{38 (link)}. Total antioxidant capacity was established using an ORAC Assay kit (CellBioLabs, San Diego, USA). CA-VI was quantified using an Enzyme-Linked ImmunoSorbent Assay kit (USCN Life Science Inc.). Plasma and salivary LPS levels were assayed according to^{60 (link)}. In brief, LPS-derived 3-hydroxymyristate was extracted from plasma and salivary samples with an organic solvent, separated by reversed phase HPLC, then quantitated by MS/MS.