Oral Enzyme Activities Profiling

Enzyme activities were measured in supragingival, subgingival and saliva samples (collected in the first recruitment, n = 15 from each group). urease and ADS activities were determined as described by Nascimento et al.^{16 (link)}. In brief, ammonia generated from incubation (37 °C, 120 min) of 125 μl oral samples (suspended plaque and saliva samples) and a 500 μl mixture [50 mM urea (Sigma-Aldrich, St. Louis, MO, USA) or L-arginine-HCL (Sigma-Aldrich), 0.5 mM Tris-maleate buffer (pH = 6.0)] were measured by Nessler’s reagent (Sigma-Aldrich) with ammonium sulfate as a standard. Meanwhile, protein content in each sample was determined by Bradford’s Assay with bovine serum albumin as a standard. urease and ADS activities were expressed as μmol ammonia produced per min and were normalized to mg of protein (μmol/min/mg). LDH activities in plaque and saliva samples were measured by the LDH Activity Assay Kit (Sigma-Aldrich) according to the manufacturer’s instructions. One unit of LDH was defined as the amount of the enzyme that catalyzed the conversion of lactate into pyruvate to generate 1.0 μmol of NADH per min. LDH activity was expressed as the amount of enzyme (U) per g of protein (U/g).