Determination of arginine catabolism by salivary pellet

To determine the formation of ammonium from arginine by oral bacteria residing in saliva and dental plaque, an arginolytic activity assay based on the work described by Nascimento et al. [2] was developed. Following the original methodology, insufficient ammonium production could be determined. To improve the method for our research goal, the following modifications were made to the original protocols: The following changes were made to the arginine catabolism assay resulting in a 1000-fold increase in sensitivity: (i) the salivary pellet was washed and concentrated five times resulting in the removal of low density compounds interfering with the assay, (ii) the pH of the Tris-maleate buffer was increased from 6.0 to 7.5 resulting in a better conversion of arginine to ammonium and (iii) the incubation time was increased to 3h to ensure that non-responders and salivary pellets low in cell numbers could yield detectable levels of ammonium. Removal of a centrifuge step from the protein determination resulted in a higher protein yield improving the accuracy of the assay. Changing from the use of the toxic, environmentally hazardous, mercury containing Nessler's reagent to a colorimetric enzyme assay achieved a safer and greener determination of ammonium concentration. (C) 2014 The Authors. Published by Elsevier B.V. All rights reserved.