Characterization of total adenosine deaminase activity (ADA) and its isoenzymes in saliva and serum in health and inflammatory conditions in four different species: an analytical and clinical validation pilot study

Abstract

Background: Measurement of adenosine deaminase (ADA) can provide information about cell-mediated immunity. This report’s objective was to study the enzymatic activity of total ADA (tADA) and its isoenzymes ADA1 and ADA2 in canine, equine, porcine, and bovine serum and saliva and their changes in different inflammatory situations in each species. Besides, an automated method for ADA2 measurement was developed and validated. Results: tADA was present in serum and saliva of healthy animals of the four species. Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) concentration of 0.47 mM was needed for ADA1 inhibition in canine and porcine samples (serum and saliva) and bovine saliva, whereas for equine saliva 0.94 mM was needed. ADA2 activity was not detected in bovine serum and was very low or absent in equine serum and bovine saliva. An automated procedure to measure ADA2 consisting of adding EHNA to a commercial reagent for tADA measurement provided repetitive (coefficients of variation < 8.8% in serum and < 10% in saliva) and accurate (linearity of serial sample dilutions with R2 > 0.90) results, being equivalent to a manual incubation of the sample with EHNA at a similar concentration. Salivary tADA, as well as ADA1 and ADA2, were higher in dogs with leishmaniosis, horses with acute abdominal disease and pigs with lameness than in healthy animals. tADA and isoenzymes in saliva showed a positive significant correlation with serum ferritin in dogs (r = 0.602, P < 0.01; r = 0.555, P < 0.05; and r = 0.632, P < 0.01; respectively for tADA, ADA1 and ADA2) and serum C-reactive protein in pigs (r = 0.700, P < 0.01, for both tADA and ADA1; r = 0.770, P < 0.001, for ADA2), whereas salivary ADA2 significantly correlated with serum amyloid A in horses (r = 0.649, P < 0.01). In cows, salivary tADA and ADA1 significantly increased after calving, correlating with total white blood cell count (r = 0.487, P < 0.05, for both tADA and ADA1). Conclusions: The activity of total ADA and its different isoenzymes, can be measured in serum and saliva of dogs, horses, pigs and cows by a simple and fast procedure described in this report. When measured in saliva, these analytes correlated with other biomarkers of inflammation and it could potentially be used as a biomarkers of inflammation and immune activation in the species of this study.