Optimization and validation of assays to estimate pancreatic esterase activity using well-characterized micellar solutions of cholesteryl oleate and tocopheryl acetate

Abstract

Studies have been carried out to determine the maximal solubilization of cholesteryl oleate and tocopheryl acetate within mixed bile salt-fatty acid micelles and to establish reproducible assays for pancreatic esterase activity using these micellar substrates. At pH 8.5, using 30 mM sodium taurocholate and 10 mM oleic acid, reproducible micellar solutions of the esters could be prepared giving micellar concentrations of 0.4 mM and 0.1 mM for tocopheryl acetate and cholesteryl oleate, respectively. Conditions were then optimized to estimate pancreatic esterase activity using these micellar-solubilized substrates. Maximal activity was obtained at pH 8.5 with 2-4 mM oleic acid and 15-30 mM sodium taurocholate, and gave coefficients of variation for the assays of 7.4% and 20.2% using tocopheryl acetate and cholesteryl oleate, respectively, as substrates. Micellar-solubilized cholesteryl oleate and tocopheryl acetate, together with a non-micellar system using p-nitrophenyl acetate, were used to estimate esterase activity in human duodenal aspirates and rat pancreatic homogenates. Esterase activity in children with cystic fibrosis was greatly reduced and paralleled tryptic and pancreatic lipase activity, which suggested that the esterase activity was pancreatic in origin. The results of this study, therefore, provide a basis for future investigations concerned with the hydrolysis and absorption of dietary esters.