Fluorimetric analysis of lipase hydrolysis of intermediate- and long-chain glycerides

Abstract

For the purpose of deducing the digestive behavior of dietary fat in the digestive organs, a fluorimetric method for the measurement of hydrolysis by porcine pancreatic lipase was performed using intermediate- and long- acyl chain glycerides as substrates. Insoluble glycerides constituted by C₁₀–C₁₆ acyl chains were mechanically dispersed in 100% buffer and hydrolyzed by porcine pancreatic lipase. After the reaction, fatty acid released by the enzyme was extracted and its carboxyl group was fluorescently labeled with 9-bromomethylacridine. The 9-acridinylmethyl derivative of the fatty acid was separated and determined by HPLC. The sensitivity of this method was about 1000 times higher than that of the titrimetric method. Only 0.5 ng of porcine pancreatic lipase was sufficient for one routine assay. This assay method was successfully applied to investigate the enzymatic properties of porcine pancreatic lipase with respect to dietary lipids. The effects of some physiological factors concerned with lipid digestion, such as bile salt and colipase, on the lipase hydrolysis were also examined. The method established in the present study could contribute to a highly sensitive assay of some hydrolases containing lipase with regard to insoluble substrates.