Electrophoretic separation of proteolytic enzymes in pancreatic juice collected with the pouch or catheter method

Abstract

Conclusion: The results of this study demonstrated that proteolytic enzymes in pancreatic juice from pigs prepared with the pouch method (PM) were nearly fully active or were fully active. When activation with enterokinase was carried out further inactivation and/or breakdown occurred for chymotrypsin C and cathodal trypsin. In addition, some inactivation and/or breakdown of proteolytic enzymes in pancreatic juice occurred during collection of pancreatic juice from PM pigs.

Methods: Samples of pancreatic juice were collected from growing pigs using either the PM or the catheter method (CM). An isolated pouch was prepared where the pancreatic duct enters the duodenum, and three pigs were fitted with a pancreatic pouch re-entrant cannula. Three different pigs had a catheter surgically inserted into the pancreatic duct. Pooled 8-h samples of pancreatic juice were analyzed before and after activation with enterokinase. Chymotrypsin, trypsin, and elastase activities were identified in pancreatic juice after separation by electrophoresis in 1% agarose gels at pH 8.6 using N-acetyl-DL-phenylalanine-beta-naphthyl ester (Ac-Phe-beta ne) as a substrate.

Results: This qualitative enzyme assay indicated that a considerable amount of chymotrypsin C, anodal trypsin, chymotrypsins A and B, elastase II, and cathodal trypsin were present in samples of nonactivated pancreatic juice from PM pigs. In contrast, the only active enzymes identified in pancreatic juice from CM pigs were very small amounts of chymotrypsin A and elastase II. The amounts of chymotrypsin C and cathodal trypsin were lower in activated than in nonactivated pancreatic juice from PM pigs. However, there were increases in the amounts of the other enzymes when pancreatic juice from PM pigs was activated. As expected, the activation of pancreatic juice from CM pigs resulted in the measurement of very high amounts of all the proteolytic enzymes. The amounts of anodal trypsin, chymotrypsins A and B, and elastase II were higher in activated pancreatic juice from CM pigs than from PM pigs.