A preliminary study of the activation of endogenous pancreatic exocrine enzymes during automated porcine islet isolation.
The activation of endogenous pancreatic enzymes during automated pancreas digestion may be detrimental to islet isolation. In this report we assessed the activation of trypsin, chymotrypsin, elastase, carboxypeptidases A and B, phospholipase A2, and lipase using a porcine model. Four islet isolations were examined. Duplicate aliquots were taken from the automated circuit at 5-min time intervals up to the completion of pancreas digestion (approx 60 min). One aliquot was activated in vitro with exogenous trypsin in order to convert the enzymes into their active non-"proform," with the exception of trypsinogen, which was activated with exogenous enterokinase. This was done to assess the percentage activation of each individual enzyme (total potentially activatable enzyme release). The extent of activation between isolations was extremely variable. During the closed (recirculating) circuit phase of pancreas digestion there were both gradual and rapid increases in the levels of enzymes released. Peak activity of enzyme activation varied from 13 to 30 min; similarly, total potentially activatable peaks occurred between 13 and 38 min. Lipase and carboxypeptidase B showed greater than 70% activation, chymotrypsin, carboxypeptidase A, and phospholipase A2 between 50% and 70% activation, and trypsin and elastase less than 20%. There were up to 30-fold differences between the four islet preparations. In summary, it is unlikely that poor islet yields are soley explained by variations between collagenases; the variable activation of endogenous pancreatic exocrine enzymes is also likely to be influential to porcine islet yields.
White
S A
SA
Department of Surgery, University of Leicester, UK.
Djaballah
H
H
Hughes
D P
DP
Roberts
D L
DL
Contractor
H H
HH
Pathak
S
S
London
N J
NJ
D000818:Animals; D001331:Automation; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000378:metabolism; D043422:Carboxypeptidases A; D002469:Cell Separation / Q000379:methods; D002918:Chymotrypsin / Q000378:metabolism; D004789:Enzyme Activation; D007515:Islets of Langerhans / Q000166:cytology*; D007700:Kinetics; D008049:Lipase / Q000378:metabolism; D010179:Pancreas / Q000166:cytology* / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D013552:Swine; D014357:Trypsin / Q000378:metabolism
D008049:Lipase; D010741:Phospholipases A; D054467:Phospholipases A2; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D043424:Carboxypeptidase B; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D014357:Trypsin