An evaluation of endogenous pancreatic enzyme levels following enzymatic digestion.
Human islet isolation consists of a digestion phase, dilution phase, and purification phase. Recent evidence suggests that inconsistencies in islet yields are attributed to the activation of endogenous enzymes of the donor pancreas during the digestion phase. Therefore, following the digestion phase, it is important to inhibit these enzymes by the addition of an inhibitor to the dilution phase. In this study, we report the endogenous pancreatic enzyme levels after the purification phase and the effects of potential inhibitors on the proteases of interest. Results at the end of the purification phase indicated that chymotrypsin retained approximately 20% of the activity observed after the digestion phase, whereas trypsin, elastase, and collagenase retained approximately 2.5%, 2%, and 3.5% of the activity, respectively, demonstrating that added inhibitors are not fully effective. Potential enzyme inhibitors, human albumin, fetal calf serum, and aprotinin, were incubated with trypsin, chymotrypsin, elastase, and collagenase and assayed for activity. Fetal calf serum and aprotinin showed strong inhibitory actions toward trypsin and chymotrypsin. Aprotinin completely inhibited the tryptic activity; however, it did not inhibit human chymotrypsin or elastase activity. Human albumin showed minimal inhibition and was shown to act as a competitive inhibitor. This study clearly demonstrates that low amounts of endogenous pancreatic enzymes remain active at the end of the human islet isolation procedure and that the added inhibitors at the dilution are not fully effective at inhibiting the enzymes.
Rose
N L
NL
Department of Surgery, University of Alberta, Edmonton, Alberta, Canada.
Palcic
M M
MM
Shapiro
A M J
AM
Lakey
J R T
JR
D002470:Cell Survival; D004247:DNA / Q000032:analysis; D010450:Endopeptidases; D006801:Humans; D006868:Hydrolysis; D007328:Insulin / Q000032:analysis / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology* / Q000378:metabolism*; D007700:Kinetics; D010179:Pancreas / Q000166:cytology / Q000201:enzymology*; D020858:Tissue and Organ Harvesting / Q000379:methods
D007328:Insulin; D004247:DNA; D010450:Endopeptidases