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Evaluation of Pefabloc as a serine protease inhibitor during human-islet isolation
Natisha L Rose1, Monica M Palcic, Lisa M H Helms
1Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
Background:
Recent evidence has suggested that inconsistencies in human-islet yields after collagenase digestion are attributed to the activation of endogenous enzymes of the cadaveric donor pancreas. Inhibition of protease activity by Pefabloc (0.4 mM; Roche Biochemicals Inc., Indianapolis, IN) has recently been shown to improve human-islet isolation after prolonged cold storage of the pancreas. In this study, we have hypothesized that this improvement was because of the inhibition of three key serine proteases.
Methods:
Twenty cadaveric pancreases were perfused in the presence (n=12) and absence (n=8) of Pefabloc added at the time of distention using a customized perfusion device. Samples were collected throughout the digestion process and were assayed for trypsin, chymotrypsin, elastase, and total protease activity.
Results:
In all cases, the enzyme activity levels remained lower in the presence of Pefabloc as compared with the control samples. There was significantly higher chymotrypsin and elastase activity in the control group, but not trypsin or total protease activity, from the time following loading of the enzyme onto the pancreas until the stopping of the enzymatic digestion phase (dilution).
Conclusions:
Pefabloc was shown to be an effective protease inhibitor throughout the entire digestion process. Pefabloc supplementation did not significantly effect the dilution time or the islet yield in this study; however, these data show that serine proteases are effectively inhibited by Pefabloc during the clinical islet process.
