title: An evaluation of endogenous pancreatic enzyme levels after human islet isolation.
abstract: BACKGROUND
  Recent evidence has suggested that inconsistencies in human islet yield and viability after collagenase digestion is attributed to the activation of endogenous enzymes of the cadaveric donor pancreas. A study of the enzyme kinetics of serine proteases throughout human islet isolations showed a significant increase in activity levels throughout the digestion period. Following the digestion, it is important to further inhibit these enzymes by the addition of an inhibitor to the dilution media.
  
  
  OBJECTIVE
  To report the levels of endogenous pancreatic enzymes remaining after human islet isolation and the effects of three potential enzyme inhibitors on the proteases.
  
  
  METHODS
  Human albumin, fetal calf serum, and the protease inhibitor aprotinin were incubated with the trypsin, chymotrypsin, elastase, and collagenase and were assayed for activity.
  
  
  RESULTS
  Results at the final stage indicated that chymotrypsin retained 21.0 +/- 7.5% (mean +/- SE; n = 20) of the activity observed at the conclusion of the enzymatic digestion phase of the isolation process, whereas trypsin, elastase, and collagenase retained 3.0 +/- 1.5%, 2.1 +/- 0.6%, and 3.9 +/- 0.9%, respectively. Fetal calf serum and aprotinin showed strong inhibitory effects against bovine pancreatic trypsin; however, they showed a weak inhibitory effect against elastase. Supplementation with aprotinin failed to inhibit human chymotrypsin and elastase. Human albumin showed minimal inhibition and was shown to serve only as a competitive inhibitor. No inhibition to collagenase was observed with human albumin, fetal calf serum, or aprotinin.
  
  
  CONCLUSIONS
  This study clearly demonstrates that low amounts of endogenous pancreatic enzymes remain active throughout the human islet isolation process and that the added inhibitors at the end of the isolation process are not fully effective at inhibiting the enzymes.
authors.affiliation: Department of Surgery, Surgical-Medical Research Institute, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Natisha L
authors.initials: NL
authors.lastname: Rose
authors.forename: Monica M
authors.initials: MM
authors.lastname: Palcic
authors.forename: Jonathan R T
authors.initials: JR
authors.lastname: Lakey
mesh_terms: D000818:Animals; D007611:Aprotinin / Q000494:pharmacology; D002417:Cattle; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D017364:Collagenases / Q000302:isolation & purification / Q000378:metabolism; D003470:Culture Media / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D005312:Fetal Blood / Q000737:chemistry; D006801:Humans; D007515:Islets of Langerhans / Q000201:enzymology*; D061965:Matrix Metalloproteinase Inhibitors; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D012709:Serum Albumin / Q000494:pharmacology; D014357:Trypsin / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000494:pharmacology
chemical_list: D003470:Culture Media; D061965:Matrix Metalloproteinase Inhibitors; D012709:Serum Albumin; D014361:Trypsin Inhibitors; D007611:Aprotinin; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D014357:Trypsin; D017364:Collagenases