Marion Hänsler

Pancreatic duct mucosa following bile salt injury in cats. Morphology, barrier function to pancreatic exocrine proteins and vulnerability by activated pancreatic juice.

We studied pancreatic duct mucosal morphology and barrier function to both activated and nonactivated pancreatic exocrine proteins following bile salt injury in cats. Prograde pancreatic duct perfusion with 15 mM glycodeoxycholate caused epithelial disruption with focal epithelial loss in the majority of animals. In these cats, flow of preanalyzed nonactivated rat pancreatic juice along the duct resulted in a selective loss of zymogens from the duct lumen as determined by two-dimensional isoelectric focusing-sodium dodecyl sulfate gel electrophoresis and reversed-phase high-performance liquid chromatography. Proteins lost had a low isoelectric point (≤5.7) and a molecular weight as large as 47,000 Da and included chymotrypsinogen 1, trypsinogens 1 and 2, and procarboxypeptidases B. Proteins with a high isoelectric point (>5.7), a high molecular weight (>47,000 Da), or both, were completely recovered from the duct lumen. Flow of activated rat pancreatic juice along a pancreatic duct with bile salt-induced epithelial disruptions caused additional morphologic alterations including an increase in epithelial destruction and occasional necrosis of the duct interstitial and vascular tissue. In some cats, in which the integrity of the ductal epithelium remained preserved following exposure to the bile salt, neither loss of rat pancreatic exocrine zymogens from the duct lumen nor degradation of the duct mucosa by activated rat pancreatic juice was observed. We conclude: (1) glycodeoxycholate-induced duct epithelial disruptions result in loss of pancreatic duct mucosal barrier function to pancreatic exocrine proteins; (2) pancreatic duct mucosal permselectivity to pancreatic exocrine zymogens following exposure to glycodeoxycholate is based on discrimination of molecular weight and charge, and the duct mucosa acts as a cationic charge selective macromolecular filter; and (3) glycodeoxycholate-induced duct epithelial disruptions render the MPD vulnerable to degradation by activated pancreatic juice. Thus, escape of pancreatic exocrine enzymes from the lumen of the MPD following bile salt injury may be due to both penetration across and/or degradation of the pancreatic duct mucosal barrier.

Peptidsynthesen mit immobilisierten Enzymen II. Immobilisiertes Trypsin, Thermolysin und Papain

Model studies were performed on the utility of covalently immobilized trypsin, thermolysin and papain for peptide bond formation. Trypsin and thermolysin catalyzed the formation of peptide bonds with nearly the same efficiency as the soluble proteases and they could be re-used successfully for further coupling experiments. The possibility of using immobilized trypsin and papain for kinetically controlled peptide bond formation was investigated. With the serine type enzyme trypsin excellent product yields were obtained starting with ester carboxyl components and an economical ratio of substrates. Experiments with the thiol protease papain were unsatisfactory because the once formed product is hydrolyzed as fast as the starting ester substrate used.

Direct conversion of porcine insulin into human insulin ester catalyzed by immobilized trypsin

Abstract Trypsin covalently bound to macroporous bead cellulose was found to have a sufficiently high activity to catalyze the direct conversion of porcine insulin into human insulin ethyl ester.

Improved detection of human pancreatic proteins separated by two-dimensional isoelectric focusing/sodium dodecyl sulphate gel electrophoresis using a combined staining procedure.

In order to assess secretory pancreatic proteins in a two-dimensional isoelectric focusing/sodium dodecyl sulphate electrophoresis gel, a highly sensitive double-staining method with Coomassie Brilliant Blue followed by silver stain was used. This combined procedure afforded more distinct spots and additional bands, particularly glycoproteins, than either silver or Coomassie Blue staining alone. As measurements of dye volumes by densitometry have shown, double staining of two-dimensional separated pancreatic proteins is up to twenty times more sensitive than the usual Coomassie Brilliant Blue staining.

Does high pancreatic duct pressure compromise the duct mucosal barrier function to pancreatic exocrine proteins

The effect of duct pressure on the barrier function of the pancreatic duct mucosa to both activated and nonactivated pancreatic exocrine enzymes was studied in a feline model. The cat main pancreatic duct was perfused from the tail to the head of the gland with rat pancreatic juice at high duct pressure (40 cm H2O). In a first experiment, nonactivated pancreatic juice was perfused. Analysis of the juice for loss of fluid volume (measurement by weight) and for loss of individual proteins (two‐dimensional isoelectric focusing/sodium dodecyl sulphate gel electrophoresis, reversed phase‐high performance liquid chromatography) after duct passage showed that pancreatic secretions were completely recovered from the duct lumen. In another experiment, applying the same pressure, the duct was perfused with activated pancreatic juice. Morphologic analysis showed a preservation of the pancreatic duct mucosal integrity immediately after duct perfusion and absence of pancreatic inflammatory lesions 24 h after duct passage. We conclude that the pancreatic duct mucosa is impermeable to the leakage of pancreatic exocrine proteins from the duct lumen at duct pressure ≥40 cm H2O. Flow of activated pancreatic juice along the pancreatic duct at duct pressure ≥40 cm H2O does not cause acute pancreatitis. These data do not support the hypothesis that leakage of pancreatic juice from the duct lumen caused by high intraductal pressure due to duct obstruction initiates acute pancreatitis.