alpha-chymotrypsin and Pancreatic-Fistula

Postoperative pancreatic fistula (POPF) is the most serious and challenging complication following gastroenterological surgery. Activated pancreatic juice leaking from the organ remnant contains proteases that attack the surrounding tissue, potentially leading to severe inflammation, tissue necrosis, and fistula formation. However, it is difficult to observe pancreatic leakage during surgery and to evaluate the protease activity of leaked fluid at the patient's bedside. This report describes a protein nanocage-based protease ratiometric sensor comprising a pancreatic protease-sensitive small heat-shock protein (HSP) 16.5, which is a naturally occurring protein in Methanococcus jannaschii that forms a spherical structure by self-assembly of 24 subunits, and a chemically conjugated donor-acceptor dye pair for Förster resonance energy transfer (FRET). The HSP-FRET probe was constructed by subunit exchange of each dye-labeled engineered HSP, resulting in a spherical nanocage of approximately 10 nm in diameter, which exhibited very high stability against degradation in blood plasma and no remarkable toxicity in mice. The efficiency of FRET was found to depend on both the dye orientation and the acceptor/donor ratio. Pancreatic proteases, including trypsin, α-chymotrypsin, and elastase, were quantitatively analyzed by fluorescence recovery with high specificity using the HSP-FRET nanoprobe. Furthermore, the HSP-FRET nanoprobe was sufficiently sensitive to detect POPF in the pancreatic juice of patients using only the naked eye within 10 min. Thus, this novel nanoprobe is proposed as an effective and convenient tool for the detection of POPF and the visualization of activated pancreatic juice during gastroenterological surgery.

Topics: Animals; Chymotrypsin; Fluorescence Resonance Energy Transfer; Gastrointestinal Tract; General Surgery; Heat-Shock Proteins, Small; Humans; Inflammation; Methanocaldococcus; Mice; Nanostructures; Pancreatic Fistula; Postoperative Complications; Quantum Dots

Topics: Animals; Chymotrypsin; Disease Models, Animal; Fluorescence; Fluorescent Dyes; Male; Pancreas; Pancreatectomy; Pancreatic Fistula; Pancreatic Juice; Swine

Pancreatic fistula (PF) remains the most serious complication after digestive surgery. It is difficult to prevent because of the inability to visualize the leakage of pancreatic juice during surgery or to evaluate the protease activity of leaked fluid, which is responsible for PF formation.. The fluorescence intensities of a chymotrypsin probe (glutaryl-phenylalanine [corrected] hydroxymethyl rhodamine green with added trypsin) in pancreatic juice and in intestinal or abdominal fluids drained after pancreatic resection were evaluated. The chymotrypsin probe was sprayed on to filter papers that had been placed on the resected pancreatic stump in patients undergoing pancreaticoduodenectomy or central pancreatectomy. The ability of this technique to visualize the leakage of pancreatic juice and predict postoperative PF formation was assessed.. The fluorescence intensity of the chymotrypsin probe in 76 fluid samples correlated positively with amylase levels (r(s) = 0.678, P < 0.001). The fluorescence patterns of the pancreatic stump were classified grossly into the three types: duct (fluorescence signal visualized only on the stump of the main pancreatic duct, 16 patients), diffuse (ductal stump and surrounding pancreatic parenchyma, 7) and negative (no fluorescence signal, 7). Symptomatic PFs developed in 13 of 23 patients with duct- or diffuse-type fluorescence, but in none of the seven patients with negative-type fluorescence (P = 0.008).. The chymotrypsin probe enabled determination of the protease activity in drained pancreatic fluid samples and allowed real-time visualization of pancreatic juice leakage during surgery.

Topics: Aged; Aged, 80 and over; Chymotrypsin; Dipeptides; Female; Fluorescence; Fluorescent Dyes; Humans; Male; Middle Aged; Pancreatectomy; Pancreatic Fistula; Pancreatic Juice; Pancreatic Neoplasms; Pancreaticoduodenectomy; Peptide Hydrolases; Rhodamines

The effect of intraluminal bile on the well-known feedback regulatory mechanism of exocrine pancreatic secretion exerted by intraluminal trypsin was investigated in conscious rats with pancreatic, biliary and duodenal fistulae. The stimulated pancreatic enzyme secretion caused by diversion of bile-pancreatic juice from the intestine was apparently suppressed by intraduodenal reintroduction of pancreatic juice or bile-pancreatic juice, while it was slightly suppressed by intraduodenal reintroduction of bile. Although additional reintroduction of bile did not alter the already suppressed pancreatic enzyme secretion by the presence of pancreatic juice in the intestine, diversion of bile stimulated the suppressed pancreatic enzyme secretion by intraluminal bile-pancreatic juice. Infusion of sodium taurocholate into the duodenum with diversion of bile-pancreatic juice effectively inhibited pancreatic enzyme secretion. The inhibitory effect seemed to be dependent on the concentration of taurocholate infused into the duodenum. The results suggest that bile and bile acid have an important role in the feedback regulatory mechanism of pancreatic enzyme secretion, at least partly directly inhibiting the secretion.

Topics: Amylases; Animals; Bile; Bile Acids and Salts; Biliary Fistula; Chymotrypsin; Duodenal Diseases; Feedback; Intestinal Fistula; Male; Pancreas; Pancreatic Fistula; Pancreatic Juice; Proteins; Rats; Rats, Inbred Strains; Taurocholic Acid; Time Factors

Topics: Acute Disease; Adult; Aged; Catheterization; Chymotrypsin; Chymotrypsinogen; Enzyme Activation; Enzyme Precursors; Female; Humans; Hydrogen-Ion Concentration; Male; Middle Aged; Pancreatic Ducts; Pancreatic Elastase; Pancreatic Fistula; Pancreatic Juice; Pancreatitis; Proteins; Temperature; Thrombin; Time Factors; Trypsin; Trypsinogen

Topics: Amylases; Animals; Bile; Biliary Fistula; Cattle; Chymotrypsin; Chymotrypsinogen; Enzyme Activation; Feedback; Glycine max; Intestines; Intubation, Gastrointestinal; Male; Pancreas; Pancreatic Fistula; Pancreatic Juice; Rats; Trypsin; Trypsin Inhibitors

Topics: Animals; Bicarbonates; Blood Volume; Cholecystokinin; Chymotrypsin; Dogs; Dose-Response Relationship, Drug; Gastric Fistula; Infusions, Parenteral; Isoproterenol; Pancreas; Pancreatic Elastase; Pancreatic Fistula; Propranolol; Secretin; Trypsin

Topics: Animals; Caseins; Cattle; Chick Embryo; Chymotrypsin; Digestion; Freeze Drying; Humans; In Vitro Techniques; Lactalbumin; Male; Ovalbumin; Pancreas; Pancreatic Fistula; Pancreatic Juice; Serum Albumin, Bovine; Swine; Trypsin Inhibitors

Topics: Adaptation, Physiological; Amylases; Animals; Chymotrypsin; Dietary Carbohydrates; Dietary Proteins; Male; Pancreas; Pancreatic Fistula; Protein Biosynthesis; Proteins; Species Specificity; Starch; Swine; Trypsin

Topics: Atropine; Chymotrypsin; Gastrointestinal Hormones; Humans; Methionine; Pancreas; Pancreatic Fistula; Pancreatic Function Tests; Pancreatic Juice; Pharmacology; Physiology; Secretin; Trypsin; Trypsinogen