Quantification of human lithostathine S2-5 forms using the antibody to the N-terminal peptide region.

Lithostathine S2-5 inhibits in vitro crystal growth of CaCO3. We developed an antibody against the peptide region responsible for inhibitory effect to determine whether lithostathine S2-5 levels are different in the pancreatic juice of patients with and without chronic pancreatitis. The antibody against the synthetic peptide of the N-terminal end of lithostathine S2-5 detected lithostathine S2-5 but not lithostathine S1 or lithostathine extracted from pancreatic calculi. Lithostathine S2-5 was detected in samples of pancreatic juice protein by immunoblotting using the specific antibody. The concentration of lithostathine S2-5 was compared between control and chronic pancreatitis groups. The mean concentrations of lithostathine S2-5 were significantly (p=0.002) lower in chronic pancreatitis, 16.3 microg/mg of total protein, than in the control, 47.1 microg/mg of total protein. A decreased concentration of lithostathine S2-5 seems to increase the risk of stone formation in the ducts during the course of chronic pancreatitis because of insufficient inhibition of CaCO3 crystal growth.

Application of pure pancreatic juice collection to the pancreatic exocrine function test.

We determined the usefulness of pure pancreatic juice collected by endoscopic procedures for assaying impaired pancreatic function in patients with chronic pancreatitis. The functional findings were compared with the degree of morphological change in endoscopic retrograde pancreatograms. Samples of pancreatic juice were collected from 23 patients with suspected chronic pancreatitis by endoscopic cannulation after a bolus intravenous injection of secretin, 100 IU, for 20 min in four 5-min samples. Volume and amylase and bicarbonate concentrations were measured. The maximum bicarbonate concentration (MBC) was found in fractions collected after 10 min. The MBC in patients with chronic pancreatitis (n = 7) was lower than in normal subjects (n = 10) (p < 0.05). When we regarded 125 mEq/L (mean - 1.5 SD) as the normal limit of MBC, the sensitivity, specificity, and overall efficiency of the pancreatic functional test using pancreatic juice to detect chronic pancreatitis were 86, 100, and 94%, respectively. In comparison with the group with normal pancreatogram findings (n = 10), the MBC, volume, amylase output, and bicarbonate output were lower in the combined group (n = 9) of Cambridge II and III (p < 0.05). MBC was the most reliable parameter in the evaluation of the exocrine pancreatic functional test using pancreatic juice samples.

Effect of a wheat amylase inhibitor on canine carbohydrate digestion, gastrointestinal function, and pancreatic growth.

BACKGROUND/AIMS: Chronic amylase inhibition might be useful to treat diabetes mellitus and obesity. Duodenal and ileal cannulas were placed in 8 dogs to determine if long-term ingestion of a wheat amylase inhibitor maintained amylase inhibition or affected gastrointestinal or metabolic function or pancreatic growth. METHODS: Five dogs were fed and 3 were not fed 1.5 g of the inhibitor with meals for 9 weeks. Postprandial and cholecystokinin octapeptide stimulated pancreatic secretion, and fecal balance studies were performed at intervals. After the experiment, the pancreas was analyzed. RESULTS: Weight loss was similar in both groups. Amylase inhibition persisted throughout the 9 weeks; it declined from 91% to 37% from the first to the sixth postprandial hour. Amylase inhibition decreased plasma glucose levels during the first hour (P < 0.05), increased carbohydrate delivery to the ileum (315 vs. 555 mg/h; P = 0.002), and increased cholecystokinin octapeptide-stimulated amylase secretion. However, amylase inhibition did not significantly change plasma concentrations of insulin, peptide YY or neurotensin, postprandial pancreatic secretion, gastrointestinal transit or pancreatic weight, and protein or DNA content. CONCLUSIONS: Prandial ingestion of 1.5 g of the inhibitor for 9 weeks reduces postprandial amylase levels enough to delay carbohydrate digestion and absorption and lower plasma glucose levels without altering pancreatic growth. This dose may be effective to treat diabetes mellitus but not obesity.

Separation of canine pancreatic juice proteins by hydrophobic interaction chromatography preserves enzyme activity.

To measure the synthesis of pancreatic enzymes requires the separation of pancreatic juice proteins. The aim of the present study was to separate amylase, lipase, and trypsinogen present in dog pancreatic juice by using a hydrophobic interaction high-performance liquid chromatography (HPLC). During a 40-min, four-stage gradient of decreasing sodium sulfate concentration, dog pancreatic juice proteins were separated into 10 peaks based on hydrophobicity. Amylase, lipase, and trypsinogen were identified in HPLC fractions by measuring enzyme activity and molecular weight. Amylase and lipase were present in separate peaks. By sodium dodecyl sulfate (SDS) gel electrophoresis, peak 10, the only peak with amylase activity had a single protein, but peak 3, containing lipase, and peak 9, containing trypsinogen, had two or more proteins. Trypsinogen activity was also detected as a main protein in peak 5 and the molecular weight of this protein, 26 kDa corresponds to that of dog trypsinogen. Trypsinogen was not identified in peak 9 by SDS gel electrophoresis because other proteins were close to this location on the gel. In summary, the proteins and activities of amylase, lipase, and two trypsinogens secreted by the dog pancreas can be separated rapidly by hydrophobic interaction liquid chromatography. Also, this one step procedure recovers 87% of amylase from dog pancreatic juice as a single protein.

Identification of immunoreactive pancreatic stone protein in pancreatic stone, pancreatic tissue, and pancreatic juice.

We first examined whether pancreatic stone protein (PSP) was present in pancreatic stone and normal pancreatic tissue. By using HPLC and Western blotting, a protein of Mr 13.5 kDa that reacted with monoclonal antibody against PSP was detected as a major component in EDTA-soluble fractions of pancreatic stone. In an in vitro experiment, this protein dose-dependently suppressed CaCO3 precipitation. PSP was immunohistochemically stained in the acinar cells of normal pancreatic tissue. Based on these findings, it seemed that PSP in pancreatic stone is probably a physiological secretory protein of the pancreas. We subsequently examined immunoreactive PSP in normal pancreatic juice by the Western blotting method. In all of the specimens, the band for immunoreactive PSP in pancreatic juice was found to correspond to 13.5 kDa, which thus agreed with that of purified PSP from a stone.