Measurement of 5-fluorouracil and its active metabolites in tissue.

The 5-fluorouracil content of serum, bile, pancreatic juice, liver, pancreas and muscle was measured by reversed-phase high-performance liquid chromatography using a mobile phase of 5 mM 1-heptanesulfonic acid in 5 mM acetic acid. Free or unmetabolized 5-fluorouracil was extracted from samples with a mixture of light petroleum-n-propanol (40:60). The active metabolites of 5-fluorouracil were hydrolyzed with hot perchloric acid to free 5-fluorouracil and the combined 5-fluorouracil content was extracted. The active metabolite fraction was calculated from the difference between the combined and the free fractions. A straight line plot of the peak areas against concentration was achieved and the detection limit was 50 ng/ml. Five minutes after stopping an intravenous infusion of 15 mg/kg of 5-fluorouracil in a dog, the serum contained only the free form, but other body fluids and tissues contained both free and metabolite fractions. The method may be useful to determine the amount of total drug in patient samples.

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.

Isolation, purification, and the amino acid sequence of a secretory trypsin inhibitor from the chicken pancreas.

A trypsin inhibitor is secreted in the pancreatic juice of the chick. Extracts from tissue have an inhibitor that corresponds to the secreted inhibitor on the basis of chromatography on DEAE-cellulose. The secretory inhibitor was purified by anion- and cation-exchange chromatography and by preparative isoelectric focusing. The purified inhibitor has 69 amino acids and is highly homologous with the secretory inhibitor from the turkey pancreas.

The clinical utility of the CA 19-9 tumor-associated antigen.

Since Koprowski and coworkers discovered the CA 19-9 antigen 10 yr ago, it has become the most useful blood test in the diagnosis and management of patients with cancer of the pancreas. With an upper limit of normal of 37 U/ml, the assay's overall sensitivity is approximately 80% and its specificity is 90%. If higher cutoffs are used, the specificity rises so that, at levels greater than 1000 U/ml, the marker's specificity approaches 100%. Acute cholangitis and cirrhosis are two benign conditions that might raise this assay significantly. This tumor-associated marker is also helpful in predicting unresectability of pancreatic adenocarcinoma, as 96% of tumors that result in blood levels greater than 1000 U/ml have been found to be unresectable. After potentially curative surgery, the CA 19-9 can help prognosticate survival. Patients who normalize their CA 19-9 postoperatively live longer than those who do not. Furthermore, the assay, when used serially, predicts recurrence of disease prior to radiographic or clinical findings. The CA 19-9 is currently the "gold" standard marker for pancreatic cancer, against which other assays in this field will be judged.

Distribution, release, and secretory activity of epidermal growth factor in the pancreas.

This study was designed to determine the distribution of immunoreactive epidermal growth factor (EGF) in the gastrointestinal tract and the action of this peptide on pancreatic secretion in vivo and in vitro. Immunoreactive EGF was found in large amounts in the salivary glands and the pancreas and in the pancreatic juice. EGF infused subcutaneously (50 micrograms/kg-h) in conscious rats with intact or removed salivary glands stimulated pancreatic protein secretion after 4 h of peptide infusion; this effect was completely prevented by the pretreatment with DL-difluoromethyl-ornithine (DFMO) (200 mg/kg), an irreversible inhibitor of activity of ornithine decarboxylase (ODC), a key enzyme in polyamine synthesis. EGF added to the incubation medium in concentrations ranging from 10(-10)-10(-6) M increased, in a concentration-dependent manner both unstimulated and stimulated by caeruelin or urecholine, amylase release from dispersed pancreatic acini obtained from rats pretreated in 3 h with EGF in a dose of 50 micrograms/kg-h. Spermine given at concentrations ranging from 10(-12)-10(-6) M to the freshly prepared rat pancreatic acini also increased amylase release in a concentration-related manner. DFMO injected in a single dose (200 mg/kg), before the infusion of EGF to the rats, completely abolished the stimulatory effect of EGF on amylase release, but failed to affect that of spermine. This study shows that 1. EGF is present in large amounts in pancreatic tissue and pancreatic juice. 2. EGF stimulates pancreatic secretion in vivo and amylase release in vitro from isolated rat pancreatic acini. 3. The activation of ODC and polyamine biosynthesis in acinar cells plays an important role in EGF-induced stimulation of pancreatic secretion.

Origin and characterization of immunoreactive somatostatin in rat pure pancreatic juice.

This study was undertaken to determine the origin and forms of immunoreactive somatostatin (SLI) present in the pancreatic juice of cannulated nonanesthetized rats. The systemic origin of somatostatin (SS) was assessed from bolus intravenous injections of either I125-Tyr1-SS-14 or synthetic SS-14 under basal and stimulated pancreatic juice secretion, and from measurements of radioactivity and SS-14 in the collected juice. Gel filtration of pooled juice samples indicated that there were no forms of SS corresponding to standards of I125-Tyr1-SS-14 or SS-14, but radioactive material eluted in the area of either I125-Tyr or free iodine. Investigation of the presence of SS-14 in the juice by radioimmunoassay (RIA) led to the identification of a 23-kDa SS-like immunoreactive protein that did not correspond to any of the three known forms of SS present in the pancreatic tissue (Pro-SS, SS-28, and SS-14). By boiling the juice before the RIA, this 23-kDa protein was no longer detectable by RIA. These data suggest that this protein may be a "putative" enzyme, degrading the SS tracer in the RIA. This possibility is supported by the observation that boiled juice lost this capacity of interfering with the RIA. In conclusion, SS in the pancreatic juice, if present, is not from systemic origin and the juice contains an active thermosensitive substance degrading the SS tracer in the RIA.

[Enzymatic determination of glandular kallikrein in human pancreatic secretions]. / Enzymatische Bestimmung von glandulärem Kallikrein im menschlichen Pankreassekret.

A method for the enzymatic determination of tissue kallikrein (EC 3.4. 21.8) in human pancreatic juice using D-Val-cyclohexyl-Ala-Arg-4-nitroaniline as substrate as well as the reaction conditions optimized for 37 degrees C are described. The reaction is characterized by the following kinetic parameters, determined according to Hanes and Eisenthal, Cornish-Bowden: KM = 2.10(-5) mol/l; Vmax = 0.800 mumol/(l.s). The coefficient of intra-assay variation (N = 10) was determined to 2.54%. The detection limit of the assay was found to be 8.63 nmol/(l.s).

Effect of short-chain fatty acids on pancreatic exocrine secretion in calves aged 2 weeks and 13 weeks.

Effects of short-chain fatty acids on pancreatic exocrine secretion were studied under anesthesia in calves within 2 weeks of age (2-wks calves) given only whole milk and milk replacement and in which rumen fermentation has not begun yet, and in calves at 13 weeks of age (13-wks calves) weaned at 40 days of age and in which rumen fermentation has already begun. Basal rate of juice flow and protein concentration and amylase activity in pancreatic juice under basal condition were significantly lower in the 2-wks calves than those in the 13-wks calves. Intravenous administrations of acetate, propionate and butyrate stimulated pancreatic juice secretion and protein and amylase output in both groups of calves. Those responses were increased with increasing carbon number in the molecule of fatty acids. Although the response of amylase output (/kg of body weight) in the 2-wks calves was significantly less than that in the 13-wks calves, the response of juice flow and protein output (/kg of body weight) in the 2-wks calves were equivalent to or greater than those in the 13-wks calves. These results indicate that the characteristic of pancreas, being stimulated by short-chain fatty acids, in calves and probably in other ruminants is not generated on the process of postnatal development, but has been already acquired before rumen fermentation begins.

Purification and molecular characterization of FAP, a feto-acinar protein associated with the differentiation of human pancreas.

This work describes the purification of FAP, a feto-acinar pancreatic protein associated with the ontogenesis, differentiation, and transformation of the human exocrine pancreas. The protein was purified to homogeneity from pancreatic juices of patients with pancreatic pathology by a two-step chromatographic procedure which consisted of size exclusion on Sephacryl S-200 and affinity on heparin-Sepharose. The final preparation gave a single band at Mr 110,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after Coomassie stain or autoradiography of the 125I-labeled protein. Immunodetection with the murine monoclonal antibody mAb J28 in nitrocellulose replicas demonstrated a main Mr 110,000 component and trace components in the Mr 100,000-80,000 range. The immunopattern was identical to that in the original crude pancreatic secretion, therefore showing that the molecular characteristics of the protein, i.e. molecular mass, microheterogeneity, and immunoreactivity, were not altered along the purification procedure. FAP was identified as an acidic protein (isoelectric point 4.2-4.8) consisting of a single polypeptide chain having no free SH residues. Analysis of the amino acid composition showed a high proline content. Twenty-two residues of the N-terminal sequence were determined. No significant homology between this peptide and other proteins was found following a search of the NBRF-18 data bank. Sugar analysis showed the presence of mannose which is consistent with N-linked carbohydrate chains and an unusual high ratio in N-acetylgalactosamine residues suggesting the presence of many O-linked carbohydrate chains. Sequential deglycosylation with neuraminidase, hexosaminidase, and O-glycanase yield a single Mr 58,000 peptide showing that, relative to a molecular mass of 110,000, the carbohydrate moiety of FAP accounts for at least 47% of its apparent Mr by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

[An experimental study of the action of 5-fluorouracil on the function and cytomorphology of the exocrine pancreas]. / Studiu experimental privind actiunea 5-fluorouracilului asupra functiei si citomorfologiei pancreasului exocrin.

The authors have taken up previous studies by Martin (1969), and by Johnson (1973), and carried out an experimental study on white rats aimed at evaluating the morpho-functional changes of cells from the exocrine pancreas under the influence of 5-fluorouracil (Ftorafur), a cytostatic drug which is a general inhibitor of protein synthesis by cells. Ftorafur was injected in amounts of 2.3 mg/100 g of body weight, and a significant reduction was noted in the secretion of bicarbonates, amylase and lipase by the pancreas of the animals. Cytologic changes were also noted in the pancreatic tissue of these animals, indicating, on the one hand, a deficient protein and enzyme synthesis by the pancreatic cells, and a blocking of the mechanism of discharge of zymogen granules, on the other hand. The most intensive morpho-functional changes were noted following repeated administration of Ftorafur, probably due to the cumulative effects of this substance at the level of secretory pancreatic cells. The authors consider that 5-fluorouracil inhibits to a considerable degree the synthesis of pancreatic enzymes, and as such it can be used as a therapeutic means for the reduction of the external secretion of the pancreas.

Measurement of proinsulin in human pancreatic juice.

In this study, we measured proinsulin in human pancreatic juice by radioimmunoassay (RIA). Affinity chromatography was used to separate proinsulin and insulin from C-peptide; and high-performance liquid chromatography, to separate proinsulin from insulin. In the RIA procedure for proinsulin we used porcine insulin antiserum as the antibody and porcine proinsulin as the standard and labelled antigen. The concentrations of proinsulin in human pancreatic juice obtained 5, 10, 15 and 20 minutes after intravenous injection of secretin were 29 +/- 6, 41 +/- 4, 35 +/- 14 and 37 +/- 17 (pmol/l +/- SD, means of 7 subjects), respectively. These values were higher than those in serum from fasted subjects, 12 +/- 4 pmol +/- SD. This work shows that proinsulin is present in human pancreatic juice.

Pancreatic pseudocyst fluid--a mixture of plasma proteins and pancreatic juice possessing a high proteolytic activity.

Pancreatic pseudocyst fluid from eight patients was examined biochemically. The fluid was found to be a mixture of plasma proteins and pancreatic juice, possessing a high proteolytic activity against high- as well as low-molecular-weight proteins. The proteolytic activity was found to be trypsin-, kallikrein- and plasmin-like. Gel filtration studies showed proteolytic activity to be present corresponding to alpha-2-macroglobulin-bound proteases and also to free proteases. Quantitative immunochemical levels were about 30-100% of normal plasma levels for alpha-2-macroglobulin, C1 inhibitor, antithrombin III and alpha-2-antiplasmin. However, there was practically no functional inhibitory capacity left in the pseudocyst fluid, except for alpha-1-protease inhibitor, which retained its inhibitory capacity. Neither native kininogen nor complement factor C3 was found: this was probably a result of the proteolytic activity. It is concluded, that a continuing proteolytic activity within the pseudocyst, although decreasing with aging of the cyst, could explain symptoms and complications caused by the pseudocyst.

Secretory pancreatic stone protein messenger RNA. Nucleotide sequence and expression in chronic calcifying pancreatitis.

The pancreatic stone protein and its secretory form (PSP-S) are inhibitors of CaCO3 crystal growth, possibly involved in the stabilization of pancreatic juice. We have established the structure of PSP-S mRNA and monitored its expression in chronic calcifying pancreatitis (CCP). A cDNA encoding pre-PSP-S has been cloned from a human pancreatic cDNA library. Its nucleotide sequence revealed that it comprised all but the 5' end of PSP-S mRNA, which was obtained by sequencing the first exon of the PSP-S gene. The complete mRNA sequence is 775 nucleotides long, including 5'- and 3'- noncoding regions of 80 and 197 nucleotides, respectively, attached to a poly(A) tail of approximately 125 nucleotides. It encodes a preprotein of 166 amino acids, including a prepeptide of 22 amino acids. No overall sequence homology was found between PSP-S and other pancreatic proteins. Some homology with several serine proteases was observed in the COOH-terminal region, however. The mRNA levels of PSP-S, trypsinogen, chymotrypsinogen, and colipase in CCP and control pancreas were compared. PSP-S mRNA was three times lower in CCP than in control, whereas the others were not altered. It was concluded that PSP-S gene expression is specifically reduced in CCP patients.

Substitution of milk protein with isolated soy protein in calf milk replacers.

The influence of replacement of milk protein by isolated soy protein on digestion and pancreatic enzyme secretion was determined in nine Holstein male calves. Calves (average weight 47 kg) were fitted with permanent re-entrant pancreatic and a T-type cannula in the distal ileum at 6 to 10 d of age. Following a 2-wk recuperation period, the calves were fed three milk replacers in a triplicated 3 x 3 latin square. Experimental diets consisted of a control, in which 100% of the CP originated from spray-dried skim milk powder (SM), and the test diets, in which 50% (SM/ISP) or 100% (ISP) of the skim milk protein was replaced by isolated soy protein. Each experimental period lasted 2 wk. Replacement of SM protein by ISP decreased (P less than .05) the digestibilities of protein and most amino acids. Ileal digestibilities of total indispensable amino acids for SM, SM/ISP and ISP diets were 82.1, 75.8 and 61.8%, respectively, and total tract digestibilities of total indispensable amino acids were 90.0, 82.6 and 74.0%, respectively. Including ISP did not affect (P greater than .05) the volume of secretion of pancreatic juice, protein or chymotrypsin; however, the secretion of trypsin decreased (P less than .05). Reduction in trypsin secretion may be responsible, in part, for the lower amino acid digestibilities in milk replacers containing isolated soy protein.

The diagnostic significance of carbohydrate antigen CA 19-9 in serum and pancreatic juice in pancreatic carcinoma.

CA 19-9 is a carbohydrate antigen isolated from human colon carcinoma cell line, and is reportedly a tumor marker for pancreatic carcinoma. In this study we determined serum CA 19-9 in 71 normal subjects, 103 patients with benign digestive diseases, 85 patients with periampullary cancers, and 160 patients with other digestive cancers. Serum CA 19-9 was elevated only in 2.3% of normals and benign digestive disease patients, whereas it was increased in 72.7%, 86.4%, and 89.5% of pancreatic, ampullary, and choledochal carcinoma patients, respectively. Of other digestive cancer patients, it was elevated in 23.8%. In addition, very high serum CA 19-9 (greater than 120 u/m) was more often observed in patients with pancreatic, ampullary, and biliary cancer patients than in GL cancer patients (54.1% vs 9.4%, p less than 0.001). In 18 normal subjects and 68 patients with benign and malignant diseases, it was found that CA 19-9 content in the pancreatic juice was significantly increased in pancreatic, ampullary, and choledochal cancer patients, whereas in chronic pancreatitis patients it was normal, indicating that it is a specific and valuable tumor marker in differential diagnosis of pancreatic cancer.

Effect of octreotide acetate on pancreatic exocrine function.

Somatostatin and its analogs have been shown to inhibit both pancreatic endocrine and exocrine function. We hypothesized that octreotide acetate (Sandostatin), a somatostatin analog, decreases the pancreatic flow rate through a peptide-mediated mechanism and alters pancreatic fluid composition by inhibiting carbonic anhydrase action and circulating peptide levels. To test this hypothesis, we collected pancreatic fluid from six patients (four with pancreatic fistulas and two with pancreatic drains after pancreatic resection). Pancreatic fluid volume and chloride, sodium, potassium, amylase, lipase, and bicarbonate levels were measured before and after octreotide acetate therapy. Octreotide acetate reduced pancreatic fluid output by a mean of 75 percent (p less than 0.05), increased chloride concentration by 21 percent (p less than 0.05), and reduced bicarbonate content by 45 percent (p less than 0.05). Sodium levels were unchanged, but the potassium concentration was increased by 14 percent (p less than 0.05). Total amylase and lipase production per 24 hours was decreased by 63 percent and 27 percent, respectively (differences not significant). Somatostatin may be useful in the treatment of established pancreatic fistulas and may be a useful prophylactic tool to prevent postoperative fistula formation.

Inhibition of the stimulated canine exocrine pancreas by amino acids and fat.

To investigate the possible exocrine pancreatic inhibitory actions of amino acids and fat, pancreatic fistula outputs and plasma concentrations of glucagon, somatostatin, and pancreatic polypeptide were measured for response to intravenous (IV) and intraduodenal nutrient administration in six dogs submaximally stimulated with cholecystokinin. Intravenous amino acids caused abrupt and significant 45% to 73% reductions in stimulated pancreatic protein, bicarbonate, and volume outputs. There were no significant associated changes in plasma hormone concentrations and no similar immediate pancreatic inhibition with IV mannitol, thus suggesting a possible direct inhibitory effect of amino acids. Intraduodenal amino acids and IV fat evoked no significant pancreatic output suppression. Intraduodenal fat rapidly caused a significant 40% to 62% reductions in stimulated outputs that were associated with an 81% rise in plasma pancreatic polypeptide concentration, suggesting a gut-mediated inhibition. We conclude that IV amino acids and intraduodenal fat both inhibited stimulated pancreatic secretion but probably by different mechanisms.