Human Pancreatic Juice Research Articles

Development of mutant human immunoreactive trypsinogen 1 (IRT1) and mutant human immunoreactive trypsinogen 2 (IRT2) for use in immunoassays

Immunoreactive Trypsinogen (IRT) is a protein-based pancreatic proenzyme that has an important role in protein digestion in humans. In human body, once IRT present in the small intestine, the proteolytic cleavage activates trypsinogen into trypsin. When IRT is in the active form, it is capable to cleave antibodies, other proteins and even itself while it is desired to use in immunoassays. According to the literature, there are three important IRT isoforms called Immunoreactive Trypsinogen 1 (IRT1), Immunoreactive Trypsinogen 2 (IRT2), and Immunoreactive Trypsinogen 3 (IRT3). However, trypsinogen 1 (cationic trypsinogen, IRT1) and trypsinogen 2 (anionic trypsinogen, IRT2) are the major isoforms in human pancreatic juice and used in the diagnosis of cystic fibrosis (CF). In this study, it is aimed to restrain its proteolytic activity with K23D mutation, which changes lysine (K) residue at the 23rd position to aspartic acid (D). Because we wanted to produce a hassle-free human recombinant immune reactive trypsinogen proenzyme which has similar antigenic properties with the native form. It is also aimed that the mutant IRTs do not exhibit proteolytic activity for the development of durable detection kits with a longer shelf life for both two isoforms. The innovation was actualized in order to use IRTs as a standard antigen in Immunoassays such as ELISA kits. The gene was synthesized as mutated and expressed in P. pastoris X-33 strain. The loss of proteolytic activity has been proven with the BAEE test. Antigenic properties of K23D IRTs and the effect of proteolytic inactivation on their performance in immunoassays were assessed with ELISA and Western Blot. In ELISA results K23D mutated IRTs showed higher signals than Wild-Type forms.

Characterization of all the lipolytic activities in pancreatin and comparison with porcine and human pancreatic juices

Porcine pancreatic extracts (PPE), also named pancreatin, are commonly used as a global source of pancreatic enzymes for enzyme replacement therapy in patients with exocrine pancreatic insufficiency. They are considered as a good substitute of human pancreatic enzymes and they have become a material of choice for in vitro models of digestion. Nevertheless, while the global PPE contents in lipase, protease and amylase activities are well characterized, little is known about individual enzymes. Here we characterized the lipase, phospholipase, cholesterol esterase and galactolipase activities of PPE and compared them with those of porcine (PPJ) and human (HPJ) pancreatic juices. The phospholipase to lipase activity ratio was similar in PPJ and HPJ, but was 4-fold lower in PPE. The galactolipase and cholesterol esterase activities were found at lower levels in PPJ compared to HPJ, and they were further reduced in PPE. The enzymes known to display these activities in HPJ, pancreatic lipase-related protein 2 (PLRP2) and carboxylester hydrolase/bile salt-stimulated lipase (CEH/BSSL), were identified in PPJ using gel filtration experiments, SDS-PAGE and LC-MS/MS analysis. The galactolipase and cholesterol esterase activities of PPE indicated that PLRP2 and CEH/BSSL are still present at low levels in this enzyme preparation, but they were not detected by mass spectrometry. Besides differences between porcine and human enzymes, the lower levels of phospholipase, galactolipase and cholesterol esterase activities in PPE are probably due to some proteolysis occurring during the production process. In conclusion, PPE do not provide a full substitution of the lipolytic enzymes present in HPJ.

Clinical Significance of Plasma Apolipoprotein-AII Isoforms as a Marker of Pancreatic Exocrine Disorder for Patients with Pancreatic Adenocarcinoma Undergoing Chemoradiotherapy, Paying Attention to Pancreatic Morphological Changes.

Background Circulating apolipoprotein-AII (apoAII-) ATQ/AT is a potential useful biomarker for early stage pancreatic ductal adenocarcinoma (PDAC), but its clinical significance in PDAC patients remains uncertain. The aim of the current study was to assess the usefulness of apoAII-ATQ/AT as a surrogate for the effect of chemoradiotherapy (CRT) and its association with pancreatic exocrine disorder, paying attention to morphological changes of the pancreas. Methods In the 264 PDAC patients who were enrolled in our CRT protocol, the following parameters were measured at specified time points before and after CRT: serum levels of albumin, total cholesterol, and amylase as indices of pancreatic exocrine function, serum levels of CA19-9, and the pancreatic morphology including tumor size (TS), main pancreatic duct diameter (MPDD), and pancreatic parenchymal volume excluding tumor volume (PPV) by using computed tomography (CT) images. Plasma apoAII-ATQ/AT levels were simultaneously measured with enzyme-linked immunosorbent assay in 4 healthy volunteers and the 44 PDAC patients before and after CRT. Plasma apoAII-ATQ/AT levels after CRT were analyzed according to small/large-MPDD and small/large-PPV groups based on their median values after CRT. Plasma samples after CRT were measured after incubation with human pancreatic juice (PJ) to examine the relevance between apoAII isoforms and circulating pancreatic enzymes. Results The serum levels of albumin, amylase, CA19-9, TS, MPDD, and PPV after CRT were significantly lower than those before CRT (median, before vs. after: 3.9 g/dl, 74 U/l, 180.2 U/ml, 58.1 mm, 4.0 mm, and 34.8 ml vs. 3.8, 59, 43.5, 55.6, 3.6, and 25.2). ApoAII-ATQ/AT levels (median, μg/ml) of PDAC patients before CRT were significantly lower than those in healthy volunteers: 32.9 vs. 61.2, and unexpectedly those after CRT significantly decreased: 14.7. The reduction rate of apoAII-ATQ/AT was not correlated with those of CA19-9 and TS, indicating that apoAII-ATQ/AT is not a tumor-specific marker. On the other hand, the patient group with large MPDD and small PV exhibited higher apoAII-ATQ levels than those with small MPDD and large PPV. The incubation of plasma samples after CRT with PJ did not alter apoAII-ATQ/AT and apoAII-AT levels but significantly decreased apoAII-ATQ levels, suggesting that circulating pancreatic enzymes markedly influenced apoAII-ATQ levels. Conclusions ApoAII-ATQ/AT levels are not useful for evaluation of clinical effect of CRT for PDAC, but apoAII isoforms are very useful to assess pancreatic exocrine disorder because pancreatic atrophy and insufficient secretion of circulating pancreatic enzymes are considered likely to influence apoAII-ATQ levels.

In vitro digestion of galactolipids from chloroplast-rich fraction (CRF) of postharvest, pea vine field residue (haulm) and spinach leaves.

The removal of intact chloroplasts from their cell wall confinement offers a novel way to obtain lipophilic nutrients from green biomass, especially carotenoids and galactolipids. These latter are the main membrane lipids in plants and they represent a major source of the essential α-linolenic acid (18:3; ALA). Nevertheless, knowledge on their digestion is still limited. We have developed a physical method of recovering a chloroplast-rich fraction (CRF) from green biomass and tested its digestibility in vitro under simulated gastrointestinal conditions. Using a two-step static model, CRF from both spinach leaves and postharvest, pea vine field residue (haulm) were first exposed to enzymes from rabbit gastric extracts and then either to pancreatic enzymes from human pancreatic juice (HPJ) or to porcine pancreatic extracts (PPE). The lipolysis of monogalactosyldiacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) was monitored by thin layer chromatography and gas chromatography of fatty acid methyl esters. For both CRF preparations, MGDG and DGDG were converted to monogalactosylmonoacylglycerol (MGMG) and digalactosylmonoacylglycerol (DGMG), respectively, during the intestinal phase and ALA was the main fatty acid released. Galactolipids were more effectively hydrolysed by HPJ than by PPE, and PPE showed a higher activity on MGDG than on DGDG. These findings may be explained by the higher levels of galactolipase activity in HPJ compared to PPE, which mainly results from pancreatic lipase-related protein 2. Thus, we showed that CRF galactolipids are well digested by pancreatic enzymes and represent an interesting vehicle for ALA supplementation in human diet.

The mucinous domain of pancreatic carboxyl-ester lipase (CEL) contains core 1/core 2 O-glycans that can be modified by ABO blood group determinants

Carboxyl-ester lipase (CEL) is a pancreatic fat-digesting enzyme associated with human disease. Rare mutations in the CEL gene cause a syndrome of pancreatic exocrine and endocrine dysfunction denoted MODY8, whereas a recombined CEL allele increases the risk for chronic pancreatitis. Moreover, CEL has been linked to pancreatic ductal adenocarcinoma (PDAC) through a postulated oncofetal CEL variant termed feto-acinar pancreatic protein (FAPP). The monoclonal antibody mAb16D10 was previously reported to detect a glycotope in the highly O-glycosylated, mucin-like C terminus of CEL/FAPP. We here assessed the expression of human CEL in malignant pancreatic lesions and cell lines. CEL was not detectably expressed in neoplastic cells, implying that FAPP is unlikely to be a glycoisoform of CEL in pancreatic cancer. Testing of the mAb16D10 antibody in glycan microarrays then demonstrated that it recognized structures containing terminal GalNAc-α1,3(Fuc-α1,2)Gal (blood group A antigen) and also repeated protein sequences containing GalNAc residues linked to Ser/Thr (Tn antigen), findings that were supported by immunostainings of human pancreatic tissue. To examine whether the CEL glycoprotein might be modified by blood group antigens, we used high-sensitivity MALDI-TOF MS to characterize the released O-glycan pool of CEL immunoprecipitated from human pancreatic juice. We found that the O-glycome of CEL consisted mainly of core 1/core 2 structures with a composition depending on the subject's FUT2 and ABO gene polymorphisms. Thus, among digestive enzymes secreted by the pancreas, CEL is a glycoprotein with some unique characteristics, supporting the view that it could serve additional biological functions to its cholesteryl esterase activity in the duodenum.

Detection and proteomic characterization of extracellular vesicles in human pancreatic juice

AimsThe prognosis of patients with pancreatic cancer has remained virtually unchanged with a high mortality rate compared to other types of cancers. An earlier detection would provide a time window of opportunity for treatment and prevention of deaths.In the present study we investigated extracellular vesicle (EV)-associated potential biomarkers for pancreatic cancer by directly assessing EV size-based subpopulations in pancreatic juice samples of patients with chronic pancreatitis or pancreatic cancer. In addition, we also studied blood plasma and pancreatic cancer cell line-derived EVs. MethodsComparative proteomic analysis was performed of 102 EV preparations from human pancreatic juices, blood, and pancreatic cancer cell lines Capan-1 and MIA PaCa-2. EV preparations were also characterized by electron microscopy, tunable resistive pulse sensing, and flow cytometry. ResultsHere we describe the presence of EVs in human pancreatic juice samples. Pancreatic juice EV-associated proteins that we identified as possible candidate markers for pancreatic cancer included mucins, such as MUC1, MUC4, MUC5AC, MUC6 and MUC16, CFTR, and MDR1 proteins. These candidate biomarkers could also be detected by flow cytometry in EVs found in pancreatic juice and those secreted by pancreatic cancer cell lines. ConclusionsTogether our data show that detection and characterization of EVs directly in pancreatic juice is feasible and may prove to be a valuable source of potential biomarkers of pancreatic cancer.

Potential Prebiotic Oligosaccharide Mixtures from Acidic Hydrolysis of Rice Bran and Cassava Pulp.

Two agricultural wastes, rice bran and cassava pulp were subjected to acidic hydrolysis by 2M sulfuric acid which resulted in hemicellulosic oligosaccharide mixtures. Monosaccharide component analysis of these mixtures revealed that the oligosaccharides of rice bran acid hydrolysate (RAHF) composed of glucose and arabinose while cassava pulp acid hydrolysate (CAHF) was found to be comprised of glucose, galactose and arabinose. Both RAHF and CAHF were able to fuel all of the tested threeLactobacillus, five Bifidobacterium and three Bacteroides strains indicating the prebiotic potential of these oligosaccharide mixtures. Moreover, Lb. gasseri grew significantly better on RAHF than on inulin, a benchmark prebiotic oligo- and polysaccharide mixture. When the digestibility of RAHF and CAHF were tested it was found that these oligosaccharide mixtures were only slightly hydrolyzed upon exposure to simulated human gastric (by less than 8%) and pancreatic juices (by less than 3%). Additionally, most sensory attributes of the above obtained oligosaccharide mixtures supplemented two model cereal drink formulations were generally not different from those of the control, while the overall acceptance was not affected significantly in one cereal drink formulation.

Slowing down fat digestion and absorption by an oxadiazolone inhibitor targeting selectively gastric lipolysis

Based on a previous study and in silico molecular docking experiments, we have designed and synthesized a new series of ten 5-Alkoxy-N-3-(3-PhenoxyPhenyl)-1,3,4-Oxadiazol-2(3H)-one derivatives (RmPPOX). These molecules were further evaluated as selective and potent inhibitors of mammalian digestive lipases: purified dog gastric lipase (DGL) and guinea pig pancreatic lipase related protein 2 (GPLRP2), as well as porcine (PPL) and human (HPL) pancreatic lipases contained in porcine pancreatic extracts (PPE) and human pancreatic juices (HPJ), respectively. These compounds were found to strongly discriminate classical pancreatic lipases (poorly inhibited) from gastric lipase (fully inhibited). Among them, the 5-(2-(Benzyloxy)ethoxy)-3-(3-PhenoxyPhenyl)-1,3,4-Oxadiazol-2(3H)-one (BemPPOX) was identified as the most potent inhibitor of DGL, even more active than the FDA-approved drug Orlistat. BemPPOX and Orlistat were further compared in vitro in the course of test meal digestion, and in vivo with a mesenteric lymph duct cannulated rat model to evaluate their respective impacts on fat absorption. While Orlistat inhibited both gastric and duodenal lipolysis and drastically reduced fat absorption in rats, BemPPOX showed a specific action on gastric lipolysis that slowed down the overall lipolysis process and led to a subsequent reduction of around 55% of the intestinal absorption of fatty acids compared to controls. All these data promote BemPPOX as a potent candidate to efficiently regulate the gastrointestinal lipolysis, and to investigate its link with satiety mechanisms and therefore develop new strategies to “fight against obesity”.

Specificity of a Polyclonal Fecal Elastase ELISA for CELA3.

IntroductionElastase is a proteolytic pancreatic enzyme that passes through the gastrointestinal tract undergoing only limited degradation. ELISA tests to determine stool elastase concentrations have therefore been developed for the diagnosis of exocrine pancreatic insufficiency. Five different isoforms of pancreatic elastase (CELA1, CELA2A, CELA2B, CELA3A, CELA3B) are encoded in the human genome. We have investigated three different polyclonal antisera that are used in a commercial fecal elastase ELISA to determine their specificity for different pancreatic elastase isoforms.Material and MethodsDifferent polyclonal rabbit antisera against human elastase peptides (BIOSERV Diagnostics GmbH, Germany) were tested by Western blot analysis of human pancreatic juice, in HEK-293 cells expressing Elastase constructs, and in the protein content of porcine pancreatin, used for treatment of exocrine pancreatic insufficiency.ResultsIn human pancreatic juice the polyclonal antisera detected proteins at the corresponding size of human pancreatic elastase isoforms (~29kDa). Transiently expressed GFP fusion protein of elastase isoform CELA3A (CELA3A-GFP), but not CELA2A (CELA2A-GFP) could be precipitated from HEK-293 cell lysates with the elastase antisera. We detected no cross-reactivity with elastases in the porcine pancreatic extracts (pancreatin) used for enzyme replacement therapy.ConclusionThe polyclonal antisera used in a commercial fecal elastase ELISA are specific for the human pancreatic elastase isoform CELA3 and do not cross-react with elastase contained in pig pancreatin. While pancreatic elastase 1 (CELA1) is not expressed in the adult human pancreas, possible differences between the other isoforms regarding their cellular expression, pathophysiological role and relevance in exocrine pancreatic insufficiency deserve further investigation.

Combined derivatization and high-performance liquid chromatography with fluorescence and ultraviolet detection for simultaneous analysis of octreotide and gabexate mesylate metabolite in human pancreatic juice samples.

A simple and sensitive method based on the combination of derivatization and high-performance liquid chromatography with ultraviolet and fluorimetric detection was developed for the simultaneous determination of octreotide and gabexate mesylate metabolite in human pancreatic juice samples. Parameters of the derivatization procedure affecting extraction efficiency were optimized. The developed method was validated according to the International Conference on Harmonization guidelines. The calibration curves were linear over a range of 0.1-15 µg/mL for octreotide and 0.20-15 µg/mL for gabexate mesylate metabolite. Derivatized products of octreotide and gabexate mesylate metabolite were separated on a Luna C18 column (4.6 × 250 mm; 5 µm particle size) using a gradient with a run time of 36 min, without further purification. The limits of detection were 0.025 and 0.05, respectively, for octreotide and gabexate mesylate metabolite. This paper reports the validation of a quantitative high performance liquid chromatography-photodiode array-fluorescence (HPLC-PDA-FL) method for the simultaneous analysis of octreotide and gabexate mesylate metabolite in pancreatic juice by protein precipitation using zinc sulfate-methanol-acetonitrile containing the derivatizing reagent, 4-fluoro-7-nitro-[2,1,3]-benzoxadiazole (NBD-F). Derivatized products of octreotide and gabexate mesylate metabolite were separated on a Luna C18 column (4.6 × 250 mm; 5 µm particle size) using a gradient with a run time of 36 min, without further purification. The method was validated over the concentration ranges 0.1-15 and 0.2-15 µg/mL for octreotide and gabexate mesylate metabolite, respectively, in human pancreatic juice. Biphalin and methyl-p-hydroxybenzoate were used as the internal standards. This method was successfully utilized to support clinical studies in humans. The results from assay validations show that the method is selective, sensitive and robust. The limit of quantification of the method was 0.1 µg/mL for octreotide and 0.2 µg/mL for gabexate mesylate metabolite, and matrix matched standard curves showed a good linearity up to 15 µg/mL. In the entire analytical range the intra- and inter-day precision (RSD%) values were respectively ≤5.9% and ≤3.1% for octreotide and ≤2.0% and ≤3.9% for gabexate mesylate metabolite. For both analytes the intra- and inter-day accuracy (bias) values ranged respectively from -6.8 to -2.5% and from -4.6 to -5.7%. This method utilizes derivatization with NBD-F and provides adequate sensitivity for both drugs.

Abstract 1897: Application of a novel DNA methylation analysis method (MSE) for mucin expression in pancreatic juices for diagnosis of pancreatic neoplasms

Abstract Mucins play crucial roles in diagnostic and prognostic prediction and in carcinogenesis and tumor invasion. In our clinicopathological investigation for mucin expression in human pancreatic ductal adenocarcinomas (PDACs) and intraductal papillary mucinous neoplasms (IPMNs), we had reported that MUC1 (pan-epithelial membrane mucin) or MUC4 (tracheobronchial mambrane mucin) expression is related with the aggressive behavior and a poor outcome of the patients, whereas MUC2 (intestinal secretory mucin) expression tends to be related with the indolent behavior and a favorable outcome of the patients. We investigated the mechanism of mucin genes expression from the DNA methylation standpoint in human cancer cell lines using the mapping of methylated cytosines by the quantitative MassARRAY analysis, followed by the confirmation with MSP analysis and a novel DNA methylation analysis method ‘methylation specific electrophoresis (MSE, international patent open: WO 2011/132798)’, and compared the results with expression levels of mucin core protein and mRNA in the cancer cells. The results obtained showed that MUC1, MUC2 and MUC4 expression is regulated by DNA methylation at their proximal 5′ flanking regions in the cancer cells. The MSE method greatly decreases the amount of input DNA, although conventional analytical methods for DNA methylation require a large amount of DNA and have low sensitivity. The lower detection limit for distinguishing different methylation status is under 0.1% and the detectable minimum amount of DNA is 20 pg, which can be obtained from only a few cells. We applied the MSE method to human pancreatic juices from 45 patients with various pancreatic lesions. The analyses for DNA methylation status of MUC1, MUC2 and MUC4 combined with image data were useful for differential diagnosis of various human pancreatic neoplasms. The examination of 45 samples of human pancreatic juices by the MSE method combined with the image examination showed the following specificity and sensitivity by quadratic discrimination analysis: PDAC, 87% and 80%; gastric-type IPMN (safe type), 88% and 77%; intestinal-type IPMN (dangerous type), 100% and 88%; respectively. The MSE analysis of human pancreatic juices may give us very useful informations for the selection of the treatment methods for pancreatic neoplasms. Citation Format: Suguru Yonezawa, Seiya Yokoyama, Michiyo Higashi. Application of a novel DNA methylation analysis method (MSE) for mucin expression in pancreatic juices for diagnosis of pancreatic neoplasms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1897. doi:10.1158/1538-7445.AM2014-1897

Diagnosis of pancreatic neoplasms using a novel method of DNA methylation analysis of mucin expression in pancreatic juice.

Mucins (MUC) play crucial roles in carcinogenesis and tumor invasion in pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasms (IPMNs). Our immunohistochemistry (IHC) studies have shown a consensus position on mucin expression profiles in pancreatic neoplasms as follows: MUC1-positive but MUC2-negative expression in PDACs; MUC1-negative but MUC2-positive expression in intestinal-type IPMNs (dangerous type); MUC1-negative and MUC2-negative expression in gastric-type IPMNs (safe type); High MUC4 expression in PDAC patients with a poor outcome; and MUC4-positive expression in intestinal-type IPMNs. We also showed that three mucin genes (MUC1, MUC2 and MUC4) expression in cancer cell line was regulated by DNA methylation. We have developed a novel ‘methylation-specific electrophoresis (MSE)’ method to analyze the DNA methylation status of mucin genes by high sensitivity and resolution. By using the MSE method, we evaluated pancreatic juice samples from 45 patients with various pancreatic lesions. The results were compared with final diagnosis of the pancreatic lesions including IHC of mucin expression in the paired pancreatic tissues. The results indicated that the DNA methylation status of MUC1, MUC2 and MUC4 in pancreatic juice matched with the mucin expression in tissue. Analyses of the DNA methylation status of MUC1, MUC2 and MUC4 were useful for differential diagnosis of human pancreatic neoplasms, with specificity and sensitivity of 87% and 80% for PDAC; 100% and 88% for intestinal-type IPMN; and 88% and 77% for gastric-type IPMN, respectively. In conclusion, MSE analysis of human pancreatic juice may provide useful information for selection of treatment for pancreatic neoplasms.

Clinical pharmacokinetics of meropenem in pancreatic juice and site-specific pharmacodynamic target attainment against Gram-negative bacteria: Dosing considerations

Background and objectiveVery few studies have revealed the dynamics of meropenem penetration into the pancreas or pancreatic juice in humans. This study of the clinical pharmacokinetics and pharmacodynamics of meropenem in human pancreatic juice was performed to establish a basis for the validation of dosing regimens for pancreatic infections. MethodsTen patients with endoscopic naso-pancreatic drainage received 500 mg meropenem over 0.5 h via intravenous infusion. Venous blood and pancreatic juice samples were collected post-infusion for up to 5.0 h and used to obtain measures of meropenem concentration. The probability of attaining the pharmacodynamic target (40% of the time above the minimum inhibitory concentration [MIC]) in pancreatic juice against MIC distributions for clinical isolates of Gram-negative bacteria was determined using a Monte Carlo simulation. ResultsThe mean maximum concentration of meropenem in pancreatic juice was 2.08 ± 0.94 μg/mL at 1.025 ± 0.18 h. The pancreatic juice/plasma ratio was 0.055 ± 0.028. A 0.5-h infusion of 500 mg meropenem every 8 h achieved a 99.4% probability of target attainment against Escherichia coli, 96.4% against Klebsiella species, 94.3% against Enterobacter species and 96.2% against Proteus species, but only 41.3% against Pseudomonas aeruginosa isolates. ConclusionIntravenous meropenem exhibits low penetrance into pancreatic juice. However, a dosing regimen of 500 mg meropenem (0.5-h infusion) every 8 h provides sufficient drug-exposure time in pancreatic juice against the four common Gram-negative bacteria populations.

In vitro effects of proteases in human pancreatic juice on stability of liquid and carrier-bound fibrin sealants

Fibrin sealants are used in pancreatic surgery to prevent leakage of pancreatic fluid and reduce associated complications. The efficacy of this approach is unclear. Fibrin clots were generated in vitro from two commercially available liquid fibrin sealants (Tissucol Duo® and Evicel®) and the carrier-bound fibrin sealant Tachosil®, and exposed to normal saline or human pancreatic fluid. Stability of the sealants was assessed by release of the fibrin and collagen degradation products, D-dimer and hydroxyproline. The effect of protease inhibitors on sealant breakdown was assessed. Clots generated from liquid fibrin sealants degraded rapidly in pancreatic fluid, but not in normal saline. D-dimer release from fibrin clots by pancreatic fluid was approximately 1700 µg/ml after 24 h and less than 20 µg/ml by saline. Pancreatic fluid, but not normal saline, degraded both the fibrin and collagen component of Tachosil®. After 6 h, mean(s.e.m.) D-dimer levels in pancreatic fluid exposed to Tachosil® were 850(183) ng/ml, compared with 60(6) ng/ml in normal saline. The mean(s.e.m.) hydroxyproline concentration in pancreatic fluid was 497(17) µg/ml after a 24-h exposure to Tachosil®, compared with 26(12) µg/ml in normal saline. Protease inhibitors significantly inhibited breakdown of liquid sealants (D-dimer levels less than 50 µg/ml after 24 h) and Tachosil® (D-dimer release 179(12) ng/ml at 6 h; hydroxyproline release 181(29) µg/ml at 24 h). Proteases in pancreatic juice effectively degrade both liquid and carrier-bound fibrin sealants in vitro. The use of these products in pancreatic surgery with the aim of preventing leakage of pancreatic fluid is not supported by this experimental study.

Penetration of meropenem into human pancreatic juice

This study examined the penetration of meropenem into pancreatic juice in patients who had undergone hepato-biliary-pancreatic surgery. The patients received a 0.5-h infusion of 500 mg meropenem. The observed maximum concentration (mean ± standard deviation, n = 5) was 39.1 ± 11.2 μg/ml at 0.5 h in plasma and 2.12 ± 0.73 μg/ml at 1.10 ± 0.14 h in pancreatic juice. The pancreatic juice/plasma ratio was 0.06 ± 0.02. The area under the drug concentration-time curve was 73.0 ± 37.5 μg•h/ml in plasma and 4.24 ± 2.77 μg•h/ml in pancreatic juice. The pancreatic juice/plasma ratio was 0.06 ± 0.01. The mean drug-exposure times above 0.125 μg/ml and 0.25 μg/ml (the minimum inhibitory concentrations (MIC) for common pathogens) in pancreatic juice were 99.4% and 87.3%, respectively, for 500 mg meropenem 3 times daily. This commonly used regimen for pancreatitis achieved the drug-exposure time target (40% of the time above the MIC) at the action site, despite the low penetrability of meropenem.

Pancreatic duct disruption following necrotising pancreatitis; various approaches to management for this challenging problem

Very few studies have revealed the dynamics of meropenem penetration into the pancreas or pancreatic juice in humans. This study of the clinical pharmacokinetics and pharmacodynamics of meropenem in human pancreatic juice was performed to establish a basis for the validation of dosing regimens for pancreatic infections.Ten patients with endoscopic naso-pancreatic drainage received 500 mg meropenem over 0.5 h via intravenous infusion. Venous blood and pancreatic juice samples were collected post-infusion for up to 5.0 h and used to obtain measures of meropenem concentration. The probability of attaining the pharmacodynamic target (40% of the time above the minimum inhibitory concentration [MIC]) in pancreatic juice against MIC distributions for clinical isolates of Gram-negative bacteria was determined using a Monte Carlo simulation.The mean maximum concentration of meropenem in pancreatic juice was 2.08 ± 0.94 μg/mL at 1.025 ± 0.18 h. The pancreatic juice/plasma ratio was 0.055 ± 0.028. A 0.5-h infusion of 500 mg meropenem every 8 h achieved a 99.4% probability of target attainment against Escherichia coli, 96.4% against Klebsiella species, 94.3% against Enterobacter species and 96.2% against Proteus species, but only 41.3% against Pseudomonas aeruginosa isolates.Intravenous meropenem exhibits low penetrance into pancreatic juice. However, a dosing regimen of 500 mg meropenem (0.5-h infusion) every 8 h provides sufficient drug-exposure time in pancreatic juice against the four common Gram-negative bacteria populations.

Screening of an α-Amylase Inhibitor Peptide by Photolinker–Peptide Array

Peptide arrays in which peptides were immobilized on cellulose membranes through photolinkers were synthesized. The peptides were subsequently detached from the arrays by ultraviolet (UV) photolysis for 3 h, and were used to search for functional peptides that inhibit the activity of α-amylase derived from human pancreatic juice. Amino acid replacement with high-molecular-size amino acids, Arg (R), Phe (F), Trp (W), or Tyr (Y), for the first and seventh residues of amylase inhibitor peptide, GHWYYRCW, as previous reported, led to enhancement of the inhibitory effect of the peptide on α-amylase. In particular, one of the resulting peptides, RHWYYRYW, showed a stronger inhibitory effect than acarbose (which is used as a hypoglycemic agent) or inhibitor peptide GHWYYRCW.

The application of methylation specific electrophoresis (MSE) to DNA methylation analysis of the 5' CpG island of mucin in cancer cells

BackgroundMethylation of CpG sites in genomic DNA plays an important role in gene regulation and especially in gene silencing. We have reported mechanisms of epigenetic regulation for expression of mucins, which are markers of malignancy potential and early detection of human neoplasms. Epigenetic changes in promoter regions appear to be the first step in expression of mucins. Thus, detection of promoter methylation status is important for early diagnosis of cancer, monitoring of tumor behavior, and evaluating the response of tumors to targeted therapy. However, conventional analytical methods for DNA methylation require a large amount of DNA and have low sensitivity.MethodsHere, we report a modified version of the bisulfite-DGGE (denaturing gradient gel electrophoresis) using a nested PCR approach. We designated this method as methylation specific electrophoresis (MSE). The MSE method is comprised of the following steps: (a) bisulfite treatment of genomic DNA, (b) amplification of the target DNA by a nested PCR approach and (c) applying to DGGE. To examine whether the MSE method is able to analyze DNA methylation of mucin genes in various samples, we apply it to DNA obtained from state cell lines, ethanol-fixed colonic crypts and human pancreatic juices.ResultThe MSE method greatly decreases the amount of input DNA. The lower detection limit for distinguishing different methylation status is < 0.1% and the detectable minimum amount of DNA is 20 pg, which can be obtained from only a few cells. We also show that MSE can be used for analysis of challenging samples such as human isolated colonic crypts or human pancreatic juices, from which only a small amount of DNA can be extracted.ConclusionsThe MSE method can provide a qualitative information of methylated sequence profile. The MSE method allows sensitive and specific analysis of the DNA methylation pattern of almost any block of multiple CpG sites. The MSE method can be applied to analysis of DNA methylation status in many different clinical samples, and this may facilitate identification of new risk markers.

The never-ending search of an acceptable compromise for pancreatic lipase standardisation

The never-ending search of an acceptable compromise for pancreatic lipase standardisation

SPINK1: A New Therapeutic Target in Cancer?

The serine protease inhibitor Kazal type 1 (SPINK1)2 was initially isolated from bovine pancreas by Kazal and colleagues. A similar protease inhibitor was later identified in human pancreatic juice and called “pancreatic secretory trypsin inhibitor.” By means of immunologic techniques, SPINK1 was later identified as a tumor-associated peptide and isolated from the urine of a patient with ovarian cancer. Hence, it was called “tumor-associated trypsin inhibitor” (TATI) (1). This name has been used in most studies of its use as a tumor marker [reviewed in (2)]. TATI was shown to be a serum and urine marker for many types of cancer, but it was not found to be superior to earlier known markers that had been identified for diagnostic use. It was an independent prognostic marker for several types of cancer, however. Tumors producing SPINK1 have been shown to also produce trypsin, and because trypsin activates matrix metalloproteinases, which have been shown to mediate cancer invasion, an association between SPINK1 production and an adverse prognosis in cancer was ascribed to its coproduction with trypsin. SPINK1 was thought to have a role in cancer similar to the one it has in the pancreas, where it protects the pancreas by inhibiting prematurely activated trypsin (2). Mechanisms by which SPINK1 could exert alternative functions were suggested by its structural similarities with epidermal growth factor (EGF): a 50% sequence homology, a molecular size of approximately 6 kDa, and the presence of 3 intrachain disulfide bridges (2). This similarity with EGF prompted studies on the ability of SPINK1 to act as a growth factor. In 1985, SPINK1 was shown to bind to surface receptors and to stimulate DNA synthesis in …