Simultaneous and sequential combination of genetic and epigenetic biomarkers for the presence of high-grade dysplasia in patients with pancreatic cyst: Discovery in cyst fluid and test in pancreatic juice.

BACKGROUND/OBJECTIVES: Screening patients with intraductal papillary mucinous neoplasms (IPMN) has the primary goal of identifying potentially curable noninvasive precursors. We aimed to evaluate the diagnostic impact of genetic and epigenetic biomarkers in the presence of noninvasive precursors. METHODS: Mutated KRAS/GNAS and methylated SOX17/TBX15/BMP3/TFPI2 DNA were assessed by droplet digital PCR in a discovery cohort of 70 surgically aspirated cyst fluids, and diagnostic performances for differentiating high-grade dysplasia (HGD) from low-grade dysplasia (LGD) was evaluated. We then tested these markers using an independent test cohort consisting of 156 serially collected pancreatic juice samples from 30 patients with IPMN. RESULTS: Mutated KRAS and GNAS are specific for IPMNs but are not helpful for the prediction of histological grades. Cyst fluids from IPMN with HGD showed higher methylation levels of SOX17 (median, 0.141 vs. 0.021; P = 0.086) and TBX15 (median, 0.030 vs. 0.003; P = 0.028) than those with LGD. The combination of all tested markers yielded a diagnostic performance with sensitivity of 69.6%, and specificity of 90.0%. Among the 30 pancreatic juice samples exhibiting the highest abundance of KRAS/GNAS mutations in each patient in the test cohort, patients with histologically proven HGD due to pancreatic resection had a significantly higher prevalence (100% vs. 31%, P = 0.018) and abundance (P = 0.037) of methylated TBX15 than those without cytohistological diagnosis undergoing surveillance. CONCLUSIONS: A simultaneous and sequential combination of mutated and methylated DNA markers in pancreatic cyst fluid and juice sample markers can help detect noninvasive pancreatic precursor neoplasms.

Systematic review and meta-analysis: Diagnostic performance of DNA alterations in pancreatic juice for the detection of pancreatic cancer.

BACKGROUND AND AIMS: Pancreatic cancer has a dismal prognosis. So far, imaging has been proven incapable of establishing an early enough diagnosis. Thus, biomarkers are urgently needed for early detection and improved survival. Our aim was to evaluate the pooled diagnostic performance of DNA alterations in pancreatic juice. METHODS: A systematic literature search was performed in EMBASE, MEDLINE Ovid, Cochrane CENTRAL and Web of Science for studies concerning the diagnostic performance of DNA alterations in pancreatic juice to differentiate patients with high-grade dysplasia or pancreatic cancer from controls. Study quality was assessed using QUADAS-2. The pooled prevalence, sensitivity, specificity and diagnostic odds ratio were calculated. RESULTS: Studies mostly concerned cell-free DNA mutations (32 studies: 939 cases, 1678 controls) and methylation patterns (14 studies: 579 cases, 467 controls). KRAS, TP53, CDKN2A, GNAS and SMAD4 mutations were evaluated most. Of these, TP53 had the highest diagnostic performance with a pooled sensitivity of 42% (95% CI: 31-54%), specificity of 98% (95%-CI: 92%-100%) and diagnostic odds ratio of 36 (95% CI: 9-133). Of DNA methylation patterns, hypermethylation of CDKN2A, NPTX2 and ppENK were studied most. Hypermethylation of NPTX2 performed best with a sensitivity of 39-70% and specificity of 94-100% for distinguishing pancreatic cancer from controls. CONCLUSIONS: This meta-analysis shows that, in pancreatic juice, the presence of distinct DNA mutations (TP53, SMAD4 or CDKN2A) and NPTX2 hypermethylation have a high specificity (close to 100%) for the presence of high-grade dysplasia or pancreatic cancer. However, the sensitivity of these DNA alterations is poor to moderate, yet may increase if they are combined in a panel.

Detection and proteomic characterization of extracellular vesicles in human pancreatic juice.

AIMS: The 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. METHODS: Comparative 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. RESULTS: Here 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. CONCLUSIONS: Together 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.

Measurement of indicator genes using global complementary DNA (cDNA) amplification, by polyadenylic acid reverse transcriptase polymerase chain reaction (poly A RT-PCR): A feasibility study using paired samples from tissue and ductal juice in patients undergoing pancreatoduodenectomy.

OBJECTIVES: The aim of this study is to compare gene expression profiles in RNA isolated from pancreatic ductal juice with the RNA expression profiles of the same genes from matched intra-operative tissue samples from pancreatic tumours. METHODS: Intra-operative sampling of pancreatic juice and collection of matched tissue samples was undertaken in patients undergoing pancreatoduodenectomy for clinically suspected pancreatic cancer and a precursor lesion, main-duct intraductal papillary mucinous neoplasm. RNA was isolated and Poly A PCR was used to globally amplify the RNA. Real-time polymerase chain reaction (RT-PCR) was used to measure expression levels of 17 genes selected from microarray studies. Spearman's rank correlation test was used to examine the relationship of gene expression between pancreatic juice and tissue. The study was approved by Regional Ethics Committee. RESULTS: Mesothelin (MSLN) showed significant correlation (p < 0.008) in expression levels between paired pancreatic juice and tissue samples in pancreas cancer. In intraductal papillary mucinous neoplasms (IPMN), Matrix Metalloproteinase 7 (MMP7), showed significant correlation (p < 0.01) in the expression levels between paired pancreatic juice and tissue samples. CONCLUSION: This study confirms that RNA analysis of paired pancreatic juice and tissue samples and establishment of cDNA using poly A PCR is technically feasible. Application of the technique to non-invasively obtained pancreatic juice during endoscopic assessment of tumours and the use of gene arrays of cancer indicator genes are the next steps in development of this technique.

Duodenal infusions of isoleucine influence pancreatic exocrine function in dairy heifers.

Four healthy Holstein heifers (235 ± 12 kg) fitted with duodenal and pancreatic cannulas were used to investigate infusion of isoleucine (Ile) on the pancreatic exocrine function in a 4 × 4 Latin square design. Three doses of Ile, 10, 20 and 30 g in 2500 ml water, respectively, were infused into the duodenum over a period of 12 h in Experiment (Exp) 1 and over 10 d in Exp 2. Hourly pancreatic juice and jugular blood were taken during the infusion period in Exp 1, and the blood samples were taken in 2-h intervals over the last 2 d in Exp 2. Compared with no Ile infusion, the Ile infusions in both experiments increased the concentration and secretion rate of the protein, activity of ɑ-amylase and trypsin and plasma cholecystokinin. The secretion rate of ɑ-amylase and the activity of trypsin linearly increased with the Ile doses. The pancreatic juice secretion linearly increased with Ile in Exp 2 but not in Exp 1. Isoleucine linearly increased plasma insulin in Exp 1, but not in Exp 2. No effects of Ile on pH of pancreatic juice, the activity of chymotrypsin and lipase and plasma glucose were found. In conclusion, duodenal Ile infusion could increase the pancreatic exocrine function of Holstein heifers, especially ɑ-amylase, and the increment appeared to be dose and time dependent.

Extracellular matrix proteins and carcinoembryonic antigen-related cell adhesion molecules characterize pancreatic duct fluid exosomes in patients with pancreatic cancer.

BACKGROUND: Exosomes are nanovesicles that have been shown to mediate carcinogenesis in pancreatic ductal adenocarcinoma (PDAC). Given the direct communication of pancreatic duct fluid with the tumor and its relative accessibility, we aimed to determine the feasibility of isolating and characterizing exosomes from pancreatic duct fluid. METHODS: Pancreatic duct fluid was collected from 26 patients with PDAC (n = 13), intraductal papillary mucinous neoplasm (IPMN) (n = 8) and other benign pancreatic diseases (n = 5) at resection. Exosomes were isolated by serial ultracentrifugation, proteins were identified by mass spectrometry, and their expression was evaluated by immunohistochemistry. RESULTS: Exosomes were isolated from all specimens with a mean concentration of 5.9 ± 1 × 108 particles/mL and most frequent size of 138 ± 9 nm. Among the top 35 proteins that were significantly associated with PDAC, multiple carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) and extracellular matrix (ECM) proteins were identified. Interestingly, CEACAM 1/5 expression by immunohistochemistry was seen only on tumor epithelia whereas tenascin C positivity was restricted to stroma, suggesting that both tumor and stromal cells contributed to exosomes. CONCLUSION: This is the first study showing that exosome isolation is feasible from pancreatic duct fluid, and that exosomal proteins may be utilized to diagnose patients with PDAC.

Analysis of amylase in duodenal juice - Automated kinetic spectrophotometric analysis versus manual colorimetric endpoint assay.

OBJECTIVE: The measurement of duodenal amylase by a colorimetric end-point assay has been the most used method for amylase activity analyses. The method is manual, time consuming and dependent on specialized equipment. In this study, we compare an automated kinetic spectrophotometric method for pancreatic amylase measurement in duodenal juice with a standardized colorimetric end-point assay. METHODS: We used specimen of duodenal juice at random from a biobank obtained by short endoscopic secretin test in patients with suspected exocrine pancreatic failure of different reasons. Duodenal juice was tested for amylase activity with a conservative manual colorimetric endpoint assay (Phadebas Amylase test, Magle AB) and an automated enzymatic kinetic spectrophotometric method using standard reagents for pancreatic amylase activity for Cobas c111 (Roche Diagnostics). RESULTS: 52 samples for assay of amylase were analyzed in pairs. Correlation between measurements with the two methods was r = 0.99 (p < 0.001), linear regression 0.99 (p < 0.001). CONCLUSION: Quantification of duodenal amylase activity with automated spectrophotometry has excellent correlation to measurements made by the manual method. This allows for standardized, center independent analyses of duodenal amylase for the assessment of acinar pancreatic function.

Cholestyramine as a promising, strong anion exchange resin for direct capture of genetic biomarkers from raw pancreatic fluids.

The ability to capture cell-free DNA from the gastrointestinal tract, in a minimally invasive manner, could enhance our ability to diagnose gastrointestinal disease, or gain a better understanding of the spatial mapping of the intestinal microbiota. We, therefore, sought to identify a class of capture agents that could directly and efficiently sequester genetic material from intestinal fluids. As a particular case study, we examined the ability to capture DNA from pancreatic secretions, for potential application in enabling the sequestration of early, genetic biomarkers of pancreatic disease. We hypothesized that the cholestyramine series of strong cation exchange resins, which are FDA approved for the treatment of high cholesterol, may be capable of capturing DNA from pancreatic secretions. We identified a particular cholestyramine resin, DOWEX 1 × 2 100-200 mesh, which is able to efficiently capture and purify DNA from pancreatic fluid. Using only 200 µL of pancreatic secretions, we are able to recover 247 ± 182 ng of amplifiable human DNA, giving an estimated pancreatic fluid DNA content of 1.23 ± 0.91 ng/µL. To our knowledge, this is the first demonstration of a material that can effectively capture and purify DNA directly from untreated pancreatic fluids. Thus, our approach could hold high utility for the in vivo capture of DNA and disease biomarkers if incorporated into an appropriate sampling device. Biotechnol. Bioeng. 2017;114: 934-938. © 2016 Wiley Periodicals, Inc.

A study of the clinical utility of a 20-minute secretin-stimulated endoscopic pancreas function test and performance according to clinical variables.

BACKGROUND AND AIMS: Direct pancreas juice testing of bicarbonate, lipase, or trypsin after stimulation by secretin or cholecystokinin is used to determine exocrine function, a surrogate for diagnosing chronic pancreatitis (CP). Endoscopic pancreas function tests (ePFTs), where a peak bicarbonate concentration (PBC) ≥80 mEq/L in pancreas juice is considered normal, are now used more frequently. In this ePFT, aspirates start 35 minutes after secretin administration because pancreas output peaks 30 minutes after secretagogue administration. The performance of ePFT in a cohort of patients with a presumptive diagnosis of CP referred to a pancreas clinic for consideration of an intervention including total pancreatectomy and islet autotransplantation was studied, compared with EUS, ERCP, histology, and consensus diagnosis. The effect of sedation, narcotic use, aspirate volume, body mass index, age, and proton pump inhibitors (PPIs) on test performance is reported. METHODS: After a test dose, synthetic human secretin was administered intravenously, and 30 minutes later sedation was achieved with midazolam and fentanyl or propofol. A gastroscope was advanced to the major papilla where 4 continuous aspiration samples were performed at 5-minute intervals in sealed bottles. PBC ≥80 mEq/L was normal. RESULTS: Eighty-one patients had ePFTs from August 2010 through October 2015. Twenty-seven patients (33%) were diagnosed with CP. Eighteen of the 27 patients with CP and 1 of the 54 patients without CP had an abnormal ePFT, producing a sensitivity of 66% (95% CI, 46.0-83.5), specificity 98% (95% CI, 90.1-99.9), positive predictive value 94.7% (95% CI, 74-99.9), and negative predictive value 85.5% (95% CI, 74.2-93.1). ERCP and PBC concordance was generally poor, but none of the patients without CP had major EUS changes, and only 3 patients with a PBC <80 mEq/L had a normal EUS. The PBC was affected by narcotics and PPI use. CONCLUSION: A 20-minute ePFT after secretin administration had a marginal sensitivity for diagnosis of CP. The diagnosis of CP should not rely on a single study and certainly not a PFT. The duodenal aspirate volume did not correlate with the PBC, which contrasts with current secretin-enhanced MRCP knowledge; therefore, further studies on this subject are warranted. Neither type of sedation, BMI, nor age affected test performance. Narcotics and PPIs may affect the PBC, so borderline results should be interpreted with caution in these groups.

In vitro digestion, antioxidant and antiacetylcholinesterase activities of two species of Ruta: Ruta chalepensis and Ruta montana.

CONTEXT: Ruta genus (Rutaceae) is abundantly used and described in the most ancient systematic records of medical practice of the Mediterranean world. In Tunisia, this genus is represented by two medicinal and aromatic shrubs: Ruta chalepensis L. and Ruta montana L. OBJECTIVE: This study investigates the antioxidant and acetylcholinesterase inhibition (AChE) activities before and after in vitro gastrointestinal metabolism of leaf decoction of R. chalepensis and R. montana. MATERIALS AND METHODS: We study, in vitro, the effect of the gastrointestinal juices gastric (1.75 mL) or pancreatic (2.5 mL) juices, on the biological activity by the measurement of the antioxidant activity and AChE inhibition during 4 h of decoction extract obtained from the leaves of the two species of Ruta. RESULTS: The results showed that the ability to inhibit the AChE enzyme was similar; being the greatest inhibitory activity exhibited by the ethanol extract (IC50 = 12 ± 1.1 µg/mL) obtained from leaves of R. chalepensis. CONCLUSION: In conclusion, we showed that there was no appreciable degradation and that the activity was kept constant after gastric and pancreatic juice digestion.

Endoscopic Pancreas Fluid Collection: Methods and Relevance for Clinical Care and Translational Science.

Pancreatic secretions have an important role in the regulation of a normal nutritional state but can be altered owing to a variety of pathophysiological mechanisms in the context of exocrine pancreatic disease. The development of an endoscopic technique for collection of pancreatic fluid, termed endoscopic pancreatic function testing, has led to improved understanding of these alterations and is particularly helpful to characterize chronic pancreatitis. In addition, investigators have found endoscopically collected pancreatic fluid to be a valuable biofluid for the purposes of translational science. Techniques such as proteomic, cytokine, genetic mutation, DNA methylation, and microRNA analyses, among others, can be utilized to gain a better understanding of the molecular characteristics of chronic pancreatitis and other pancreatic diseases. Endoscopic collection of pancreatic fluid is safe and relatively straightforward, permitting opportunities for longitudinal analysis of these translational markers throughout the course of disease. This manuscript summarizes our current knowledge of pancreatic fluid, with an emphasis on proper techniques for sample collection and handling, its clinical utility, and preliminary observations in translational science.

Automated spectrophotometric bicarbonate analysis in duodenal juice compared to the back titration method.

OBJECTIVES: We have recently evaluated a short endoscopic secretin test for exocrine pancreatic function. Bicarbonate concentration in duodenal juice is an important parameter in this test. Measurement of bicarbonate by back titration as the gold standard method is time consuming, expensive and technically difficult, thus a simplified method is warranted. We aimed to evaluate an automated spectrophotometric method in samples spanning the effective range of bicarbonate concentrations in duodenal juice. We also evaluated if freezing of samples before analyses would affect its results. METHODS: Patients routinely examined with short endoscopic secretin test suspected to have decreased pancreatic function of various reasons were included. Bicarbonate in duodenal juice was quantified by back titration and automatic spectrophotometry. Both fresh and thawed samples were analysed spectrophotometrically. RESULTS: 177 samples from 71 patients were analysed. Correlation coefficient of all measurements was r = 0.98 (p < 0.001). Correlation coefficient of fresh versus frozen samples conducted with automatic spectrophotometry (n = 25): r = 0.96 (p < 0.001) CONCLUSIONS: The measurement of bicarbonate in fresh and thawed samples by automatic spectrophotometrical analysis correlates excellent with the back titration gold standard. This is a major simplification of direct pancreas function testing, and allows a wider distribution of bicarbonate testing in duodenal juice. Extreme values for Bicarbonate concentration achieved by the autoanalyser method have to be interpreted with caution.

A meta-analysis of the diagnostic value of detecting K-ras mutation in pancreatic juice as a molecular marker for pancreatic cancer.

BACKGROUND: K-ras codon 12 mutation is one of the earliest genetic changes in the development of pancreatic cancer (PC) and accurate detection of K-ras mutations is gaining increasing attention in the field of molecular diagnosis. METHODS: Original research articles which evaluated the diagnostic accuracy of K-ras mutation detection in PC were selected. Data were presented as forest plots and summary receiver operating characteristic curve analysis was used to summarize the overall test performance. RESULTS: We assessed 16 studies from 15 published articles. The pooled sensitivity and specificity were 59% (95%CI: 54%-64%) and 87% (95%CI: 84%-89%), respectively. The pooled positive likelihood ratio and negative likelihood ratio were 4.13 (95%CI: 2.73-6.25) and 0.42 (95%CI: 0.32-0.56), respectively, and the pooled diagnostic odds ratio was 13.66 (95% CI: 7.25-25.74). CONCLUSIONS: Our results indicate that the analysis of K-ras mutations in pancreatic juice has a considerable diagnostic value in PC. Further studies with rigorous design, large sample size, and multi-regional co-operation are needed.

Pancreatic adenocarcinomas without secondary signs on multiphasic multidetector CT: association with clinical and histopathologic features.

PURPOSE: To determine the clinical, histopathologic and imaging features of pancreatic adenocarcinomas without secondary signs on dynamic CT. MATERIALS AND METHODS: Seventy patients (mean age 70 years) with histologically proven pancreatic adenocarcinoma underwent preoperative contrast material-enhanced multiphasic multidetector CT before pancreatic resection. In each patient, clinical data including carbohydrate antigen 19-9, frequency of isoattenuating tumours, and presence of secondary signs and histopathologic findings such as tumour location, tumour stage, and microscopic infiltrative growth grade were evaluated. RESULTS: Ten tumours (14%) were without secondary signs, and 60 (86%) were with secondary signs. Tumours without and with secondary signs were located in the uncinate process in 5 (50%) and 3 (5%), head in 3 (30%) and 29 (48%), body in 2 (20%) and 22 (37%), and tail in 0 (0%) and 6 (10%), respectively (p = .001). The frequency of isoattenuating pancreatic adenocarcinomas without secondary signs was significantly higher than those with secondary signs (p = 0.034). The tumour stage of pancreatic adenocarcinomas without secondary signs was earlier than that in tumours with secondary signs (p = 0.041). CONCLUSIONS: Pancreatic adenocarcinomas without secondary signs is characterized by the presence of uncinate and isoattenuating tumours and earlier tumour stage compared to tumours with secondary signs. KEY POINTS: Frequency of pancreatic adenocarcinomas without secondary signs on multiphasic CT is 14 . Pancreatic adenocarcinomas without secondary signs are common in the uncinate process. Pancreatic adenocarcinomas without secondary signs are common in isoattenuating tumours. Pancreatic adenocarcinomas without secondary signs are characterized by earlier-stage tumours.

The exocrine pancreas: the acinar-ductal tango in physiology and pathophysiology.

There are many reviews of pancreatic acinar cell function and also of pancreatic duct function, but there is an almost total absence of synthetic reviews bringing the integrated functions of these two vitally and mutually interdependent cells together. This is what we have attempted to do in this chapter. In the first part, we review the normal integrated function of the acinar-ductal system, with particular emphasis on how regulation of one type of cell also influences the other cell type. In the second part, we review a range of pathological processes, particularly those involved in acute pancreatitis (AP), an often-fatal human disease in which the pancreas digests itself, in order to explore how malfunction of one of the cell types adversely affects the function of the other.

KRAS and guanine nucleotide-binding protein mutations in pancreatic juice collected from the duodenum of patients at high risk for neoplasia undergoing endoscopic ultrasound.

BACKGROUND & AIMS: Pancreatic imaging can identify neoplastic cysts but not microscopic neoplasms. Mutation analysis of pancreatic fluid after secretin stimulation might identify microscopic neoplasias in the pancreatic duct system. We determined the prevalence of mutations in KRAS and guanine nucleotide-binding protein α-stimulating genes in pancreatic juice from subjects undergoing endoscopic ultrasound for suspected pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasms, or pancreatic adenocarcinoma. METHODS: Secretin-stimulated juice samples were collected from the duodenum of 272 subjects enrolled in Cancer of the Pancreas Screening studies; 194 subjects were screened because of a family history of, or genetic predisposition to, pancreatic cancer, and 78 subjects were evaluated for pancreatic cancer (n = 30) or other disorders (controls: pancreatic cysts, pancreatitis, or normal pancreata, n = 48). Mutations were detected by digital high-resolution melt-curve analysis and pyrosequencing. The number of replicates containing a mutation determined the mutation score. RESULTS: KRAS mutations were detected in pancreatic juice from larger percentages of subjects with pancreatic cancer (73%) or undergoing cancer screening (50%) than controls (19%) (P = .0005). A greater proportion of patients with pancreatic cancer had at least 1 KRAS mutation detected 3 or more times (47%) than screened subjects (21%) or controls (6%, P = .002). Among screened subjects, mutations in KRAS (but not guanine nucleotide-binding protein α-stimulating) were found in similar percentages of patients with or without pancreatic cysts. However, a greater proportion of patients older than age 50 years had KRAS mutations (54.6%) than younger patients (36.3%) (P = .032); the older subjects also had more mutations in KRAS (P = .02). CONCLUSIONS: Mutations in KRAS are detected in pancreatic juice from the duodenum of 73% of patients with pancreatic cancer, and 50% of asymptomatic individuals with a high risk for pancreatic cancer. However, KRAS mutations were detected in pancreatic juice from 19% of controls. Mutations detected in individuals without pancreatic abnormalities, based on imaging analyses, likely arise from small pancreatic intraepithelial neoplasia lesions. ClinicalTrials.gov no: NCT00438906 and NCT00714701.

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.

Visualization of the leakage of pancreatic juice using a chymotrypsin-activated fluorescent probe.

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Exocrine pancreatic function in hepatocyte nuclear factor 1ß-maturity-onset diabetes of the young (HNF1B-MODY) is only moderately reduced: compensatory hypersecretion from a hypoplastic pancreas.

OBJECTIVES: To examine the exocrine pancreatic function in carriers of the hepatocyte nuclear factor 1ß gene (HNF1B) mutation by direct testing. METHODS: Patients with HNF1B mutations and control subjects were assessed using rapid endoscopic secretin tests and secretin-stimulated magnetic resonance imaging. Seven patients and 25 controls underwent endoscopy, while eight patients and 20 controls had magnetic resonance imaging. Ductal function was assessed according to peak bicarbonate concentrations and acinar function was assessed according to peak digestive enzyme activities in secretin-stimulated duodenal juice. The association of pancreatic exocrine function and diabetes status with pancreatic gland volume was examined. RESULTS: The mean increase in secretin-stimulated duodenal fluid was smaller in patients than controls (4.0 vs 6.4 ml/min; P = 0.003). We found lower ductal function in patients than controls (median peak bicarbonate concentration: 73 vs 116 mEq/L; P < 0.001) and lower acinar function (median peak lipase activity: 6.4 vs 33.5 kU/ml; P = 0.01; median peak elastase activity: 0.056 vs 0.130 U/ml; P = 0.01). Pancreatic fluid volume outputs correlated significantly with pancreatic gland volumes (r² = 0.71, P = 0.008) in patients. The total fluid output to pancreatic gland volume ratios were higher in patients than controls (4.5 vs 1.3 ml/cm³; P = 0.03), suggesting compensatory hypersecretion in the remaining gland. CONCLUSION: Carriers of the HNF1B mutation have lower exocrine pancreatic function involving both ductal and acinar cells. Compensatory hypersecretion suggests that the small pancreas of HNF1B mutation carriers is attributable to hypoplasia, not atrophy.

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.