It is a proenzyme that is converted into active form trypsin by the enzyme enterokinase. Solution: Trypsinogen is present in pancreatic juice. The digestive juices are secreted by different organs, vary widely in chemical composition, and play different roles in the digestive process.14. They include saliva, gastric juice, pancreatic juice, bile, and intestinal juice. The digestive juices are the secretions of the digestive tract that break down food.

Out of the 5В promoters analyzed, APC (71%) and HRH2 (65%) were the most frequently methylated in PC juice. Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations were also tested with alleleвЂ‘specific quantitative polymerase chain reaction amplification. The methylation status of the APC, HRH2, CDH13, SPARC and ENвЂ‘1 promoters was analyzed using methylation specificвЂ‘melting curve analysis (MSвЂ‘MCA). A total of 135В pancreatic juices obtained from 85В pancreatic cancer (PC), 26В ampullary carcinoma (AC), 10В intraductal papillary mucinous neoplasm (IPMN) and 14В chronic pancreatitis (CP) patients were analyzed. The aim of the present study was to assess their prevalence in pancreatic juice of carcinomas of the pancreas and periampullary area. The present authors previously demonstrated that DNA hypermethylation of adenomatous polyposis coli (APC), histamine receptor H2 (HRH2), cadherinВ 13 (CDH13), secreted protein acidic and cysteine rich (SPARC) and engrailed-1 (ENвЂ‘1) promoters is frequently detected in pancreatic tumor cells.

TheUse of epigenetic abnormalities as biomarkers is based on theirRelative high frequency and the development of methodologies thatCan sensitively detect methylation even when cancer cells are aCandidate genes for these purposes should ideally have a highPrevalence of hypermethylation in tumors cells not being present inThe absence of disease. In this regard, genetic alterations canBe more easily detected in DNA extracted from pancreatic juiceThis may well reflect a field effect within the pancreas or, whenThe tumor is already evident, the possibility that exfoliated cellsOnly represent part of a tumor that is intrinsically heterogeneousAberrant promoter methylation of tumor suppressorGenes is a frequent and early event in multiple tumors, includingCarcinomas of the pancreas and the periampullary area ( 12, 13). InFact, KRAS mutations have been detected in variableProportion (0вЂ“60%) in ductal lesions in CP samples and even inPancreatic juice can be considered a good surrogateOf the status of the pancreatic duct epithelium, since it containsExfoliated cells from all areas of the pancreas ( 10). The high frequency (75вЂ“100%) ofKirsten rat sarcoma viral oncogene homolog (KRAS) mutationsIn PC ( 5, 6), has lead to extensive research aimed atEstablishing their role as diagnostic markers for this disease.However, when assessing pancreatic juice, its clinical utility isHampered by a lack of specificity ( 7вЂ“ 9) linked toThe detection of KRAS mutations in a significant proportionOf patients with chronic pancreatitis (CP) ( 8, 10). Intraductal papillary mucinous neoplasms(IPMNs) are a distinct subset of tumors that are increasinglyRecognized and may be cured if they are timely detected ( 3).Early detection of PC is the best and currently onlyOption to improve its dismal prognosis ( 4). AmpullaryTumors are also aggressive, although to a lesser extent ( 3).

Quantitative polymerase chain reaction (PCR) withTemperature dissociation or MCA was used to assess the differenceIn melting temperatures between methylated and unmethylatedSamples. A total of 800 ng genomic DNA wereTreated with sodium bisulfite according to the manufacturer'sProtocol (EZ-DNA Methylation-Goldв„ў kit Zymo Research Corporation,Irvine, CA, USA). Methylation assessmentAssess the methylation status of EN-1, HRH2, SPARC, APC andCDH13 gene promoters. AModified standard phenol-chloroform method was used to optimize DNANP9 and NP18 cell lines (previously generated from pancreaticDuctal adenocarcinoma biopsies in the Translational ResearchLaboratory, Duran i Reynals Hospital, Barcelona, Spain) used asControls and biopsies was performed using the saline methodWith 2-propanol after mechanical disgregation and overnightDigestion with proteinase K at 53вЊ. DNA extraction wasDirectly performed from the PPJ with no further processing. Sample processing and DNAThe intraoperative pure pancreatic juice (PPJ) wasSnap frozen immediately after collection.

Primer sequences, annealingTemperature and CpG residues targeted in each reaction are detailedAmplified with the REPLI-g kit (Qiagen, Inc., Valencia, CA, USA)Was used as unmethylated control. For qPCR, either 25 ngBisulfite-modified DNA or 1 Вµl pre-amplified DNA was used as aTemplate, using a LightCycler В® 486 (Roche AppliedScience, Penzberg, Germany). PCR was performed using IMMOLASEв„ў DNAPolymerase (Bioline, London, UK).

The primers used did not target CpGResidues amenable for methylation, thus resulting in unbiasedAnd PCR conditions for DNA hypermethylation analysisMutations analyzed, and probe and primer sequences5вЂІ-GCCTGCTGAAAATGACTGAATATAAACT-3вЂІ reverse primer codon 12,5вЂІ-GCTGTATCGTCAAGGCACTCTT-3вЂІ reverse primer codon 13,5вЂІ-GAATTAGCTGTATCGTCAAGGCACT-3вЂІ.

Pancreatic Juices