Roles of ursodeoxycholic acid in the bile biochemistry and metabolomics in patients with choledocholithiasis: a prospective study.

INTRODUCTION: Recurrence after the endoscopic treatment of common bile duct stones (CBDS) is related to bile metabolism and bile compositions. Ursodeoxycholic acid (UDCA) has been proved effective in reducing the recurrence of CBDS. However, the detailed effects of UDCA on bile metabolism are still not extensively explored. OBJECTIVES: This study aimed to analyze the role of UDCA in patients with choledocholithiasis (CDC) from the perspective of biochemistry and metabolomics. METHODS: A total of 89 patients with CDC who underwent endoscopic retrograde cholangiopancreatography were prospectively examined and randomly assigned to control and UDCA groups. The biochemical detections (cholesterol, bilirubin, and so on) were performed on the collected bile. Moreover, the metabolomics analysis was conducted based on bile from 20 patients in the UDCA group. RESULTS: The bile levels of cholesterol and endotoxins significantly decreased after UDCA treatment. Regarding bile metabolomics, the levels of 25 metabolites changed significantly after UDCA treatment. The pathway enrichment analysis showed that the UDCA addition evoked a common response related to phenylalanine, tyrosine, and tryptophan biosynthesis; phenylalanine metabolism; arachidonic acid metabolism; and terpenoid backbone biosynthesis. CONCLUSIONS: UDCA treatment within a short time interval (7 days) did not improve the circulating laboratory values in patients with CDC who had undergone endoscopy surgery. However, relevant decreases in the bile levels of cholesterol and endotoxin were observed. UDCA evoked a common response related to lipid metabolism and amino acid metabolism, which probably reduced the bile level of cholesterol, protected hepatocytes, and corrected the abnormality of lipid metabolism caused by CDC.

Loss of cellular FLICE-inhibitory protein promotes acute cholestatic liver injury and inflammation from bile duct ligation.

Cholestatic liver injury results from impaired bile flow or metabolism and promotes hepatic inflammation and fibrogenesis. Toxic bile acids that accumulate in cholestasis induce apoptosis and contribute to early cholestatic liver injury, which is amplified by accompanying inflammation. The aim of the current study was to evaluate the role of the antiapoptotic caspase 8-homolog cellular FLICE-inhibitory (cFLIP) protein during acute cholestatic liver injury. Transgenic mice exhibiting hepatocyte-specific deletion of cFLIP (cFLIP-/-) were used for in vivo and in vitro analysis of cholestatic liver injury using bile duct ligation (BDL) and the addition of bile acids ex vivo. Loss of cFLIP in hepatocytes promoted acute cholestatic liver injury early after BDL, which was characterized by a rapid release of proinflammatory and chemotactic cytokines (TNF, IL-6, IL-1ß, CCL2, CXCL1, and CXCL2), an increased presence of CD68+ macrophages and an influx of neutrophils in the liver, and resulting apoptotic and necrotic hepatocyte cell death. Mechanistically, liver injury in cFLIP-/- mice was aggravated by reactive oxygen species, and sustained activation of the JNK signaling pathway. In parallel, cytoprotective NF-κB p65, A20, and the MAPK p38 were inhibited. Increased injury in cFLIP-/- mice was accompanied by activation of hepatic stellate cells and profibrogenic regulators. The antagonistic caspase 8-homolog cFLIP is a critical regulator of acute, cholestatic liver injury. NEW & NOTEWORTHY The current paper explores the role of a classical modulator of hepatocellular apoptosis in early, cholestatic liver injury. These include activation of NF-κB and MAPK signaling, production of inflammatory cytokines, and recruitment of neutrophils in response to cholestasis. Because these signaling pathways are currently exploited in clinical trials for the treatment of nonalcoholic steatohepatitis and cirrhosis, the current data will help in the development of novel pharmacological options in these indications.

Pathological features of primary sclerosing cholangitis identified by bile proteomic analysis.

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease of unknown origin. Previous bile proteomic analyses in patients with PSC have revealed changes in disease activity specific to malignant transformation. In this study, we established a reference bile duct-derived bile proteome for PSC that can be used to evaluate biliary pathophysiology. Samples were collected from patients with PSC or with choledocholithiasis (control) (n=6 each). Furthermore, patients with PSC-associated cholangiocarcinoma (CC) and with CC without concomitant PSC were analyzed. None of the patients showed signs of inflammation or infection based on clinical and laboratory examinations. Proteins overexpressed in patients with PSC relative to control patients were detected by two-dimensional difference gel electrophoresis and identified by liquid chromatography-tandem mass spectrometry. Functional proteomic analysis was performed using STRING software. A total of 101 proteins were overexpressed in the bile fluid of patients with PSC but not in those of controls; the majority of these were predicted to be intracellular and related to the ribosomal and proteasomal pathways. On the other hand, 91 proteins were found only in the bile fluid of controls; most were derived from the extracellular space and were linked to cell adhesion, the complement system, and the coagulation cascade. In addition, proteins associated with inflammation and the innate immune response-e.g., cluster of differentiation 14, annexin-2, and components of the complement system-were upregulated in PSC. The most prominent pathways in PSC/CC-patients were inflammation associated cytokine and chemokine pathways, whereas in CC-patients the Wnt signaling pathway was upregulated. In PSC/CC-patients DIGE-analysis revealed biliary CD14 and Annexin-4 expression, among others, as the most prominent protein that discriminates between both cohorts. Thus, the bile-duct bile proteome of patients with PSC shows disease-specific changes associated with inflammation and the innate immune response even in the absence of obvious clinical signs of cholangitis, malignancy, or inflammation. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni and Peter Jansen.

Effect of Acupoint Catgut Embedding at Yanglingquan (GB34) on the Bile Metabolism of Patients with Choledocholithiasis after Surgery.

Importance: Choledocholithiasis, or bile duct gallstones, is effectively treated with surgery, which does not prevent relapse. A common adjuvant therapy is the stimulation of the Yanglingquan point (GB34). Acupoint catgut embedding (ACE), an acupoint stimulation therapy, may be a better treatment for choledocholithiasis. Objectives: To investigate the effect of ACE in stimulating GB34 on bile metabolism and its possible mechanism via metabonomics. Methods: In this study, we used ultrahigh performance liquid chromatographyquadrupole time-of-flight mass spectrometry (UHPLC-MS/MS) to analyze the changes in bile metabolites, metabolic pathways, and liver function indicators in 16 patients with choledocholithiasis before and after ACE stimulation. Results: We identified 10 metabolites that exhibited significant differences in the bile before and after ACE, six of which significantly increased and four that significantly decreased. Moreover, six liver function indicators showed a downward trend. We identified related metabolic pathways as glycerophospholipid metabolism, steroid biosynthesis, and the citrate cycle (TCA cycle). Conclusions and Relevance: This study shows that ACE stimulation of GB34 can effectively help treat choledocholithiasis, which may be clinically applicable to ACE.

Vascular endothelial growth factor levels in bile distinguishes pancreatic cancer from other etiologies of biliary stricture: a pilot study.

BACKGROUND: Determining the benign or malignant nature of biliary strictures can be challenging. Vascular endothelial growth factor (VEGF) plays an important role in tumor angiogenesis. OBJECTIVE: The purpose of this study was to investigate whether VEGF levels in bile aspirated during endoscopic retrograde cholangiography (ERCP) can distinguish pancreatic cancer from other causes of biliary stricture. METHODS: Bile was directly aspirated in 53 consecutive patients from March 2012 to October 2012 during ERCP from the common bile duct including 15 with pancreatic cancer, 18 with primary sclerosing cholangitis (PSC), nine with cholangiocarcinoma (CCA), and 11 with benign biliary conditions (sphincter of Oddi and choledocholihiasis). Levels of VEGF in bile were measured. The diagnostic performance was then validated in a second, independent validation cohort of 18 patients (pancreatic cancer n = 10, benign n = 8). RESULTS: A total of 53 consecutive patients were recruited. The median bile VEGF levels were significantly elevated in patients with pancreatic cancer (1.9 ng/ml (interquartile range [IQR] 0.7, 2.2) compared to those with benign biliary conditions (0.3 ng/ml [IQR 0.2, 0.6]; p < 0.001), PSC (0.7 ng/ml [IQR 0.5, 0.9]; p = 0.02) or CCA (0.4 ng/ml [IQR 0.1, 0.5]; p < 0.001). A VEGF cut-off value of 0.5 ng/ml distinguished pancreatic cancer from CCA with a sensitivity and specificity of 93.3 and 88.9 %, respectively, and area under curve (AUC) of 0.93, and from benign conditions with a sensitivity and specificity of 93.3 and 72.7 %, respectively, with AUC of 0.89. The diagnostic accuracy of biliary VEGF was confirmed in the second independent validation cohort. CONCLUSIONS: This study suggests that measurement of biliary VEGF-1 levels distinguishes patients with pancreatic cancer from other etiologies of biliary stricture. This may be particularly relevant in approaching patients with indeterminate biliary stricture.

Hepatolithiasis with secondary cholangitis and supernumerary left hepatic lobe.

We report the case of a 31-year old woman with recurrent cholangitis secondary to hepatolithiasis. The stones were composed of calcium bilirubinate. The patient also had a supernumerary hepatic lobe connected to the inferior aspect of the segment III of the liver. The role of the supernumerary hepatic lobe in the development of hepatolithiasis is unclear and may be coincidental.

Endocrine cells in the human common bile duct in patients with obstructive jaundice.

BACKGROUND/AIMS: There was no data about endocrine cells in the extrahepatic bile duct in secondary cholangitis due to obstructive jaundice. The aim of the present study is to investigate immunohistochemically the endocrine cell types in the lower part of the human common bile duct in biopsy samples, collected during drainage because of complete or incomplete obstruction, caused mainly by stones. We explained the presence of various hormone-producing endocrine cells in this region with the regulation of physiological and pathological processes there. METHODOLOGY: We used light and electron microscopic immunohistochemistry. RESULTS: More gastrin-positive, somatostatin-positive, secretin-positive, serotonin-positive, chromogranin- A-positive and synaptophysin-positive endocrine cells were found compared to control preparations. CONCLUSIONS: Occurrence of endocrine cells may relate to disturbed bile flow and to formation of calculi. Endocrine cell hyperplasia may be related to longstanding inflammation as in chronic cholecystitis and all secreted hormones from the described ECs can support pathologic process in the choledochus, i.e. inflammation, increased mucus secretion, fibrosis, muscle contraction, etc. We may state that various ECs (similar to those in duodenum) present in the lower part of the large bile duct and their hormones exert action on physiology (motility, secretion) and pathology (inflammation and fibrosis) in that part of the biliary tree.

Differences in phosphatidylcholine and bile acids in bile from Egyptian and UK patients with and without cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CC) is a fatal malignancy, the incidence of which is increasing worldwide, with substantial regional variation. Current diagnostic techniques to distinguish benign from malignant biliary disease are unsatisfactory. Metabolic profiling of bile may help to differentiate benign from malignant disease. No previous studies have compared the metabolic profiles of bile from two geographically and racially distinct groups of CC patients. OBJECTIVES: This study aimed to compare metabolic profiles of bile, using in vitro proton magnetic resonance spectroscopy, from CC patients from Egypt and the UK, and from patients with CC and patients with non-malignant biliary disease. METHODS: A total of 29 bile samples, collected at cholangiography, were analysed using an 11.7-T system. Samples were from eight CC patients in either Egypt (n = 4) or the UK (n = 4) and 21 patients with benign biliary disease (choledocholithiasis [n = 8], sphincter of Oddi dysfunction [n = 8], primary sclerosing cholangitis [n = 5]). RESULTS: Bile phosphatidylcholine (PtC) was significantly reduced in CC patients. Egyptian CC patients had significantly lower biliary PtC levels compared with UK patients. Taurine- and glycine-conjugated bile acids (H-26 and H-25 protons, respectively) were significantly elevated in bile from patients with CC compared with bile from patients with benign diseases (P = 0.013 and P < 0.01, respectively). CONCLUSIONS: Biliary PtC levels potentially differentiate CC from benign biliary disease. Reduced biliary PtC in Egyptian compared with UK patients may reflect underlying carcinogenic mechanisms.

Efficacy of Lidan Tang on high-fat-diet induced hepatolithiasis in mice and possible mechanism.

OBJECTIVE: To investigate efficacy of Lidan Tang (LDT) on gallstone induced by high fat diet in mice, and to study its underlying mechanism. METHODS: Mice were fed with high fat diet every day and treated with LDT (9.01 times of human clinic dosage). Mice were randomly divided into 6 groups as control group, gallstone model group (high-fat diet), positive control ursodeoxycholic acid (UDCA) group (80 mg·kg-1·d-1, i.g.), LDT low dose group (6 kg/d, i.g.), LDT middle dose group (12 kg/d, i.g.), and LDT high dose group (24 kg/d, i.g.). The whole experiment was lasted for 4 weeks. The levels of ALT, AST, LDH, CHO, HDL-C and LDL-C in serum were measured, the pathological sections were observed by hematoxylin-eosin staining, the activities of antioxidant enzymes were measured by kits, and the proteins related to oxidative stress and lipid transport were detected by Western blot analysis. RESULTS: LDT could significantly reduce the contents of ALT and AST in serum and improve the pathological tissue of liver. LDT could significantly reduce the content of MDA and LPO, and increase the level of GSH and GSH-PX in liver tissue. The data of Western blot showed that LDT had antioxidant effect promoting Keap1/Nrf2 pathway and regulated the process of lipid transport, which was statistically significant. In addition, LDT treatment inhibited the expression of ATP-binding cassette transports ABCG5/8 in liver, and reduced cholesterol transport from the hepatocytes to the gallbladder. CONCLUSION: LDT has protective effect on gallstones induced by high fat diet in mice, which might be based on the protective effect on liver, including enhancing the antioxidant capacity of liver and reducing the production of lipid peroxides.

Is routine MR cholangiopancreatography (MRCP) justified prior to cholecystectomy?

PURPOSE: We investigated routinely the bile ducts by magnetic resonance cholangiopancreaticography (MRCP) prior to cholecystectomy. The aim of this study was to analyze the rate of clinically inapparent common bile duct (CBD) stones, the predictive value of elevated liver enzymes for CBD stones, and the influence of the radiological results on the perioperative management. METHODS: In this prospective study, 465 patients were cholecystectomized within 18 months, mainly laparoscopically. Preoperative MRCP was performed in 454 patients. RESULTS: With MRCP screening, clinically silent CBD stones were found in 4%. Elevated liver enzymes have only a poor predictive value for the presence of CBD stones (positive predictive value, 21%; negative predictive value, 96%). Compared to the recent literature, the postoperative morbidity in this study was low (0 bile duct injury, 0.4% residual gallstones). CONCLUSIONS: Although MRCP is diagnostically useful in the perioperative management in some cases, its routine use in the DRG-era may not be justified due to the costs.

MDR3 mutations associated with intrahepatic and gallbladder cholesterol cholelithiasis: an update.

BACKGROUND: The recurrent microlithiasis represents one of the most frequent clinical forms of lithiasis of the bile ducts. This affection is characterized by the presence of cholesterolic microgallstones on hepatic canaliculars, and belongs to a heterogeneous group of autosomal recessive liver disorders. Radiological diagnosis can be confirmed by analysis of MDR3 gene, coding a protein involved in physiologic translocation of phospholipids in bile. Discovery of MDR3 mutations is of particular interest, since normally associated with good effectiveness of medication by ursodesoxycholic acid. AIM: To review MDR3 mutations in humans associated with recurrent cholesterol microlithiasis and to suggest a practical approach for MDR3 gene analysis. RESULTS: 48 mutations of MDR3 gene have been reported in humans to date, from which 43 (89.5%) in the coding region, and 5 splice site mutations have been associated to cholesterol cholelithiasis. 21 (43.8%) of the 43 precited mutations are located in only 8 exons on 28, near transmembrane or nucleotide binding domains of the protein. From the 22 remaining described mutations, 9 (18.8%) are restricted to exon 14. We suggest therefore to start analysis of MDR3 gene by screening exons 6, 7, 9, 10, 12, 14, 17, 23 and 24 with an appropriate protocol in this diagnosis associated with effective treatment. In conclusion such therapeutic orientation is valuable, since recurrent cholesterolic microlithiasis occurs relatively early in life, and by the fact that recurrence of symptoms may occur despite cholecystectomy, or shock-wave therapy.

Promoter methylation of INK4a/ARF as detected in bile-significance for the differential diagnosis in biliary disease.

PURPOSE: There is a need to enhance endobiliary cytotechniques by molecular marker lesions. This is of special significance for patients with primary sclerosing cholangitis, a disease predisposing for the development of cholangiocarcinoma. The INK4a/ADP ribosylation factor (ARF) locus encodes two tumor suppressor genes: p16INK4a and p14ARF. p16INK4a has been shown to be of major significance in cholangiocarcinoma. EXPERIMENTAL DESIGN: In an effort to evaluate the potential diagnostic role of p16INK4a and p14ARF promoter methylation in biliary disease, endoscopical obtained bile specimens of 71 patients were analyzed (26 choledocholithiasis, 6 with normal results, 23 bile duct carcinoma, 5 gall bladder carcinoma). Eleven patients with primary sclerosing cholangitis were enrolled. RESULTS: Merely 6% of specimens (2 of 32) obtained from patients without evidence for malignant biliary disease but 53.5% of malignancies (15 of 28) showed p16 promoter methylation (p14: 3 and 46.2%, respectively). The concordance of methylation rates detected in either bile or tissue specimens was high. In primary sclerosing cholangitis, a similar prevalence of methylation was detected as in malignant disease. CONCLUSIONS: This study demonstrates: (a) a high frequency and specificity of INK4a/ARF methylation in malignant biliary disease compared with mere cholangitis; and (b) the capability to detect these alterations reliably in endoscopically obtained bile. Thus, INK4a/ARF's promoter methylation status represents a candidate marker for the endoscopic diagnosis of biliary disease.

Abnormal liver function tests in the symptomatic pregnant patient: the local experience in Singapore.

INTRODUCTION: The causes of abnormal liver function tests in pregnancy are varied and may or may not be pregnancy-related. Often, the diagnosis can be difficult. This study looked at the causes of deranged liver function tests in obstetric patients with significant symptoms and signs. MATERIALS AND METHODS: Data from 50 cases of abnormal liver function tests in pregnant patients, who presented from 1998 to 2001, were analysed. Their presenting symptoms included persistent vomiting (48%), pruritus (14%), jaundice (26%), upper abdominal discomfort (24%) and hypertension (46%). RESULTS: Pregnancy-related causes accounted for 84% of the abnormal liver function tests. Abnormal liver function tests occurred more frequently in the first (34%) and third (58%) trimesters than in the second trimester (8%). Hyperemesis gravidarum (94%) and partial haemolysis, elevated liver enzymes and low platelets (HELLP) syndrome (31%) were the commonest causes in the first and third trimesters respectively. Hepatitis B flare resulted in 2 maternal deaths. Seven patients with pre-eclampsia toxaemia, acute fatty liver of pregnancy or partial/complete HELLP syndrome had their liver function tests measured sequentially before and after delivery. All of them showed rapid improvement postpartum with their alanine aminotransferase (ALT) dropping 50% within 3 days. CONCLUSIONS: The majority of patients with abnormal liver function tests had a cause related to pregnancy, and pregnancy-related causes in the third trimester improved rapidly postpartum. Hepatitis B flare was a significant non-obstetric cause leading to maternal mortality. This diagnosis must therefore be considered in ethnic groups where the incidence of chronic hepatitis B infection is high, especially in chronic hepatitis B carriers with suspected pregnancy-related disease who deteriorate postpartum.

[Dynamic of nonspecific markers of endogenous intoxication under the influence of enteral probe nutrition in patients with choledocholithiasis]. / Dynamika nespetsyfichnykh markeriv endohennoï intoksykatsiï pid vplyvom enteral'noho kharchuvannia u khvorykh na kholedokholitiaz.

Results of the dynamic investigation of the endogenous intoxication nonspecific markers in 107 patients suffering choledocholithiasis were presented. Under the influence of enteral probe nutrition, using oligosized mixture Peptamen (Nestle), in patients of the main group the endogenous intoxication nonspecific markers normalization had occurred faster than in a control group.

[Reduction of severity of endogenous intoxication in patients with choledocholithiasis under the influence of the probe enteral nutrition]. / Zmenshennia tiazhkosti endohennoï intoksykatsiï u khvorykh na kholedokholitiaz pid vplyvom enteral'noho zondovoho kharchuvannia.

Results of study of the endogenous intoxication severity (EI) in 107 patients for choledocholithiasis (CL) were presented. In complex of treatment the probe enteral nutrition (PEN) was applied. Diagnosis of EI according to cellular indexes of intoxication and the peptides level of the average molecular mass (PAMM) in blood for wavelengths 254 and 280 nm was conducted. There was established, that leukocytic index of intoxication, calculated according to formula Himich-Kostyuchenko, have strong correlational causation with index of PAMM and may be applied for diagnosis and control of efficacy of the EI correction in patients with CL. PEN with application of oligopeptide mixture "Peptamen" (Nestle) had promoted effective correction of EI, what have allowed to lower frequency of postoperative complications by 11% and to shorten the treatment duration of patients after operation by 4 days.

Correlation of inflammation parameters and biochemical markers of cholestasis with the intensity of lipid peroxidation in patients with choledocholithiasis.

BACKGROUND/AIM: During choledocholitiasis inflammatory oxidant stress involves the promotion of mitochondrial dysfunction through an intracellular oxidant stress in hepatocytes leading mainly to necrosis and less to apoptosis. The product of oxidative stress, malondialdehyde (MDA), is extremely cytotoxic and damages cell membranes and intracellular macromolecules. The toxicity of MDA is based on its ability to act as a mutagenic agent in a cell. Therefore, the aim of this prospective study was to establish correlation of the parameters of inflammation and biochemical markers of cholestasis with the intensity of oxidative stress in pathogenesis of liver function disorders. METHODS: Seventy adult subjects of either sex included in the study were devided into two groups: I--40 patients with obstructive icterus caused by choledocholithiasis, and II--30 healthy individuals. All the participants were subjected to a clinical, laboratory and ultrasonic check-up at the Internal Department of the Military Hospital in Nis. The parameters of oxidative stress: MDA, a measure of lipid peroxidation, and inflammation parameters: C-reactive protein (CRP), fibrinogen, albumins, number of leukocytes (Leu), granulocytes (Gr), lymphocytes (Ly) and monocytes (Mo) and biochemical markers of cholestasis: activity of gamma-glutamyltransferase (gamma-GT) and alkaline phosphatase (AP) enzymes, the level of total, direct and indirect bilirubin were determined by standard biochemical methods. RESULTS: Lower values of albumin (p < 0.001), and significantly higher values of fibrinogen (p < 0.05) and CRP (p < 0.001) were found in the blood of the patients with cholestasis due to choledocholithiasis in relation to the controls. Significantly higher values of Leu (p < 0.01) and Gr (p < 0.001) with decreasing number of Ly (p < 0.001) and Mo (p < 0.001) were found in blood of the patients with cholestasis due to choledocholithiasis in relation to the control. Similarly, higher values of gamma-GT, and AP (p < 0.001), as well as the level of total, direct and indirect bilirubin (p < 0.001) were found in blood of the patients with cholestasis due to choledocholithiasis in relation to the controls. The concentration of MDA (p < 0.001) was increased in the patients with choledocholithiasis in relation to the controls. There was a significant positive linear correlation of the number of leukocytes (r = 0.51, p < 0.05) and the concentration of total (r = 0.87, p < 0.01), direct (r = 0.85, p < 0.01) and indirect (r = 0.88, p < 0.01) bilirubin with the concentration of MDA in the group of patients with choledocholithiasis. CONCLUSION: Neutrophils and the levels of total, direct and indirect bilirubin have a significant positive linear correlation with the level of lipid peroxidation in patients with choledocholithiasis. Neutrophilia and hiperbilirubinemia observed in this way represent important parameters in estimating the level of liver tissue damage in choledocholithiasis.

Transthyretin, identified by proteomics, is overabundant in pancreatic juice from pancreatic carcinoma and originates from pancreatic islets.

Analyses of pancreatic juice by proteomics have identified many proteins that are overabundant in pancreatic cancer (PC) juice. The mechanism by which secretion of these proteins occur remains unclear. Pancreatic juice was collected from patients with three pancreatic diseases: PC, chronic pancreatitis (CP), and simple choledocholithiasis (CDS), and analyzed by 2-DE, MALDI-TOF/MS, and Western blot. Five PC cell lines, 30 PC tissues and their corresponding adjacent pancreatic tissues were used to validate the expression of genes which code for overabundant proteins in PC juice. The mRNA and protein levels were measured by RT-PCR and immunohistochemistry, respectively. Using proteomics, it was demonstrated that the protein transthyretin (TTR) was upregulated more than 2-fold in PC juice compared with CP and CDS, while apolipoprotein A-I, lithostathine, and regenerating islet-derived 1 beta precursor were downregulated more than 2-fold. Western blots confirmed that TTR was overabundant in the PC juice. However, TTR mRNA was not detected in any of the five PC cell lines, and was only detected in islet cells. By microscopy, it was shown that islet architecture was almost completely destroyed, and the islet's maximum diameter appeared larger in PC tissues than in normal. Some overabundant proteins in PC juice, such as TTR expressed only in islets, leak into the pancreatic ductal system due to hyperplasia and architectural damage in PC tissues. The destruction of organ and tissue architecture by tumor growth may result in novel tumor markers even if the markers are not secreted directly by tumor cells.

Relationship of CA 19-9 with choledocholithiasis and cholangitis.

BACKGROUND/AIMS: It is well known that increased concentrations of CA 19-9 can be found in benign disease of the liver, pancreas and biliary tract, especially in cases with gallstone disease with cholangitis. The aim of this study was to investigate the relation of CA 19-9 with the number and size of the stones, cholangitis and biliary obstruction in patients with choledocholithiasis. METHODS: Seventy patients with radiologically proven choledocholithiasis were studied. Endoscopic retrograde cholangiopancreatography, sphincterotomy and stone extraction were applied to all patients. In each case, the parameters recorded included the levels of CA 19-9 and other laboratory tests before and after endoscopic retrograde cholangiopancreatography and the results of imaging techniques and immunoserologic tests. The correlations of these parameters were determined by SPSS 17 package program for statistical analysis. RESULTS: Elevation of CA 19-9 was found in 32 patients (46%), while 8 patients (11%) had extraordinarily high levels (>1000 U/ml). CA 19-9 levels were correlated with serum alkaline phosphatase (r=0.5, p<0.01), gamma glutamyl transpeptidase (r=0.5, p<0.01) and bilirubin (r=0.4, p<0.01) levels but not with aspartate aminotransferase or alanine aminotransferase levels. There was also no association between serum CA 19-9 levels and the number and size of stones. Six patients had cholangitis. CA 19-9 levels were found higher in patients with cholangitis than others (100% vs. 41%, p<0.01) as well as alkaline phosphatase, gamma glutamyl transpeptidase and bilirubin levels. After stone extraction, CA 19-9 levels started to decrease and reached normal values 1-28 days later. CONCLUSION: In conclusion, CA 19-9 levels are associated with biliary obstruction and cholangitis but not with the number and size of stones in patients with choledocholithiasis.

Identification of pancreatic juice proteins as biomarkers of pancreatic cancer.

Pancreatic juice is a potential source of proteins associated with pancreatic cancer (PC) due to the proximity of ducts to tumor tissue. Therefore, screening of proteins in pancreatic juice from PC patients may identify new PC biomarkers. We analyzed pancreatic juice from patients with pancreatic diseases including PC, chronic pancreatitis (CP) and simple choledocholithiasis (CDS) by 2-DE. Protein spots from PC patients that changed >2-fold compared with both CP and CDS were selected and identified by mass spectrometry (MS). mRNA levels were measured by QRT-PCR in PC cell lines, PC tissues and adjacent pancreatic normal (PN) tissues. Relationships between mRNA levels in PC tissues and their clinical characteristics and promoter methylation were analyzed in PC cell lines and tissues. We found that four proteins were significantly changed in PC compared to CP and simple CDS. Two proteins were up-regulated, serine proteinase-2 (PRSS2) preproprotein and pancreatic lipase-related protein-1 (PLRP1), and two proteins were down-regulated, chymotrypsinogen B (CTRB) precursor and elastase 3B (ELA3B) preproprotein. In all PC cell lines, PRSS 2 mRNA levels were elevated, while PLRP 1 mRNA was detected in 4/5 cell lines. ELA3B mRNA was undetectable in all cell lines, but CTRB mRNA was detected in 2/5 cell lines. In PC tissues compared to PN, levels of PRSS2 mRNA were significantly higher, ELA3B significantly lower, and PLRP1 and CTRB not significantly different. Elevated PRSS2 mRNA levels correlated with high T stage. The ELA3B gene promoter had higher methylation in PC cell lines and tissues compared with PN tissues, and correlated with low ELA3B gene expression. In conclusion, comparative proteomic analysis of pancreatic juice from PC patients is a powerful method to find new PC biomarkers. Hyperexpression of the PRSS2 gene and hypermethylation of ELA3B gene promoter were associated with PC, raising the possibility of their application as new biomarkers in PC diagnosis and screening.

Prostaglandin E receptors in bile ducts of hepatolithiasis patients and the pathobiological significance for cholangitis.

BACKGROUND & AIMS: In hepatolithiasis, chronic proliferative cholangitis may influence the progression of the disease. Prostaglandin (PG) E(2) experimentally causes morphologic changes to intrahepatic bile ducts, analogous to the changes found in cholangitis. This study was designed to gain an understanding of the involvement of PGE(2) and PGE receptor (EP) subtypes in the development of cholangitis. METHODS: The expression levels of secretory-type group IIA phospholipase A(2) (sPLA(2)-IIA) and cyclooxygenase (COX)-2 as well as EP subtypes were determined in the bile ducts with change of cholangitis. In in vitro experiments, growth promotion and mucin secretagogue properties of biliary epithelial cells in response to EP-selective agonists or antagonists were studied. RESULTS: The messenger RNA (mRNA) level of sPLA(2)-IIA and the protein and mRNA levels of COX-2 were significantly increased in the bile ducts of patients with hepatolithiasis compared with the levels of the bile ducts of control subjects. These changes were associated with a concomitant increase in PGE(2) and total mucin concentrations in the bile. The mRNAs of EP subtypes EP(2), EP(3), and EP(4) but not EP(1) were amplified in the bile ducts. Treatment with an EP(4)-selective agonist (ONO-AE1-329) caused a dose-dependent increase in DNA synthesis, colony number, and mucin secretion in the cells. Conversely, treatment with an EP(4)-selective antagonist (ONO-AE3-208) abolished the biological effects of PGE(2) on the cells. CONCLUSIONS: In hepatolithiasis, an enhanced synthesis of sPLA(2)-/COX-2-derived PGE(2) and its actions mediated via the EP(4) receptor in the bile ducts may be of pathobiological significance for chronic proliferative cholangitis.