Variants and haplotypes in Flap endonuclease 1 and risk of gallbladder cancer and gallstones: a population-based study in China.

The role of FEN1 genetic variants on gallstone and gallbladder cancer susceptibility is unknown. FEN1 SNPs were genotyped using the polymerase chain reaction-restriction fragment length polymorphism method in blood samples from 341 gallbladder cancer patients and 339 healthy controls. The distribution of FEN1-69G > A genotypes among controls (AA, 20.6%; GA, 47.2% and GG 32.2%) was significantly different from that among gallbladder cancer cases (AA, 11.1%; GA, 48.1% and GG, 40.8%), significantly increased association with gallbladder cancer was observed for subjects with both the FEN1-69G > A GA (OR = 1.73, 95% CI = 1.01-2.63) and the FEN1-69G > A GG (OR = 2.29, 95% CI = 1.31-3.9). The distribution of FEN1 -4150T genotypes among controls (TT, 21.8%;GT, 49.3% and GG 28.9%) was significantly different from that among gallbladder cancer cases (TT, 12.9%; GT, 48.4% and GG 38.7%), significantly increased association with gallbladder cancer was observed for subjects with both the FEN1-4150T GT(OR = 1.93, 95% CI = 1.04-2.91) and the FEN1-4150T GG(OR = 2.56, 95% CI = 1.37-5.39). A significant trend towards increased association with gallbladder cancer was observed with potentially higher-risk FEN1-69G > A genotypes (P < 0.001, χ2 trend test) and FEN14150G > T (P < 0.001, χ2 trend test) in gallstone presence but not in gallstone absence (P = 0.81, P = 0.89, respectively). In conclusion, this study revealed firstly that FEN1 polymorphisms and haplotypes are associated with gallbladder cancer risk.

PON3 knockout mice are susceptible to obesity, gallstone formation, and atherosclerosis.

We report the engineering and characterization of paraoxonase-3 knockout mice (Pon3KO). The mice were generally healthy but exhibited quantitative alterations in bile acid metabolism and a 37% increased body weight compared to the wild-type mice on a high fat diet. PON3 was enriched in the mitochondria-associated membrane fraction of hepatocytes. PON3 deficiency resulted in impaired mitochondrial respiration, increased mitochondrial superoxide levels, and increased hepatic expression of inflammatory genes. PON3 deficiency did not influence atherosclerosis development on an apolipoprotein E null hyperlipidemic background, but it did lead to a significant 60% increase in atherosclerotic lesion size in Pon3KO mice on the C57BL/6J background when fed a cholate-cholesterol diet. On the diet, the Pon3KO had significantly increased plasma intermediate-density lipoprotein/LDL cholesterol and bile acid levels. They also exhibited significantly elevated levels of hepatotoxicity markers in circulation, a 58% increase in gallstone weight, a 40% increase in hepatic cholesterol level, and increased mortality. Furthermore, Pon3KO mice exhibited decreased hepatic bile acid synthesis and decreased bile acid levels in the small intestine compared with wild-type mice. Our study suggests a role for PON3 in the metabolism of lipid and bile acid as well as protection against atherosclerosis, gallstone disease, and obesity.

TG-interacting factor 1 acts as a transcriptional repressor of sterol O-acyltransferase 2.

Acat2 [gene name: sterol O-acyltransferase 2 (SOAT2)] esterifies cholesterol in enterocytes and hepatocytes. This study aims to identify repressor elements in the human SOAT2 promoter and evaluate their in vivo relevance. We identified TG-interacting factor 1 (Tgif1) to function as an important repressor of SOAT2. Tgif1 could also block the induction of the SOAT2 promoter activity by hepatocyte nuclear factor 1α and 4α. Women have ∼ 30% higher hepatic TGIF1 mRNA compared with men. Depletion of Tgif1 in mice increased the hepatic Soat2 expression and resulted in higher hepatic lipid accumulation and plasma cholesterol levels. Tgif1 is a new player in human cholesterol metabolism.

Gallstone disease in Swedish twins is associated with the Gilbert variant of UGT1A1.

BACKGROUND & AIMS: The Gilbert syndrome-associated functional TATA box variant UGT1A1*28 (A(TA)7TAA) was found to increase susceptibility to pigment gallstone formation in patients with haemolytic anaemia. Further studies in extensive cohorts demonstrated an increased risk of this variant for cholesterol gallstone disease (GD). We now investigated this polymorphism as a determinant of symptomatic GD in Swedish twins. METHODS: The Swedish Twin Registry was merged with the Hospital Discharge and Causes of Death Registries and searched for GD-related diagnoses among monozygotic (MZ) twins living in the Stockholm area. In addition, we screened the TwinGene database for GD. In total, we found 44 MZ twin pairs with and eight MZ twins without GD to be evaluable. GD-free twins from TwinGene (109 concordantly MZ and 126 independent DZ) served as controls. UGT1A1*28 genotyping was performed using TaqMan assays. RESULTS: Overall, 58 and 8 of 106 twins with GD were hetero- and homozygous UGT1A1 risk allele carriers respectively. The case-control association tests showed a significantly (P < 0.05) increased risk of developing GD (OR = 1.62, 95% CI 1.00-2.63) in heterozygotes carriers and in addition, a trend (P = 0.075) for an increased risk among carriers (OR = 1.52, 95% CI 0.97-2.44) of the risk allele. CONCLUSION: These data from Swedish twins confirm the Gilbert variant as risk factor for GD. Our observation is in line with nucleation in bilirubin supersaturated bile representing an initial step in cholelithogenesis.

Evaluation of lymphocytes CD4+ and CD8+ and expression of ZAP-70 kinase on CD3+ and CD19+ lymphocytes in obese patients undergoing laparoscopic cholecystectomy.

BACKGROUND: Obesity has become a global epidemic and a leading metabolic disease in the world. Laparoscopic surgeries may influence the function of the immunologic system. The percentages of CD4+ and CD8+ T lymphocyte cells have been described as prognostic factors for patients undergoing abdominal surgeries. This study aimed to evaluate the changes in CD4+ and CD8+ T lymphocyte cells, the ratio of CD4+ to CD8+ cells, and the ZAP-70 kinase expression on T CD3+ and B CD19+ cells in obese and normal-weight individuals undergoing laparoscopic cholecystectomy (LC). METHODS: The study group consisted of 46 asymptomatic patients with gallstones shown by ultrasound examination but without signs of any gallbladder complications. The patients underwent planned LC. Blood samples were obtained at three times, and the percentages of studied cells were measured by flow cytometry. Patients were enrolled to two groups: N group (body mass index [BMI], ≤ 25 kg/m(2)) and O group (BMI, ≥ 30 kg/m(2)). For statistical analysis, the Mann-Whitney U test and the Wilcoxon matched-pairs signed-ranks test were used. All p values lower than 0.05 were considered significant. RESULTS: The percentage of CD4+ T cells did not differ between the N and O groups before or after the surgery. Only in the N group did the percentage of CD4+ lymphocytes increase from 0 to 48 h. A higher percentage of CD8+ lymphocytes was observed in the O group postoperatively than in the N group. Differences of ZAP-70 kinase expression in the O group were observed at 24 and 48 h of the study. Decreased expression of ZAP-70 kinase was shown in the N group at both 0-24 and 24-48 h. In the O group, this tendency was noted at 24-48 h. CONCLUSIONS: Immunologic activation after LC was confirmed in both weight groups. However, higher modulation, more typical for open surgeries, was observed in the obese group.

Disruption of the murine protein kinase Cbeta gene promotes gallstone formation and alters biliary lipid and hepatic cholesterol metabolism.

The protein kinase C (PKC) family of Ca(2+) and/or lipid-activated serine-threonine protein kinases is implicated in the pathogenesis of obesity and insulin resistance. We recently reported that protein kinase Cß (PKCß), a calcium-, diacylglycerol-, and phospholipid-dependent kinase, is critical for maintaining whole body triglyceride homeostasis. We now report that PKCß deficiency has profound effects on murine hepatic cholesterol metabolism, including hypersensitivity to diet-induced gallstone formation. The incidence of gallstones increased from 9% in control mice to 95% in PKCß(-/-) mice. Gallstone formation in the mutant mice was accompanied by hyposecretion of bile acids with no alteration in fecal bile acid excretion, increased biliary cholesterol saturation and hydrophobicity indices, as well as hepatic p42/44(MAPK) activation, all of which enhance susceptibility to gallstone formation. Lithogenic diet-fed PKCß(-/-) mice also displayed decreased expression of hepatic cholesterol-7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8b1). Finally, feeding a modified lithogenic diet supplemented with milk fat, instead of cocoa butter, both increased the severity of and shortened the interval for gallstone formation in PKCß(-/-) mice and was associated with dramatic increases in cholesterol saturation and hydrophobicity indices. Taken together, the findings reveal a hitherto unrecognized role of PKCß in fine tuning diet-induced cholesterol and bile acid homeostasis, thus identifying PKCß as a major physiological regulator of both triglyceride and cholesterol homeostasis.

Study of serum lipase, alpha-amylase and pancreatic amylose in gall-stone diseases.

Silent gall-stone causes significant morbidity and mortality and its incidence in India as well as in whole world is on the rise. It has positive correlation with development of carcinoma gall bladder. So far no predictive study has been done to show its correlation with biochemical markers. The present study has been aimed to establish whether simple enzymatic markers can predict association with cholelithiasis. Study group has been selected from the patients attending general surgery OPD of a tertiary healthcare centre with complaints of vague abdominal pain, flatulence and dyspepsia. A total of 61 cases (male = 18, female = 43) were studied and data matched with age and sex matched control. The biochemical markers studied are serum alkaline phosphatase, serum lipase, serum alpha-amylase and serum pancreatic amylase. Patients with obstructive cholelithiasis, duct stones, pancreatic insufficiency and malignancy are excluded from the study. The results were analysed by Student's t-test. Alkaline phosphatase in all the above mentioned cases was not significantly different from the control group (40 female, 21 male healthy individuals). A significant association was found out with serum alpha-amylase (p < 0.05) and a highly significant association was found out with pancreatic amylase (p < 0.001). Results of serum lipase however were inconclusive (p = 0.1). Pancreatic amylase can be estimated at a reasonable cost and costwise may prove to be a marker of gall-stone diseases which are in many cases silent preventing further complications and chances of Malignancy especially where alkaline phosphatase isinconclusive.

ACAT2 and human hepatic cholesterol metabolism: identification of important gender-related differences in normolipidemic, non-obese Chinese patients.

OBJECTIVE: ACAT2 is a major cholesterol esterification enzyme specifically expressed in hepatocytes and may control the amount of hepatic free (unesterified) cholesterol available for secretion into bile or into HDL. This study aims to further elucidate physiologic roles of ACAT2 in human hepatic cholesterol metabolism. METHODS AND RESULTS: Liver biopsies from 40 normolipidemic, non-obese gallstone patients including some gallstone-free patients (female/male, 18/22) were collected and analyzed for microsomal ACAT2 activity, protein and mRNA expression. Plasma HDL-cholesterol (HDL-C) was significantly higher in females than in males, while triglycerides were significantly lower. ACAT2 activity in females was significantly lower than observed in males, regardless of the presence of gallstone disease. Moreover, the activity of ACAT2 correlated negatively with plasma levels of HDL-C (r=-0.57, P<0.05) and with Apo AI (r=-0.49, P<0.05). CONCLUSION: This is the first description of a gender-related difference in hepatic ACAT2 activity in normolipidemic non-obese Chinese patients suggesting a possible role for ACAT2 in the regulation of cholesterol metabolism in humans. The negative correlation between ACAT2 activity and HDL-C or Apo AI may reflect this regulation. Since ACAT2 activity generally has been found to be pro-atherogenic in animal models, the observed sex-related difference may contribute to female protection from complications of coronary heart disease (CHD).

Cholesterol 7alpha-hydrolase (CYP7A1) c.-278A>C promoter polymorphism in gallstone disease patients.

There is growing evidence that gallstone formation may be genetically determined. Cholesterol 7alpha-hydrolase (CYP7A1) is an enzyme that catalyzes the first, rate-limiting reaction of cholesterol catabolic pathway. Recently, a common c.-278A>C polymorphism (rs3808607:G>T) has been described in CYP7A1 gene, associated with altered plasma lipid levels. The aim of this study was to verify the finding that CYP7A1 polymorphism may be associated with gallstone disease. Frequency and distribution of the studied alleles did not differ significantly between the patients (-278C; minor allele frequency: 0.45) and the controls (0.48). No significant gender-related differences of allele frequencies or distribution were noted. We conclude that CYP7A1 promoter polymorphism is not a valuable marker of gallstone disease susceptibility in a Polish population.

Hypocholesterolemic effects of fatty acid bile acid conjugates (FABACs) in mice.

Fatty acid bile acid conjugates (FABACs) prevent and dissolve cholesterol gallstones and prevent diet induced fatty liver, in mice. The present studies aimed to test their hypocholesterolemic effects in mice. Gallstone susceptible (C57L/J) mice, on high fat (HFD) or regular diet (RD), were treated with the conjugate of cholic acid with arachidic acid (FABAC; Aramchol). FABAC reduced the elevated plasma cholesterol levels induced by the HFD. In C57L/J mice, FABAC reduced plasma cholesterol by 50% (p<0.001). In mice fed HFD, hepatic cholesterol synthesis was reduced, whereas CYP7A1 activity and expression were increased by FABAC. The ratio of fecal bile acids/neutral sterols was increased, as was the total fecal sterol excretion. In conclusion, FABACs markedly reduce elevated plasma cholesterol in mice by reducing the hepatic synthesis of cholesterol, in conjunction with an increase of its catabolism and excretion from the body.

Hyperamylasemia induced by percutaneous papillary balloon dilatation for symptomatic choledocholithiasis.

BACKGROUND/AIMS: We investigated the relationship between percutaneous papillary balloon dilatation (PPBD) and hyperamylasemia after PPBD. METHODOLOGY: We studied the rate of pancreatitis and asymptomatic hyperamylasemia after PPBD for choledocholithiasis in 64 symptomatic patients. Pancreatitis was defined as epigastric pain combined with at least a 3-fold rise in serum amylase at 24 hours after PPBD. Asymptomatic hyperamylasemia was defined as a rise in serum amylase (normal range, 50 to 160 IU/L) without epigastric pain. RESULTS: The stones were successfully pushed out into the duodenum in all patients. Three patients developed post-PPBD pancreatitis, graded moderate in one and mild in two. Serum amylase values were elevated over the normal upper limit in 21 patients, 33%, over 3-fold in 10, 16% over 1000 IU/L in 6, 9%. Asymptomatic hyperamylasemia was observed in 18 patients. The amylase value after PPBD was elevated to more than 160 IU/L in 44% (17/39) of patients 80 years old or under vs. 16% (4/25) of patients older than 80 and in 23% (10/44) of patients with intrahepatic bile duct dilatation on admission vs. 55% (11/20) of patients without it, with a significant difference, respectively (p<0.05). The amylase value after PPBD was elevated to more than 1000 IU/L in 15% (6/39) of patients 80 years old or under vs. 0% (0/25) of patients older than 80 and in 29% (4/14) of patients with bile duct stones having a horizontal diameter of 8mm or smaller and 4% (2/50) of patients with stones larger than 8mm (p < 0.05 and p<0.01, respectively). CONCLUSIONS: We believe that postoperative continuous decompression of the bile duct by PPBD is reliable and that it contributed to the prevention of severe pancreatitis. We conclude that PPBD can be performed more safely in symptomatic patients older than 80 with choledocholithiasis with intrahepatic bile duct dilatation at the time of admission.

Structural examination of tryptase-, and VIP-positive mast cells in the common bile duct of patients with lithiasis.

The morphology of tryptase-, and vasoactive intestinal polypeptide (VIP)-positive mast cells was examined immunohistochemically in 38 common bile ducts collected from patients with secondary chronic cholangitis and varying degrees of inflammatory activity. Mast cells numbers in chronic exacerbated and chronic sclerotic cholangitis were significantly higher as compared with those in controls (72.4 cells/mm2 and 25.2 cells/mm2 vs. 5.9 cells/mm2; p < 0.0001, Student's t test). The increased number of tryptase-positive mast cells in chronic exacerbated cholangitis correlated with the severeness of inflammatory infiltration. In cases of chronic exacerbated cholangitis, the increased number of mast cells was detected in conjunction with active fibroplasia. In chronic sclerotic cholangitis mast cells were lower in number as compared with exacerbated cholangitis and were observed in relation with inactive fibrosis. Numerous VIP-positive mast cells were found in all patients with cholangitis. Ultrastructural immunocytochemistry showed tryptase positivity to be localized over either electron-dense or particulate granules with a mean diameter of 0.261+/-0.073 microm or 0.171+/-0.053 microm, respectively. VIP positivity was formed as a finely or coarsely granular pattern over larger electron-dense granules of 0.475+/-0.14 microm in diameter. Tryptase-positive mast cells were located mainly in and around surface and glandular epithelium. The involvement of tryptase- and VIP-positive mast cells in inflammation, fibrosis and epithelial reactions in the common bile duct is discussed.

Pigment gallstone pathogenesis: slime production by biliary bacteria is more important than beta-glucuronidase production.

Pigment stones are thought to form as a result of deconjugation of bilirubin by bacterial beta-glucuronidase, which results in precipitation of calcium bilirubinate. Calcium bilirubinate is then aggregated into stones by an anionic glycoprotein. Slime (glycocalyx), an anionic glycoprotein produced by bacteria causing foreign body infections, has been implicated in the formation of the precipitate that blocks biliary stents. We previously showed that bacteria are present within the pigment portions of gallstones and postulated a bacterial role in pigment stone formation through beta-glucuronidase or slime production. Ninety-one biliary bacterial isolates from 61 patients and 12 control stool organisms were tested for their production of beta-glucuronidase and slime. The average slime production was 42 for biliary bacteria and 2.5 for stool bacteria (P <0.001). Overall, 73% of biliary bacteria and 8% of stool bacteria produced slime (optical density >3). In contrast, only 38% of biliary bacteria produced beta-glucuronidase. Eighty-two percent of all patients, 90% of patients with common bile duct (CBD) stones, 100% of patients with primary CBD stones, and 93% of patients with biliary tubes had one or more bacterial species in their stones that produced slime. By comparison, only 47% of all patients, 60% of patients with CBD stones, 62% of patients with primary CBD stones, and 50% of patients with biliary tubes had one or more bacteria that produced beta-glucuronidase. Most biliary bacteria produced slime, and slime production correlated better than beta-glucuronidase production did with stone formation and the presence of biliary tubes or stents. Patients with primary CBD stones and biliary tubes had the highest incidence of slime production. These findings suggest that bacterial slime is important in gallstone formation and the blockage of biliary tubes.

The role of serum liver enzymes in the diagnosis of choledocholithiasis.

BACKGROUND/AIMS: The introduction of laparoscopic cholecystectomy has increased the need for preoperative diagnosis of common bile duct stones. The purpose of this study is to verify the sensitivity of the liver function tests in the detection of duct stones. METHODOLOGY: We evaluated 438 patients (223 retrospectively and 215 prospectively) who underwent endoscopic papillotomy for bile duct stones in two different services. In every case, blood samples for liver function tests levels were collected prior to endoscopic retrograde cholangiopancreatography. RESULTS: The most sensitive test was gamma-glutamyl transpeptidase, that was abnormal in 92.2% of the cases. Alkaline phosphatase was elevated in 74.7% of the patients with choledocholithiasis. The least sensitive parameter was AST, altered in only 50.8% of times. The sensitivity of all liver tests for the diagnosis of choledochal stones taken together was 94.3%. CONCLUSIONS: Liver function tests are very sensitive in the detection of common bile duct stones, however these blood tests are in the normal range of about 5% of endoscopically treated patients.

[Alpha-L-fucosidase activity in patients with ductal cholelithiasis]. / Alfa-L-fukozydaza u chorych z przewodowymi postaciami kamicy zólciowej.

Alpha-L-fucosidase (ALF), AST, ALT and GGT activities were measured in blood serum of 36 patients with recurrent cholelithiasis (group I), 32 patients with ductal and/or bladder cholelithiasis (group II), 24 patients with focal changes in the liver (group III) and in 22 patients without disturbances of gastrointestinal tract (control group). A statistically significant increase in ALF activity was found in the patients with recurrent cholelithiasis as compared to the control group (p < 0.001), the patients of group II (p < 0.001) and the patients of group III (p < 0.05). The AST and ALT activities were higher both in group I and in group II than in the control group (p < 0.001 and p < 0.02 for group I and group II, respectively), whereas the mean GGT activity was significantly higher in all three patients groups as compared to the control group (p < 0.001 in all cases). On the basis of the observed differences in the activities of the enzymes studied it was postulated that the determination od ALF activity in blood serum of patients with cholelithiasis may provide the means for early diagnosis of the predisposition to recurrent stones.

A randomised trial of selective or routine on-table cholangiography.

To assess the value of pre- and peroperative indicators of common bile duct (CBD) stones, 167 patients undergoing cholecystectomy were randomised to receive either routine (R) or selective (S) on-table cholangiography (OTC). In all, 81/84 patients in the R group and 22/78 in the S group had OTCs (P < 0.0001). In the R group 11/81 were positive (one false-positive) and in the S group 7/22 were positive (P < 0.05). CBD stones were present in 10/81 in the R group and 7/78 in the S group (P = NS). In the R group 8/10 and in the S group 5/7 had CBD stones diagnosed preoperatively by ultrasound scanning (USS), giving USS a positive predictive value (PPV) of 100% and a sensitivity of 71.4% in the S group. A raised preoperative alkaline phosphatase (ALP) was found in 1/10 in the R group and 1/7 in the S group (PPV = 33.3%, sensitivity = 14.3%). In the R group 1/10 and in the S group 1/7 (PPV = 25%, sensitivity = 14.3%) were found to have a dilated CBD during operation suggesting CBD stones. There have been no cases of symptomatic unsuspected retained CBD stones during follow-up to date. Our study has demonstrated that selective use of OTCs can be safely employed in open cholecystectomies. Over 85% (6/7 in the S group and 15/17 overall) of CBD stones can be diagnosed preoperatively by USS and liver function tests, with USS having the greatest PPV and sensitivity.

[Bile composition and antibiotic excretion. Observations with T-drainage]. / Gallenzusammensetzung und Antibiotikaausscheidung. Beobachtungen an der T-Drainage.

Patients comparable in disease, therapy and serum bilirubin concentration were either treated with mezlocillin intravenously or not at all. The bile of each patient was collected either from a T-drainage or from a percutaneously placed drainage into the bile ducts. The concentrations of GGT and AP, which were liberated by destroyed liver cells, and of bilirubin and mezlocillin, which were secreted actively, were analysed. Those patients who had normal serum bilirubin concentrations had a significantly higher biliary bilirubin excretion than those with high serum bilirubin level. The maximum excretion was after 4 hours. While the biliary concentration of bilirubin decreased, the concentration of secreted mezlocillin increased. Due to destroyed liver cells those patients with pathologically elevated blood bilirubin levels had a 50-fold lower mezlocillin excretion than those with normal blood values.

Clinical meaning of GGT activity in follow-up of patients with alcohol-related liver injury and cholestasis.

The dynamics of GGT was investigated in three groups of patients after removing some primary causes of GGT increase. Group A included 34 patients with alcohol-related liver disease, group B included 16 patients with alcoholic liver injury and cholestasis, caused by concomitant alcoholic pancreatitis and group C included 17 patients with extrahepatic cholestasis, caused by choledocholithiasis. Follow-up assays of GGT were performed on the 7th, 14th and 30th days. Our results showed that the dynamics of GGT was more rapid after removing the cause for cholestasis than in stopping alcohol consumption in patients with chronic liver diseases. On the 14th day more than a 50% decrease in GGT activity was noted in 20% of the patients from groups A and B and in almost all cases from group C. On the 30th day, the reference range of GGT was not attained by any of the patients with liver disease nor in five patients from group C. No significant correlation was found between the severity of liver damage and the extent of GGT increase at the beginning and at the end of the follow-up period.