Intermittent Secretion of Abnormal Bile in Patients with Cholesterol Gall Stones

Gall bladder and hepatic bile was sampled from 66 patients undergoing elective operations on the biliary tract. Fifty-one patients had cholesterol gall stones but only 59% of these were found to have bile which was supersaturated with cholesterol. Repeated sampling of hepatic bile from patients with T-tubes showed that the secretion of supersaturated bile was intermittent.These results indicate that it is impossible to separate patients with cholesterol stones from controls simply by examination of the lipid composition of their bile, since an appreciable number of bile samples from patients with cholesterol stones were unsaturated.The fact that cholesterol gall stones form when the bile is supersaturated with cholesterol only intermittently suggests that the gall bladder may also have a part in their formation.     

Complete Radio Frequency Ablation of Hepatocellular Carcinoma Adjacent to the Main Bile Duct and Blood Vessels Between the First and the Second Hepatic Portal

In cases where hepatocellular carcinoma cannot be surgically removed either due to the capacity of hepatic functional reserve or the special location of the tumor, a radiofrequency ablation (RFA) is recognized to be an effective and minimally invasive treatment. However, when the tumor is adjacent to the main bile duct and blood vessels, it is feared that due to the “heat-sink effect” of the blood and the possible damage to the duct and blood vessels, complete tumor ablation is hard to achieve. We report here a case of complete RFA of hepatocellular carcinoma, adjacent to the main bile duct and blood vessels between the first and the second hepatic portal, with emphasis on the safety of the approach for complete ablation of the tumor.     

Variation in Hepatic Bile Composition Following Cholecystectomy in Patients with Previous Gallstones

The composition of fasting hepatic bile was analyzed in 63 samples from 8 patients following cholecystectomy to determine if bile was lithogenic in patients with previous cholesterol gallstones after removal of the gallbladder. Bile specimens were obtained from t-tubes over a 7-20 day study period following re-establishment of the enterohepatic circulation. Bile composition varied on a day to day basis in each patient. 18 of 63 samples were lithogenic according to criteria of Admirand and Small while 35 of 63 samples were lithogenic according to criteria of Hegardt and Dam. Variations in the composition of hepatic bile appeared related to changes in the excretion rate of bile acids. These studies demonstrate that hepatic bile may be lithogenic after cholecystectomy and indicate that factors other than sequestration of the bile acid pool in the gallbladder influence the enterohepatic circulation of bile acids and the lithogenicity of bile.     

Improved method for bile collection in unrestrained conscious rats.

We describe an improved method for continuous collection of bile from unrestrained rats. Use of an externally accessible, continuous-loop cannula when cannulating the common bile duct allows for full recovery from anesthetic effects and maintenance of a normal bile salt pool until the cannula loop is cut. Bile resulting from the cut cannula is diverted into a surgically implanted glass collection vessel and removed periodically via an externalized sampling port. Bile flow over a 24-hour collection period averaged 0.98 +/- 0.04 ml/hr (Mean +/- SEM, n = 9) with no gross pathological changes noted upon necropsy. This technique offers the capability of reestablishing conditions as close to physiologic as possible postsurgery to minimize potential artifacts during bile collection.     

Substrate specificity of the ileal and the hepatic Na(+)/bile acid cotransporters of the rabbit. I. Transport studies with membrane vesicles and cell lines expressing the cloned transporters.

The substrate specificity of the ileal and the hepatic Na(+)/bile acid cotransporters was determined using brush border membrane vesicles and CHO cell lines permanently expressing the Na(+)/bile acid cotransporters from rabbit ileum or rabbit liver. The hepatic transporter showed a remarkably broad specificity for interaction with cholephilic compounds in contrast to the ileal system. The anion transport inhibitor diisothiocyanostilbene disulfonate (DIDS) is a strong inhibitor of the hepatic Na(+)/bile acid cotransporter, but does not show any affinity to its ileal counterpart. Inhibition studies and uptake measurements with about 40 different bile acid analogues differing in the number, position, and stereochemistry of the hydroxyl groups at the steroid nucleus resulted in clear structure;-activity relationships for the ileal and hepatic bile acid transporters. The affinity to the ileal and hepatic Na(+)/bile acid cotransport systems and the uptake rates by cell lines expressing those transporters as well as rabbit ileal brush border membrane vesicles is primarily determined by the substituents on the steroid nucleus. Two hydroxy groups at position 3, 7, or 12 are optimal whereas the presence of three hydroxy groups decreased affinity. Vicinal hydroxy groups at positions 6 and 7 or a shift of the 7-hydroxy group to the 6-position significantly decreased the affinity to the ileal transporter in contrast to the hepatic system. 6-Hydroxylated bile acid derivatives are preferred substrates of the hepatic Na(+)/bile acid cotransporter. Surprisingly, the 3alpha-hydroxy group being present in all natural bile acids is not essential for high affinity interaction with the ileal and the hepatic bile acid transporter. The 3alpha-hydroxy group seems to be necessary for optimal transport of a bile acid across the hepatocyte canalicular membrane. A modification of bile acids at the 3-position therefore conserves the bile acid character thus determining the 3-position of bile acids as the ideal position for drug targeting strategies using bile acid transport pathways.     

Hepatic overexpression of cholesteryl ester hydrolase enhances cholesterol elimination and in vivo reverse cholesterol transport

Neutral cholesteryl ester hydrolase (CEH)-mediated hydrolysis of cellular cholesteryl esters (CEs) is required not only to generate free cholesterol (FC) for efflux from macrophages but also to release FC from lipoprotein-delivered CE in the liver for bile acid synthesis or direct secretion into the bile. We hypothesized that hepatic expression of CEH would regulate the hydrolysis of lipoprotein-derived CE and enhance reverse cholesterol transport (RCT). Adenoviral-mediated CEH overexpression led to a significant increase in bile acid output. To assess the role of hepatic CEH in promoting flux of cholesterol from macrophages to feces, cholesterol-loaded and [(3)H]cholesterol-labeled J774 macrophages were injected intraperitoneally into mice and the appearance of [(3)H]cholesterol in gallbladder bile and feces over 48 h was quantified. Mice overexpressing CEH had significantly higher [(3)H]cholesterol radiolabel in bile and feces, and it was associated with bile acids. This CEH-mediated increased movement of [(3)H]cholesterol from macrophages to bile acids and feces was significantly attenuated in SR-BI(-/-) mice. These studies demonstrate that similar to macrophage CEH that rate-limits the first step, hepatic CEH regulates the last step of RCT by promoting the flux of cholesterol entering the liver via SR-BI and increasing hepatic bile acid output.     

The transport of lipoprotein cholesterol into bile: a reassessment of kinetic studies in the experimental animal

Studies were undertaken to assess the contribution of lipoprotein cholesterol to bile and to determine whether already-existent hepatic free cholesterol and the free cholesterol which is newly generated from the hydrolysis of hepatic cholesteryl esters are equally available for secretion into bile or constitute metabolically separate pools. Rats with a bile fistula were injected with an intravenous bolus of high-density lipoprotein recombinants containing free [14C]cholesterol and [3H]cholesteryl esters. Results showed (1) that bile free [14C]cholesterol secretion was a constant and linear proportion of the whole liver free [14C]cholesterol pool, (2) that secretion into bile of free [3H]cholesterol was in direct proportion to the rate of hydrolysis of hepatic [3H]cholesteryl esters, and (3) that rates of biliary cholesterol secretion were very similar when secretion was calculated using the specific activity of free [14C]cholesterol and free [3H]cholesterol in the entire liver to 'label' the precursor free cholesterol pool. Furthermore, rates of secretion that were calculated using either isotope closely approximated the mass of free cholesterol that was directly measured in bile. Results thus indicate that because of equilibration and extensive dilution by the large pool of already-existent free cholesterol, the transport of isotopic cholesterol from lipoproteins cannot be used to directly assess the contribution of lipoprotein cholesterol to the cholesterol that is secreted in bile. These studies further suggest that the totality of preformed free cholesterol in the liver is in metabolic equilibrium in one single kinetic pool and that all hepatic free cholesterol is potentially available for secretion into bile.     

Binding of bile acids, organic anions, and fatty acids by bovine intestinal Z protein.

Z protein from bovine small intestinal mucosa was purified and its binding affinities for bile acids, organic anions, and fatty acids were compared with those of bovine hepatic Z protein. Purification of Z protein from intestinal and hepatic cytosol was performed by gel filtration, chromatofocusing, and hydroxyapatite chromatography. Both purified proteins had the same molecular weight (Mr 14,000) and eluted from a chromatofocused gel at about pH 10. Binding studies were performed by the competitive displacement of 8-anilinonaphthalene-1-sulfonic acid and by equilibrium dialysis. Binding affinities for bile acids, organic anions, and fatty acids were very similar between intestinal and hepatic Z proteins. Although the real physiologic role of Z protein remains to be further elucidated, these data indicate that intestinal Z protein participates in the mechanism of intracellular bile acid transfer in enterocytes.     

The extracorporeal bile duct: a new model for determination of bile flow and bile composition in the intact rat

A new model is described which allows measurement of bile flow and sampling of bile in the intact rat with a physiologically functioning sphincter of Oddi. A number of metabolic parameters have been followed to show that animals with such an "extracorporeal bile duct" (EBD) behave as intact controls. Especially, there was no difference in the increase in body weight or hepatic fatty acid and cholesterol synthesis between EBD animals and intact controls. The amount of bile salts circulating through the biliary tract amounted to 30.5+/-1.5mumol . 100 g body wt-1 . hr-1, when diurnal variations were averaged. Animals adapted to a restricted feeding regimen showed a significant increase of bile flow and of biliary bile salt and cholesterol excretion during feeding (10AM-3 PM); these parameters reached their maximum 3 hours after onset of food intake.     

The metabolism of the isolated perfused canine liver

An isolated liver perfusion was used for metabolic interrelation studies in our laboratory. The liver slices, after a 2-hr perfusion period in various pretreated groups, were also studied for carbohydrate metabolism. It was found that aerobic and anaerobic metabolism of liver slices treated with deoxycorticosterone acetate, testosterone, and partial ligation of the thoracic inferior vena cava, were the same as in normal livers. We also observed depression of the glycolytic pathway for utilizing exogenous fructose in the group pretreated with carbon tetrachloride and common bile duct ligation. An increase in oxygen ocnsumption in common bile duct-pretreated animals was also observed. Such studies suggest that hepatic metabolic performance in vitro or after perfusion cannot, therefore, provide infallible information on the prior presence of important host drug treatments in hepatic disease states. Such features may complicate donor considerations in hepatic transplantation patients.     

Common bile duct obstruction caused by the hydatid daughter cysts

Echinococcosis is a human parasitary disease. In 2002, 29 new cases of liver echinococcosis were recorded in Croatia. Liver is the most common site of hydatid cysts. Nine patients with echinoccocal liver disease were operated in our department in 2002. Here we present a case where a patient with verified hydatid cyst in the left liver lobe developed high fever, jaundice, nausea, vomiting and pain in the upper abdomen. The symptoms were initially ascribed to the acute cholangitis. After unsuccessful antibiotic treatment, computerized tomography and endoscopic retrograde cholangiopancreatography (ERCP) were performed, demonstrating daughter cysts in the common bile duct. During ERCP, papilotomy was made and daughter cysts were extracted. Hydatid cyst was surgically removed, and a communication between the cyst and left hepatic duct was noted during surgery. Pericystectomy, choledochotomy, removal of remaining daughter cysts from the common bile duct, and sutures of left hepatic duct were performed. The patient recovered fully after the surgery. One of the possible complications of the liver hydatid cysts is the communication between cyst and the biliary tree. Such communications are usually asymptomatic, but symptoms can also mimic acute cholangitis and jaundice, which may lead to the misdiagnosis of the patient's condition.     

四氯化碳、酒精与四氯化碳联合、胆管结扎致SD大鼠肝纤维化病理学比较

目的研究四氯化碳、酒精与四氯化碳联合、胆管结扎致SD大鼠肝纤维化模型肝脏的病理学改变,初步探讨肝纤维化发病机制。方法四氯化碳组SD大鼠以3 mg/kg的剂量（首次剂量加倍）皮下注射50%四氯化碳（四氯化碳∶橄榄油=1∶1）,每周2次,连续注射6周;酒精与四氯化碳联合组SD大鼠每日按照10 mL/kg剂量灌服酒精混合物（酒精∶吡唑∶玉米油=10 mL∶25 mg∶2 mL）,同时每周2次按0.3 mL/kg剂量给予腹腔注射四氯化碳∶橄榄油（1∶3）,连续造模60 d;胆管结扎组大鼠按10 mg/kg体重腹腔注射3%戊巴比妥钠麻醉,腹部皮肤消毒,无菌操作沿腹部正中线剪开腹腔,分离出胆管,在胆管近端和远端2处结扎胆管,28 d后结束实验。试验结束后麻醉动物,解剖取动物肝脏组织,用10%福尔马林固定,进行病理学检查。结果四氯化碳致SD大鼠肝纤维化模型表现为弥漫性脂肪肝、肝炎、肝纤维化;酒精与四氯化碳联合致SD大鼠肝纤维化模型表现为酒精性脂肪肝、肝炎、肝纤维化;胆管结扎致SD大鼠肝纤维化模型表现为胆管增生、肝炎、肝纤维化。结论这3种方法都可以引起大鼠肝脏发生纤维化,其中胆管结扎致SD大鼠肝纤维化造模方法适合于临床胆汁淤积所致肝纤维化的模型建立,其它两种方法适合于化学性、病毒性肝炎引起的肝纤维化模型的建立,可根据不同的实验目的选择不同的方法构建相应的动物模型。     

Characterization of rat liver bile acid acyl glucuronosyltransferase

Recent studies have suggested that bile acid acyl glucuronides form covalently bound protein adducts which may cause hypersensitivity reactions and increased morbidity in patients. Although the preferential biosynthesis of the acyl glucuronides has been known, the characterization of hepatic bile acid acyl glucuronosyltransferase has not yet been clearly elucidated. We have investigated the substrate specificity of the hepatic bile acid acyl glucuronosyltransferase using five common bile acids as substrates. The glucuronidation rate was dependent on the number of the hydroxy group on the steroid nucleus and mono-hydroxylated lithocholic acid, the more lipophilic common bile acid, was most effectively metabolized into its acyl glucuronide. The tri-hydroxylated cholic acid, the more water-soluble common bile acid, barely transformed into its glucuronide. Results showed decreasing of the initial velocity of the acyl glucuronidation with increasing of the concentration of substrate, lithocholic acid, owing to the substrate inhibition of the hepatic bile acid acyl glucuronosyltransferase. The substrate analogues, glycine and taurine conjugated bile acids, which exist in the body fluids in high concentrations, also inhibited the enzyme's activity. In addition, enzymatic reaction products, bile acid acyl glucuronides, also inhibited the activity. These inhibitory mechanisms may be responsible for the low concentration of bile acid acyl glucuronides in urine and may be an important detoxification system in the body.     

Increases in biliary cholesterol-to-bile acid ratio in pregnant hamsters fed low and high levels of cholesterol

Gallstones develop when the secretion of cholesterol is elevated compared with the secretion of bile acids into bile. One of the risk factors for the formation of gallstones is pregnancy. Because the pregnancy-induced increase in hepatic cholesterol synthesis rates could play a critical role in the development of cholesterol stones, the aim of the present study was to determine whether stone formation, as assessed by the ratio of cholesterol to bile acids in bile, could be ablated by blocking the pregnancy-induced increase in hepatic sterol synthesis rates. Golden Syrian hamsters were fed either ground chow or chow supplemented with 0.5% cholesterol for 3 wk and studied in the nonpregnant state or in late gestation. In chow-fed animals, a 1.6-fold increase in the ratio of cholesterol to bile acids occurred simultaneously with a sevenfold increase in hepatic sterol synthesis rate and a ninefold increase in the amount of newly synthesized cholesterol secreted into the bile in late gestation. In the cholesterol-fed dams, an increase in the ratio of cholesterol to bile acids occurred even with the lack of induction of hepatic sterol synthesis rates during pregnancy. Thus it appears that the marked induction of hepatic sterol synthesis rates during gestation is not essential for the pregnancy-induced cholesterol saturation of bile when cholesterol is fed to animals.     

Bile acids in the rat: studies in experimental occlusion of the bile duct

Bile acids in the plasma, urine, and small intestine of adult male rats with occluded bile ducts have been studied using a method of high specificity for their determination. After bile duct ligation cholic acid rapidly accumulates in the plasma for 8 hr, remains high for a further 8 hr, and subsequently diminishes; bile acids disappear from the small intestine. During the first 12 hr after bile duct ligation the excretion of trihydroxy acids in the urine was 10 times that of the dihydroxy acids. Subsequently the two excretion rates became equal. Because bile acids have been implicated in the etiology of hepatic damage following bile duct ligation, studies have been made of the effect on the liver of removing (with cholestyramine) and supplementing (with cholic acid) the intestinal bile acid pool. The addition of cholestyramine to the stock diet prevented the rise in trihydroxy bile acids after bile duct ligation, but did not prevent the development of histological abnormalities in the liver. Supplementing the diet with cholic acid raised the plasma cholic acid levels but had little effect on the hepatic histological findings.     

A novel bile duct cannulation method with tail cuff exteriorization allowing continuous intravenous infusion and enterohepatic recirculation in the unrestrained rat.

A variety of bile duct cannulation methods have been used in the study of biliary excretion in the rat. We now report the validation and use of one such method. In this method, the common bile duct and duodenum were cannulated, the free ends of the cannulae tunnelled through the abdominal wall, passed through a trochar and exteriorized at the ventral aspect of the tail. A purpose-designed stainless steel tail cuff was then attached, to protect the cannulae from the rat. The cannulae were passed through the top of a metabolism cage and attached to a dual swivel that allows the rat freedom of movement within the metabolism cage. Where necessary an additional cannula could be placed in the femoral vein to allow infusion of test material or blood sampling. The results demonstrate that the method is robust and that its use allows a reliable correlation between surgically prepared and intact animals, as physiological parameters are allowed to return to normal prior to inclusion of the animals in the study. The technique allows the animals a great deal of freedom and, as such, is considered to minimize stress associated with the procedure. This fact is reflected in the reliability and reproducibility of the data obtained over the wide range of studies that have been conducted using this method. This method has been in use for over 4 years at Inveresk and this paper describes the authors' experience with the method to date.     

Multiplicity of Hepatic Excretory Mechanisms for Organic Anions

Previous studies based upon competition between different organic anions for biliary excretion in vivo have suggested that all organic anions share a common hepatic secretory mechanism. Corriedale sheep with an inherited defect in organic anion excretion by the liver were used to study this problem directly without the need for competition studies, the results of which are difficult to analyze. Maximal biliary excretion of sulfobromphthalein (BSP) in mutant Corriedale sheep was less than 7% of that observed in normal sheep whereas maximal biliary excretion of taurocholate, the major organic anion in sheep bile, was not different in mutant and normal sheep. Taurocholate infusion enhanced maximal hepatic excretion of BSP in normal but not in mutant sheep. These studies of an inheritable disorder which appears to be identical to the Dubin-Johnson syndrome in man, demonstrate that taurocholate excretion requires at least one step in biliary excretion which is not required by other organic anions such as bile pigment, porphyrins, drugs, and dyes.     

The biliary excretion of sulfbromophthalein in the pig

The characteristics of the hepatic metabolism of Sulfbromophthalein (BSP) have not been described previously for the pig. This is an important deficiency, since the pig is particularly suitable for studies of hepatic physiology and pharmacology which might apply to man. The aim of these experiments was to establish the pattern of serum clearance and biliary excretion of BSP and to determine that dose which would produce a maximal concentration in bile. A dose response and pattern of biliary excretion of BSP was studied at three dose levels administered either as a single bolus of a continuous infusion. All experiments were performed in conscious, conditioned pigs. The patterns of serum clearance and biliary excretion were found to be similar to other laboratory animals and to man. Maximary biliary concentration of BSP was achieved by a single bolus of 5-9 mumol/kg or a constant infusion of 0-59 mumol/kg/min. At these dose levels no significant alteration in bile flow was demonstrated nor was there any correlation between bile flow and BSP excretion. Supra-maximal doses produced a significant increase in bile flow and with these doses there was a significant positive correlation between bile flow and BSP excretion.     

Radioassay of bile acid coenzyme A:glycine/taurine: N-acyltransferase using an n-butanol solvent extraction procedure

A rapid, specific, and sensitive radioassay for measuring bile acid CoA:glycine/taurine: N-acyltransferase (EC 2.3.1) has been developed. In this assay, 3H-labeled amino acids (glycine or taurine) are conjugated with unlabeled bile acid CoA derivatives to form 3H-labeled bile acid amidates. Following incubation, the 3H-labeled bile acid amidate is separated from the unreacted amino acid by an n-butanol extraction method. The extraction procedure was developed by evaluating the effects of buffer concentration and pH on the recovery of radiolabeled bile acid amidate standards in the presence of human hepatic cytosol. Highest recovery (greater than 90%) of bile acid amidate standards occurred under acidic conditions (pH 2) in the presence of 1% (w/v) SDS. When the radioassay and accompanying n-butanol extraction procedure were utilized to study the amidation of glycine or taurine with cholic acid in human hepatic cytosol, a single peak of radioactivity corresponding with either authentic glycocholate or taurocholate was detected in the n-butanol phase by high-performance liquid chromatography. This assay for bile acid CoA:glycine/taurine: N-acyltransferase activity was linear with incubation time and protein concentration. This assay should be useful in the biochemical studies of this enzyme, as well as in the examination of bile acid amidation in clinical liver specimens.