Normal Bile Flow Research Articles

The Hardest Job in Medicine

DOI: 10.1200/JCO.2009.25.1371 This was not how I was supposed to meet my new patient Bruce. I had been called recently by his internist, who alerted me to his situation: vigorous man in his early 60s, a lifetime of good health, came to see the doctor for mild abdominal discomfort and weight loss, a computed tomography scan showed tumors in the liver and the pancreas, the diagnosis appeared to be pancreatic cancer that had spread to the liver, an incurable situation. A blood marker of that cancer, named CA 19-9, was very elevated at 200,000 (normal less than 35), solidifying the diagnosis. Bruce was set to see me, a medical oncologist, in my office in a few days to discuss his treatment options. But, it was not to be. Cancer makes the appointments, patients and doctors follow fast afoot. I received the call from Bruce’s doctor while in the hospital at the end of a long day. “Bruce was admitted last night through emergency. He’s in severe pain, deeply jaundiced, the disease is just growing so fast. Nice man. Lovely family. Tough situation. They’re waiting to see you.” Click. As the dial tone droned on, it blanked out my thoughts and vibrated in synch, like a tuning fork, with my sinking spirit. Eventually, I put down the receiver and got in motion, steeling myself to do the hardest job in medicine. I found Bruce in bed, sleepy from the morphine dripping in his vein to kill the pain, yellow from the bilirubin building up in his bloodstream. His wife Joan was seated, her upper body draped over him like a blanket. They greeted me cordially but I immediately palpated their fear of what I was going to say. I pulled up a chair, pulled out paper and pen, and discussed and diagrammed how Bruce’s cancer had spread from the pancreas to the liver, how the tumors in the liver were causing his pain and impeding the normal flow of bile, leading to his jaundice. I told them how the elevated CA 19-9 was consistent with the diagnosis of pancreatic cancer but that we would get a biopsy to prove it. The lighting in the room was soft. The conversation was calm; there was a give and take, some levity and the initial forging of an intense bond. Then Joan asked about the CA 19-9. “Was it rechecked? What is it now?” I hesitated, and they saw it. I did not want to deflate them further. “Not important,” I said and tried to move on. Joan looked me squarely in the eye and said, “I want to know.” “700,000” was my reply (it was the highest I had ever seen). “Oh my god,” she said as the emotional dam broke, giving way to the pent up terror and shock that needed to be released. The hardest job in medicine just got a whole lot harder. As Bruce drifted off on a morphine cloud, Joan asked to talk with me outside of the room. “What you’re telling me is that my husband’s got cancer and it’s the worst of the worst. How much time? How much time does my husband have?” she implored. “Every cancer has an average survival and then a much broader range, with some people living shorter, others living far beyond the average; every patient is unique,” I said. “How much time?” she pressed. “The average is 6 months, but I’m not even sure he will make it out of the hospital. We have to hope that the treatment slows the growth of the cancer so that he improves enough to be discharged. That is the first hurdle. Pancreatic cancer is a very difficult cancer to treat. It might not happen, and he could succumb.” She asked, “If we did nothing?” “Days” was my reply. We spent time talking about Bruce, their life and family and some about mine. We agreed to keep Bruce comfortable above all else. I told her, I needed to tell Bruce much of what I just told her, and I did. His response, “just do what has to be done. I’m going to make it.” I recently saw Bruce in my office, a year and a half after our initial conversation. He comes for weekly chemotherapy treatments, works full time, has traveled abroad, and looks fit. Is he cured? No. How long will he live? I don’t know. How did this happen? I cannot say. From time to time, Joan drops in to bear hug me, look me in the eye and say “I love you.” The hardest job in medicine is sometimes the very best.

Development of a model for long-term bile collection in Göttingen minipigs

Studying metabolism and elimination of exogenous substances includes investigation of potential biliary excretion of metabolites of the parent compound. Repeated sampling of bile for analysis in relation to dosing is therefore necessary, which requires surgical catheterisation of the bile duct. The Gottingen minipig is a commonly used species within nonclinical drug research due to the high level of similarity to humans. This also includes similarities in many drug metabolism pathways, as several porcine cytochromeP450’smetabolise the same test substrates as the human enzymes. For long termbile collection in consciousGottingenminipigswe have developed a catheterisation procedure allowing for repeated sampling, without disturbing the normal bile flow into the intestinal lumen, that otherwise could result in digestive and nutritional disturbances over time. A T-tube catheter with two side arms was inserted into the bile duct of four minipigs. The side arm towards the duodenum contained a conical diaphragm. Inflation of the diaphragm caused obstruction of normal bile flow, diverting the bile into the side arm towards the liver. Both side arms were tunnelled to the back and connected to vascular access ports, placed subcutaneously. This enclosed system allowed for bile collection when puncturing one port while inflating the diaphragm through the second port. The catheters were used for repeated bile collection in relation to two dosing occasions and remained patent for twoweeks. Study results and practical aspects of the procedure will be presented, including suggestions for method improvement.

1H and 31P NMR studies indicate reduced bile constituents in patients with biliary obstruction and infection

Patients with extrahepatic biliary obstruction present with impairment of the normal bile flow, with jaundice and cholangitis as common complications. (1)H and (31)P NMR quantitative analysis of bile specimens from patients with extrahepatic biliary obstruction (n = 80) (with/without jaundice and cholangitis separately and together) was carried out for the chief biliary constituents to determine the relationship between biliary constituents and jaundice (serum bilirubin concentration >or=1.0 mg/dL) and cholangitis (total leucocyte count >11,000 cells/mm(3) and/or fever >38.5 degrees C with/without bile culture positivity). Compared with controls (patients without jaundice and cholangitis), median indices of the chief biliary constituents (total bile acids, cholesterol, phosphatidylcholine and inorganic phosphate) were significantly suppressed in patients with cholangitis and/or jaundice. Quantities of total bile acids, cholesterol and phosphatidylcholine correlated negatively with the quantity of bilirubin and with cholangitis, i.e. total leucocyte count. Suppression of biliary constituents correlated significantly with the severity of jaundice and cholangitis. The decrease in biliary constituents in the presence of jaundice and cholangitis is possibly the result of downregulation of the function of transporters located at the canalicular side of hepatocytes, leading to their suppressed indices in bile. This information may have implications in the examination of bile for clinical studies.

Type III Choledochal Cyst Variant: First Description of a “Closed Choledochocele” with Biliary and Pancreatic Drainage Via the Accessory Duct

Type III choledochal cysts, also called choledochoceles, are rare congenital or acquired malformations of the terminal biliary tree of unknown etiology. These cysts can predispose a patient to biliary colic, obstructive jaundice, or idiopathic recurrent pancreatitis through an obstructive mechanism as the normal flow of bile is disrupted. They have been described in two anatomic variants 1) both the pancreatic and bile ducts drain into the choledochocele 2) the choledochocele is a cystic outpouching into the duodenum proximal to a normal ampulla. Endoscopic ultrasound and ERCP are primary tools to help diagnosis these anomalous formations and needle-knife sphincterotomy has recently been used to treat these cysts in symptomatic patients. We report the case of a 23 year-old white female with no previous past medical history who developed acute pancreatitis two weeks after completing an unremarkable first pregnancy. On EUS, she was found to have a Type III choledochocele with no apparent drainage into the duodenum. Bile was seen draining from the accessory duct. Subsequent ERCP via the minor papilla revealed communication of the ventral pancreatic and common bile ducts within a choledochocele which did not communicate with the duodenum. The bile duct appeared to empty retrograde into the ventral pancreatic duct and subsequently out to the duodenum via the accessory duct. To facilitate biliary and pancreatic duct drainage, the choledochocele was accessed and opened with a needle-knife followed by biliary sphincterotomy and temporary plastic biliary stent placement. Six weeks following the procedure, she continues to feel well. This case represents the first description of a “closed choledochocele” with biliary and pancreatic drainage via the accessory duct.[figure1]Figure

Effect of α‐asarone and a derivative on lipids, bile flow and Na+/K+‐ATPase in ethinyl estradiol‐induced cholestasis in the rat

Administration of ethinyl estradiol (EE), a widely used component of oral contraceptives, has been associated with impairment of bile flow and the capacity to excrete organic anions in man and experimental animals. alpha-Asarone (2,4,5-trimethoxypropenylbenzene) and 2-methoxy-4-(2-propenyl) phenoxyacetic acid (MPPA) have shown hypolipidemic effects. In addition to these effects, we decided to evaluate the properties of these compounds on EE-induced cholestasis. Wistar male rats were injected subcutaneously with 10 mg/kg of EE for 5 days; simultaneously, alpha-asarone or MPPA were also administered and appropriate controls were performed. alpha-asarone and MPPA decreased plasma and bile cholesterol. EE diminished triglycerides total, low-density lipoprotein, high-density lipoprotein and bile cholesterol. MPPA further decreased these lipid parameters. Alkaline phosphatase (an enzyme marker of cholestasis) was increased after administration of EE, but this effect was prevented significantly by alpha-asarone or MPPA administration. Bile flow was importantly decreased by EE and increased by alpha-asarone alone. Furthermore, alpha-asarone or MPPA preserved the normal bile flow in EE-treated rats. EE inhibited the activity of the Na(+)/K(+)-ATPase, while both alpha-asarone and MPPA preserved this enzyme activity. Na(+)/K(+)-ATPase is involved in Na(+)-coupled uptake of bile acids into hepatocytes and, therefore, ultimately is the driving force for the generation of bile flow. Therefore, the anticholestatic effects of alpha-asarone and MPPA, described herein by the first time, may be due to its ability to preserve ATPase activity. This enzyme is negatively regulated by membrane cholesterol, thus the hypolipidemic effects of the compounds tested may be responsible for Na(+)/K(+)-ATPase activity and bile flow maintenance.

Proteomic Analysis of Ischemia-Reperfusion Injury upon Human Liver Transplantation Reveals the Protective Role of IQGAP1

Ischemia-reperfusion injury (IRI) represents a major determinant of liver transplantation. IRI-induced graft dysfunction is related to biliary damage, partly due to a loss of bile canaliculi (BC) integrity associated with a dramatic remodeling of actin cytoskeleton. However, the molecular mechanisms associated with these events remain poorly characterized. Using liver biopsies collected during the early phases of organ procurement (ischemia) and transplantation (reperfusion), we characterized the global patterns of expression and phosphorylation of cytoskeleton-related proteins during hepatic IRI. This targeted functional proteomic approach, which combined protein expression pattern profiling and phosphoprotein enrichment followed by mass spectrometry analysis, allowed us to identify IQGAP1, a Cdc42/Rac1 effector, as a potential regulator of actin cytoskeleton remodeling and maintenance of BC integrity. Cell fractionation and immunohistochemistry revealed that IQGAP1 expression and localization were affected upon IRI and related to actin reorganization. Furthermore using an IRI model in human hepatoma cells, we demonstrated that IQGAP1 silencing decreased the basal level of actin polymerization at BC periphery, reflecting a defect in BC structure coincident with reduced cellular resistance to IRI. In summary, this study uncovered new mechanistic insights into the global regulation of IRI-induced cytoskeleton remodeling and led to the identification of IQGAP1 as a regulator of BC structure. IQGAP1 therefore represents a potential target for the design of new organ preservation strategies to improve transplantation outcome.

Isotope dilution analysis for the determination of zinc in blood samples of diabetic patients

Radioanalytical techniques have been applied in a study on (subclinical) hepatitis in Dobermann dogs. Cu levels in transcutaneous obtained full liver biopsies were quantified using INAA and 66 Cu. A copper excretion study was performed in a group of completely normal Dobermann dogs and in six Dobermanns with elevated copper levels and suffering from subclinical hepatitis. 64 Cu 2+ was used to assess whether a normal bile flow and thus excretion was possible. All subjects underwent separately from this 64 Cu 2+ excretion study a 99m Tc-Bis-IDA scintigraphy. The Cu levels in liver of these hepatitis patients vary from 600 to 1600 mg . kg -1 whereas 100-300 mg . kg -1 is probably normal for healthy Dobermann dogs. These Cu levels match fully with histopathology results and clinical observations. Measurements during a 2 years' evaluation period showed a clear relation between the existence of subclinical hepatitis and increased Cu levels without a sign for cholestasis or an abnormal bile flow.

Chemical mediators of gallbladder dysmotility.

In order to accomplish its contribution to the digestive process, the gallbladder must contract appropriately during its emptying phases and it must be able to relax adequately for filling to occur. A variety of neuro-hormonal inputs to gallbladder smooth muscle coordinate the gallbladder emptying process with other events occurring in the bowel. Gallbladder dysmotility can disrupt the normal flow of bile to the small bowel, resulting in digestive dysfunction. In addition to this, alterations in gallbladder motility may play a role in pathological conditions, such as cholesterol gallstone formation and cholecystitis. It is still not entirely clear whether impaired gallbladder emptying is a cause or consequence of cholesterol gallstones, but recent experimental evidences demonstrate that cholesterol can directly affect the plasma membrane of gallbladder smooth muscle cells to cause impaired contraction. In addition, gallbladder emptying is impaired in acute gallbladder inflammation, probably as the result of the deleterious neural and muscular actions of inflammatory mediators such as reactive oxygen species, prostaglandins and histamine. It should also be noted that opiate treatments in critically ill patients can reduce gallbladder motility by inhibiting neurotransmitter release, and may contribute to the onset of acalculous cholecystitis, which is associated with significant morbidity in these patients.

Is bile flow reduced in patients with hypothyroidism?

Background. Disturbances in the sphincter of Oddi (SO) function may prevent normal bile flow and thus enhance the probability of common bile duct stone (CBDS) formation. We have previously shown increased prevalence of diagnosed hypothyroidism in CBDS patients, thyroxine (T4) -induced inhibition of the SO contractility both in animal and in human experiments ex vivo, and reduced bile flow to duodenum in hypothyroid rats. The aim of the present study was to investigate human biliary dynamics in relation to altered thyroid gland function. Methods. Eight female patients, 1 with diagnosed untreated hypothyroidism and 7 with total thyroidectomy performed due to thyroid cancer, were studied in hypothyroid stage and again after thyroxine replacement therapy in euthyroid stage, with quantitative 99mTc HIDA cholescintigraphy (QC), biliary ultrasonography, and serum determinations. Each patient served as her own control in the 2 stages of the study. Results. In QC, maximal uptake of 99mTc HIDA was not changed in hypothyroidism compared to euthyroidism. The first appearance of radioactivity to large bile ducts at the hepatic hilum remained unchanged in the 2 stages of the study. Hepatic clearance of 99mTc HIDA was decreased at 45 minutes (28% [11-38] vs 50% [33-54]; P =.028; median and range) and at 60 minutes (55% [28-80] vs 69% [61-79]; P =.028; median and range) and hilum-duodenal transit time increased by 31% compared to euthyroid stage. In US no changes were seen in gall bladder or bile ducts in the 2 stages of the study. Serum hypercholesterolemia was observed in the hypothyroid stage. Conclusions. We conclude that hypothyroidism may result in delayed emptying of the biliary tract, as studied with QC. In addition to the changes in bile composition and excretion rate suggested before to take place in hypothyroidism, according to the present study changes in biliary emptying also may be included in the probable causes for the increased prevalence of CBDS in hypothyroidism. This may be due to the absence of the prorelaxing effect of thyroxine on SO, which we have shown before to exist ex vivo. (Surgery 2003;133:288-93)

Biliary decompression reduces the susceptibility to ethanol-induced ulcer in jaundiced rats

We investigated the gastric response to an ulcerogenic irritant and the change in gastric functions in an experimental rat model of obstructive jaundice, with or without biliary drainage. After biliary obstruction for 14 days, rats with ligated bile duct (BDL) were randomly divided into three groups: BDL group without biliary drainage, BDL followed by choledochoduodenostomy (CD) or a choledochovesical fistula (CVF). The gastric functions were evaluated 2 weeks after the surgery. Gastric damage, induced by orogastric administration of ethanol, was evaluated 30 min later using a lesion index and microscopic scoring was then performed on fixed stomachs. Basal gastric acid secretion was measured by the pyloric ligation method.The lesion index and maximum lesion depth did not differ in the BDL and sham groups, while they were significantly reduced in the CD group. Gastric acid output and secretory volume were reduced in the BDL group compared to the sham group, while these reductions were abolished in the CD group. Afferent denervation with capsaicin further reduced the ulcer index in the later group. Our data suggest that gastric mucosal susceptibility to injury is dependent on the normal flow of bile into the duodenal lumen, which appears to be a requirement for adaptive gastric cytoprotection.

Mechanism of the Prorelaxing Effect of Thyroxine on the Sphincter of Oddi

Background: Disturbances in the function of sphincter of Oddi (SO) may prevent normal bile flow and thus enhance the probability of common bile duct stone (CBDS) formation. Previously, we have shown increased prevalence of hypothyroidism in CBDS patients. Methods: In animal (pig) experiments, thyroxine (T 4 ) and triiodothyronine have a specific inhibitory effect on SO contractility, which raises the possibility that the lack of this prorelaxing effect in hypothyroidism could, at least in part, explain the increased prevalence of CBDS. The aims of the present study were to investigate, whether human SO reacts similarly to T 4 , and to study the mechanisms of the T 4 prorelaxing effect. Results: We found that T 4 had similar inhibitory effects on both human and pig SO contractions. The T 4 effect was dosedependent, and maximum was observed in 30 min. The maximal prorelaxing effect was achieved with 0.1 nM T 4 concentration, the effect of the physiological T 4 concentration (0.01 nM) being about half of the maximal effect. Addition of α -adrenoceptor antagonist phentolamine, β -adrenoceptor antagonist propranolol, nitric oxide (NO)-synthesis inhibitor L-NAME, nerve conductance blocker tetrodotoxin, or cyclooxygenase inhibitor diclofenac did not affect the T 4 -induced inhibition of contraction. Addition of transcription inhibitor actinomycin D or translation inhibitor cyclophosphamide partially reversed the T 4 -induced inhibition of contraction. Addition of K + channel blocker glibenclamide totally reversed the T 4 -induced inhibition of contraction. In Western blotting, the thyroid hormone receptor (TR) antibody recognized 53 kDa and 58 kDa proteins, corresponding to β 1 and β 2 isoforms of TR, in the human SO tissue. Conclusions: We conclude that T 4 has a direct prorelaxing effect on human SO that expresses TR β 1 and β 2. This effect is mediated through a transcriptional mechanism that requires new mRNA and protein synthesis and subsequently leads to the activation of K + channels.

Stimulation of choleresis by insulin-like growth factor-I in rats.

Insulin-like growth factor-I (IGF-I) is an endogenous growth factor which is mainly produced in the liver. The functions of IGF-I can be summarized as growth-promoting and insulin-like metabolic actions. In the present study, the effect of IGF-I on bile flow and bile acid secretion was investigated in rats. In normal rats bile flow was significantly increased by single exogenous administration of IGF-I, and by 1 week treatment of IGF-I, both bile flow and bile acid secretion were significantly increased. Moreover, to further understand the relationship between IGF-I and bile acid secretion, hypophysectomized rats were next used. We found that the decreases in bile flow and bile acid secretion observed in rats after hypophysectomy, as well as the decrease in the endogenous level of IGF-I in the blood, were partially reversed by 1 week exogenous IGF-I treatment. Overall, this study showed that IGF-I stimulates choleresis associated with an elevation of bile acid secretion in both normal and hypophysectomized rats when exogenously administered, suggesting the importance of IGF-I in the stimulation of choleresis in vivo.

Hepatobiliary and intestinal clearance of amphiphilic cationic drugs in mice in which both mdr1a and mdr1b genes have been disrupted.

1. We have used mice with homozygously disrupted mdr1a and mdr1b genes (mdr1a/1b (-/-) mice) to study the role of the mdr1-type P-glycoprotein (P-gp) in the elimination of cationic amphiphilic compounds from the body. These mice lack drug-transporting P-gps, but show no physiological abnormalities under laboratory conditions and have normal bile flow. 2. 3H-labelled cationic drugs were administered intravenously (i.v.) to mice as a single bolus dose and the disposition of the studied cationic drugs was investigated by focusing on drug secretion into bile, intestinal lumen and urine. 3. Hepatobiliary secretion of the investigated cationic drugs was profoundly reduced in mice devoid of the mdr1-type P-gps. In fact, the cumulative biliary output, measured during 1 h, of the small type 1 compounds tri-butylmethyl ammonium (TBuMA) and azidoprocainamide methoiodide (APM), as well as of the more bulky type 2 cationic drug vecuronium, was reduced by at least 70% in the mdrla/lb (-/-) mice compared to wild-type. 4. The intestinal secretion of TBuMA, APM and vecuronium was also profoundly reduced in mdrla/lb (-/-) mice compared to wild-type mice. The absence of the mdrl-type P-gp resulted in virtual elimination of intestinal secretion of TBuMA and APM (>90% reduced as compared to wild-type (P=0.0001 and 0.0022, respectively)). The intestinal secretion of the type 2 cation drug vecuronium was reduced by 58% (P=0.0004) compared to the wild-type mice. 5. Increased renal clearances of both the type 1 compounds TBuMA and APM and also of the type 2 cationic compound vecuronium in the mdrla/lb (-/-) mice were observed. Furthermore, the balance between hepatic, intestinal and renal clearances of small type 1 organic cations clearly shifted towards a predominant role for renal clearance. Increased renal clearance may be explained by (over)expression of additional mechanisms for renal organic cation secretion, alternatively they may also point to an as yet undefined role of P-glycoprotein in kidney physiology and renal secretory function. 6. We conclude that the elimination from the body of a broad spectrum of cationic amphiphilic drugs via liver and intestine, is largely dictated by the activity of mdrl-type P-glycoproteins.

Contribution of the murine mdr1a P-glycoprotein to hepatobiliary and intestinal elimination of cationic drugs as measured in mice with an mdr1a gene disruption.

In the mouse, both the mdr1a and the mdr1b gene encode drug-transporting P-glycoproteins. The mdr1a P-glycoprotein is expressed in epithelial cells of, among others, the liver and the intestine. Furthermore, the mdr1b gene product is found in the liver but is not detectable in the intestine. To establish the potential involvement of P-glycoprotein in the elimination of cationic amphiphilic drugs from the body, we investigated biliary, intestinal, and urinary excretion in mice with a homozygous disruption of the mdr1a gene (mdr1a(-/-) mice). These mice are fully viable under laboratory conditions and have normal bile flow. Cumulative biliary excretion (expressed as percent of the intravenously administered dose excreted over a 1-hour period) of several cationic compounds was decreased as follows in mdr1a(-/-) mice compared with the wild-type animals: tri-n-butylmethylammonium (TBuMA), 0.7% versus 2.1%; azidoprocainamide methoiodide (APM), 3.8% versus 7.6%; and vecuronium, 22.7% versus 41.3%. The luminal secretion of both TBuMA and APM in the small intestine was profoundly decreased, respectively 4.6-fold (1.8% vs. 8.2% in the wild-type) and 7.9-fold (1.6% vs. 10.3% in the wild-type) in mdr1a(-/-) mice. Thus mdr1a P-glycoprotein contributes substantially to the removal of a wide variety of cationic agents from the body through intestinal and hepatobiliary secretion, but it evidently acts in concert with other transport system(s). These processes probably provide a protective mechanism limiting the overall rate of absorption as well as the bioavailability of potentially toxic organic amines.

Disruption of actin organization by cytochalasin D does not impair biliary secretion of organic anions in the rat.

The bile canaliculi of hepatocytes contract spontaneously, and it is hypothesized that this canalicular motility provides a propulsive force for normal intrahepatic bile flow. Cytochalasin disrupts actin polymerization, inhibits contraction, and decreases bile flow. We investigated whether this cholestasis was associated with impaired canalicular secretion. Isolated rat hepatocyte doublets, with and without incubation with 2 mumol/L cytochalasin D (cytD), were superfused, under first-order conditions, to steady state with fluorescein isothiocyanate-labeled glycocholic acid (FITC-GC) and carboxy-4',5'-dimethylfluorescein diacetate (CMFD), which are fluorescent substrates for the bile acid and the nonbile acid organic anion transport pathways, respectively. Fluorescent microscopic images were quantified and the data analyzed by noncompartmental and compartmental kinetic methods. cytD dilated the canalicular spaces fivefold but did not change the proportion of doublets that secreted either probe. Cytochalasin did not affect the mean cellular transit times of FITC-GC (2.8 and 2.5 minutes for control and cytochalasin-treated groups, respectively) and of carboxy-4',5'-dimethylfluorescein (3.8 and 3.7 minutes, respectively). Analysis with a three-compartment model gave estimates of the rate constants for canalicular secretion: 0.21 +/- 0.04 and 0.22 +/- 0.03 min-1 in control and treated cells, respectively, for FITC-GC, and 0.14 +/- 0.01 and 0.16 +/- 0.02 min-1, respectively, for carboxy-dimethylfluorescein. When kinetics are first-order, the canalicular secretion of organic anions is not altered by actin disruptive agents, suggesting that actin filaments do not modulate the function or distribution of these transporters. This suggests that impaired contractility rather than impaired canalicular secretion is the mechanism of cytD-induced cholestasis.

Effects of wortmannin, a novel myosin light-chain kinase inhibitor, on bile canalicular contraction in vitro and in vivo.

The cytoskeletal system is believed to play an important role in normal bile formation. The effects of wortmannin, a new myosin light-chain kinase inhibitor, on bile canalicular contraction and bile flow have been observed. The bile canalicular contraction of cultured hepatocyte doublets was investigated, using an image analyzer with a phase contrast microscope, and the intracellular Ca2+ concentration was measured, using microscopic fluorometry. We also investigated bile flow by in vivo intraportal infusion of the drug in rats. Treatment with wortmannin inhibited norepinephrine-induced canalicular contraction and caused a decrease in bile flow without changing systematic and portal blood pressure. Morphologic examination of the electron microscopic study showed that most bile canaliculi were dilated, with loss of microvilli, but no other apparent damage was seen in parenchymal hepatocytes. These data suggest that the integrity of the phosphorylation system of myosin is essential for normal bile flow.

In vivo microdialysis sampling in the bile, blood, and liver of rats to study the disposition of phenol.

Methods for continuous in vivo sampling in the bile, blood, and liver extracellular fluid are described. These methods are based on microdialysis sampling in anesthetized rats. A new flow-through microdialysis probe is described for sampling bile while maintaining normal bile flow. All three sites are simultaneously and continuously sampled to provide concentration-time profiles at multiple sites in a single experimental animal. This technique is demonstrated by studying the hepatic metabolism and biliary excretion of phenol in rats. Following an i.v. infusion of phenol, the major hepatic metabolite was found to be phenyl-glucuronide. Hydroquinone and 2-glutathionyl-hydroquinone were also detected but at lower concentrations. A similar pattern of metabolites was found in the bile and blood. For all of the metabolites, bile concentrations are higher than liver concentrations, indicating that the metabolites are actively excreted into the bile.

Enterohepatic circulation is essential for regular cycling of duodenal migrating motor complexes in dogs

The role of enterohepatic circulation and specific bile acids in the initiation and caudad migration of duodenal migrating motor complexes (MMCs) was investigated in conscious dogs. All dogs had spontaneous duodenal MMCs that migrated to the terminal ileum when bile flow was intact. During the first 3 days after total external biliary diversion, no MMCs originated in the duodenum. Instead, all MMCs originated in the jejunum and migrated to the ileum. During the next 4 days of total external biliary diversion, 81% of the MMCs originated in the jejunum and 19% in the duodenum. When normal bile flow was restored after 9 days of total external biliary diversion, regular duodenal MMCs resumed after a delay of 126 ± 27 minutes. Perfusion of individual bile acids or dogs' own bile, but not saline or alkaline solution, into the duodenum or perfusion of dogs' own bile directly into the ileum during total external biliary diversion restarted duodenal MMCs with a time lag of about 2 hours. The authors conclude that intact enterohepatic circulation is essential for the initiation of regular duodenal MMCs.

Pathogenesis of lithocholate-induced intrahepatic cholestasis: Role of glucuronidation and hydroxylation of lithocholate

It has been shown that lithocholic glucuronide is more cholestatic than lithocholic acid (LCA), as well as its taurine and glycine conjugates. Furthermore, LCA hydroxylation is thought to be a major detoxifying mechanism. Therefore, the role of LCA glucuronidation and hydroxylation was investigated during the development of LCA-induced cholestasis and recovery from it. Male rats received a bolus intravenous injection of [14C]LCA (12 mumol/100 g body weight) and bile samples were collected every 30 min for 5 h. Bile flow (BF) was reduced immediately after LCA injection, dropping to 40% of basal BF at 60 min. It then started to increase, reaching normal bile flow values at 3.5 h. Morphologically, canalicular lesions were dominant at 60 min and virtually absent at 2 h. At 60 min (maximal cholestasis), 30% of the LCA injected was secreted in bile, 20% was found in plasma while the other 50% was recovered in the liver and distributed mainly in plasma membranes, microsomes and cytosol. At the end of the experiment (normal BF), 20% of the LCA injected was still in the liver but was present mainly in the cytosol. In bile, within 30 min after injection, 46% of the LCA secreted was lithocholic glucuronide, 24% was conjugated with taurine and glycine, and 21% was in the form of hydroxylated bile acids. During the recovery period, lithocholic glucuronide secretion decreased to 18-25%. Taurine and glycine conjugate secretion increased to a maximum of 43% at 60 min, after which it was reduced to 21-28%. In contrast, hydroxylated metabolites were elevated during the recovery periods, reaching a maximum (45%) at 120 min and remaining constant thereafter. These results suggest that: (i) LCA binding to plasma membranes and microsomes appeared to correlate with the development of cholestasis; (ii) LCA glucuronidation may initiate and/or contribute to LCA-induced cholestasis; and (iii) hydroxylation predominates during recovery from cholestasis.

Cystic fibrosis: bile secretion.

Bile is an isotonic aqueous solution of bile acids, cholesterol, phospholipids, bile pigments and inorganic electrolytes. It is secreted by the hepatocytes into the bile canaliculi and modified in the bile ductules or ducts. The three main processes identified in the generation of bile flow are: 1. Active transport of bile acids from blood into bile canaliculi. This is responsible of the bile acid-dependent canalicular bile flow. Coupling between water flow and bile acid secretion is probably effected mainly through an osmotic mechanism. There is evidence that water flows (at least in part) through the interhepatocytic junctions. The bile acid-dependent flow accounts for 30 to 60% of spontaneous basal bile flow. 2. A canalicular, bile acid-independent secretion, probably due to transport into bile of organic solutes (glutathione) and inorganic electrolytes. This fraction of bile flow is stimulated by phenobarbital. It represents 30 to 60% of basal bile flow. Normal canalicular bile flow also depends on the integrity of intracellular cytoskeletal organelles, mostly microfilaments. 3. Reabsorption and secretion of fluid and inorganic electrolytes by the ductules and ducts. Secretion chiefly occurs in response to secretin and represents 30% of basal bile flow. Although several ion transport systems have been identified on the biliary epithelial cells (in particular a Na+/H+ exchange, a Na+:HCO3- symport and a CI-/HCO3- exchange), the cellular mechanism of secretion is not known. Abnormalities of bile duct function may account for the liver disease of cystic fibrosis, but these abnormalities have not been characterized.