Huong-Thu Ton-Nu

Hepatocyte transport of bile acids and organic anions in endotoxemic rats: Impaired uptake and secretion

BACKGROUND & AIMS In sepsis, intrahepatic cholestasis occurs frequently, suggesting impaired hepatocyte transport of bile acids and organic anions. The aim of the study was to define the magnitude, time course, and the site of impaired biliary secretion in a rat sepsis model. METHODS Maximal transport for two bile acids (cholyltaurine and chenodeoxycholyltaurine) and two organic anions (sulfobromophthalein and sulfolithocholyltaurine) was measured in isolated perfused livers at various times after lipopolysaccharide injection. Basolateral and canalicular liver plasma membrane vesicles were used to characterize the impairment in hepatocyte transport. RESULTS Maximal hepatocyte transport was reduced for all compounds by 60%-81% compared with controls. Bile acid-independent bile flow was reduced by 51%. Impairment was maximal 12 hours after endotoxin injection and recovered thereafter. In basolateral plasma membrane vesicles, sodium-dependent transport for bile acids was reduced by 36%-47%. Sodium-independent transport of organic anions was reduced by 40%-55%. Adenosine triphosphate-stimulated transport was greatly decreased in canalicular vesicles prepared from endotoxemic animals for all four compounds probably because of a reduced number of transport molecules, based on kinetic studies. CONCLUSIONS Basolateral and canalicular bile acid and organic anion transport are markedly impaired in endotoxemia. These mechanisms may contribute to the cholestasis of sepsis.

Activation of mast cells by bile acids.

To test whether bile acids interact with mast cells, dilute, aqueous solutions of five pure unconjugated natural bile acids and their corresponding glycine or taurine conjugates were incubated with murine PT-18 cells (a mast cell line functionally and cytochemically similar to mucosal mast cells) or with freshly isolated rat peritoneal mast cells. Bile acid solutions ranged in concentration from 0.3 to 10 mmol/L; histamine release was assessed by a fluorimetric assay, and cell lysis by cytosolic enzyme (lactate dehydrogenase) release. Lipophilic, dihydroxy bile acids (chenodeoxycholic acid and deoxycholic acid as well as their glycine and taurine conjugates) caused histamine release in a dose-related manner; cholic acid and its conjugates caused much less or no histamine release. Two hydrophilic bile acids (ursodeoxycholic acid and ursocholic acid and their conjugates) were virtually devoid of activity. Histamine release, which was independent of extracellular Ca2+, occurred at 0.3 mmol/L, well below the critical micellization concentration. For a given concentration, unconjugated bile acids and glycine-conjugated bile acids induced more histamine release than taurine-conjugated bile acids; maximal release was observed at 3 mmol/L for lipophilic, dihydroxy bile acids. To test whether bile acids could also cause histamine release from cutaneous mast cells in vivo, rats were injected intradermally with bile acid solutions and histamine release assessed by capillary leakage of Evans blue dye. Cutaneous blueing was greater with cytotoxic bile acids, chenodeoxycholyglycine or deoxycholylglycine, than with ursodeoxycholylglycine and was inhibited by prior antihistamine treatment. Histamine release correlated highly and positively with lipophilicity and with bile acid surface activity. It was concluded that lipophilic but not hydrophilic bile acids possess concentration-dependent cytotoxicity toward mast cells causing histamine release, that unconjugated and glycine-conjugated bile acids are more potent than taurine-conjugated bile acids, and that mast cell histamine release is highly correlated with lipophilicity of bile acids as well as their surface activity.

Transport, metabolism, and effect of chronic feeding of cholylsarcosine, a conjugated bile acid resistant to deconjugation and dehydroxylation

To test the effect in rodents of chronic ingestion of a bile acid resistant to deconjugation, cholylsarcosine was synthesized and its transport, metabolism, and effect on biliary bile acid and biliary lipid composition were determined in rabbits, hamsters, and rats. Cholylsarcosine was shown to be well absorbed from the ileum but underwent little absorption from the jejunum or colon. When cholylsarcosine was administered in the diet at 140 mumol/kg.day, it was well absorbed and underwent little biotransformation during enterohepatic cycling; however, both bacterial deconjugation and dehydroxylation (without deconjugation) occurred to a small extent. With chronic feeding, cholylsarcosine accumulated to compose 24%-29% of circulating bile acids in all 3 rodent species. It was rapidly lost from the enterohepatic circulation, with a daily fractional turnover rate of 75%-150%, depending on the species. Cholylsarcosine caused no change in liver tests or hepatic morphology and did not influence biliary lipid secretion. When cholyltaurine was fed, it was also absorbed, but, in contrast to cholylsarcosine, was rapidly deconjugated and dehydroxylated to form deoxycholic acid. The deoxycholic acid accumulated in the enterohepatic circulation, as evidenced by a slow fractional turnover rate of 26%-40% per day, depending on the species. It is concluded that cholylsarcosine is absorbed from the ileum, has an enterohepatic circulation, does not undergo appreciable deconjugation or dehydroxylation in these rodents, and is nontoxic. In the rodent, the circulating bile acids can be somewhat enriched when a bile acid resistant to deconjugation is ingested; but the effect on the steady state biliary bile acid composition is less than that obtained when cholyltaurine is administered because cholyltaurine is biotransformed to deoxycholic acid, which in turn is absorbed and has its own efficient enterohepatic circulation.

Physicochemical and physiological properties of 5α-cyprinol sulfate, the toxic bile salt of cyprinid fish

5α-Cyprinol sulfate was isolated from bile of the Asiatic carp, Cyprinus carpio. 5α-Cyprinol sulfate was surface active and formed micelles; its critical micellization concentration (CMC) in 0.15 M Na+ using the maximum bubble pressure device was 1.5 mM; by dye solubilization, its CMC was ∼4 mM. At concentrations >1 mM, 5α-cyprinol sulfate solubilized monooleylglycerol efficiently (2.1 molecules per mol micellar bile salt). When infused intravenously into the anesthetized rat, 5α-cyprinol sulfate was hemolytic, cholestatic, and toxic. In the isolated rat liver, it underwent little biotransformation and was poorly transported (Tmax ≅ 0.5 μmol/min/kg) as compared with taurocholate. 5α-Cyprinol, its bile alcohol moiety, was oxidized to its corresponding C27 bile acid and to allocholic acid (the latter was then conjugated with taurine); these metabolites were efficiently transported. 5α-Cyprinol sulfate inhibited taurocholate uptake in COS-7 cells transfected with rat asbt, the apical bile salt transporter of the ileal enterocyte. 5α-Cyprinol had limited aqueous solubility (0.3 mM) and was poorly absorbed from the perfused rat jejunum or ileum. Sampling of carp intestinal content indicated that 5α-cyprinol sulfate was present at micellar concentrations, and that it did not undergo hydrolysis during intestinal transit. These studies indicate that 5α-cyprinol sulfate is an excellent digestive detergent and suggest that a micellar phase is present during digestion in cyprinid fish.

Active absorption of conjugated bile acids in vivo

Active transport of conjugated bile acids by the distal ileum is required for efficient enterohepatic cycling of bile acids. Experiments were performed in the rat to obtain accurate values for Tmax and Michaelis constant (Km) of the absorptive area of the rat ileum and to define the structural specificity of the transport system. The distal fifth (20 cm) of the small intestine from an anesthetized animal with a biliary fistula was perfused using solutions of 10 taurine-conjugated bile acids; a flow rate was used that was sufficiently high such that unstirred water layer effects were negligible and the intraluminal concentration remained unchanged throughout the perfused segment. The absorption rate was equated with the rate of hepatic bile acid secretion. Values of Tmax (mumol/min.kg) were markedly influenced by bile acid structure: cholyltaurine, 12.9; ursocholyltaurine, 9.6; ursodeoxycholyl taurine, 5.0; and lagodeoxycholyl-(3 alpha,12 beta-dihydroxy-cholanoic acid)-taurine, 1.2. Decreasing the length of the side chain of ursodeoxycholate conjugates from 8 to 6 carbon atoms was associated with a modest increase in Tmax values from 5.0 to 9.1 mumols/min.kg. Values of Km correlated with Tmax values and ranged from 0.5 to 5 mmol/L, being highest for those bile acids that were best transported. The Tmax for cholyltaurine transport was not reached when the intraluminal concentration was as high as its critical micellization concentration, precluding the definition of its Tmax; however, for ursocholyltaurine, with a critical micellization concentration of 40 mmol/L, saturation of transport was clearly shown. Kinetic parameters could not be obtained for two common dihydroxy conjugates (chenodeoxycholyltaurine and deoxycholyltaurine) because at a transport rate of 2 mumols/min.kg systemic toxicity and death occurred. These studies define the maximal transport capacity of the rat ileum for taurine-conjugated bile acids; they indicate that the ileal transport system in the rat is of low affinity and high capacity for taurine conjugates of hydrophilic bile acids, and they show that both nuclear substituents and side chain length influence the transport rate of taurine-conjugated bile acids.

Use of a fluorescent bile acid to enhance visualization of the biliary tract and bile leaks during laparoscopic surgery in rabbits

BackgroundWe set out to determine whether intravenously administered cholylglycylaminofluorescein (CGF), a fluorescent bile acid, would enhance the visualization of the biliary tract and bile leaks in rabbits undergoing laparoscopic cholecystectomy (LC).MethodsCGF was infused at doses of 1, 5, and 10 mg/kg b.w. Biliary recovery was determined spectrophotometrically (six rabbits). For LC (seven rabbits), a blue (fluorescein) filter was attached to the light source, and a fluorescein-emission filter was attached to the charge coupled device (CCD) camera. The biliary tract and bile leak (made by incising the gallbladder) was observed under standard and fluorescent illumination.ResultsApple-green fluorescence appeared in 2 min and persisted for 30–60 min, enhancing visualization of bile duct anatomy as well as the bile leak. Biliary recovery of CGF at 90 min was high (86–96% of the infused dose).ConclusionIn rabbits, CGF is secreted quantitatively in bile, induces biliary fluorescence, and enhances visualization of the bile ducts and bile leaks when viewed with appropriate filters.