Naoyo Sano

Effects of organic anions and bile acids on biliary lipid excretion in hyperbilirubinemic mutant Sprague-Dawley rats

The effects of organic anions and bile acids on biliary lipid excretion were studied in EHBR, a hyperbilirubinemic mutant Sprague-Dawley rat. A marked delay in the biliary excretion of BSP, cefpiramide, rose bengal and ursodeoxycholate-disulfate was observed in these animals. The marked decrease in the biliary excretion of phospholipids and cholesterol and the uncoupling of biliary bile acids and lipids that occurred after the administration of BSP, cefpiramide and ursodeoxycholate-disulfate in control Sprague-Dawley rats was absent in EHBR. Rose bengal did not change biliary lipid excretion in either the control Sprague-Dawley rats or the EHBR. Although taurocholate markedly increased bile flow and biliary bile acid excretion in both types of rats, the increase in biliary lipid excretion observed in the control Sprague-Dawley rats was absent in EHBR. These findings indicate that EHBR have an impairment of hepatic lipid transfer that is enhanced by bile acids, possibly at the level of intracellular vesicular lipid transport.

Effects of ursodeoxycholate, its glucuronide and disulfate and β-muricholate on biliary bicarbonate concentration and biliary lipid excretion

We previously reported that high-dose ursodeoxycholate (UDC) infusion in rats resulted in extensive glucuronidation of UDC, and speculated that the glucuronidation causes bicarbonate-rich hypercholeresis induced by UDC (Takikawa, H., Sano, N., Narita, T. and Yamanaka, M. Hepatology 1990; 11: 743-749). To test this hypothesis, UDC, UDC-3-O-glucuronide, UDC-3,7-disulfate and beta-muricholate were separately and intravenously infused into rats (1 mumol/min per 100 g), and biliary bicarbonate concentration was measured. The effects of these bile acids on biliary lipid secretion were also studied. All four bile acids increased bile flow and biliary bile acid excretion. UDC and beta-muricholate significantly increased biliary bicarbonate concentration, whereas UDC glucuronide and disulfate did not. Independence of UDC glucuronide excretion and biliary bicarbonate concentration was also confirmed in EHBR, a hyperbilirubinemic mutant Sprague-Dawley rat. In this case biliary bicarbonate concentration also increased in spite of the absence of UDC glucuronide in the bile after UDC infusion. Biliary phospholipid secretion was increased with UDC, unchanged with beta-muricholate, and decreased with UDC glucuronide and disulfate. Biliary cholesterol secretion was increased with UDC, unchanged with beta-muricholate and UDC glucuronide, and decreased with UDC disulfate. These data indicate that glucuronidation is not the cause of bicarbonate-rich hypercholeresis induced by UDC but that glucuronidation and sulfation change the effect of UDC on biliary lipid secretion.

Estradiol-17β-glucuronide-induced cholestasis : effects of ursodeoxycholate-3-O-glucuronide and 3,7-disulfate

The effect of the co-infusion of ursodeoxycholate and its taurine conjugate, 3-O-glucuronide and 3,7-disulfate on estradiol-17 beta-glucuronide-induced cholestasis was examined. Estradiol-17 beta-glucuronide was intravenously administered to bile-drained rats at a rate of 0.075 mumol/min/100 g for 20 min. Co-infusion of ursodeoxycholate and its conjugates was simultaneously begun at a rate of 0.2 mumol/min/100 g and continued for 120 min. Ursodeoxycholate failed to improve and tauroursodeoxycholate only partially improved estradiol-17 beta-glucuronide-induced cholestasis between 20 and 40 min, although both bile acids increased bile flow after 80 min. Tauroursodeoxycholate increased biliary estradiol-17 beta-glucuronide excretion. Ursodeoxycholate-3-O-glucuronide completely inhibited cholestasis induced by estradiol-17 beta-glucuronide without changing biliary estradiol-17 beta-glucuronide excretion. Although ursodeoxycholate-3,7-disulfate had only a minor effect on cholestasis, it increased biliary excretion of estradiol-17 beta-glucuronide. In the Eizai hyperbilirubinuria rat (EHBR), a hyperbilirubinemic mutant Sprague-Dawley rat, the same dose of estradiol-17 beta-glucuronide failed to induce cholestasis with a marked delay in biliary excretion of estradiol-17 beta-glucuronide. In summary, ursodeoxycholate-3-O-glucuronide is more effective than tauroursodeoxycholate in inhibiting estradiol-17 beta-glucuronide-induced cholestasis and ursodoexycholate-3,7-disulfate had little effect. However, the unexpected effects of ursodeoxycholate-3-O-glucuronide and 3,7-disulfate on excretion of estradiol-17 beta-glucuronide suggest that the interaction of these anions at the canalicular membrane is complicated, with interaction occurring at more than two pathways of the biliary excretion of these anions.

Comparison of biliary excretion and metabolism of lithocholic acid and its sulfate and glucoronide conjugates in rats

Biliary excretion and biotransformation of tracer doses of [14C]lithocholic acid and its sulfate and glucuronide intravenously injected into bile-drainaged rats were compared. Biliary excretion efficiency was in the order of unconjugate sulfate glucuronide and all conjugates were completely excreted into bile within 60 min after injection. Only tracer doses of radioactivity were found in the liver and urine. About 90% of radiolabeled bile acids in bile were conjugated with taurine immediately after injection of lithocholic acid, whereas lithocholic acid-glucuronide was only partly conjugated with taurine all the time (less than 6%) and excreted into bile mainly as native compound. In the first 10 min, 66% of lithocholic acid-sulfate was conjugated with taurine and it gradually proceeded up to 87%. Hydroxylation at C-6 and C-7 positions of lithocholic acid proceeded time-dependently up to 45%. No hydroxylation was observed with lithocholic acid-sulfate or glucuronide. Differences of biliary excretion rate of these conjugates may be one of the reasons for the delayed decrease of sulfated and glucuronidated bile acids in serum after bile drainage to patients with obstructive jaundice of during the recovery of acute hepatitis than non-esterified bile acids.

Changes in biliary excretory mechanisms in bile duct-ligated rat

The effects of bile duct ligation on biliary excretion of bile acids, glutathione, and lipids were studied in the rat. The bile duct of the rat was ligated for three days. The biliary bile acid excretion after bile duct cannulation was higher at first, but after 90 min became lower than that in the control rat. The bile flow in the bile duct-ligated rat was higher after bile duct cannulation and gradually decreased to the same level as in the control rat. Biliary glutathione excretion, which has been suggested to be a driving force for the bile acid-independent canalicular bile flow, was markedly decreased in the bile duct-ligated rat. The mannitol clearance was increased and the bile ductules showed proliferation in the bile duct-ligated rat, suggesting an increase in the ductular bile flow. Biliary excretion of lithocholate glucuronide was more markedly impaired than that of taurocholate. When taurocholate was infused at higher rates, which increases bile flow and biliary excretion of bile acid and lipids in the control rat, biliary bile acid and lipid excretion remained constant in the bile duct-ligated rat. These findings indicate that, in the bile duct-ligated rat, the ductular bile flow was increased and bile acid-independent canalicular bile flow was decreased and that, although the biliary excretion of bile acids was not as impaired as that of organic anions, the capacity of bile acid and lipid excretion was markedly decreased.

Colchicine inhibits lithocholate-3-O-glucuronide-induced cholestasis in rats

BACKGROUND/AIMS It has been suggested that vesicular transport of bile acids in hepatocytes occurs, especially at high-dose loads. METHODS The effect was studied of colchicine, a vesicular transport inhibitor, on lithocholate-3-O-glucuronide-induced cholestasis in rats. Cholestasis was induced by an intravenous infusion of lithocholate-3-O-glucoronide at the rate of 0.1 mumol.min-1.100 g-1 for 40 min. RESULTS Colchicine treatment almost completely inhibited cholestasis and increased biliary excretion of lithocholate-3-O-glucoronide, whereas lumicolchicine had no effect. Treatment with vinblastine, another vesicular transport inhibitor, also reduced the cholestasis. Colchicine did not affect biliary excretion of taurocholate infused at the rate of 0.3 mumol.min-1.100 g-1 for 40 min, but markedly inhibited its biliary excretion when infused at the rate of 1.5 mumol.min-1.100 g-1 for 40 min. Colchicine had no effect on biliary excretion of tauroursodeoxycholate (1.5 mumol.min-1.100 g-1 for 40 min), lithocholate-3-sulfate (0.3 mumol.min-1.100 g-1 for 40 min), or a trace amount of lithocholate-3-O-glucuronide. CONCLUSIONS These findings indicate that lithocholate-3-O-glucoronide-induced cholestasis is caused by its increased access to the vesicular transport pathway, possibly beyond the capacity of the transport by the cytosolic binders, and that the transport of lithocholate-3-O-glucoronide via the vesicular pathway induces cholestasis. Furthermore, the contribution of the vesicular pathway to hepatic transport may be different among bile acids, and lithocholate-3-O-glucuronide seems to have higher accessibility to this transport system.

Mechanisms of biliary excretion of lithocholate-3-sulfate in Eisai hyperbilirubinemic rats (EHBR)

Biliary excretion of lithocholate-3-sulfate is markedly impaired in EHBR. To examine the mechanism of biliary lithocholate-3-sulfate excretion in EHBR, the effects of colchicine treatment, a vesicular transport inhibitor, and infusion of taurocholate and organic anions were studied in EHBR and Sprague-Dawley rats. Colchicine treatment and taurocholate infusion had no effect on biliary lithocholate-3-sulfate excretion in EHBR, suggesting that biliary lithocholate-3-sulfate excretion is not mediated by the vesicular transport or by the bile acid excretory pathway. In control Sprague-Dawley rats, both sulfobromophthalein and dibromosulfophthalein infusion inhibited biliary lithocholate-3-sulfate excretion. In contrast, in EHBR dibromosulfophthalein infusion inhibited biliary lithocholate-3-sulfate excretion but BSP infusion did not. Indocyanine green and pravastatin infusion did not affect biliary lithocholate-3-sulfate excretion but pravastatin infusion had no effect in EHBR. These findings indicate that, whether physiologically important or not, two or more excretory pathways for organic anions exist at the canalicular membrane other than the ATP-dependent one.

Lithocholate-3-O-glucuronide-induced cholestasis. A study with congenital hyperbilirubinemic rats and effects of ursodeoxycholate conjugates.

The mechanism of lithocholate-3-O-glucuronide-induced cholestasis is unknown. In this study, we investigated the cholestatic effects of this agent in a congenital hyperbilirubinemic rat, EHBR. We also studied the effects of ursodeoxycholate-3-O-glucuronide and tauroursodeoxycholate on lithocholate-3-O-glucuronide-induced cholestasis in rats. Lithocholate-3-O-glucuronide, administered at the rate of 0.1 μmol/min/100 g for 40 min, a cholestatic dose in control rats, failed to cause cholestasis in EHBR, and biliary lithocholate-3-O-glucuronide excretion was delayed. Biliary concentrations of this agent did not correlate with the severity of cholestasis. Both tauroursodeoxycholate and ursodeoxycholate-3-O-glucuronide, infused at the rate of 0.2 μmol/min/100 g for 120 min, completely inhibited cholestasis induced by lithocholate-3-O-glucuronide administered at the rate of 0.1 μmol/min/100 g for 40 min. Only tauroursodeoxycholate enhanced biliary lithocholate-3-O-glucuronide excretion. These findings indicate that lithocholate-3-O-glucuronide-induced cholestasis is induced by damage at the level of the bile canalicular membrane. Ursodeoxycholate-3-O-glucuronide inhibits this cholestasis, possibly by inhibiting the access of lithocholate-3-O-glucuronide to the bile canalicular membrane.

Effects of lipopolysaccharide on the biliary excretion of bile acids and organic anions in rats

Background: Lipopolysaccharide is known to be a cause of cholestasis associated with sepsis. It has also recently been reported to down‐regulate the basolateral and canalicular transporters. The aim of the present study was to examine simultaneously the effect of lipopolysaccharide on the biliary excretion of typical substrates of bile salt export pump and multidrug resistance protein 2 in vivo, and the effect of lipopolysaccharide on the amount of these transporters.

Biliary Excretion of Phenolphthalein Sulfate in Rats

Glucuronide and glutathione conjugates have been reported to be substrates of multidrug resistance protein 2 (Mrp2), whereas sulfates of nonbile acid organic anions have never been reported as substrates of Mrp2. To further examine the substrate specificity of Mrp2, we examined the effects of bile acid sulfates on the biliary excretion of phenolphthalein sulfate in rats. The biliary excretion of phenolphthalein sulfate was markedly delayed in Eisai hyperbilirubinemic rats, an Mrp2-deficient strain, and was markedly inhibited by taurolithocholate-3-sulfate. The biliary excretion of leukotriene C4 metabolites and sulfobromophthalein was inhibited by phenolphthalein sulfate infusion to some extent. These findings suggest that phenolphthalein sulfate is a unique sulfated nonbile acid organic anion which is a substrate of Mrp2.