Hajime Takikawa

ATP-dependent Transport of Bile Salts by Rat Multidrug Resistance-associated Protein 3 (Mrp3)

We have previously shown that cloned rat multidrug resistance-associated protein 3 (Mrp3) has the ability to transport organic anions such as 17β-estradiol 17-β-d-glucuronide (E217βG) and has a different substrate specificity from MRP1 and MRP2 in that glutathione conjugates are poor substrates for Mrp3 (Hirohashi, T., Suzuki, H., and Sugiyama, Y. (1999) J. Biol. Chem. 274, 15181–15185). In the present study, the involvement of Mrp3 in the transport of endogenous bile salts was investigated using membrane vesicles from LLC-PK1 cells transfected with rat Mrp3 cDNA. The ATP-dependent uptake of [3H]taurocholate (TC), [14C]glycocholate (GC), [3H]taurochenodeoxycholate-3-sulfate (TCDC-S), and [3H]taurolithocholate-3-sulfate (TLC-S) was markedly stimulated by Mrp3 transfection in LLC-PK1 cells. The extent of Mrp3-mediated transport of bile salts was in the order, TLC-S > TCDC-S > TC > GC. The K m andV max values for the uptake of TC and TLC-S wereK m = 15.9 ± 4.9 μm andV max = 50.1 ± 9.3 pmol/min/mg of protein and K m = 3.06 ± 0.57 μm andV max = 161.9 ± 21.7 pmol/min/mg of protein, respectively. At 55 nm[3H]E217βG and 1.2 μm[3H]TC, the apparent K m values for ATP were 1.36 and 0.66 mm, respectively. TC, GC, and TCDC-S inhibited the transport of [3H]E217βG and [3H]TC to the same extent with an apparent IC50 of ∼10 μm. TLC-S inhibited the uptake of [3H]E217βG and [3H]TC most potently (IC50 of ∼1 μm) among the bile salts examined, whereas cholate weakly inhibited the uptake (IC50 ∼75 μm). Although TC and GC are transported by bile salt export pump/sister of P-glycoprotein, but not by MRP2, and TCDC-S and TLC-S are transported by MRP2, but not by bile salt export pump/sister of P-glycoprotein, it was found that Mrp3 accepts all these bile salts as substrates. This information, together with the finding that MRP3 is extensively expressed on the basolateral membrane of human cholangiocytes, suggests that MRP3/Mrp3 plays a significant role in the cholehepatic circulation of bile salts.

Urinary concentrations of bile acid glucuronides and sulfates in hepatobiliary diseases

SummaryUrinary bile acids in normal subjects and patients with obstructive jaundice and liver cirrhosis were quantitated by mass fragmentography after separation into nonglucuronidated-nonsulfated, glucuronidated and sulfated fractions. Mean values of total bile acids in urine were as follows: Control subjects (n=7), 1.90 ±0.67; obstructive jaundice (n=9), 77.90 ±40.39; liver cirrhosis, compensated (n=6), 15.14 ±8.97, and decompensated (n=6), 11.84 ±9.32 (mean ±SD, mg/day). The percentages of each conjugate was 19–29% in the non glucuronidated-nonsulfated fraction, 6–14% in the glucuronidated fraction and 60–74% in the sulfated frac tion. Bile acids in urine and serum correlated well in each fraction (r=0.82-0.84, p<0.001). The clearance of the three conjugates was the highest in the sulfates, and the clearance of glucuronides was higher than that of non-esterified bile acids. The glucuronidation and sulfation of bile acids play an important role in the detoxication of bile acids by excreting them into urine, especially in patients with elevated serum bile acids.

Effects of organic anions and bile acid conjugates on biliary excretion of pravastatin in the rat

AbstractPurpose. Biliary organic anion excretion is mediated by an ATP-dependent primary active transporter, a so-called canalicular multispecific organic anion transporter (cMOAT). As there appear to be many canalicular organic anion transports, we examined the effects of various organic anions and bile acid conjugates on the biliary excretion of pravastatin in rats. Methods. [l4C]pravastatin was intravenously injected into rats with bile drainage in the presence and absence of the continuous infusion of organic anions and bile acids, and radioactivity of its biliary excretion was studied. Results. Biliary excretion of [14C]pravastatin was markedly inhibited by sulfobromophthalein-glutathione, taurolithocholate-3-sulfate, ursodeoxycholate-3,7-sulfate, and ursodeoxycholate-3-O-glucuronide. In contrast, dibromosulfophthalein only slightly inhibited biliary pravastatin excretion, and cefpiramide did not affect biliary pravastatin excretion. Conclusions. These findings further support the multiplicity of canalicular organic anion transport, and pravastatin is considered to be excreted through a canalicular transporter which is absent in EHBR in addition to through cMOAT.

Cholestasis induced by lithocholate and its glucuronide: their biliary excretion and metabolism

We studied the effects of the infusion of lithocholate and lithocholate-3-sulfate and 3-glucuronide in rats (0.29 mumol/min per 100 g body weight for 40 min) on bile flow, together with their biliary excretion and metabolism. Lithocholate-glucuronide had a higher cholestatic effect than lithocholate, whereas lithocholate-sulfate had almost no effect on bile flow. Lithocholate was mainly converted to taurine or glucuronide conjugates in the bile, serum and liver and hydroxylation of the tauro-conjugate proceeded. Lithocholate-sulfate was almost completely excreted in the bile, mainly as tauro-conjugate. Lithocholate-glucuronide was excreted in bile almost without conjugation, while some taurine conjugation occurred in the serum and liver. These results suggest that the poor biotransformation of lithocholate-glucuronide is related to its higher cholestatic potency than lithocholate.

Determination of sulfated and nonsulfated bile acids in serum by mass fragmentography

A Sep-Pak C18 cartridge was used for purification of bile acids from serum. Three kinds of deuterium labeled internal standards were required for accurate measurement of individual sulfated and nonsulfated bile acids. These internal standards were added to the serum before its application to the cartridge. Separation of sulfated and nonsulfated bile acids was performed on piperidinohydroxypropyl Sephadex LH-20 column chromatography. The nonsulfate fraction was submitted to alkaline hydrolysis, and the sulfate fraction to solvolysis followed by alkaline hydrolysis. Each fraction was converted to the hexafluoroisopropyl-trifluoroacetyl derivatives and quantitated by mass fragmentography. The recovery of each bile acid sulfate was quite satisfactory. In fasting healthy subjects the mean of total nonsulfated bile acids in serum was 1.324 micrograms/ml, and that of total sulfated bile acids was 0.450 micrograms/ml. Sulfated lithocholic acid comprised a large part of sulfated bile acids in healthy subjects.

Superoxide anion generating capacity and lysosomal enzyme activities of Kupffer cells in galactosamine induced hepatitis.

SummaryTo elucidate the function of the reticuloendothelial system of liver in hepatic injury, we investigated the effect of endotoxins on Superoxide anion (O2-) generating capacity and lysosomal enzyme activities of Kupffer cells isolated from rats treated with galactosamine (Gal N), with Gal N supplemented with polymyxin B (Polymyxin B-Gal N), with lipopolysaccharide (LPS) and from control rats. After collagenase digestion of the liver and centrifugation over metrizamide gradient, Kupffer cells were prepared by the dish adherence procedure. O2- production by the cells was examined as chemiluminescence during phagocytosis of latex particles and Β-glucuronidase activities were analyzed. High titers of endotoxemia were detected in LPS and Gal N rats by Iimulus test, while a low endotoxemia titer was found in Polymyxin B-Gal N rats. Hepatocyte damage was found in Gal N rats, but little was recognized in LPS and Polymyxin B-Gal N rats. In the latter groups, Kupffer cells, activated by endotoxins, showed the enhancement of chemiluminescence and a release of lysosomal enzyme. Though lysosomal enzyme was released from Kupffer cells in Gal N rats, chemiluminescence was slightly suppressed in spite of the high titer of endotoxemia. These results appear to be related to the consumption of O2- during liver injury. The functional state of Kupffer cells was thus changed by the grade of endotoxemia and hepatic injury.

Effect of organic anions and bile acid conjugates on biliary excretion of taurine-conjugated bile acid sulfates in the rat

Biliary organic anion excretion is mediated by an ATP-dependent primary active transporter, canalicular multispecific organic anion transporter/multidrug resistance protein 2. On the other hand, a multiplicity of canalicular organic anion transporter/multidrug resistance protein 2 has been suggested. Therefore, to examine the effect of hydrophobicity on the substrate specificity of canalicular multispecific organic anion transporter/multidrug resistance protein 2, we examined the effect of organic anions and bile acid conjugates on biliary excretion of three taurine-conjugated bile acid sulfates with different hydrophobicity, taurolithocholate-3-sulfate, taurochenodeoxycholate3-sulfate, and taurocholate-3-sulfate in rats. Biliary excretions of these bile acid conjugates were delayed in Eisai hyperbilirubinemic rats. Biliary excretion of these bile acid conjugates was inhibited by sulfobromophthalein, whereas biliary excretion and taurocholate-3-sulfate was not inhibited by phenolphthalein glucuronide. Taurolithocholate-3-sulfate and ursodeoxycholate-3-glucuronide decreased biliary excretion of taurochenodeoxycholate-3-sulfate and taurocholate-3-sulfate, but ursodeoxycholate-3,7-disulfate did not affect biliary excretion of taurochenodeoxycholate-3-sulfate and taurocholate-3-sulfate. These findings indicate that very hydrophilic organic anions are not good substrates of canalicular multispecific organic anion transporter/multidrug resistance protein 2.

Glucuronidated and sulfated bile acids in serum of patients with acute hepatitis

The concentrations of glucuronidated and sulfated bile acids in the serum of 15 patients with acute hepatitis were determined by mass fragmentography. Total serum bile acid levels were 13.79–444.10 μmol/liter, and the percentages of glucuronidated and sulfated bile acids were in the wide ranges of 1.7–33% and 2.4–49%, respectively. In four of the five patients for whom serum bile acids were analyzed serially from the acute to the recovery stages of acute hepatitis, the decrease of the glucuronidated and sulfated bile acids was slower than that of nonglucuronidated, nonsulfated bile acids. Thus, the relative proportion of the glucuronides and sulfates in total bile acids apparently increased during the recovery phase. The mechanism for the relative predominance of bile acid esters in serum during recovery is unknown but might reflect an improved excretion of the nonesterified bile acids into bile after the rapid recovery of intrahepatic cholestasis.

Serum concentrations of glucuronidated and sulfated bile acids in children with cholestasis

Serum concentrations of nonglucuronidated-nonsulfated, glucuronidated, and sulfated bile acids in 9 control children and 16 children with cholestasis were quantitated by mass fragmentography. Total bile acid levels in control children were 19.55 +/- 2.78 mumol/liter (mean +/- SEM), and glucuronidated and sulfated bile acids comprised 2.6 +/- 0.5 and 17 +/- 3.1%, respectively. In 9 patients with congenital biliary atrasia, total bile acid levels were 167.34 +/- 11.18 mumole/liter of which 2.1 +/- 0.3% were glucuronidated and 15 +/- 1.4% were sulfated. Lithocholic and 3 beta-hydroxy-5-cholenoic acids, which have hepatotoxic effects, were presented in only small amounts in cholestatic children, and they were almost all glucuronidated or sulfated. The percentages of glucuronidated bile acids in control and cholestatic children were lower than in healthy and cholestatic adults, which may be explained by the lower activity of UDP-glucuronyltransferase in neonatal liver.

Effects of Organic Anions and Vinblastine on Biliary Excretion of Erythromycin in Rats

Since little is known about the mechanism of biliary excretion of cationic drugs, biliary excretion of erythromycin was studied in rats. Infusion of sulfobromophthalein and taurocholate significantly decreased biliary erythromycin excretion, whereas infusion of dibromosulfophthalein, cefpiramide, ursodeoxycholate-3-O-glucuronide and taurolithocholate-3-sulfate had no effect on biliary excretion of erythromycin. Vinblastine significantly inhibited biliary erythromycin excretion. Phenothiazine treatment significantly increased biliary erythromycin excretion. However, erythromycin infusion did not affect biliary vinblastine excretion. These findings indicate a multiplicity of biliary excretory pathways for organic cations; at least one additonal pathway may exist for organic cations apart from P-glycoprotein.