Impaired uptake of conjugated bile acids and hepatitis b virus pres1-binding in na(+) -taurocholate cotransporting polypeptide knockout mice.

UNLABELLED: The Na(+) -taurocholate cotransporting polypeptide (NTCP) mediates uptake of conjugated bile acids (BAs) and is localized at the basolateral membrane of hepatocytes. It has recently been recognized as the receptor mediating hepatocyte-specific entry of hepatitis B virus and hepatitis delta virus. Myrcludex B, a peptide inhibitor of hepatitis B virus entry, is assumed to specifically target NTCP. Here, we investigated BA transport and Myrcludex B binding in the first Slc10a1-knockout mouse model (Slc10a1 encodes NTCP). Primary Slc10a1(-/-) hepatocytes showed absence of sodium-dependent taurocholic acid uptake, whereas sodium-independent taurocholic acid uptake was unchanged. In vivo, this was manifested as a decreased serum BA clearance in all knockout mice. In a subset of mice, NTCP deficiency resulted in markedly elevated total serum BA concentrations, mainly composed of conjugated BAs. The hypercholanemic phenotype was rapidly triggered by a diet supplemented with ursodeoxycholic acid. Biliary BA output remained intact, while fecal BA excretion was reduced in hypercholanemic Slc10a1(-/-) mice, explained by increased Asbt and Ostα/ß expression. These mice further showed reduced Asbt expression in the kidney and increased renal BA excretion. Hepatic uptake of conjugated BAs was potentially affected by down-regulation of OATP1A1 and up-regulation of OATP1A4. Furthermore, sodium-dependent taurocholic acid uptake was inhibited by Myrcludex B in wild-type hepatocytes, while Slc10a1(-/-) hepatocytes were insensitive to Myrcludex B. Finally, positron emission tomography showed a complete abrogation of hepatic binding of labeled Myrcludex B in Slc10a1(-/-) mice. CONCLUSION: The Slc10a1-knockout mouse model supports the central role of NTCP in hepatic uptake of conjugated BAs and hepatitis B virus preS1/Myrcludex B binding in vivo; the NTCP-independent hepatic BA uptake machinery maintains a (slower) enterohepatic circulation of BAs, although it is occasionally insufficient to clear BAs from the circulation.

Regulation of renal tubular bile acid transport in the early phase of an obstructive cholestasis in the rat.

BACKGROUND/AIMS: In obstructive liver diseases, urinary excretion of bile acids is markedly enhanced. The mechanism of this effect is not entirely clear. The aim of the present study was to assess the glomerular and tubular factors involved in the renal handling of bile acids during the early phase of an obstructive cholestasis induced by a 24-hour bile duct ligation in rats. METHODS: In addition to conventional clearance experiments, taurocholate transport was studied on proximal tubular cells freshly isolated from rat kidneys. The expression of the taurocholate transport protein was determined in these cells by immunoblotting. RESULTS: Glomerular filtration rate and arterial blood pressure were not significantly affected by the cholestasis induced. The (3)H-taurocholate and (3)H-cholate clearances significantly increased whereas the fractional tubular reabsorption rates of these bile acids significantly fell. Probenecid did not affect the (3)H-taurocholate and (3)H-cholate clearances. By employing S0960, a specific inhibitor of the sodium-dependent bile salt transporter ASBT, it could be shown that this transporter mediates (3)H-taurocholate uptake into the proximal tubular cells. The bile duct ligation caused a significant decrease of the V(max) value of the taurocholate transporter indicating a downregulation of this transporter in cholestasis. The downregulation occurred without a change of the ASBT protein content of the proximal tubular cells. CONCLUSION: The results demonstrate that there is a functional adaptive downregulation of renal tubular bile acid transport enhancing renal clearance of bile acids during the early phase of an obstructive cholestasis.

Gender difference in the urinary excretion of organic anions in rats.

This study was aimed at clarifying the gender differences in the urinary excretion of organic anions and the gene expression of organic anion transporters in rats. The renal clearance with regard to the plasma concentration (CL(urine,p)) of taurocholate, dibromosulfophthalein (DBSP), and zenarestat, all substrates and/or inhibitors of organic anion transporting polypeptide 1 (Oatp1), was much higher in female than in male rats. The following results imply that the transport system(s) for the reabsorption of zenarestat across the luminal side exhibits a gender difference: 1) the renal uptake clearance assessed by an in vivo integration plot analysis of zenarestat from the blood side does not show any clear gender differences; 2) the renal clearance with regard to the kidney concentration (CL(urine,k)) of zenarestat in female rats was approximately 30 times higher than in male rats; and 3) both CL(urine,p) and CL(urine,k) were increased in male rats by the coinfusion of DBSP, which is an inhibitor of organic anion transporters. Northern and Western blot analyses confirmed a previous finding that the gene expression of Oatp1, which is localized at the apical plasma membrane of the kidney, was much higher in the kidneys of male rats. Overall, a gender difference in urinary excretion is commonly observed for several organic anions, including Oatp1 substrates and inhibitors, and Oatp1 and/or transporters that have a similar substrate specificity to Oatp1 could be involved in such a phenomenon involving its substrates.

Ursocholic acid, a hydrophilic bile acid, fails to improve liver function parameters in primary biliary cirrhosis: comparison with ursodeoxycholic acid.

OBJECTIVE: To compare the effect of short term feeding of ursocholic acid, a hydrophilic bile acid, as the unconjugated acid and the taurine conjugate, on clinical and biochemical features and bile acid metabolism with that of ursodeoxycholic acid in four patients with primary biliary cirrhosis. METHODS: Four patients with stage II primary biliary cirrhosis were studied. Two were fed ursocholic acid (900 mg/day), and two were given tauroursocholate (900 mg/day) in three divided doses. After 1 month, all patients were given 900 mg/day of ursodeoxycholic acid. Fasting serum, bile, and 24-hour urine levels were measured before and at the end of ursocholic acid and tauroursocholate feeding and after 1 month of ursodeoxycholic acid feeding. Clinical and biochemical symptoms were measured by routine hospital methods, and bile acids were measured by gas-liquid chromatography. RESULTS: One month of ursocholic acid or tauroursocholate feeding did not improve clinical or biochemical findings in any patient. Approximately 21-25% ursocholic acid was present in the serum and bile, with substantial metabolism to deoxycholic acid. Increased ursocholic acid was excreted in the urine. In comparison, ursodeoxycholic acid improved biochemical parameters and was 45-65% enriched in the serum and bile. CONCLUSION: Ursocholic acid as the free bile acid or as taurine conjugate, although more hydrophilic, is poorly enriched in serum and bile and is ineffective in patients with primary biliary cirrhosis.

Measurement of conjugated 1 beta-hydroxycholic acid in urine of newborns by specific radioimmunoassay.

Anti-tauro 1 beta-hydroxycholic acid antisera were prepared by immunizing rabbits with N-(1 beta,3 alpha,7 alpha,12 alpha-tetrahydroxy-5 beta-cholan-24-oyl)glycine bovine serum albumin conjugate. The immunoglobulin G fraction was obtained by ammonium sulfate precipitation, followed by diethylaminoethyl cellulose column chromatography. The antibody was characterized using [2-3H]tauro 1 beta-hydroxycholic acid which has a high affinity (Ka = 1.09 x 10(9) M-1) and reasonable specificity. Cross-reactivity for glyco 1 beta-hydroxycholic acid was 100% and those for other 1 beta-hydroxylated bile acids ranged from 4.32 to 29.6%. Concentrations of conjugated 1 beta-hydroxycholic acid in urine of newborns at 0-20 d after birth were determined by radioimmunoassay to be significant (0.2-11.1 micrograms/ml), exhibiting a tendency to increase during the 20 d after birth.

N-acetylglucosaminides. A new type of bile acid conjugate in man.

Bile acids were extracted from human urine and were separated into groups of nonamidated and glycine- and taurine-conjugated compounds. Each group was subfractionated in a reversed-phase high performance liquid chromatography system, and the fractions were analyzed by negative ion fast atom bombardment mass spectrometry and also by gas chromatography-mass spectrometry after enzymatic removal of glycine and taurine moieties. The major glycosides of the non-amidated bile acids were more polar than reference bile acid glucosides and gave quasimolecular ions at m/z 592, 594, and 610 consistent with N-acetylglucosaminides of unsaturated dihydroxy and saturated di- and trihydroxy bile acids. Gas chromatography-mass spectrometry analyses of methyl ester trimethylsilyl ether derivatives showed fragments typical for N-acetylglucosaminides (m/z 173 and 186) in addition to those also given by glucosides (m/z 204 and 217). The N-acetylglucosaminides were inert toward alpha- and beta-glucosidase but were cleaved completely with N-acetylglucosaminidase. The released sugar moiety was identified as N-acetylglucosamine. One of the liberated bile acids was identified as ursodeoxycholic acid. The other acids were not identical to any known primary or secondary bile acid in humans. Fast atom bombardment mass spectrometry analyses of the glycine-and taurine-conjugated bile acid glycosides only showed ions consistent with the presence of glucosides (m/z 626 and 676). These compounds were sensitive only toward beta-glucosidase which liberated a trihydroxy bile acid as the major compound. Based on the recover of 13C- and 14C-labeled chenodeoxycholic acid glucoside added as internal standard, the daily excretion of nonamidated bile acid glycosides was estimated to be about 137 micrograms or 0.29 mumol, N-acetylglucosaminides constituting about 90%. The daily excretion of the glucosides of amidated bile acids was about 150 micrograms or 0.25 mumol, glycine conjugates constituting about 90%.

Sulfated and nonsulfated bile acids in urine of patients with biliary atresia: analysis of bile acids by high-performance liquid chromatography.

To elucidate urinary bile acid patterns in patients with biliary atresia (BA), 15 sulfated and nonsulfated bile acids in urine were separately measured by high-performance liquid chromatography. This relatively simple technique for fluorescence detection utilizes the enzyme 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) to reveal urinary bile acid patterns. By this method, recovery rates of sulfated and nonsulfated bile acids in urine were satisfactory, and this analysis was shown to be applicable to clinical situations. In 10 patients with BA, the mean level of total bile acids in urine (23.35 +/- 18.51 mumol/day) was seven times higher than the mean level in eight normal infants (3.05 +/- 2.05 mumol/day). In the infants with BA, the mean level of total sulfated bile acids was about half of the total bile acid level. The main components of urinary nonsulfated bile acids in BA were glycocholic acid (6.21 +/- 5.55 mumol/day) and taurocholic acid (2.28 +/- 1.33 mumol/day), whereas the main components of the urinary sulfated bile acids were glycochenodeoxycholic acid (4.58 +/- 6.97 mumol/day) and taurochenodeoxycholic acid (3.67 +/- 3.54 mumol/day). Chenodeoxycholic acid, which is relatively toxic to the liver, may more easily be conjugated with sulfate and, hence, excreted into urine at a faster rate than cholic acid. Marked individual variations in urinary bile acid patterns were observed not only in BA patients but also in normal controls.

Enhancement of the urinary excretion of non-sulphated and sulphated radioactive bile acids by sodium acetate in the bile duct obstructed rat.

The renal clearances of [14C]glycocholate, [14C]taurocholate and [3H]glycochenodeoxycholate-3-sulphate were determined in bile duct obstructed rats. Comparisons of the bile acid clearances with glomerular filtration rates (GFR) indicate that most of the filtered bile acids are reabsorbed. Inhibition studies with p-aminohippurate (PAH) and probenecid suggest that a proportion of the bile acids in urine is secreted. Attempts were made to increase the renal clearance of the bile acids by the administration of pharmacological agents. An infusion of sodium acetate (0.3 mol/l) increased the clearance of the radioactive bile acids and augmented the urinary excretion of endogenous 3 alpha-hydroxy bile acids and reduced their concentration in plasma.

Unconjugated, glycine-conjugated, taurine-conjugated bile acid nonsulfates and sulfates in urine of young infants with cholestasis.

A direct assay system for conjugated bile acids using an enzymatic procedure and high-performance liquid chromatography was used for the analysis of urinary bile acid profiles in young infants with intrahepatic cholestasis (idiopathic neonatal hepatitis syndrome) or extra-hepatic biliary atresia. The major urinary bile acids were cholate and chenodeoxycholate conjugates, but a small amount of deoxycholate and 3 beta-hydroxy-5-cholenate conjugates were detected. Although there was no significant difference in total bile acid excretion between patients with intrahepatic cholestasis and extrahepatic biliary atresia, mean ratios of cholate to chenodeoxycholate and sulfated to total urinary bile acids were different between the two groups examined (5.63 +/- 2.83 vs. 2.50 +/- 1.25, p less than 0.05, 15.8 +/- 9.9 vs. 34.5 +/- 9.9%, p less than 0.005). The proportion of taurine-conjugated chenodeoxycholate in the sulfate fraction to the total bile acid was lower in intrahepatic cholestasis, compared with that in biliary atresia (7.7 +/- 7.5 vs 22.7% +/- 7.8%, p less than 0.005). The greater ratio of cholate to chenodeoxycholate and the reduced excretion of sulfated urinary bile acids in intrahepatic cholestasis was due to decreased taurine-conjugated chenodeoxycholate sulfate excretion.

Metabolism of steroid and amino acid moieties of conjugated bile acids in man. 3. Cholyltaurine (taurocholic acid).

After oral administration of [2,4-(3)H]-cholyl[(35)S]taurine to eight healthy subjects with indwelling nasoduodenal tubes, the specific activity of the cholyl and taurine moieties and the distribution of radioactivity in biliary bile acid and urinary metabolites, as well as total urinary and fecal (35)S and (3)H, were measured at intervals for 4-8 days. Similar measurements were made after [(35)S]taurine was given orally or intravenously or instilled into the distal intestine. The daily fractional turnover rate of the taurine moiety of cholyltaurine was low and similar to that of the cholyl moiety, indicating that deconjugation occurring during enterohepatic cycling was less than half that previously observed for glycine-conjugated bile acids. Some of the cholyl moiety was absorbed but, since reconjugation occurred predominantly with glycine, little reincorporation into the cholyltaurine pool was observed. Some of the taurine moiety was also absorbed intact but entered large taurine pools, and little reincorporation into the cholyltaurine pool was seen. Oral administration of taurine expanded the cholyltaurine pool and induced a decrease in the fractional turnover rate of the cholyl moiety of cholyltaurine, interpreted to indicate a greater reincorporation of the cholyl moiety because of increased reconjugation with taurine. Taurine moiety not absorbed as taurine appeared to be absorbed largely as sulfate which, like taurine, entered large endogenous pools. Little fecal excretion of (35)S occurred. (35)S was excreted in urine as taurine and sulfate, and excretion in the first 24 h (as percentage of administered dose) correlated highly (r = 0.93) with the daily fractional turnover rate of the taurine moiety. When taurine was instilled into the distal intestine, it appeared as such in plasma, but the more distal the site of instillation, the greater the fraction of urinary (35)S present as sulfate. The [(35)S]sulfate appeared to have come from bacterial degradation of [(35)S]taurine because, when [(35)S]taurine was given intravenously, (35)S was excreted in urine chiefly as [(35)S]taurine with little SO(4)=(-)[(35)S] being present.