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Metabolism and enterohepatic circulation of a new conjugated bile acid analogue: Ursodeoxycholyl-n-benzyltaurine
320A
853
AASLD ABSTRACTS
NTCP IS A CANDIDATE FOR THE CARRIER THAT MEDIATES BILE ACID TRANSPORT IN RAT LIVER MICROSOMES. F.Watanabe & A.Reuben. Liver Service. Medical University of South Carolina. Background: Bile acids are translocated within hepatocytes by cytoplasmic diffusion, and by carrier-mediated transport in the endoplasmic reticulum and Golgi apparatus. We have previously isolated a 52kD putative carrier from rat liver microsomal membranes using glycocholate-sepharose affinity chromatography (Hepatology 14:147A). Aim: To identify the 52kD putative carrier for microsomal bile acid transport. Methods and Results: Microsomes were isolated from rat liver hom0genates by sucrose density gradient Centrifugation; membranes were separated from secretory and non-integral membrane proteins by alkaline lysis. Microsomal membrane proteins were solubilized with octyl glucoside. 2D SDS-PAGE of glycocholate-sepharose affinity-purified microsomal membrane proteins showed the pl of previously identified 52kD proteins to be 4.5-5.5. Microsomal membrane proteins of 52kD and pl 4.55.5 were then isolated using preparative isoelectric focusing, preparative SDS-PAGE and electroelution. Amino acid sequencing and i m m u n o b l o t t i n g identified UDPglucuronosyltransferase (UGT) 2B2 as the major component of the acidic 52kD fraction. However, immunoblotting of the 52kD acidic proteins with a fusion protein antiserum to Ntcp (the basolateral liver plasma membrane Na÷-bile acid cotransporter, Ananthanarayan et al, Am. J. Physiol 1994; 267: G637) detected Ntcp immunoreactivity. After glycopeptidase F deglycosylation of the 52kD acidic proteins, Ntcp immunoreactivity shifted from 52 to 33kD, whereas UGT2B2 did not change. Conclusion: A 52kD acidic fraction of rat liver microsomal membranes contains both UGT2B2 and Ntcp immunoreactivity, the presence of which could account for microsomal bile acid binding and transport, respectively.
8 5 5 METABOLISM AND ENTEROHEPATIC CIRCULATION OF A NEW CONJUGATED BILE ACID ANALOGUE: URSODEOXYCHOLYLN-BENZYLTAURINE. E. Frick, L.R. Hagey, CD.Schtein~,art. H-T. Ton-Nu, U. Bolder, C. Cerr~, R.T.Stravitz* and A.F. Hofmann Dept. of Medicine, U. California, San Diego, CA and *Medical College of Virginia, Richmond, VA Ursodeoxycholic acid (UDCA) is now widely used in the treatment of cholestatic liver disease. We sought to prepare a new conjugate of UDCA that would I) be resistant to bacterial 7-dehydroxylation to lithocholic acid 2) have no effect on bile acid (BA)biosynthesis and 3) inhibit ileal transport of endogenous conjugated BA (to upregulate cholesterol biosynthesis and hepatocyte LDL receptors). Methods: N-benzyl-tautine, prepared in 2 steps from tautine and banzyl bromide, was conjugated using conventional methods with UDCA and [24-14C]-UDCA to give ursodeoxycholyl-N-banzyltaurine (UDC-NBT) andits 14Clabelled malogue. Cholesterol 7tz-hydroxylase (C7AH) mRNA was measured in cultured rat hepatocytes by dot blot hybridization. Hepatic transport and metabolism were defined in anesthetized biliary fistula animals. Ileal peffusion used high flow rates to maintain a constant intraluminal conc~atrstion; absorption was equated with biliary recovery. The chemical form of radioactivity was assessed by chromatography. Results: UDC-NBT (up to 50 IxM) hadno effect on C7AH mRNA levels in isolated rat hepatocytes. In the biliary fistula rat, the T ~ for hepatic trmsport was 9 rtmol/min-kg [versus 17 for Cholyltanfine (C-tan)] and in the hamster was 5 ~tmoYmin-kg(versus 30 for Ctau). In the peffused rat ileum, UDC-NBT (2 mM) caused 43 % 4- 8.5 (M:eSD) reversible inhibition of C-tau (2 raM) transport. Under similar conditions UDCtau inhibited C-tau transport by 19% ± 2.8. UDC-NBT remained unchanged during ileal and hepatic ~arteport. In chronic feeding studies in hamsters, fecal radioactivity was largely (>90%) in the form of UDC-NBT, indicating the compound was resistmt to deconjugation-dehydroxylation during anterohepafic cycling. Condusiom: UDC-NBT appears to be superior to UDC-tau in that it is not converted to lithocholic acid and inhibits ileal transport of andoganous bile adds to a greater extant. These properties suggest the compound has potential utility in the (raatmant ofliver diseases and hypercholesterolemia. This research was funded in part by the Falk Foundation e.V., Freiburg, Germany.
HEPATOLOGYOctober 1995
854 INFLAMMATORY CYTOKINE REGULATION OF SODIUM DEPENDENT TAUROCHOLATE TRANSPORTER (Ntep) GENE EXPRESSION IN THE LIVER. R.M. Green and J.L Gollan. Brigham & Women's Hospital, Harvard Medical School, Boston, MA. We have recently dvraonstrat~ that tbe administration of tumor necrosis factor-a (TNFa), an important mediator of bepatic regeneration and sepsis, reduces mouse liver Ntcp mRNA levels. Since TNFct stimulates the production of other inflammatory cytokines, we hav©investigated the dt'ects of interloukin113 (IL-l[3) or intvrlankin-6 (IL-6) on Ntcp mRNA levels. C57BL/6 mice were treated with intrap~toneal TNFa, IL-l[3, or IL-6, and mRNA was isolated at various time periods. Northern blot analysis was performed using probes for Ntcp and Serum amyloid A (SAA), an acute phase reactant. A ubiquitin probe was used to validate the intw,rity of the mRNA and confirm equal RNA loading. Although TNF¢ (6gg/25g i.p.) administration caused a significant time-d~t decrea~ of stony-state Ntcp levels, which was maximal after 6 hours, SAA mRNA levels increased markedly in a similar fin~-depandant maoner. These concomitant effects suggest that the reduction in Ntcp mRNA is due to a regulated process, as opposed to noaspeeifie cellular cytotoxicity. Administration of IL-II3 (lpg/25g i.p.) caused a reduction of Ntcp mRNA levels in a manner similar to that observed with TNFa. Mice treated with IL-6 (l-10 p~/25g i.p.), however, ~xhibited no change in steady-state levels of Ntcp mRNA. In contrast, these doses of IL-6 stimulated a marked increase of SAA mRNA, indicating that these were physiologically active doses. SAA mRNA was undetectable in the saline treated control mine, yet highly abundant following IL-6 stimulation. Conclusions: These data indicate that TNFa and 113deema~ gone expression of Nwp. These e~zts are not due to the in vivo production of 11.,-6 which occurs following TNFct or IL-II3 administration. Morcov~, this decrease in mRNA expression appears to be a regulated process, rather than a nonepecific xeeponse. We postulate that TNFa and IL-I[3 can decrease theexpression of hepatecyte transporters aad thus potentially are important mediators of the cholestasis associated with inflammatory diseases.
856 E L E C T R O S P R A Y
IONIZATION-MASS SPECTROMETRY (ESI-MS) OF BILE SALTS (BS) IN P H Y S I O L O G I C A L FLUIDS. S. Barnes. L. Coward, M. Kirk & J.M. Crawford. Dept. Pharmacol., UAB, Birmingham, AL 35294 and Dept. Pathology, Brigham & Women's Hospital, Boston, MA 02115. ESI-MS is a soft ionization technique ideally suited to measurement of biological compounds. In this study we have examined the sensitivity limit for the detection of BS and the linear range of response using HPLC-ESI-MS, and have applied the method to the measurement of BS in several physiological fluids from rats. Analyses were carried out using gradient eintion (30-100% methanol in 1% acetic acid)on a 10 cm x 2.1 mm i.d. Cs reversed-phase HPLC column; eluates were passed into the ESI interface of a PE-Sciex API III triple quadrupole mass spectrometer. Negative ion spectra were recorded. Results: In ESI spectra, BS gave rise only to the molecular ions [M-H]-. The lowest amount of cholyltanrine (30 pmol) injected onto the HPLC column gave a signal-to-noise ratio of 10:1. The response curve for cholyltaurine was linear from 30 - 500 pine1 injected. Above that, the response curve underwent a sudden change of slope suggesting that the ion evaporation process was effected by the transition from a sprayed droplet solution containing monomer to one containing micelles, since at the time when ions are formed, evaporation reduces droplet volume by 50-fold. The slopes of the response curves for unconjugated BS and glycine and taurine conjugated BS were similar. The high sensitivity of ESI-MS enabled analysis of BS in bile without the necessity of extraction - biles were merely diluted 20-fold in 30% aqueous methanol containing 1% acetic acid. Urines were analyzed without dilution. Sera (500 pl) were subjected to solid phase extraction; extracts were reconstituted in 50 I.d of starting solvent. In cases where BS were intravenously infused into rats, the serum concentrations were high enough to necessitate dilution of the sample to prevent overloading of the detector. Summary_ & Conclusion: ESI=MS is ideally suited to the analysis of BS, giving a specific, highly sensitive and quantitative method without the need for sample recovery and chemical derivatization. Since ESI is dependent on solute concentration in the eluate, 3-5 btl/min flow rates used for reversed-phase HPLC on 300 Ilm i.d. capillary columns (as used in the analysis of peptides) will result in enhanced sensitivity for BS into the frnol range.
853
AASLD ABSTRACTS
NTCP IS A CANDIDATE FOR THE CARRIER THAT MEDIATES BILE ACID TRANSPORT IN RAT LIVER MICROSOMES. F.Watanabe & A.Reuben. Liver Service. Medical University of South Carolina. Background: Bile acids are translocated within hepatocytes by cytoplasmic diffusion, and by carrier-mediated transport in the endoplasmic reticulum and Golgi apparatus. We have previously isolated a 52kD putative carrier from rat liver microsomal membranes using glycocholate-sepharose affinity chromatography (Hepatology 14:147A). Aim: To identify the 52kD putative carrier for microsomal bile acid transport. Methods and Results: Microsomes were isolated from rat liver hom0genates by sucrose density gradient Centrifugation; membranes were separated from secretory and non-integral membrane proteins by alkaline lysis. Microsomal membrane proteins were solubilized with octyl glucoside. 2D SDS-PAGE of glycocholate-sepharose affinity-purified microsomal membrane proteins showed the pl of previously identified 52kD proteins to be 4.5-5.5. Microsomal membrane proteins of 52kD and pl 4.55.5 were then isolated using preparative isoelectric focusing, preparative SDS-PAGE and electroelution. Amino acid sequencing and i m m u n o b l o t t i n g identified UDPglucuronosyltransferase (UGT) 2B2 as the major component of the acidic 52kD fraction. However, immunoblotting of the 52kD acidic proteins with a fusion protein antiserum to Ntcp (the basolateral liver plasma membrane Na÷-bile acid cotransporter, Ananthanarayan et al, Am. J. Physiol 1994; 267: G637) detected Ntcp immunoreactivity. After glycopeptidase F deglycosylation of the 52kD acidic proteins, Ntcp immunoreactivity shifted from 52 to 33kD, whereas UGT2B2 did not change. Conclusion: A 52kD acidic fraction of rat liver microsomal membranes contains both UGT2B2 and Ntcp immunoreactivity, the presence of which could account for microsomal bile acid binding and transport, respectively.
8 5 5 METABOLISM AND ENTEROHEPATIC CIRCULATION OF A NEW CONJUGATED BILE ACID ANALOGUE: URSODEOXYCHOLYLN-BENZYLTAURINE. E. Frick, L.R. Hagey, CD.Schtein~,art. H-T. Ton-Nu, U. Bolder, C. Cerr~, R.T.Stravitz* and A.F. Hofmann Dept. of Medicine, U. California, San Diego, CA and *Medical College of Virginia, Richmond, VA Ursodeoxycholic acid (UDCA) is now widely used in the treatment of cholestatic liver disease. We sought to prepare a new conjugate of UDCA that would I) be resistant to bacterial 7-dehydroxylation to lithocholic acid 2) have no effect on bile acid (BA)biosynthesis and 3) inhibit ileal transport of endogenous conjugated BA (to upregulate cholesterol biosynthesis and hepatocyte LDL receptors). Methods: N-benzyl-tautine, prepared in 2 steps from tautine and banzyl bromide, was conjugated using conventional methods with UDCA and [24-14C]-UDCA to give ursodeoxycholyl-N-banzyltaurine (UDC-NBT) andits 14Clabelled malogue. Cholesterol 7tz-hydroxylase (C7AH) mRNA was measured in cultured rat hepatocytes by dot blot hybridization. Hepatic transport and metabolism were defined in anesthetized biliary fistula animals. Ileal peffusion used high flow rates to maintain a constant intraluminal conc~atrstion; absorption was equated with biliary recovery. The chemical form of radioactivity was assessed by chromatography. Results: UDC-NBT (up to 50 IxM) hadno effect on C7AH mRNA levels in isolated rat hepatocytes. In the biliary fistula rat, the T ~ for hepatic trmsport was 9 rtmol/min-kg [versus 17 for Cholyltanfine (C-tan)] and in the hamster was 5 ~tmoYmin-kg(versus 30 for Ctau). In the peffused rat ileum, UDC-NBT (2 mM) caused 43 % 4- 8.5 (M:eSD) reversible inhibition of C-tau (2 raM) transport. Under similar conditions UDCtau inhibited C-tau transport by 19% ± 2.8. UDC-NBT remained unchanged during ileal and hepatic ~arteport. In chronic feeding studies in hamsters, fecal radioactivity was largely (>90%) in the form of UDC-NBT, indicating the compound was resistmt to deconjugation-dehydroxylation during anterohepafic cycling. Condusiom: UDC-NBT appears to be superior to UDC-tau in that it is not converted to lithocholic acid and inhibits ileal transport of andoganous bile adds to a greater extant. These properties suggest the compound has potential utility in the (raatmant ofliver diseases and hypercholesterolemia. This research was funded in part by the Falk Foundation e.V., Freiburg, Germany.
HEPATOLOGYOctober 1995
854 INFLAMMATORY CYTOKINE REGULATION OF SODIUM DEPENDENT TAUROCHOLATE TRANSPORTER (Ntep) GENE EXPRESSION IN THE LIVER. R.M. Green and J.L Gollan. Brigham & Women's Hospital, Harvard Medical School, Boston, MA. We have recently dvraonstrat~ that tbe administration of tumor necrosis factor-a (TNFa), an important mediator of bepatic regeneration and sepsis, reduces mouse liver Ntcp mRNA levels. Since TNFct stimulates the production of other inflammatory cytokines, we hav©investigated the dt'ects of interloukin113 (IL-l[3) or intvrlankin-6 (IL-6) on Ntcp mRNA levels. C57BL/6 mice were treated with intrap~toneal TNFa, IL-l[3, or IL-6, and mRNA was isolated at various time periods. Northern blot analysis was performed using probes for Ntcp and Serum amyloid A (SAA), an acute phase reactant. A ubiquitin probe was used to validate the intw,rity of the mRNA and confirm equal RNA loading. Although TNF¢ (6gg/25g i.p.) administration caused a significant time-d~t decrea~ of stony-state Ntcp levels, which was maximal after 6 hours, SAA mRNA levels increased markedly in a similar fin~-depandant maoner. These concomitant effects suggest that the reduction in Ntcp mRNA is due to a regulated process, as opposed to noaspeeifie cellular cytotoxicity. Administration of IL-II3 (lpg/25g i.p.) caused a reduction of Ntcp mRNA levels in a manner similar to that observed with TNFa. Mice treated with IL-6 (l-10 p~/25g i.p.), however, ~xhibited no change in steady-state levels of Ntcp mRNA. In contrast, these doses of IL-6 stimulated a marked increase of SAA mRNA, indicating that these were physiologically active doses. SAA mRNA was undetectable in the saline treated control mine, yet highly abundant following IL-6 stimulation. Conclusions: These data indicate that TNFa and 113deema~ gone expression of Nwp. These e~zts are not due to the in vivo production of 11.,-6 which occurs following TNFct or IL-II3 administration. Morcov~, this decrease in mRNA expression appears to be a regulated process, rather than a nonepecific xeeponse. We postulate that TNFa and IL-I[3 can decrease theexpression of hepatecyte transporters aad thus potentially are important mediators of the cholestasis associated with inflammatory diseases.
856 E L E C T R O S P R A Y
IONIZATION-MASS SPECTROMETRY (ESI-MS) OF BILE SALTS (BS) IN P H Y S I O L O G I C A L FLUIDS. S. Barnes. L. Coward, M. Kirk & J.M. Crawford. Dept. Pharmacol., UAB, Birmingham, AL 35294 and Dept. Pathology, Brigham & Women's Hospital, Boston, MA 02115. ESI-MS is a soft ionization technique ideally suited to measurement of biological compounds. In this study we have examined the sensitivity limit for the detection of BS and the linear range of response using HPLC-ESI-MS, and have applied the method to the measurement of BS in several physiological fluids from rats. Analyses were carried out using gradient eintion (30-100% methanol in 1% acetic acid)on a 10 cm x 2.1 mm i.d. Cs reversed-phase HPLC column; eluates were passed into the ESI interface of a PE-Sciex API III triple quadrupole mass spectrometer. Negative ion spectra were recorded. Results: In ESI spectra, BS gave rise only to the molecular ions [M-H]-. The lowest amount of cholyltanrine (30 pmol) injected onto the HPLC column gave a signal-to-noise ratio of 10:1. The response curve for cholyltaurine was linear from 30 - 500 pine1 injected. Above that, the response curve underwent a sudden change of slope suggesting that the ion evaporation process was effected by the transition from a sprayed droplet solution containing monomer to one containing micelles, since at the time when ions are formed, evaporation reduces droplet volume by 50-fold. The slopes of the response curves for unconjugated BS and glycine and taurine conjugated BS were similar. The high sensitivity of ESI-MS enabled analysis of BS in bile without the necessity of extraction - biles were merely diluted 20-fold in 30% aqueous methanol containing 1% acetic acid. Urines were analyzed without dilution. Sera (500 pl) were subjected to solid phase extraction; extracts were reconstituted in 50 I.d of starting solvent. In cases where BS were intravenously infused into rats, the serum concentrations were high enough to necessitate dilution of the sample to prevent overloading of the detector. Summary_ & Conclusion: ESI=MS is ideally suited to the analysis of BS, giving a specific, highly sensitive and quantitative method without the need for sample recovery and chemical derivatization. Since ESI is dependent on solute concentration in the eluate, 3-5 btl/min flow rates used for reversed-phase HPLC on 300 Ilm i.d. capillary columns (as used in the analysis of peptides) will result in enhanced sensitivity for BS into the frnol range.