Sulfated Bile Acids Research Articles

Uninary sulfated bile acids: A new simple urine test for cholestasis in infants and children

Background/Purpose: Measurement of urinary sulfated bile acid (USBA) has been reported as a simple urine test that reflects the degree of cholestasis. The authors report the diagnostic value of this new laboratory test in various cholestatic conditions affecting infants and children. Methods: A urine sample was collected from 4 surgical neonates with parenteral nutrition–induced cholestasis and 48 patients with biliary atresia (BA). USBA was measured by direct enzymatic assay. Results: In 3 patients receiving parenteral nutrition, USBA increased with caloric gains. For one surgical patient, a decrease in calories because of liver dysfunction resulted in a decrease of USBA, closely reflecting the fluctuations of caloric intake. In patients with BA, a significant positive correlation was noted between serum bile acid and USBA (r = 0.85; P <.01). Ten of 14 febrile episodes in 6 patients with liver dysfunction and increased C-reactive protein showed elevated USBA, thus diagnosed as cholangitis. Four febrile episodes caused by viral infection showed no elevation of USBA. Conclusions: USBA is a simple and sensitive noninvasive test reflecting the degree of cholestasis in infants and children. USBA correlated highly with serum bile acid levels and may be helpful in diagnosis of cholestasis caused by cholangitis without blood sampling. J Pediatr Surg 37:1707-1708. Copyright 2002, Elsevier Science (USA). All rights reserved.

Hepatobiliary transport of bile acids and organic anions.

Recent studies have elucidated the mechanism and regulation of hepatic transport of bile acids and organic anions. Bile acids are taken up into hepatocytes by basolateral transporters, Na+-dependently by Na+/taurocholate cotransporting polypeptide (NTCP) and Na+-independently by organic anion transporting polypeptide (OATP) families. Organic anions are taken up into hepatocytes by OATP families. These compounds are then transported in hepatocytes bound to cytosolic binders, and subjected to transport by ATP binding cassette (ABC) transporters at the canalicular membrane. Amidated bile acids are excreted into bile by bile salt export pump (BSEP), and organic anions and bile acid sulfates and glucuronides are excreted by multidrug resistance protein 2 (MRP2). Hepatic transporters are downregulated under cholestasis in rats and humans, except for MRP3, a basolateral ABC transporter, which is upregulated and may have a role in removing bile acids and organic anions from hepatocytes to the blood under cholestatic conditions. Nuclear receptors, which bind bile acids, have been shown to regulate the expression of hepatic transporters. Farnesoid X receptor (FXR), which downregulates CYP7A1, the rate-limiting enzyme of bile acid biosynthesis, upregulates BSEP and downregulates NTCP. MRP2 is upregulated by both FXR and pregnane X receptor (PXR), which upregulates CYP3A.

Transport of the sulfated, amidated bile acid, sulfolithocholyltaurine, into rat hepatocytes is mediated by Oatp1 and Oatp2

The uptake of the sulfated bile acid sulfolithocholyltaurine (SLCT) was investigated in isolated rat hepatocytes and in HeLa cells transfected with complementary DNAs (cDNAs) of organic anion transporting polypeptides (Oatps) 1 and 2 cloned from rat liver. In hepatocytes, transport of SLCT was greatly reduced by bromosulfophthalein (BSP), estrone sulfate, the precursor bile acids cholyltaurine and lithocholyltaurine, and 4,4'-diisothiocyanostilbene-2-2'-disulfonic acid (DIDS). However, SLCT transport was insensitive to 4-methylumbelliferyl sulfate, harmol sulfate, digoxin, fexofenadine, and lack of sodium ion. Because the estimation of kinetic constants was enhanced with use of inhibitors, BSP (1-50 μmol/L) was added to isolated rat hepatocytes to assess the various transport components for SLCT uptake. The resulting data showed a nonsaturable pathway and at least 2 pathways of different Michaelis-Menten constants ( K m ) (70 and 6 μmol/L) and similar maximum velocities ( V max ) (1.73 and 1.2 nmol/min/mg protein) and inhibition constants of 0.63 and 10.3 μmol/L for BSP. In expression systems, SLCT was taken up by Oatp1 and Oatp2 expressed in HeLa cells with similar K m values (12.6 ± 6.2 and 14.6 ± 1.9 μmol/L). These K m values were comparable to that observed for the high-affinity pathway in rat hepatocytes. In conclusion, the results suggest that transport of SLCT into rat liver is mediated in part by Oatp1 and Oatp2, high-affinity pathways, a lower-affinity pathway of unknown origin, and a nonsaturable pathway that is compatible with a transport system of high K m and/or passive diffusion. (H EPATOLOGY 2002;35:1031-1040.)

Identification of unusual 7-oxygenated bile acid sulfates in a patient with Niemann-Pick disease, type C

Niemann-Pick disease, type C, was diagnosed in a 3-month-old boy with hepatosplenomegaly, mild signs of cholestasis, hepatic inflammation and extramedullary erythropoesis, together with chronic airway disease. He developed muscular hypotonia, psychomotor retardation, rickets, and signs of peripheral neuropathy. The patient was found to excrete abnormal amounts of unusual bile acids in urine at 3 and 5 months of age. These acids were shown to have a 3β-hydroxy-Δ5 structure and to carry an oxo or hydroxy group at C-7. They were sulfated at C-3 and nonamidated or conjugated with glycine or taurine at C-24. Part of the 7-hydroxy acids, presumably the 7β-hydroxylated one, was also conjugated with N-acetylhexosamine, probably N-acetylglucosamine, at the 7-hydroxy group. Possible metabolic pathways for the formation of the 7-oxo and 7β-hydroxycholenoic acids are discussed. Based on previous data concerning the effects of 3β-hydroxy-Δ5 bile acids on bile acid transport, it is suggested that the formation of such bile acids is responsible for the cholestasis in this patient.—Alvelius, G., O. Hjalmarson, W. J. Griffiths, I. Björkhem, and J. Sjövall. Identification of unusual 7-oxygenated bile acid sulfates in a patient with Niemann-Pick disease, type C. J. Lipid Res. 2001. 42: 1571-1577.

Biliary excretion of tauroursodeoxycholate-3-sulfate in the rat.

Biliary organic anion excretion is mediated by an ATP-dependent primary active transporter, multidrug resistance protein 2. On the other hand, a multiplicity of canalicular organic anion transport has been suggested. Ursodeoxycholic acid, the 7beta-epimer of chenodeoxycholic acid, is clinically used for various hepatobiliary diseases. In our previous study, the contribution of multidrug resistance protein 2 for biliary excretion of taurine-conjugated bile acid sulfates depended on the numbers of hydroxyl residue. Therefore, to further examine the effect of hydrophobicity on the substrate specificity of multidrug resistance protein 2, we examined the effect of bile acid conjugates and organic anions on biliary excretion of tauroursodeoxycholate-3-sulfate, taurine and sulfonate-conjugated ursodeoxycholic acid, in rats. Biliary tauroursodeoxycholate-3-sulfate excretions was markedly delayed in Eisai hyperbilirubinemic rats. Taurolithocholate-3-sulfate inhibited but ursodeoxycholate-3,7-disulfate did not affect biliary tauroursodeoxycholate-3-sulfate excretion. Biliary tauroursodeoxycholate-3-sulfate excretion was inhibited by sulfobromophthalein, but was not inhibited by dibromosulfophthalein and cefpiramide. These findings indicate that tauroursodeoxycholate-3-sulfate is very specific for multidrug resistance protein 2.

Flow injection spectrophotometric determination of sulfated bile acids in urine with immobilized enzyme reactors using water soluble tetrazolium blue-5

A simple assay of sulfated bile acid (SBA) in urine using flow injection (FI)-spectrophotometry with immobilized enzyme reactors is proposed. The system consists of an injection valve, a switch valve, two immobilized enzyme reactors and a UV–VIS detector with a flow cell. The multi-step enzymatic reactions will occur when an injected sample containing SBA passes through the immobilized enzyme reactors. First, SBA will desulfate under catalysis of immobilized bile acid sulphate sulfatase (BSS), to form 3β-hydroxyl bile acids; the produced 3β-hydroxyl bile acid reacts with nicotinamide adenine dinucleotide (NAD +) under catalysis of co-immobilized 3β-hydroxylsteroid dehydrogenase (3β-HSD), and is converted to the 3-ketosteroid. Meanwhile, β-NAD + is converted to reduced nicotinamide adenine dinucleotide (NADH). Then by catalysis of immobilized diaphorase, NADH reacts with a novel reagent called “water soluble tetrazolium blue-5” (WST-5) to generate a blue diformazan dye, which is detected at 550 nm. By using FI-spectrophotometry manifold and optimized conditions, we have obtained a linear response for 1–75 μM glycolithocholate sulfate (GLCA-S) with a correlation coefficient of 0.999 and an analytical rate of 15 samples per hour. The R.S.D. was less than 1%. The recoveries (91–108%) of GLCA-S added into urine were satisfactory and the assay correlated well with the manual UBASTEC method. Therefore, it will be applicable for urine tests on patients suffering from hepatobiliary disease.

Characterization of bile acid transport mediated by multidrug resistance associated protein 2 and bile salt export pump

Biliary excretion of certain bile acids is mediated by multidrug resistance associated protein 2 (Mrp2) and the bile salt export pump (Bsep). In the present study, the transport properties of several bile acids were characterized in canalicular membrane vesicles (CMVs) isolated from Sprague–Dawley (SD) rats and Eisai hyperbilirubinemic rats (EHBR) whose Mrp2 function is hereditarily defective and in membrane vesicles isolated from Sf9 cells infected with recombinant baculovirus containing cDNAs encoding Mrp2 and Bsep. ATP-dependent uptake of [3H]taurochenodeoxycholate sulfate (TCDC-S) (Km=8.8 μM) and [3H]taurolithocholate sulfate (TLC-S) (Km=1.5 μM) was observed in CMVs from SD rats, but not from EHBR. In addition, ATP-dependent uptake of [3H]TLC-S (Km=3.9 μM) and [3H]taurocholate (TC) (Km=7.5 μM) was also observed in Mrp2- and Bsep-expressing Sf9 membrane vesicles, respectively. TCDC-S and TLC-S inhibited the ATP-dependent TC uptake into CMVs from SD rats with IC50 values of 4.6 μM and 1.2 μM, respectively. In contrast, the corresponding values for Sf9 cells expressing Bsep were 59 and 62 μM, respectively, which were similar to those determined in CMVs from EHBR (68 and 33 μM, respectively). By co-expressing Mrp2 with Bsep in Sf9 cells, IC50 values for membrane vesicles from these cells shifted to values comparable with those in CMVs from SD rats (4.6 and 1.2 μM). Moreover, in membrane vesicles where both Mrp2 and Bsep are co-expressed, preincubation with the sulfated bile acids potentiated their inhibitory effect on Bsep-mediated TC transport. These results can be accounted for by assuming that the sulfated bile acids trans-inhibit the Bsep-mediated transport of TC.

Biochemical and physiological evidence that bile acids produced and released by lake char (Salvelinus namaycush) function as chemical signals.

It has been hypothesized that faeces of juvenile lake char (Salvelinus namaycush) may contain chemical cues that mediate behaviour of conspecifics. However, our knowledge of bile acids naturally produced and released by fish is limited. Using HPLC, we fractionated bile acids produced and released by lake char and examined their stimulatory effectiveness using electro-olfactogram recordings. Taurocholic acid, taurochenodeoxycholic acid, taurooxocholanic acid, taurooxodeoxycholic acid 3alpha-sulphate, trace amounts of taurolithocholic acid and an unidentified sulphated bile steroid were found in bile and faeces. Bile acids were either taurine amidated or sulphated, or both. Lake char released an average of 4 nmol min(-1) bile acids per kilogram of body weight into their tank water. Urinary bile acids accounted for only a small portion of total bile acids released into water. Water and faeces contained higher proportion of taurochenodeoxycholic acid and sulphated bile acids (relative to taurocholic acid) than bile. The electro-olfactogram recordings demonstrated that bile acids released by lake char were detectable by their olfactory system at nanomolar concentrations, which is well below the levels of bile acids released into water. The exquisite olfactory sensitivity of lake char to water-borne bile acids released by their conspecifics is consistent with a role for these compounds as important chemical signals.

Development of a determination method for sulfated bile acids in urine

This dissertation gives a brief description of two new approachs for the assay of sulfated bile acid(SBA)in urine based on flow-injection chemiluminescence and spectrophotometry with immobilized enzyme reactors.The analytical procedure of FIA-chemiluminescence is as follows.SBAs take place in the reaction of desulphation under a catalytical action of a bile salt sulfatase immobilized in the column and formed 3β-hydroxyl bile acids;the formed 3β-hydroxyl bile acids react with nicotinamide adenine dinucleotide(β-NAD+)under the catalysis of 3β-hydroxysteroid dehydrogenase coimmobilized in the same column, and are converted to 3-ketosteroid.At the same time, β-NAD+ is changed into reduced nicotinamide adenine dinucleotide(NADH).Looking like a chain reaction, 1-MPMS is taken as an electron mediator and reacts immediately with NADH coexisting in the carrier solution, and is turned into 1-MPMSH2;the formed 1-MPMSH2 again reacts with dissolved oxygen existing in the carrier solution, and produces hydrogen peroxide.Last, the hydrogen peroxide reacts with the luminol reagent, and gives out light in the presence of POD.SBA can be determined by the luminous intensity.Based on this principle, an approach was established for the determination of SBAs.Its sampling frequency is 30 sample/h, and the relative standard deviation(RSD)is smaller than 2.2%.It can be used to determine SBAs in the range of 0.1∼12μM.Flow-injection spectrophotometry with immobilized enzyme reactors is based on a part of the principle mentioned above, namely, after β-NAD+ is converted to NADH, by catalysis of diaphorase immobilized, NADH reacts with a novel colouring reagent, called Water Soluble Tetrazolium Blue(WST-5), to generate a blue diformazan dye.Last, SBA is determined from the absorbance of the formed formazan(λmax=550nm).By using the manifold and optimized conditions, the author has obtained the linear response in the range of 1∼75μM GLCA-S with a correlation coefficient of 0.999 and an analytical rate of 15 sample/h.RSD was less than 1%.The recoveries(91∼108%)of GLCA-S added into urine were satisfactory and the assay had a good correlativity with manual the UBASTEC method.As a consequence, based on the method, a new-type of analytical instrument for the assay of SBAs in urine has been developed.The author also thinks that this method is very useful for routine analyses in a clinic laboratory and long-period monitoring to patients with acute hepatitis, liver cirrhosis, and intra-and extra-hepatic biliary obstruction urinary.

Different pathways of canalicular secretion of sulfated and non-sulfated fluorescent bile acids: a study in isolated hepatocyte couplets and TR− rats

Background/Aims: Fluorescent bile acids have proved useful for characterizing bile salt transport mechanisms. The aim of this study was to further validate the use of lysyl-fluorescein conjugated bile acid analogues as surrogate bile acids.Methods: We analyzed biliary excretion kinetics of cholyl lysyl fluorescein (CLF), lithocholyl lysyl fluorescein (LLF) and sulfo-lithocholyl lysyl fluorescein (sLLF), both in the isolated rat hepatocyte couplet model and in TR− rats with a selective canalicular transport defect of non-bile acid organic anions.Results: CLF and LLF, which like their natural nonsulfated bile acid congeners are expected to behandled by the canalicular bile salt export pump, were transferred into the bile canaliculus much faster than sLLF, a putative substrate for the canalicular multispecific organic anion transporter in both the in vivo and the in vitro models employed. The contention that different transport systems are involved in sulfated and non-sulfated lysyl fluorescein conjugated bile acids biliary excretion was supported further by studies using TR− rats, in which the cumulative biliary excretion of sLLF was reduced to 6% as compared with that of normal Wistar rats, in good agreement with values for its naturally-occurring radiolabeled parent compound sulfoglycolithocholate. In contrast, CLF and LLF were reduced to 66% and 52%, similar values to these for their congeners, [14C] glycocholate and [14C] lithocholate.Conclusion: The close similarity in behavior of lysyl fluorescein conjugated bile acids to that of their naturally-occurring parent compounds in these different models gives support for both sulfated and nonsulfated lysyl fluorescein conjugated bile acids as substitute molecules for studies of bile acid transport.

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, taurochenodeoxycholate-3-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.

BILE ACID REGURGITATION INTO THE STOMACH IS A COMMON FINDING IN CYSTIC FIBROSIS (CF)

105 During studies of gastrointestinal motor activity, the content of the stomach was aspirated continuously in seven patients with cystic fibrosis (CF), aged 23-46 years. All patients had normal liver function tests but two were treated with ursodeoxycholic acid (10-15 mg/kg/day) due to previously slightly increased serum activities of transaminases. The median bilirubin concentration in the gastric contents was 3.5 µmol/l (range 1.2-15.4), and was significantly correlated to the bile acid concentration, r=0.96 (p=0.0006). In previously investigated control subjects only trace amounts of bilirubin have been found. In five of the patients the bile acid pattern was analysed by GLC/MS, total concentration ranging 0.15 to 7.2 µmol/l, median 0.9µmol/l. The bile acids were mainly conjugated with glycine and taurine, medians (range) being 93 (87-97) and 7 (1-13)%, respectively. Unconjugated and sulphated bile acids were only found in ≤ 1%. The primary bile acids, cholic and chenodeoxycholic acids, constituted 75 (71-93)% of the bile acids, cholic acid being the predominating bile acid. Secondary bile acids were found in vary varying amounts, being <1 to 26% of all bile acids (median 5%). The amount of ursodeoxycholic acid was <1 to 2% except in the two patients who had been treated with ursodeoxycholic acid, in whom the amounts were 18 and 25% of all bile acids, respectively. Interestingly tetrahydroxylated bile acids, previously only reported in the urine in cholestasis, were found in all of the patients, representing <1 to 10% not correlated to the presence or amount of ursodeoxycholic acid. Another unexpected finding, usually found in faeces as bacterial degradation products or in the urine mainly related to cholestasis, was ketonic bile acids, present in all patients in amounts of <1-10% of the bile acids. Our findings of a complicated bile acid pattern of regurgitated bile in the stomach challenge our limited knowledge about the pathophysiology of gastrointestinal reflux - a common finding in CF - and also if some bacterial degradation of bile acids might occur already in the upper gastrointestinal tract of CF patients.

In VitroMorphological and Functional Characterization of Isolated Porcine Hepatocytes for Extracorporeal Liver Support: Bile Acid Uptake and Conjugation

Recently, researchers have focused on the use of bioartificial liver (BAL) to support patients with fulminant hepatic failure (FHF). We have developed a cell-based BAL, consisting of porcine hepatocytes in a hollow-fiber bioreactor. To better characterize BAL metabolic functionsin vitro,bioreactors were inoculated with 48-h-cultured, microcarrier-attached hepatocytes and perifused with recirculating human plasma that contained either 1 μCi of [24-14C] plasma-enriched cholate or 1 μCi of [24-14C] plasma-enriched taurocholate. Bile acids were sampled hourly and separated into four fractions (unconjugated, glycoconjugated, tauroconjugated, and sulfated) for radioactivity determination. Following 3 h perifusion, the glycoconjugated and sulfated bile acid fractions in the bioreactor extrafiber space were significantly elevated when compared to the recirculating plasma. During perifusion with taurocholate-enriched plasma, a relative decrease in the tauroconjugated fraction and an increase in the glycoconjugated fraction were observed. Cholate was accumulated by hepatocytes to a level threefold lower than taurocholate; however, a significant proportion of radioactivity (<25%) was detected in the glycoconjugated fraction. Ultrastructural examination of microcarrier-attached hepatocytes illustrated that the features typical of metabolically active liver cells were maintained. Our data demonstrate the ability of BAL to clear bile acids from the circulation, to accumulate cholate and taurocholate, and to conjugate a substantial amount of cholic acid.

採血のいらない肝機能検査 酵素法による尿中硫酸抱合型胆汁酸の測定

採血のいらない肝機能検査 酵素法による尿中硫酸抱合型胆汁酸の測定

Effects of bile acid conjugates on biliary excretion of estradiol-17-glucuronide in the rat

Estradiol-17 β -glucuronide is a cholestatic agent and is considered to be related to the pathogenesis of intrahepatic cholestasis of pregnancy. We previously reported that biliary estradiol-17 β -glucuronide excretion was delayed in Eisai hyperbililubinemic rats (EHBR) and inhibited by sulfobromophthalein whereas dibromosulfophthalein had not effect on biliary estradiol-17 β -glucuronide excretion. In the present study, we examined the effects of bile acid conjugates on biliary excretion of tracer doses of [ 3 H]estradiol-17 β -glucuronide and [ 14 C]estradiol intravenously injected to rats. Biliary excretion of estradiol-17 β -glucuronide and estradiol metabolites was significantly delayed by the infusion of taurolithocholate-3-sulfate or ursodeoxycholate-3-O-glucuronide, but not by ursodeoxycholate-3,7-disulfate. These findings indicate that estradiol-17 β -glucuronide is excreted into bile at least partially by the canalicular multispecific organic anion transporter which is common for sulfobromophthaleinglutathione and some bile acid sulfates and glucuronides.

Lack of biliary excretion of Cd linked to an inherent defect of the canalicular isoform of multidrug resistance protein (cMrp) does not abnormally stimulate accumulation of Cd in the Eisai hyperbilirubinemic (EHB) rat liver.

A new mutant, the Eisai hyperbilirubinemic (EHB) rat, shows no inherent expression of the canalicular isoform of the multidrug resistance protein (cMrp) in the liver. It has defective biliary secretion of organic anions such as bilirubin glucuronides, bromosulfophthalein (BSP), cysteinyl leukotrienes, glutathione (GSH) and bile acid sulfate and glucuronides. When rats were injected intravenously with CdCl2, biliary excretion of Cd over 30 min was 0.28% and 0.004% of the total dose in Sprague-Dawley (SD) and EHB rats, respectively. Six SD rats and five EHB rats were fed a diet containing Cd. Bile Cd was detected at the level of 2 ng/20 min in SD rats, but not in EHB rats. There was no significant difference of hepatic Cd concentration between SD and EHB rats. Furthermore, there were no significant differences of renal and intestinal Cd, and hepatic and renal metallothionein (MT) concentrations between the SD and EHB groups. Biliary excretion of reduced-GSH for 20 min was 1.3 +/- 0.3 mg and 3.6 +/- 0.9 micrograms in SD and EHB rats, respectively. Our results suggest that hepatobiliary excretion of exogenous Cd is mediated mainly via carrier transport, including a cMrp or GSH carrier, but that the lack of the transport pathway does not contribute to abnormal accumulation of Cd in the liver.

Bile acid transport across the hepatocyte canalicular membrane.

Transport of bile acids across the canalicular membrane of the hepatocyte provides the primary motive force for generation of bile flow and is rate limiting in the vectorial movement of bile acids from blood to bile. Several distinct carriers for bile acids have been defined based on physiological studies in isolated hepatocytes, membrane vesicles, hepatocyte couples, and the perfused rat liver including membrane potential-driven and ATP-dependent mechanisms. Several groups have isolated and functionally reconstituted a canalicular bile acid transport protein of M(r) approximately 110 kDa. The ATP-dependent mechanism for secretion of monovalent bile acids appears to be mediated by a yet to be identified protein of the ATP binding cassette family of transporters. However, it remains conjectural whether the ATP-dependent and membrane potential-driven components of canalicular bile acid transport are mediated by one or more transport proteins. Bile acid sulfates and glucuronides are substrates for the canalicular multispecific organic anion transporter whose activity has recently been associated with the multidrug resistance-associated protein.

The synthesis of taurine‐conjugated bile acids and bile acid sulfates labeled with 14C or 3H in the taurine moiety

Studies of bile acid transport systems require radio-labeled taurine-conjugated bile acids with high specific activity. An established synthetic procedure was optimized to provide mild, fast, and effective conjugation of radio-labeled taurine with different types of bile acids, including those with labile 7α-hydroxy-3-oxo-Δ4 or 3β,7α-dihydroxy-Δ5 structures. Taurine labeled with 14C or 3H was reacted with excess bile acid anhydride formed from the tributylamine salt and ethylchloroformate (2/1 M/M) in aqueous dioxane for 15 min at room temperature. The yields were higher than 95% and less than 2% side products were formed. Bile acid sulfates were conjugated with 14C- or 3H- labeled taurine by using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline as the coupling reagent. The products were effectively purified by chromatography of the sodium salts on Sephadex LH-20. The yields of taurine-conjugated bile acid sulfates were 65–70%. © 1997 John Wiley & Sons, Ltd.

The synthesis of taurine-conjugated bile acids and bile acid sulfates labeled with 14C or 3H in the taurine moiety

Studies of bile acid transport systems require radio-labeled taurine-conjugated bile acids with high specific activity. An established synthetic procedure was optimized to provide mild, fast, and effective conjugation of radio-labeled taurine with different types of bile acids, including those with labile 7α-hydroxy-3-oxo-Δ4 or 3β,7α-dihydroxy-Δ5 structures. Taurine labeled with 14C or 3H was reacted with excess bile acid anhydride formed from the tributylamine salt and ethylchloroformate (2/1 M/M) in aqueous dioxane for 15 min at room temperature. The yields were higher than 95% and less than 2% side products were formed. Bile acid sulfates were conjugated with 14C- or 3H- labeled taurine by using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline as the coupling reagent. The products were effectively purified by chromatography of the sodium salts on Sephadex LH-20. The yields of taurine-conjugated bile acid sulfates were 65–70%. © 1997 John Wiley & Sons, Ltd.

A novel assay for determination of sulfated bile acids in urine by use of flow-injection chemiluminescence principle with immobilized enzymes

Multistep enzymatic reactions take place when sulfated bile acids (SBAs) pass through an immobilized enzyme reactor. First, SBAs take place in the reaction of desulfation under catalytical action of a bile salt sulfatase immobilized in the reactor and formed 3β-hydroxyl bile acids. Formed 3β-hydroxyl bile acids react with nicotinamide adenine dinucleotide (β-NAD+) under catalysis of another enzyme of 3β-hydroxysteroid dehydrogenase coimmobilized in the column and are converted to 3-ketosteroid. At the same time, β-NAD+ is changed into reduced nicotinamide adenine dinucleotide (NADH). Looking as if chain reaction, 1-MPMS taken as an electron mediator reacts immediately with NADH coexisting in the carrier solution and is turned into 1-MPMSH2. Formed 1-MPMSH2 again reacts with dissolved oxygen existing in the carrier solution and produces hydrogen peroxide. Last, the hydrogen peroxide reacts with the luminol reagent and gives out light in the presence of POD. Consequently, SBA can be determined by the luminous intensity. Based on the above-mentioned principle, applying the technology of flow-injection analysis, we established an accurate, simple, less time-consuming and sensitive approach for the determination of SBAs. According to the FIA manifold and experimental results optimized in the work, a new type of analytical instrument for the clinical assay of SBAs in urine or blood can be developed. We also think this method is very useful for routine analysis in clinical laboratory and long-period monitoring to patients with acute hepatitis, liver cirrhosis, and intra- and extrahepatic biliary obstruction of the urinary tract. Sampling frequency of the method is 30 samples/h, and its relative standard deviation is smaller than 2.2%. It can be used to determine SBAs in the ranges of 0.1–2.5 or 2–25 μM. © 1997 John Wiley & Sons, Inc. Lab Robotics and Automation 9: 69–79, 1997.