Taurine Conjugates Of Bile Acids Research Articles

Identification of Taurine-Responsive Genes in Murine Liver Using the Cdo1-Null Mouse Model.

The cysteine dioxygenase (Cdo1)-null mouse is unable to synthesize hypotaurine and taurine by the cysteine/cysteine sulfinate pathway and has very low taurine levels in all tissues. The lack of taurine is associated with a lack of taurine conjugation of bile acids, a dramatic increase in the total and unconjugated hepatic bile acid pools, and an increase in betaine and other molecules that serve as organic osmolytes. We used the Cdo1-mouse model to determine the effects of taurine deficiency on expression of proteins involved in sulfur amino acid and bile acid metabolism. We identified cysteine sulfinic acid decarboxylase (Csad), betaine:homocysteine methytransferase (Bhmt), cholesterol 7α-hydroxylase (Cyp7a1), and cytochrome P450 3A11 (Cyp3a11) as genes whose hepatic expression is strongly regulated in response to taurine depletion in the Cdo1-null mouse. Dietary taurine supplementation of Cdo1-null mice restored hepatic levels of these four proteins and their respective mRNAs to wild-type levels, whereas dietary taurine supplementation had no effect on abundance of these proteins or mRNAs in wild-type mice.

A fluorescent analogue of tauroursodeoxycholic acid reduces ER stress and is cytoprotective

Tauroursodeoxycholic acid (TUDCA) is a cytoprotective ER stress inhibitor and chemical chaperone. It has therapeutic potential in a wide array of diseases but a specific macromolecular target or molecular mechanism of action remains obscure. This Letter describes an effective new synthetic approach to taurine conjugation of bile acids which we used to prepare 3α-dansyl TUDCA (4) as a probe for TUDCA actions.As a model of ER stress we used the hepatocarcinoma cell line HUH7 and stimulation with either deoxycholic acid (DCA, 200μM) or tunicamycin (5μg/ml) and measured levels of Bip/GRP78, ATF4, CHOP and XBP1s/XBP1u. Compound 4 was more effective than UDCA at inhibiting ER stress markers and had similar effects to TUDCA. In a model of cholestasis using the cytotoxic DCA to induce apoptosis, pretreatment with 4 prevented cell death similarly to TUDCA whereas the unconjugated clinically used UDCA had no effect.3α-Dansyl TUDCA (4) appears to be a suitable reporter for TUDCA effects on ER stress and related cytoprotective activity.

Bile acids: the role of peroxisomes

It is well established that peroxisomes play a crucial role in de novo bile acid synthesis. Studies in patients with a peroxisomal disorder have been indispensable for the elucidation of the precise role of peroxisomes. Several peroxisomal disorders are associated with distinct bile acid abnormalities and each disorder has a characteristic pattern of abnormal bile acids that accumulate, which is often used for diagnostic purposes. The patients have also been important for determining the pathophysiological consequences of defects in bile acid biosynthesis. In this review, we will discuss all the peroxisomal steps involved in bile acid synthesis and the bile acid abnormalities in patients with peroxisomal disorders. We will show the results of bile acid measurements in several tissues from patients, including brain, and we will discuss the toxicity and the pathological effects of the abnormal bile acids.

Determination of taurine and hypotaurine in animal tissues by reversed-phase high-performance liquid chromatography after derivatization with dabsyl chloride.

Taurine and sulfate are major end products of L-cysteine metabolism in mammals1, and they play various roles as tissue constituents and physiologically important compounds. Taurine is found in high concentrations in skeletal and heart muscle and brain2. It is utilized for the synthesis of taurine conjugates of bile acids. It is believed that most taurine is produced via the cysteinesulfinate pathway3and cysteamine pathway4. In both pathways, hypotaurine is an obligatory precursor leading to taurine. Hypotaurine content in animal tissues is far less than the taurine content. However, it has been reported that hypotaurine is found in rat brain5, accumulates in the liver of cysteine-administered rats3and in the regenerating rat liver6, increases in blood of rats injected with L-cysteine7, and is excreted in the urine of rats fed L-cystine8. When hypotaurine was injected into rats, urinary taurine excretion increased in a dose-dependent manner9.KeywordsPeak Area RatioSulfonyl ChlorideTaurine ContentAutomatic Amino Acid AnalyzerTaurine ConjugateThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Synthesis and intestinal metabolism of ursodeoxycholic acid conjugate with an antiinflammatory agent, 5-aminosalicylic acid

5-Aminosalicylic acid conjugate of ursodeoxycholic acid was synthesized in above 90% yield by adding a basic solution of 5-aminosalicylic acid into the mixed anhydride formed with ursodeoxycholic acid and ethyl chloroformate. The 5-aminosalicylic acid conjugate of ursodeoxycholic acid was poorly secreted into the bile and was deconjugated with cholylglycine hydrolase and Clostridium perfringens, that deconjugate naturally occurring glycine and taurine conjugates of bile acids. However, ursodeoxycholic acid 5-aminosalicylic acid conjugate was not absorbed from the duodenum but was concentrated in the colon where it was partially hydrolyzed by the intestinal bacteria to ursodeoxycholic acid and 5-aminosalicylic acid. We believe that this unique conjugation of ursodeoxycholic acid with 5-aminosalicylic acid may facilitate the transport of both 5-aminosalicylic acid and ursodeoxycholic acid to the colon and may be useful for the treatment of colonic inflammatory bowel diseases, ulcerative colitis and Crohn's disease.—Batta, A. K., G. S. Tint, G. Xu, S. Shefer, and G. Salen. Synthesis and intestinal metabolism of ursodeoxycholic acid conjugate with an antiinflammatory agent, 5-aminosalicylic acid.

Cloning, expression, and chromosomal localization of mouse liver bile acid CoA:amino acid N-acyltransferase

A mouse liver lambda Zap XR cDNA library was screened using the coding region of human bile acid CoA:amino acid N-acyltransferase (BAT) cDNA as a probe. Ten positive clones were isolated and purified, two of which apparently possessed complete open reading frames for BAT based on sequence analysis of the ends of the cDNAs. One clone (mBAT#9) was selected for sequence analysis and characterization. mBAT#9 is 1869 basepairs in length and the full-length cDNA possesses a 189 basepair 5'-nontranslated region, an open-reading frame of 1260 basepairs, and a 404 basepair 3'-nontranslated region followed by a poly(A) tail. The open-reading frame codes for a 420 amino acid protein with a calculated molecular mass of 46,525 daltons. The structural gene for mBAT was mapped to mouse Chromosome 4. The amino acid sequence of mBAT is 69% identical and 84% similar to that of hBAT, and 86% identical and 95% similar to that of kan-1, a putative rat liver BAT. Enzymatically active mBAT was expressed in E. coli using the bacterial expression vector pKK233-2. Immunoblot analysis of expressed mBAT with rabbit anti-human BAT polyclonal antibodies detected a single protein with a molecular mass of approximately 45,000 daltons. Cytosol from cells transformed with mBAT#9/pKK233-2 possessed significant amounts of BAT-catalyzed conjugating activity with taurine as substrate but the expressed enzyme did not use glycine or fluoro-beta-alanine as substrates. The K(m) value for taurine was 1.9 mM +/- 0.1 mM in reactions with cholyl CoA as a cosubstrate. The specificity of mBAT for taurine as a substrate was confirmed by the demonstration, using HPLC-electrospray ionization mass spectrometry, that mouse gallbladder bile contained only taurine conjugates of bile acids. The identification of the types of amino acid conjugates of bile acids present in mouse bile had not been previously reported. These results indicate that a taurine-specific form of BAT has been cloned and expressed from mouse liver.

Effect of bile acids on the growth and differentiation of cultured human keratinocytes.

Sixteen bile acids were tested at a concentration of 50 microM for their effect on growth of preconfluent cultures of proliferating keratinocytes. Monohydroxy bile acids (3-beta-hydroxy-delta 5-cholenate and lithocholate) stopped the accumulation of protein, dramatically decreased DNA content and led to a 90% loss of cell viability. Deoxycholate (DOC) and chenodeoxycholate inhibited protein accumulation and blocked increases in DNA content, without affecting cell viability. DOC had measurable growth-retarding effects at concentrations as low as 15 microM, and lithocholate at 2 microM. The glycine and taurine conjugates of bile acids were significantly less effective inhibitors of growth, as was the sulfate conjugate of lithocholic acid. DOC and chenodeoxycholate at 25-50 microM enhanced the differentiation-specific increase in particulate transglutaminase activity by as much as 80% over 6 days. Lithocholate had a similar effect at 5 microM. Glycine and taurine conjugates of DOC had a similar effect but were less potent; tauroursodeoxycholate had no effect. The data indicate that bile acids, at levels seen in obstructive biliary disease, can lead to a down-regulation of keratinocyte growth and an up-regulation of differentiation.

Assay for taurine conjugates of bile acids in serum by reversed-phase high-performance liquid chromatography.

The purpose of this study was to develop a new high-performance liquid chromatographic (HPLC) procedure for quantifying taurine conjugates of bile acids in serum. The technique involved three basic steps. The first removed free amino acids via solid-phase extraction of the serum. The second step involved the reaction of the extracted serum with the enzyme choloylglycine hydrolase, which liberated the taurine from the conjugated bile acids. The third step was the reversed-phase HPLC separation of o-phthalicdicarboxaldehyde derivatives of taurine. The assay provides a simple technique for determination of the total amount of taurine-conjugated bile acids in serum.

Glycine and taurine conjugation of bile acids by a single enzyme. Molecular cloning and expression of human liver bile acid CoA:amino acid N-acyltransferase.

In order to establish whether a single enzyme in human liver was capable of conjugating bile acids with both glycine and taurine, a cDNA encoding human liver bile acid-CoA:amino acid N-acyltransferase (hBAT) has been isolated and characterized. A specific immunoaffinity-purified rabbit anti-hBAT polyclonal antibody was used to screen a lambda Zap XR human liver cDNA library resulting in the isolation of two unique clones. hBAT8 and hBAT9 (1669 and 1491 base pairs in length, respectively) were isolated following screening of 4 x 10(5) clones of the cDNA library. Restriction mapping and sequence analysis demonstrated that the cDNAs were identical except hBAT8 contained an additional 178 bases of 5' sequence; hBAT8 was completely sequenced, characterized, and used for all subsequent studies. hBAT8 consisted of a 184-nucleotide 5'-nontranslated region, an open reading frame of 1,254 bases predicting a protein of 418 amino acids with a molecular mass of 46,296 Da, and a 3'-nontranslated region of 209 nucleotides followed by a poly(A)+ tail. The identity of the cDNA was confirmed by the following findings: 1) the open reading frame began with an ATG codon and was followed by a nucleotide sequence which, when translated, corresponded exactly to the first 17 NH2-terminal amino acids of purified human liver BAT; 2) cytosol of Escherichia coli XL1-Blue cells transfected with hBAT8 subcloned into an expression vector, pKK233-2, demonstrated significant enzymatic activity for the conjugation of both taurine and glycine with cholic acid; 3) bacterial expression of hBAT8 generated a protein that comigrated with hBAT from human liver during SDS-polyacrylamide gel electrophoresis and cross-reacted with a specific polyclonal rabbit anti-hBAT antibody during immunoblot analysis; 4) kinetic characteristics of the expressed enzyme were very similar to those reported for purified liver BAT. These data demonstrate that a single cDNA is present in human liver which codes for a protein capable of catalyzing the conjugation of cholic acid with both glycine and taurine.

Pre-column derivatization of free bile acids for high-performance liquid chromatographic and gas chromatographic—mass spectrometric analysis

Pentachlorophenyl (PCP) esters of five free bile acids (FBA) were obtained by reacting the FBA and Kovacs' complex (KC) in a 1:8 molar ratio in acetone at 65°C, and were purified by column chromatography on silica gel. The esters were crystallized from benzene—hexane, derivatized as trimethylsilyl ethers for gas chromatography on a DB-1 capillary column and for gas chromatography—mass spectrometry with a DB-5 column, and mass spectrometry (MS) in the electron-impact (EI) positive-ion mode at 70 eV. The reaction is specific for FBA even in the presence of glycine and taurine conjugates of bile acids. The PCP esters were treated with benzylamine in chloroform or methanol to produce N-benzyl derivatives of FBA. The N-benzylamides were separated by high-performance liquid chromatography (HPLC) on a 4-μm Nova-Pak C 18 column, studied by thermospray—LC—MS, and in the direct insertion probe—EI positive-ion mode.

Altered Glycine and Taurine Conjugation of Bile Acids following Aluminum Administration to Rats

Aluminum contaminates components of intravenous nutrient solutions and accumulates in the liver with parenteral feeding. Abnormalities in hepatic function associated with aluminum accumulation include increased serum bile acid concentration and glucuronyl transferase activity and reduced mixed function oxidase levels and bile flow. Whether there are other biochemical responses of the liver to aluminum is unclear. We report the effects of aluminum administration on bile acid conjugation in rats given aluminum intravenously as follows: group I, 1 mg/kg/day for 14 days; group II, 5 mg/kg/day for 14 days; and group III, 5 mg/kg/day for 7 days. Taurine‐conjugated bile acids were reduced and glycine/ taurine elevated in all groups compared with pair‐fed controls. Glycine/taurine was greater in group II versus III and varied directly with serum bile acid concentration. These findings suggest that aluminum administration is associated with decreased taurine conjugation of bile acids, a phenomenon that may be associated with cholestasis.

Altered glycine and taurine conjugation of bile acids following aluminum administration to rats.

Aluminum contaminates components of intravenous nutrient solutions and accumulates in the liver with parenteral feeding. Abnormalities in hepatic function associated with aluminum accumulation include increased serum bile acid concentration and glucuronyl transferase activity and reduced mixed function oxidase levels and bile flow. Whether there are other biochemical responses of the liver to aluminum is unclear. We report the effects of aluminum administration on bile acid conjugation in rats given aluminum intravenously as follows: group I, 1 mg/kg/day for 14 days; group II, 5 mg/kg/day for 14 days; and group III, 5 mg/kg/day for 7 days. Taurine-conjugated bile acids were reduced and glycine/taurine elevated in all groups compared with pair-fed controls. Glycine/taurine was greater in group II versus III and varied directly with serum bile acid concentration. These findings suggest that aluminum administration is associated with decreased taurine conjugation of bile acids, a phenomenon that may be associated with cholestasis.

77 SPECIES DEPENDENCE OF THE HYPERCHOLESTEROLEMIC EFFECT OF DIETARY CASEIN

We have hypothesized that casein, a phosphorylated protein, binds to an intestinal calcium-phosphate (CaP.) sediment and thus inhibits the efflux of bile acids from the enterohepatic cycle (EHC); consequently serum cholesterol may increase. This hypothesis has been supported by in vitro studies, which showed that unconjugated and glycine-conjugated bile acids bind to insoluble CaPi. This binding is inhibited by casein but not by dephosphorylated casein. In vivo studies with rabbits showed that dietary casein immediately inhibited the fecal output of bile acids. This inhibition preceded casein-induced hypercholesterolemia, indicating a cause-and-effect relationship. When rabbits were fed dephosphorylated casein, none of these casein-specific effects was observed. This indicates that the hypercholesterolemic effect of casein is due to its phosphorylation state. In species (like rat and man) with a high activity of intestinal phosphatase and a significant taurine conjugation of bile acids, casein neither affects the EHC of bile acids nor the serum cholesterol concentration. Results indicate that in these species the mucosal enzyme alkaline phosphatase dephosphorylates dietary casein, thus preventing hypercholesterolemia.

Development and validation of a method for measuring the glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography

We developed and validated a simple method for measuring the individual glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography with a C 18 reversed-phase column using an isocratic solvent system of acidified methanol—potassium phosphate. Without preliminary derivatization or purification, complete separation of the ten major conjugated bile acids present in bile could be achieved in 65 min. Total bile acid concentrations were identical when measured enzymatically and by summing the individual bile acids determined by high-performance liquid chromatography. Bile acid composition determined by gas—liquid chromatography correlated with results by high-performance liquid chromatography. Finally, measurements of individual glycine and taurine conjugates in human bile and in mixtures of bile acid standards by high-performance liquid chromatography and thin-layer chromatography gave similar results. This high-performance liquid chromatographic system permits simultaneous quantification of total and individual bile acids and their glycine and taurine conjugates in bile.

Feeding the low-birth weight infant: V. Effects of taurine, cholesterol, and human milk on bile acid kinetics

This study was conducted to compare the influence of diet on the physiologic changes in bile acid kinetics, intraluminal bile acid concentrations, conjugation patterns, and nutrient lipid absorption, which occur postnatally. Preterm infants, 31–35 wk gestation, were fed one of four diets: (a) human milk pasteurized at 62 °C for 30 min, 55% from a pooled source, 35% from the infant's own mother, with the remainder (∼10%) being fresh human milk; (b) an adapted infant formula (F1; (c) F1 supplemented with taurine. 30 μmol/dl. (F2); and (d) F1 with both taurine. 30 μmol/dl, and cholesterol, 9.0 mg/dl, to a total of 12.7 mg/dl, the levels found in human milk (F3). In all infants, the bile acid pool size increased nearly twofold between 11 and 35 days, irrespective of dietary regimens. Taurine conjugation of bile acids predominated in all infants at 11 days of age and at 35 days in those infants fed human milk or the taurine-supplemented formulas. In taurine-supplemented formulas, the conjugation pattern did not influence bile acid kinetics. However, the bile acid pool and intraluminal bile acid concentrations were significantly greater in infants fed human milk at all ages, suggesting that human milk feeding, per se, uniquely influences intestinal and possibly hepatic function independent of developmental factors.

Effects of bile acids and their glycine conjugates on gamma-glutamyl transpeptidase.

Glycine and taurine conjugates of bile acids modulate gamma-glutamyl transpeptidase by interacting with the cysteinylglycine binding site (acceptor site) of the enzyme. These compounds stimulate hydrolysis of glutamine and S-methylglutathione and the rate of the inactivation of the enzyme by the gamma-glutamyl site-directed reagent, AT-125 (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid). Transpeptidation between S-methylglutathione and methionine was inhibited by these compounds. These effects resemble those caused by hippurate; the glycine derivatives of bile acids, however, exhibit a much greater affinity for transpeptidase than hippurate. Cholate, as shown previously for benzoate, also seems to bind to a portion of the acceptor site as indicated by its effects on S-methylglutathione utilization and AT-125-dependent inactivation of the enzyme. The Kd values for cholate and benzoate are, however, at least one order of magnitude larger than those for their respective glycine derivatives. The acceptor site-directed modulators increase the affinity of the enzyme for AT-125 and kinetic and binding studies show that binding of gamma-glutamyl site-directed reagents increases the affinity of the enzyme for cholate. These results thus indicate cooperative interactions between the gamma-glutamyl donor and acceptor binding domains of the transpeptidase active center.

The patterns of glycine and taurine conjugates of bile acids in serum in hepatobiliary disease.

Fasting serum concentrations of glycine and taurine conjugates of cholic, chenodeoxycholic, and deoxycholic acid were measured with a high-pressure liquid chromatography-enzymatic assay in patients with hepatobiliary disease. The total glycine to taurine ratio was significantly lower in extrahepatic cholestasis (median 1.1) than in cirrhosis (median, 2.0) and controls (median, 1.7). In patients with cirrhosis the ratio was significantly correlated with the S-bilirubins, P-coagulation factors (II + VII + X), and S-total conjugated bile acids. Because of large overlaps of the ratio between the groups the glycine to taurine ratio is of hardly any diagnostic value. The ratio of cholic acid conjugates to chenodeoxycholic acid conjugates was over 1.5 in 10 of 12 cholestasis patients and below this value in all but 1 patient with cirrhosis; the separation of the groups was not improved by splitting the ratio in glycine and taurine conjugates. This study does not suggest that separate determination of glycine and taurine conjugates of bile acids in serum adds diagnostic information in hepatobiliary disease.

Hepatic Taurine Concentration and Dietary Taurine as Regulators of Bile Acid Conjugation with Taurine

Taurine concentration in liver biopsies taken from 20 patients undergoing cholecystectomy or laparotomy for obstructive jaundice correlated with percentage of taurine-conjugated bile acids in hepatic bile. In biliary obstruction, taurine concentrations in muscle did not parallel high hepatic taurine concentrations, suggesting selective hepatic taurine accumulation in biliary obstruction. In fasting subjects with an intact bile acid enterohepatic circulation, per cent taurine-conjugated bile acids in bile was the same as per cent taurine conjugation of bile acids by the liver. Ingestion of small amounts of taurine (250 mg) can increase the per cent taurine conjugation by the liver. Of 10 subjects, 7 increased per cent taurine conjugation of bile acids by the liver by 2.5 to 10% at 2 1/2 hr after intraduodenal taurine administration. We conclude that hepatic taurine concentration is a major determinant of per cent taurine conjugation of bile acids by the liver in man. In the fasting subject with an intact enterohepatic circulation, per cent taurine-conjugated bile acid in the bile acid pool is very close to the per cent taurine conjugation of bile acids by the liver. Hepatic bile acid conjugation pattern may differ from that of the bile acid pool as a result of taurine ingested with meals, but the deviation is small, and acute alteration of the per cent taurine conjugation in the bile acid pool does not occur.

Duodenal bile acid conjugation patterns and dietary sulphur amino acids in the newborn.

Patterns of glycine and taurine conjugation of bile acids in duodenal juice have been studied in 20 low birth weight babies at 12 and 22 days of age. They were fed from birth on one of three feeds containing different amounts of taurine and its precursors, cysteine and methionone. Taurine conjugation predominated in those fed on human milk throughout the first three weeks of life. In those fed on SMA or S26 infant feeding formulae, both of which contain very little taurine, concentrations of glycine conjugates were found to exceed those of taurine conjugates by the 12th day. The low glycine-taurine conjugate ratios previously described in newborn babies are, therefore, probably related to the taurine content of the milk which they received. Glycine-taurine conjugate ratios less than 1.0 occur in breast fed infants during the first three weeks of life.

Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from rat liver

An in vitro study of bile acid-CoA:amino acid N-acyltransferase activity of rat liver was undertaken in order to determine whether separate amino acid-specific enzymes catalyzed the formation of glycine and taurine conjugates of bile acids as postulated by others. Polyacrylamide gel electrophoresis of 200-fold purified enzyme localized the glycine- and taurine-dependent activities to a single band. Both activities were optimal at pH 7.8 and showed similar loss of activity at pH 6.0, pH 9.0, in the presence of 5,5'-dithiobis(2-nitrobenzoic acid), and at temperatures exceeding 50 degrees. With the purified fraction, Km for glycine was 31 mM and Km for taurine was 0.8 mM. Km for several bile acid-CoA substrates was approximately 20 micron and independent of the amino acid acceptor. Only amino acids with terminal alpha- or beta-amino groups were active as acyl acceptors. Acyl donors were limited to bile acid-CoA derivatives. The data support the conclusion that the rat has a single bile acid-CoA:amino acid N-acyltransferase. The substrate kinetics are consistent with previous observations that taurine conjugates predominate in rat bile at normal hepatocellular concentrations of glycine and taurine.