Bile Acid Metabolites Research Articles

Synthesis of 3α,7α,14α‐Trihydroxy‐5β‐cholan‐24‐oic Acid: A Potential Primary Bile Acid in Vertebrates.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Interactions between Hepatic Mrp4 and Sult2a as Revealed by the Constitutive Androstane Receptor and Mrp4 Knockout Mice

The ABC transporter, Mrp4, transports the sulfated steroid DHEA-s, and sulfated bile acids interact with Mrp4 with high affinity. Hepatic Mrp4 levels are low, but increase under cholestatic conditions. We therefore inferred that up-regulation of Mrp4 during cholestasis is a compensatory mechanism to protect the liver from accumulation of hydrophobic bile acids. We determined that the nuclear receptor CAR is required to coordinately up-regulate hepatic expression of Mrp4 and an enzyme known to sulfate hydroxy-bile acids and steroids, Sult2a1. CAR activators increased Mrp4 and Sult2a1 expression in primary human hepatocytes and HepG2, a human liver cell line. Sult2a1 was down-regulated in Mrp4-null mice, further indicating an inter-relation between Mrp4 and Sult2a1 gene expression. Based on the hydrophilic nature of sulfated bile acids and the Mrp4 capability to transport sulfated steroids, our findings suggest that Mrp4 and Sult2a1 participate in an integrated pathway mediating elimination of sulfated steroid and bile acid metabolites from the liver.

Synthesis of 3alpha,7alpha,14alpha-trihydroxy-5beta-cholan-24-oic acid: a potential primary bile acid in vertebrates.

A method for the synthesis of 3alpha,7alpha,14alpha-trihydroxy-5beta-cholan-24-oic acid which is a possible candidate of bile acid metabolite in vertebrates was developed. The principal reactions involved were 1). stereoselective remote-hydroxylation of methyl ursodeoxycholate diacetate with dimethyldioxirane, 2). site-selective protection at C-3 by tert-butyldimethylsilylation of the resulting 3alpha,7alpha,14alpha-trihydroxy ester, 3). oxidation of the diol with pyridinium dichromate adsorbed on activated alumina, 4). stereoselective reduction of the 7-ketone with zinc borohydride, and 5). cleavage of the protecting group at C-3 with p-toluenesulfonic acid. A facile elimination of the 14alpha-hydroxy group under an acidic or neutral condition is also described. The synthetic reference compound is now available for comparison with unidentified biliary bile acids detected in vertebrate bile.

Cholesterol and Bile Acids Regulate Xenosensor Signaling in Drug-mediated Induction of Cytochromes P450

Cytochromes P450 (CYP) constitute the major enzymatic system for metabolism of xenobiotics. Here we demonstrate that transcriptional activation of CYPs by the drug-sensing nuclear receptors pregnane X receptor, constitutive androstane receptor, and the chicken xenobiotic receptor (CXR) can be modulated by endogenous cholesterol and bile acids. Bile acids induce the chicken drug-activated CYP2H1 via CXR, whereas the hydroxylated metabolites of bile acids and oxysterols inhibit drug induction. The cholesterol-sensing liver X receptor competes with CXR, pregnane X receptor, or constitutive androstane receptor for regulation of drug-responsive enhancers from chicken CYP2H1, human CYP3A4, or human CYP2B6, respectively. Thus, not only cholesterol 7 alpha-hydroxylase (CYP7A1), but also drug-inducible CYPs, are diametrically affected by these receptors. Our findings reveal new insights into the increasingly complex network of nuclear receptors regulating lipid homeostasis and drug metabolism.

Potential bile acid metabolites. 24. An efficient synthesis of carboxyl-linked glucosides and their chemical properties.

A facile and efficient synthesis of the carboxyl-linked glucosides of bile acids is described. Direct esterification of unprotected bile acids with 2,3,4,6-tetra-O-benzyl-D-glucopyranose in pyridine in the presence of 2-chloro-1,3,5-trinitrobenzene as a coupling agent afforded a mixture of the alpha- and beta-anomers (ca. 1:3) of the 1-O-acyl-D-glucoside benzyl ethers of bile acids, which was separated effectively on a C18 reversed-phase chromatography column (isolated yields of alpha- and beta-anomers are 4-9% and 12-19%, respectively). Subsequent hydrogenolysis of the alpha- and beta-acyl glucoside benzyl ethers on a 10% Pd-C catalyst in acetic acid/methanol/EtOAc (1:2:2, by vol) at 35 degrees C under atmospheric pressure gave the corresponding free esters in good yields (79-89%). Chemical specificities such as facile hydrolysis and transesterification of the acyl glucosides in various solvents were also discussed.

Effects of internal biliary bypass on the regulation of hepatic cholesterol 7alpha-hydroxylase activity in rats.

In order to re-evaluate the importance of the enterohepatic circulation of bile acid in the regulation of the activities of hepatic cholesterol 7alpha-hydroxylase, bile acid metabolism was examined in internal biliary bypass models of rats. A polyethylene tube was inserted into the common bile duct and another side of the tube was placed in the duodenum (DD), lower jejunum (JD), cecum (CeD), or transverse colon (CoD) as internal biliary bypass models and in the urinary bladder as an external biliary drainage (ED). After bile diversion for 7 days in each group, hepatic cholesterol 7alpha-hydroxylase activities, bile acid concentrations in bile, serum, and portal vein, biliary bile acid compositions, and intestinal absorption rates of infused labeled taurocholic acid were analyzed. Hepatic cholesterol 7alpha-hydroxylase activity was similar in the JD group compared with the DD group, however, it was significantly up-regulated in the CeD (227% of the DD group), CoD (312%), and ED groups (316%). Biliary, serum, and portal bile acid concentrations were not significantly changed in the DD, JD, and CeD groups but those were significantly lower in the CoD and ED groups compared with the DD group. The proportion of the secondary bile acids was significantly increased in the CeD group and was decreased in the CoD and ED groups. The absorption rate of taurocholic acid was almost 100%, 56%, and 23% in the JD, CeD, and the CoD group, respectively. As the cholesterol 7alpha-hydroxylase activity was not significantly changed in the JD group and the predominance of secondary bile acids did not suppress the enzyme activity in the CeD group, the luminal factor, which is absorbed in the presence of bile acids, and the bile acid metabolites are not likely the regulatory factor. The cholesterol 7alpha-hydroxylase activity seems to be primarily regulated by the intestinal absorption of bile acids and partly by the intestinal mucosal factor which is linked to the intestinal bile acid absorption.

Isoursodeoxycholic acid: metabolism and therapeutic effects in primary biliary cirrhosis

Significant amounts of ursodeoxycholic acid (UDCA) used for the treatment of patients with primary biliary cirrhosis (PBC) become epimerized at C-3 to isoUDCA. We investigated the metabolism of isoUDCA and a possible pharmacologic effect in five patients (51.4 ± 5.8 years old; 3 females, 2 males) with PBC and persistent elevations of γ-glutamyl transpeptidase (γ-GT) and alkaline phosphatase despite treatment with UDCA for more than one year. Serum samples were analyzed for bile acid metabolites and surrogate markers of cholestasis in 4-week intervals after 1 g/d UDCA, wash-out, 0.5 g/d isoUDCA, 0.75 g/d isoUDCA, 0.75 g/d UDCA, and two further periods with 1 g/d UDCA. Bile acids in urine were analyzed after wash-out, 0.5 and 0.75 g/d isoUDCA, and 0.75 and 1 g/d UDCA. During wash-out, AST, AP, and γ-GT rose significantly (P < 0.05) but reversed to previous levels during the first isoUDCA period, with 0.5 g/d only. No further improvements were observed after increasing the dose of isoUDCA or switching back to UDCA. In serum, the relative amounts of isoUDCA and UDCA were 8.1 ± 7.4% and 16.2 ± 6.4% during 0.5 g/d isoUDCA, 6.2 ± 2.5% and 45.0 ± 4.1% during 0.75 g/d isoUDCA, and 0.5–3% and 56.4–60.0%, respectively, during UDCA. In urine, UDCA was the predominant bile acid both during isoUDCA and UDCA medications. The similar serum enrichment and urinary excretion of UDCA during administration of either isoUDCA or UDCA together with low concentrations of the intermediate of isomerization, 3-dehydro-UDCA, indicate a first-pass epimerization of isoUDCA to UDCA in the liver. Approximately 25% of serum isoUDCA and 10% of serum UDCA were conjugated with either glucuronic acid or N-acetylglucosamine, indicating hepatic formation and systemic secretion of glycosidic conjugates.In PBC patients, isoUDCA becomes isomerized to UDCA and has similar effects on surrogate markers of cholestasis. Thus, isoUDCA has pro-drug characteristics.

Monoclonal antibodies generated against an affinity-labeled immune complex of an anti-bile acid metabolite antibody: an approach to noncompetitive hapten immunoassays based on anti-idiotype or anti-metatype antibodies

Conventional immunoassays for haptens such as steroids and synthetic drugs are dependent on the competitive reaction between an unlabeled antigen (analyte) and a labeled antigen against a limited amount of anti-hapten antibody. Although noncompetitive immunoassay procedures such as two-site immunometric assays offer a much higher sensitivity, direct application of this principle to haptens has been difficult due to their small molecular mass precluding simultaneous binding by two antibody molecules. Here, we have attempted to develop a noncompetitive immunoassay system based on anti-idiotype or anti-metatype antibodies. Ursodeoxycholic acid 7- N-acetylglucosaminide (UDCA 7-NAG), which is a bile acid metabolite (molecular weight, 595.8), was selected as the model hapten. A/J mice were immunized with a monoclonal antibody against UDCA 7-NAG, which had been affinity-labeled with a relevant hapten derivative. The fusion between the immune spleen cells and P3/NS1/1-Ag4-1 myeloma cells yielded four kinds of α-type and two kinds of β-type monoclonal anti-idiotype antibodies, each recognizing the framework region and paratope of the anti-hapten antibody. The use of a selected combination between α-type and β-type antibodies together with the anti-hapten antibody provided a noncompetitive assay system with a subfemtomole order sensitivity (detection limit, 118 amol) and a practical specificity.

More hydrophilic bacterial bile acid metabolites are concentrated in the aqueous phase of human stool

More hydrophilic bacterial bile acid metabolites are concentrated in the aqueous phase of human stool

The identification of unusual bile acid metabolites by tandem mass spectrometry: use of low‐energy collision‐induced dissociation to produce informative spectra

The identification of unusual bile acid metabolites by tandem mass spectrometry: use of low‐energy collision‐induced dissociation to produce informative spectra

The identification of unusual bile acid metabolites by tandem mass spectrometry: use of low-energy collision-induced dissociation to produce informative spectra

The identification of unusual bile acid metabolites by tandem mass spectrometry: use of low-energy collision-induced dissociation to produce informative spectra

The identification of unusual bile acid metabolites by tandem mass spectrometry: use of low-energy collision-induced dissociation to produce informative spectra.

The use of collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) has been shown to produce fragmentation that is useful for the structural analysis of bile acids and their conjugates. Low-energy CID using a triple quadrupole has been used to help characterise bile acid identity but the majority of work has been conducted using high-energy CID on specialised instrumentation. This paper describes the use of low-energy CID as a rapid method for identification of urinary bile acids and presents some examples of its use in the diagnosis of liver disease in infants. These include the differential diagnosis of peroxisomal disorders, identification of compounds (e.g. 3 beta,7 alpha-dihydroxy-5-cholenoic acid 3-sulphate) indicative of 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase/isomerase deficiency and the confirmation of the identity of an unusual bile acid series consisting of different conjugates of lithocholic acid. The use of lithium cationisation and derivatisation with aminosulfonic acids for the analysis of unconjugated and glycine-conjugated bile acids has also been evaluated.

Characterization of the inhibitory effects of bile acids on very-low-density lipoprotein secretion by rat hepatocytes in primary culture.

In this study the effects of bile acids and other organic anions on the secretion of very-low-density lipoproteins (VLDLs) were evaluated in rat hepatocytes in primary culture. Incubation of cells with portal blood concentrations (10-200 microM) of bile acids resulted in dose-dependent suppression of secretion of VLDL-associated [3H]triglyceride (TG) formed from [3H]glycerol, and also of TG mass. The degree of the inhibition was highly correlated with intracellular bile acid concentration. Prolonged incubation with 100 microM extracellular taurocholic acid (TC) decreased the secretion of [3H]TG and TG mass to 35% and 50% of the controls respectively. Cellular content of mass and of [3H]TG during prolonged incubation with TC were about 20% and 60% higher than the controls respectively. The inhibitory effect remained for at least 24 h in the presence of TC without altering VLDL-lipid and VLDL-apolipoprotein compositions or the size distribution of the particles. Secretion of apoB-100 and of apoB-48 was inhibited to a similar extent. Cells largely lost their capacity to accumulate bile acids intracellularly after 48 h in culture. In these cells TC was unable to exert its suppressive effects. Taurine and glycine conjugates of all common bile acids were capable of suppressing [3H]TG secretion. Trihydroxylated (cholic acid) and various dihydroxylated (deoxycholic, chenodeoxycholic and ursodeoxycholic acids) bile acids had similar capacities in this respect, suggesting that their common sterol-3 alpha-OH structure is required for the suppressive effect. Neither non-bile acid organic anions, e.g. bilirubin ditaurate and dibromosulphthalein, nor dianionic bile acid metabolites, e.g. sulphated taurolithocholic acid and lithocholate-3-O-glucuronide, showed any effect on [3H]TG secretion. These results indicate that bile acids might play a physiological role in regulating VLDL production by the liver, especially in the postprandial state when their enterohepatic circulation is stimulated.

Faecal bile acid excretion during detoxification in patients with alcohol abuse.

We wanted to ascertain whether alcohol abuse is associated with a changed faecal bile acid excretion. Faecal bile acid excretion was studied in seven chronic alcoholic subjects when admitted to hospital for detoxification. Bile acids in faeces and serum were quantified by gas-liquid chromatography and identified by mass spectrometry. Daily faecal bile acid excretion was on average 948 mumol in the patients at admission, compared with 400 mumol in eight healthy controls (P < 0.01). The daily faecal bile acid excretion decreased slowly during the detoxification period but was still higher in the patients than in controls after 9 days of detoxification (P < 0.05). Faecal bile acid excretion varied greatly between the different patients, with poor correlation to diarrhoea. In one patient the faecal bile acid excretion was remarkably high, amounting to an average of 6800 mumol day-1 during the first 3 days. The percentage metabolites of total faecal bile acids in the patients did not differ significantly from that of controls. The faecal wet weight was higher in the patients than in the controls, although significantly so only after the first 3 days of detoxification. Faecal bile acid excretion is increased after alcohol abuse. A gradual decrease in faecal bile acid excretion was observed during the 9-day observation period when the subjects abstained from alcohol.

Potential bile acid metabolites. 23. Syntheses of 3-glucosides of nonamidated and glycine- and taurine-amidated bile acids.

The 3-glucosides of nonamidated lithocholic, chenodeoxycholic, ursodeoxycholic, deoxycholic, and cholic acids, and their double conjugate forms with glycine and taurine were synthesized. The key reactions used were 1) beta-D- glucosidation at C-3 by the Koenigs-Knorr condensation reaction of 3 alpha-hydroxylated bile acid methyl (or p-nitrophenyl) esters with 1 alpha-bromo-1-deoxy-2, 3, 4, 6-tetra-O-acetyl-D-glucopyranose in the presence of cadmium carbonate in refluxing benzene; 2) indirect and direct amidations at C-24 by the activated p-nitrophenyl ester and by the diethylphosphorylcyanide methods, respectively, using glycinate ester and taurine as coupling agents; and 3) simultaneous alkaline hydrolysis of the hydroxyl-protecting and ester groups in both the sugar and aglycone moieties.

Reply To: Δ4-3-Oxosteroid 5β-reductase deficiency and neonatal hemochromatosis

Dr. Clayton's letter raises pertinent points with regard to the interpretation of the finding of increased urinary excretion of Δ4-3- oxo bile acids. These metabolites can be detected in a variety of severe cholestatic liver disorders. The interpretation of their presence in liver failure is much more complex. Currently the finding of high proportions of Δ4-3-oxo bile acids and markedly reduced or absent primary bile acids is used as supportive evidence of a quantitative defect in Δ4-3-oxosteroid 5β-reductase enzyme activity. This pattern of urinary bile acid metabolites is not necessarily a simple reflection of liver dysfunction. Fast atom bombardment ionization-mass spectrometry analyses of urine from many patients with liver failure, including four with the clinical diagnosis of neonatal hemochromatosis, did not reveal this bile acid profile. An additional five patients have been identified who had a clinical diagnosis of neonatal hemochromatosis, and had a bile acid metabolite pattern similar to the two patients that we described.1Shneider BL Setchell KDR Whitington PF Neilson KA Suchy FJ. Δ4-3-Oxosteroid 5β-reductase deficiency causing neonatal liver failure and hemochromatosis.J PEDIATR. 1994; 124: 234-238Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar Definitive diagnosis of a primary genetic deficiency of the enzyme is hampered by the fact that it is not expressed in fibroblasts. When possible we have attempted to obtain additional evidence for an enzyme defect by immunoblotting for the 38 kd protein with monoclonal antibodies against the enzyme.2Russell DW Setchell KDR Bile acid biosynthesis.Biochemistry. 1992; 31: 4737-4749Crossref PubMed Scopus (644) Google Scholar As pointed out by Dr. Clayton, the finding of an absence of immunodetectable protein may be caused by a generalized destruction of hepatic proteins. The finding of a truncated protein in our first patient is intriguing.1Shneider BL Setchell KDR Whitington PF Neilson KA Suchy FJ. Δ4-3-Oxosteroid 5β-reductase deficiency causing neonatal liver failure and hemochromatosis.J PEDIATR. 1994; 124: 234-238Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar This feature is more consistent with a primary defect, rather than simple nonspecific loss of proteins as a result of hepatocellular injury. At present, confirmatory evidence of a primary enzyme defect can best be established from the observation that an interruption of primary bile acid therapy in these patients, after liver function has returned to normal, results in a reemergence of Δ4-3-oxo bile acids as major metabolites in urine. A human complementary DNA encoding 5β-reductase was recently sequenced, and molecular genetic techniques should soon be available to aid in clarifying the role of a primary genetic deficiency of this enzyme in the pathogenesis of liver failure.3Kondo K-H Kai M-H Setoguchi Y et al.Cloning and expression of cDNA of human Δ4-3-oxosteroid 5β-reductase and substrate specificity of the expressed enzyme.Eur J Biochem. 1994; 219: 357-363Crossref PubMed Scopus (121) Google Scholar The role of biliary excretion of iron has been persistently underestimated and poorly studied, especially in the newborn period. Examination of the studies of Hultcrantz et al.4Hultcrantz R Angelin B Bjorn-Rasmussen E Ewerth S Einarsson K Biliary excretion of iron and ferritin in idiopathic hemochromatosis.Gastroenterology. 1989; 96: 1539-1545PubMed Google Scholar indicates that normal biliary excretion amounts to between 0.4 and 2.3 mg of iron/day, in the range of normal uptake of iron. We agree that the finding of 5β-reductase deficiency should not be the end of the diagnostic evaluation in an infant with neonatal liver failure, but it should be part of the evaluation. Only through systematic evaluation of infants with neonatal liver failure will we determine the significance of this finding, in particular its relationship to the development of neonatal hemochromatosis. Unfortunately, at this time pediatric hepatologists are faced with very difficult therapeutic decisions. More exact molecular probes may simplify those decisions in the future. 9/35/58922

Advances in microbial biotechnology of bile acids

The recent advances in microbial biotechnology of production of bile acid metabolites helped to identify a number of neutral and acidic steroidal compounds useful as drugs and drug intermediates on a scale which would not have been possible by classical chemical transformations. Microbial transformations viz., hydroxylation, dehydroxylation, reduction of the carbonyl moieties, epimerization, side-chain metabolism, introduction of carbon-carbon double bonds into the steroid nucleus, deconjugation of bile acid conjugates carried out by various microorganisms for production of useful metabolites with special reference to newer techniques including cell immobilization and transposon mutagenesis for selective transformations are reviewed. The different pathways of microbial degradation of bile acids leading to the formation of various products are discussed. A compilation of the metabolites formed by various microorganisms from the bile acids or their conjugates and reported during the period 1979–1992 is also provided.

Interactions between nutrition and the intestinal microflora.

The human gut is well colonized by a wide variety of bacteria. In the industrial nations, the stomach and small intestine have a sparse flora protected by gastric acid and a highly propulsive motility. The flora of the small intestine increases along its length and particularly in the terminal ileum (Drasar & Hill, 1974). The large intestine has a very rich, mainly anaerobic, flora capable of fermenting carbohydrate and protein, and metabolizing a wide and diverse range of endogenous and exogenous molecules such as bile acids, fats and drugs. In the less-developed countries the small intestine has heavier populations of bacteria and these may be a factor in the higher occurrence of diarrhoea and malnutrition (Gorbach et al. 1970; Gracey, 1979). The interactions between nutrition and the intestinal microflora are complex. Bacteria in the gut may affect digestion and absorption, the products of bacterial fermentation may provide nutrients or affect the well-being of the host, but diet may also affect the survival and metabolism of the bacteria. The products of bacterial metabolism may be beneficial, for example butyrate which may have anti-neoplastic properties (Augeron & Laboisse, 1984), or they may be potentially harmful, for example bile acid metabolites which may be co-carcinogenic (Owen et af. 1987). The interaction between the intestinal microflora and nutrition begins at birth and changes with age and disease.

Potential bile acid metabolites. 20. A new synthetic route to stereoisomeric 3,6,-dihydroxy- and 6-hydroxy-5α-cholanoic acids

An improved procedure for the syntheses of stereoisomeric 3,6,-dihydroxy- and 6-hydroxy-5α-cholanoic acids (and their methyl esters) is described. The principal reactions emplyoed are those reported in the preceding paper of this series, with the commercially available hyodeoxycholic acid as starting material. The final step in the procedure is the reduction of the key 5α C-6 ketones with either the stereoselective equatorial reagent, Li/NH 3/MeOH, or the axial reagent, Zn(BH 4) 2). The results of analysis of the prepared 6-monohydroxylated and 3,6-dihydroxylated stereoisomers by thin-layer chromatographic, high performance liquid chromatographic and gas-liquid chromatographic mobilities, and 1H and 13C nuclear magnetic resonance spectra are discussed along with the dat for the corresponding compounds in the 5β-series. (Steroids 58: 362–369, 1993)

Potential bile acid metabolites. 19. The epimeric 3α,6,7β-trihydroxy-and 3α,6,7β,12α-tetrahydroxy-5α-cholanoic acids

Syntheses by a new procedure of the known 3α,6α,7β- and 3α,6β,7β-trihydroxy-5α-cholanoic acids, and of the once-reported analog 3α,6α,7β,12α-, as well as the new 3α,6β,7β,12α-tetrahydroxy-5α-cholanoic acids, are described. Key intermediates of the syntheses are the 6-oxo-7β-ols of the respective 5α-cholanoic acids (and their methyl esters) prepared by allomerization at C-5 of appropriate 6-bromo-7-oxo derivatives of the corresponding 5β-acids. Successful reduction of the 6,7-ketols to the desired products depended on the proper choice of reagents, either Zn(BH 4) 2 or Li/NH 3/MeOH. (Steroids 58:148–152, 1993)