Amidated Bile Acids Research Articles

Bile acid‐CoA ligase deficiency—a new inborn error of bile acid metabolism

Born at 27 weeks gestation, a child of consanguineous parents of Pakistani origin required prolonged parenteral nutrition. She developed jaundice, with extensive fibrosis and architectural distortion at liver biopsy; jaundice resolved with supportive care. Serum γ-glutamyl transpeptidase values were within normal ranges. The bile acids in her plasma and urine were >85% unconjugated (non-amidated). Two genes encoding bile-acid amidation enzymes were sequenced. No mutations were found in BAAT, encoding bile acid-CoA : aminoacid N-acyl transferase. The patient was homozygous for the missense mutation c.1012C > T in SLC27A5, predicted to alter a highly conserved amino-acid residue (p.H338Y) in bile acid-CoA ligase (BACL). She also was homozygous for the missense mutation c.1772A > G in ABCB11, predicted to alter a highly conserved amino-acid residue (p.N591S) in bile salt export pump (BSEP). BACL is essential for reconjugation of bile acids deconjugated by gut bacteria, and BSEP is essential for hepatocyte-canaliculus export of conjugated bile acids. A female sibling born at term had the same bile-acid phenotype and SLC27A5 genotype, without clinical liver disease. She was heterozygous for the c.1772A > G ABCB11 mutation. This is the first report of a mutation in SLC27A5. The amidation defect may have contributed to cholestatic liver disease in the setting of prematurity, parenteral nutrition, and homozygosity for an ABCB11 mutation.

The Human UGT1A3 Enzyme Conjugates Norursodeoxycholic Acid into a C23-ester Glucuronide in the Liver

Norursodeoxycholic acid (norUDCA) exhibits efficient anti-cholestatic properties in an animal model of sclerosing cholangitis. norUDCA is eliminated as a C(23)-ester glucuronide (norUDCA-23G) in humans. The present study aimed at identifying the human UDP-glucuronosyltransferase (UGT) enzyme(s) involved in hepatic norUDCA glucuronidation and at evaluating the consequences of single nucleotide polymorphisms in the coding region of UGT genes on norUDCA-23G formation. The effects of norUDCA on the formation of the cholestatic lithocholic acid-glucuronide derivative and of rifampicin on hepatic norUDCA glucuronidation were also explored. In vitro glucuronidation assays were performed with microsomes from human tissues (liver and intestine) and HEK293 cells expressing human UGT enzymes and variant allozymes. UGT1A3 was identified as the major hepatic UGT enzyme catalyzing the formation of norUDCA-23G. Correlation studies using samples from a human liver bank (n = 16) indicated that the level of UGT1A3 protein is a strong determinant of in vitro norUDCA glucuronidation. Analyses of the norUDCA-conjugating activity by 11 UGT1A3 variant allozymes identified three phenotypes with high, low, and intermediate capacity. norUDCA is also identified as a competitive inhibitor for the hepatic formation of the pro-cholestatic lithocholic acid-glucuronide derivative, whereas norUDCA glucuronidation is weakly stimulated by rifampicin. This study identifies human UGT1A3 as the major enzyme for the hepatic norUDCA glucuronidation and supports that some coding polymorphisms affecting the conjugating activity of UGT1A3 in vitro may alter the pharmacokinetic properties of norUDCA in cholestasis treatment.

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.

Proteomics Characterization of Mouse Kidney Peroxisomes by Tandem Mass Spectrometry and Protein Correlation Profiling

The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by alpha- and beta-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals. To fulfil this vast array of metabolic functions, peroxisomes accommodate approximately 50 different enzymes at least as identified until now. Interest in peroxisomes has been fueled by the discovery of a group of genetic diseases in humans, which are caused by either a defect in peroxisome biogenesis or the deficient activity of a distinct peroxisomal enzyme or transporter. Although this research has greatly improved our understanding of peroxisomes and their role in mammalian metabolism, deeper insight into biochemistry and functions of peroxisomes is required to expand our knowledge of this low abundance but vital organelle. In this work, we used classical subcellular fractionation in combination with MS-based proteomics methodologies to characterize the proteome of mouse kidney peroxisomes. We could identify virtually all known components involved in peroxisomal metabolism and biogenesis. Moreover through protein localization studies by using a quantitative MS screen combined with statistical analyses, we identified 15 new peroxisomal candidates. Of these, we further investigated five candidates by immunocytochemistry, which confirmed their localization in peroxisomes. As a result of this joint effort, we believe to have compiled the so far most comprehensive protein catalogue of mammalian peroxisomes.

Quantification and regulation of the subcellular distribution of bile acid coenzyme A:amino acid N-acyltransferase activity in rat liver

Bile acid coenzyme A:amino acid N-acyltransferase (BAT) is responsible for the amidation of bile acids with the amino acids glycine and taurine. To quantify total BAT activity in liver subcellular organelles, livers from young adult male and female Sprague-Dawley rats were fractionated into multiple subcellular compartments. In male and female rats, 65-75% of total liver BAT activity was found in the cytosol, 15-17% was found in the peroxisomes, and 5-10% was found in the heavy mitochondrial fraction. After clofibrate treatment, male rats displayed an increase in peroxisomal BAT specific activity and a decrease in cytosolic BAT specific activity, whereas females showed an opposite response. However, there was no overall change in BAT specific activity in whole liver homogenate. Treatment with rosiglitazone or cholestyramine had no effect on BAT activity in any subcellular compartment. These experiments indicate that the majority of BAT activity in the rat liver resides in the cytosol. Approximately 15% of BAT activity is present in the peroxisomal matrix. These data support the novel finding that clofibrate treatment does not directly regulate BAT activity but does alter the subcellular localization of BAT.

Human cecal bile acids: concentration and spectrum

To obtain information on the concentration and spectrum of bile acids in human cecal content, samples were obtained from 19 persons who had died an unnatural death from causes such as trauma, homicide, suicide, or drug overdose. Bile acid concentration was measured via an enzymatic assay for 3alpha-hydroxy bile acids; bile acid classes were determined by electrospray ionization mass spectrometry and individual bile acids by gas chromatography mass spectrometry and liquid chromatography mass spectrometry. The 3alpha-hydroxy bile acid concentration (mumol bile acid/ml cecal content) was 0.4 +/- 0.2 mM (mean +/- SD); the total 3-hydroxy bile acid concentration was 0.6 +/- 0.3 mM. The aqueous concentration of bile acids (supernatant after centrifugation) was identical, indicating that most bile acids were in solution. By liquid chromatography mass spectrometry, bile acids were mostly in unconjugated form (90 +/- 9%, mean +/- SD); sulfated, nonamidated bile acids were 7 +/- 5%, and nonsulfated amidated bile acids (glycine or taurine conjugates) were 3 +/- 7%. By gas chromatography mass spectrometry, 10 bile acids were identified: deoxycholic (34 +/- 16%), lithocholic (26 +/- 10%), and ursodeoxycholic (6 +/- 9), as well as their primary bile acid precursors cholic (6 +/- 9%) and chenodeoxycholic acid (7 +/- 8%). In addition, 3beta-hydroxy derivatives of some or all of these bile acids were present and averaged 27 +/- 18% of total bile acids, indicating that 3beta-hydroxy bile acids are normal constituents of cecal content. In the human cecum, deconjugation and dehydroxylation of bile acids are nearly complete, resulting in most bile acids being in unconjugated form at submicellar and subsecretory concentrations.

Differential regulation of cytosolic and peroxisomal bile acid amidation by PPARα activation favors the formation of unconjugated bile acids

In human liver, unconjugated bile acids can be formed by the action of bile acid-CoA thioesterases (BACTEs), whereas bile acid conjugation with taurine or glycine (amidation) is catalyzed by bile acid-CoA:amino acid N-acyltransferases (BACATs). Both pathways exist in peroxisomes and cytosol. Bile acid amidation facilitates biliary excretion, whereas the accumulation of unconjugated bile acids may become hepatotoxic. We hypothesized that the formation of unconjugated and conjugated bile acids from their common substrate bile acid-CoA thioesters by BACTE and BACAT is regulated via the peroxisome proliferator-activated receptor alpha (PPARalpha). Livers from wild-type and PPARalpha-null mice either untreated or treated with the PPARalpha activator WY-14,643 were analyzed for BACTE and BACAT expression. The total liver capacity of taurochenodeoxycholate and taurocholate formation was decreased in WY-14,643-treated wild-type mice by 60% and 40%, respectively, but not in PPARalpha-null mice. Suppression of the peroxisomal BACAT activity was responsible for the decrease in liver capacity, whereas cytosolic BACAT activity was essentially unchanged by the treatment. In both cytosol and peroxisomes, the BACTE activities and protein levels were upregulated 5- to 10-fold by the treatment. These effects caused by WY-14,643 treatment were abolished in PPARalpha-null mice. The results from this study suggest that an increased formation of unconjugated bile acids occurs during PPARalpha activation.

Bile Acid Amides Derived from Chiral Amino Alcohols: Novel Antimicrobials and Antifungals.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Bile acid amides derived from chiral amino alcohols: novel antimicrobials and antifungals

Cholic and deoxycholic acid amides 10– 17 have been synthesised from (1 R,2 R)-1-phenyl-2-amino-1,3-propanediol 2, (1 S,2 S)-1-phenyl-2-amino-1,3-propanediol 4, (1 R,2 R)-1- para-nitrophenyl-2-amino-1,3-propanediol 3, (1 S,2 S)-1- para-nitrophenyl-2-amino-1,3-propanediol 5. Amide 12 derived from N-succinimidyl ester 9 of deoxycholic acid and (1 R,2 R)-1-phenyl-2-amino-1,3-propanediol 2, found to be active against Cryptococcus neoformans and the amide 17 obtained from N-succinimidyl ester 9 of deoxycholic acid and (1 S,2 S)-1- para-nitrophenyl-2-amino-1,3-propanediol 5, is found to be potent against various gram-positive bacteria.

Rat liver bile acid CoA:amino acid N-acyltransferase: expression, characterization, and peroxisomal localization

Bile acid CoA:amino acid N-acyltransferase (BAT) is responsible for the amidation of bile acids with the amino acids taurine and glycine. Rat liver BAT (rBAT) cDNA was isolated from a rat liver lambdaZAP cDNA library and expressed in Sf9 insect cells using a baculoviral vector. rBAT displayed 65% amino acid sequence homology with human BAT (hBAT) and 85% homology with mouse BAT (mBAT). Similar to hBAT, expressed rBAT was capable of forming both taurine and glycine conjugates with cholyl-CoA. mBAT, which is highly homologous to rBAT, forms only taurine conjugated bile acids (Falany, C. N., H. Fortinberry, E. H. Leiter, and S. Barnes. 1997. Cloning and expression of mouse liver bile acid CoA: Amino acid N-acyltransferase. J. Lipid Res. 38: 86-95). Immunoblot analysis of rat tissues detected rBAT only in rat liver cytosol following homogenization and ultracentrifugation. Subcellular localization of rBAT detected activity and immunoreactive protein in both cytosol and isolated peroxisomes. Rat bile acid CoA ligase (rBAL), the enzyme responsible for the formation of bile acid CoA esters, was detected only in rat liver microsomes. Treatment of rats with clofibrate, a known peroxisomal proliferator, significantly induced rBAT activity, message, and immunoreactive protein in rat liver. Peroxisomal membrane protein-70, a marker for peroxisomes, was also induced by clofibrate, whereas rBAL activity and protein amount were not affected. In summary, rBAT is capable of forming both taurine and glycine bile acid conjugates and the enzyme is localized primarily in peroxisomes in rat liver.

Farnesoid X Receptor Regulates Bile Acid-Amino Acid Conjugation

The farnesoid X receptor (FXR; NR1H4) regulates bile acid and lipid homeostasis by acting as an intracellular bile acid-sensing transcription factor. Several identified FXR target genes serve critical roles in the synthesis and transport of bile acids as well as in lipid metabolism. Here we used Affymetrix micro-array and Northern analysis to demonstrate that two enzymes involved in conjugation of bile acids to taurine and glycine, namely bile acid-CoA synthetase (BACS) and bile acid-CoA: amino acid N-acetyltransferase (BAT) are induced by FXR in rat liver. Analysis of the human BACS and BAT genes revealed the presence of functional response elements in the proximal promoter of BACS and in the intronic region between exons 1 and 2 of the BAT gene. The response elements resemble the consensus FXR binding site consisting of two nuclear receptor half-sites organized as an inverted repeat and separated by a single nucleotide (IR-1). These response elements directly bind FXR/retinoid X receptor (RXR) heterodimers and confer the activity of FXR ligands in transient transfection experiments. Further mutational analysis confirms that the IR-1 sequence of the BACS and BAT genes mediate transactivation by FXR/RXR heterodimers. Finally, Fisher rats treated with the synthetic FXR ligand GW4064 clearly show increased transcript levels of both the BACS and BAT mRNA. These studies demonstrate a mechanism by which FXR regulates bile acid amidation, a critical component of the enterohepatic circulation of bile acids.

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.

Bile acid derivatives of 5-amino-1,3,4-thiadiazole-2-sulfonamide as new carbonic anhydrase inhibitors: synthesis and investigation of inhibition effects

Bile acid amides (cholan-24-amides) of 5-substituted 1,3,4-thiadiazole-2-sulfonamide have been prepared from lithocholic, deoxycholic, cholic and dehydrocholic acids. Besides, the alcohol functional groups on the cholane ring systems were protected with acetyl group. Amides of the protected cholanes of lithocholic and cholic acids were also synthesized. Later, inhibition effects of these compounds on human carbonic anhydrase isozymes (HCA-I and II) have been investigated in vitro. For the most active compounds, inhibition constants ranged from 66 to 190nM for HCA-II with I50 (molarity of inhibitor producing a 50% inhibition of CA activity). In addition, in vivo studies were performed for the synthesized compounds in Sprague–Dawley rats. The compounds (11 and 18) showed especially significant inhibition efficacy (p<0.001).

Complete 1H and 13C resonance assignments of glycosidic conjugates of non‐amidated and amidated bile acids

AbstractComplete 1H and 13C resonance assignments on the basis of 1D and 2D NMR were made for seven naturally occurring variants of the 3α‐, 6α‐ and 7β‐O‐β‐D‐glycosidic conjugates (glucosides, N‐acetylglucosaminides and glucuronides) of non‐amidated and glycine‐ and taurine‐amidated bile acids related to lithocholic, hyodeoxycholic, chenodeoxycholic, ursodeoxycolic, deoxycholic and cholic acids. The 1H and 13C NMR signals serving to characterize each of the glycosidic conjugates of bile acids and the effects of the O‐β‐D‐glucosidation, O‐β‐D‐N‐acetylglucosaminidation and O‐β‐D‐glucuronidation at the 3‐, 6‐ or 7‐position in the 5β‐steroid nucleus on the 1H and 13C resonances were clarified and used in the structural elucidation of these bile acid glycosides. Copyright © 2001 John Wiley &amp; Sons, Ltd.

Subcellular organization of bile acid amidation in human liver: a key issue in regulating the biosynthesis of bile salts

To extend our knowledge of how the synthesis of free bile acids and bile salts is regulated within the hepatocyte, bile acid-CoA:amino acid N-acyltransferase and bile acid-CoA thioesterase activities were measured in subcellular fractions of human liver homogenates. Some bile acids, both conjugated and unconjugated, have been reported to be natural ligands for the farnesoid X receptor (FXR), an orphan nuclear receptor. The conversion of [14C]choloyl-CoA and [14C]chenodeoxycholoyl-CoA into the corresponding tauro- and glyco-bile acids or the free bile acids was measured after high-pressure liquid radiochromatography. There was an enrichment of the N-acyltransferase in the cytosolic and the peroxisomal fraction. Bile acid-CoA thioesterase activities were enriched in the cytosolic, peroxisomal, and mitochondrial fractions. The highest amidation activities of both choloyl-CoA and chenodeoxycholoyl-CoA were found in the peroxisomal fraction (15–58 nmol/mg protein/min). The Km was higher for glycine than taurine both in cytosol and the peroxisomal fraction.These results show that the peroxisomal de novo synthesis of bile acids is rate limiting for peroxisomal amidation, and the microsomal bile acid-CoA synthetase is rate limiting for the cytosolic amidation. The peroxisomal location may explain the predominance of glyco-bile acids in human bile. Both a cytosolic and a peroxisomal bile acid-CoA thioesterase may influence the intracellular levels of free and conjugated bile acids.

The Enzymatic Formation of Novel Bile Acid Primary Amides

Bifunctional peptidylglycine α-amidating monooxygenase (PAM) catalyzes the copper-, ascorbate-, and O2-dependent cleavage of C-terminal glycine-extended peptides and N-acylglycines to the corresponding amides and glyoxylate. The α-amidated peptides and the long-chain acylamides are hormones in humans and other mammals. Bile acid glycine conjugates are also substrates for PAM leading to the formation of bile acid amides. The (VMAX/Km)app values for the bile acid glycine conjugates are comparable to other known PAM substrates. The highest (VMAX/Km)app value, 3.1 ± 0.12 × 105 M−1 s−1 for 3-sulfolithocholylglycine, is 6.7-fold higher than that for d-Tyr–Val–Gly, a representative peptide substrate. The time course for O2 consumption and glyoxylate production indicates that bile acid glycine conjugate amidation is a two-step reaction. The bile acid glycine conjugate is first converted to an N-bile acyl-α-hydroxyglycine intermediate which is ultimately dealkylated to the bile acid amide and glyoxylate. The enzymatically produced bile acid amides and the carbinolamide intermediates were characterized by mass spectrometry and two-dimensional 1H–13C heteronuclear multiple quantum coherence NMR.

Presence of choloyl- and chenodeoxycholoyl-coenzyme A thioesterase activity in human liver

In human liver homogenate the formation of bile acid-CoA thioesters is localized both to the microsomal fraction catalysed by an ATP-dependent synthetase and to the peroxisomal fraction catalysed by the thiolase in the last step of the beta-oxidative cleavage of the 5beta-cholestanoyl side chain. The cytosolic bile acid-CoA:amino acid N-acyltransferase catalyse the conjugation of the CoA-activated bile acids with taurine or glycine prior to secretion into bile. The formation of bile acid-CoA esters is considered the rate-limiting step in bile acid amidation. So far, a bile acid-CoA cleaving activity has not been assessed in the research of bile acid amidation in human liver. In this work, a bile acid-CoA cleaving activity has been demonstrated at a rate that may influence the concentration of bile acid-CoA thioesters, free bile acids and amidated bile acids within the hepatocyte. Recently, it was shown that free chenodeoxycholic acid, formed by the thioesterase, is the physiological ligand of the farnesoid X receptor. A multiorganelle distribution of the bile acid-CoA hydrolytic activity was found. In the postnuclear fraction of human liver homogenate, apparent Km and Vmax for the cleavage of choloyl-CoA were 7.7 x 10-5 mol/L and 3.6 nmol x mg-1 x min-1 respectively. The corresponding values for chenodeoxycholoyl-CoA cleavage were 7.1 x 10-5 mol/L and 4.8 nmol x mg-1 x min-1. Hydrolytic activities were detected in the microsomal and the peroxisomal fractions where the bile acid-CoA esters are formed as well as in cytosol housing the N-acyltransferase activity. Compared to the bile acid-CoA synthetase activities, the hydrolytic activities were considerably higher, both in the postnuclear fraction and in the microsomal fraction. The thioesterase activities were in the same range as detected for the N-acyltransferase activities both in the postnuclear fraction and in the cytosolic fraction. The mere presence of thioesterase in microsomes, peroxisomes and cytosol seems counterproductive to bile acid amidation. The thioesterases may have an indirect regulatory function on the bile acid synthesis and are important for the regulation of bile acid synthesis by providing free chenodeoxycholic acid, the most potent activator of the farnesoid X receptor.

Relevance of serum bile acid profile in the diagnosis of intrahepatic cholestasis of pregnancy in an high incidence area: Portugal

Objective(s): The present work was conducted to clarify the relevance of usual liver function tests, and define the most predictive serum bile acid profile for diagnosis of intrahepatic cholestasis of pregnancy (ICP). Study design: This study comprised 20 healthy nonpregnant women and 77 pregnant women in the last trimester of pregnancy, from which 38 were normal pregnancies, and 39 suffered from ICP. Liver function tests were evaluated by routine laboratory techniques, conjugated bile acids were analysed by high-performance liquid chromatography, and unconjugated forms were measured by an enzymatic–fluorimetric assay. Results: During the third trimester in normal pregnancy, increased concentration of conjugated species affected all primary bile acids, although only significantly for glycocholic acid. Moreover, deoxycholic acid proportion decreased when compared with healthy nonpregnant women. Important ICP-induced changes in serum profiles of amidated bile acids were observed, involving both a marked increase in cholic acid concentration and a shift towards a higher proportion of taurine-conjugated species. Among routine liver tests, alanine aminotransferase and conjugated bilirubin were the most common indicators of ICP. Conclusion(s): In the early diagnosis and follow-up of ICP, the most predictive and accurate markers (efficiency 100%) were: (i) TBA concentration in serum >11.0 μmol l −1: (ii) cholic/chenodeoxycholic acid ratio >1.5 and cholic acid percentage >42%: (iii) glycine/taurine bile acid ratio <1.0 or glycocholic acid concentration >2.0 μmol l −1. Accurate diagnosis based on sensitive biochemical markers followed by appropriate treatment may improve both pregnancy outcome and newborn prognosis.

Studies on N-nitroso bile acid amides in relation to their possible role in gastrointestinal cancer.

Cancers of the gastrointestinal tract account for a large proportion of neoplastic diseases which afflict humans. The etiology of gastrointestinal cancer has been attributed in part to exogenous carcinogens, such as food substances and environmental pollutants. Recent hypotheses suggest that carcinogens may arise endogenously. Evidence suggests that some bile acids and their isomeric metabolites may be involved in the pathogenesis of colon cancer. However, the mechanisms responsible for their cancer-promoting effect is not clear. We and others propose that one mechanism for the mitogenic effects of bile acids may be N-nitrosation of their glycine and taurine amides; human gastric aspirates do contain small quantities of N-nitroso compounds of other substrates. Many foods contain nitrites and nitrates, which can react with bile acid amides to form N-nitroso derivatives. Our recent studies demonstrated the potential for N-nitroso conjugate formation from ursodeoxycholic acid, a 7 beta-epimer of chenodeoxycholic acid used as a drug Actigall to dissolve gallstones. The N-nitroso derivative of this compound, a direct-acting carcinogen, has a long half-life and, once nitrosated is stable enough to survive passage through the gastrointestinal tract. We describe the synthesis of N-nitrosated derivatives of various bile acid conjugates and mechanisms of decomposition of (Z)- and (E)-bile acid diazoates. Studies of the effects of enzymes such as cholylglycine hydrolase on the N-nitroso bile acid conjugates and their reaction with DNA are also described. These studies may have important implications in the interplay of diet with endogenous substrates in the etiology of cancers of the stomach, liver, and colon.

Influence of hydroxylation and conjugation in cross-inhibition of bile acid transport across the human trophoblast basal membrane

Taurocholate (TC) transport across the basal plasma membrane of the human trophoblast is a carrier-mediated process, whose specificity is probably not restricted to TC. The aim of this work was to gain further insight into the role of hydroxylation and conjugation in the behavior of the carrier system vs. bile acid (BA) species. Radiolabeled TC transport by basal plasma membrane (BPM) vesicles obtained from human term placenta was measured by a rapid filtration technique. Glycocholate (GC), taurochenodeoxycholate (TCDC) and taurodeoxycholate (TDC) inhibited TC binding to BPM. These bile acids complete with TC for the binding sites. Symmetry properties for GC- and TCDC-induced inhibition of TC transport was found in experiments where GC or TCDC were at the cis-side of the membrane (uptake and efflux experiments). GC and TCDC-induced inhibition seems to be of mixed type. By contrast, TDC was observed to affect TC transport differently, depending on whether the experiments addressed uptake or efflux. At the intracellular side of the membrane (uptake), TDC induced a marked increase in both V max and K t . However, at the fetal side (efflux) a significant reduction in both V max and K t was found. In spite of these peculiarities, the values for K i were very close for GC, TCDC and TDC at the intracellular side but not at the fetal side, where the decreasing order for K i was GC > TCDC > TDC . TC uptake by BPM vesicles was not modified in the presence of a wide range of estrone sulfate concentrations (0.002–1.0 mM). In summary, these results indicate that a particular bile acid molecular structure is necessary for steroid-related compounds to interact with the bile acid carrier located in BPM. They also suggest that changes in the number and position of hydroxy groups, as well as in the amino-acid moiety in amidated bile acids modify the behavior of the carrier, which may play an important role in the net vectorial transfer of bile acids across the placenta.