Trihydroxy Bile Acids Research Articles

Further evidence for blood-to-brain influx of unconjugated bile acids by passive diffusion: Determination of their brain-to-serum concentration ratios in rats by LC/MS/MS

Bile acids (BAs) reside in the brain and are probably involved in some neurological disorders. The view that most of unconjugated BAs in the brain are derived across the blood–brain barrier from the periphery by passive diffusion depending on their hydrophobicity is currently dominant, but some studies have made conflicting claims. In this study, the correlation analysis between the rat brain and serum levels of unconjugated BAs with a wider range of hydrophobicity was conducted to obtain further evidence about the blood-to-brain influx of unconjugated BAs by passive diffusion. We first developed the precise, accurate and matrix effect-free LC/ESI-MS/MS methods for quantifying eight major unconjugated BAs in the rat brain and serum. Derivatization was employed for increasing the assay sensitivity and specificity. The analysis using these methods reproduced the strong positive correlations between the brain and serum levels, and significant higher concentrations in the serum than in the brain for all the unconjugated BAs. The BA with the higher logPow (hydrophobicity) had the higher brain-to-serum concentration ratio (mono- > di- > trihydroxy BAs). Furthermore, the hydrophobicity was considered as the stronger factor for the blood-to-brain influx of the BAs than the serum protein binding ratio. Thus, this study provided further evidence supporting that passive diffusion is the major mechanism for the blood-to-brain influx of the unconjugated BAs.

Analysis of the Electron Density of a Water Molecule Encapsulated by Two Cholic Acid Residues

Cholic acid is a trihydroxy bile acid with a nice peculiarity: the average distance between the oxygen atoms (O7 and O12) of the hydroxy groups located at C7 and C12 carbon atoms is 4.5 Å, a value which perfectly matches with the O/O tetrahedral edge distance in Ih ice. In the solid phase, they are involved in the formation of hydrogen bonds with other cholic acid units and solvents. This fact was satisfactorily used for designing a cholic dimer which encapsulates one single water molecule between two cholic residues, its oxygen atom (Ow) being exactly located at the centroid of a distorted tetrahedron formed by the four steroid hydroxy groups. The water molecule participates in four hydrogen bonds, with the water simultaneously being an acceptor from the 2 O12 (hydrogen lengths are 2.177 Å and 2.114 Å) and a donor towards the 2 O7 (hydrogen bond lengths are 1.866 Å and 1.920 Å). These facts suggest that this system can be a nice model for the theoretical study of the formation of ice-like structures. These are frequently proposed to describe the water structure found in a plethora of systems (water interfaces, metal complexes, solubilized hydrophobic species, proteins, and confined carbon nanotubes). The above tetrahedral structure is proposed as a reference model for those systems, and the results obtained from the application of the atoms in molecules theory are presented here. Furthermore, the structure of the whole system allows a division into two interesting subsystems in which water is the acceptor of one hydrogen bond and the donor of another. The analysis of the calculated electron density is performed through its gradient vector and the Laplacian. The calculation of the complexation energy used correction of the basis set superposition error (BSSE) with the counterpoise method. As expected, four critical points located in the H…O bond paths were identified. All calculated parameters obey the proposed criteria for hydrogen bonds. The total energy for the interaction in the tetrahedral structure is 54.29 kJ/mol, while the summation obtained of the two independent subsystems and the one between the alkyl rings without water is only 2.5 kJ/mol higher. This concordance, together with the calculated values for the electron density, the Laplacian of the electron density, and the lengths of the oxygen atom and the hydrogen atom (involved in the formation of each hydrogen bond) to the hydrogen bond critical point, suggests that each pair of hydrogen bonds can be considered independent of each other.

The urinary bile acid profiling analysis of asymptomatic hypercholanemia of pregnancy: A pseudo-targeted metabolomics study

BackgroundAsymptomatic hypercholanemia of pregnancy (AHP) is a controversial hypercholanemia, which is difficult to distinguish from intrahepatic cholestasis of pregnancy (ICP). Our aim is to elucidate the characteristics of urinary bile acid (BA) profiling of women with AHP and to find potential biomarkers for the diagnosis and differential diagnosis of AHP. MethodsWe developed a pseudo-targeted approach to perform metabolomics analysis of bile acids (BAs) using ultra-high performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Urinary BAs profiles were compared among AHP women (n = 20), ICP patients (n = 33) and normal controls (n = 35). ResultsThe profiling of urinary BAs was significantly different among the AHP, ICP and control groups. Compared to the control group, the AHP group had higher levels of four possible sulfated BAs and trihydroxy BAs, including the species of muricholic acid (MCA), cholic acid (CA) and six possible BAs, whereas, 20 possible sulfated BAs, taurochenodeoxycholic acid (TCDCA), tetrahydrocannabinolic acid (THCA), and seven possible BAs were significantly lower in the AHP group than those in the ICP group. Based on the receiver operating characteristic (ROC) analysis, glycocholic acid (GCA) combined with T-ω-MCA were found to be the potential combination biomarker for the diagnosis (area under the curve was 0.960) of AHP, and mono-S, Gtri-S-2 combined with TLCA-S were found to be the potential combination biomarker for the differential diagnosis (area under the curve was 0.990) of AHP and ICP. ConclusionsThe metabolisms of urinary Bas were altered in the AHP group compared with the ICP group and the control group. Urinary BA profiling analysis can serve as an effective tool for the diagnosis of AHP and the differential diagnosis of AHP and ICP.

Bile acid patterns in commercially available oxgall powders used for the evaluation of the bile tolerance ability of potential probiotics.

This study aimed to analyze the bile acid patterns in commercially available oxgall powders used for evaluation of the bile tolerance ability of probiotic bacteria. Qxgall powders purchased from Sigma-Aldrich, Oxoid and BD Difco were dissolved in distilled water, and analyzed. Conjugated bile acids were profiled by ion-pair high-performance liquid chromatography (HPLC), free bile acids were detected as their p-bromophenacyl ester derivatives using reversed-phase HPLC after extraction with acetic ether, and total bile acids were analyzed by enzymatic-colorimetric assay. The results showed that 9 individual bile acids (i.e., taurocholic acid, glycocholic acid, taurodeoxycholic acid, glycodeoxycholic acid, taurochenodeoxycholic acid, glycochenodeoxycholic acid, cholic acid, chenodeoxycholic acid, deoxycholic acid) were present in each of the oxgall powders tested. The content of total bile acid among the three oxgall powders was similar; however, the relative contents of the individual bile acids among these oxgall powders were significantly different (P < 0.001). The oxgall powder from Sigma-Aldrich was closer to human bile in the ratios of glycine-conjugated bile acids to taurine-conjugated bile acids, dihydroxy bile acids to trihydroxy bile acids, and free bile acids to conjugated bile acids than the other powders were. It was concluded that the oxgall powder from Sigma-Aldrich should be used instead of those from Oxoid and BD Difco to evaluate the bile tolerance ability of probiotic bacteria as human bile model.

History of hepatic bile formation: old problems, new approaches.

Studies of hepatic bile formation reported in 1958 established that it was an osmotically generated water flow. Intravenous infusion of sodium taurocholate established a high correlation between hepatic bile flow and bile acid excretion. Secretin, a hormone that stimulates bicarbonate secretion, was also found to increase hepatic bile flow. The sources of the water entering the biliary system with these two stimuli were differentiated by the use of mannitol. An increase in its excretion parallels the increase in bile flow in response to bile acids but not secretin, which led to a quantitative distinction between canalicular and ductular water flow. The finding of aquaglyceroporin-9 in the basolateral surface of the hepatocyte accounted for the rapid entry of mannitol into hepatocytes and its exclusion from water movement in the ductules where aquaporin-1 is present. Electron microscopy demonstrated that bile acids generate the formation of vesicles that contain lecithin and cholesterol after their receptor-mediated canalicular transport. Biophysical studies established that the osmotic effect of bile acids varies with their concentration and also with the proportion of mono-, di-, and trihydroxy bile acids and provides a basis for understanding their physiological effects. Because of the varying osmotic effect of bile acids, it is difficult to quantify bile acid independent flow generated by other solutes, such as glutathione, which enters the biliary system. Monohydroxy bile acids, by markedly increasing aggregation number, severely reduce water flow. Developing biomarkers for the noninvasive assessment of normal hepatic bile flow remains an elusive goal that merits further study.

Determination of bile acids in pig liver, pig kidney and bovine liver by gas chromatography-chemical ionization tandem mass spectrometry with total ion chromatograms and extraction ion chromatograms

An effective method has been developed for quantitative determination of six bile acids including lithocholic acid (LCA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), hydodeoxycholic acid (HDCA), cholic acid (CA) and ursodeoxycholic acid (UDCA) in biological tissues including pig liver, pig kidney and bovine liver by gas chromatography-chemical ionization/tandem mass spectrometry (GC-CI/MS/MS). Camphor-10-sulphonic acid (CSA) was proposed as effective catalyst for bile acid derivatization. Reactions were accelerated ultrasonically. The effects of different catalysts and reaction times on derivatization efficiency were evaluated and optimized. Bile acids were determined as methyl ester-trimethylsilyl ether and methyl ester-acetate derivatives. The efficiency of trimethylsilylation and acetylation was evaluated. Trimethylsilylation was done with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) as the trimethylsilyl donating reagent in a ultrasonic bath for 20min. Acetylation was done in pyridine with acetic anhydride at 40–45°C for 4h. The former reaction was faster than the latter. Thus, trimethylsilylation was employed for the quantitative analysis. Negligible interferences from sterols in biological matrices were observed when the biological samples were treated with solid phase extraction before GC-CI/MS/MS. The linearity, reproducibility, detection limit and recovery were evaluated under the optimized conditions. Satisfactory results were obtained when bile acid derivatives of LCA, CDCA, HDCA, and UDCA were determined with total ion chromatograms (TIC) while DCA and CA were determined with extracted ion chromatograms (EIC), respectively. The detection limits (S/N=3) for six bile acids in biological tissues were ranging from 0.40 to 1.6ng/mL and the recoveries indicated that the proposed method was feasible for the determination of trace bile acids in the biological samples studied. The experimental results for the animal tissues purchased from five different markets were compared. Interestingly, all of the six bile acids were present in pig liver while only the dihydroxy bile acids, DCA, CDCA and HDCA were found in pig kidney. In addition to DCA and CDCA, trihydroxy bile acid, CA, are the major bile acids in bovine liver.

Synthesis, aggregation behavior and cholesterol solubilization studies of 16-epi-pythocholic acid (3α,12α,16β-trihydroxy-5β-cholan-24-oic acid)

Synthesis, aggregation behavior and in vitro cholesterol solubilization studies of 16-epi-pythocholic acid (3 alpha,12 alpha,16 beta-trihydroxy-5 beta-cholan-24-oic acid, EPCA) are reported. The synthesis of this unnatural epimer of pythocholic acid (3 alpha,12 alpha,16 alpha-trihydroxy-5 beta-cholan-24-oic acid, PCA) involves a series of simple and selective chemical transformations with an overall yield of 21% starting from readily available cholic acid (CA). The critical micellar concentration (CMC) of 16-epi-pythocholate in aqueous media was determined using pyrene as a fluorescent probe. In vitro cholesterol solubilization ability was evaluated using anhydrous cholesterol and results were compared with those of other natural di- and trihydroxy bile acids. These studies showed that 16-epi-pythocholic acid (16 beta-hydroxy-deoxycholic acid) behaves similar to cholic acid (CA) and avicholic acid (3 alpha,7 alpha,16 alpha-trihydroxy-5 beta-cholan-24-oic acid, ACA) in its aggregation behavior and cholesterol dissolution properties.

A new, major C27 biliary bile acid in the Red-winged tinamou (Rhynchotus rufescens):(25R)-1β,3α,7α-trihydroxy-5β-cholestan-27-oic acid

The chemical structures of the three major bile acids present in the gallbladder bile of the Red-winged tinamou (Rhynchotus rufescens), an early evolving, ground-living bird related to ratites, were determined. Bile acids were isolated by preparative reversed-phase HPLC. Two of the compounds were identified as the taurine N-acylamidates of 25R-3alpha,7alpha-dihydroxy-5beta-cholestan-27-oic acid (constituting 22% of biliary bile acids) and 25R-3alpha,7alpha,12alpha-trihydroxy-5beta-cholestan-27-oic acid (constituting 51%). The remaining compound, constituting 21% of biliary bile acids, was an unknown C27 bile acid. Its structure was elucidated by LC/ESI-MS/MS and NMR and shown to be the taurine conjugate of 25R-1beta, 3alpha, 7alpha-trihydroxy-5beta-cholestan-27-oic acid, a C27 trihydroxy bile acid not previously reported. Although C27 bile acids with a 1beta-hydroxyl group have been identified as trace bile acids in the alligator, this is the first report of a major biliary C27 bile acid possessing a 1beta-hydroxyl group.

Constitutive Androstane Receptor-Mediated Changes in Bile Acid Composition Contributes to Hepatoprotection from Lithocholic Acid-Induced Liver Injury in Mice

Pharmacological activation of the constitutive androstane receptor (CAR) protects the liver during cholestasis. The current study evaluates how activation of CAR influences genes involved in bile acid biosynthesis as a mechanism of hepatoprotection during bile acid-induced liver injury. CAR activators phenobarbital (PB) and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) or corn oil (CO) were administered to C57BL/6 wild-type (WT) and CAR knockout (CAR-null) mice before and during induction of intrahepatic cholestasis using the secondary bile acid, lithocholic acid (LCA). In LCA-treated WT and all the CAR-null groups (excluding controls), histology revealed severe multifocal necrosis. This pathology was absent in WT mice pretreated with PB and TCPOBOP, indicating CAR-dependent hepatoprotection. Decreases in total hepatic bile acids and hepatic monohydroxy, dihydroxy, and trihydroxy bile acids in PB- and TCPOBOP-pretreated WT mice correlated with hepatoprotection. In comparison, concentrations of monohydroxylated and dihydroxylated bile acids were increased in all the treated CAR-null mice compared with CO controls. Along with several other enzymes (Cyp7b1, Cyp27a1, Cyp39a1), Cyp8b1 expression was increased in hepatoprotected mice, which could be suggestive of a shift in the bile acid biosynthesis pathway toward the formation of less toxic bile acids. In CAR-null mice, these changes in gene expression were not different among treatment groups. These results suggest CAR mediates a shift in bile acid biosynthesis toward the formation of less toxic bile acids, as well as a decrease in hepatic bile acid concentrations. We propose that these combined CAR-mediated effects may contribute to the hepatoprotection observed during LCA-induced liver injury.

Species-Specific and Age-Dependent Bile Acid Composition: Aspects on CYP8B and CYP4A Subfamilies in Bile Acid Biosynthesis

The present review aims to give an overview of the cytochrome P450 8B (CYP8B) and cytochrome P450 4A (CYP4A) subfamilies in relation to biosynthesis of bile acids, in particular trihydroxy bile acids. Trihydroxy bile acids are basically required in most species and have an impact on cholesterol and lipid metabolism. The primary trihydroxy bile acid in most mammals is cholic acid. Some species produce other important trihydroxy bile acids, for example the adult pig which produce hyocholic acid instead of cholic acid. The position of the third hydroxyl group in cholic acid and hyocholic acid, 12alpha or 6alpha position, respectively, has a profound effect on the hydrophilic-hydrophobic property of the trihydroxy bile acids. The CYP8B subfamily is required for introduction of the 12alpha-hydroxyl group in cholic acid biosynthesis. The enzyme responsible for 6alpha-hydroxylation in hyocholic acid biosynthesis, however, varies among species. This review will discuss, in particular, porcine members of the CYP8B and CYP4A subfamilies because interesting findings regarding members of these subfamilies have recently been recognized in this species. CYP8B1 was for a long time believed to be absent in the pig but was recently found to be expressed in fetal pig liver. The enzyme catalyzing the 6alpha-hydroxylation in hyocholic acid biosynthesis in pig was found to be an atypical member of the CYP4A subfamily, denoted CYP4A21. The review presents bile acid biosynthesis in view of these findings and discusses physiochemical properties and developmental-dependent aspects related cholic acid and hyocholic acid biosynthesis.

Hepatobiliary effects of cholic and lithocholic acids: experimental study in hamsters

Etiopathogenesis of biliary atresia remains unknown. Among several theories, one proposes that the disorder may be caused by the toxic effect of monohydroxy bile acids on fetal and neonatal hepatobiliary system. In this paper we evaluated toxic effects produced by ingestion of cholic acid, a trihydroxy bile acid, and lithocholic acid, a monohydroxy bile acid in the hepatobiliary system of a hamster during gestational and perinatal periods. A diet composed by 0.5% cholic acid and 0.25% lithocholic acid was administrated to pregnant hamsters. Liver and bile ducts of the adult and newborn animals were analyzed to point out the changes induced by these acids after birth. Because hamsters and humans have a similar bile metabolism, these animals were eligible for the study. The ingestion of 0.5% lithocholic acid, during hamster's gestation, caused maternal intense ductal/ductular proliferation, inflammatory signs, hepatic cells degeneration and regeneration, hyperplasia of extra hepatic ducts epithelium, and abortion. Both 0.5% cholic acid and 0.25% lithocholic acid ingested by pregnant hamsters, caused ductal/ductular proliferation and hepatobiliary inflammatory damage in a different degree of intensity in adult animals and mild intensity in the young; and also the number of the young was reduced in the litter. We found that the ingestion of these bile acids by hamsters, during gestational period caused different degrees of toxicity on maternal and neonatal hepatobiliary systems. The histopathologic findings observed in biliary atresia patients could not be found in newborn hamsters. New experimental models are needed in the attempt to establish a correlation of these acids with neonatal cholestatic diseases.

Cholestatic liver disease in adults may be due to an inherited defect in bile acid biosynthesis

An increasing number of treatable inborn errors of bile acid synthesis have been described, primarily in infants with severe cholestatic liver disease. The present patient, whose two older siblings had died from progressive cholestatic liver disease, developed neonatal cholestasis and rickets but recovered during the childhood years and follow-up was terminated at 12 years of age. The patient presented again at 26 years of age with jaundice and pathological liver function tests. This was normalized upon treatment with ursodeoxycholic acid. Electrospray mass spectrometry of urine showed predominance of unsaturated bile acids, characteristic of 3beta-hydroxy-Delta5-C27-steroid dehydrogenase/isomerase (HSD3B7) deficiency. The activity of HSD3B7 in cultured fibroblasts was less than 5% of normal. A single homozygous mutation was found in exon 4 leading to an amino acid exchange (S162R) and loss of enzyme activity. This case illustrates that infants with an inherited absence of HSD3B7 may survive the neonatal period of life and childhood without treatment with bile acids. A low level of sulphation of the abnormal trihydroxy bile acid formed as a result of enzyme deficiency may be of importance for survival. The possibility that liver disease presenting in the adult may be due to a mutation in the HSD3B7 gene should be considered, especially in cases with familial occurrence of liver disease and earlier periods of liver dysfunction.

Effect of tibolone on turnover of cholesterol to bile acids in ovariectomized rats

Tibolone is used in breast cancer therapy in women. The most important aspect of this therapy concerns its influence on bone and lipid metabolism. Several studies have reported the effect of tibolone on plasma lipids. Approximately 40% of total cholesterol removal occurs by its conversion to bile acids, a process that takes place in the liver. The aim of the study was to evaluate the influence of tibolone on the conversion of cholesterol to bile acids in estrogen-deficient rats. Thirty female Wistar rats were divided into three groups: sham-operated controls, ovariectomized controls, and ovariectomized rats treated with tibolone. After 42 days of drug administration, bile was collected under anesthesia after administration of radioactive 4-C cholesterol. Bile was assayed for total C radioactivity; C bile acids were determined after thin-layer chromatography separation by using the isotopic technique. The ovariectomy decreased the concentrations of C-radioactive bile, taurocholic acid, and cholic acid. The administration of tibolone significantly increased the excretion of C-radioactive bile and C-radioactive bile acids as compared with ovariectomized controls. Moreover, the therapy with tibolone significantly increased the concentrations of C taurocholic acid, C glycocholic acid, cholic acid, and taurochenodeoxycholic plus taurodeoxycholic acids. The results of the present study suggest that tibolone may increase the concentrations of cholesterol and trihydroxy bile acids in bile. These changes may be associated with an increased risk of gallstone generation.

Complementary Stimulation of Hepatobiliary Transport and Detoxification Systems by Rifampicin and Ursodeoxycholic Acid in Humans

Rifampicin (RIFA) and ursodeoxycholic acid (UDCA) improve symptoms and biochemical markers of liver injury in cholestatic liver diseases by largely unknown mechanisms. We aimed to study the molecular mechanisms of action of these drugs in humans. Thirty otherwise healthy gallstone patients scheduled for cholestectomy were randomized to RIFA (600 mg/day for 1 week) or UDCA (1 g/day for 3 weeks) or no medication before surgery. Routine biochemistry, lipids, and surrogate markers for P450 activity (4beta-hydroxy cholesterol, 4beta-OH-C) and bile acid synthesis (7alpha-hydroxy-4-cholesten-3-one, C-4) were measured in serum. Bile acids were analyzed in serum, urine, and bile. A wedge liver biopsy specimen was taken to study expression of hepatobiliary ABC transporters as well as detoxification enzymes and regulatory transcription factors. RIFA enhanced bile acid detoxification as well as bilirubin conjugation and excretion as reflected by enhanced expression of CYP3A4, UGT1A1, and MRP2. These molecular effects were paralleled by decreased bilirubin and deoxycholic acid concentrations in serum and decreased lithocholic and deoxycholic acid concentrations in bile. UDCA on the other hand stimulated the expression of BSEP, MDR3, and MRP4. UDCA became the predominant bile acid after UDCA treatment and lowered the biliary cholesterol saturation index. RIFA enhances bile acid detoxification as well as bilirubin conjugation and export systems, whereas UDCA stimulates the expression of transporters for canalicular and basolateral bile acid export as well as the canalicular phospholipid flippase. These independent but complementary effects may justify a combination of both agents for the treatment of cholestatic liver diseases.

Effect of tamoxifen and raloxifene on cholesterol transformation to bile acids in ovariectomized rats

Tamoxifen is used as adjuvant therapy for prevention and treatment of metastatic breast cancer, whereas raloxoifene is approved for osteoporosis and is being investigated for breast cancer prevention. The most important aspects of these therapies are focused on their influence on bone and lipid metabolism. Several studies have reported the effect of tamoxifen and raloxifene on plasma lipids. Approximately 40% of total cholesterol removal occurs by conversion to bile acids, a process that takes place in the liver. The aim of the present study was to evaluate the influence of tamoxifen and raloxifene on the conversion of cholesterol to bile acids in estrogen-deficient in rats. The study included 40 female Wistar rats, divided into four groups: sham-operated controls, ovariectomized controls, ovariectomized rats treated with tamoxifen and ovariectomized rats treated with raloxifene. After 42 days of drug administration, bile was collected under anesthesia following administration of radioactive [4-14C]cholesterol and assayed for total 14C radioactivity; 14C-labeled bile acids were determined by the use of isotopic techniques after initial separation by thin-layer chromatography. Ovariectomy caused decreased excretion of bile and bile acids. Administration of tamoxifen and raloxifene significantly reduced the excretion of 14C-labeled bile and bile acids compared with ovariectomized controls. Tamoxifen therapy significantly reduced the excretion of taurocholic acid and glycochenodeoxycholic plus glycodeoxycholic acids, and excretion of cholic, litocholic and chenodeoxycholic plus deoxycholic acids was increased. Raloxifene significantly reduced the excretion of taurocholic, glycocholic and litocholic acids, taurochenodeoxycholic plus taurodeoxycholic acids, as well as chenodeoxycholic plus deoxycholic acids. The results of the present study suggest that tamoxifen and raloxifene did not reverse the changes in bile composition induced by estrogen deficiency. The decreased concentration of trihydroxy bile acids during therapy with raloxifene might decrease the risk of gallstone formation, although this hypothesis requires further investigation.

Interfacial properties of most monofluorinated bile acids deviate markedly from the natural congeners: studies with the Langmuir-Pockels surface balance

We characterized the air-water interfacial properties of four monofluorinated bile acids alone and in binary mixtures with a common lecithin, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), using an automated Langmuir-Pockels surface balance. We compared 7alpha-fluoromurocholic acid (FMCA), 7alpha-fluorohyodeoxycholic acid (FHDCA), 6alpha-fluoroursodeoxycholic acid (FUDCA), and 6alpha-fluorochenodeoxycholic acid (FCDCA) with their natural dihydroxy homologs, murocholic acid (MCA), hyodeoxycholic acid (HDCA), ursodeoxycholic acid (UDCA), and chenodeoxycholic acid (CDCA). For further comparison, two trihydroxy bile acids, 3alpha,6beta,7alpha-trihydroxycholanoic acid [alpha-muricholic acid (alpha-MCA)] and 3alpha,6alpha,7beta-trihydroxycholanoic acid [omega-muricholic acid (omega-MCA)], with isologous OH polar functions to FMCA and FUDCA were also studied. Pressure-area isotherms of MCA, HDCA, UDCA, CDCA, and FMCA displayed sharp collapse points. In contrast, FHDCA, FUDCA, and FCDCA formed monolayers that were less stable than the trihydroxy bile acids, displaying second-order phase transitions in their isotherms. All natural and fluorinated bile acids condensed mixed monolayers with POPC, with maximal effects at molar bile acid concentrations between 30 and 50 mol%. Examination of molecular models revealed that the 7alpha-F atom of the interfacially stable FMCA projects away from the 6beta-OH function, resulting in minimal steric interactions, whereas in FHDCA, FUDCA, and FCDCA, close vicinal interactions between OH and F polar functions result in progressive bulk solubility upon monolayer compression. These results provide a framework for designing F-modified bile acids to mimic or diverge from the natural compounds in vivo.

Micellar aggregates and hydrogels from phosphonobile salts

The aggregation properties of novel bile acid analogs-phosphonobile salts (PBS)-have been studied. The critical micellar concentration of 23 and 24-phosphonobile salts were measured using fluorescence and 31P NMR methods. All the ten synthesized phosphonobile salts formed gels at different pH ranges in water. The pH range at which individual PBSs could gelate water was narrow and influenced by the number and conformation of hydroxyl groups. A reversible thermochromic system has been developed (with 23-phosphonodeoxycholate at pH 3.3), which changes color upon gelation. The investigation of the first hydrogels derived from trihydroxy bile acid analogs 1 and 6 was made using fluorescence, 31P NMR, X-ray crystallography, circular dichroism and SEM. The present studies reveal that the gel network consists of a chiral, fibrous structure possessing hydrophobic interiors.

Detoxification of Lithocholic Acid, A Toxic Bile Acid: Relevance to Drug Hepatotoxicity

Lithocholic acid, a monohydroxy, secondary bile acid, is formed by bacterial 7‐dehydroxylation of the primary bile acid chenodeoxycholic acid (CDCA) and of the secondary bile acid ursodeoxycholic acid (UDCA). Lithocholic acid and its precursor CDCA are toxic when fed to the rabbit, rhesus monkey, and baboon, but not when CDCA, as well as UDCA, is used for therapeutic purposes in man. Older studies showed that the species specific toxicity of lithocholic acid could be explained by efficient sulfation of lithocholic acid in man and in chimpanzee, but not in the rabbit, rhesus monkey, or baboon. Rodents detoxify lithocholic acid by hydroxylation, but this does not occur in species in which it is toxic. Recent studies suggest that lithocholic acid induces its own detoxification by activating nuclear receptors to promote transcription of genes encoding sulfotransferase. In addition, work with CaCoi2 cells suggest that lithocholic acid may undergo sulfation in the enterocyte and be effluxed back into the intestinal lumen. The evolution of trihydroxy bile acids in vertebrates may have occurred to decrease the formation of lithocholic acid. Lithocholic acid is a rare example of a toxic endobiotic; a variety of mechanisms have evolved to solve the problem of efficient detoxification.

Gene structure of pig sterol 12α-hydroxylase (CYP8B1) and expression in fetal liver: comparison with expression of taurochenodeoxycholic acid 6α-hydroxylase (CYP4A21)

Cholic acid is the major trihydroxy bile acid formed in most mammals. The domestic pig ( Sus scrofa) is an exception. The bile of adult pig is devoid of cholic acid whereas hyocholic acid is found in amounts equal to that of cholic acid in humans. The pathway leading to formation of hyocholic acid is believed to be species-specific and to have evolved in the pig to compensate for a nonexistent or deficient cholic acid biosynthesis. However, a high level of cholic acid has recently been found in the bile of fetal pig. Here we describe that a gene encoding the key enzyme in cholic acid biosynthesis, the sterol 12α-hydroxylase (CYP8B1), is in fact present in the pig genome. The deduced amino acid sequence shows 81% identity to the human and rabbit orthologues. CYP8B1 mRNA is expressed at significant levels in fetal pig liver. Both CYP8B1 and the key enzyme in hyocholic acid formation, taurochenodeoxycholic acid 6α-hydroxylase (CYP4A21), were found to be expressed in pig liver in a developmental-dependent but opposite fashion.

Substrate specificities of rat oatp1 and ntcp: implications for hepatic organic anion uptake

Transport of a series of 3H-radiolabeled C23, C24, and C27 bile acid derivatives was compared and contrasted in HeLa cell lines stably transfected with rat Na+/taurocholate cotransporting polypeptide (ntcp) or organic anion transporting polypeptide 1 (oatp1) in which expression was under regulation of a zinc-inducible promoter. Similar uptake patterns were observed for both ntcp and oatp1, except that unconjugated hyodeoxycholate was a substrate of oatp1 but not ntcp. Conjugated bile acids were transported better than nonconjugated bile acids, and the configuration of the hydroxyl groups (alpha or beta) had little influence on uptake. Although cholic and 23 norcholic acids were transported by ntcp and oatp1, other unconjugated bile acids (chenodeoxycholic, ursodeoxycholic) were not. In contrast to ntcp, oatp1-mediated uptake of the trihydroxy bile acids taurocholate and glycocholate was four- to eightfold below that of the corresponding dihydroxy conjugates. Ntcp mediated high affinity, sodium-dependent transport of [35S]sulfobromophthalein with a Km similar to that of oatp1-mediated transport of [35S]sulfobromophthalein (Km = 3.7 vs. 3.3 muM, respectively). In addition, for both transporters, uptake of sulfobromophthalein and taurocholic acid showed mutual competitive inhibition. These results indicate that the substrate specificity of ntcp is considerably broader than previously suspected and caution the extrapolation of transport data obtained in vitro to physiological function in vivo.