Bile acid patterns in meconium are influenced by cholestasis of pregnancy and not altered by ursodeoxycholic acid treatment.
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BACKGROUND: Data on meconium bile acid composition in newborn babies of patients with intrahepatic cholestasis of pregnancy (ICP) are relatively scant, and changes that occur on ursodeoxycholic acid (UDCA) administration have not been evaluated. AIMS: To investigate bile acid profiles in meconium of neonates from untreated and UDCA treated patients with ICP. Maternal serum bile acid composition was also analysed both at diagnosis and delivery to determine whether this influences the concentration and proportion of bile acids in the meconium. PATIENTS/METHODS: The population included eight healthy pregnant women and 16 patients with ICP, nine of which received UDCA (12.5-15.0 mg/kg body weight/day) for 15+/-4 days until parturition. Bile acids were assessed in the meconium by gas chromatography-mass spectrometry and in maternal serum by high performance liquid chromatography. RESULTS: Total bile acid and cholic acid concentrations in the meconium were increased (p<0.01) in newborns from patients with ICP (13.5 (5.1) and 8.4 (4.1) micromol/g respectively; mean (SEM)) as compared with controls (2.0 (0.5) and 0.8 (0.3) micromol/g respectively), reflecting the total bile acid and cholic acid levels in the maternal serum (r = 0.85 and r = 0.84, p<0.01). After UDCA administration, total bile acid concentrations decreased in the mother ( approximately 3-fold, p<0. 05) but not in the meconium. UDCA concentration in the meconium showed only a 2-fold increase after treatment, despite the much greater increase in the maternal serum (p<0.01). Lithocholic acid concentration in the meconium was not increased by UDCA treatment. CONCLUSIONS: UDCA administration does not influence the concentration and proportion of bile acids in the meconium, which in turn are altered by ICP. Moreover, this beneficial treatment for the mother does not increase meconium levels of potentially toxic metabolites of UDCA such as lithocholic acid. (+info)
Effect of ursodeoxycholic acid administration in patients with acute viral hepatitis: a pilot study.
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BACKGROUND: Ursodeoxycholic acid (UDCA) is able to improve biochemical markers of cholestasis, with a parallel decrease in transaminases, in various cholestatic liver diseases. AIM: To evaluate the effects of UDCA administration on acute viral hepatitis-related cholestasis and the course of acute viral hepatitis. METHODS: Seventy-nine consecutive patients with acute viral hepatitis (HBV: 43, HCV: 11, HAV: 15, HEV: 3, Non A-E: 7) were randomized to receive either UDCA for 3 weeks or no treatment. Liver biochemistry and serum bile acid determinations were run at weekly intervals. RESULTS: No significant differences were observed in mean percentage decreases in transaminases between treated and untreated patients. By contrast, cholestatic indexes decreased significantly more quickly in patients treated with UDCA than in controls, and this effect was more evident in patients with increasing alanine transaminase levels at admission. After a peak at the end of the first week of therapy, serum levels of conjugated ursodeoxycholic acid (CUDCA) showed a gradual decrease. Conjugated cholic acid (CCA) and chenodeoxycholic acid (CCDCA) showed a progressive decrease with the resolution of viral hepatitis, but no influence of UDCA administration was observed. CONCLUSIONS: Our study demonstrates that UDCA significantly improves cholestatic indices in patients with acute viral hepatitis, but this effect does not seem to affect the course of the illness. (+info)
Sulphation of lithocholic acid in the colon-carcinoma cell line CaCo-2.
(11/334)
High levels of bile acids in the colon may correlate with an increased risk of colon cancer, but the underlying mechanisms are not known. Proteoglycan structures have been shown to change when human colon cells differentiate in vitro. The expression of [(35)S]sulphated molecules was used as a phenotypic marker to study the effects of bile acids on the human-colon-carcinoma cell line CaCo-2. [(35)S]sulphated compounds were isolated from the medium of cell fractions of cells metabolically labelled with [(35)S]sulphate in the absence and presence of cholic acid, deoxycholic acid, chenodeoxycholic acid and lithocholic acid (LA). Labelled molecules were analysed by gel chromatography, HPLC and SDS/PAGE in combination with chemical and enzymic methods. The expression of (35)S-labelled proteoglycans was not affected by any of the bile acids tested. However, the level of sulphated metabolites increased 7-18-fold in different experiments during a 22 h labelling period in the presence of an LA concentration of 10 microg/ml (26.6 nmol/ml) compared with controls. Further analyses showed that this was due, at least in part, to the sulphation of LA itself. This sulphation of LA was a rapid process followed by secretion back to the medium. Brefeldin A did not reduce the sulphation of LA, indicating that this conversion takes place in the cytosol, rather than in the Golgi apparatus of the CaCo-2 cells. LA in colon may be sulphated efficiently by the colonocytes to reduce the toxic effects of this particular bile acid. Sulphation may possibly be an important protective mechanism in the colon. (+info)
Comparison of the effects of bile acids on cell viability and DNA synthesis by rat hepatocytes in primary culture.
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Bile acid-induced inhibition of DNA synthesis by the regenerating rat liver in the absence of other manifestation of impairment in liver cell viability has been reported. Because in experiments carried out on in vivo models bile acids are rapidly taken up and secreted into bile, it is difficult to establish steady concentrations to which the hepatocytes are exposed. Thus, in this work, a dose-response study was carried out to investigate the in vitro cytotoxic effect of major unconjugated and tauro- (T) or glyco- (G) conjugated bile acids and to compare this as regards their ability to inhibit DNA synthesis. Viability of hepatocytes in primary culture was measured by Neutral red uptake and formazan formation after 6 h exposure of cells to bile acids. The rate of DNA synthesis was determined by radiolabeled thymidine incorporation into DNA. Incubation of hepatocytes with different bile acid species - cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), in the range of 10-1000 microM - revealed that toxicity was stronger for the unconjugated forms of CDCA and DCA than for CA and UDCA. Conjugation markedly reduced the effects of bile acids on cell viability. By contrast, the ability to inhibit radiolabeled thymidine incorporation into DNA was only slightly lower for taurodeoxycholic acid (TDCA) and glycodeoxycholic acid (GDCA) than for DCA. When the effect of these bile acids on DNA synthesis and cell viability was compared, a clear dissociation was observed. Radiolabeled thymidine incorporation into DNA was significantly decreased (-50%) at TDCA concentrations at which cell viability was not affected. Lack of a cause-effect relationship between both processes was further supported by the fact that well-known hepatoprotective compounds, such as tauroursodeoxycholic acid (TUDCA) and S-adenosylmethionine (SAMe) failed to prevent the effect of bile acids on DNA synthesis. In summary, our results indicate that bile acid-induced reduction of DNA synthesis does not require previous decreases in hepatocyte viability. This suggests the existence of a high sensitivity to bile acids of cellular mechanisms that may affect the rate of DNA repair and/or proliferation, which is of particular interest regarding the role of bile acids in the etiology of certain types of cancer. (+info)
Regulation of 25- and 27-hydroxylation side chain cleavage pathways for cholic acid biosynthesis in humans, rabbits, and mice. Assay of enzyme activities by high-resolution gas chromatography;-mass spectrometry.
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In classic cholic acid biosynthesis, a series of ring modifications of cholesterol precede side chain cleavage and yield 5beta-cholestane-3alpha, 7alpha, 12alpha-triol. Side chain reactions of the triol then proceed either by the mitochondrial 27-hydroxylation pathway or by the microsomal 25-hydroxylation pathway. We have developed specific and precise assay methods to measure the activities of key enzymes in both pathways, 5beta-cholestane-3alpha, 7alpha, 12alpha-triol 25- and 27-hydroxylases and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases. The extracts from either the mitochondrial or microsomal incubation mixtures were purified by means of a disposable silica cartridge column, derivatized into trimethylsilyl ethers, and quantified by gas chromatography;-mass spectrometry with selected-ion monitoring in a high resolution mode. Compared with the addition of substrates in acetone, those in 2-hydroxypropyl-beta-cyclodextrin increased mitochondrial triol 27-hydroxylase activity 132% but decreased activities of the enzymes in microsomal 25-hydroxylation pathway (triol 25-hydroxylase and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases) 13;-60% in human liver. The enzyme activities in both pathways were generally 2- to 4-times higher in mouse and rabbit livers compared with human liver. In all species, microsomal triol 25-hydroxylase activities were 4- to 11-times larger than mitochondrial triol 27-hydroxylase activities but the activities of tetrol 24S-hydroxylase were similar to triol 27-hydroxylase activities in our assay conditions. The regulation of both pathways in rabbit liver was studied after bile acid synthesis was perturbed. Cholesterol feeding up-regulated enzyme activities involved in both 25- (64;-142%) and 27- (77%) hydroxylation pathways, while bile drainage up-regulated only the enzymes in the 25-hydroxylation pathway (178;-371%). Using these new assays, we demonstrated that the 25- and 27-hydroxylation pathways for cholic acid biosynthesis are more active in mouse and rabbit than human livers and are separately regulated in rabbit liver. (+info)
Alpha 1-fetoprotein transcription factor is required for the expression of sterol 12alpha -hydroxylase, the specific enzyme for cholic acid synthesis. Potential role in the bile acid-mediated regulation of gene transcription.
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Cholesterol conversion to bile acids occurs via the "classic" (neutral) or the "alternative" (acidic) bile acid biosynthesis pathways. Sterol 12alpha-hydroxylase/CYP8b1 is the specific enzyme required for cholic acid synthesis. The levels of this enzyme determine the ratio of cholic acid to chenodeoxycholic acid and thus the hydrophobicity of the circulating bile acid pool. Expression of the 12alpha-hydroxylase gene is tightly down-regulated by hydrophobic bile acids. In this study, we report the characterization of two DNA elements that are required for both the 12alpha-hydroxylase promoter activity and bile acid-mediated regulation. Mutation of these elements suppresses 12alpha-hydroxylase promoter activity. Mutations of any other part of the promoter do not alter substantially the promoter activity or alter regulation by bile acids relative to the wild type promoter. These two DNA elements bind alpha(1)-fetoprotein transcription factor (FTF), a member of the nuclear receptor family. We also show that overexpression of FTF in a non-liver cell line activates the sterol 12alpha-hydroxylase promoter. These studies demonstrate the crucial role of FTF for the expression and regulation of a critical gene in the bile acid biosynthetic pathways. (+info)
The human liver-specific homolog of very long-chain acyl-CoA synthetase is cholate:CoA ligase.
(15/334)
Unconjugated bile acids must be activated to their CoA thioesters before conjugation to taurine or glycine can occur. A human homolog of very long-chain acyl-CoA synthetase, hVLCS-H2, has two requisite properties of a bile acid:CoA ligase, liver specificity and an endoplasmic reticulum subcellular localization. We investigated the ability of this enzyme to activate the primary bile acid, cholic acid, to its CoA derivative. When expressed in COS-1 cells, hVLCS-H2 exhibited cholate:CoA ligase (choloyl-CoA synthetase) activity with both non-isotopic and radioactive assays. Other long- and very long-chain acyl-CoA synthetases were incapable of activating cholate. Endogenous choloyl-CoA synthetase activity was also detected in liver-derived HepG2 cells but not in kidney-derived COS-1 cells. Our results are consistent with a role for hVLCS-H2 in the re-activation and re-conjugation of bile acids entering liver from the enterohepatic circulation rather than in de novo bile acid synthesis. (+info)
Interaction of three-way DNA junctions with steroids.
(16/334)
DNA aptamers that bind to cholic acid were previously isolated by an in vitro selection method. Secondary structural prediction and deletion-mutant experiments suggested that the cholic-acid binding regions of 19 sequenced clones could form three-way-junction structures. In this article, the secondary structures of the sequenced clones and the structural requirements for binding to cholic acid were evaluated. A course of mutational-analysis and chemical-modification experiments provided strong support for the predicted secondary structure and also indicated that the binding site is located at the branching point of the three-way junction. Sequence analysis revealed that the sequences of the three base pairs flanking the junction of the three stems are highly conserved among selected clones. The evaluation of the relative binding of several bile acids and structurally related steroids with the aptamer was also carried out. The results revealed a broad range of selectivity and preference for hydrophobic steroids rather than for cholic acid upon binding, indicating that the binding is driven by a hydrophobic interaction. The experimental results reported here allowed us to propose a structural model of a binding site formed by three Watson-Crick base pairs. (+info)