Calcium binding by bile acids: in vitro studies using a calcium ion electrode

In this study, we compared in vitro calcium binding by the taurine and glycine conjugates of the major bile acids in human bile: cholic (CA), chenodeoxycholic (CDCA) and deoxycholic (DCA) acids, together with the cholelitholytic bile acids ursodeoxycholic (UDCA) and ursocholic (UCA) acids. At physiological total calcium (CaTOT) (1-15 mM) and bile acid (BA) (10-50 mM) concentrations, all the bile acids caused concentration-dependent falls in [Ca2+], suggesting calcium binding. Except for glycine-conjugated CDCA, all the other calcium-bile acid complexes were soluble in 150 mM NaCl. The calcium binding affinities followed the pattern: dihydroxy (CDCA, UDCA and DCA) greater than trihydroxy (CA and UCA) bile acids, and glycine conjugates greater than taurine conjugates. The glycine conjugate of UDCA, which increases during UDCA treatment, had the highest calcium binding affinity. Ten-20 mM phospholipid modestly increased calcium binding by CA conjugates, but not by CDCA, UDCA, and DCA conjugates. Phospholipid also prevented the precipitation of glyco-CDCA in the presence of calcium. Bile acid-calcium biding was pH-independent over the range 6.5-8.5. The different calcium binding affinities of the major biliary bile acids may partly explain their varying effects on biliary calcium secretion. The results also suggest that neither precipitation of calcium-bile acid complexes nor impaired calcium binding by bile acids is important in the pathogenesis of human calcium gallstone formation.     

Structure-activity relationship of bile acids and bile acid analogs in regard to FXR activation

The farnesoid X receptor (FXR) is a bile acid-activated nuclear receptor that plays a major role in bile acid and cholesterol metabolism. To obtain an insight into the structure-activity relationships of FXR ligands, we investigated the functional roles of structural elements in the physiological ligands chenodeoxycholic acid [CDCA; (3alpha,7alpha)], cholic acid [CA; (3alpha,7alpha,12alpha)], deoxycholic acid [DCA; (3alpha,12alpha)], and lithocholic acid (3alpha) in regard to FXR activation in a cell-based FXR response element-driven luciferase assay and an in vitro coactivator association assay. Conversion of the carboxyl group of CDCA or CA to an alcohol did not greatly diminish their ability to activate FXR. In contrast, the 7beta-epimers of the alcohols were inactive, indicating that the bile alcohols retained the ligand properties of the original bile acids and that the 7beta-hydroxyl group diminished their FXR-activating effect. Similarly, hydroxyl epimers of DCA exhibited decreased activity compared with DCA, indicating a negative effect of 3beta- or 12beta-hydroxyl groups. Introduction of an alkyl group at the 7beta- or 3beta-position of CDCA resulted in diminished FXR activation in the following order of alkyl groups: 7-ethyl=7-propyl>3-methyl>7-methyl. These results indicate that bulky substituents, whether hydroxyl groups or alkyl residues, at the beta-position of cholanoids decrease their ability to activate FXR.     

Reversal of clofibrate-induced cholesterol oversaturation of bile with chenodeoxycholic acid.

Giving clofibrate 2 g daily to seven patients significantly increased the biliary cholesterol concentration while the proportion of bile acids fell. Five patients on established clofibrate treatment were given 750 mg of chenodeoxycholic acid (CDCA) daily for one month. Biliary lipid analysis after the CDCA treatment showed a significant fall in the proportion of cholesterol and a rise in that of bile acids. The serum lipid concentrations, which had already been reduced by diet and clofibrate, showed a further significant reduction after the introduction of CDCA. This study suggests that CDCA may be usefully combined with clofibrate to reverse the tendency towards cholesterol saturation of bile and enhance the effect of lowering serum lipid concentrations.     

Effects of feeding bile acids and a bile acid sequestrant on hepatic bile acid composition in mice

An improved ultra performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) method was established for the simultaneous analysis of various bile acids (BA) and applied to investigate liver BA content in C57BL/6 mice fed 1% cholic acid (CA), 0.3% deoxycholic acid (DCA), 0.3% chenodeoxycholic acid (CDCA), 0.3% lithocholic acid (LCA), 3% ursodeoxycholic acid (UDCA), or 2% cholestyramine (resin). Results indicate that mice have a remarkable ability to maintain liver BA concentrations. The BA profiles in mouse livers were similar between CA and DCA feedings, as well as between CDCA and LCA feedings. The mRNA expression of Cytochrome P450 7a1 (Cyp7a1) was suppressed by all BA feedings, whereas Cyp7b1 was suppressed only by CA and UDCA feedings. Gender differences in liver BA composition were observed after feeding CA, DCA, CDCA, and LCA, but they were not prominent after feeding UDCA. Sulfation of CA and CDCA was found at the 7-OH position, and it was increased by feeding CA or CDCA more in male than female mice. In contrast, sulfation of LCA and taurolithocholic acid (TLCA) was female-predominant, and it was increased by feeding UDCA and LCA. In summary, the present systematic study on BA metabolism in mice will aid in interpreting BA-mediated gene regulation and hepatotoxicity.     

Bile acids stimulate PKCalpha autophosphorylation and activation: role in the attenuation of prostaglandin E1-induced cAMP production in human dermal fibroblasts

The aim was to identify the specific PKC isoform(s) and their mechanism of activation responsible for the modulation of cAMP production by bile acids in human dermal fibroblasts. Stimulation of fibroblasts with 25-100 microM of chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA) led to YFP-PKCalpha and YFP-PKCdelta translocation in 30-60 min followed by a transient 24- to 48-h downregulation of the total PKCalpha, PKCdelta, and PKCepsilon protein expression by 30-50%, without affecting that of PKCzeta. Increased plasma membrane translocation of PKCalpha was associated with an increased PKCalpha phosphorylation, whereas increased PKCdelta translocation to the perinuclear domain was associated with an increased accumulation of phospho-PKCdelta Thr505 and Tyr311 in the nucleus. The PKCalpha specificity on the attenuation of cAMP production by CDCA was demonstrated with PKC downregulation or inhibition, as well as PKC isoform dominant-negative mutants. Under these same conditions, neither phosphatidylinositol 3-kinase, p38 MAP kinase, p42/44 MAP kinase, nor PKA inhibitors had any significant effect on the CDCA-induced cAMP production attenuation. CDCA concentrations as low as 10 microM stimulated PKCalpha autophosphorylation in vitro. This bile acid effect required phosphatidylserine and was completely abolished by the presence of G?6976. CDCA at concentrations less than 50 microM enhanced the PKCalpha activation induced by PMA, whereas greater CDCA concentrations reduced the PMA-induced PKCalpha activation. CDCA alone did not affect PKCalpha activity in vitro. In conclusion, although CDCA and UDCA activate different PKC isoforms, PKCalpha plays a major role in the bile acid-induced inhibition of cAMP synthesis in fibroblasts. This study emphasizes potential consequences of increased systemic bile acid concentrations and cellular bile acid accumulation in extrahepatic tissues during cholestatic liver diseases.     

Isolation and chemical synthesis of a major, novel biliary bile acid in the common wombat (Vombatus ursinus): 15alpha-hydroxylithocholic acid

The major bile acids present in the gallbladder bile of the common Australian wombat (Vombatus ursinus) were isolated by preparative HPLC and identified by NMR as the taurine N-acylamidates of chenodeoxycholic acid (CDCA) and 15alpha-hydroxylithocholic acid (3alpha,15alpha-dihydroxy-5beta-cholan-24-oic acid). Taurine-conjugated CDCA constituted 78% of biliary bile acids, and (taurine-conjugated) 15alpha-hydroxylithocholic acid constituted 11%. Proof of structure of the latter compound was obtained by its synthesis from CDCA via a Delta14 intermediate. The synthesis of its C-15 epimer, 15beta-hydroxylithocholic acid (3alpha,15beta-dihydroxy-5beta-cholan-24-oic acid), is also reported. The taurine conjugate of 15alpha-hydroxylithocholic acid was synthesized and shown to have chromatographic and spectroscopic properties identical to those of the compound isolated from bile. It is likely that 15alpha-hydroxylithocholic acid is synthesized in the wombat hepatocyte by 15alpha-hydroxylation of lithocholic acid that was formed by bacterial 7alpha-dehydroxylation of CDCA in the distal intestine. Thus, the wombat appears to use 15alpha-hydroxylation as a novel detoxification mechanism for lithocholic acid.     

LC/ESI-tandem mass spectrometric determination of bile acid 3-sulfates in human urine 3beta-Sulfooxy-12alpha-hydroxy-5beta-cholanoic acid is an abundant nonamidated sulfate

We developed a highly sensitive and quantitative method to detect bile acid 3-sulfates in human urine employing liquid chromatography/electrospray ionization-tandem mass spectrometry. This method allows simultaneous analysis of bile acid 3-sulfates, including nonamidated, glycine-, and taurine-conjugated bile acids, cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), and lithocholic acid (LCA), using selected reaction monitoring (SRM) analysis. The method was applied to analyze bile acid 3-sulfates in human urine from healthy volunteers. The results indicated an unknown compound with the nonamidated common bile acid 3-sulfates on the chromatogram obtained by the selected reaction monitoring analysis. By comparison of the retention behavior and MS/MS spectrum of the unknown peak with the authentic specimen, the unknown compound was identified as 3beta,12alpha-dihydroxy-5beta-cholanoic acid 3-sulfate.     

Presence of protein-bound unconjugated bile acids in the cytoplasmic fraction of rat brain

Using liquid chromatography/electrospray ionization mass spectrometry, we have found three unconjugated bile acids [cholic acid (CA), chenodeoxycholic acid (CDCA), and deoxycholic acid (DCA)] in the rat brain cytoplasmic fraction. CDCA was detected only upon extraction with high concentrations of guanidine, indicating that it is bound noncovalently to protein in the brain. The most abundant of the three, it was present at a concentration of 1.6 nmol/g wet weight (approximately 15 mg of protein) of brain, corresponding to almost 30 times its serum concentration. CA and DCA were present at 1/30th the concentration of CDCA. Bile acids conjugated with amino acids, sulfuric acid, and glucuronic acid were not detected. These data clearly demonstrate that unconjugated CDCA and, to a lesser extent, CA and DCA, exists in the rat brain.     

Profiling circulating and urinary bile acids in patients with biliary obstruction before and after biliary stenting

Bile acids are considered as extremely toxic at the high concentrations reached during bile duct obstruction, but each acid displays variable cytotoxic properties. This study investigates how biliary obstruction and restoration of bile flow interferes with urinary and circulating levels of 17 common bile acids. Bile acids (conjugated and unconjugated) were quantified by liquid chromatography coupled with tandem mass spectrometry in serum and urine samples from 17 patients (8 men and 9 women) with biliary obstruction, before and after biliary stenting. Results were compared with serum concentrations measured in 40 age- and sex-paired control donors (20 men and 20 women). The total circulating bile acid concentration increases from 2.7 μM in control donors to 156.9 μM in untreated patients with biliary stenosis. Serum taurocholic and glycocholic acids exhibit 304- and 241-fold accumulations in patients with biliary obstruction compared to controls. The enrichment in chenodeoxycholic acid species reached a maximum of only 39-fold, while all secondary and 6α-hydroxylated species--except taurolithocholic acids--were either unchanged or significantly reduced. Stenting was efficient in restoring an almost normal circulating profile and in reducing urinary bile acids. Conclusion: These results demonstrate that biliary obstruction affects differentially the circulating and/or urinary levels of the various bile acids. The observation that the most drastically affected acids correspond to the less toxic species supports the activation of self-protecting mechanisms aimed at limiting the inherent toxicity of bile acids in face of biliary obstruction.     

Bile acids modulate tight junction structure and barrier function of Caco-2 monolayers via EGFR activation

Intestinal and systemic illnesses have been linked to increased gut permeability. Bile acids, whose luminal profile can be altered in human disease, modulate intestinal paracellular permeability. We investigated the mechanism by which selected bile acids increase gut permeability using a validated in vitro model. Human intestinal Caco-2 cells were grown in monolayers and challenged with a panel of bile acids. Transepithelial electrical resistance and luminal-to-basolateral fluxes of 10-kDa Cascade blue-conjugated dextran were used to monitor paracellular permeability. Immunoprecipitation and immunoblot analyses were employed to investigate the intracellular pathway. Redistribution of tight junction proteins was studied by confocal laser microscopy. Micromolar concentrations of cholic acid, deoxycholic acid (DCA), and chenodeoxycholic acid (CDCA) but not ursodeoxycholic acid decreased transepithelial electrical resistance and increased dextran flux in a reversible fashion. Coincubation of 50 muM CDCA or DCA with EGF, anti-EGF monoclonal antibody, or specific src inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP-2) abolished the effect. A concentration of 50 muM of either CDCA or DCA also induced EGF receptor phosphorylation, occludin dephosphorylation, and occludin redistribution at the tight junction level in the same time frame and in a reversible fashion. We conclude that selected bile acids modulate intestinal permeability via EGF receptor autophosphorylation, occludin dephosphorylation, and rearrangement at the tight junction level. The effect is mediated by the src family kinases and is abolished by EGF treatment. These data also support the role of bile acids in the genesis of necrotizing enterocolitis and the protective effect of EGF treatment.     

Characterization of conjugated and unconjugated bile acid transport via human organic solute transporter α/β

Bile acids are biosynthesized from cholesterol in hepatocytes and usually localize in the enterohepatic circulation system. This system is regulated by several transporters that are expressed in the liver and intestine. Organic solute transporter (OST) α/β, which is known as a bidirectional transporter for some organic anions, contributes to the transport of bile acids; however, the transport properties of individual bile acids are not well understood. In this study, we investigated the transport properties of five bile acids (cholic acid [CA], chenodeoxycholic acid [CDCA], deoxycholic acid [DCA], ursodeoxycholic acid [UDCA], and lithocholic acid [LCA]) together with their glycine and taurine conjugates mediated by OSTα/β. Of the unconjugated bile acids, CA, CDCA, DCA, and LCA were taken up by OSTαβ/MDCKII cells more rapidly than mock cells, but no significant increase in the uptake of UDCA was observed. On the contrary, all glycine- and taurine-conjugated bile acids showed a significant increase in the uptake by OSTαβ/MDCKII cells. Saturable OSTα/β-mediated transports of CDCA, DCA, glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), glycolithocholic acid (GLCA), taurochenodeoxycholic acid (TCDCA), and taurolithocholic acid (TLCA) were observed. The apparent Michaelis constants of CDCA, DCA, GCDCA, GDCA, GLCA, TCDCA, and TLCA for OSTα/β were 23.0 ± 4.0, 14.9 ± 1.9, 864.2 ± 80.7, 586.4 ± 43.2, 12.8 ± 0.5, 723.7 ± 4.8, and 23.9 ± 0.3 μM, respectively. However, the transport of other bile acids was not saturable. Our results indicate that OSTα/β has a low affinity but a high capacity for transporting bile acids.     

Effects of bile acids on proliferation and production of proteinase activity of Uronema marinum (Ciliophora: Scuticociliatida)

Little is known about the effects of bile acids in relation to infectivity on the biological characteristics of Uronema marinum, a serious opportunistic parasite of farmed olive flounder Paralichthys olivaceus. In this study, we examined the effects of bile acids on the proliferation of U. marinum and on proteinase production in vitro. Proliferation of U, marinum was significantly enhanced by lithocholic acid (LCA) at 30 and 60 pmol, and by chenodeoxycholic acid (CDCA) at 0.06 pmol. In contrast, a significant decrease in proliferation was observed with cholic acid (CA) at 30 and 60 micromol, and with deoxycholic acid (DCA) at all amounts used. Proteinase production from live U. marinum was significantly increased by LCA, whereas CA significantly decreased proteinase production. CDCA and DCA had no effect on proteinase production. Although the types and concentrations of bile acids in the faeces of olive flounder are not known, the present results suggest that bile acids in the culturing water might influence the proliferation and production of proteinases in U. marinum, resulting in an increased possibility of scuticociliatosis in olive flounder farms.     

Deoxycholic and chenodeoxycholic bile acids induce apoptosis via oxidative stress in human colon adenocarcinoma cells

The continuous exposure of the colonic epithelium to high concentrations of bile acids may exert cytotoxic effects and has been related to pathogenesis of colon cancer. A better knowledge of the mechanisms by which bile acids induce toxicity is still required and may be useful for the development of new therapeutic strategies. We have studied the effect of deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) treatments in BCS-TC2 human colon adenocarcinoma cells. Both bile acids promote cell death, being this effect higher for CDCA. Apoptosis is detected after 30 min–2 h of treatment, as observed by cell detachment, loss of membrane asymmetry, internucleosomal DNA degradation, appearance of mitochondrial transition permeability (MPT), and caspase and Bax activation. At longer treatment times, apoptosis is followed in vitro by secondary necrosis due to impaired mitochondrial activity and ATP depletion. Bile acid-induced apoptosis is a result of oxidative stress with increased ROS generation mainly by activation of plasma membrane enzymes, such as NAD(P)H oxidases and, to a lower extent, PLA₂. These effects lead to a loss of mitochondrial potential and release of pro-apoptotic factors to the cytosol, which is confirmed by activation of caspase-9 and -3, but not caspase-8. This initial apoptotic steps promote cleavage of Bcl-2, allowing Bax activation and formation of additional pores in the mitochondrial membrane that amplify the apoptotic signal.     

Corticosteroids, receptors, and the organ-specific functions of 11 beta-hydroxysteroid dehydrogenase.

Reversible oxidation of the biologically active corticosteroids to the inactive 11-dehydrocorticosteroids is catalyzed by 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). The properties of the enzyme based on clinical observations of individuals with defective 11 beta HSD expression, and laboratory studies of the properties and behavior of the enzyme, are consistent with separate 11 beta-dehydrogenase and 11-oxoreductase species. However, recombinant enzyme expressed in mammalian cells retain both activities, leading to the conclusion that 11 beta HSD is a unique, reversible enzyme. 11 beta HSD is present in most tissues, but its specific functions in most tissues are unknown. How the enzyme may mediate corticosteroid-receptor interaction is illustrated by studies using kidney, testis, and brain. In kidney, 11 beta HSD prevents glucocorticoids from competing inappropriately with aldosterone for mineralocorticoid receptor (MR). Lack of enzyme in humans due to natural causes or inhibition by pharmacological agents results in maximum activation of MR by glucocorticoids, leading to the clinical symptoms of apparent mineralocorticoid excess. Leydig cells of the testes synthesize testosterone, a process that is suppressed by events initiated by the binding of corticosteroid to glucocorticoid receptors (GR). Depletion of active steroid mediated by 11 beta HSD may initiate testosterone production at puberty and affect testosterone production during adult life, as for example during periods of stress. The heterogeneous distribution of MR and GR in the brain reflects the specific regional effects of glucocorticoids and mineralocorticoids on neural function. Colocalization of 11 beta HSD and corticosteroid receptors in brain may be important in controlling the specificity of corticosteroid interaction with GR and MR. The patterns of 11 beta HSD-steroid-receptor interaction illustrated with these three tissues may provide models applicable to other tissues in which corticosteroid receptors and 11 beta HSD coexist.     

Modulation of nuclear steroid receptors by ursodeoxycholic acid inhibits TGF-beta1-induced E2F-1/p53-mediated apoptosis of rat hepatocytes

We have recently shown that both ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA) prevent transforming growth factor beta1 (TGF-beta1)-induced hepatocyte apoptosis by modulating the E2F-1/p53/Bax pathway. In addition, activation of glucocorticoid (GR) and mineralocorticoid receptors (MR) inhibits apoptosis in various systems. UDCA induces a ligand-independent activation of the GR, thus potentially regulating a number of targets. In this study, we investigated the role of GR and MR during TGF-beta1-induced hepatocyte apoptosis, and identified additional antiapoptotic targets for UDCA. Our results showed that in primary hepatocytes, TGF-beta1 induced 40-50% decreases in gr and mr mRNA expression (p < 0.01), together with up to 10-fold reductions in their protein levels (p < 0.01). Notably, pretreatment with UDCA resulted in a significant upregulation of nuclear steroid receptors (p < 0.05), which coincided with 2- and 3-fold increases in the level of GR and MR nuclear translocation, respectively, when compared with that of TGF-beta1 alone (p < 0.05). Similarly, TUDCA induced GR and MR nuclear translocations (p < 0.05) and markedly prevented MR protein changes associated with TGF-beta1 (p < 0.05) without affecting GR protein levels. Moreover, when interference RNA was used to inhibit GR and MR, UDCA no longer protected hepatocytes against TGF-beta1-induced apoptosis. In fact, the protective effect of UDCA in TGF-beta1-associated caspase activation decreased from 65 to <10% when GR or MR function was blocked. Finally, the TGF-beta1-induced E2F-1/Mdm-2/p53 apoptotic pathway, normally inhibited by UDCA, was not regulated by the bile acid after GR or MR silencing. These results demonstrate that UDCA protects against apoptosis through an additional pathway that involves nuclear receptors GR and MR as key factors. Further, the E2F-1/Mdm-2/p53 apoptotic pathway appears to be a prime target for UDCA-induced steroid receptor activation.