Tertiary Bile Acid Research Articles

Characterization of a novel bile acid-based delivery platform for microencapsulated pancreatic β-cells

Introduction In a recent study, we confirmed good chemical and physical compatibility of microencapsulated pancreatic β-cells using a novel formulation of low viscosity sodium alginate (LVSA), Poly-L-Ornithine (PLO), and the tertiary bile acid, ursodeoxycholic acid (UDCA). This study aimed to investigate the effect of UDCA on the morphology, swelling, stability, and size of these new microcapsules. It also aimed to evaluate cell viability in the microcapsules following UDCA addition. Materials and methods Microencapsulation was carried out using a Büchi-based system. Two (LVSA-PLO, control and LVSA-PLO-UDCA, test) pancreatic β-cells microcapsules were prepared at a constant ratio of 10:1:3, respectively. The microcapsules’ morphology, cell viability, swelling characteristics, stability, mechanical strength, Zeta potential, and size analysis were examined. The cell contents in each microcapsule and the microencapsulation efficiency were also examined. Results The addition of UDCA did not affect the microcapsules’ morphology, stability, size, or the microencapsulation efficiency. However, UDCA enhanced cell viability in the microcapsules 24 h after microencapsulation (p < 0.01), reduced swelling (p < 0.05), reduced Zeta potential (− 73 ± 2 to − 54 ± 2 mV, p < 0.01), and increased mechanical strength of the microcapsules (p < 0.05) at the end of the 24-h experimental period. Discussion and conclusion UDCA increased β-cell viability in the microcapsules without affecting the microcapsules’ size, morphology, or stability. It also increased the microcapsules’ resistance to swelling and optimized their mechanical strength. Our findings suggest potential benefits of the bile acid UDCA in β-cell microencapsulation.

Cholestatic pregnancy is associated with reduced placental 11βHSD2 expression

IntroductionIntrahepatic Cholestasis of Pregnancy (ICP) is associated with an increased risk of fetal morbidity and mortality and is characterised by elevated bile acids in the maternal and fetal compartments. Bile acids have been shown to attenuate renal 11βHSD2 expression and, given the protective role of placental 11βHSD2 in preventing fetal exposure to excessive maternal cortisol, we aimed to establish whether raised serum bile acids in ICP influence placental 11βHSD2 expression. MethodsPlacental tissue from human and murine cholestatic pregnancy was evaluated for changes in 11βHSD2 mRNA expression compared to uncomplicated pregnancy using quantitative PCR. Parallel in vitro studies were performed using BeWo choriocarcinoma cells to assess the effect of different bile acid species on 11βHSD2 gene expression and whether concurrent UDCA administration can reverse any bile acid induced changes. ResultsPlacental 11βHSD2 mRNA expression was reduced in human and murine cholestatic pregnancy. In BeWo cells, treatment with the primary bile acid CDCA resulted in reduced 11βHSD2 gene expression, while treatment with other primary bile acids had no significant effect. Furthermore, the tertiary bile acid UDCA, used in the treatment of ICP did not significantly affect 11βHSD2 mRNA levels either alone, or when co-administered with CDCA. DiscussionUnder cholestatic conditions placental 11βHSD2 mRNA is reduced. Studies in BeWo choriocarcinoma cells demonstrated that CDCA is likely to be the specific bile acid that mediates this effect. UDCA, the main bile acid used to treat cholestasis, did not reduce placental 11βHSD2 expression, further supporting its use in the management of ICP.

Abstract 4002: Differential regulation of the ERGF-MAPK pathway by deoxycholic acid (DCA) and Ursodeoxycholic acid (UDCA) in colon cancer.

Abstract Obesity and a western diet are risk factors for the development of colorectal cancer and correlate with high levels of bile acids. Specifically, the secondary bile acid deoxycholic acid (DCA) has been shown to act as a tumor promoter in animal studies. Interestingly, the tertiary bile acid Ursodeoxycholic acid (UDCA) was found to have chemopreventive activity in a clinical trial conducted in 1285 individuals with recently resected colorectal adenomas. The mechanism(s) of DCA's tumor promoting effects and UDCA's chemopreventive activities are unclear, however DCA and UDCA have been previously shown to regulate EGFR-MAPK signaling in a differential manner. Because this mitogenic pathway has been shown to play a role in colon cancer cell proliferation and survival, we hypothesize that DCA's tumor promoting effect may stem from its regulation of this mitogenic pathway. We therefore examined the role of DCA in the regulation of the EGFR MAPK pathway in human colon cancer cells as compared to UDCA. Our findings show that DCA can induce ERK1/2 phosphorylation in an EGFR-independent manner in HT-29 colon adenocarcinoma cells, and that this is at least partially dependent on PI3K activation. We also determined that UDCA pretreatment was able to suppress both EGF and DCA-induced EGFR-MAP signaling. By elucidating the mechanism by which DCA and UDCA differentially regulate colon cancer proliferation and survival, we may be able to skew the signaling in colon cancer cells to favor chemoprevention as opposed to tumorgenesis. Citation Format: Sara M. Centuori. Differential regulation of the ERGF-MAPK pathway by deoxycholic acid (DCA) and Ursodeoxycholic acid (UDCA) in colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4002. doi:10.1158/1538-7445.AM2013-4002

Treatment of NASH with ursodeoxycholic acid: Cons

Non-alcoholic steatohepatitis (NASH) has a prevalence of 1% in Western countries. Its causes as well as its medical treatment are, to date, still debated. Recently, studies of agents suggested to have antiapoptotic, insulin-sensitizing or anti-inflammatory effects in patients with NASH have been conducted, one of which is ursodeoxycholic acid (UDCA), a tertiary bile acid. Between 1994 and 2008, four prospective randomized, double-blind, placebo-controlled studies of the treatment of NASH with UDCA were conducted. The first study, by Lindor et al., compared the impact of 13-15 mg/kg/day of UDCA to a placebo. The second study by Dufour et al. had an additional third arm that administered combination therapy with UDCA and vitamin E. The third and fourth studies by Leuschner et al. and by Ratziu et al. evaluated high doses of UDCA at 25-35 mg/kg/day, and used liver biopsies and serum liver enzyme levels to evaluate the impact of UDCA. With the exception of Ratziu et al.'s study, which was lacking a second liver biopsy, none of these studies showed any significant differences in the treatment of NASH with UDCA compared with a placebo. However, Dufour et al. did observe a significant improvement of NASH with the combination (UDCA/VitE) vs placebo therapy, whereas UDCA monotherapy was not effective in the treatment of NASH. Nevertheless, the effects of other bile acids and combination therapies need to be explored.

New fluorescent bile acids: Synthesis, chemical characterization, and disastereoselective uptake by Caco-2 cells of 3-deoxy 3-NBD-amino deoxycholic and ursodeoxycholic acid

Deoxycholic acid (DCA), a secondary bile acid (BA), and ursodeoxycholic acid (UDCA), a tertiary BA, cause opposing effects in vivo and in cell suspensions. Fluorescent analogues of DCA and UDCA could help investigate important questions about their cellular interactions and distribution.We have prepared a set of isomeric 3α- and 3β-amino analogues of UDCA and DCA and derivatised these with the discrete fluorophore, 4-nitrobenzo-2-oxa-1,3-diazol (NBD), forming the corresponding four fluorescent adducts. These absorb in the range 465–470nm and fluoresce at approx. 535nm.In order to determine the ability of the new fluorescent bile acids to mimic the parents, their uptake was studied using monolayers of Caco-2 cells, which are known to express multiple proteins of the organic anion-transporting peptide (OATP) subfamily of transporters. Cellular uptake was monitored over time at 4 and 37°C to distinguish between passive and active transport.All four BA analogues were taken up but in a strikingly stereo- and structure-specific manner, suggesting highly discriminatory interactions with transporter protein(s). The α-analogues of DCA and to a lesser extent UDCA were actively transported, whereas the β-analogues were not. The active transport process was saturable, with Michaelis–Menten constants for 3α-NBD DCA (5) being Km=42.27±12.98μM and Vmax=2.8±0.4nmol/(mg protein∗min) and for 3α-NBD UDCA (3) Km=28.20±7.45μM and Vmax=1.8±0.2nmol/(mg protein∗min). These fluorescent bile acids are promising agents for investigating questions of bile acid biology and for detection of bile acids and related organic anion transport processes.

Structure–function analysis of the tertiary bile acid TUDCA for the resolution of endoplasmic reticulum stress in intestinal epithelial cells

Inflammatory bowel diseases (IBD) are chronically relapsing and immune-mediated disorders of the gastrointestinal tract. Endoplasmic reticulum (ER) stress mechanisms in the epithelium have been demonstrated to be implemented into the pathogenesis of intestinal inflammation. Chemical chaperones have been demonstrated to exhibit beneficial effects in various diseases associated with ER stress mechanisms by prohibiting the unfolded protein response (UPR). In a structure–function analysis, we tested the potential of the conjugated bile salt sodium tauroursodeoxycholate (TUDCA), naturally present in the small bowel, to resolve ER stress in intestinal epithelial cells.TUDCA efficiently inhibited the expression of UPR dependent genes like GRP78 triggered by the ER stressor tunicamycin in the small intestinal epithelial cell line Mode-K. TUDCA inhibited upstream signaling events in all three branches of the UPR cascade and diminished binding of UPR activated transcription factors to the grp78 promoter. A structure–function analysis revealed that UDCA but not its conjugation partner taurine, known as a chemical chaperone, is responsible for the inhibition of GRP78 induction and that UDCA is 10 times more effective than its taurine conjugate. This inhibitory effect was confirmed in a cell free assay, where TUDCA and UDCA but not taurine effectively inhibited the aggregation of thermally denatured BSA.We conclude that TUDCA and UDCA are potent anti-aggregants for the resolution of ER stress in intestinal epithelial cells and should be considered as a potential drug target to resolve ER stress mechanisms underlying the pathology of IBD.

The role of secondary bile acids in neoplastic development in the oesophagus

Bile acids have been demonstrated, through the use of animal models and clinical association studies, to play a role in neoplastic development in Barrett's metaplasia. How specific bile acids promote neoplasia is as yet unknown, as are the exact identities of the important bile acid subtypes. The combination of bile subtype with appropriate pH is critical, as pH alters bile acid activity enormously. Hence glycine-conjugated bile acids are involved in neoplastic development at acidic pH (pH ~4), and unconjugated bile acids are involved in neoplastic development at more neutral pH (~6). Bile acids (at the appropriate pH) are potent DNA-damaging agents, due to the induction of ROS (reactive oxygen species), which are mainly induced by bile-induced damage to mitochondrial membranes, allowing leakage of ROS into the cytosol. These ROS also induce pro-survival signalling pathways [e.g. via PKC (protein kinase C)-dependent NF-kappaB (nuclear factor kappaB) activity]. Interestingly, NOS (nitric oxide synthase), through induction of NO may exacerbate this NF-kappaB activity and form a positive-feedback loop to amplify the activation of NF-kappaB by deoxycholic acid in particular. This combination of induced DNA damage and cell survival by bile acids is of major importance in neoplasia. Antioxidants and the tertiary bile acid UDCA (ursodeoxycholic acid) can block bile-induced DNA damage and bile-induced NF-kappaB activity, and should be considered in chemopreventative strategies.

Bile acids modulate the Golgi membrane fission process via a protein kinase Cη and protein kinase D-dependent pathway in colonic epithelial cells

Deoxycholic acid (DCA) is a secondary bile acid that modulates signalling pathways in epithelial cells. DCA has been implicated in pathogenesis of colon carcinoma, particularly by activation of the protein kinase C (PKC) pathway. Ursodeoxycholic acid (UDCA), a tertiary bile acid, has been observed to have chemopreventive effects. The aim of this study was to investigate the effect of DCA and UDCA on the subcellular localization and activity of PKCeta and its downstream effects on Golgi structure in a colon cancer cell model. PKCeta expression was localized to the Golgi in HCT116 colon cancer cells. DCA induced fragmentation of the Golgi in these cells following activation of PKCeta and its downstream effector protein kinase D (PKD). Pretreatment of cells with UDCA or a glucocorticoid, dexamethasone, inhibited DCA-induced PKCeta/PKD activation and Golgi fragmentation. Knockdown of glucocorticoid receptor (GR) expression using small interfering RNA or inhibition using the GR antagonist mifepristone attenuated the inhibitory effect of UDCA on Golgi fragmentation. Elevated serum and faecal levels of DCA have been previously reported in patients with ulcerative colitis (UC) and colon cancer. Analysis of Golgi architecture in vivo using tissue microarrays revealed Golgi fragmentation in UC and colorectal cancer tissue. We have demonstrated that DCA can disrupt the structure of the Golgi, an organelle critical for normal cell function. Inhibition of this DCA-induced Golgi fragmentation by UDCA was mediated via the GR. This represents a potential mechanism of observed chemopreventive effects of UDCA in benign and malignant disease of the colon.

Abstract A32: Proteomic signatures of deoxycholic acid and ursodeoxycholic acid treated human colon cancer cells

Abstract A32 Dietary factors play an important role in the pathogenesis of colorectal cancer (CRC), due in part to the influence of the bile acids. While the secondary bile acid, deoxycholic acid (DCA), has been associated with tumor promotion, one tertiary bile acid in particular, ursodeoxycholic acid (UDCA), has been demonstrated to exert chemopreventive properties in the colon. Although its efficacy against high-grade adenomas has already been demonstrated in phase III clinical trials, mechanisms by which UDCA suppresses colon tumorigenesis remain unclear. In order to gain further insight into the tumor modifying properties of the bile acids, we compared the effects of DCA and UDCA on human colon tumor cells (CaCo-2) using proteomic analyses. In the following study, CaCo-2 cells grown at 70% confluence were treated with either 10 µM deoxycholic acid (DCA) or 1 mM ursodeoxycholic acid (UDCA) for 72 hours. For the proteomic analysis, protein was first fractionated by PF2D isochromatofocusing on a Beckman Coulter ProteomeLab PF2D platform. Fractions corresponding to a linear gradient between pH 8.0 and 4.0 were collected and processed through an automated autoloader for further separation by HPRP-PF2D reverse phase chromatography. Two dimensional protein expression maps displaying isoelectric point (pI) versus hydrophobicity were generated by the ProteoView/DeltaVue software package. Pairwise analysis of these complex protein chromatograms demonstrated common peak profiles (signatures) as well as unique peaks (fingerprints) characteristic of UDCA or DCA treatment. One of these protein peaks that exhibited a differential expression pattern between the two drug treatments was identified by MALDI and tandem mass spectrometry sequencing as heat-shock protein 60 (Hsp60). Furthermore, this protein was found to be less oxidized upon treatment with UDCA, suggesting the attenuation of oxidative stress present in cancer cells. Further analyses on the role of Hsp60 as well as resultant diminished oxidative stress will enable the discovery of potential mechanisms by which DCA and UDCA control cellular pathways. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A32.

Kolestazda Safra Asidlerinin Etki Mekanizmaları: Ursodeoksikolik Asidin Tedavideki Yeri

Cytotoxic effects of bile acids are related to their chemical structure and concentration The degree of cytotoxicity increases proportionally with the degree of hydrophobicity Thus the lithocholic acid is accepted to be most cystotoxic followed by deoxycholic acid chenodeoxycholic acid cholic acid and ursodeoxycholic acid Bile acids can induce bile duct injury and ductular proliferation fibrosis and cirrhosis Ursodeoxycholic acid which is a tertiary bile acid reduces intestinal bile acid reabsorption induces an increase in the hepatic bile acid excretion stabilizes cells membranes and interferes with the function of cystotoxic T lymphocytes Thus it is found to be beneficial in a variety of cholestatic diseases of adults and children Key words: Cholestasis Bile Acids Ursodeoxycholic Acid

Ursodeoxycholate–Induced Hypercholeresis in Cirrhotic Rats: Further Evidence for Cholehepatic Shunting

The aim of the investigation was to explore whether ursodeoxycholate, a tertiary bile acid with potential for treatment of chronic cholestasis in cirrhotic liver disease, has the same physiological effects in cirrhotic as in normal rats. Furthermore, we wanted to investigate whether ductular proliferation, as it occurred in this situation, increases the bicarbonate stimulatory effect of ursodeoxycholate. Rats (n = 16) were rendered cirrhotic by continuous exposure to phenobarbital-carbon tetrachloride; untreated animals (n = 13) served as controls. In cirrhotic rats in vivo, ursodeoxycholate (20 mumoles/min/kg) stimulated bile salt secretion and bile flow less than in controls. Nevertheless, the increment in ursodeoxycholate-induced biliary bicarbonate--the bicarbonate stimulatory potency--was increased by 29% in cirrhotic animals (0.55 +/- 0.08 mmol vs. 0.71 +/- 0.11 mmol; p < 0.05). This finding could be related to ductular proliferation because the volume fraction of bile ductules, determined stereologically, increased from 0.3% +/- 0.1% to 2.7% +/- 0.6% in cirrhotic rats (p < 0.005). To explore further the behavior of ductules during ursodeoxycholate stimulation, we carried out experiments in the in situ perfused rat liver. In the portally perfused organ, replacement of bicarbonate by tricine-acetate abolished ursodeoxycholate-induced hypercholeresis. In the dually perfused organ (perfusion of both portal vein and hepatic artery) perfusion of the hepatic artery with bicarbonate-containing buffer, ursodeoxycholate had a similar stimulatory effect as in vivo in both control and cirrhotic rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Ursodeoxycholate has no beneficial effect on liver function or histology in biliary cirrhosis in the rat

In different cholestatic conditions, the beneficial effects of the tertiary bile acid, ursodeoxycholate, have been described. It is unclear, however, whether ursodeoxycholate also affects the functional and structural alterations induced by chronic biliary obstruction. Therefore, we studied the effect of ursodeoxycholate (100 mg/kg/day) on microsomal function as assessed in vivo by the aminopyrine breath test, on portal hypertension and on the structural composition of the liver in rats with chronic (3-week) biliary obstruction. Hepatic composition was assessed stereologically. Ursodeoxycholate had no effect on any of the parameters measured. We conclude that this form of treatment does not affect advanced liver disease due to common bile duct obstruction. This finding supports one of the proposed mechanisms of action of ursodeoxycholate, namely that it interferes with the ileal absorption of more toxic endogenous bile salts.

Effects of ursodeoxycholic acid treatment on nutrition and liver function in patients with cystic fibrosis and longstanding cholestasis.

The prevalence of biliary and hepatic diseases is increasing in patients with cystic fibrosis as more of them reach adult life. There is no effective treatment or method of preventing cholestasis in cystic fibrosis, although beneficial effects have been ascribed to the tertiary bile acid, ursodeoxycholate, in other forms of chronic cholestasis. We evaluated prospectively the effects of a six month course of ursodeoxycholate (15-20 mg/kg per day) in eight, mostly adult, patients with cystic fibrosis and chronic cholestasis. Bile acid treatment improved inflammatory activity (average decrease in alanine aminotransferase, 60%, p less than 0.005) and cholestasis (alkaline phosphatase, 47%; p less than 0.01) in all patients. Quantitative liver function, measured by 45 minute sulphobromophthalein retention and by the 14C-aminopyrine breath test, improved in all patients while galactose elimination capacity showed a slight decrease. Patients' nutritional state improved as evidenced by a 1.8 kg weight gain and an increase in muscle mass suggested by a 26% increase in 24 hour urinary creatinine excretion. Steatorrhea was not affected by bile acid treatment. Ursodeoxycholic acid may be beneficial in the treatment of chronic cholestasis in cystic fibrosis by improving liver function and also the patient's nutritional state.

Intestinal Absorption of Bile Salts

Bile acids are secreted from the liver into the duodenum where they aid in the digestion and absorption of dietary lipids. Absorption of bile acids occurs through both ionic and nonionic diffusion in the jejunum and colon and through an active sodium ion-dependent carrier mechanism in the ileum. The prima, y bile acids synthesized in the liver can be converted by intestinal bacteria into secondary and tertiary bile acids. Bile acids may also be conjugated with glycine or taurine which results in an increase in the hydrophilicity and solubility of these compounds at physiological pH. The amount of passive diffusion of bile acids that occurs across the brush border membrane along the length of the entire intestine depends upon the ratio of ionized to nonionized bile acids coupled with the bile salt concentration and the individual permeability coefficients of monomers. Active transport of both conjugated and nonconjugated species of bile acids depends upon the presence of a single negative charge on the side chain. Maximal transport rates for bile acids are related to the number of hydroxyl groups present while the Michaelis-Menten constant for transport is dependent upon whether or not the bile acid is conjugated. Although active uptake of bile acids from the ileum has been considered the major route for bile salt absorption in the small intestine, the mechanism may actually be responsible for only a small proportion of the total bile acid pool absorbed from the lumen.