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Background & Aims Bile acid transporters maintain bile acid homeostasis. Little is known about the functions of some transporters in cholestasis or their regulatory mechanism. We investigated the hepatic expression of solute carrier organic anion transporter family member 3A1 (SLCO3A1, also called OATP3A1) and assessed its functions during development of cholestasis. Methods We measured levels of OATP3A1 protein and messenger RNA and localized the protein in liver tissues from 22 patients with cholestasis and 21 patients without cholestasis, using real-time quantitative polymerase chain reaction, immunoblot, and immunofluorescence analyses. We performed experiments with Slco3a1-knockout and C57BL/6J (control) mice. Mice and Sprague-Dawley rats underwent bile duct ligation (BDL) or a sham operation. Some mice were placed on a 1% cholic acid (CA) diet to induce cholestasis or on a control diet. Serum and liver tissues were collected and analyzed; hepatic levels of bile acids and 7-α-C4 were measured using liquid chromatography/mass spectrometry. Human primary hepatocytes and hepatoma (PLC/PRF/5) cell lines were used to study mechanisms that regulate OATP3A1 expression and transport. Results Hepatic levels of OATP3A1 messenger RNA and protein were significantly increased in liver tissues from patients with cholestasis and from rodents with BDL or 1% CA diet–induced cholestasis. Levels of fibroblast growth factor 19 (FGF19, FGF15 in rodents) were also increased in liver tissues from patients and rodents with cholestasis. FGF19 signaling activated the Sp1 transcription factor and nuclear factor κB to increase expression of OATP3A1 in hepatocytes; we found binding sites for these factors in the SLCO3A1 promoter. Slco3a1-knockout mice had shorter survival times and increased hepatic levels of bile acid, and they developed more liver injury after the 1% CA diet or BDL than control mice. In hepatoma cell lines, we found OATP3A1 to take prostaglandin E2 and thyroxine into cells and efflux bile acids. Conclusions We found levels of OATP3A1 to be increased in cholestatic liver tissues from patients and rodents compared with healthy liver tissues. We show that OATP3A1 functions as a bile acid efflux transporter that is up-regulated as an adaptive response to cholestasis.

Bile acids, the major products of hepatic cholesterol catabolism, are indispensable for lipid absorption and cholesterol metabolism. Systemic bile acid metabolism is tightly regulated by various nuclear receptors, including farnesoid X receptor (FXR; Nat Rev Gastroenterol Hepatol 2014;11:55–67). In humans, approximately 500 mg of cholesterol are converted into bile acids every day via the classical pathway initiated by CYP7A1 or an alternative pathway through CYP7B1 (Nat Rev Drug Discov 2008;7:678–693). CYP8B1 is responsible for cholic acid synthesis (J Clin Invest 2002;110:1191–1200). FXR signaling regulates bile acid synthetic pathways by repressing both Cyp7a1 and Cyp8b1 expression. FXR-mediated small heterodimer partner (Shp) induction is a mechanism of the repression of Cyp7a1 expression. However, bile acid synthetic pathways are also regulated by SHP-independent mechanisms. In a recent article published in Cell Metabolism, de Aguiar Vallim et al discovered a novel FXR-regulated transcriptional repressor, MafG (V-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog G) as an important regulator of negative feedback mechanism of bile acids. MafG belongs to small MAF proteins, which is a bidirectional transcriptional regulator. On 1 hand, MafG interacts with transcription factors Nrf2 and HIF1α to increase their transcriptional activity (Biochim Biophys Acta 2008; 1783:1847–1856; FEBS Lett 2008;582:2357–2364). On the other hand, MafG binds to DNA as a homodimer (or sometimes as a heterodimer), which leads to transcriptional repression owing to a lack of transactivation domain (Mol Cell Biol 2006;26:4652–4663). To find the repression mechanism of bile acid synthesis genes by FXR, de Aguiar Vallim et al focused on transcriptional repressors, such as MafG. The ChIP-seq analysis of hepatic FXR from 2 different datasets determined four putative transcriptional repressor genes expressed in the liver MafG, cysteine-rich intestinal protein 2 (Crips2), zinc finger protein 385a (Zfp385a), and Shp. In vivo overexpression of MafG, introduced by injection of adenoviral vector, repressed the expression of Cyp8b1, but not Cyp7a1, in the liver. Crip2 or Zfp385a overexpression did not change either Cyp7a1 or Cyp8b1 expression. Interestingly, MafG overexpression changed the composition of bile acid pool, but not the pool size. The analysis of the hepatic gene profile showed the significant reductions in the expression of bile acid synthesis genes including Cyp7b1 and Cyp27a1 by MafG overexpression. In contrast, the loss-of-gene studies using antisense oligonucleotides or in MafG heterozygous mice showed increased expression of bile acid synthesis genes including Cyp8b1, Cyp7b1, and Cyp27a1, but not Cyp7a1. This confirms that MafG is a repressor of cholic acid synthesis as well as a key regulator of bile acid metabolism. Then, the study investigated to identify MafG binding sites (MAREs) in bile acid synthetic genes through MafG ChIP-seq analysis. The ChIP-seq analysis identified MAREs at loci for multiple bile acid synthetic genes, including Cyp8b1, Cyp27a1, Cyp7b1, Acox2, Akr1d1, Akr1c14, Ntcp, Hsd17b4, and Scp2, but not Cyp7a1. Taken together, the present study demonstrates that FXR signaling regulates bile acid synthesis gene expression through induction of a transcriptional repressor MafG, a novel FXR target gene. MafG binds to MAREs on the promoter and/or intronic regions of multiple bile acid synthetic genes and suppresses their transcription, thereby regulating the bile acid synthesis.

Background & Aims Fecal microbiota transplantation (FMT) is effective for treating recurrent Clostridioides difficile infection (CDI), but there are concerns about its long-term safety. Understanding the mechanisms of the effects of FMT could help us design safer, targeted therapies. We aimed to identify microbial metabolites that are important for C difficile growth. Methods We used a CDI chemostat model as a tool to study the effects of FMT in vitro. The following analyses were performed: C difficile plate counts, 16S rRNA gene sequencing, proton nuclear magnetic resonance spectroscopy, and ultra-performance liquid chromatography and mass spectrometry bile acid profiling. FMT mixtures were prepared using fresh fecal samples provided by donors enrolled in an FMT program in the United Kingdom. Results from chemostat experiments were validated using human stool samples, C difficile batch cultures, and C57BL/6 mice with CDI. Human stool samples were collected from 16 patients with recurrent CDI and healthy donors (n = 5) participating in an FMT trial in Canada. Results In the CDI chemostat model, clindamycin decreased valerate and deoxycholic acid concentrations and increased C difficile total viable counts and valerate precursors, taurocholic acid, and succinate concentrations. After we stopped adding clindamycin, levels of bile acids and succinate recovered, whereas levels of valerate and valerate precursors did not. In the CDI chemostat model, FMT increased valerate concentrations and decreased C difficile total viable counts (94% decrease), spore counts (86% decrease), and valerate precursor concentrations; concentrations of bile acids were unchanged. In stool samples from patients with CDI, valerate was depleted before FMT but restored after FMT. Clostridioides difficile batch cultures confirmed that valerate decreased vegetative growth, and that taurocholic acid was required for germination but had no effect on vegetative growth. Clostridioides difficile total viable counts were decreased by 95% in mice with CDI given glycerol trivalerate compared with phosphate buffered saline. Conclusions We identified valerate as a metabolite that is depleted with clindamycin and only recovered with FMT. Valerate is a target for a rationally designed recurrent CDI therapy.

Background & aims: Progressive familial intrahepatic cholestasis (PFIC) is characterized by pruritus, intrahepatic cholestasis, low serum γ-glutamyltransferase levels, and characteristic "Byler bile" on electron microscopy. Many patients require liver transplantation, but partial external biliary diversion (PEBD) has shown therapeutic promise. However, the effect of PEBD on liver morphology and bile composition has not been evaluated. Methods: We reviewed liver biopsy specimens from 3 children with low γ-glutamyltransferase PFIC before and after PEBD. Follow-up liver biopsies were performed 9–60 months after PEBD. Light and electron microscopic features were scored blindly. Biliary bile acid composition was analyzed by gas chromatography—mass spectrometry before and after PEBD in 1 patient and after PEBD in 2 patients. Results: Following PEBD, all patients improved clinically. Preoperative biopsy specimens showed characteristic features of PFIC, including portal fibrosis, chronic inflammation, cholestasis, giant cell transformation, and central venous mural sclerosis. Ultrastructural findings included coarse, granular canalicular Byler bile, effaced canalicular microvilli, and proliferative pericanalicular microfilaments. Following diversion, histology showed almost complete resolution of cholestasis, portal fibrosis, and inflammation with resolution of ultrastructural abnormalities. Biliary bile acids before PEBD consisted predominantly of cholic acid. After PEBD, the proportion of chenodeoxycholic acid increased significantly in 1 patient and was above the PFIC range in a second patient. Conclusions: The resolution of hepatic morphologic abnormalities following PEBD supports PEBD as an effective therapy for PFIC. The improved biliary bile acid composition suggests enhanced bile acid secretion after PEBD, perhaps by induction of alternative canalicular transport proteins.

ObjectiveBile acids are regulators of lipid and glucose metabolism, and modulate inflammation in the liver and other tissues. Primary bile acids such as cholic acid and chenodeoxycholic acid (CDCA) are produced in the liver, and converted into secondary bile acids such as deoxycholic acid (DCA) and lithocholic acid by gut microbiota. Here we investigated the possible roles of bile acids in non-alcoholic fatty liver disease (NAFLD) pathogenesis and the impact of the gut microbiome on bile acid signalling in NAFLD.DesignSerum bile acid levels and fibroblast growth factor 19 (FGF19), liver gene expression profiles and gut microbiome compositions were determined in patients with NAFLD, high-fat diet-fed rats and their controls.ResultsSerum concentrations of primary and secondary bile acids were increased in patients with NAFLD. In per cent, the farnesoid X receptor (FXR) antagonistic DCA was increased, while the agonistic CDCA was decreased in NAFLD. Increased mRNA expression for cytochrome P450 7A1, Na+-taurocholate cotransporting polypeptide and paraoxonase 1, no change in mRNA expression for small heterodimer partner and bile salt export pump, and reduced serum FGF19 were evidence of impaired FXR and fibroblast growth factor receptor 4 (FGFR4)-mediated signalling in NAFLD. Taurine and glycine metabolising bacteria were increased in the gut of patients with NAFLD, reflecting increased secondary bile acid production. Similar changes in liver gene expression and the gut microbiome were observed in high-fat diet-fed rats.ConclusionsThe serum bile acid profile, the hepatic gene expression pattern and the gut microbiome composition consistently support an elevated bile acid production in NAFLD. The increased proportion of FXR antagonistic bile acid explains, at least in part, the suppression of hepatic FXR-mediated and FGFR4-mediated signalling. Our study suggests that future NAFLD intervention may target the components of FXR signalling, including the bile acid converting gut microbiome.

The rate of 12alpha-hydroxylation of bile acid intermediates is believed to determine the ratio of cholic acid (CA) to chenodeoxycholic acid (CDCA) biosynthesis and the overall hydrophobicity of the bile acid pool. The aim of this study was to determine the effects of the level of expression of sterol 12alpha-hydroxylase (CYP8b1) and cholesterol 7alpha-hydroxylase (CYP7a1) on rates of CA biosynthesis and bile acid pool composition.Expression of CYP8b1 and CYP7a1 was accomplished through infection of primary rat hepatocytes (PRH) or intact male SD rats with replication-defective recombinant adenoviruses encoding either CYP8b1 or CYP7a1.Increased expression of CYP7a1 over basal levels in PRH dramatically increased bile acid biosynthesis (586% +/- 82%, P0.001) but did not alter the ratio of CA to CDCA. Conversely, increased expression of CYP8b1 in vitro had no significant effect on the rates of total bile acid synthesis but significantly increased (4.1-fold) the rates of CA biosynthesis, resulting in an increase in the CA-CDCA ratio from 1:6.6 to 2.8:1. In whole rats, increased CYP8b1 expression over basal levels markedly increased the CA in the bile acid pool from 36% +/- 3.4% to 50% +/- 2.9% in 5 days. CDCA and its muricholic acid derivatives decreased from 64% +/- 3.4% to 50% +/- 2.9%.Increased expression of CYP8b1 led to a marked increase in CA biosynthesis both in PRH and in whole animals. CYP8b1 is capable of 12alpha-hydroxylating bile acid intermediates from both the classic and acidic pathways.

Background & Aims: The mechanism for abnormal hepatic bile acid transport was investigated in an 18-month-old Amish boy who presented with pruritus, poor growth, and severe bleeding episodes. Serum bilirubin, γ-glutamyltranspeptidase, and cholesterol levels were normal, but prothrombin time and partial thromboplastin time were prolonged and bone alkaline phosphatase level was elevated. Methods and Results: Cholic acid plus chenodeoxycholic acid levels measured by capillary gas-chromatography were 32 times higher than control in serum (34.7 vs. 1.1 ± 0.4 μg/dL) but were not detected in liver and were reduced in gallbladder bile. Treatment with ursodiol, a more hydrophilic bile acid, improved pruritus, produced 37% weight gain, and after 2 years reduced serum primary bile acid concentrations about 85%, while accounting for 71% of serum and 24% of biliary bile acid conjugates. On ursodiol therapy, hepatic bile acid synthesis was enhanced 2-fold compared with controls, and microscopy revealed chronic hepatitis without cholestasis. Three younger sisters with elevated serum bile acids responded positively to ursodiol. Microsatellite markers for the FIC1 (gene for Byler's disease) region in these 4 children were inconsistent with linkage to FIC1 . Conclusions: Conjugated cholic acid and chenodeoxycholic acid were synthesized in the liver and secreted into bile but could not reenter the liver from portal blood and accumulated in serum. In contrast, unconjugated ursodiol entered the liver and was conjugated and secreted into bile. Thus, the enterohepatic circulation of all conjugated bile acids was interrupted at the hepatic sinusoidal basolateral membrane. Unconjugated ursodiol bypassed the hepatic uptake block to enlarge the biliary and intestinal bile acid pools. A mutation in FIC1 recognized among the Amish and linkage of the disorder to FIC1 were excluded. GASTROENTEROLOGY 2000;119:188-195

BACKGROUND & AIMS: Bile acids exert cellular and molecular effects in the liver, but little is known about tissue concentrations. The aim of this study was to characterize bile acid composition in human and rat liver tissue and hepatocyte nuclei and examine the effects of experimental cholestasis and bile acid administration. METHODS: Bile acids were measured by gas chromatography-mass spectrometry. RESULTS: Liver tissue concentrations of sham-operated rats were 130.8 +/- 21.3 nmol/g, representing 2%-4% of the bile acid pool; cholic and delta 22- beta-muricholic acids were the major bile acids identified. Concentrations increased 7-8-fold with bile duct ligation; deoxycholate and hyodeoxycholate disappeared. Lithocholate concentrations were higher in ligated rats (6.4 +/- 0.4 vs. 3.9 +/- 0.5 nmol/g for sham- operated). Total bile acid concentrations in human liver tissue were 61.6 +/- 29.7 nmol/g and comprised mainly chenodeoxycholic and cholic acids. Concentrations were higher during ursodeoxycholate or tauroursodeoxycholate administration (157.2 +/- 45.6 and 161.6 +/- 43.4 nmol/g, respectively), and liver tissue was enriched 30% in ursodeoxycholate at the expense of hydrophobic bile acids. Bile acids were identified in rat hepatic nuclei (50-110 pmol/4 x 10(7) nuclei), accounting for > 0.1% of liver tissue levels. CONCLUSIONS: Human and rat liver tissue bile acid concentrations are low, increase with bile acid administration or bile duct ligation, and account for only a small fraction of the bile acid pool. (Gastroenterology 1997 Jan;112(1):226-35)

Background & Aims Bile salts inhibit their own production by inducing the nuclear receptor small heterodimer partner (SHP) (encoded by NR0B2 ), which contributes to repression of the gene encoding cholesterol 7α-hydroxylase ( CYP7A1 ), a key enzyme for the control of bile salt synthesis. On the other hand, bile salts stimulate hepatic synthesis of nitric oxide. We investigated the role of nitric oxide signaling in the control of CYP7A1 expression and the involvement in this process of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which participates in intracellular propagation of nitric oxide signals. Methods We studied the effects of inhibitors of nitric oxide synthesis (L-NG-nitroarginine methyl ester [L-NAME]) or protein nitrosylation (via dithiothreitol) on bile salt homeostasis in male Wistar rats placed on a cholate-rich diet for 5 days and in cultured primary hepatocytes. S-nitrosylation of GAPDH was assessed using a biotin-switch assay. Interacions of SHP with other proteins and with the Cyp7a1 promoter sequence were studied using immunoprecipitation and chromatin immunoprecipitation (ChIP) assays. We reduced the GAPDH levels in H35 cells with small interfering RNAs. GAPDH nitrosylation was assessed in normal and cholestatic rat and human livers. Results Rats placed on cholate-rich diets and given L-NAME had increased intrahepatic and biliary levels of bile salts, and deficiency in repression of CYP7A1 (at the messenger RNA and protein levels) in liver tissue, despite preserved induction of SHP. In cultured hepatocytes, L-NAME or dithiothreitol blocked cholate-induced down-regulation of CYP7A1 without impairing SHP up-regulation. In hepatocytes, cholate promoted S-nitrosylation of GAPDH and its translocation to the nucleus, accompanied by S-nitrosylation of histone deacetylase 2 (HDAC2) and Sirtuin 1 (SIRT1), deacetylases that participate, respectively, in the formation of Cyp7a1 and Shp repressor complexes. Knockdown of GAPDH prevented repression of CYP7A1 by cholate, and blocking nuclear transport of nitrosylated GAPDH reduced cholate-induced nitrosylation of HDAC2 and SIRT1; this effect was accompanied by abrogation of Cyp7a1 repression. Cholate induced binding of SHP to HDAC2 and its recruitment to the Cyp7a1 promoter; these processes were inhibited by blocking nitric oxide synthesis. Levels of nitrosylated GAPDH and nitrosylated HDAC2 were increased in cholestatic human and rat livers reflecting increased concentrations of bile salts in these conditions. Conclusions In rat liver, excess levels of bile salts activate a GAPDH-mediated transnitrosylation cascade that provides feedback inhibition of bile salt synthesis.

Background & Aims Sirtuin 1 (SIRT1), the most conserved mammalian oxidized nicotinamide adenine dinucleotide–dependent protein deacetylase, is an important metabolic sensor in many tissues. However, little is known about its role in the small intestine, which absorbs and senses nutrients. We investigated the functions of intestinal SIRT1 in systemic bile acid and cholesterol metabolism in mice. Methods SIRT1 was specifically deleted from the intestines of mice using the flox-Villin-Cre system (SIRT1 iKO mice). Intestinal and hepatic tissues were collected, and bile acid absorption was analyzed using the everted gut sac experiment. Systemic bile acid metabolism was studied in SIRT1 iKO and flox control mice placed on standard diets, diets containing 0.5% cholic acid or 1.25% cholesterol, or lithogenic diets. Results SIRT1 iKO mice had reduced intestinal farnesoid X receptor (FXR) signaling via hepatocyte nuclear factor 1α (HNF-1α) compared with controls, which reduced expression of the bile acid transporter genes Asbt and Mcf2l (encodes Ost) and absorption of ileal bile acids. SIRT1 regulated HNF-1α/FXR signaling partially through dimerization cofactor of HNF-1a (Dcoh2) Dcoh2, which increases dimerization of HNF-1α. SIRT1 was found to deacetylate Dcoh2, promoting its interaction with HNF-1α and inducing DNA binding by HNF-1α. Intestine-specific deletion of SIRT1 increased hepatic bile acid biosynthesis, reduced hepatic accumulation of bile acids, and protected animals from liver damage from a diet high in levels of bile acids. Conclusions Intestinal SIRT1, a key nutrient sensor, is required for ileal bile acid absorption and systemic bile acid homeostasis in mice. We delineated the mechanism of metabolic regulation of HNF-1α/FXR signaling. Reagents designed to inhibit intestinal SIRT1 might be developed to treat bile acid–related diseases such as cholestasis.

BACKGROUND & AIMS: Excessive deoxycholic acid (DCA) in the bile acid pool with cholesterol supersaturation of bile is prevalent in patients with cholesterol gallstones (CGs). This study examined whether this is caused by enhanced conversion of cholic acid (CA) to DCA by intestinal bacteria. METHODS: Ten patients with CGs with DCA excess (DCA/CA pool ratio, > 1.5) and 10 patients with low DCA (ratio, RESULTS: Patients with DCA excess and low DCA differed (P CONCLUSIONS: Increased CA-7 alpha-dehydroxylation activity of the intestinal microflora may be an important factor for CG formation or growth in these patients. (Gastroenterology 1996 Dec;111(6):1611-20)

Background & Aims Alterations in the gastrointestinal microbiota have been associated with metabolic diseases. However, little is known about host factors that induce changes in gastrointestinal bacterial populations. We investigated the role of bile acids in this process because of their strong antimicrobial activities, specifically the effects of cholic acid administration on the composition of the gut microbiota in a rat model. Methods Rats were fed diets supplemented with different concentrations of cholic acid for 10 days. We used 16S ribosomal RNA gene clone library sequencing and fluorescence in situ hybridization to characterize the composition of the cecal microbiota of the different diet groups. Bile acids in feces, organic acids in cecal contents, and some blood parameters were also analyzed. Results Administration of cholic acid induced phylum-level alterations in the composition of the gut microbiota; Firmicutes predominated at the expense of Bacteroidetes. Cholic acid feeding simplified the composition of the microbiota, with outgrowth of several bacteria in the classes Clostridia and Erysipelotrichi. Externally administered cholic acid was efficiently transformed into deoxycholic acid by a bacterial 7α-dehydroxylation reaction. Serum levels of adiponectin decreased significantly in rats given the cholic acid diet. Conclusions Cholic acid regulates the composition of gut microbiota in rats, inducing similar changes to those induced by high-fat diets. These findings improve our understanding of the relationship between metabolic diseases and the composition of the gastrointestinal microbiota.

Background: The hepatic secretion of supersaturated bile and gallbladder stasis are key events in cholesterol gallstone formation. The therapeutic value of cisapride, a prokinetic agent, was assessed in ground squirrels on a 1% cholesterol diet. Methods: Biliary lipid secretion was measured directly and bile salt pool size assessed by isotope dilution ([ 14 C]cholic acid). Gallbladder contraction was measured in vitro in response to cholecystokinin (CCK). Results: Cholesterol-fed animals had a combined hepatic secretory defect (a 53% decrease in bile salt secretion and also a 31% increase in cholesterol secretion). Adding cisapride restored bile salt secretion to control levels but did not affect cholesterol secretion. In cholesterol-fed animals, the cholesterol saturation index of gallbladder bile more than doubled and cholesterol crystals developed; cisapride markedly reduced cholesterol saturation, thus preventing crystal formation. Gallbladder contractility, measured in vitro in response to CCK, decreased 23% in animals on the 1% cholesterol diet; cisapride restored the CCK dose-response curve to normal. The bile salt pool as assessed by isotope dilution was similar in all groups. Conclusions: Thus, lithogenic bile develops in this model because of reduced bile salt secretion and increased cholesterol secretion. Cisapride renders biliary lipid composition towards normal by enhancing gallbladder (and possibly intestinal) motility and cycling of the bile salt pool, thereby increasing bile salt secretion.

Epidemiological and animal studies have suggested that the secondary bile acid deoxycholic acid is cocarcinogenic in colorectal cancer, but this hypothesis was not confirmed by case-control studies investigating fecal bile acids.Individual serum bile acid concentrations were investigated in 25 men and 25 women with colorectal adenomas and in an equal number of age- and sex-matched controls by gas-liquid chromatography.Deoxycholic acid levels were significantly higher in the sera of men with colorectal adenomas (1.70 +/- 0.59 vs. 1.16 +/- 0.39 mumol/L, P0.0005) and in a combined analysis of both sexes (1.47 +/- 0.78 vs. 1.08 +/- 0.39 mumol/L, P0.0025). Six- and 12-month follow-up measurements of deoxycholic acid concentrations in a subgroup of 22 men and 17 women showed higher serum levels in men with adenomas, indicating that measurement of deoxycholic acid concentration may be a reliable parameter to investigate its pathogenetic role in colonic neoplasia.The data of this study support the hypothesis that deoxycholic acid may play a role in the pathogenesis of colorectal cancer.

The regulatory and secretory properties of cholylsarcosine (C-sar), a synthetic conjugated bile acid analogue that resists deconjugation and dehydroxylation, were compared with those of the natural conjugates of cholic acid. After continuous intraduodenal infusion of cholylsarcosine (C-sar), cholyltaurine (C-tau), or cholylglycine (C-gly) at 36 mumol/100 g.h, the infused bile acid in each case became the predominant biliary bile acid. After 48 hours, infusion of C-sar, C-tau, and C-gly suppressed the activity of cholesterol 7 alpha-hydroxylase (C7 alpha H; rate-limiting for bile acid synthesis) by 65%, 78%, and 92%, respectively, compared with biliary fistula controls. After C-sar infusion, levels of C7 alpha H protein, messenger RNA, and transcriptional activity were depressed to the same extent as specific activity, indicating that C-sar, like C-tau, down-regulates C7 alpha H principally at the level of gene transcription. All three bile acids also suppressed activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (rate-limiting for cholesterol synthesis). Both short- and long-term, the three cholyl conjugates caused similar increases in bile flow and in biliary secretion of cholesterol and phospholipid. It is concluded that in the rat, cholyl conjugates per se can suppress cholesterol and bile acid biosynthesis without prior conversion to deoxycholate. The effects of C-sar on hepatic cholesterol and bile acid synthesis as well as on induced bile flow and biliary lipid secretion are essentially identical to those of the naturally occurring cholyl conjugates.

To test whether bile acids interact with mast cells, dilute, aqueous solutions of five pure unconjugated natural bile acids and their corresponding glycine or taurine conjugates were incubated with murine PT-18 cells (a mast cell line functionally and cytochemically similar to mucosal mast cells) or with freshly isolated rat peritoneal mast cells. Bile acid solutions ranged in concentration from 0.3 to 10 mmol/L; histamine release was assessed by a fluorimetric assay, and cell lysis by cytosolic enzyme (lactate dehydrogenase) release. Lipophilic, dihydroxy bile acids (chenodeoxycholic acid and deoxycholic acid as well as their glycine and taurine conjugates) caused histamine release in a dose-related manner; cholic acid and its conjugates caused much less or no histamine release. Two hydrophilic bile acids (ursodeoxycholic acid and ursocholic acid and their conjugates) were virtually devoid of activity. Histamine release, which was independent of extracellular Ca2+, occurred at 0.3 mmol/L, well below the critical micellization concentration. For a given concentration, unconjugated bile acids and glycine-conjugated bile acids induced more histamine release than taurine-conjugated bile acids; maximal release was observed at 3 mmol/L for lipophilic, dihydroxy bile acids. To test whether bile acids could also cause histamine release from cutaneous mast cells in vivo, rats were injected intradermally with bile acid solutions and histamine release assessed by capillary leakage of Evan's blue dye. Cutaneous blueing was greater with cytotoxic bile acids, chenodeoxycholyglycine or deoxycholylglycine, than with ursodeoxycholylglycine and was inhibited by prior antihistamine treatment. Histamine release correlated highly and positively with lipophilicity and with bile acid surface activity. It was concluded that lipophilic but not hydrophilic bile acids possess concentration-dependent cytotoxicity toward mast cells causing histamine release, that unconjugated and glycine-conjugated bile acids are more potent than taurine-conjugated bile acids, and that mast cell histamine release is highly correlated with lipophilicity of bile acids as well as their surface activity.

Obesity is a condition associated with an increased frequency of gallstone disease. This study attempted to evaluate the comparative effects of two gallstone-dissolving agents, chenodeoxycholic acid and ursodeoxycholic acid, on bile acid metabolism and biliary lipid secretion in obese subjects in order to identify the bile acid of choice in preventing and treating gallstone disease in obesity. Twenty obese subjects (greater than 120% ideal body wt) were randomly treated with ursodeoxycholic acid (10 mg.kg-1.day-1.1 mo-1) and then with chenodeoxycholic acid (15 mg.kg-1.day-1.1 mo-1) or with chenodeoxycholic acid first and then with ursodeoxycholic acid. Patients 1-10 were studied while eating an unrestricted weight-maintenance diet, whereas patients 11-20 were eating a 1080-kcal/d hypocaloric diet. Biliary lipid composition, cholesterol saturation index, and biliary bile acid pattern were evaluated in all subjects before and after each treatment period; in subjects 6-10 and 16-20, biliary lipid secretion rates and bile acid pool size were also evaluated. Both ursodeoxycholic acid and chenodeoxycholic acid decreased cholesterol outputs and cholesterol saturation index. However, during the weight-maintenance period the decrease induced by chenodeoxycholic acid was not significant. Biliary cholesterol outputs and cholesterol saturation index were always lower during ursodeoxycholic acid administration than during chenodeoxycholic acid therapy. Ursodeoxycholic acid levels during ursodeoxycholic acid administration and chenodeoxycholic acid levels during chenodeoxycholic acid administration increased in bile to 50% and 77%, respectively, of total bile acid levels. Bile acid pool size remained unchanged during chenodeoxycholic acid administration and was significantly reduced by ursodeoxycholic acid administration during the weight-reduction period. In conclusion, ursodeoxycholic acid in obese subjects seems more effective than chenodeoxycholic acid, at least during weight maintenance, in reducing cholesterol saturation of bile. This effect is related to a significant decrease of biliary cholesterol output.

Background & Aims Hyperhomocysteinemia is often associated with liver and metabolic diseases. We studied nuclear receptors that mediate oscillatory control of homocysteine homeostasis in mice. Methods We studied mice with disruptions in Nr0b2 (called small heterodimer partner [SHP]-null mice), betaine-homocysteine S-methyltransferase ( Bhmt ), or both genes (BHMT-null/SHP-null mice), along with mice with wild-type copies of these genes (controls). Hyperhomocysteinemia was induced by feeding mice alcohol (National Institute on Alcohol Abuse and Alcoholism binge model) or chow diets along with water containing 0.18% DL-homocysteine. Some mice were placed on diets containing cholic acid (1%) or cholestyramine (2%) or high-fat diets (60%). Serum and livers were collected during a 24-hour light−dark cycle and analyzed by RNA-seq, metabolomic, and quantitative polymerase chain reaction, immunoblot, and chromatin immunoprecipitation assays. Results SHP-null mice had altered timing in expression of genes that regulate homocysteine metabolism compared with control mice. Oscillatory production of S-adenosylmethionine, betaine, choline, phosphocholine, glyceophosphocholine, cystathionine, cysteine, hydrogen sulfide, glutathione disulfide, and glutathione, differed between SHP-null mice and control mice. SHP inhibited transcriptional activation of Bhmt and cystathionine γ-lyase by FOXA1. Expression of Bhmt and cystathionine γ-lyase was decreased when mice were fed cholic acid but increased when they were placed on diets containing cholestyramine or high-fat content. Diets containing ethanol or homocysteine induced hyperhomocysteinemia and glucose intolerance in control, but not SHP-null, mice. In BHMT-null and BHMT-null/SHP-null mice fed a control liquid, lipid vacuoles were observed in livers. Ethanol feeding induced accumulation of macrovesicular lipid vacuoles to the greatest extent in BHMT-null and BHMT-null/SHP-null mice. Conclusions Disruption of Shp in mice alters timing of expression of genes that regulate homocysteine metabolism and the liver responses to ethanol and homocysteine. SHP inhibits the transcriptional activation of Bhmt and cystathionine γ-lyase by FOXA1.

Background & Aims: The gel-forming mucins play an important role in the early stage of cholesterol gallstone formation. We investigated whether the gallbladder epithelial mucin encoded by mucin gene 1 (MUC1) influences susceptibility to gallstones. Methods: Gallbladder motility and cholesterol absorption, gallstones and expression of the mucin genes in gallbladders, and secretion rates and compositions of biliary lipids were determined in male C57BL/6J mice transgenic for the human MUC1 gene (MUC1.Tg) and wild-type mice before (day 0, on chow) and at 4 weeks on a lithogenic diet containing 1% cholesterol and 0.5% cholic acid. Results: On chow, expression levels of the gallbladder mucin genes were essentially similar between MUC1.Tg and wild-type mice. The lithogenic diet induced 3-fold higher expression levels of Muc1, Muc3, Muc4, Muc5ac, and Muc5b messenger RNA in MUC1.Tg mice compared with wild-type mice. Gallbladder cholesterol absorption and size were significantly greater in MUC1.Tg mice than in wild-type mice regardless of whether the chow or the lithogenic diet was fed. Gallbladder emptying in response to exogenously administered cholecystokinin-8 was significantly reduced in MUC1.Tg mice but not in wild-type mice. At 4 weeks on the lithogenic diet, mucin accumulation was found in all MUC1Tg mice and in 60% of wild-type mice. Consequently, these alterations greatly accelerated cholesterol crystallization and gallstone formation in MUC1.Tg mice. However, biliary lipid secretion rates and cholesterol saturation indices of gallbladder biles were comparable in MUC1.Tg and wild-type mice. Conclusions: Increased gallbladder epithelial MUC1 mucin enhances cholelithogenesis by promoting gallbladder cholesterol absorption and impairing gallbladder motility in mice.

Background & Aims: 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. Methods: 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 (4β-hydroxy cholesterol, 4β-OH-C) and bile acid synthesis (7α-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. Results: 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. Conclusions: 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.

Background & Aims: Inborn errors of bile acid metabolism may present as neonatal cholestasis and fat-soluble vitamin malabsorption or as late onset chronic liver disease. Our aim was to fully characterize a defect in bile acid synthesis in a 2-week-old African-American girl presenting with coagulopathy, vitamin D and E deficiencies, and mild cholestasis and in her sibling, whose liver had been used for orthotopic liver transplantation (OLT). Methods: Bile acids were measured by mass spectrometry in urine, bile, serum, and feces of the patient and in urine from the unrelated recipient. Results: Liver biopsy specimens showed neonatal hepatitis with giant cell transformation and hepatocyte necrosis; peroxisomes were reduced in number. High concentrations of (25R)3α,7α,12α-trihydroxy-5β-cholestanoic acid in the urine, bile, and serum established a pattern similar to that of Zellweger syndrome and identical to the Alligator mississippiensis. Serum phytanic acid was normal, whereas pristanic acid was markedly elevated. Biochemical, MRI, and neurologic findings were inconsistent with a generalized defect of peroxisomal function and were unique. Analysis of the urine from the recipient of the deceased sibling's liver confirmed the same bile acid synthetic defect. A deficiency in 2-methylacyl-CoA racemase, which is essential for conversion of (25R)THCA to its 25S-isomer, the substrate to initiate peroxisomal β-oxidation to primary bile acids, was confirmed by DNA analysis revealing a missense mutation (S52P) in the gene encoding this enzyme. Long-term treatment with cholic acid normalized liver enzymes and prevented progression of symptoms. Conclusions: This genetic defect further highlights bile acid synthetic defects as a cause of neonatal cholestasis. GASTROENTEROLOGY 2003;124:217-232

role of bile acids in the progression of HE, mice were fed a diet enriched in the bile acid sequestrant cholestyramine, or the bile acids cholic acid (CA), deoxycholic acid (DCA) or ursodeoxycholic acid (UDCA) for 3 days prior to AOM injection. Subsequent assessment of neurological decline and liver damage was performed in these mice. The dose of bile acids given has previously been shown to not cause liver damage. Results: Total bile acids were increased in the brains of mice after AOM injection compared to controls. In parallel, fluorescently-derived bile acids were increased in the brains of AOM mice, suggesting that bile acids are entering from the periphery. Cholestyramine feeding reduced both the serum and brain bile acid levels and delayed the neurological decline associated with acute liver failure without any significant differences in liver damage. In contrast, feeding with CA and DCA worsened the AOM-induced neurological decline and significantly decreased the time taken to reach coma, with DCA having the greatest effect while UDCA feeding had no effect. Conclusions: These data indicate that circulating bile acids may be pathological in the brain during the course of HE, although precisely how they are dysregulating normal brain function is unknown. Strategies to minimize the total bile acid content in the serum may improve the outcome of patients with neurological symptoms associated with liver failure.

Prolonged large bowel transit, and an increase in the proportion of deoxycholic acid (DCA), have been implicated in the pathogenesis of cholesterol gallstones-including those developing in acromegalics treated with octreotide. However, there are few data on the effects of intestinal transit on bile acid kinetics.We therefore measured the kinetics of DCA and cholic acid (CA) using stable isotopes, serum sampling, and mass spectrometry. The results were related to mouth-to-caecum (MCTT) and large bowel transit times (LBTTs) in 4 groups of 8 individuals: (1) non-acromegalic controls, (2) acromegalics untreated with octreotide, (3) acromegalics on long-term octreotide, and (4) patients with constipation. Paired, before and during octreotide, studies were performed in 5 acromegalics.In the unpaired and paired studies, octreotide significantly prolonged MCTT and LBTT. In the paired studies, the octreotide-induced prolongation of LBTT caused an increase in the DCA input rate (6.4 +/- 2.8 to 12 +/- 2.6 micromol. kg. d, P0.05) and pool size (18 +/- 12 to 40 +/- 13 micromol/kg, P0.05), and a decrease in CA pool size (45 +/- 15 to 25 +/- 11 micromol/kg, P0.05). Furthermore, during octreotide treatment, the mean conversion of 13C-CA to 13C-DCA (micromoles) was greater (P0.05) on study days 3, 4, and 5. There were also positive linear relationships between LBTT and DCA input rate (r = 0.78), pool size (r = 0.82, P0.001), and a weak (r = -0.49) negative linear relationship between LBTT and CA pool size (P0.01).These data support the hypothesis that, by increasing DCA formation and absorption, prolongation of large bowel transit is a pathogenic factor in the formation of octreotide-induced gallstones.

(basolateral) and canalicular (apical) membranes. Tight junctions (zonula occludens) between hepatocytes maintain the polarity and prevent the mixing of canalicular and sinusoidal contents. 2,3 In addition to being the site of bile acid synthesis, the hepatocyte plays a key role in bile secretion, with the clearance of bile acids from the portal circulation and their transport to the canalicular lumen. The secretion of bile is maintained by two different mechanisms. One is bile acid dependent, with a linear correlation between the secretion of bile and that of bile acids. The other mechanism of bile secretion is bile acid independent and mainly results from the flux of inorganic ions (see Graf 4 for review). The proportion of the respective bile acid‐dependent and ‐independent components of bile secretion varies with animal species. However, in humans, bile secretion is mainly bile acid driven. Loss or alteration of bile secretion is associated with various cholestatic hepatobiliary disorders, such as primary sclerosing cholangitis, primary biliary cirrhosis, drug-induced cholestasis, autoimmune chronic active hepatitis, and alcoholic liver disease. The morbidity associated with chronic cholestatic conditions is significant and may necessitate liver transplantation. 5,6 Cholic acid (CA) and chenodeoxycholic acid (CDCA) are the two primary bile acids in humans. 7 After conjugation mainly to glycine (G) and taurine (T), bile acids are secreted into bile. More than 95% of the bile acid pool is reabsorbed from the intestine and transported to the liver bound mainly to albumin and, to a lesser

Ursodeoxycholic acid treatment (600 mg/day) was evaluated in a patient with asymptomatic primary sclerosing cholangitis. Serum levels of biliary enzymes decreased to normal ranges within 1 month's treatment and remained normal for 26 months. Serum chenodeoxycholic acid had been replaced by ursodeoxycholic acid, and hepatic copper metabolism, assessed by x-ray probe analysis, improved during the treatment. However, neither biliary tract sclerosis nor portal tract pathology changed with the treatment. These observations suggest that ursodeoxycholic acid protects the liver in primary sclerosing cholangitis by improving the metabolism of bile acid and copper.

Choledochal cysts are uncommon congenital or acquired lesions of the biliary tree. The incidence of biliary tract carcinoma in patients with choledochal cysts is 5–35 times greater than that of the general population. Factors responsible for the increased risk of carcinoma are unknown. The case of a young woman who underwent excision of a choledochal cyst 16 years after initial diagnosis and treatment by choledochocystduodenostomy is reported. Metaplasia of the epithelial lining of the cyst was found in the resected specimen. The relative composition of bile acids in cyst contents was as follows: lithocholate, 2%; deoxycholate, 88%; chenodeoxycholate, 5%; and cholate, 5%. Virtually all bile acids were recovered in unconjugated form. In contrast, the bile acid composition of hepatic bile was as follows: lithocholate, 0%; deoxycholate, 34%; chenodeoxycholate, 43%; and cholate, 23%. Bile acids were fully conjugated. These data suggest that stasis of bile within choledochal cysts contributes to bacterial overgrowth and generation of unconjugated secondary bile acids.

Background & Aims: 11β-Hydroxysteroid dehydrogenase (11β-OHSD) enzymes are responsible for the interconversion of active 11β-hydroxycorticosteroids into inactive 11-ketoglucocorticosteroids and by that mechanism regulate the intracellular access of the steroids to the cognate receptor. A down-regulation of the shuttle of active to inactive glucocorticoids enhances access of glucocorticosteroids to both the glucocorticoid and the mineralocorticoid receptors. In liver cirrhosis, enhanced mineralocorticoid and glucocorticoid effects are observed. We therefore investigated the impact of liver cirrhosis after bile duct ligation on the transcription and activity of 11β-OHSD1 and 11β-OHSD2 in the corresponding tissues. Methods: Messenger RNA from 11β-OHSD1 and 11β-OHSD2 was assessed by reverse-transcription polymerase chain reaction; activity was assessed by measuring the interconversion of corticosterone to dehydrocorticosterone. The effect of bile and bile salts was determined using COS-1 cells transfected with 11β-OHSD1 or 11β-OHSD2. Results: In liver tissue, the messenger RNA ratios of 11β-OHSD1 to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels and, in kidney tissue, the ratios of 11β-OHSD2 to GAPDH levels decreased after induction of liver cirrhosis. The 11β-OHSD activities were correspondingly reduced. Bile and individual bile salts inhibited 11β-OHSD1 and 11β-OHSD2 oxidative activity in transfected COS-1 cells. Conclusions: These findings indicate that in liver cirrhosis the mineralocorticoid and glucocorticoid receptor–protecting effects by the 11β-OHSD isoenzymes are down-regulated and that by the same mechanism the glucocorticoid and mineralocorticoid effects are enhanced. GASTROENTEROLOGY 1998;114:175-184

BACKGROUND & AIMS: Cholesterol degradation to bile acids occurs via "classic" or "alternative" bile acid biosynthetic pathways. The aim of this study was to assess the contributions of these two pathways to total bile acid synthesis in vivo. METHODS: Rats with biliary fistulas were infused with squalestatin for 24 and 48 hours; specific activities of cholesterol 7 alpha-hydroxylase (C7 alpha H) and sterol 27- hydroxylase (S27H) and rates of bile acid synthesis were determined. RESULTS: Continuous squalestatin infusion (15 micrograms/h) decreased C7 alpha H specific activities to 4% and 12% of paired biliary fistula controls at 24 and 48 hours, respectively (P < 0.05) without any changes in S27H specific activities (82% and 95% of controls). At 24 hours, bile acid synthesis decreased to 43% (P < 0.05) but returned to 87% at 48 hours (P = NS). Cholic acid synthesis decreased at 24 hours but returned to control levels at 48 hours. Similar changes in C7 alpha H, S27H, and bile acid synthesis were observed in primary rat hepatocytes after addition of squalestatin (1.0 mumol/L). CONCLUSIONS: In the face of persistent suppression of C7 alpha H and the classic pathway, an alternative pathway becomes a main pathway of bile acid synthesis capable of generating cholic and chenodeoxycholic acids. The observed induction of bile acid synthesis via an alternative pathway or pathways represents an important mechanism for maintenance of cholesterol homeostasis in the rat. (Gastroenterology 1997 Dec;113(6):1949-57)

Cholesterol feeding unexpectedly inhibits cholesterol 7 alpha-hydroxylase in rabbits. The aim of this study was to explore the mechanism.Twenty male New Zealand white rabbits were fed regular chow with and without 2% cholesterol for 10 days followed by 7 days of bile drainage. The activities of hepatic cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase that control bile acid synthesis in classic and alternative pathways were related to the size and composition of bile acid pool.After feeding cholesterol, plasma and hepatic cholesterol concentrations increased, the bile acid pool doubled (from 254 +/- 44 to 533 +/- 51 mg; P0.001), cholesterol 7 alpha-hydroxylase activity decreased 68% (P0.01), but sterol 27-hydroxylase activity increased 66% (P0.05) with increased cholic acid synthesis (P0.01). Bile drainage in the cholesterol-fed rabbits depleted the bile acid pool and stimulated down-regulated cholesterol 7 alpha-hydroxylase activity 11.4-fold (P0.001), although hepatic cholesterol remained elevated. Hepatic sterol 27-hydroxylase activity was unaffected.Feeding cholesterol increased hepatic cholesterol and stimulated sterol 27-hydroxylase and alternative bile acid synthesis, which expanded the bile acid pool and inhibited cholesterol 7 alpha-hydroxylase in rabbits. In distinction, hepatic sterol 27-hydroxylase was insensitive to changes in the bile acid pool.

The ileal sodium-dependent bile acid transporter reclaims bile acids from the intestinal lumen to preserve their enterohepatic recirculation. The present studies sought to determine the possible role of enteric bile acids in the molecular regulation of the apical bile acid transporter in rat ileal mucosa.Paired rats were fed a control diet or control diet plus cholic acid (1%) or ursodeoxycholic acid (1%) for 10 days. Other paired rats underwent biliary diversion for 72 hours, followed by intraduodenal infusion of taurocholate or fluid/electrolytes. Transporter protein, messenger RNA (mRNA), and activity were determined in the distal 15 cm of ileal mucosa.Transporter protein and mRNA levels in cholic acid-fed rats increased approximately threefold above levels in paired rats fed the control diet (P0.02). Similarly, sodium-dependent [3H]taurocholate uptake into membrane vesicles from cholic acid-fed rats increased twofold above uptake into vesicles from control-fed rats because of a twofold increase in maximal transport velocity. In biliary-diverted rats (72-96 hours), transporter protein decreased to 57% +/- 5% of paired controls with intact enterohepatic circulation (P0.0001). The intraduodenal infusion of taurocholate (24 hours) in biliary-diverted rats resulted in a time-dependent reinduction of transporter protein expression (3.5-fold).The expression of the ileal apical bile acid transporter is induced at a pretranslational level by free or taurine-conjugated cholic acid within the small intestine.

Background & Aims The final step in bile acid synthesis involves conjugation with glycine and taurine, which promotes a high intraluminal micellar concentration to facilitate lipid absorption. We investigated the clinical, biochemical, molecular, and morphologic features of a genetic defect in bile acid conjugation in 10 pediatric patients with fat-soluble vitamin deficiency, some with growth failure or transient neonatal cholestatic hepatitis. Methods We identified the genetic defect that causes this disorder using mass spectrometry analysis of urine, bile, and serum samples and sequence analysis of the genes encoding bile acid-CoA:amino acid N -acyltransferase (BAAT) and bile acid-CoA ligase (SLC27A5). Results Levels of urinary bile acids were increased (432 ± 248 μmol/L) and predominantly excreted in unconjugated forms (79.4% ± 3.9%) and as sulfates and glucuronides. Glycine or taurine conjugates were absent in the urine, bile, and serum. Unconjugated bile acids accounted for 95.7% ± 5.8% of the bile acids in duodenal bile, with cholic acid accounting for 82.4% ± 5.5% of the total. Duodenal bile acid concentrations were 12.1 ± 5.9 mmol/L, which is too low for efficient lipid absorption. The biochemical profile was consistent with defective bile acid amidation. Molecular analysis of BAAT confirmed 4 different homozygous mutations in 8 patients tested. Conclusions Based on a study of 10 pediatric patients, genetic defects that disrupt bile acid amidation cause fat-soluble vitamin deficiency and growth failure, indicating the importance of bile acid conjugation in lipid absorption. Some patients developed liver disease with features of a cholangiopathy. These findings indicate that patients with idiopathic neonatal cholestasis or later onset of unexplained fat-soluble vitamin deficiency should be screened for defects in bile acid conjugation.

Background & Aims: The mechanisms causing liver injury in cholestatic diseases are unclear. The hypothesis that accumulation of hydrophobic bile acids in hepatocytes during cholestasis leads to generation of oxygen free radicals and oxidative injury was tested. The aim of this study was to determine if hydrophobic bile acid toxicity is associated with increased hydroperoxide generation in isolated rat hepatocytes and mitochondria. Methods: Hepatocytes were exposed to taurochenodeoxycholic acid (TCDC; 0–2000 μmol/L) or taurocholic acid (TC; 1000 μmol/L), and cellular injury, intracellular hydroperoxide generation, and thiobarbituric acid-reacting substances (TBARS) were measured. Isolated mitochondria were incubated with 400 μmol/L chenodeoxycholic acid or 400 μmol/L cholic acid, and hydroperoxide generation was measured fluorometrically. Results: Hepatocyte injury, hydroperoxide generation, and TBARS increased over 4 hours on exposure to TCDC but not TC. Hydroperoxide generation preceded hepatocyte injury and accumulation of TBARS. Preincubation of hepatocytes with the antioxidant, d -α-tocopheryl succinate, completely abrogated cellular injury, hydroperoxide, and TBARS generation. Hydroperoxide generation was increased in mitochondria exposed to chenodeoxycholic acid. Conclusions: Intracellular generation of hydroperoxides by mitochondria appears to be an early event in hydrophobic bile acid-induced hepatocyte toxicity. Antioxidants may be of benefit in cholestasis.

Bile Acid Malabsorption (BAM) resulting from bile acid overproduction or reduced transport can increase fecal bile acids and cause chronic diarrhea. BAM is thought to account for up to 30% of patients diagnosed with irritable bowel syndrome (IBS-D). Current methods of diagnosing BAM are limited; it is likely under-diagnosed. Our objective was to develop a non-invasive method of detecting BAM by synthesizing and testing a multi-fluorinated bile acid (MFBA) that can be used for fluorine magnetic resonance imaging (MRI) of bile acid enterohepatic circulation. A prototype MFBA was synthesized by conjugating trifluoroacetyl lysine to cholic acid (19F-CA-lys). This MFBA was tested for transport and inhibition of the apical sodium dependent bile acid transporter (ASBT) using stably transfected MDCK cell monolayers and of Na+/taurocholate cotransporting polypeptide (NTCP) using stably transfected HEK cell monolayers. In Vitro, 19F-CA-lys was a potent inhibitor and substrate of both ASBT (Ki = 20.0±3.9 μM, Kt = 39.4±23.8 μM, normalized Vmax =0.853 ± 0.197) and NTCP (Ki = 2.93±0.60 μM, Kt = 8.99 ± 2.79 μM, normalized Vmax = 0.281±0.052). Stability of 19F-CA-lys (5 μM) was measured after treatment with Caco-2 cell homogenates, rat plasma, rat liver microsomal S9 pools, simulated intestinal fluid with pancreatic enzymes, 1 M HCl, buffers at pH 7.4 and 6.8, and choloylglycine hydrolase (CGH) (N=3 for each condition). Except for CGH, 19F-CA-lys was stable in each condition. After 1 h in CGH, 60.4±4.0% 19F-CA-lys remained compared to 2.4±0.5% naturally-occurring taurocholic acid (P

Background: An increasing amount of evidence suggests that consumption of non-aspirin NSAIDs is associated with injury to the lower gut in humans, as has been established in rodent model systems. NSAID-induced lower gut injury may in fact explain the linkage between NSAID consumption and the development of ileitis, diverticulitis and inflammatory bowel disease. Evidence from our and other labs indicates that NSAID-induced injury to the lower gut is dependent on the presence of bile, based upon In Vitro (synthetic membranes and GI cells in culture) and In Vivo (rodent) studies, and we have hypothesized that NSAIDs may form toxic mixed micelles with bile acids (BAs). Aim: To test this hypothesis that NSAIDs and BAs spontaneously form mixed micellar aggregates we used a combination of powerful computational and spectroscopic techniques to gain insights into atomic associations between cholic acid (CA) and ibuprofen (IBU). Methods & Results: Using Molecular Dynamics (MD) simulations in tandem with Nuclear Magnetic Resonance (NMR) spectroscopy, we found that CA and IBU form mixed micelles in a concentration dependent manner, so that multiple CA molecules form ‘pseudo-rings' into which IBUs insert their hydrophobic tails. This ‘β-face-to-tail' packing between the steroid rings of CA and the alkyl carbons of IBU promotes the hydrophobic interaction of these two classes of amphipathic molecules. We also found that the IBU-CA mixed micelles have much larger sizes and higher net surface charges than simple CA micelles, the total charge being the sum of the single negative charge arising from each of the CA and IBU molecules. Furthermore, the IBU-CA mixed micelles adopt amore spherical shape that differs from the oblate shape formed by the pure CA clusters. These results highlight the power of a combined NMR/MD approach for characterizing the molecular interactions in the binarymixtures of NSAIDs and BAs. Conclusions: Our combined NMR/MD analysis yielded compelling structural evidence that NSAIDs and BAs form mixed micelles at concentrations greater than or equal to the CMC of the two species, which likely occur in the small intestinal lumen. The significance of the altered micellar morphology and charge density of these BA/NSAID mixed micelles to the drugs' cytotoxicity to the epithelium of the lower gut is an important area for future investigation. (Supported by NIH grants RC1 DK086304 and P30 DK56338.)

Background/Aims: Supersaturation of bile with cholesterol, rapid nucleation of cholesterol crystals, and/or incomplete emptying of the gallbladder are believed to be required for gallstone formation. The importance of these factors for the recurrence of gallbladder stones was studied. Methods: Twenty patients, untreated after successful shock wave therapy, were studied in a matched case-control design for bile acid turnover, composition of duodenal bile, and gallbladder emptying. In 10 of them, gallstones had recurred within 12 ± 2 months (X ± SEM); the other 10 had been free of stones since 22 ± 3 months. Results: In each group, duodenal bile was supersaturated with cholesterol in 8 of 10 patients and showed abnormal nucleation time of cholesterol crystals in half of the patients. Patients with recurrent stones had smaller pool sizes of cholic acid (−43%) and enhanced conversion of cholic acid to deoxycholic acid. The odds for stone recurrence were ninefold increased in the presence of excessive deoxycholic acid (exceeding cholic acid) in the bile acid pool or incomplete emptying of the gallbladder (residual volume >5 mL) in response to cholecystokinin. The odds ratio was over 20-fold increased when incomplete emptying of the gallbladder coincided with supersaturated bile or with excessive deoxycholic acid. Conclusions: Enhanced conversion of cholic acid to deoxycholic acid and incomplete emptying of the gallbladder could be important cofactors for the recurrence of gallstones.

BACKGROUND/AIM: Evaluation of bile acids (BA) is a useful method for assessing changes of the intestinal flora in patients with ulcerative colitis (UC). During enterohepatic circulation, conjugated BA is deconjugated to free BA by intestinal bacteria. The presence of intestinal microflora (Clostridium and Eubacterium) leads to 7 alpha-dehydroxylation of cholic acid (CA) and chenodeoxycholic acid (CDCA), yielding deoxycholic acid (DCA) and lithocholic acid, respectively. It was reported that the Lachnospiraceae subgroup of Firmicutes (including Clostridium) are decreased in the colon of UC patients compared to controls without inflammatory bowel disease. We have already reported that the serum%CDCA is significantly higher in patients with UC than in healthy volunteers (HV), while serum%DCA is significantly lower in UC patients than in HV, and these changes do not depend on the activity or extent of UC. The aim of the present study was to elucidate the effects of probiotics in patients with UC by examining the serum BA profile. PATIENTS/METHODS: The study population was 27 patients in whom UC was diagnosed from endoscopic and histological findings. All patients underwent ileocolonoscopy with appropriate biopsies. They were divided into the following 2 groups based on endoscopic findings: 15 patients with distal UC (dUC) and diffuse changes extending from the rectum to the splenic flexure, and 12 patients with more extensive UC or pancolitis (pUC). Treatment was givenwithmesalazine or salazosulfapyridine (5-ASA) and all patients achieved remission. After entering remission, they were treated with by 5-ASA plus the probiotic Clostridium butyricum Miyairi (MIYA) (3 g/day) for 4 weeks. The control group was composed of 8 HV. Routine laboratory tests were performed on the basis of clinical need, while fasting serum samples for measurement of BA were obtained before and after treatment with 5-ASA plus MIYA for 4 weeks. Serum BA fractions were analyzed by HPLC. RESULTS: There were no significant differences of serum total BA among the 3 groups. Before treatment, %CDCA was significantly higher in the dUC and pUC groups than in the HV group, while %DCA was significantly lower than in the HV group. In the pUC group, %CDCA was significantly higher and %DCA was significantly lower than in the HV group after 4 weeks of probiotic treatment. In the dUC group, however, there was no significant difference of %CDCA or %DCA after 4 weeks compared with the HV group. There were no significant differences in the ratio of conjugated BA to total serum BA among the three groups. CONCLUSIONS: These results suggest that intestinal bacteria involved in the deconjugation of BA are restored when patients achieve remission of UC by treatment with 5-ASA, and thatMIYA restoredmicroflora involved in 7 alpha-dehydroxylation in the dUC group, but not in the pUC group.

Background: Cholylsarcosine, the synthetic conjugate of cholic acid and sarcosine, is resistant to deconjugation-dehydroxylation during enterohepatic cycling in rodents and improves lipid absorption in a canine model of intestinal bile acid deficiency caused by distal intestinal resection. Experiments were performed to define its metabolism and effect on biliary secretion in humans. Methods: The circulating bile acid pool was labeled with [ 14 C]cholylsarcosine, and its turnover rate and biotransformation were determined by sampling bile daily. Cholylsarcosine (or cholyltaurine) was infused into the duodenum for 8 hours to define its effect on bile flow and biliary lipid secretion. Results: Cholylsarcosine was lost rapidly from the enterohepatic circulation with a t12 of 0.5 days. The compound was not biotransformed by hepatic or bacterial enzymes. Cholylsarcosine had choleretic activity similar to that of cholyltaurine but induced more phospholipid and cholesterol secretion than cholyltaurine in four of five subjects. Infusion of cholylsarcosine (or cholyltaurine) at a rate averaging 0.6 μmol · min −1 · kg −1 gave a biliary recovery of 0.2 μmol · min −1 · kg −1 ; this value is the T max for active ileal transport of conjugated bile acids in humans. Laboratory tests for liver injury remained within normal limits. Conclusions: In humans, cholylsarcosine is not metabolized, is nontoxic, and has similar effects on biliary secretion as cholyltaurine. It appears safe to test in long-term studies the effect of cholylsarcosine on bile acid-deficiency states in humans.

Objectives: Bile acids inhibit contraction of the gallbladder and intestine through the G-protein-coupled bile acid receptor (GPBAR). Perfusion of the esophagus with bile and acid (HCl) decreases lower esophageal sphincter (LES) pressure. The effects of bile acids on LES motility are not clear. The purpose of the present study was to investigate the effects of bile acids on LES motility in vitro. Materials and Methods: We measured the relaxation of muscle strips isolated from guinea pig and rat LES caused by bile acids or the selective GPBAR agonist RG-239. Reverse transcription polymerase chain reaction (RT-PCR) was performed to determine GPBAR expression in rat LES. Results: In carbachol-contracted guinea pig LES strips, lithocholic acid (LCA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), and cholic acid (CA) produced relaxation in a concentration-dependent

Ursodeoxycholic acid (UDCA) dissolves cholesterol gallstones and improves liver function test results in patients with cholestatic liver diseases. Its absorption was studied in patients who had complete extrahepatic biliary obstruction caused by pancreatic carcinoma but no intestinal or liver disease. Six patients received 500 mg chenodeoxycholic acid (CDCA) or 250-2000 mg UDCA in capsules in single oral doses in random order, with an interval of 2 days between the different treatment regimens. In the control period the patients excreted into bile 382.3 +/- 108.0 mumol CDCA (mean +/- SD) and 1866.7 +/- 172.6 mumol cholic acid per 24 hours. After administration of 1273.6 mumol (500 mg) CDCA, biliary excretion of this bile acid increased to 1370.9 +/- 185.7 mumol/24 h, indicating an intestinal absorption rate of 77.6% +/- 9.8%. After oral administration of 636.8 mumol (250 mg), 1273.6 mumol (500 mg), 2547.2 mumol (1000 mg), and 5094.4 mumol (2000 mg) of UDCA, the respective absorption rates were 60.3% +/- 7.4%, 47.7% +/- 9.0%, 30.7% +/- 7.5%, and 20.8% +/- 3.9%, and whereas in the control period no UDCA was detected in the bile, the UDCA percentages measured were 14.6% +/- 8.2%, 19.6% +/- 9.1%, 23.1% +/- 11.3%, and 27.4% +/- 12.1%. The coadministration of CDCA did not enhance the absorption of UDCA. The data indicate that absorption of orally administered CDCA is almost complete, whereas UDCA absorption is incomplete. With increasing doses UDCA absorption decreases. To achieve absorption of adequate amounts of UDCA, high and/or multiple doses are needed.

The effects of various species of bile salts (chenodeoxycholate, deoxycholate, ursodeoxycholate and cholate, and their taurine and glycine conjugates) on pancreatic exocrine secretion were studied in conscious rats with external bile and pancreatic fistulae. For examination of the stimulatory effects of bile salts, bile and pancreatic juice were collected for a basal period of 90 minutes and returned to the intestine, and then solutions of bile salts (60 mmol/ L) were infused intraduodenally at a rate of 1 mL/h for 2 hours. For examination of their inhibitory effects, pancreatic secretion was stimulated by exclusion of the bile and pancreatic juice; and then solutions of the bile salts were again infused intraduodenally. Chenodeoxycholate, glycochenodeoxycholate, ursodeoxycholate, deoxycholate, and its conjugates (glycodeoxycholate and taurodeoxycholate) significantly increased the fluid, bicarbonate and protein outputs, and bicarbonate concentration, with decrease in protein concentration. These increases were partially inhibited by infusion of either a cholecystokinin antagonist or secretin antibody. In contrast, cholate, taurocholate, tauroursodeoxycholate, glycoursodeoxycholate, and taurochenodeoxycholate inhibited pancreatic secretion and increase in the plasma cholecystokinin concentration produced by exclusion of bile and pancreatic juice. Thus, some bile salts, including taurocholate and taurochenodeoxycholate (major bile salts in rat bile) inhibited pancreatic secretion and cholecystokinin release, whereas some other bile salts increased pancreatic secretion via cholecystokinin release and secretin release.

Background & Aims Oral bile acid replacement has been shown to be an effective therapy in primary bile acid synthesis defects, but to date there have been no reports of the long-term effects of this therapy. The aim of the study was to evaluate the long-term effectiveness and safety of cholic acid (CA) therapy. Methods Fifteen patients with either 3β-hydroxy-Δ 5 -C 27 -steroid oxidoreductase (3β-HSD) (n = 13) or Δ 4 -3-oxosteroid 5β-reductase (Δ 4 -3-oxo-R) (n = 2) deficiency confirmed by mass spectrometry and gene sequencing received oral CA and were followed up prospectively. Results CA therapy was started at a median age of 3.9 years (range, 0.3–13.1 years). The median follow-up with treatment was 12.4 years (range, 5.6–15 years). The mean daily dose of CA was initially 13 mg/kg and was 6 mg/kg at last evaluation. During CA therapy, physical examination findings, laboratory test results, and findings on sonography normalized. Mass spectrometry analysis of urine showed that excretion of the atypical metabolites was reduced by 500-fold and 30-fold in 3β-HSD and Δ 4 -3-oxo-R deficiency, respectively, and total urinary bile acid excretion decreased dramatically. Liver biopsies performed in 14 patients after at least 5 years of CA therapy showed marked improvement, especially in patients with the 3β-HSD deficiency. CA was well tolerated with all children developing normally, including 2 women having 4 normal pregnancies during treatment. Conclusions Oral CA therapy is a safe and effective long-term treatment of the most common primary bile acid synthesis defects.

Introduction: Lithogenic diet and infection with enterohepatic helicobacter are important for cholesterol gallstone formation in mice. Cholesterol gallstone formation in humans is associated with slowed intestinal transit, which increases cholesterol absorption and prolithogenic deoxycholate formation. Here we studied the effects of a lithogenic diet as well as enterohepatic helicobacter infection on small intestinal transit in inbred mice to determine if either or both promote prolonged small intestinal transit in mice. Methods: Male C57L/ J mice were infected with H. hepaticus or left uninfected and fed either a lithogenic diet (containing 15% dairy triglycerides, 1% cholesterol and 0.5% cholic acid) or standard mouse chow (

Hydrophobic bile acids have been shown to be hepatotoxic, whereas treatment with ursodeoxycholic acid, a hydrophilic bile acid, has improved liver function indices in patients with chronic liver disease. Taurine administration has also been suggested to be useful for chronic hepatitis, taurine-conjugated bile acids being more hydrophilic than glycine-conjugated bile acids. To determine if taurine and ursodeoxycholic acid are beneficial and if their effects are additive, a double-blind, randomized trial was designed comparing the effects of ursodeoxycholic acid, taurine, and a combination of the two on indices of liver injury in 24 patients with chronic hepatitis. They were assigned at random to two of the four following treatments: ursodeoxycholic acid (600 mg/day), taurine (1.5 g/day), ursodeoxycholic acid plus taurine (600 mg + 1.5 g/day) or placebo, given in two successive cycles of 2 mo each, according to a balanced incomplete-block design. Ursodeoxycholic acid became the predominant biliary bile acid when administered alone or in combination with taurine, and taurine conjugate levels increased during taurine administration. Ursodeoxycholic acid reduced aspartate aminotransferase (35%), alanine aminotransferase (33%), and gamma-glutamyl transpeptidase (41%), whereas taurine alone did not. The addition of taurine to ursodeoxycholic acid produced only minor changes in the effects of ursodeoxycholic acid alone. Results were confirmed by the administration of ursodeoxycholic acid, in a successive open phase of the study, to the entire patient population, which was large enough for different subsets of patients to be compared. Serum bile acids were measured at entry and during the open phase: primary bile acids did not change, whereas ursodeoxycholic acid levels increased from trace amounts to very high levels, especially in patients with more severe histological disease. It is concluded that ursodeoxycholic acid, but not taurine, improves enzymatic indices of liver injury in chronic hepatitis.

The aim of the present study is to examine the efficacy of 6-hydroxy substituted bile acids on the prevention of cholesterol gallstones in a new hamster model of cholesterol cholelithiasis. Male golden Syrian hamsters were fed a nutritionally adequate semipurified lithogenic diet consisting of casein, cornstarch, soluble starch, butterfat, corn oil, and cellulose plus 0.3% cholesterol. Six different bile acids were added to this diet at the 0.05% level: chenodeoxycholic acid, ursodeoxycholic acid, hyodeoxycholic acid, murideoxycholic acid, 6 beta-methyl-hyodeoxycholic acid, and 6 alpha-methyl-murideoxycholic acid. At the end of the 6-wk feeding period, the control group receiving the lithogenic diet had a 55% incidence of gallstones. It was found that all bile acids had inhibited the formation of cholesterol gallstones; complete prevention of gallstones was observed with all 4 3,6-dihydroxy bile acids, whereas chenodeoxycholic acid and ursodeoxycholic acid were somewhat less effective (80% and 75% prevention, respectively). The accumulation of cholesterol in serum and liver induced by the lithogenic diet was inhibited to some extent by all of the bile acids; hyodeoxycholic acid, murideoxycholic acid, and 6 beta-methyl hyodeoxycholic acid were most effective in this respect. The administered bile acids tended to predominate in bile in the case of chenodeoxycholic acid, hyodeoxycholic acid, and 6 beta-methyl-hyodeoxycholic acid. In contrast, ursodeoxycholic acid seemed to be converted to chenodeoxycholic acid and murideoxycholic acid to hyodeoxycholic acid. Only 4% of the 6-methyl analogue of murideoxycholic acid, 6 alpha-methyl-murideoxycholic acid, was recovered in gallbladder bile. These experiments show that the new hamster model of cholesterol cholelithiasis is suitable for gallstone-prevention studies. It was not possible to draw definite conclusions concerning the mechanism of action of the administered bile acids on the basis of cholesterol saturation or the presence of liquid crystals. The detailed mechanism of gallstone prevention by hydrophilic bile acids in this model remains to be elucidated.

The effects of ursodeoxycholic acid and chenodeoxycholic acid on the small-intestinal absorption of endogenous bile acids were studied in patients with ileostomies who served as a model to investigate small-intestinal absorption in humans. In the control period, the eight patients excreted 327 +/- 91 (mean +/- standard error of the mean) mumol/8 h cholic acid and 214 +/- 38 mumol/8 h chenodeoxycholic acid by their ileal fluid. Following ursodeoxycholic acid administration (500 mg), ileal excretion of cholic acid increased to 517 +/- 96 mumol/8 h, and that of chenodeoxycholic acid increased to 337 +/- 42 mumol/8 h, indicating decreased absorption of these bile acids. Following chenodeoxycholic acid administration (500 mg), no significant increase of cholic acid excretion was observed, whereas chenodeoxycholic acid excretion increased as expected. It is concluded that following ursodeoxycholic acid administration the absorption of common bile acids from the small intestine decreases markedly. This effect of ursodeoxycholic acid on intestinal absorption of common bile acids probably is responsible for the decrease of their plasma concentrations, the reduction of their pool sizes, the increase of their fractional turnover rates, and most likely also contributes to the increased hepatic synthesis of cholic acid.