Beta-muricholic Acid Research Articles

Core biomarkers analysis benefit for diagnosis on human intrahepatic cholestasis of pregnancy

BackgroundThe pregnant women with intrahepatic cholestasis were at high risk of fetal distress, preterm birth and unexpected stillbirth. Intrahepatic cholestasis of pregnancy (ICP) was mainly caused by disorder of bile acid metabolism, whereas the specific mechanism was obscure.MethodsWe performed proteomics analysis of 10 ICP specimens and 10 placenta specimens from patients without ICP through data-independent acquisition (DIA) technique to disclose differentially expressed proteins. We executed metabolomic analysis of 30 ICP specimens and 30 placenta specimens from patients without ICP through UPLC-MS/MS to identify differentially expressed metabolites. Enrichment and correlation analysis was used to obtain the direct molecular insights of ICP development. The ICP rat models were constructed to validate pathological features.ResultsThe heatmap of proteomics analysis showed the top 30 up-regulated and 30 down-regulated proteins. The metabolomic analysis revealed 20 richer and 4 less abundant metabolites in ICP samples compared with placenta specimens from patients without ICP, and enrichment pathways by these metabolites included primary bile acid biosynthesis, cholesterol metabolism, bile secretion, nicotinate and nicotinamide metabolism, purine metabolism and metabolic pathways. Combined analysis of multiple omics results demonstrated that bile acids such as Glycohyocholic acid, Glycine deoxycholic acid, beta-Muricholic acid, Noncholic acid, cholic acid, Gamma-Mercholic Acid, alpha-Muricholic acid and Glycochenodeoxycholic Aicd were significantly associated with the expression of GLRX3, MYL1, MYH7, PGGT1B, ACTG1, SP3, LACTB2, C2CD5, APBB2, IPO9, MYH2, PPP3CC, PIN1, BLOC1S1, DNAJC7, RASAL2 and ATCN3 etc. The core protein ACAT2 was involved in lipid metabolic process and animal model showed that ACAT2 was up-regulated in placenta and liver of pregnant rats and fetal rats. The neonates had low birth weight and Safranin O-Fast green FCF staining of animal models showed that poor osteogenic and chondrogenic differentiation of fetal rats.ConclusionMultiple metabolites-alpha-Muricholic acid, beta-Muricholic acid, Glycine deoxycholic acid and Glycochenodeoxycholic Acid etc. were perfect biomarkers to predict occurrence of ICP. Bile acids were significantly associated with varieties of protein expression and these proteins were differentially expressed in ICP samples. Our study provided several biomarkers for ICP detection and potential therapeutic targets for ICP development.

The dysregulation of biliary tract microflora is closely related to primary choledocholithiasis: a multicenter study

Bile microecology changes play an important role in the occurrence and development of choledocholithiasis. At present, there is no clear report on the difference of bile microecology between asymptomatic patients with gallbladder polyps and choledocholithiasis. This study compared bile microecology between gallbladder polyp patients and patients with choledocholithiasis to identify risk factors for primary choledocholithiasis. This study was conducted in 3 hospitals in different regions of China. Bile samples from 26 patients with gallbladder polyps and 31 patients with choledocholithiasis were collected by laparoscopic cholecystectomy and endoscopic retrograde choledocholithiasis cholangiography (ERCP), respectively. The collected samples were used for 16S ribosomal RNA sequencing and liquid chromatography mass spectrometry analysis. The α-diversity of bile microecological colonies was similar between gallbladder polyp and choledocholithiasis, but the β-diversity was different. Firmicutes, Proteobacteri, Bacteroidota and Actinobacteriota are the most common phyla in the gallbladder polyp group and choledocholithiasis group. However, compared with the gallbladder polyp patients, the abundance of Actinobacteriota has significantly lower in the choledocholithiasis group. At the genera level, the abundance of a variety of bacteria varies between the two groups, and Enterococcus was significantly elevated in choledocholithiasis group. In addition, bile biofilm formation—Pseudomonas aeruginosa was more metabolically active in the choledocholithiasis group, which was closely related to stone formation. The analysis of metabolites showed that a variety of metabolites decreased in the choledocholithiasis group, and the concentration of beta-muricholic acid decreased most significantly. For the first time, our study compared the bile of gallbladder polyp patients with patients with choledocholithiasis, and suggested that the change in the abundance of Actinobacteriota and Enterococcus were closely related to choledocholithiasis. The role of Pseudomonas aeruginosa biofilm in the formation of choledocholithiasis was discovered for the first time, and some prevention schemes for choledocholithiasis were discussed, which has important biological and medical significance.

Alterations of the gut microbiota and metabolites by ShenZhu TiaoPi granule alleviates hyperglycemia in GK rats.

ShenZhu TiaoPi granule (STG) is a compound prescription that is used in Chinese medicine for the treatment of type 2 diabetes mellitus (T2DM). Previous studies have indicated a hypoglycaemic effect, but the underlying mechanism remains unclear. Goto-Kakizaki (GK) rats were used to establish an in vivo T2DM model (Mod). The metformin (Met) and STG treatment time was 12 weeks. Fasting blood glucose (FBG) and insulin levels and the area under the glucose curve (GAUC) were measured. Intestinal pathology and permeability were observed. Microbial diversity analysis and metabolomics were used to investigate the underlying mechanisms. Compared with the Con group, the T2DM Mod group presented significant differences in weight, FBG, GAUC, and homeostasis model assessment-insulin resistance (HOMA-IR) indices (p < 0.01). Met and STG improved these indicators (p < 0.01). The pathological morphology and zonula occludens 1 protein levels in the intestines of the Mod group of rats were altered, leading to increases in the lipopolysaccharide (LPS) and interleukin-1β (IL-1β) levels. In the Met and STG groups, the intestinal conditions improved, and the LPS and IL-1β levels significantly decreased (p < 0.01). Changes in the gut microbiota and metabolites occurred in the Mod group. In the STG group, the abundance of Intestinimonas increased, and the abundance of Eubacterium coprostanoligenes decreased significantly (p < 0.05). Moreover, STG also altered 2-deoxyglucose, beta-muricholic acid and dioxolithocholic acid production. In addition, the main metabolic pathways affected by STG were bile acid biosynthesis and cholesterol metabolism. Intestinimonas, D-maltose_and_alpha-lactose may be potential biomarkers for the effects of STG. STG alleviates hyperglycaemia via the gut microbiota and metabolites in GK rats.

B-197 Separation of Bile Acid Muricholic Acid Enantiomers by C18 Reversed-phase HPLC as a Function of Component Polarity Values of the Mobile Phase

Abstract Objective Optimize the reversed phase HPLC technique for the separation of alpha-muricholic acid and beta-muricholic bile acid enantiomers, by investigating the effects of the mobile phase’s three polarity components [hydrogen bond donor acidity (α), hydrogen bond donor basicity (β) and dipolarity/polarizability (π)] on the separation of the enantiomers. Background The importance of drug enantiomers in clinical pharmacology is well recognized, where often only one of the enantiomer pair effectively binds to the target protein site to exert its therapeutic effect. Same for some endogenous compounds, only one of the enantiomer pair molecules binds to an enzyme or receptor to exert its physiological action, while the other molecule is inactive. It is essential for the analytical method to separately determine the two compounds of the pair. Usual HPLC techniques for separating enantiomers employ specialized chiral columns. In this work, steroid isomers are separated on a C18 column, with the separation controlled by mobile phase (MP) component polarity adjusted with different proportions of binary organic modifiers added to the MP. Methods The bile acid enantiomers alpha-muricholic acid and beta-muricholic acid were separated on a C18 column (150 mm × 2.1 mm, 3.5 μm, 40 °C) using two different organic modifiers in the MP at a flow rate of 0.2 mL/min, with ESI-LC/MS detection. The isocratic MP consisted of 0.1% formic acid in 100% water for MP A and 0.1% formic acid and five or more different proportions (separate runs) of the two organic modifiers for MP B to adjust MP polarity. MP component polarities was determined from: 1) the literature values for the total polarity (P) for each of the pure solvents in the MP; 2) published values of the fraction of the total polarity for the polarity components (α + β + π = 1) for each solvent; and 3) the fraction proportion of each solvent in the MP. The experimentally determined selectivity factor is then plotted vs the polarity component value of the MP. Results When the selectivity factor vs the values of the MP polarity components was plotted for six different organic modifier pair combinations on the same plot, the summed dipolarity/polarizability (and basicity polarity (β) components of the MP showed reasonable correlation (R2 = 0.72), displaying mostly a linear superimposed trend for all the organic modifier pairs, while the acidity component (α, R2 = 0.13) and total polarity (P) (R2 = 0.002) of the MP did not. Thus, the π + β summed MP polarities is a significant determiner of selectivity, irrespective of the organic modifier pair used. The bile acid enantiomers were baseline separated, with a selectivity factor of 1.3 being the highest value obtained. Conclusion For the laboratory determination of steroid enantiomers, our results suggest that calculated component polarity values of the MP can be used to direct laboratorians for achieving desired separation of steroid enantiomers (as opposed to a trial-and-error approach for achieving the separation). This study shows that the separation of these bile acid enantiomers significantly depends on the summed π and β polarities of the MP, with poor correlation for total polarity (P) and α.

The effect of long-term moderate exercise on myocardial metabolome in rats.

Regular moderate physical exercise is beneficial for the cardiovascular system. Our prior study has demonstrated a long-term moderate exercise (4-week of 60-min 74.0% V̇O2max treadmill running) is optimal in protecting from exhaustive exercise-induced cardiac ischemic injury. This study is aimed to investigate the effect of long-term moderate exercise on myocardial metabolome in rats. Thirteen male Sprague-Dawley rats were randomly assigned into the control group (C) and the long-term moderate exercise group (E). The targeted metabolomics of the myocardium was analyzed by ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) system. Results showed that the metabolites categories of bile acids (BAs), fatty acids (FAs), and phenylpropanoic acids were significantly decreased. The biosynthesis of unsaturated FAs pathway was significantly downregulated. The altered metabolites in the E Group included decreased FAs (pentadecanoic acid, 10Z-heptadecenoic acid, dihomo-gamma-linolenic acid, docosahexaenoic acid, docosapentaenoic acid, and 10Z-nonadecenoic acid), decreased BAs (chenodeoxycholic acid and beta-muricholic acid), decreased organic acids (glycolic acid and 2-hydroxyglutaric acid), decreased carbohydrate (N-acetylneuraminic acid, Neu5Ac), decreased amino acids (α-aminobutyric acid and norvaline), decreased phenylpropanoic acids (hydroxyphenyllactic acid), and benzoic acids (4-hydroxybenzoic acid and phthalic acid). The results indicated that long-term moderate exercise has promoted lipids utilization in myocardium while exerted little influence on carbohydrate metabolism and diminished many detrimental metabolites. Notably, decrease of myocardial carbohydrate Neu5Ac after long-term moderate exercise might predict a prospective metabolomics biomarker for cardioprotection. This research has displayed the effect of long-term moderate exercise on myocardial metabolomic profiling in rats and indicated some promising metabolites which can be applied for exercise benefits in future.

Dietary carbon loaded with nano-ZnO alters the gut microbiota community to mediate bile acid metabolism and potentiate intestinal immune function in fattening beef cattle

BackgroundTo our knowledge, carbon loaded with nano-ZnO (NZnOC) represents a new nutritional additive for the animal husbandry industry. However, the mechanism by which NZnOC mediates beef cattle growth and intestinal health is not fully understood. This study aimed to investigate the effects of carbon loaded with nano-ZnO (NZnOC) supplementation on growth performance, gut microbiota, bile acid (BAs) metabolism and intestinal immunity in fattening cattle. Twenty cattle (16 ± 0.95 months) were randomly assigned to two dietary groups: CON (control, without feed additive) and NZnOC (diet supplemented with 80 mg NZnOC/kg diet dry matter basic) for 60 d. The colon digesta microbiota composition and BAs concentration were determined by microbiota metagenomics and gas chromatography methods, respectively.ResultsThe results showed that the NZnOC-supplemented cattle had greater final weight, average daily gain and gain-to-feed ratio than those in the CON group. Cattle fed the NZnOC diet had a higher relative abundance of the secondary BAs synthesizing phyla Firmicutes, Tenericutes and Actinobacteria than those fed the CON diet. Dietary supplementation with NZnOC increased the relative abundance of the secondary BAs synthesis microbiota genera Clostridium, Ruminococcus, Eubacterium, and Brevibacillus in colon digesta. Cattle fed the NZnOC diet had increased activities of 3α-hydroxysteroid dehydrogenase (EC: 1.1.1.52) and bile acid-CoA ligase BaiB (EC: 6.2.1.7) in the colon digesta compared with those fed the CON diet. The primary BAs taurocholic acid, taurochenodeoxycholic acid and taurodeoxycholate acid were significantly decreased by dietary NZnOC supplementation, while the secondary BAs deoxycholic acid, taurolithocholic acid, beta-muricholic acid, 12-ketolithocholic acid and ursodeoxycholic acid were significantly increased. Dietary supplementation with NZnOC increased the mRNA abundance of G protein-coupled bile acid receptor 1, protein kinase cAMP-activated catalytic subunit alpha, cyclic-AMP response element binding protein 1 and interleukin (IL)-10 in the colon mucosa of cattle, while the mRNA abundance of tumor necrosis factor and IL-1β were significantly decreased.ConclusionsIn summary, dietary supplementation with NZnOC can facilitate the growth performance and intestinal immune function of cattle by improving BAs metabolism. NZnOC can be supplemented in the diet as a safe regulator of gut microbiota and as a feed additive in the ruminants industry.

Total glycosides contribute to the anti-diarrheal effects of Qiwei Baizhu Powder via regulating gut microbiota and bile acids

Qiwei Baizhu Powder (QWBZP) is a traditional Chinese medicine formula for treating diarrhea induced by various causes. It elicits an anti-diarrheal effect by regulating the gut microbiota (diversity, structure, and abundance). However, the contribution of different components in the QWBZP decoction to this effect remains unclear. In this study, we used the QWBZP decoction as a reference standard to investigate the effects of total glycosides (TGs) extracted from QWBZP decoction on the gut microbiota and bile acid metabolism in mice with antibiotic-associated diarrhea (AAD). The results of 16S rRNA gene sequencing and liquid chromatography-mass spectrometry (LC-MS) analysis showed that the effect of total glycosides of Qiwei Baizhu Powder (QWBZP-TG) on specific intestinal bacteria and bile acids was similar to that of the QWBZP decoction, but the intensity of this effect was more significant in the case of QWBZP-TG. The QWBZP decoction and QWBZP-TG promoted the proliferation of Lactobacillus and inhibited the growth of Proteus, Clostridium, Eubacterium, Facklamia, and Escherichia in AAD mice. They also increased the levels of deoxycholic acid and beta-muricholic acid and decreased those of taurocholate acid, tauro-alpha-muricholic acid, and tauro-beta-muricholic acid in AAD mice. Lactobacillus was the key bacterial genus responding to QWBZP-TG. Thus, this study provides novel insights into the bioactive components of QWBZP and their contribution to its effects.

A strain of Bacteroides thetaiotaomicron attenuates colonization of Clostridioides difficile and affects intestinal microbiota and bile acids profile in a mouse model

Clostridioides difficile infection (CDI) is a growing global public health threat. While fecal microbiota transplantation (FMT) is an effective therapy for CDI, a number of challenges limit its application. Studies suggest that probiotics may be a promising alternative therapy. In the current study, we evaluated whether Bacteroides thetaiotaomicron (B. thetaiotaomicron) would inhibit colonization of toxigenic BI/NAP1/027 C. difficile in a mouse model. We found that B. thetaiotaomicron administration decreased the copies of C. difficile and inhibited inflammation in the colon. 16S rRNA sequencing showed that B. thetaiotaomicron administration was associated with a significantly increased relative abundance of Bacteroidetes and decreased level of Proteobacteria, leading to the reversal of the effect of antibiotics treatment and C. difficile infection on microbiota. B. thetaiotaomicron administration was associated with increases in the concentrations of alpha-muricholic acid, beta-muricholic acid, 12 ketolithocholic acid, and deoxycholic acid which are known to inhibit the growth of C. difficile, as well as reductions in the level of taurocholic acid, which promotes germination of C. difficile. Altered profile of major high abundance bile acids by B. thetaiotaomicron administration was similar to that with FMT treatment. Based on these results, we proposed the concept of “the ratio of promotion/inhibition BAs” which would advance our understanding of the relation of C. difficile and BAs.

Effect of the prebiotic fiber inulin on cholesterol metabolism in wildtype mice

Dietary non-digestible carbohydrates are perceived to improve health via gut microbiota-dependent generation of products such as short-chain fatty acids (SCFA). In addition, SCFA are also precursors for lipid and cholesterol synthesis potentially resulting in unwanted effects on lipid metabolism. Inulin is a widely used model prebiotic dietary fiber. Inconsistent reports on the effects of inulin on cholesterol homeostasis have emerged in humans and preclinical models. To clarify this issue, the present study aimed to provide an in-depth characterization of the effects of short-chain (sc)- and long-chain (lc)- inulin on cholesterol synthesis, absorption and elimination in mice. Feeding wildtype C57BL/6J mice diets supplemented with 10% (w/w) of either sc- or lc-inulin for two weeks resulted in approximately 2.5-fold higher fecal SCFA levels (P < 0.01) compared with controls, but had no significant effects on plasma and liver lipids. Subtle shifts in fecal and plasma bile acid species were detected with beta-muricholic acid increasing significantly in plasma of the inulin fed groups (1.7-fold, P < 0.05). However, neither sc-inulin nor lc-inulin affected intestinal cholesterol absorption, mass fecal cholesterol excretion or trans-intestinal cholesterol excretion (TICE). Combined, our data demonstrate that sc- and lc-inulin have no adverse effects on cholesterol metabolism in mice despite increased generation of SCFA.

Different effects of ursodeoxycholic acid on intrahepatic cholestasis in acute and recovery stages induced by alpha-naphthylisothiocyanate in mice

The aim of this study was to determine the effect of ursodeoxycholic acid (UDCA) on the alpha-naphthylisothiocyanate (ANIT)-induced acute and recovery stage of cholestasis model mice. In the acute stage of model mice, pretreatment with UDCA (25, 50, and 100 mg·kg−1, ig) for 12 days prior to ANIT administration (50 mg·kg−1, ig) resulted in the dramatic increase in serum biochemistry, with aggrevation of bile infarcts and hepatocyte necrosis. The elevation of beta-muricholic acid (β-MCA), cholic acid (CA), and taurocholic acid (TCA) in serum and liver, and reduction of these bile acids (BAs) in bile was observed. In contrast, in the recovery stage of model mice, treatment with UDCA (25, 50, and 100 mg·kg−1, ig) for 7 days after ANIT administration (50 mg·kg−1, ig) resulted in the significant decrease in levels of serum alanine aminotransferase (ALT) and total bile acid (TBA). Liver injury was attenuated, and the levels of TBA, CA, TCA, and β-MCA in the liver were significantly decreased. Additionally, UDCA can upregulate expression of BSEP, but it cannot upregulate expression of AE2. UDCA, which induced BSEP to increase bile acid-dependent bile flow, aggravated cholestasis and liver injury when the bile duct was obstructed in the acute stage of injury in model mice. In contrast, UDCA alleviated cholestasis and liver injury induced by ANIT when the obstruction was improved in the recovery stage.

Quantitative profiling of 19 bile acids in rat plasma, liver, bile and different intestinal section contents to investigate bile acid homeostasis and the application of temporal variation of endogenous bile acids

Bile acid homeostasis is maintained by liver synthesis, bile duct secretion, microbial metabolism and intestinal reabsorption into the blood. When drug insults result in liver damage, the variances of bile acids (BAs) are related to the physiological status of the liver. Here, we established a method to simultaneously quantify 19 BAs in rat plasma, liver, bile and different intestinal section contents (duodenum, jejunum, ileum, cecum and colon) using high-performance liquid chromatography-tandem mass spectrometry (LC–MS/MS) to reveal the pattern of bile acid homeostasis in the enterohepatic circulation of bile acids in physiological situations. Dynamic changes in bile acid composition appeared throughout the enterohepatic circulation of the BAs; taurine- and glycine-conjugated BAs and free BAs had different dynamic homeostasis levels in the circulatory system. cholic acid (CA), beta-muricholic acid (beta-MCA), lithocholic acid (LCA), glycocholic acid (GCA) and taurocholic acid (TCA) greatly fluctuated in the bile acid pool under physiological conditions. Taurine- and glycine-conjugated bile acids constituted more than 90% in the bile and liver, whereas GCA and TCA accounted for more than half of the total bile acids and the secretion of bile mainly via conjugating with taurine. While over 80% of BAs in plasma were unconjugated bile acids, CA and HDCA were the most abundant elements. Unconjugated bile acids constituted more than 90% in the intestine, and CA, beta-MCA and HDCA were the top three bile acids in the duodenum, jejunum and ileum content, but LCA and HDCA were highest in the cecum and colon content. As the main secondary bile acid converted by microflora in the intestine, LCA was enriched in the cecum and DCA mostly in the colon. As endogenous substances, the concentrations of plasma BAs were closely related to time rhythm and diet. In conclusion, analyzing detailed BA profiles in the enterohepatic circulation of bile acids in a single run is possible using LC–MS/MS. Based on the physiological characteristics of the metabolic profiling of 19 BAs in the total bile acid pool and the time rhythm variation of the endogenous bile acids, this study provided a new valuable method and theoretical basis for the clinical research of bile acid homeostasis.

Enterobacteria-mediated deconjugation of taurocholic acid enhances ileal farnesoid X receptor signaling

Enterobacteria are known to deconjugate amino acid-conjugated bile acids in the intestine. Administration of ampicillin (ABPC; 3 days, 100mg/kg) decreased the expression of ileal farnesoid X receptor (Fxr) target genes, and increased the levels of total bile acids in the intestinal lumen. The primary tauro-conjugates of cholic acid (TCA) and beta-muricholic acid (TβMCA) levels were increased, whereas the primary unconjugates, cholic acid (CA) and beta-muricholic acid (βMCA), levels decreased to below detectable levels (<0.01μmol) in ABPC-treated mice. The effects of individual bile acid on expression of the ileal farnesoid X receptor target genes were examined in ABPC-treated mice. The expression of ileal farnesoid X receptor target genes in ABPC-treated mice was clearly enhanced after CA (500mg/kg), but not TCA (500mg/kg) cotreatment. Their levels in control mice were enhanced after either CA or TCA-cotreatment. Unconjugated CA levels in the intestinal lumen and portal vein were increased in both ABPC-treated and control mice. Reduced ileal Fgf15 and Shp mRNA levels in ABPC-treated mice were also increased after CA (100mg/kg) cotreatment at which luminal CA levels was restored to the level in controls, but was unaffected by βMCA (100mg/kg) cotreatment. In addition, no increase in ileal Shp, Ibabp or Ostα mRNA levels was observed even after CA (500mg/kg) cotreatment in ABPC-treated farnesoid X receptor-null mice despite increased CA levels in the intestinal lumen. These results suggest the role of enterobacteria in bile acid-mediated enhancement of ileal farnesoid X receptor signaling by TCA deconjugation.

Gender-Divergent Profile of Bile Acid Homeostasis during Aging of Mice

Aging is a physiological process with a progressive decline of adaptation and functional capacity of the body. Bile acids (BAs) have been recognized as signaling molecules regulating the homeostasis of glucose, lipid, and energy. The current study characterizes the age-related changes of individual BA concentrations by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in serum and liver of male and female C57BL/6 mice from 3 to 27 months of age. Total BA concentrations in serum increased 340% from 3 to 27 months in female mice, whereas they remained relatively constant with age in male mice. During aging, male and female mice shared the following changes: (1) BA concentrations in liver remained relatively constant; (2) the proportions of beta-muricholic acid (βMCA) increased and deoxycholic acid (DCA) decreased between 3 and 27 months in serum and liver; and (3) total BAs in serum and liver became more hydrophilic between 3 and 27 months. In female mice, (1) the mRNAs of hepatic BA uptake transporters, the Na+/taurocholate cotransporting polypeptide (Ntcp) and the organic anion transporting polypeptide 1b2 (Oatp1b2), decreased after 12 months, and similar trends were observed for their proteins; (2) the mRNA of the rate-limiting enzyme for BA synthesis, cholesterol 7α-hydroxylase (Cyp7a1), increased from 3 to 9 months and remained high thereafter. However, in male mice, Ntcp, Oatp1b2, and Cyp7a1 mRNAs remained relatively constant with age. In summary, the current study shows gender-divergent profiles of BA concentrations and composition in serum and liver of mice during aging, which is likely due to the gender-divergent expression of BA transporters Ntcp and Oatp1b2 as well as the synthetic enzyme Cyp7a1.

Two loci on chromosome 9 control bile acid composition: evidence that a strong candidate gene, Cyp8b1, is not the culprit

An intercross between C57BL/6J and CASA/Rk mice was used to study the genetics of biliary bile acid composition. In parental strains, male C57BL/6J mice had significantly higher cholic acid (CA; 14%) and lower beta-muricholic acid (betaMC; 27%) than CASA/Rk mice, whereas females did not differ. However, quantitative trait locus analysis of F2 mice revealed no significant chromosome 9 loci in males but loci in females on chromosome 9 for percentage CA (%CA) at 72 centimorgan (cM) [logarithm of the odds (LOD) 5.89] and %betaMC at 54 cM (LOD 4.09). Chromosome 9 congenic and subcongenic strains representing CASA/Rk intervals 38-73 cM (9KK) and 68-73 cM (9DKK) on the C57BL/6J background were made. In 9KK and 9DKK males, %CA was increased and %betaMC was unchanged, whereas in 9KK but not 9DKK females, %CA was increased and %betaMC was decreased. Sterol 12alpha-hydroxylase (Cyp8b1) channels bile acid precursors into CA and maps at chromosome 9 (73 cM). However, there was no significant difference in Cyp8b1 mRNA or enzymatic activity between parental mice, parental-congenic-subcongenic mice, or high-low biliary %CA F2 mice. In summary, two chromosome 9 loci control sexually dimorphic effects on biliary bile acid composition: a distal (68-73 cM) major determinant in males, and a more proximal (38-68 cM) major determinant in females. In this intercross, Cyp8b1, a strong candidate, does not appear to be responsible.

Oxidative stress and apoptosis in fetal rat liver induced by maternal cholestasis. Protective effect of ursodeoxycholic acid

The sensitivity of fetal rat liver to maternal obstructive cholestasis during pregnancy (OCP), and the effect of ursodeoxycholic acid (UDCA) were investigated. UDCA was administered (i.g. 0.6 mg/kg b.wt./day) from day 14 to day 21 of pregnancy after maternal common bile duct ligation. Impairment in the activity of antioxidant enzymes, levels of total glutathione and GSH/GSSG ratio and the degrees of lipid peroxidation and protein carbonylation were similar in livers of OCP mothers and fetuses at term, despite hypercholanemia was milder in fetuses. Treatment of OCP rats with UDCA reduced maternal and fetal liver oxidative stress. Although maternal hypercholanemia was not corrected, fetal serum concentrations of major bile acids (except UDCA and beta-muricholic acid) were reduced. Fetal liver expression of key enzyme in bile acid synthesis, Cyp7a1, Cyp27 and Cyp8b1 was not affected by OCP or UDCA treatment. In OCP fetal livers, the relative expression of Bax-alpha and Bcl-2 and the activity of caspase-3, but not caspase-8, were increased. These changes were markedly reduced in fetuses of OCP animals treated with UDCA. OCP induced moderate fetal hypercholanemia but marked liver oxidative stress and apoptosis that were partly prevented by treatment of pregnant rats with UDCA.

Chemical synthesis of (22E)-3α,6β,7β-trihydroxy-5β-chol-22-en-24-oic acid and its taurine and glycine conjugates: a major bile acid in the rat

A method for the synthesis of Delta(22)-beta-muricholic acid (Delta(22)-beta-MCA), (22E)-3 alpha,6 beta,7 beta-trihydroxy-5 beta-chol-22-en-24-oic acid, and its taurine and glycine conjugates (Delta(22)-beta-muricholyltaurine and Delta(22)-beta-muricholylglycine) is described. The key intermediate, 3 alpha,6 beta,7 beta-triformyloxy-23,24-dinor-5 beta-cholan-22-al, was prepared from beta-muricholic acid (beta-MCA) via the 24-nor-22-ene and 24-nor-22,23-diol derivatives. Wittig reaction of the aldehyde with (carbomethoxymethylene) triphenylphosphorane and subsequent hydrolysis gave (unconjugated) Delta(22)-beta-MCA. Condensation reaction of the unconjugated acid with taurine or glycine methyl ester using diethylphosphorocyanide yielded the naturally occurring taurine or glycine conjugate (N-acylamidate) of Delta(22)-beta-MCA. These synthetic reference compounds are now available for investigation of the metabolism of beta-MCA by bacterial and hepatic enzymes in the rat and should also be useful as substrates for reductive deuteration or tritiation to give the 22,23-(2)H or (3)H-beta-MCA.

Effect of β-muricholic acid on the prevention and dissolution of cholesterol gallstones in C57L/J mice

This study investigated whether beta-muricholic acid, a natural trihydroxy hydrophilic bile acid of rodents, acts as a biliary cholesterol-desaturating agent to prevent cholesterol gallstones and if it facilitates the dissolution of gallstones compared with ursodeoxycholic acid (UDCA). For gallstone prevention study, gallstone-susceptible male C57L mice were fed 8 weeks with a lithogenic diet (2% cholesterol and 0.5% cholic acid) with or without 0.5% UDCA or beta-muricholic acid. For gallstone dissolution study, additional groups of mice that have formed gallstones were fed chow with or without 0.5% beta-muricholic acid or UDCA for 8 weeks. One hundred percent of mice fed the lithogenic diet formed cholesterol gallstones. Addition of beta-muricholic acid and UDCA decreased gallstone prevalence to 20% and 50% through significantly reducing biliary secretion rate, saturation index, and intestinal absorption of cholesterol, as well as inducing phase boundary shift and an enlarged Region E that prevented the transition of cholesterol from its liquid crystalline phase to solid crystals and stones. Eight weeks of beta-muricholic acid and UDCA administration produced complete gallstone dissolution rates of 100% and 60% compared with the chow (10%). We conclude that beta-muricholic acid is more effective than UDCA in treating or preventing diet-induced or experimental cholesterol gallstones in mice.

Absence of cecal secondary bile acids in gnotobiotic mice associated with two human intestinal bacteria with the ability to dehydroxylate bile acids in vitro.

Germ-free mice were orally inoculated with human intestinal 7alpha-dehydroxylating bacterial strains to evaluate their ability to transform bile acids in vivo. Three weeks after inoculation of the bacteria, cecal bile acids were examined. Among free-form bile acids, only beta-muricholic acid was detected in the cecal contents of gnotobiotic mice associated with Bacteroides distasonis strain K-5. No secondary bile acid was observed in the cecal contents of any of the gnotobiotic mice associated with 7alpha-dehydroxylating bacteria, Clostridium species strain TO-931 or Eubacterium species strain 36S.

Formation of hyodeoxycholic acid from muricholic acid and hyocholic acid by an unidentified gram-positive rod termed HDCA-1 isolated from rat intestinal microflora.

From the rat intestinal microflora we isolated a gram-positive rod, termed HDCA-1, that is a member of a not previously described genomic species and that is able to transform the 3alpha,6beta, 7beta-trihydroxy bile acid beta-muricholic acid into hyodeoxycholic acid (3alpha,6alpha-dihydroxy acid) by dehydroxylation of the 7beta-hydroxy group and epimerization of the 6beta-hydroxy group into a 6alpha-hydroxy group. Other bile acids that were also transformed into hyodeoxycholic acid were hyocholic acid (3alpha, 6alpha,7alpha-trihydroxy acid), alpha-muricholic acid (3alpha,6beta, 7alpha-trihydroxy acid), and omega-muricholic acid (3alpha,6alpha, 7beta-trihydroxy acid). The strain HDCA-1 could not be grown unless a nonconjugated 7-hydroxylated bile acid and an unidentified growth factor produced by a Ruminococcus productus strain that was also isolated from the intestinal microflora were added to the culture medium. Germfree rats selectively associated with the strain HDCA-1 plus a bile acid-deconjugating strain and the growth factor-producing R. productus strain converted beta-muricholic acid almost completely into hyodeoxycholic acid.

Bile acid synthesis in primary cultures of rat and human hepatocytes.

The regulation of hepatic bile acid formation is incompletely understood. Primary cultures of mammalian hepatocytes offer an opportunity to examine putative regulatory factors in relative isolation. Using rat and human hepatocytes in primary culture, we examined bile acid composition and the expression of the rate-limiting enzyme of formation, cholesterol 7alpha-hydroxylase. Control rat hepatocytes showed a declining bile acid production over 4 days, from 156 +/- 24 ng/mL (67% cholic acid) on day 1 to 55 +/- 11 ng/mL (55% cholic acid) on day 4. In addition to cholic acid, chenodeoxycholic acid, alpha-muricholic acid, and beta-muricholic acid were formed. Treatment with triidothyronine (T3) or dexamethasone alone had no significant effect on bile acid production. A combination of T3 and dexamethasone significantly increased the total bile acid production on day 4 (224 +/- 54 ng/mL) and resulted in a marked change in composition to 23% cholic acid and 77% non-12alpha-hydroxylated bile acids. Control rat hepatocytes had a cholesterol 7alpha-hydroxylase activity of 3.3 +/- 0.6 pmol/mg protein/min after 4 days in culture. Cells treated with the combination of dexamethasone and T3 had an activity of 16.4 +/- 3.6 pmol/mg protein/min. The cholesterol 7alpha-hydroxylase messenger RNA (mRNA) levels, determined by solution hybridization after 4 days of culture, showed results similar to those for the activity data; control cells had 5.3 +/- 0.9 cpm/microg total nucleic acids (tNAs). T3 or dexamethasone-treated cells did not differ from control cells, whereas the combination of T3 and dexamethasone increased the mRNA levels to 20.6 +/- 2.8 cpm/microg tNAs. In human hepatocytes, isolated from donor liver, bile acid formation increased from 206 +/- 79 ng/mL on day 2 to 1490 +/- 594 ng/mL on day 6 and then declined slightly. Cholic acid and chenodeoxycholic acid were formed, constituting about 80% and 20%, respectively. The combined addition of T3 and dexamethasone had a tendency to decrease rather than increase bile acid formation. Also, mRNA levels of the cholesterol 7alpha-hydroxylase increased severalfold in the human hepatocytes from day 2 to day 4 and then declined. The addition of T3 or dexamethasone did not effect the mRNA levels in any consistent way. It is noteworthy that the capacity of the cultured human hepatocytes to produce bile acids was higher than that of cultured rat hepatocytes, in spite of the fact that the production of bile acids in rat liver is 3- to 5-fold higher than that in human liver in vivo. It is also evident that while hormonal factors appear to regulate bile acid synthesis in the rat, no evidence for this was found in human hepatocytes. As the composition of bile acids secreted by human hepatocytes in primary culture closely resembles that found in vivo, this represents a useful model for further studies of the synthesis and regulation of bile acids.