Cholic Acid Levels Research Articles

Turnover of Bile Acids in Liver, Serum and Caecal Content by High-Fat Diet Feeding Affects Hepatic Steatosis in Rats

Abstract Background Bile acids (BAs) participate in lipid absorption and serve as metabolic regulatory factors in gut-liver communication. To date, there are no studies on the systemic patterns of BAs in the serum, liver, and gut in the same non-alcoholic fatty liver disease (NAFLD) model. Methods A targeted metabolomics approach and 16S rRNA sequencing were used to identify the profile of BAs and connection between BAs and microbiota. The role and mechanism of altered BAs on hepatic steatosis were investigated. Findings In the liver, the composition of taurocholic acid (TCA) was increased, but taurohyodeoxycholic acid (THDCA) and ursodeoxycholic acid (UDCA) were decreased. In the gut, the deconjugated form of TCA (cholic acid (CA)) was increased, while the deconjugated forms of THDCA (α-hyodeoxycholic acid (HDCA)) and ω-muricholic acid (ωMCA) were decreased. In the serum, the composition of TCA was increased, while both HDCA and THDCA were decreased. THDCA induced the gene expression of apolipoprotein, bile secretion-related proteins, and cytochrome P450 family but suppressed inflammatory response genes expression in steatotic hepatocytes by RNAseq analysis. THDCA ameliorated neutral lipid accumulation and improved insulin sensitivity in primary rat hepatocytes. The decreased HDCA level correlated with the level of Bacteroidetes, while the level of CA correlated with the levels of Firmicutes and Verrucomicrobia but correlated inversely with Bacteroidetes. Conclusion BAs profiles in the serum, liver and caecal content were altered in a rat NAFLD model, which may affect hepatic lipid accumulation and correlate with gut dysbiosis.

Hypocholesterolaemic action of Lactobacillus plantarum VJC38 in rats fed a cholesterol-enriched diet

This study was conducted to evaluate hypocholesterolaemic activity of probiotic strains Lactobacillus plantarum VJC38 and VJI21 in Wistar albino rats fed a cholesterol-enriched diet. The experimental animals were divided into five groups (n = 6) viz., normal diet control group (NDC), hypercholesterolaemic diet (HD) control group (HDC), HD supplemented with 3 × 108 CFU/ml of L. plantarum VJC38 group (HD-C38), HD supplemented with 3 × 108 CFU/ml of L. plantarum VJI21 group (HD-I21), and HD supplemented with 3 × 108 CFU/ml of L. rhamnosus GG group (HD-GG) as positive control. Animals were administered bacterial culture by oral gavage once daily for 45 days. After trial, animals were sacrificed and blood samples were collected. Serum total cholesterol (T-CHO), triglyceride (TG), high-density lipoprotein (HDL) cholesterol, glucose, glutamyl pyruvate transaminase (GPT), and glutamyl oxaloacetate transaminase (GOT) levels were determined. Serum low-density lipoprotein (LDL) cholesterol levels were estimated using the Friedewald’s equation. Liver and fecal lipid contents and fecal cholic acids were measured. Serum T-CHO levels were significantly decreased by 15.6 and 17.4% in the HD-GG and HD-C38 groups, respectively, but not in the HD-I21 group compared with HDC group (P < 0.05). HD-GG and HD-C38 groups showed 26.3 and 27.2% reduction in serum LDL cholesterol, respectively when compared with HDC group (P < 0.05). Serum LDL cholesterol levels in HD-I21 group were not significantly different from HDC group. Serum TG levels in the HD-GG and HD-C38 were decreased by 14.2 and 22.8%, respectively compared with HDC group (P < 0.05). Liver T-CHO and TG levels in the HD-GG, HD-C38, and HD-I21 were reduced significantly compared with the HDC group (P < 0.05). Atherogenic coefficient values of HD-GG, HD-C38, and HD-21 were significantly decreased compared with HDC group (P < 0.05). Serum GPT levels in the HD-GG, HD-C38, and HD-I21 were decreased by 20.6, 10.9, and 20.6%, respectively, vs. the HDC group. Serum GOT levels were not significantly different among the groups. Serum glucose levels were significantly low in HD-GG, HD-C38, and HD-I21 compared with HDC group (P < 0.05). Fecal cholesterol and cholic acid levels were significantly higher in the HD-C38 and HD-GG groups than other groups (P < 0.05). This study suggests that L. plantarum VJC38 exhibits hypocholesterolaemic effect through hydrolysis of conjugated bile acids in the small intestine and excretion of cholesterol in feces. Lactobacillus plantarum VJC38 could be used as a potential cholesterol-lowering probiotic after validation of the hypocholesterolaemic activity in placebo-controlled human clinical trials.

Fecal Microbiota Transplantation Can Alleviate Gastrointestinal Transit in Rats with High-Fat Diet-Induced Obesity via Regulation of Serotonin Biosynthesis.

Aim We tested the hypothesis that fecal microbiota transplantation (FMT) could regulate the biotransformation of bile acids, such as deoxycholic acid (DCA) and cholic acid (CA), which in turn regulate the biosynthesis of serotonin in the gut and relieve gastrointestinal dysmotility in high-fat diet- (HFD-) induced obesity in rats. Methods Male Sprague-Dawley rats were randomly divided into the control diet group, HFD group, and HFD-fed with receiving FMT. HFD was fed for 12 weeks. At the end of two-week HFD, FMT was carried out for two weeks. The gastrointestinal transit, serotonin concentration, the expression of tryptophan hydroxylase 1 (TPH1) and serotonin reuptake transporter (SERT), and the levels of bile acids in intestinal contents were examined. Results Compared with the control group, the gastrointestinal transit and small intestinal serotonin concentration of HFD-fed rats were increased. In HFD-fed rats, TPH1 protein expression was increased significantly, while SERT protein expression was decreased, but not significant. The levels of CA and DCA in intestinal contents were also significantly increased in HFD-fed rats compared with the control group. After HFD-fed rats receiving FMT treatment, the gastrointestinal transit, small intestinal serotonin concentration, and TPH1 expression were decreased, while SERT expression was not affected. Moreover, the levels of CA and DCA in intestinal contents were also decreased. Conclusions FMT could alleviate small intestinal transit in the HFD-fed rats by regulating the serotonin biosynthesis. In this process, CA and DCA may be related to the regulation of synthesis of serotonin.

ZYDG2, a Potent, Selective, and Safe GPR40 Agonist for Treatment of Type 2 Diabetes

GPR40 is a G-protein-coupled receptor predominantly expressed in pancreatic β-cells. Agonists of GPR40 are known to stimulate insulin secretion and reduces circulating glucose levels in a glucose-dependent manner. TAK-875, a GPR40 small molecule agonist developed by Takeda Pharmaceuticals showed antidiabetic efficacy in animals and humans, but its development was terminated in phase 3 due to adverse liver effects. ZYDG2 is a safer GPR40 agonist that has shown desirable profile in preclinical studies. ZYDG2 was identified as a potent and selective agonist for GPR40, exhibiting EC50 of 13 nM and 41 nM for hGPR40, in HEK-293 cell-based Ca2+ mobilization assay and IP1 ELISA assays respectively. It increased insulin secretion and showed dose-dependent improvement in glucose tolerance test in n-STZ Wistar rats, DIO mice and db/db mice. It also showed anti-hyperglycemic effects in rats unresponsive to sulfonylureas. No tachyphylaxis was observed after repeated administration for 15 weeks in n-STZ rats. ZYDG2 showed glucose-stimulated insulin secretion as revealed by significant increase in glucose infusion rate in hyperglycemic clamp study using SD rats. ZYDG2 treatment was associated with significant rise in plasma total GLP1 levels in nondiabetic and diabetic animal models which was not observed in case of TAK-875. Since TAK-875 was terminated due to hepatotoxicity associated with bile acid transporter inhibition, this aspect was studied in details. TAK-875 inhibited the hBSEP and hMRP2 at efficacy concentrations whereas ZYDG2 does not show any inhibition (up to 300 µM) for hBSEP and hMRP2. TAK-875 also showed significantly increased cholic acid levels in Sprague Dawley rat which was not shown by ZYDG2. The maximum tolerated dose for ZYDG2 was 2000 mg/kg and 28 days NOAEL was 300 mg/kg. This clearly demonstrates that ZYDG2 has a great potential to become a safe and effective candidate for treatment of type 2 diabetes. Disclosure M.R. Jain: None. S.R. Giri: None. C.J. Trivedi: None. B.B. Bhoi: None. A.C. Rath: None. R.M. Rathod: None. R. Sundar: None. D. Bandyopadhyay: None. R. Ramdhave: None. G.D. Patel: None. B.K. Srivastava: None. R.C. Desai: None.

Metabolomics-based evidence of the hypoglycemic effect of Ge-Gen-Jiao-Tai-Wan in type 2 diabetic rats via UHPLC-QTOF/MS analysis

Ethnopharmacological relevanceGe-Gen-Jiao-Tai-Wan (GGJTW) formula, derived from traditional Chinese herbal medicine, is composed of Pueraria montana var. lobata (Willd.) Sanjappa & Pradeep (Ge-Gen in Chinese), Coptis chinensis Franch (Huang-Lian), and Cinnamomum cassia (L.) J. Presl (Rou-Gui). GGJTW is used for treatment of diabetes in China, reflecting the potent hypoglycemic effect of its ingredients. However, little is known of the hypoglycemic effect of GGJTW and the underlying metabolic mechanism. Aim of the studyThis study aimed to investigate the hypoglycemic effect of GGJTW in type 2 diabetic rats and the metabolic mechanism of action. Materials and methodsUltra high-performance liquid chromatography coupled with quadrupole-time-of-flight tandem mass spectrometry (UHPLC-QTOF/MS)-based metabolomics approach was used for monitoring hyperglycaemia induced by high-sugar high-fat fodder and streptozotocin (STZ), and the protective effect of GGJTW. Dynamic fasting blood glucose (FBG) levels, body weight, and biochemical parameters, including lipid levels, hepatic-renal function, and hepatic histopathology were used to confirm the hyperglycaemic toxicity and attenuation effects. An orthogonal partial least squared-discriminant analysis (OPLS-DA) approach highlighted significant differences in the metabolome of the healthy control, diabetic, and drug-treated rats. The metabolomics pathway analysis (MetPA) and Kyoto encyclopedia of genes and genomes (KEGG) database were used to investigate the underlying metabolic pathways. ResultsMetabolic profiling revealed 37 metabolites as the most potential biomarker metabolites distinguishing GGJTW-treated rats from model rats. Most of the metabolites were primarily associated with bile acid metabolism and lipid metabolism. The most critical pathway was primary bile acid biosynthesis pathway involving the up-regulation of the levels of cholic acid (CA), chenodeoxycholic acid (CDCA), taurocholic acid (TCA), glycocholic acid (GCA), taurochenodesoxycholic acid (TCDCA), and taurine. ConclusionsThe significantly-altered metabolite levels indicated the hypoglycemic effect of GGJTW on diabetic rats and the underlying metabolic mechanism. This study will be meaningful for the clinical application of GGJTW and valuable for further exploration of the mechanism.

Free oxysterols and bile acids including conjugates - Simultaneous quantification in human plasma and cerebrospinal fluid by liquid chromatography-tandem mass spectrometry

A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI(+)-MS/MS) assay was developed and qualified for analyzing 35 analytes of the cholesterol metabolism, including free cholesterol, 17 free, non-esterified oxysterols and 17 free and conjugated bile acids in plasma and cerebrospinal fluid. As internal standards, 25 commercially available stable deuterium-labeled analogs of the analytes were used. Pre-analytical investigations included stability tests of analyte concentrations affected by different anticoagulation additives: lithium heparin-, citrate-, EDTA-K3-stabilized plasma and serum, and the stability in EDTA whole blood at RT. This LC-ESI(+)-MS/MS method was successfully applied for the analysis of paired serum/cerebrospinal fluid samples of patients with and without blood-brain barrier disturbance, as well as of 100 plasma samples of a LIFE-Adult study sub-cohort.A fast and simple sample preparation including protein precipitation and on-line solid-phase extraction was developed. As little as 55 μL of human plasma/serum or cerebrospinal fluid were needed for the analysis. It was possible to separate isomeric oxysterols and bile acids within 23 min using a C18 core-shell column. The assay is capable of quantifying in a linear range of 0.8–250 ng mL−1 for free hydroxycholesterols, 0.2–10 ng mL−1 for dihydroxycholesterols, 0.2–500 ng mL−1 for bile acids and 16–2000 μg mL−1 for cholesterol with acceptable accuracy and precision.In cerebrospinal fluid one free oxysterols, five free and five conjugated bile acids could be quantified. No significant differences between patients with and without blood-brain barrier disturbance were obtained.In the LIFE-Adult sub-cohort two free oxysterols, four free and seven conjugated bile acids could be quantified in EDTA plasma. Men showed significantly higher concentrations of 26-OHC than women (p = 0.035). Furthermore, in women lower levels of cholic acid, glycocholic acid, glycodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, glycoursodeoxycholic acid, glycolithocholic acid and higher levels of taurocholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid/hyodeoxycholic acid were quantified.

Metformin impacts cecal bile acid profiles in mice

Bile acids (BAs) are major components of bile synthesized from cholesterol and take part in the digestion of dietary lipids, as well as having signaling functions. They undergo extensive microbial metabolism inside the gastrointestinal tract. Here, we present a method of ultra-high pressure liquid chromatography coupled to ion trap mass spectrometry for quantification of 45 BAs in mouse cecum. The system was validated in regard to sensitivity with limits of detection and quantification (0.6–24.9 nM), interday accuracy (102.4%), interday precision (15.2%), recovery rate (74.7%), matrix effect (98.2%) and carry-over effect (<1.1%). Afterwards, we applied our method to investigate the effect of metformin on BA profiles. Diabetic mice were treated with metformin for 1 day or 14 days. One day of treatment resulted in a significant increase of total BA concentration (2.7-fold increase; db/db metformin 5.32 μmol/g, db/db control mice 1.95 μmol/g), most notable in levels of 7-oxodeoxycholic, 3-dehydrocholic and cholic acid. We observed only minor impact on BA metabolism after 14 days of metformin treatment, compared to the single treatment. Furthermore, healthy wild type mice had elevated concentrations of allocholic and ω-muricholic acid compared to diabetic mice. Our method proved the applicability of profiling BAs in cecum to investigate intestinal BA metabolism in diabetes and pharmacological applications.

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.

A Simple and Novel Electrochemical Sensor Based on Phosphomolybdic‐Polypyrrole Film Modified Electrode for Highly Sensitive Detection of Cholic Acid

AbstractA simpe electrochemical sensor for detection of cholic acid (CA) was designed by modifying phosphomolybdate (PMo12) doped polypyrrole (PPy) film on glassy carbon electrode (PMo12‐PPy/GCE). The electrochemical behavior of CA on PMo12‐PPy/GCE was investigated by cyclic voltammetry and 0.5 order differential voltammetry. The results indicated that CA had high inhibitory activity toward the peak currents of PMo12‐PPy/GCE. The reduction peak currents were linearly related to the logarithmic value of the concentration of CA from 1.0×10−7 to 1.0×10−3 mol/L with a low detection limit of 1.0×10−8 mol/L. The developed sensor exhibited excellent sensitivity, selectivity and stability for detection of CA, and it could be successfully applied to detect the level of CA in the urine samples. Moreover, the response mechanism of CA on the PMo12‐PPy/GCE was discussed in detail.

Cholic acid therapy in Zellweger spectrum disorders.

IntroductionZellweger spectrum disorders (ZSDs) are characterized by a failure in peroxisome formation, caused by autosomal recessive mutations in different PEX genes. At least some of the progressive and irreversible clinical abnormalities in patients with a ZSD, particularly liver dysfunction, are likely caused by the accumulation of toxic bile acid intermediates. We investigated whether cholic acid supplementation can suppress bile acid synthesis, reduce accumulation of toxic bile acid intermediates and improve liver function in these patients.MethodsAn open label, pretest-posttest design study was conducted including 19 patients with a ZSD. Participants were followed longitudinally during a period of 2.5 years prior to the start of the intervention. Subsequently, all patients received oral cholic acid and were followed during 9 months of treatment. Bile acids, peroxisomal metabolites, liver function and liver stiffness were measured at baseline and 4, 12 and 36 weeks after start of cholic acid treatment.ResultsDuring cholic acid treatment, bile acid synthesis decreased in the majority of patients. Reduced levels of bile acid intermediates were found in plasma and excretion of bile acid intermediates in urine was diminished. In patients with advanced liver disease (n = 4), cholic acid treatment resulted in increased levels of plasma transaminases, bilirubin and cholic acid with only a minor reduction in bile acid intermediates.ConclusionsOral cholic acid therapy can be used in the majority of patients with a ZSD, leading to at least partial suppression of bile acid synthesis. However, caution is needed in patients with advanced liver disease due to possible hepatotoxic effects.Electronic supplementary materialThe online version of this article (doi:10.1007/s10545-016-9962-9) contains supplementary material, which is available to authorized users.

Bile Acids and Dysbiosis in Non-Alcoholic Fatty Liver Disease.

Background & AimsNon-alcoholic fatty liver disease (NAFLD) is characterized by dysbiosis. The bidirectional effects between intestinal microbiota (IM) and bile acids (BA) suggest that dysbiosis may be accompanied by an altered bile acid (BA) homeostasis, which in turn can contribute to the metabolic dysregulation seen in NAFLD. This study sought to examine BA homeostasis in patients with NAFLD and to relate that with IM data.MethodsThis was a prospective, cross-sectional study of adults with biopsy-confirmed NAFLD (non-alcoholic fatty liver: NAFL or non-alcoholic steatohepatitis: NASH) and healthy controls (HC). Clinical and laboratory data, stool samples and 7-day food records were collected. Fecal BA profiles, serum markers of BA synthesis 7-alpha-hydroxy-4-cholesten-3-one (C4) and intestinal BA signalling, as well as IM composition were assessed.Results53 subjects were included: 25 HC, 12 NAFL and 16 NASH. Levels of total fecal BA, cholic acid (CA), chenodeoxycholic acid (CDCA) and BA synthesis were higher in patients with NASH compared to HC (p<0.05 for all comparisons). The primary to secondary BA ratio was higher in NASH compared to HC (p = 0.004), but ratio of conjugated to unconjugated BAs was not different between the groups. Bacteroidetes and Clostridium leptum counts were decreased in in a subset of 16 patients with NASH compared to 25 HC, after adjusting for body mass index and weight-adjusted calorie intake (p = 0.028 and p = 0.030, respectively). C. leptum was positively correlated with fecal unconjugated lithocholic acid (LCA) (r = 0.526, p = 0.003) and inversely with unconjugated CA (r = -0.669, p<0.0001) and unconjugated CDCA (r = - 0.630, p<0.0001). FGF19 levels were not different between the groups (p = 0.114).ConclusionsIn adults with NAFLD, dysbiosis is associated with altered BA homeostasis, which renders them at increased risk of hepatic injury.

Beneficial effects of voglibose administration on body weight and lipid metabolism &lt;i&gt;via&lt;/i&gt; gastrointestinal bile acid modification

This study was designed with the goal of examining the effects of voglibose administration on body weight and lipid metabolism and underlying mechanism high fat diet-induced obese mice. Male C57BL/6 mice were randomly assigned to one of four groups: a control diet (CTL), high-fat diet (HF), high-fat diet supplemented with voglibose (VO), and high fat diet pair-fed group (PF). After 12 weeks, the following characteristics were investigated: serum lipid and glucose levels, serum polar metabolite profiles, and expression levels of genes involved in lipid and bile acid metabolism. In addition, pyrosequencing was used to analyze the composition of gut microbiota found in feces. Total body weight gain was significantly lower in the VO group than in the CTL, HF, and PF groups. The VO group exhibited improved metabolic profiles including those of blood glucose, triglyceride, and total cholesterol levels. The 12-week voglibose administration decreased the ratio of Firmicutes to Bacteroidetes found in feces. Circulating levels of taurocholic and cholic acid were significantly higher in the VO group than in the HF and CTL groups. Deoxycholic acid levels tended to be higher in the VO group than in the HF group. Voglibose administration downregulated expression levels of CYP8B1 and HNF4α genes and upregulated those of PGC1α, whereas FXRα was not affected. Voglibose administration elicits changes in the composition of the intestinal microbiota and circulating metabolites, which ultimately has systemic effects on body weight and lipid metabolism in mice.

Special features of bile acids spectral composition in patients with hyperuricemia in combination with obesity and non-alcoholic steatohepatitis

Aim. To study changes of the bile acids spectral composition in patients with hyperuricemia combined with obesity and non-alcoholic steatohepatitis using thin-layer chromatography.Materials and methods. We examined 146 patients separated in two groups. The main group included 84 patients with hyperurecimia combined with obesity and non-alcoholic steatohepatitis. 62 patients with non-alcoholic steatohepatitis without additional factors of metabolic syndrome formed the control group. The non-alcoholic steatohepatitis (NASH) was diagnosed on the base of criteria of exclusion of the chronic diffuse disease of liver of viral, autoimmune, inherited and medicamental genesis as a cause of cytolytic syndrome and also increase of exogeneity and decrease of sound conductivity of the liver according to the results of ultrasound examination.Results. Examined patients with hyperurecemia combined with NASH and obesity demonstrated the reliable increase of cholic acid level in cystic bile in 2,9 times (р&lt;0,001) and deoxycholic acid level in 2,6 times (р&lt;0,001). We observed decrease of taurocholic acid in cystic bile in 1,4 times (р&lt;0,001) and decrease of glycocholic acid in 2,1 times (р&lt;0,01). We noticed an increase of index of taurohenodeoxycholic and taurodeoxycholic acids mixture in 1,5 times (р&lt;0,05) and also glycohenodeoxycholic and glycodeoxycholic ones in 1,3 times (р&lt;0,01).Conclusions. So during the research there were demonstrated changes of spectral composition of bile acids in patients with hyperuricemia combined with obesity and non-alcoholic steatohepatitis. There was demonstrated an importance of defining the bile acids spectrum with the method of thin-layer chromatography for further prevention of cholelithiasis development

Oxidative stress markers, secondary bile acids and sulfated bile acids classify the clinical liver injury type: Promising diagnostic biomarkers for cholestasis

Clinicians sometimes encounter difficulty in choosing a therapeutic strategy due to the uncertainty regarding the type of liver injury. In particular, cholestasis is difficult to diagnose by conventional markers at an early stage of disease. The aim of this study was to identify promising biomarkers for distinguishing the symptom-based types of liver injury (e.g. hepatocellular injury, cholestasis), which was derived from a rigorously statistical perspective. The associations between diagnostic biomarkers (e.g. bile acid components, oxidative stress markers and liver fibrosis markers) and the liver injury types were assessed by a multiple logistic regression analysis using 304 blood samples from patients with liver disease. As a result, reductions in the lithocholic acid (LCA) and deoxycholic acid (DCA) levels, and elevation of the serum sulfated bile acid (SSBA), liver fibrosis marker IV collagen (type IV collagen), hyaluronic acid (HA) and reactive oxygen species (ROS) levels were all significantly associated with cholestasis. On the other hand, elevations in the LCA and type IV collagen levels, and a reduction in the ursodeoxy cholic acid (UDCA) level, were significantly associated with hepatocellular injury. The receiver operating characteristic (ROC) analyses showed that the largest area under the ROC curve (AUC) was found for ROS, followed by DCA, HA, LCA, SSBA and type IV collagen in the cholestatic-type cases. These results indicated that ROS, the secondary bile acid levels such as DCA and LCA, and SSBA are promising biomarkers for cholestasis and for classifying the type of liver injuries. This comprehensive approach will allow for an accurate diagnosis, which will facilitate the selection of an appropriate therapy at the onset of disease.

Serum Metabolomics Reveals Cholic Acid, Chenodeoxycholic Acid, and Taurochenodeoxycholic Acid as Potential Biomarkers for Hypothyroid Rats

Hypothyroidism decreases energy metabolism including carbohydrate and lipid metabolism and protein synthesis, due to reduced serum levels of the thyroid hormones thyroxine (T4) and triiodothyronine (T3). Although many endogenous serum metabolites are influenced by hypothyroidism, serum metabolomic profiling has rarely been applied to the study of hypothyroidism. In the present study, we investigated potential biomarkers for hypothyroidism using serum metabolomics, and then measured serum levels of these endogenous metabolites using an analytical method: ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. There was a significant difference in the metabolic profiles of thyroidectomized (Tx) and normal rats. We found that many bile acid (BA) levels were significantly changed in serum of Tx rats. Simultaneous measurement of 12 different BAs in serum revealed that cholic acid (CA), chenodeoxycholic acid (CDCA), and taurochenodeoxycholic acid (TCDCA) levels significantly increased in Tx rats by approximately 25-fold, 11-fold, and 3-fold, respectively, compared with those of control rats. In Tx rats with active hormone T3 replacement, serum T3 levels were returned to physiological levels. However, these changes in BA levels were maintained at a high level. These results indicate that T3 replacement does not normalize the thyroid hormonal milieu. Thus, increased CA, CDCA, and TCDCA levels in serum after Tx may be a homeostatic response to not only T3 but also pro-thyroid hormone T4 deficiency. This study is the first to report that CA, CDCA, and TCDCA may be potential biomarkers for hypothyroidism and the efficacy of thyroid hormone replacement therapy in hypothyroidism.

Impact of physiological levels of chenodeoxycholic acid supplementation on intestinal and hepatic bile acid and cholesterol metabolism in Cyp7a1-deficient mice

Mice deficient in cholesterol 7α-hydroxylase (Cyp7a1) have a diminished bile acid pool (BAP) and therefore represent a useful model for investigating the metabolic effects of restoring the pool with a specific BA. Previously we carried out such studies in Cyp7a1−/− mice fed physiological levels of cholic acid (CA) and achieved BAP restoration, along with an increased CA enrichment, at a dietary level of just 0.03% (w/w). Here we demonstrate that in Cyp7a1−/− mice fed chenodeoxycholic acid (CDCA) at a level of 0.06% (w/w), the BAP was restored to normal size and became substantially enriched with muricholic acid (MCA) (>70%), leaving the combined contribution of CA and CDCA to be <15%. This resulted in a partial to complete reversal of the main changes in cholesterol and BA metabolism associated with Cyp7a1 deficiency such as an elevated rate of intestinal sterol synthesis, an enhanced level of mRNA for Cyp8b1 in the liver, and depressed mRNA levels for Ibabp, Shp and Fgf15 in the distal small intestine. When Cyp7a1−/− and matching Cyp7a1+/+ mice were fed a diet with added cholesterol (0.2%) (w/w), either alone, or also containing CDCA (0.06%) (w/w) or CA (0.03%) (w/w) for 18days, the hepatic total cholesterol concentrations (mg/g) in the Cyp7a1−/− mice were 26.9±3.7, 16.4±0.9 and 47.6±1.9, respectively, vs. 4.9±0.4, 5.0±0.7 and 6.4±1.9, respectively in the corresponding Cyp7a1+/+ controls. These data affirm the importance of using moderate levels of dietary BA supplementation to elicit changes in hepatic cholesterol metabolism through shifts in BAP size and composition.

Diet supplementation with cholic acid promotes intestinal epithelial proliferation in rats exposed to γ-radiation

Consumption of a high-fat diet increases some secondary bile acids (BAs) such as deoxycholic acid (DCA) in feces. DCA is derived from cholic acid (CA), a primary BA. We evaluated intestinal epithelial proliferation and BA metabolism in response to oral administration of cholic acid (CA) in rats to determine the influence of a CA diet on the responses of gut epithelia to γ-rays. WKAH/HkmSlc rats were divided into two dietary groups: control diet or CA-supplemented (2g/kg diet) diet. Some of the rats from each group were irradiated with γ-rays, and epithelial cell proliferation in the colon was analyzed histochemically. Unirradiated CA-fed rats had high levels of DCA and CA in the sera, as well as the presence of taurocholic acid in their feces. Significant increases were observed in both epithelial proliferation and the number of epithelial cells in the colon of the CA-fed rats, and this effect was observed at 8 weeks after γ-ray exposure. Furthermore, extracts from both cecal contents and sera of the unirradiated CA-fed rats promoted proliferation of IEC-6 cells. These results indicate that BAs in enterohepatic circulation promote proliferation and survival of the intestinal epithelium after receiving DNA damage.

Decreased bile-acid synthesis in livers of hepatocyte-conditional NADPH-cytochrome P450 reductase-null mice results in increased bile acids in serum.

NADPH-cytochrome P450 reductase (Cpr) is essential for the function of microsomal cytochrome P450 monooxygenases (P450), including those P450s involved in bile acid (BA) synthesis. Mice with hepatocyte-specific deletion of NADPH-cytochrome P450 reductase (H-Cpr-null) have been engineered to understand the in vivo function of hepatic P450s in the metabolism of xenobiotics and endogenous compounds. However, the impact of hepatic Cpr on BA homeostasis is not clear. The present study revealed that H-Cpr-null mice had a 60% decrease in total BA concentration in liver, whereas the total BA concentration in serum was almost doubled. The decreased level of cholic acid (CA) in both serum and livers of H-Cpr-null mice is likely due to diminished enzyme activity of Cyp8b1 that is essential for CA biosynthesis. Feedback mechanisms responsible for the reduced liver BA concentrations and/or increased serum BA concentrations in H-Cpr-null mice included the following: 1) enhanced alternative BA synthesis pathway, as evidenced by the fact that classic BA synthesis is diminished but chenodeoxycholic acid still increases in both serum and livers of H-Cpr-null mice; 2) inhibition of farnesoid X receptor activation, which increased the mRNA of Cyp7a1 and 8b1; 3) induction of intestinal BA transporters to facilitate BA absorption from the intestine to the circulation; 4) induction of hepatic multidrug resistance-associated protein transporters to increase BA efflux from the liver to blood; and 5) increased generation of secondary BAs. In summary, the present study reveals an important contribution of the alternative BA synthesis pathway and BA transporters in regulating BA concentrations in H-Cpr-null mice.

Development of a highly sensitive MIP based-QCM nanosensor for selective determination of cholic acid level in body fluids

Determination of cholic acid is very important and necessary in body fluids due to its both pharmaceutical and clinical significance. In this study, a quartz crystal microbalance (QCM) nanosensor, which is imprinted cholic acid, has been developed for the assignation of cholic acid. The cholic acid selective memories have been generated on QCM electrode surface by using molecularly imprinted polymer (MIP) based on methacryloylamidohistidine-copper (II) (MAH-Cu(II)) pre-organized monomer. The cholic acid imprinted nanosensor was characterized by atomic force microscopy (AFM) and then analytical performance of the cholic acid imprinted QCM nanosensor was studied. The detection limit was found to be 0.0065μM with linear range of 0.01–1000μM. Moreover, the high value of Langmuir constant (b) (7.3*105) obtained by Langmuir graph showed that the cholic acid imprinted nanosensor had quite strong binding sites affinity. At the last step of this procedure, cholic acid levels in body fluids were determined by the prepared imprinted QCM nanosensor.

A Bayes Study of Bile Acid Constituents on Cholelithiasis and Carcinoma of the Gallbladder

Background: Significantly high concentration of secondary bile acids and low concentration of primary acids are reported by the surgeons in patients with gallbladder carcinoma (GBC) compared to control and cholelithiasis groups. Aim: To examine the effect of primary and secondary bile acids on the development of cholelithiasis and GBC and to investigate the association, if any, among the two groups of bile acids. Methods: The study uses two groups of patients at a time selected in accordance with some pre-fixed inclusion and exclusion criteria. Informed consent was obtained from all patients. The demographic characteristics such as mean age, sex ratio and body mass index, etc. are obtained for the selected groups of patients. The study defines dichotomous responses and the four bile acid constituents, namely cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and lithocholic acid (LCA), as the predictors. It then assumes logistic regression model to associate the binary responses with the predictors by using probability scores. Bayes analysis is developed using Markov chain Monte Carlo (MCMC) pack in R software for the posterior simulation. Results: Twenty one cholelithiasis patients and twenty patients in each of control and GBC groups are studied. It is seen that a unit decrement in the level of CA (CDCA) increases the log (odds ratio) for cholelithiasis by an amount of 0.49 (0.14) and odds ratio by almost 1.5 (1.12). Similarly, a unit increment in the level of DCA (LCA) provides the log (odds ratio) for cholelithiasis as 0.18 (1.3) and odds ratio as 1.16 (2.95). Comparing GBC with control population, it is noted that a unit decrease in the level of CA (CDCA) in the control population increases the log (odds ratio) for GBC by an amount of 1.16(0.26) and odds ratio by almost 2.63 (1.24) times. Similarly, the log(odds ratio) for GBC increases by 0.77(1.94) and the odds ratio increases by 1.9 (5.0) for the unit increment in the level of DCA(LCA). Conclusions: The study observes relatively high variations in the primary and secondary bile acids in the cholelithiasis and GBC groups as compared with the control group. It, in turn, reflects strong association among the two categories of bile acids in gallbladder diseases.