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Background: Dysregulation of bile acids (BAs), as important signalling molecules in regulating lipid and glucose metabolism, contributes to the development of non-alcoholic fatty liver disease (NAFLD). However, static BA profiles during fasting may obscure certain pathogenetic aspects. In this study, we investigate the dynamic alterations of BAs in response to an oral glucose tolerance test (OGTT) among children with NAFLD., Methods: We recruited 230 subjects, including children with overweight/obesity, or complicated with NAFLD, and healthy controls. Serum BAs, 7-hydroxy-4-cholesten-3-one (C4) and fibroblast growth factor 19 (FGF19) were quantified during OGTT. Clinical markers related to liver function, lipid metabolism and glucose metabolism were assessed at baseline or during OGTT., Findings: Conjugated BAs increased while unconjugated ones decreased after glucose uptake. Most BAs were blunted in response to glucose in NAFLD (p > .05); only glycine and taurine-conjugated chenodeoxycholic acid (CDCA) and cholic acid (CA) were responsive (p < .05). Primary BAs were significantly increased while secondary BAs were decreased in NAFLD. C4 and FGF19 were significantly increased while their ratio FGF19/C4 ratio was decreased in NAFLD. The dynamic pattern of CDCA and taurine-conjugated hyocholic acid (THCA) species was closely correlated with glucose (correlation coefficient r = .175 and -.233, p < .05), insulin (r = .327 and -.236, p < .05) and c-peptide (r = .318 and -.238, p < .05). Among which, CDCA was positively associated with liver fat content in NAFLD (r = .438, p < .05). Additionally, glycochenodeoxycholic acid (GCDCA), CDCA and THCA were potential biomarkers to discriminate paediatric NAFLD from healthy controls and children with obesity., Interpretation: This study provides novel insights into the dynamics of BAs during OGTT in paediatric NAFLD. The observed variations in CDCA and HCA species were associated with liver dysfunction, dyslipidaemia and dysglycaemia, highlighting their potential roles as promising diagnostic and therapeutic targets in NAFLD., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Introduction: Bile acid diarrhoea (BAD) can occur due to disruption to the enterohepatic circulation such as following cholecystectomy. However, the mechanism behind this is as yet unknown. The aim of this study was to determine the rate of post-cholecystectomy diarrhoea and to assess whether FGF19 within the gallbladder was associated with the development of BAD., Methods: This was a prospective case-control study in which patients were assessed pre- and post- cholecystectomy (study group) and compared with patients also having laparoscopic surgery but not cholecystectomy (control group). Their bowel habits and a GIQLI questionnaire was performed to compare the pre- and post-operative condition of the two groups. Gallbladder tissue sample was tested for FGF19 and PPARα in the study group patients. A subset had serum lipid levels, FGF19 and C4 measurements., Results: Gallbladder PPAR α was found to have a significant correlation with stool consistency, with the lower the PPARα concentration the higher the Bristol stool chart number (i.e. looser stool). There were no significant correlation when assessing the effect of gallbladder FGF19 concentration on bowel habit, stool consistency, lipid levels, BMI or smoking. The study group showed a significant increase in triglycerides post-operatively, however there were no changes in cholesterol, HDL and LDL levels. Correlation of the increased triglyceride levels with stool consistency and frequency showed no significant results DISCUSSION AND CONCLUSION: We did not find any direct evidence that FGF19 levels within the gallbladder impact the development of post-cholecystectomy diarrhoea. There was however a significant increase in triglycerides postoperatively. There was also no correlation of bowel habits with PPARα suggesting the observed rise is independent of this pathway. Further work is required particularly relating to the gut microbiome to further investigate this condition., (© 2024. The Author(s).)

Background: Bile acid malabsorption is common in microscopic colitis, irritable bowel syndrome with diarrhea, and inflammatory bowel disease. We investigated the diagnostic accuracy of 7-alfa-hydroxy-4-cholesten-3-one and compared it with fibroblast growth factor-19 as biomarkers for bile acid malabsorption., Methods: We enrolled consecutively 109 chronic diarrhea patients with standard laboratory tests, fecal calprotectin, and endoscopy separated into six groups: n = 30 with active inflammatory bowel disease, n = 21 with inflammatory bowel disease in remission reporting >3 bowel movements per day, n = 21 with inflammatory bowel disease after surgery, n = 23 with irritable bowel syndrome with diarrhea, n = 14 with microscopic colitis and 11 healthy subjects (controls). We defined bile acid malabsorption as >3 bowel movements and lower fibroblast growth factor-19 (<60 pg/ml)., Results: Median levels of 7-alfa-hydroxy-4-cholesten-3-one in inflammatory bowel disease active were 53.1 ng/ml, inflammatory bowel disease remission were 52.2 ng/ml, inflammatory bowel disease after surgery were 85.7 ng/ml, irritable bowel syndrome with diarrhea were 7.5 ng/ml, microscopic colitis were 69.3 ng/ml, and healthy controls were 3.7 ng/ml. We estimate a 7-alfa-hydroxy-4-cholesten-3-one cutoff of 48.9 ng/ml with 82.6% sensitivity and 84.3% specificity for detecting bile acid malabsorption. Both 7-alfa-hydroxy-4-cholesten-3-one >48.9 ng/ml and fibroblast growth factor-19 (<60 pg/ml) were found in 52% of the patients, compared with those 8% of patients below this 7-alfa-hydroxy-4-cholesten-3-one cutoff (P < 0.001). Serum 7-alfa-hydroxy-4-cholesten-3-one correlated with the number of bowel movements/day (r = -0.709; P < 0.001) and correlated inversely with fibroblast growth factor-19 (r = -0.741; P < 0.001)., Conclusions: Serum 7-alfa-hydroxy-4-cholesten-3-one above 48.9 ng/ml and fibroblast growth factor-19 below 60 pg/ml identify patients with diarrhea likely attributable to bile acid malabsorption with high diagnostic accuracy and they can be used as screening biomarkers for bile acid malabsorption in microscopic colitis and inflammatory bowel disease., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Introduction: The diagnosis of bile acid diarrhea is often missed because the availability of the seleno-taurohomocholic acid (SeHCAT) test is limited. We aimed to compare the biomarkers 7α-hydroxy-4-cholesten-3-one (C4) and fibroblast growth factor 19 (FGF19) with the SeHCAT test., Methods: Patients with chronic diarrhea without intestinal resection referred for SeHCAT were prospectively recruited for this diagnostic accuracy study. Blood was sampled at fasting and after a stimulation meal with chenodeoxycholic acid. SeHCAT retention ≤10% defined bile acid diarrhea and >10% defined miscellaneous diarrhea. Receiver operating characteristics (ROC) were analyzed with SeHCAT as the gold standard. www.clinicaltrials.gov (NCT03059537)., Results: Patients with bile acid diarrhea (n = 26) had mean C4 of 30 ng/mL (95% confidence interval: 19-46) vs 8 (7-11; P < 0.001) in the miscellaneous diarrhea group (n = 45). Area under the ROC curve (ROCAUC) for C4 was 0.83 (0.72-0.93). C4 < 15 ng/mL had 85% (74%-96%) negative predictive value; C4 > 48 ng/mL had 82% (59%-100%) positive predictive value. Twenty patients had C4 values 15-48 ng/mL, of whom 11/20 had SeHCAT ≤10%. Median fasting FGF19 was 72 pg/mL (interquartile range: 53-146) vs 119 (84-240) (P = 0.004); ROCAUC was 0.71 (0.58-0.83). Stimulated FGF19 responses did not differ (P = 0.54)., Discussion: We identified C4 thresholds with clinically useful predictive values for the diagnosis of and screening for bile acid diarrhea in patients with chronic watery diarrhea. Further validation of the cutoff values with the placebo-controlled effect of sequestrant therapy is warranted (see Visual Abstract, Supplementary Digital Content 2, http://links.lww.com/AJG/B603).

Bile acids (BAs) are the central signals in enterohepatic communication, and they also integrate microbiota-derived signals into enterohepatic signaling. The tissue distribution and signaling pathways activated by BAs through natural receptors, farsenoid X receptor and G protein-coupled BA receptor 1 (GPBAR1, also known as Takeda G-coupled receptor 5), have led to a greater understanding of the mechanisms and potential therapeutic agents. BA diarrhea is most commonly encountered in ileal resection or disease, in idiopathic disorders (with presentation similar to functional diarrhea or irritable bowel syndrome with diarrhea), and in association with malabsorption such as chronic pancreatitis or celiac disease. Diagnosis of BA diarrhea is based on Se-homocholic acid taurine retention, 48-hour fecal BA excretion, or serum 7αC4; the latter being a marker of hepatic BA synthesis. BA diarrhea tends to be associated with higher body mass index, increased stool weight and stool fat, and acceleration of colonic transit. Biochemical markers of increased BA synthesis or excretion are available through reference laboratories. Current treatment of BA diarrhea is based on BA sequestrants, and, in the future, it is anticipated that farsenoid X receptor agonists may also be effective. The optimal conditions for an empiric trial with BA sequestrants as a diagnostic test are still unclear. However, such therapeutic trials are widely used in clinical practice. Some national guidelines recommend definitive diagnosis of BA diarrhea over empirical trial.

Background & Aims: Volixibat is an inhibitor of the apical sodium-dependent bile acid transporter (ASBT) that has been hypothesized to improve non-alcoholic steatohepatitis (NASH) by blocking bile acid reuptake and stimulating hepatic bile acid production. We studied the safety, tolerability and efficacy of volixibat in patients with NASH., Methods: In this double-blind, phase II dose-finding study, adults with ≥5% steatosis and NASH without cirrhosis (N = 197) were randomized to receive volixibat (5, 10 or 20 mg) or placebo once daily for 48 weeks. The endpoints of a predefined interim analysis (n = 80), at week 24, were: ≥5% reduction in MRI-proton density fat fraction and ≥20% reduction in serum alanine aminotransferase levels. The primary endpoint was a ≥2-point reduction in non-alcoholic fatty liver disease activity score without worsening fibrosis at week 48., Results: Volixibat did not meet either interim endpoint; the study was terminated owing to lack of efficacy. In participants receiving any volixibat dose, mean serum 7-alpha-hydroxy-4-cholesten-3-one (C4; a biomarker of bile acid synthesis) increased from baseline to week 24 (+38.5 ng/ml [SD 53.18]), with concomitant decreases in serum total cholesterol (-14.5 mg/dl [SD 28.32]) and low-density lipoprotein cholesterol (-16.1 mg/dl [SD 25.31]). These changes were generally dose-dependent. On histological analysis, a greater proportion of participants receiving placebo (38.5%, n = 5/13) than volixibat (30.0%, n = 9/30) met the primary endpoint. Treatment-emergent adverse events (TEAEs) were mainly mild or moderate. No serious TEAEs were related to volixibat. Diarrhoea was the most common TEAE overall and the most common TEAE leading to discontinuation., Conclusions: Increased serum C4 and decreased serum cholesterol levels provide evidence of target engagement. However, inhibition of ASBT by volixibat did not elicit a liver-related therapeutic benefit in adults with NASH., Lay Summary: A medicine called volixibat has previously been shown to reduce cholesterol levels in the blood. This study investigated whether volixibat could reduce the amount of fat in the liver and reduce liver injury in adults with an advanced form of non-alcoholic fatty liver disease. Volixibat did not reduce the amount of fat in the liver, nor did it have any other beneficial effect on liver injury. Participants in the study generally tolerated the side effects of volixibat and, as in previous studies, the main side effect was diarrhoea. These results show that volixibat is not an effective treatment for people with fatty liver disease., Clinical Trial Identifier: NCT02787304., Competing Interests: Conflict of interest PNN reports grants from Boehringer Ingelheim and Novo Nordisk and consultancy work (for the University of Birmingham) for Boehringer Ingelheim, Gilead Sciences Inc., Intercept Pharmaceuticals, Novo Nordisk, Pfizer, Poxel SA and Shire International. MP was an employee of Shire International (a Takeda company) during the conduct of the study. BF reports support from Shire International (a Takeda company). AS reports advisory board fees from Novartis and grants from Allergan, Cirius Therapeutics, Conatus Pharmaceuticals Inc., GENFIT, Gilead Sciences Inc., Intercept Pharmaceuticals, Madrigal Pharmaceuticals, Novartis, Novo Nordisk and Zydus Cadila. TH reports grants from Shire International (a Takeda company). H-ML was an employee of Shire International (a Takeda company) during the conduct of the study. MYS, HS, RH, PM, ZK and GPA have nothing to disclose. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: The bile acid (BA) pathway plays a role in regulation of food intake and glucose metabolism, based mainly on findings in animal models. Our aim was to determine whether the BA pathway is altered and correctable in human obesity and diabetes., Methods: We conducted 3 investigations: 1) BA receptor pathways were studied in NCI-H716 enteroendocrine cell (EEC) line, whole human colonic mucosal tissue and in human colonic EEC isolated by Fluorescence-activated Cell Sorting (ex vivo) from endoscopically-obtained biopsies colon mucosa; 2) We characterized the BA pathway in 307 participants by measuring during fasting and postprandial levels of FGF19, 7αC4 and serum BA; 3) In a placebo-controlled, double-blind, randomised, 28-day trial, we studied the effect of ileo-colonic delivery of conjugated BAs (IC-CBAS) on glucose metabolism, incretins, and lipids, in participants with obesity and diabetes., Findings: Human colonic GLP-1-producing EECs express TGR5, and upon treatment with bile acids in vitro, human EEC differentially expressed GLP-1 at the protein and mRNA level. In Ussing Chamber, GLP-1 release was stimulated by Taurocholic acid in either the apical or basolateral compartment. FGF19 was decreased in obesity and diabetes compared to controls. When compared to placebo, IC-CBAS significantly decreased postprandial glucose, fructosamine, fasting insulin, fasting LDL, and postprandial FGF19 and increased postprandial GLP-1 and C-peptide. Increase in faecal BA was associated with weight loss and with decreased fructosamine., Interpretations: In humans, BA signalling machinery is expressed in colonic EECs, deficient in obesity and diabetes, and when stimulated with IC-CBAS, improved glucose homeostasis. ClinicalTrials.gov number, NCT02871882, NCT02033876., Funding: Research support and drug was provided by Satiogen Pharmaceuticals (San Diego, CA). AA, MC, and NFL report grants (AA- C-Sig P30DK84567, K23 DK114460; MC- NIH R01 DK67071; NFL- R01 DK057993) from the NIH. JR was supported by an Early Career Grant from Society for Endocrinology., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Patients with Crohn's disease (CD) often have bile acid diarrhea (BAD), due to bile acid malabsorption following ileal resection (IR). Bile acid malabsorption increases production of 7α-hydroxy-4-cholesten-3-one (C4), a bile acid precursor. We investigated relationships between serum concentrations of C4 and BAD in patients with CD., Methods: We collected demographic data, serum samples, and information on the presence of diarrhea (>3 liquid bowel movements/day), as well as clinical, endoscopic, and histologic scores from 26 patients with CD and IR, 21 patients with CD without IR, and 37 patients with ulcerative colitis (UC). We compared serum concentrations of C4 and fibroblast growth factor 19 (FGF19) between groups. We performed area under the receiver operating characteristic curve (AUROC) analysis to identify the optimal cutoff C4 concentrations for the diagnosis of diarrhea attributable to bile acid malabsorption (BAD), defined as diarrhea and a serum concentration of FGF19 <60 pg/mL., Results: Patients with UC had a median serum C4 concentration of 11.8 ng/mL, whereas patients with CD and IR with ileitis (documented endoscopically) had a median concentration of 100.0 ng/mL (P compared to UC < .0001) and patients with CD and IR without ileitis had a median concentration of 51.6 ng/mL (P compared to UC < .001). Patients with CD without IR did not have a significantly higher median concentration of C4 than patients with UC (P = .71), regardless of ileitis (P = .34). When endoscopic findings were confirmed histologically, similar results were found to analyses using endoscopic findings alone. A higher proportion of patients with active UC had diarrhea (72.0% vs 0 patients with inactive UC; P < .001), but their median concentrations of C4 did not differ significantly from that of patients with inactive UC (12.1 ng/mL vs 9.7 ng/mL; P = .3). A cutoff concentration of C4 of 48.3 ng/mL or greater identified patients with diarrhea attributable to bile acid malabsorption with 90.9% sensitivity, 84.4% specificity, and an AUROC 0.94. A significantly higher proportion of patients with concentrations of C4 above this cutoff had BAD (50.0%) than below this cutoff (1.8%) (P < .001). When we analyzed only patients with diarrhea, a C4 cutoff of 48.3 ng/mL identified those with low FGF19 concentrations (<60 pg/mL) with 91% sensitivity and 95.5% specificity (AUROC, 0.99). Above this cutoff, 83.3% of patients had a serum concentration of FGF19 <60 pg/mL compared to 4.5% below this threshold (P < .0001). C4 concentrations correlated with the number of daily bowel movements (r = 0.41; P = .004) and correlated inversely with FGF19 concentrations (r = -0.72; P<.0001)., Conclusion: We observed significantly increased serum concentrations of C4 in patients with CD with IR, compared to patients with UC. A cutoff concentration of C4 above 48.3 ng/mL identifies patients with diarrhea likely attributable to bile acid malabsorption (BAD) with an AUROC value of 0.94. Increased serum levels of bile acid precursors identify patients with diarrhea and a low serum concentration of FGF19, and concentrations of C4 correlate with daily liquid bowel movements and correlate inversely with FGF19 concentrations. C4 may be a biomarker to identify patients with diarrhea attributable to bile acid malabsorption., (Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background & Aims: The nuclear farnesoid X receptor (FXR) agonist obeticholic acid (OCA) has been developed for the treatment of liver diseases. We aimed to determine whether OCA treatment increases the risk of gallstone formation., Methods: Twenty patients awaiting laparoscopic cholecystectomy were randomized to treatment with OCA (25 mg/day) or placebo for 3 weeks until the day before surgery. Serum bile acids (BAs), the BA synthesis marker C4 (7α-hydroxy-4-cholesten-3-one), and fibroblast growth factor 19 (FGF19) were measured before and after treatment. During surgery, biopsies from the liver and the whole bile-filled gallbladder were collected for analyses of gene expression, biliary lipids and FGF19., Results: In serum, OCA increased FGF19 (from 95.0 ± 8.5 to 234.4 ± 35.6 ng/L) and decreased C4 (from 31.4 ± 22.8 to 2.8 ± 4.0 nmol/L) and endogenous BAs (from 1,312.2 ± 236.2 to 517.7 ± 178.9 nmol/L; all p <0.05). At surgery, BAs in gallbladder bile were lower in patients that received OCA than in controls (OCA, 77.9 ± 53.6 mmol/L; placebo, 196.4 ± 99.3 mmol/L; p <0.01), resulting in a higher cholesterol saturation index (OCA, 2.8 ± 1.1; placebo, 1.8 ± 0.8; p <0.05). In addition, hydrophobic OCA conjugates accounted for 13.6 ± 5.0% of gallbladder BAs after OCA treatment, resulting in a higher hydrophobicity index (OCA, 0.43 ± 0.09; placebo, 0.34 ± 0.07, p <0.05). Gallbladder FGF19 levels were 3-fold higher in OCA patients than in controls (OCA, 40.3 ± 16.5 ng/L; placebo, 13.5 ± 13.1 ng/ml; p <0.005). Gene expression analysis indicated that FGF19 mainly originated from the gallbladder epithelium., Conclusions: Our results show for the first time an enrichment of FGF19 in human bile after OCA treatment. In accordance with its murine homolog FGF15, FGF19 might trigger relaxation and filling of the gallbladder which, in combination with increased cholesterol saturation and BA hydrophobicity, would enhance the risk of gallstone development., Lay Summary: Obeticholic acid increased human gallbladder cholesterol saturation and bile acid hydrophobicity, both decreasing cholesterol solubility in bile. Together with increased hepatobiliary levels of fibroblast growth factor 19, our findings suggest that pharmacological activation of the farnesoid X receptor increases the risk of gallstone formation. Clinical trial number: NCT01625026., (Copyright © 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: NGM282, an engineered analogue of the gut hormone FGF19, improves hepatic steatosis and fibrosis biomarkers in patients with non-alcoholic steatohepatitis (NASH). However, NGM282 increases serum cholesterol levels by inhibiting CYP7A1, which encodes the rate-limiting enzyme in the conversion of cholesterol to bile acids. Herein, we investigate whether administration of a statin can manage the cholesterol increase seen in patients with NASH receiving treatment with NGM282., Methods: In this phase II, open-label, multicenter study, patients with biopsy-confirmed NASH were treated with subcutaneous NGM282 once daily for 12 weeks. After 2 weeks, rosuvastatin was added in stepwise, biweekly incremental doses to a maximum of 40 mg daily. Both drugs were continued until the end of treatment at week 12. We evaluated plasma lipids, lipoprotein particles and liver fat content., Results: In 66 patients who received NGM282 0.3 mg (n = 23), NGM282 1 mg (n = 21), or NGM282 3 mg (n = 22), circulating cholesterol increased from baseline at week 2. Initiation of rosuvastatin resulted in rapid decline in plasma levels of total cholesterol and low-density lipoprotein cholesterol. At week 12, reductions from baseline in total cholesterol levels of up to 18% (p <0.001), low-density lipoprotein cholesterol of up to 28% (p <0.001), triglycerides of up to 34% (p <0.001) and an increase in high-density lipoprotein cholesterol of up to 16% (p <0.001), with similar changes in lipoprotein particles, were observed in these patients. Robust decreases from baseline in 7alpha-hydroxy-4-cholesten-3-one (p <0.001) and liver fat content (p <0.001) were also observed. Rosuvastatin was safe and well-tolerated when co-administered with NGM282 in patients with NASH., Conclusions: In this multicenter study, NGM282-associated elevation of cholesterol was effectively managed with rosuvastatin. Co-administration of rosuvastatin with NGM282 may be a reasonable strategy to optimize the cardiovascular risk profile in patients with NASH., Lay Summary: Non-alcoholic steatohepatitis (NASH) represents a large and growing public health concern with no approved therapy. NGM282, an engineered analogue of the gut hormone FGF19, reduces liver fat, liver injury and inflammation in patients with NASH. However, NGM282 increases cholesterol levels. Here we show that co-administration of a statin can manage the cholesterol increase seen in patients with NASH receiving treatment with NGM282, producing a favorable overall lipid profile., (Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Primary sclerosing cholangitis (PSC) is an inflammatory, cholestatic and progressively fibrotic liver disease devoid of effective medical intervention. NGM282, an engineered, non-tumorigenic FGF19 analogue, potently regulates CYP7A1-mediated bile acid homeostasis. We assessed the activity and safety of NGM282 in patients with PSC., Methods: In this double-blind, placebo-controlled phase II trial, 62 patients who had PSC confirmed by cholangiography or biopsy and an elevated alkaline phosphatase (ALP) >1.5 × the upper limit of normal were randomly assigned 1:1:1 to receive NGM282 1 mg, 3 mg or placebo once daily for 12 weeks. The primary outcome was the change in ALP from baseline to week 12. Secondary and exploratory outcomes included changes in serum biomarkers of bile acid metabolism and fibrosis. Efficacy analysis was by intention-to-treat., Results: At 12 weeks, there were no significant differences in the mean change from baseline in ALP between the NGM282 and placebo groups, and therefore, the primary endpoint was not met. However, NGM282 significantly reduced levels of 7alpha-hydroxy-4-cholesten-3-one (a marker of hepatic CYP7A1 activity, LS mean differences -6.2 ng/ml (95% CI -10.7 to -1.7; p = 0.008) and -9.4 ng/ml (-14.0 to -4.9; p <0.001) in the NGM282 1 mg and 3 mg groups, respectively, compared with placebo) and bile acids. Importantly, fibrosis biomarkers that predict transplant-free survival, including Enhanced Liver Fibrosis score and Pro-C3, were significantly improved following NGM282 treatment. Most adverse events were mild to moderate in severity, with gastrointestinal symptoms more frequent in the NGM282 treatment groups., Conclusions: In patients with PSC, NGM282 potently inhibited bile acid synthesis and decreased fibrosis markers, without significantly affecting ALP levels., Lay Summary: We present for the first time, the clinical and laboratory effects of a first-in-class, engineered analogue of the endocrine hormone FGF19 in patients with primary sclerosing cholangitis (PSC). By incorporating non-invasive markers of fibrosis, beyond standard liver injury markers, we show that NGM282 impacted on fibrosis turnover and hepatic inflammation without changing alkaline phosphatase. Our findings demonstrate the complexities of using highly potent rational agents in PSC, and furthermore challenge the dogma about what the appropriate endpoints should be for trials in PSC., (Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Objective: Disruption of the enterohepatic circulation of bile acids (BAs) is part of the gastrointestinal phenotype of cystic fibrosis (CF). Ivacaftor (VX-770), a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator, improves pulmonary function in CF patients with class III gating mutations. We studied the effect of ivacaftor on the enterohepatic circulation by assessing markers of BA homeostasis and their changes in CF patients., Methods: In CF patients with an S1251N mutation (N = 16; age 9-35 years S125N study/NTR4873) or a G551D mutation (N = 101; age 10-24 years; GOAL study/ NCT01521338) we analyzed plasma fibroblast growth factor 19 (FGF19) and 7α-hydroxy-4-cholesten-3-one (C4) levels, surrogate markers for intestinal BA absorption and hepatic synthesis, respectively, before and after treatment with ivacaftor., Results: At baseline, median FGF19 was lower (52% and 53%, P < .001) and median C4 higher (350% and 364%, P < .001), respectively, for the S1251 N and G551D mutation patient groups compared to healthy controls. Treatment with ivacaftor significantly increased FGF19 and reduced C4 levels towards normalization in both cohorts but this did not correlate with CFTR function in other organs, as measured by sweat chloride levels or pulmonary function., Conclusions: We demonstrate that patients with CFTR gating mutations display interruption of the enterohepatic circulation of BAs reflected by lower FGF19 and elevated C4 levels. Treatment with ivacaftor partially restored this disruption of BA homeostasis. The improvement did not correlate with established outcome measures of CF, suggesting involvement of modulating factors of CFTR correction in different organs., (Copyright © 2018 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Background: Non-alcoholic fatty liver disease (NAFLD) may be associated with changes in bile acid (BA) metabolism. Hepatic BA production, measured by serum levels of the precursor 7α-hydroxy-4-cholesten-3-one (C4), is regulated by the farnesoid-X-receptor (FXR)-dependent ileal hormone fibroblast growth factor 19 (FGF19). Low FGF19 and high C4 are features of chronic BA diarrhea. Obeticholic acid, an FXR agonist, stimulates FGF19 and has shown therapeutic potential in both BA diarrhea and in NAFLD. We hypothesized there are associations of FGF19, C4 and BA diarrhea with NAFLD., Methods and Findings: 127 patients with known NAFLD were recruited prospectively. Clinical features, including metformin use, markers of NAFLD severity and BA synthesis were analyzed. The overall incidence of chronic diarrhea was 25%, with features of BA diarrhea in 12%. FGF19 negatively correlated with C4 (rs = -0.43, p = 0.001) and with alanine aminotransferase (rs = -0.22, p = 0.03), but not with either NAFLD fibrosis or Fibroscan scores. High C4 was associated with a higher NAFLD fibrosis score (p < 0.05), and with diarrhea (p = 0.001). The median NAFLD fibrosis score was higher in those with diarrhea (p = 0.002). Metformin use, in 44% overall, was particularly associated with diarrhea (in 36% vs 17%, p = 0.02), and a lower median FGF19 (74 vs 105 pg/mL, p < 0.05)., Conclusions: Increased hepatic BA production and diarrhea, but not low FGF19, were associated with increased NAFLD fibrosis score, indicating dysregulation of the FXR-FGF19 axis and suggesting hepatic FGF19 resistance. Metformin use was an important factor in a subgroup, lowering FGF19, and resulting in bile acid diarrhea., Competing Interests: We have read the journal's policy and the authors of this manuscript have the following competing interests: JW has been a consultant, speaker or advisory board member for Intercept Pharmaceuticals, GE Healthcare, Novartis, Albireo, Falk, Enyo and Metacrine. Funding for this study was received in the form of an institutional award to support RA from a Intercept Pharmaceuticals. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

GSK2330672 is an inhibitor of the ileal bile acid transporter, designed to have minimal systemic exposure, and is under development as a potential therapeutic for pruritus associated with primary biliary cholangitis and other cholestatic liver diseases. A phase 1, double-blind, placebo-controlled, 4-period crossover study was conducted to evaluate the safety, tolerability, and pharmacokinetic/pharmacodynamic characteristics of GSK2330672 in healthy Japanese participants. Sixteen healthy male participants received single oral doses of GSK2330672 (10-180 mg) or placebo in each period. No serious adverse events and no adverse events leading to study discontinuation or withdrawal were reported. Drug-related adverse events reported included gastrointestinal symptoms (mostly diarrhea) and positive fecal occult blood tests, and were all mild and resolved without any interventions. GSK2330672 was undetectable in the majority of participants' plasma. Pharmacodynamic observations included a tendency for total serum bile acids to reduce and for serum 7α-hydroxy-4-cholesten-3-one, a key intermediate of bile acid synthesis, to increase with increasing doses of GSK2330672. In the context of recently published indications of potential efficacy for cholestatic pruritus in non-Japanese populations, these data support further evaluations of GSK2330672 in Japanese patients., (© 2018, The American College of Clinical Pharmacology.)

Background and Objective: Danggui-Shaoyao-San (DSS), a famous Chinese formula, has been widely used to treat gynecological disorders since ancient times and has recently showed efficacy in treating Alzheimer's disease. Butylidenephthalide (BDPH) and alisol B (ALI) are recognized as the primary active ingredients of DSS. The objectives of the present study were to evaluate the pharmacokinetic comparative study of BDPH and ALI in herbal extracts and their purified forms., Method: A sensitive and specific high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) methodology was developed to determine the concentration level of BDPH and ALI in rat plasma. This approach enables a real-time pharmacokinetics profiling of BDPH and ALI in DSS extracts as well as their purified forms in rats after oral administration., Results: The validated method showed an evident linearity over a wide range of dosages (r > 0.99) with sensitivity down to 1.0 ng/mL for each analyte. The extraction recovery of the analyte ranged from 80.8 to 99.1%. The pharmacokinetic parameters were significantly different in herbal extracts and their purified forms. The results showed that the absorption of both BDPH and ALI from DSS extracts was significantly greater compared with their purified forms., Conclusions: A highly specific, sensitive and rapid HPLC-MS/MS method was developed and applied for the determination of BDPH and ALI in rat plasma. It was found that BDPH and ALI had higher bioavailability in the DSS extract compared with their purified forms.

Bile acid (BA) synthesis is regulated through suppression of hepatic cholesterol 7α-hydroxylase via farnesoid X receptor (FXR) activation in hepatocytes and/or enterocytes; in enterocytes, this process requires FGF19 signaling. To study these pathways, we quantified markers of BA synthesis (7α-hydroxy-4-cholesten-3-one [C4]) and cholesterol production (lathosterol), fibroblast growth factor (FGF)19, and BAs in serum from healthy male volunteers given 1 oral dose of the nonsteroidal FXR agonist Px-102 (0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 1.12 mg/kg, 2.25 mg/kg, 3.38 mg/kg, or 4.5 mg/kg). After 8 hours, serum levels of C4 decreased by 80% in volunteers given 0.15 mg/kg, whereas serum levels of FGF19 were unchanged. Serum levels of FGF19 increased significantly, in a dose-dependent manner, in volunteers given >0.3 mg/kg Px-102, up to as much as 1600%, whereas C4 levels remained significantly reduced (by >80%). For all doses, FGF19 levels returned to normal 24 hours after administration of Px-102. Serum levels of C4 decreased before levels of FGF19 levels increased, and were still reduced by 95% 24 hours after the highest dose (4.5 mg/kg) of Px-102, even though levels of FGF19 had returned to baseline. Our findings indicate that activation of hepatic FXR is able to suppress BA synthesis, independent of FGF19., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Aims: Elobixibat is a minimally absorbed ileal bile acid transporter inhibitor. This study aimed to investigate the safety, tolerability, efficacy, pharmacokinetics and pharmacodynamics of elobixibat in Japanese patients with chronic constipation., Methods: This study consisted of single-dose and multiple-dose tests with a dose-escalating design. Sixty patients including females and males were randomized into five dose levels of elobixibat (2.5, 5, 10, 15 or 20 mg, n = 10 per level) and corresponding placebo (n = 2 per group). A crossover design was used to examine food effect in single-dose test. Patients received test tablets once daily for 14 days in multiple-dose test. We assessed pharmacokinetic-dose proportionality, levels of serum high- and low-density lipoprotein cholesterol and plasma 7α-hydroxy-4-cholesten-3-one (C4), food effect and sex-specific effect. Adverse events and bowel functions such as bowel movements, stool consistency and straining were also evaluated., Results: Food consumption reduced systemic exposure by around 80% [e.g. least squares mean (ratio of breakfast/no breakfast) maximum plasma concentration: 0.2085 (90% confidence interval, 0.1371-0.3172) at 15 mg] while increased plasma C4 level (P < 0.001). In the multiple-dose test, elobixibat reduced low-density lipoprotein cholesterol and increased C4 whilst unaltering high-density lipoprotein cholesterol level. The increased spontaneous bowel movement frequency was correlated with higher dosage and higher C4 level (R 2  = 0.5929 at Week 2). Adverse events were mainly gastrointestinal symptoms, most of which were mild., Conclusions: Elobixibat should be taken before breakfast. Once-daily administration of elobixibat was found to be safe and tolerated up to 20 mg in female and male patients with chronic constipation., (© 2018 EA Pharma Co., Ltd. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background: Non-alcoholic fatty liver disease (NAFLD) is common both in obese and overweight patients. Fibroblast growth factor 19 (FGF19), an intestinal hormone, could play a role in the complex pathogenesis of NAFLD. The aim of our study was to investigate responses of FGF19 and bile acid (BA) synthesis after a body weight-adjusted oral fat tolerance test (OFTT) in overweight and obese NAFLD patients., Methods: For this study, we recruited 26 NAFLD patients; 14 overweight (median BMI 28.3 kg/m 2 ), 12 obese (35.3 kg/m 2 ) and 16 healthy controls (24.2 kg/m 2 ). All individuals received 1 g fat (Calogen®) per kg body weight orally. Serum concentrations of FGF19 were determined by ELISA. Concentrations of BAs and BA synthesis marker 7α-hydroxy-4-cholesten-3-one (C4) were measured by gas chromatography-mass spectrometry and high-performance liquid chromatography, respectively; all at 0 (baseline), 2, 4 and 6 h during the OFTT., Results: BMI correlated negatively with fasting FGF19 concentrations (rho = - 0.439, p = 0.004). FGF19 levels of obese NAFLD patients were significantly (p = 0.01) lower in the fasting state (median 116.0 vs. 178.5 pg/ml), whereas overweight NAFLD patients had significantly (p = 0.004) lower FGF19 concentrations 2 h after the fat load (median 163.0 vs. 244.5 pg/ml), and lowest values at all postprandial time points as compared to controls. Baseline BA concentrations correlated positively with FGF19 values (rho = 0.306, p = 0.048). In all groups, we observed BA increases during the OFTT with a peak at 2 h but no change in C4 levels in overweight/obese NAFLD patients., Conclusions: Reduced basal gastrointestinal FGF19 secretion and decreased postprandial response to oral fat together with blunted effect on BA synthesis indicate alterations in intestinal or hepatic FXR signaling in overweight and obese NAFLD subjects. The precise mechanism of FGF19 signaling after oral fat load needs further evaluation., Trial Registration: We have registered the trial retrospectively on 30 Jan 2018 at the German clinical trials register ( http://www.drks.de /), and the following number has been assigned DRKS00013942 .

Background: Accumulation of toxic free cholesterol in hepatocytes may cause hepatic inflammation and fibrosis. Volixibat inhibits bile acid reuptake via the apical sodium bile acid transporter located on the luminal surface of the ileum. The resulting increase in bile acid synthesis from cholesterol could be beneficial in patients with non-alcoholic steatohepatitis. This adaptive dose-finding study investigated the safety, tolerability, pharmacodynamics, and pharmacokinetics of volixibat., Methods: Overweight and obese adults were randomised 3:1 to double-blind volixibat or placebo, respectively, for 12 days. Volixibat was initiated at a once-daily dose of 20 mg, 40 mg or 80 mg. Based on the assessment of predefined safety events, volixibat dosing was either escalated or reduced. Other dose regimens (titrations and twice-daily dosing) were also evaluated. Assessments included safety, tolerability, stool hardness, faecal bile acid (FBA) excretion, and serum levels of 7α-hydroxy-4-cholesten-3-one (C4) and lipids., Results: All 84 randomised participants (volixibat, 63; placebo, 21) completed the study, with no serious adverse events at doses of up to 80 mg per day (maximum assessed dose). The median number of daily bowel evacuations increased from 1 (range 0-4) to 2 (0-8) during volixibat treatment, and stool was looser with volixibat than placebo. Volixibat was minimally absorbed; serum levels were rarely quantifiable at any dose or sampling time point, thereby precluding pharmacokinetic analyses. Mean daily FBA excretion was 930.61 μmol (standard deviation [SD] 468.965) with volixibat and 224.75 μmol (195.403) with placebo; effects were maximal at volixibat doses ≥20 mg/day. Mean serum C4 concentrations at day 12 were 98.767 ng/mL (standard deviation, 61.5841) with volixibat and 16.497 ng/mL (12.9150) with placebo. Total and low-density lipoprotein cholesterol levels decreased in the volixibat group, with median changes of - 0.70 mmol/L (range - 2.8 to 0.4) and - 0.6990 mmol/L (- 3.341 to 0.570), respectively., Conclusions: This study indicates that maximal inhibition of bile acid reabsorption, as assessed by FBA excretion, occurs at volixibat doses of ≥20 mg/day in obese and overweight adults, without appreciable change in gastrointestinal tolerability. These findings guided dose selection for an ongoing phase 2 study in patients with non-alcoholic steatohepatitis., Trial Registration: ClinicalTrials.gov identifier: NCT02287779 (registration first received 6 November 2014).

We attempted to clarify the therapeutic capability of antagonists of the farnesoid X receptor (FXR), a nuclear receptor that regulates lipid and bile acid metabolism. Herein, we report the antidyslipidemic effects of a novel synthesized FXR antagonist, compound-T1, utilizing a dyslipidemic hamster model. Compound-T1 selectively inhibited chenodeoxycholic acid-induced FXR activation (IC 50 , 2.1 nmol·L -1 ). A hamster model of diet-induced hyperlipidemia was prepared to investigate the antidyslipidemic effects of compound-T1 through comparative studies of the nonstatin lipid-modulating agents ezetimibe, cholestyramine, and torcetrapib. In the hamster model, compound-T1 (6 mg·kg -1 ·day -1 , p.o.) increased the level of plasma high-density lipoprotein (HDL)-cholesterol (+22.2%) and decreased the levels of plasma non-HDL-cholesterol (-43.6%) and triglycerides (-31.1%). Compound-T1 also increased hepatic cholesterol 7α-hydroxylase expression and fecal bile acid excretion, and decreased hepatic cholesterol content. Moreover, the hamster model could reflect clinical results of other nonstatin agents. Torcetrapib especially increased large HDL particles compared with compound-T1. Additionally, in the human hepatoma Huh-7 cells, compound-T1 enhanced apolipoprotein A-I secretion at a concentration close to its IC 50 value for FXR. Our results indicated the usefulness of the hamster model in evaluating FXR antagonists and nonstatin agents. Notably, compound-T1 exhibited beneficial effects on both blood non-HDL-cholesterol and HDL-cholesterol, which are thought to involve enhancement of cholesterol catabolism and apolipoprotein A-I production. These findings aid the understanding of the antidyslipidemic potential of FXR antagonists with a unique lipid and bile acid modulation.

Background: Imbalance of bile acids (BA) homeostasis in the gastrointestinal tract can lead to chronic diarrhea or constipation when BA in the colon are in excess or low, respectively. Since both disturbances of bowel function can result from other etiologies, identifying BA imbalance is important to tailor treatment strategies. Serum concentrations of 7-alpha-hydroxy-4-cholesten-3-one (7aC4), a precursor in bile acid synthesis, reflect BA homeostasis. Here we describe a method to accurately measure serum 7aC4 and evaluate the clinical utility in patients with diarrhea or constipation phenotypes., Methods: Serum 7aC4 is measured after acetonitrile protein precipitation using C18 liquid chromatography, tandem mass spectrometry, and deuterium-labeled 7aC4 internal standard. Assay performance including linearity, precision, and accuracy was assessed using waste serum samples. The reference interval was established in healthy individuals without BA-altering conditions or medications. Clinical performance was assessed in patients with irritable bowel syndrome., Results: The method precisely and accurately measured 7aC4 in human serum from 1.4-338ng/mL with no ion suppression or interference from related 7-keto-cholesterol. Central 95th percentile reference interval was 2.5-63.2ng/mL. Lower serum 7aC4 was found in patients with constipation with sensitivity/specificity of 79%/55% compared to healthy controls. Higher 7aC4 was found in patients with bile acid diarrhea (BAD) compared to those without BAD with sensitivity/specificity of 82%/53%., Conclusions: We have developed a sensitive and precise assay for measuring the concentration of 7aC4 in serum. The assay can be used to screen for diarrhea caused by bile acid malabsorption., (Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: Pathogenesis in non-alcoholic steatohepatitis (NASH) involves abnormal cholesterol metabolism and hepatic accumulation of toxic free cholesterol. Apical sodium-dependent bile acid transporter (ASBT) inhibition in the terminal ileum may facilitate removal of free cholesterol from the liver by reducing recirculation of bile acids (BAs) to the liver, thereby stimulating new BA synthesis from cholesterol. The aim of this phase 1 study in adult healthy volunteers (HVs) and patients with type 2 diabetes mellitus (T2DM) was to assess the safety, tolerability, pharmacokinetics and pharmacodynamics of ASBT inhibition with volixibat (SHP626; formerly LUM002)., Methods: Participants were randomised 3:1 to receive once-daily oral volixibat (0.5 mg, 1 mg, 5 mg or 10 mg) or placebo for 28 days in two cohorts (HV and T2DM). Assessments included safety, faecal BA and serum 7α-hydroxy-4-cholesten-3-one (C4; BA synthesis biomarker)., Results: Sixty-one individuals were randomised (HVs: placebo, n = 12; volixibat, n = 38; T2DM: placebo, n = 3; volixibat, n = 8). No deaths or treatment-related serious adverse events were reported. Mild or moderate gastrointestinal adverse events were those most frequently reported with volixibat. With volixibat, mean total faecal BA excretion on day 28 was ~1.6-3.2 times higher in HVs (643.73-1239.3 μmol/24 h) and ~8 times higher in T2DM (1786.0 μmol/24 h) than with placebo (HVs: 386.93 μmol/24 h; T2DM: 220.00 μmol/24 h). With volixibat, mean C4 concentrations increased by ~1.3-5.3-fold from baseline to day 28 in HVs and by twofold in T2DM., Conclusions: Volixibat was generally well tolerated. Increased faecal BA excretion and serum C4 levels support the mechanistic rationale for exploring ASBT inhibition in NASH. The study was registered with the Dutch clinical trial authority (Centrale Commissie Mensgebonden Onderzoek; trial registration number NL44732.056.13; registered 24 May 2013).

Underlying mechanisms responsible for the cholesterol-lowering effect of β-glucan have been proposed, yet have not been fully demonstrated. The primary aim of this study was to determine whether the consumption of barley β-glucan lowers cholesterol by affecting the cholesterol absorption, cholesterol synthesis or bile acid synthesis. In addition, this study was aimed to assess whether the underlying mechanisms are related to cholesterol 7α hydroxylase (CYP7A1) SNP rs3808607 as proposed by us earlier. In a controlled, randomised, cross-over study, participants with mild hypercholesterolaemia (n 30) were randomly assigned to receive breakfast containing 3 g high-molecular weight (HMW), 5 g low-molecular weight (LMW), 3 g LMW barley β-glucan or a control diet, each for 5 weeks. Cholesterol absorption was determined by assessing the enrichment of circulating 13C-cholesterol over 96 h following oral administration; fractional rate of synthesis for cholesterol was assessed by measuring the incorporation rate of 2H derived from deuterium oxide within the body water pool into the erythrocyte cholesterol pool over 24 h; bile acid synthesis was determined by measuring serum 7α-hydroxy-4-cholesten-3-one concentrations. Consumption of 3 g HMW β-glucan decreased total cholesterol (TC) levels (P=0·029), but did not affect cholesterol absorption (P=0·25) or cholesterol synthesis (P=0·14). Increased bile acid synthesis after consumption of 3 g HMW β-glucan was observed in all participants (P=0·049), and more pronounced in individuals carrying homozygous G of rs3808607 (P=0·033). In addition, a linear relationship between log (viscosity) of β-glucan and serum 7α-HC concentration was observed in homozygous G allele carriers. Results indicate that increased bile acid synthesis rather than inhibition of cholesterol absorption or synthesis may be responsible for the cholesterol-lowering effect of barley β-glucan. The pronounced TC reduction in G allele carriers of rs3808607 observed in the previous study may be due to enhanced bile acid synthesis in response to high-viscosity β-glucan consumption in those individuals.

7α-hydroxy-4-cholesten-3-one (C4) is an oxidative enzymatic product of cholesterol metabolism via cholesterol 7α-hydroxylase, an enzyme also known as cholesterol 7-alpha-monooxygenase or cytochrome P450 7A1 (CYP7A1). C4 is a stable intermediate in the rate limiting pathway of bile acid biosynthesis. Previous studies showed that plasma C4 levels correlated with CYP7A1 enzymatic activity and could serve as a biomarker for bile acid synthesis. Here we developed and qualified a simple and robust high-throughput method using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to quantify C4 in rat and monkey plasma. As C4 being an endogenous compound, this method used calibration standards in 50/50: acetonitrile/water (v/v). In order to mimic the incurred samples, quality control samples were prepared in the authentic plasma. Stable isotope labeled C4 (C4-d 7 ) was used as the internal standard. The sample volume for analysis was 20μL and the sample preparation method was protein precipitation with acetonitrile. The average endogenous C4 concentrations, from 10 different lots of rat and monkey plasma, were 53.0±16.5ng/mL and 6.8±5.6ng/mL, respectively. Based on these observed endogenous C4 levels, the calibration curve ranges were established at 1-200ng/mL and 0.5-100ng/mL for rat assay and monkey assay, respectively. The method was qualified with acceptable accuracy, precision, linearity, and specificity. Matrix effect, recovery, and plasma stability of bench-top, freeze-thaw, and long-term frozen storage were also evaluated. The method has been successfully applied to pre-clinical studies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objectives: To assess the association between biomarkers of thyroid status and 5α-stanols in patients with sitosterolemia treated with ezetimibe (EZE)., Study Design: Eight patients with sitosterolemia (16-56 years of age) were studied during 14 weeks off EZE therapy and 14 weeks on EZE (10 mg/day). Serum thyroid biomarkers (free triiodothyronine [FT3], free thyroxine [FT4], FT3/FT4 ratio, thyroid-stimulating hormone), 5α-stanols (sitostanol and cholestanol), and cholestanol precursors (total cholesterol and its synthesis marker lathosterol, and 7α-hydroxy-4-cholesten-3-one cholestenol) were measured at baseline and during the 14 weeks off EZE and on EZE., Results: EZE increased FT3/FT4 (10% ± 4%; P = .02). EZE reduced plasma and red blood cells sitostanol (-38% ± 6% and -20% ± 4%; all P < .05) and cholestanol (-18% ± 6% and -13% ± 3%; all P < .05). The change in plasma cholestanol level on EZE inversely correlated with the change in FT3/FT4 (r = -0.86; P = .01). EZE lowered total cholesterol (P < .0001) and did not affect 7α-hydroxy-4-cholesten-3-one cholestanol. EZE increased (P < .0001) lathosterol initially, but the level was not sustained, resulting in similar levels at week 14 off EZE and on EZE., Conclusion: In patients with STSL, 5α-stanols levels might be associated with thyroid function. EZE reduces circulating 5α-stanols while increasing FT3/FT4, implying increased conversion of T4 to T3, thus possibly improving thyroid hormone status., Trial Registration: ClinicalTrials.govNCT01584206., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The unfolded protein response (UPR) is an adaptive response to endoplasmic reticulum stress and the inositol-requiring enzyme 1α/X-box binding protein 1 (IRE1α/XBP1) pathway of the UPR is important in lipid metabolism. However, its role in bile acid metabolism remains unknown. We demonstrate that liver-specific Xbp1 knockout (LS- Xbp1 -/- ) mice had a 45% reduction in total bile acid pool. LS- Xbp1 -/- mice had lower serum 7α-hydroxy-4-cholesten-3-one (C4) levels compared with Xbp1 fl/fl mice, indicating reduced cholesterol 7α-hydroxylase (CYP7A1) synthetic activity. This occurred without reductions of hepatic CYP7A1 protein expression. Feeding LS- Xbp1 -/- mice cholestyramine increased hepatic CYP7A1 protein expression to levels 2-fold and 8-fold greater than cholestyramine-fed and chow-fed Xbp1 fl/fl mice, respectively. However, serum C4 levels remained unchanged and were lower than both groups of Xbp1 fl/fl mice. In contrast, although feeding LS- Xbp1 -/- mice cholesterol did not increase CYP7A1 expression, serum C4 levels increased significantly up to levels similar to chow-fed Xbp1 fl/fl mice and the total bile acid pool normalized. In conclusion, loss of hepatic XBP1 decreased the bile acid pool and CYP7A1 synthetic activity. Cholesterol feeding, but not induction of CYP7A1 with cholestyramine, increased CYP7A1 synthetic activity and corrected the genotype-specific total bile acid pools. These data demonstrate a novel role of IRE1α/XBP1 regulating bile acid metabolism., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

We confirmed previous findings by a Japanese group that there is an accumulation of 7α-hydroxy-3-oxo-4-cholestenoic acid (7-Hoca) in human subdural hematomas. The accumulation correlated with the time from the bleeding to the sample collection. We present evidence that these accumulations are likely to be caused by the strong affinity of 7-Hoca to albumin and the marked difference between plasma and brain with respect to levels of albumin. In the circulation, 80-90% of 7-Hoca is bound to albumin with a ratio between the steroid acid and albumin of ∼1.4 ng/mg. In cerebrospinal fluid (CSF), the ratio between 7-Hoca and albumin is ∼30 ng/mg. When albumin or hemolyzed blood in a dialysis bag was exposed to CSF, there was a flux of 7-Hoca from CSF to the albumin. We suggest that the major explanation for accumulation of 7-Hoca in subdural hematoma is a flux from the brain into the hematoma due to the high affinity to albumin and the high capacity of 7-Hoca to pass biomembranes. We discuss the possibility that the markedly different ratios between 7-Hoca and albumin in circulation and brain can explain the flux of 7-Hoca from the brain into circulation against a concentration gradient., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

Goals: To perform an exploratory pilot study of all-trans retinoic acid (ATRA) combined with ursodeoxycholic acid (UDCA) in patients with primary sclerosing cholangitis (PSC)., Background: PSC is a progressive disorder for which there is no accepted therapy. Studies in human hepatocyte cultures and in animal models of cholestasis indicate that ATRA might have beneficial effects in cholestatic disorders., Study: ATRA (45 mg/m/d, divided and given twice daily) was combined with moderate-dose UDCA in patients with PSC who had incomplete response to UDCA monotherapy. The combination was administered for 12 weeks, followed by a 12-week washout in which patients returned to UDCA monotherapy. We measured alkaline phosphatase (ALP), alanine aminotransferase (ALT), bilirubin, cholesterol, bile acids, and the bile acid intermediate 7α-hydroxy-4-cholesten-3-one (C4) at baseline, week 12, and after washout., Results: Fifteen patients completed 12 weeks of therapy. The addition of ATRA to UDCA reduced the median serum ALP levels (277±211 to 243±225 U/L, P=0.09) although this, the primary endpoint, did not reach significance. In contrast, median serum ALT (76±55 to 46±32 U/L, P=0.001) and C4 (9.8±19 to 7.9±11 ng/mL, P=0.03) levels significantly decreased. After washout, ALP and C4 levels nonsignificantly increased, whereas ALT levels significantly increased (46±32 to 74±74, P=0.0006), returning to baseline., Conclusions: In this human pilot study, the combination of ATRA and UDCA did not achieve the primary endpoint (ALP); however, it significantly reduced ALT and the bile acid intermediate C4. ATRA appears to inhibit bile acid synthesis and reduce markers of inflammation, making it a potential candidate for further study in PSC (NCT 01456468).

Background: Bile salts likely contribute to liver injury in patients with primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC). Fibroblast growth factor 19 (FGF19) is a bile salt-induced enterokine with hepatoprotective potential as it suppresses de novo bile salt synthesis. Here, we evaluated the bile salt receptor FXR/FGF19 gut-liver axis in PSC and PBC patients., Methods: Fasted patients with PSC (n = 12) and PBC (n = 10), and healthy controls (HC; n = 10) were orally challenged with the natural FXR agonist chenodeoxycholic acid (CDCA 15 mg/kg). Blood was sampled hourly until 8 h afterwards. Serum FGF19 and bile salt excursions were determined. Serum levels of 7α-hydroxy-4-cholesten-3-one (C4), reflecting bile salt synthesis, were measured as a biomarker of FGF19 response., Results: Baseline serum FGF19 levels were comparable between groups, while fasted bile salt levels in PSC patients were elevated. Upon CDCA challenge, HC and PBC patients showed a serum FGF19 peak after 4 h followed by a decline. PSC patients showed a prolonged and elevated serum FGF19 response up to 8 h, combined with a sustained serum elevation of CDCA and other bile salts. In general, C4 levels declined following FGF19 elevation. In PSC patients with less favorable prognosis, baseline C4 levels were drastically suppressed and did not further decline., Conclusion: Following an oral CDCA challenge, PSC patients showed an impaired clearance of CDCA and a prolonged serum FGF19 response. FXR agonist therapy in PSC could cause prolonged exposure to elevated levels of FGF19, and we propose careful monitoring for detrimental side effects in patient studies., Competing Interests: Serge Zweers, Elisabeth de Vries, Martin Lenicek, Dagmar Tolenaars, Rudi de Waart, Kiran Koelfat, Albert Groen, Steven Olde Damink, Ulrich Beuers, Cyriel Ponsioen, Peter Jansen, Frank Schaap declare that they have no conflict of interest. This study was supported by grants from the Dutch Digestive Diseases Foundation (WO#08-69), Dutch Society for Gastroenterology (Gastrostart 2012-10), Norwegian PSC Consortium (to Peter Jansen and Frank Schaap) and the German Crohn’s and Ulcerative Colitis Association (to Ulrich Beuers). Informed consent in studies with human subjects All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008. Informed consent was obtained from all patients for being included in the study.

The treatment of hypercholesterolemia with bile acid (BA) sequestrants results in upregulation of BA synthesis through the classical pathway initiated by cholesterol 7alpha-hydroxylase (CYP7A1). To characterize the detailed dynamics of serum lipid and BA concentrations and the BA synthesis rate in response to treatment with BA sequestrants and to determine whether the -203A/C promoter polymorphism of the CYP7A1 encoding gene (CYP7A1) affects such a response, this pilot study was carried out in healthy men (8 homozygous for the -203A allele and 8 homozygous for the -203C allele of CYP7A1). The subjects were treated for 28 days with colesevelam and blood was drawn for analysis before and on days 1, 3, 7, 14 and 28 of treatment. The response of lipids, BA, fibroblast growth factor-19 (FGF19) and 7alpha-hydroxy-4-cholesten-3-one (C4) to colesevelam did not differ between carriers of -203A and -203C alleles; their data were then aggregated for further analysis. Colesevelam treatment caused immediate suppression of FGF19 concentration and a fivefold increase in CYP7A1 activity, as assessed from C4 concentration, followed by a 17 % decrease in LDL-cholesterol. Although total plasma BA concentrations were not affected, the ratio of cholic acid/total BA rose from 0.25+/-0.10 to 0.44+/-0.16 during treatment at the expense of decreases in chenodeoxycholic and deoxycholic acid.

Aim: Serum 7α-hydroxy-cholesten-3-one (C4) has been reported as a biomarker to assess CYP7A1 enzyme activity and bile acid synthesis. To support a clinical program, a sensitive and reliable assay without derivatization was required for the analysis of C4 in human serum. Methodology & results: A systematic approach was used to optimize mass spectrometry, LC and sample extraction conditions, therefore, significantly improved assay sensitivity, and achieved the required quantification limit without derivatization. A surrogate matrix approach was used to overcome the interference from endogenous C4. A stable isotope-labeled C4 was used as internal standard. The samples were extracted using a simple protein precipitation method with 2% formic acid in acetonitrile., Conclusion: A simple, fast, sensitive and robust UHPLC-MS/MS method for the quantification of 0.50 ng/ml C4 in 100 µl human serum was developed and fit for purpose validated. The method was successfully applied to the bioanalysis of C4 in a clinical study.

Objective: To evaluate the impacts of clinical, pathological, and laboratory factors on oncological outcomes of patients with T3N0M0 renal cell carcinoma., Methods: The clinical data, laboratory exam results, and follow-up outcomes of 182 patients with T3N0M0 renal cell carcinoma who underwent nephrectomy from 2007 to 2012 in Peking University First Hospital were retrospectively collected. The 5-year cancer-specific survival and 5-year recurrence-free survival of all the patients were calculated using Kaplan-Meier method, and the statistical significance between the survival curves were compared using the Log-rank test. Variables with significant differences in the univariate analysis were subjected to the multivariate analysis by Cox regression model. All the comparisons were conducted using two-tailed test and P<0.05 was considered statistically significant., Results: A total of 182 patients were included in this study. Of all the 182 patients, 126 were male (69.23%) and 56 were female (30.77%). The mean age was (56.75±12.45) years. The median follow-up time was 48 months (3-99 months). At the end of the follow-up, 50 patients (27.47%) died due to the disease after a median of 29.74 months and 59 patients (32.42%) had tumor recurrence after a median of 22.12 months. The 5-year cancer-specific survival of all patients was 68.30% (95% CI: 60.16%-75.84%); the 5-year recurrence-free survival was 60.70% (95% CI: 53.16%-68.84%). In the univariate analysis, diabetes mellitus, tumor invasion status, Fuhrman grade, serum album, serum cholestenone, anemia, and neutrophils percentage were associated with the cancer-specific survival and Fuhrman grade, serum album and anemia were associated with the recurrence-free survival. Variables with significant differences on univariate analysis were included in Cox multivariate regression analysis. Multivariate Logistic regression analysis showed that diabetes mellitus (HR=2.434, 95% CI: 1.243-4.769, P=0.010), hypoalbuminemia (HR=2.188, 95% CI: 1.074-1.074, P=0.031), and anemia (HR=3.320, 95% CI: 1.839-5.991, P<0.001) were independent risk factors significantly associated with cancer-specific survival; and higher Fuhrman grade (HR=2.552, 95% CI: 1.433-4.545, P=0.001), anemia (HR=2.535, 95% CI: 1.497-4.293, P=0.001) were independent factors significantly associated with recurrence-free survival., Conclusion: Diabetes mellitus, hypoalbuminemia, and anemia were independent risk factors significantly associated with cancer-specific survival of T3N0M0 renal cell carcinoma patients; higher Fuhrman grade and anemia were independent risk factors significantly associated with tumor recurrence of T3N0M0 renal cell carcinoma patients.

The serum concentration of 7α-hydroxy-4-cholesten-3-one (C4), a marker of cholesterol 7α-hydroxylase activity, has recently become an attractive diagnostic tool for researchers interested in cholesterol and bile acid metabolism. The rapidly increasing demand of C4 measurement led to the development of various fast, mostly mass spectrometry-based analytical methods. Our aim was to compare four simple (i.e., not requiring solid phase extraction) extraction procedures (two "one-phase", and two "two-phase") in terms of basic analytical performance and their labouriousness. All methods exhibited comparable extraction recoveries (ranging from 88 to 97%) and intra-assay precision (variation coefficients below 10%), and failed in the removal of phospholipids. Although marked differences were observed in desalting and deproteination, all methods can be considered satisfactory. Simple acetonitrile precipitation can be recommended if a fast extraction and minimal hands-on time is preferred; while two-phase ammonium sulphate:acetonitrile extraction should be chosen when maximal deproteination is required., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Aim: To determine whether the promoter polymorphism -203A>C of cholesterol-7α-hydroxylase encoding gene (CYP7A1) affects diurnal variation in CYP7A1 enzyme activity., Methods: The study included 16 healthy male volunteers - 8 homozygous for -203A and 8 homozygous for the -203C allele of CYP7A1. Three 15-hour examinations (from 7am to 10pm) were carried out for each of the participants: after one-day treatment with cholestyramine; after one-day treatment with chenodeoxycholic acid (CDCA); and a control examination without any treatment. The plasma concentration of 7α-hydroxy-4-cholesten-3-one (C4), a marker of CYP7A1 activity, was determined in all the experiments at 90-min intervals., Results: CYP7A1 activity was up-regulated after treatment with cholestyramine and suppressed after treatment with CDCA. There were no differences between -203A and -203C allele carriers in the response of enzyme activity to both drugs. In the control experiment, -203A allele carriers displayed diurnal variation in enzyme activity, whereas CYP7A1 activity did not change in -203C allele carriers. These results were confirmed by modeling the dynamics of C4 using polynomial regression., Conclusion: The promoter polymorphism of the CYP7A1 gene has a pronounced impact on diurnal variation in CYP7A1 activity.

Background: Reabsorption of bile acids from the intestine by ileal bile acid transporter is pivotal for the enterohepatic circulation of BAs and sterol homoeostasis., Aim: To assess tolerability and study, bile acid metabolism in a phase 1 trial with the selective ileal bile acid transporter inhibitor A4250., Methods: A randomised double-blind, single-ascending dose (SAD) and multiple-ascending-dose study consisting of five cohorts comprising 40 individuals with a single administration of A4250 (0.1, 0.3, 1, 3, or 10 mg) or placebo and three cohorts comprising 24 individuals with a 1-week administration of A4250 (1 or 3 mg once daily or 1.5 mg twice daily) or placebo. For the multiple-ascending-dose study, bile acids were measured by HPLC-MS in plasma and faeces, and fibroblast growth factor 19 (FGF19) and 7α-hydroxy-4-cholesten-3-one (C4) were measured in plasma., Results: No serious adverse events occurred and all participants finished the trial per protocol. At the end of the multiple-ascending-dose study, plasma total bile acids and FGF19 decreased by 47% and 76%, respectively, at 3 mg/day (P < 0.01), and by 15% and 16%, respectively, at 1.5 mg twice daily (P < 0.05). Plasma C4 and faecal bile acids increased at all dose regimens, by 555%, 664%, 292% and 338%, 421%, 420%, respectively (P < 0.01-0.05). The primary bile acids cholic and chenodeoxycholic acids constituted the majority of faecal bile acids in the A4250-treated groups., Conclusions: A4250 is well tolerated. By blocking ileal bile acid transporter in the terminal ileum, it highly efficiently interrupts the enterohepatic circulation of BAs, and should be of benefit to patients with cholestatic liver diseases. Clinical Trial registration EudraCT 2013-001175-21., (© 2015 John Wiley & Sons Ltd.)

Huntington's disease is a fatal human neurodegenerative disorder caused by a CAG repeat expansion in the HTT gene, which translates into a mutant huntingtin protein. A key event in the molecular pathogenesis of Huntington's disease is the proteolytic cleavage of mutant huntingtin, leading to the accumulation of toxic protein fragments. Mutant huntingtin cleavage has been linked to the overactivation of proteases due to mitochondrial dysfunction and calcium derangements. Here, we investigated the therapeutic potential of olesoxime, a mitochondria-targeting, neuroprotective compound, in the BACHD rat model of Huntington's disease. BACHD rats were treated with olesoxime via the food for 12 months. In vivo analysis covered motor impairments, cognitive deficits, mood disturbances and brain atrophy. Ex vivo analyses addressed olesoxime's effect on mutant huntingtin aggregation and cleavage, as well as brain mitochondria function. Olesoxime improved cognitive and psychiatric phenotypes, and ameliorated cortical thinning in the BACHD rat. The treatment reduced cerebral mutant huntingtin aggregates and nuclear accumulation. Further analysis revealed a cortex-specific overactivation of calpain in untreated BACHD rats. Treated BACHD rats instead showed significantly reduced levels of mutant huntingtin fragments due to the suppression of calpain-mediated cleavage. In addition, olesoxime reduced the amount of mutant huntingtin fragments associated with mitochondria, restored a respiration deficit, and enhanced the expression of fusion and outer-membrane transport proteins. In conclusion, we discovered the calpain proteolytic system, a key player in Huntington's disease and other neurodegenerative disorders, as a target of olesoxime. Our findings suggest that olesoxime exerts its beneficial effects by improving mitochondrial function, which results in reduced calpain activation. The observed alleviation of behavioural and neuropathological phenotypes encourages further investigations on the use of olesoxime as a therapeutic for Huntington's disease., (© The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Elobixibat is a minimally absorbed ileal bile acid (BA) transporter (IBAT) inhibitor in development against chronic constipation (CC) and constipation-predominant Irritable Bowel Syndrome (IBS-C). CC is associated with an increased risk for cardiovascular disease and type2 diabetes mellitus. The objectives of this study were to evaluate metabolic effects of elobixibat. Effects on plasma lipids and BA synthesis were evaluated utilizing a 4-week, placebo-controlled study in patients with dyslipidemia while changes of glucagon-like peptide-1 (GLP-1) by elobixibat was assayed in samples from a 14 day high-dose elobixibat study in patients with CC., Methods: Thirty-six dyslipidemic patients, 21 females, mean age 63 years, were randomized to 2.5 mg or 5 mg elobixibat or placebo once daily for four weeks. The primary endpoint was the change in low density lipoprotein (LDL) cholesterol. Secondary endpoints included other lipid parameters and serum 7α-hydroxy-4-cholesten-3-one (C4), a marker of BA (bile acid) synthesis. Another study, in 36 patients with CC treated with high dose elobixibat; 15 mg or 20 mg/day or placebo for 14 days, was evaluated for changes in GLP-1., Results: In the dyslipidemia study LDL cholesterol was reduced by 7.4 % (p = 0.044), and the LDL/HDL ratio was decreased by 18 % (p = 0.004). Serum C4 increased, indicating that BA synthesis was induced. No serious adverse events were recorded. In the CC study, GLP-1 increased significantly in both the 15 mg (20.7 ± 2.4 pmol/L; p = 0.03) and the 20 mg group (25.6 ± 4.9 pmol/L; p = 0.02)., Conclusions: Elobixibat reduces LDL cholesterol and LDL/HDL ratio and increase circulating peak GLP-1 levels, the latter in line with increased intestinal BA mediated responses in humans., Trial Registrations: ClinicalTrial.gov: NCT01069783 and NCT01038687 .

Background: About one-third of patients with IBS-diarrhoea (irritable bowel syndrome-D) have evidence of increased bile acid synthesis or excretion., Aims: To assess effects of the bile acid sequestrant, colesevelam, on faecal excretion of BAs, hepatic BA synthesis and diarrhoea in IBS-D; to appraise whether individual or random stool samples accurately reflect 48-h total faecal bile acid excretion and proportions of the main bile acids excreted and to study the faecal fat excretion in response to colesevelam., Methods: A single-centre, unblinded, single-dose trial of effects of colesevelam, 1875 mg [3 tablets (625 mg tablets)] orally, twice daily, for 10 days on total 48-h faecal bile acid excretion and fasting serum C4 (7α-hydroxy-4-cholesten-3-one; surrogate of hepatic bile acid synthesis). Stool diaries documented bowel functions for 8 days prior and 8 days during colesevelam treatment. Stool 48-h samples and fasting serum were collected for faecal fat, faecal bile acid and serum C4., Results: Colesevelam was associated with significantly increased faecal total bile acid excretion and deoxycholic acid excretion, increased serum C4 and more solid stool consistency. There was a significant inverse correlation between number of bowel movements per week and the total bile acid sequestered into stool during the last 48 h of treatment. Random stool samples did not accurately reflect 48-h total or individual faecal bile acid excretion. Sequestration of bile acids by colesevelam did not increase faecal fat., Conclusions: Colesevelam increases delivery of bile acids to stool while improving stool consistency, and increases hepatic bile acid synthesis, avoiding steatorrhoea in patients with IBS-D. Overall effects are consistent with luminal bile acid sequestration by colesevelam., (© 2015 John Wiley & Sons Ltd.)

Background and Aims: Bile acid malabsorption (BAM)-associated diarrhea is an important clinical issue in patients with Crohn's disease (CD). We analyzed the efficacy and safety of the bile acid sequestrant colesevelam for treatment of BAM-associated diarrhea in CD patients in a randomized, double-blind, placebo-controlled study., Methods: The primary endpoint was the proportion of patients with >30% reduction of liquid stools/day from baseline to termination visit at week 4. Secondary endpoints were reduction of the number of liquid stools/day, improvement of stool consistency and quality of life., Results: 26 patients were analyzed in the intention-to-treat (ITT) analysis. The primary endpoint was reached by 10 patients (69.7%) in the colesevelam group compared to 3 patients (27.3%) in the placebo group (risk difference RD=.394, 95%CI:[-0.012; 0.706]; P=.0566). In the per-protocol analysis (n=22), the risk difference was statistically significant (RD=.470, 95%CI:[0.018; 0.788], P(H0: RD=0)=0.0364; 95% CI:[1.3;54.7]). Regarding secondary endpoints, in the ITT population colesevelam-treated patients had a significant reduction of liquid stools/day at week 4 (median 5.0 to 2.0; P=0.01), while patients treated with placebo had no significant reduction (median 4.0 to 3.0; P=0.42). Significantly more patients in the colesevelam group had improvement of stool consistency of at least one level in the Bristol stool chart, as compared to the placebo group (P=0.003)., Conclusions: We found significant differences in favor for colesevelam treatment compared to placebo treatment for CD patients with BAM regarding the reduction of the number of liquid stools/day and stool consistency. ClinicalTrials.gov number: NCT01203254., (Copyright © 2014 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.)

Objectives: Approximately 25% of patients with irritable bowel syndrome-diarrhea (IBS-D) have increased total fecal bile acids (BA) and serum C4 (surrogate for BA synthesis). BA synthesis-related genes (KLB and FGFR4) are associated with colonic transit (CT) in IBS-D. Our aims were: (i) to compare phenotype and pathophysiology in IBS-D patients with increased or normal fecal excretion or synthesis of BA; and (ii) to explore association of variations in two candidate bile-acid synthesis genes (KLB and FGFR4) in these two subgroups of IBS-D., Methods: A total of 64 IBS-D patients underwent on one occasion: fasting serum C4 and FGF19, total fecal fat and BA excretion, CT, intestinal and colonic permeability, and candidate genotyping (rs17618244 (KLB), rs351855 (FGFR4)). Colonic sensation and tone were measured in 47 of the IBS-D patients. IBS-D subgroups were identified by fecal BA >2,337 mM per 48 h or by serum C4 >47.1 ng/ml., Results: IBS-D patients with fecal BA >2,337 mM per 48 h (19/54) had significantly greater body mass index, fecal fat, percent chenodeoxycholic acid (CDCA) in feces, and intestinal permeability, and borderline increased CT (P=0.13). Those IBS-D patients with serum C4 >47.1 ng/ml (13/54) had increased total fecal BA excretion and borderline increased colonic permeability. Variants in genes involved in feedback regulation of BA synthesis (KLB, P=0.06 and FGFR4, P=0.09) were potentially associated with the subgroup with elevated serum C4., Conclusions: IBS-D with increased BA excretion or synthesis is associated with significant pathophysiological changes relative to patients with normal BA profile. BA diarrhea is identified more effectively with total fecal BA than with serum C4.

The pathobiology of irritable bowel syndrome (IBS) is multifaceted. We aimed to identify candidate genes predisposing to quantitative traits in IBS. In 30 healthy volunteers, 30 IBS-constipation, and 64 IBS-diarrhea patients, we measured bowel symptoms, bile acid (BA) synthesis (serum 7α-hydroxy-4-cholesten-3-one and FGF19), fecal BA and fat, colonic transit (CT by scintigraphy), and intestinal permeability (IP by 2-sugar excretion). We assessed associations of candidate genes controlling BA metabolism (KLB rs17618244 and FGFR4 rs351855), BA receptor (GPBAR1 rs11554825), serotonin (5-HT) reuptake (SLC6A4 through rs4795541 which encodes for the 44-bp insert in 5HTTLPR), or immune activation (TNFSF15 rs4263839) with three primary quantitative traits of interest: colonic transit, BA synthesis, and fecal BA excretion. There were significant associations between fecal BA and CT at 48 h (r = 0.43; P < 0.001) and IP (r = 0.23; P = 0.015). GPBAR1 genotype was associated with CT48 (P = 0.003) and total fecal BA [P = 0.030, false detection rate (FDR) P = 0.033]. Faster CT48 observed with both CC and TT GPBAR1 genotypes was due to significant interaction with G allele of KLB, which increases BA synthesis and excretion. Other univariate associations (P < 0.05, without FDR correction) observed between GPBAR1 and symptom phenotype and gas sensation ratings support the role of GPBAR1 receptor. Associations between SLC6A4 and stool consistency, ease of passage, postprandial colonic tone, and total fecal BA excretion provide data in support of future hypothesis-testing studies. Genetic control of GPBAR1 receptor predisposing to pathobiological mechanisms in IBS provides evidence from humans in support of the importance of GPBAR1 to colonic motor and secretory functions demonstrated in animal studies., (Copyright © 2014 the American Physiological Society.)

Second-generation antipsychotics (SGAs) are commonly used to treat schizophrenia. However, SGAs cause metabolic disturbances that can manifest as metabolic syndrome (MetS) in a subset of patients. The causes for these metabolic disturbances remain unclear. We performed a comprehensive metabolomic profiling of 60 schizophrenia patients undergoing treatment with SGAs that puts them at high (clozapine, olanzapine), medium (quetiapine, risperidone), or low (ziprasidone, aripiprazole) risk for developing MetS, compared to a cohort of 20 healthy controls. Multiplex immunoassays were used to measure 13 metabolic hormones and adipokines in plasma. Mass spectrometry was used to determine levels of lipids and polar metabolites in 29 patients and 10 controls. We found that levels of insulin and tumor necrosis factor alpha (TNF-α) were significantly higher (p < 0.005) in patients at medium and high risk for MetS, compared to controls. These molecules are known to be increased in individuals with high body fat content and obesity. On the other hand, adiponectin, a molecule responsible for control of food intake and body weight, was significantly decreased in patients at medium and high risk for MetS (p < 0.005). Further, levels of dyacylglycerides (DG), tryacylglycerides (TG) and cholestenone were increased, whereas α-Ketoglutarate and malate, important mediators of the tricarboxylic acid (TCA) cycle, were significantly decreased in patients compared to controls. Our studies suggest that high- and medium-risk SGAs are associated with disruption of energy metabolism pathways. These findings may shed light on the molecular underpinnings of antipsychotic-induced MetS and aid in design of novel therapeutic approaches to reduce the side effects associated with these drugs.

Hepatic accumulation of bile acids is central to the pathogenesis of cholestatic liver diseases. Endocrine hormone fibroblast growth factor 19 (FGF19) may reduce hepatic bile acid levels through modulation of bile acid synthesis and prevent subsequent liver damage. However, FGF19 has also been implicated in hepatocellular carcinogenesis, and consequently, the potential risk from prolonged exposure to supraphysiological levels of the hormone represents a major hurdle for developing an FGF19-based therapy. We describe a nontumorigenic FGF19 variant, M70, which regulates bile acid metabolism and, through inhibition of bile acid synthesis and reduction of excess hepatic bile acid accumulation, protects mice from liver injury induced by either extrahepatic or intrahepatic cholestasis. Administration of M70 in healthy human volunteers potently reduces serum levels of 7α-hydroxy-4-cholesten-3-one, a surrogate marker for the hepatic activity of cholesterol 7α-hydroxylase (CYP7A1), the enzyme responsible for catalyzing the first and rate-limiting step in the classical bile acid synthetic pathway. This study provides direct evidence for the regulation of bile acid metabolism by FGF19 pathway in humans. On the basis of these results, the development of nontumorigenic FGF19 variants capable of modulating CYP7A1 expression represents an effective approach for the prevention and treatment of cholestatic liver diseases as well as potentially for other disorders associated with bile acid dysregulation., (Copyright © 2014, American Association for the Advancement of Science.)

Aims: We previously reported anti-dyslipidemic effects of a farnesoid X receptor antagonist in monkeys. In this study, we compared the cholesterol-lowering effects of single and combined administration of a farnesoid X receptor antagonist, compound-T8, and the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor atorvastatin in a guinea pig model., Main Methods: Plasma levels of 7α-hydroxy-4-cholesten-3-one, a marker of hepatic cholesterol 7α-hydroxylase activity, were measured after a single administration of compound-T8. The effects of compound-T8 or atorvastatin on plasma cholesterol levels and low-density lipoprotein (LDL) clearance were investigated after 14 or 16 days of repeated dosing, respectively. Fractional catabolic rate of plasma LDL was estimated by intravenous injection of DiI-labeled human LDL. The cholesterol-lowering effects of combination therapy were investigated after 7 days of repeated treatment., Key Findings: Compound-T8 (10 and 30 mg/kg) increased plasma 7α-hydroxy-4-cholesten-3-one levels in a dose-dependent manner. Single administration of compound-T8 (30 mg/kg) and atorvastatin (30 mg/kg) reduced plasma non-high-density lipoprotein (non-HDL) cholesterol levels by 48% and 46%, respectively, and increased clearance of plasma DiI-labeled LDL by 29% and 35%, respectively. Compound-T8 (10mg/kg) or atorvastatin (10mg/kg) reduced non-HDL cholesterol levels by 19% and 25%, respectively, and combination therapy showed an additive effect and lowered cholesterol levels by 48%., Significance: Similar to atorvastatin, compound-T8 reduced plasma non-HDL cholesterol levels accompanied with accelerated LDL clearance in guinea pigs. Combination therapy additively decreased plasma non-HDL cholesterol levels. Therefore, monotherapy with a farnesoid X receptor antagonist and combination therapy of a farnesoid X receptor antagonist with atorvastatin would be attractive dyslipidemia treatment options., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: The modifications of cholesterol metabolism associated with aging are ill-defined. The objective of this study was to define age-associated alterations of the different metabolic pathways controlling cholesterol homeostasis by analyzing circulating sterols., Methods: We analyzed serum samples collected from 201 adult (75 male, 126 female) subjects within the epidemiological MICOL study (Multicentrica Italiana Colelitiasi). The age range was 38-79 years; 103 had evidence of gallstones. The concentrations of the different sterols, recognized as markers of the main pathways of cholesterol homeostasis, were analyzed by gas chromatography-mass spectrometry, including lathosterol (synthesis), campesterol and sitosterol (absorption), and 7α-hydroxy-4-cholesten-3-one (degradation to bile acids)., Results: A significant direct correlation was detected between age and cholesterol levels (r =0.34, P<0.01). The lathosterol/cholesterol ratio was lower in older age quartiles (P<0.05 by analysis of variance), with an inverse correlation between the lathosterol/cholesterol ratio and age (r=-0.32, P<0.01). Such correlation was particularly evident in females. The campesterol/cholesterol and sitosterol/cholesterol ratios were inversely correlated with aging in control, but not in gallstone patients. The levels of 7α-hydroxy-4-cholesten-3-one were not correlated with age., Conclusion: These data show a reduction of cholesterol synthesis with aging which is associated with increased circulating cholesterol levels. The finding might be related to a reduced metabolic need for cholesterol in advancing age, leading to a downregulation of the main mechanisms of cholesterol intake in the liver. A different age-related behavior was observed in gallstone-free versus gallstone patients regarding cholesterol absorption. The possible implications in terms of the pharmacological management of hypercholesterolemia in the elderly remain to be defined.

Total parenteral nutrition (TPN) is associated with the development of parenteral nutrition-associated liver disease (PNALD) in infants. Fish oil-based lipid emulsions can reverse PNALD, yet it is unknown if they can prevent PNALD. We studied preterm pigs administered TPN for 14 days with either 100% soybean oil (IL), 100% fish oil (OV), or a mixture of soybean oil, medium chain triglycerides (MCTs), olive oil, and fish oil (SL); a group was fed formula enterally (ENT). In TPN-fed pigs, serum direct bilirubin, gamma glutamyl transferase (GGT), and plasma bile acids increased after the 14 day treatment but were highest in IL pigs. All TPN pigs had suppressed hepatic expression of farnesoid X receptor (FXR), cholesterol 7-hydroxylase (CYP7A1), and plasma 7α-hydroxy-4-cholesten-3-one (C4) concentrations, yet hepatic CYP7A1 protein abundance was increased only in the IL versus ENT group. Organic solute transporter alpha (OSTα) gene expression was the highest in the IL group and paralleled plasma bile acid levels. In cultured hepatocytes, bile acid-induced bile salt export pump (BSEP) expression was inhibited by phytosterol treatment. We show that TPN-fed pigs given soybean oil developed cholestasis and steatosis that was prevented with both OV and SL emulsions. Due to the presence of phytosterols in the SL emulsion, the differences in cholestasis and liver injury among lipid emulsion groups in vivo were weakly correlated with plasma and hepatic phytosterol content.

Cerebrotendinous xanthomatosis (CTX) is a rare, difficult-to-diagnose genetic disorder of bile acid (BA) synthesis that can cause progressive neurological damage and premature death. Detection of CTX in the newborn period would be beneficial because an effective oral therapy for CTX is available to prevent disease progression. There is no suitable test to screen newborn dried bloodspots (DBS) for CTX. Blood screening for CTX is currently performed by GC-MS measurement of elevated 5α-cholestanol. We present here LC-ESI/MS/MS methodology utilizing keto derivatization with (O-(3-trimethylammonium-propyl) hydroxylamine) reagent to enable sensitive detection of ketosterol BA precursors that accumulate in CTX. The availability of isotopically enriched derivatization reagent allowed ready tagging of ketosterols to generate internal standards for isotope dilution quantification. Ketosterols were quantified and their utility as markers for CTX was compared with 5α-cholestanol. 7α,12α-Dihydroxy-4-cholesten-3-one provided the best discrimination between CTX and unaffected samples. In two CTX, newborn DBS concentrations of this ketosterol (120-214 ng/ml) were ∼10-fold higher than in unaffected newborn DBS (16.4 ± 6.0 ng/ml), such that quantification of this ketosterol provides a test with potential to screen newborn DBS for CTX. Early detection and intervention through newborn screening would greatly benefit those affected with CTX by preventing morbidity and mortality.

Objective: To establish age-related reference values for 7-alpha-hydroxy-4-cholesten-3-one (C4) in a pediatric population and to investigate bile acid malabsorption in children with short bowel syndrome (SBS)., Study Design: Serum was obtained between 8:00 a.m. and 11:00 a.m. from 100 healthy children (52% males, 9 months to 18 years of age) after 10 hours of fasting. Pediatric patients with SBS served as disease controls (n = 12). Following solid-phase extraction and purification, C4 was determined by high-performance liquid chromatography using a ultraviolet detector at a wavelength of 241 nm. The upper limit of normal for C4 concentrations was defined as the mean plus 2 SD of the log-normal distribution., Results: The mean concentration and SD of C4 in healthy children was 22.8 ± 15.8 ng/mL with no relation to age or sex and an upper limit of normal of 66.5 ng/mL. Normal C4 values were found in 97 of 100 healthy children, and all 12 patients with SBS had C4 concentrations above 100 ng/mL (mean 299.6 ± 167.8 ng/mL; range 105.7-562.1 ng/mL, P < .0001 compared with controls)., Conclusions: The determined upper limit of normal for C4 concentration in healthy children corresponds to previously published levels in healthy adults and is independent of age and sex. The consistently elevated C4 concentrations in our patients with SBS confirm the reliability of this noninvasive, nonisotopic method to assess bile acid malabsorption in children., (Copyright © 2013 Mosby, Inc. All rights reserved.)

Background: Bile acid diarrhoea is a common, under-diagnosed cause of chronic watery diarrhoea, responding to specific treatment with bile acid sequestrants. We previously showed patients with bile acid diarrhoea have lower median levels compared with healthy controls, of the ileal hormone fibroblast growth factor 19 (FGF19), which regulates bile acid synthesis., Aim: To measure serum FGF19 and SeHCAT retention prospectively in patients with chronic diarrhoea., Methods: One hundred and fifty-two consecutive patients were grouped according to (75) Se-homocholic acid taurine (SeHCAT) 7-day retention: normal (>15%) in 72 (47%) diarrhoea controls; ≤15% in 54 (36%) with primary bile acid diarrhoea, and in 26 (17%) with secondary bile acid diarrhoea. Fasting blood was assayed for FGF19, 7α-hydroxy-4-cholesten-3-one (C4) and total bile acids., Results: FGF19 was significantly lower in the primary bile acid diarrhoea group compared with the diarrhoea control group (median 147 vs. 225 pg/mL, P < 0.001), and also in the secondary group (P < 0.006). FGF19 and SeHCAT values were positively correlated (rs = 0.44, P < 0.001); both were inversely related to C4. Other significant relationships included SeHCAT and body mass index (BMI)(P = 0.02), and FGF19 with age (P < 0.01). The negative and positive predictive values of FGF19 ≤ 145 pg/mL for a SeHCAT <10% were 82% and 61%, respectively, and were generally improved in an index including BMI, age and C4. In a subset of 28 primary patients, limited data suggested that FGF19 could predict response to sequestrant therapy., Conclusions: Reduced fibroblast growth factor 19 is a feature of bile acid diarrhoea. Further studies will fully define its role in predicting the response of these patients to therapy., (© 2013 John Wiley & Sons Ltd.)

Ergosta-4,6,8(14),22-tetraen-3-one (ergone) isolated from Polyporus umbellatus possesses a variety of pharmacological activities in vivo and in vitro, including cytotoxic, diuretic, and immunosuppressive effect. The interaction of cerium ions (Ce(3+)) with ergone was studied by fluorescence and absorption spectroscopy. Spectra data revealed that Ce(3+) ions exhibited emission maxima around 350 nm when the excitation wavelength was fixed at 255 or 290 nm, and the fluorescence of Ce(3+) ions was quenched by the addition of ergone, indicating that a Ce(3+)-ergone complex was formed. According to the modified Benesi-Hildebrand equation, the binding constant of interaction of Ce(3+) ions with ergone was obtained at room temperature. Based on this, a sensitive spectrofluorometric method using Ce(3+) ions as a probe was applied for the identification and quantification of ergone in rat plasma, feces, and urine. The linear ranges of the calibration curves were 1.31 to 4.50 μM for plasma, 1.12-9.87 μM for feces, and 1.28-3.42 μM for urine, and the ergone recoveries were found to be 97.1 ± 0.9%, 98.2 ± 0.7% and 96.5 ± 1.4% for plasma, feces, and urine, respectively. The intraday and inter-day relative standard deviations were less than 9.7%. The proposed spectrofluorometric method is simple and rapid for the quantitative determination of ergone in rat plasma, feces, and urine, and it is affordable for most laboratories because it has few requirements and uses low cost, easy to operate equipment.