A novel LC-MS/MS method for simultaneous estimation of obeticholic acid, glyco-obeticholic acid, tauro-obeticholic acid in human plasma and its application to a pharmacokinetic study.

A rapid, robust, simple, selective, and sensitive liquid chromatography-tandem mass spectrometry method was developed for the simultaneous estimation of obeticholic acid and its two pharmacologically active metabolites, glyco-obeticholic acid, and tauro-obeticholic acid in human plasma. The analytes and their heavy stable isotope-labeled internal standards were extracted from 250 µL human plasma by a solid-phase extraction technique. The method linearity was established over a concentration range of 0.410 to 120.466 ng/mL for obeticholic acid, 0.414 to 121.708 ng/mL for glyco-obeticholic acid, and 0.255 to 75.101 ng/mL for tauro-obeticholic acid. The method was fully validated as per current guidelines on bioanalytical method validation of "United States of Food and Drug Administration" and "European Medicines Agency." The method was successfully applied to study the pharmacokinetics of obeticholic acid, glyco-obeticholic acid, and tauro-obeticholic acid following oral administration of obeticholic acid tablets to healthy male volunteers. All the measured concentrations were within calibration curve ranges.

Sensitivity and Specificity of Biochemical Tests for Diagnosis of Intrahepatic Cholestasis of Pregnancy.

BACKGROUND AND AIM: Intrahepatic cholestasis of pregnancy (ICP) is linked with increased risk of fetal complications. An accurate diagnostic test is needed to diagnose this disorder on time. We aimed to assess sensitivity and specificity of laboratory tests used for diagnosis of intrahepatic cholestasis of pregnancy and determine more reliable cut-off values of transaminases. MATERIAL AND METHODS: Sixty one symptomatic patients with ICP and 29 healthy pregnant women were included in the retrospective analysis. RESULTS: ICP patients had higher total bile acids (TBA) levels than healthy women (32 vs. 6; P < 0.0001) due to increase in cholic acid (CA) and chenodeoxycholic acid (CDCA). CA/CDCA ratio was significantly higher in ICP patients compared to healthy pregnant women (1.13 vs. 0.68; P < 0.00002). TBA, CA, CDCA and CA/CDCA ratio demonstrate the following sensitivity (94%, 96%, 89%, 71.9%) and specificity (63%, 63%, 59%, 79.3%, respectively) for ICP diagnosis. Lowering cut-off values for ALT (31 U/L) and AST (30 U/L) resulted only in minimal increase of sensitivity to 92.2% vs. 90.1% for ALT and to 92.2%, vs. 90.6% for AST. CONCLUSION: The present study did not reveal any single specific and sensitive marker for reliable diagnosis of ICP. Establishment of lower cut-off values for transaminases activity might only minimally increase the accuracy of diagnosing ICP.

Distinct Plasma Bile Acid Profiles of Biliary Atresia and Neonatal Hepatitis Syndrome.

Biliary atresia (BA) is a severe chronic cholestasis disorder of infants that leads to death if not treated on time. Neonatal hepatitis syndrome (NHS) is another leading cause of neonatal cholestasis confounding the diagnosis of BA. Recent studies indicate that altered bile acid metabolism is closely associated with liver injury and cholestasis. In this study, we systematically measured the bile acid metabolome in plasma of BA, NHS, and healthy controls. Liver bile acids were also measured using biopsy samples from 48 BA and 16 NHS infants undergoing operative cholangiography as well as 5 normal adjacent nontumor liver tissues taken from hepatoblastoma patients as controls. Both BA and NHS samples had significantly elevated bile acid levels in plasma compared to normal controls. BA patients showed a distinct bile acid profile characterized by the higher taurochenodeoxycholic acid (TCDCA) level and lower chenodeoxycholic acid (CDCA) level than those in NHS patients. The ratio of TCDCA to CDCA in plasma was significantly higher in BA compared to healthy infants (p < 0.001) or NHS (p < 0.001). The area under receiver operating characteristic curve for TCDCA/CDCA to differentiate BA from NHS was 0.923 (95% CI: 0.862-0.984). These findings were supported by significantly altered expression levels of bile acid transporters and nuclear receptors in liver including farnesoid X receptor (FXR), small heterodimer partner (SHP), bile salt export pump (BSEP), and multidrug resistant protein 3 (MDR3) in BA compared to NHS. Taken together, the plasma bile acid profiles are distinct in BA, NHS, and normal infants, as characterized by the ratio of TCDCA/CDCA differentially distributed among the three groups of infants.

Simultaneous determination of Obeticholic acid and its two major metabolites in human plasma after administration of Obeticholic acid tablets using ultra-high performance liquid chromatography tandem mass spectrometry.

Obeticholic acid (OCA), a semisynthetic bile acid derivative, was approved for its therapeutic use in primary biliary cirrhosis. OCA has a enterohepatic circulation and host-gut microbiota metabolic interaction, which produce various metabolites. Such metabolites, especially structural isomers of OCA, together with the need to achieve idea lower limit of quantitation (LLOQ) with minimum matrix interference, bring about significant difficulties to the bioanalysis of OCA. Herein, by applying a combination of solid-phase extraction (SPE) and ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), we introduced an approach for the bioanalysis of OCA along with its two major metabolites-glyco-OCA (GOA) and tauro-OCA (TOA) in human plasma, the full validation results of which showed excellent performance. The quantitative range is 0.2506 âˆ¼ 100.2 ng/mL for OCA, 0.2500 âˆ¼ 100.0 ng/mL for GOA, as well as 0.1250 âˆ¼ 50.00 ng/mL for TOA, respectively. This method was successfully applied to the pharmacokinetic studies in healthy subjects following administration of OCA tablets.

Bile acid concentration reference ranges in a pregnant Latina population.

OBJECTIVE: The total bile acid (TBA) concentration criterion for diagnosing intrahepatic cholestasis of pregnancy varies in the published literature. The purpose of this study was to establish pregnancy-specific reference ranges for the TBA concentration among Latina women. STUDY DESIGN: Self-identified Latina women (n = 211) over 18 years of age with a singleton pregnancy were recruited and had random serum samples drawn during the second and third trimesters. The total and fractionated bile acid concentrations were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and reference ranges were calculated. Laboratory-provided general reference ranges from a general population of adult men and nonpregnant women were used for comparison. RESULTS: The TBA reference range for our Latina pregnant population (<8.5 µmol/L) was markedly lower than the laboratory-provided reference range (4.5 to 19.2 µmol/L). CONCLUSION: These data suggest that the upper TBA concentration reference range in our Latina pregnant population is 8.5 µmol/L, based on LC-MS/MS measurements.

Effect of probiotics on serum bile acids in patients with ulcerative colitis.

BACKGROUND/AIMS: Evaluation of bile acids (BA) is useful for assessing the changes of intestinal flora in patients with ulcerative colitis (UC). During enterohepatic circulation, the intestinal micro flora cause 7 alpha-dehydroxylation of cholic acid (CA) and chenodeoxycholic acid (CDCA), yielding deoxycholic acid(DCA) and lithocholic acid, respectively. The aim of the present study was to investigate the effects of probiotics in patients with UC by examining changes of the serum BA profile. METHODOLOGY: Twenty-seven patients were divided into the following 2 groups based on endoscopic findings: Fifteen patients with distal UC (dUC group) and 12 patients with pancolitis (pUC group). After treatment with mesalazine or salazosulfapyridine (5-ASA), all patients achieved remission. Then they were given 5-ASA plus the probiotic Clostridium butyricum Miyairi (3.0 g/day) for 4 weeks. RESULTS: After 4 weeks of probiotic treatment, %CDCA was significantly higher and %DCA was significantly lower in the pUC group than in the HV group. In contrast, the dUC group showed no significant differences of %CDCA or %DCA from the HV group after 4 weeks. CONCLUSIONS: Probiotic therapy restored intestinal flora involved in 7 alpha-dehydroxylation in the dUC group, but not in the pUC group.

High-dose ursodeoxycholic acid is associated with the development of colorectal neoplasia in patients with ulcerative colitis and primary sclerosing cholangitis.

OBJECTIVES: Some studies have suggested that ursodeoxycholic acid (UDCA) may have a chemopreventive effect on the development of colorectal neoplasia in patients with ulcerative colitis (UC) and primary sclerosing cholangitis (PSC). We examined the effects of high-dose (28-30 mg/kg/day) UDCA on the development of colorectal neoplasia in patients with UC and PSC. METHODS: Patients with UC and PSC enrolled in a prior, multicenter randomized placebo-controlled trial of high-dose UDCA were evaluated for the development of colorectal neoplasia. Patients with UC and PSC who received UDCA were compared with those who received placebo. We reviewed the pathology and colonoscopy reports for the development of low-grade or high-grade dysplasia or colorectal cancer. RESULTS: Fifty-six subjects were followed for a total of 235 patient years. Baseline characteristics (including duration of PSC and UC, medications, patient age, family history of colorectal cancer, and smoking status) were similar for both the groups. Patients who received high-dose UDCA had a significantly higher risk of developing colorectal neoplasia (dysplasia and cancer) during the study compared with those who received placebo (hazard ratio: 4.44, 95% confidence interval: 1.30-20.10, P=0.02). CONCLUSIONS: Long-term use of high-dose UDCA is associated with an increased risk of colorectal neoplasia in patients with UC and PSC.

Bioequivalence and Pharmacokinetic Profiles of Generic and Branded Obeticholic Acid in Healthy Chinese Subjects Under Fasting and Fed Conditions.

OBJECTIVES: This study was conducted to evaluate the bioequivalence (BE) of a generic form of obeticholic acid (OCA) and OcalivaTM under fasting and fed conditions and to determine the effects of food on the pharmacokinetic (PK) profiles of OCA in healthy Chinese subjects. METHODS: A randomized, single-dose, three-sequence, three-period, partial replicated crossover study was conducted with a 21-day washout interval between periods under fasting (n=48) and fed (n=48) conditions. Blood samples for OCA and its metabolites Glyco-OCA and Tauro-OCA were collected up to 168 hours after administration in each period. PK parameters were calculated using the non-compartmental method. Geometric mean ratios for PK parameters of the test to reference drug under fasting and fed conditions and their 90% confidence intervals were estimated. Safety evaluations were carried out all through the study. RESULTS: A total of 91 subjects completed the study with 45 in a fasted state and 46 receiving a high-fat diet. There were no serious or unexpected drug-related adverse events occurring during the study. There was no significant difference in the main PK parameters of the two preparations, irrespective of the fasting or fed conditions. Under fasting and fed conditions, the SWR of lnCmax, lnAUC0-t and lnAUC0-∞ were 0.445, 0.370, 0.448, 0.340, 0.168, and 0.180, respectively. Thus, the average BE or the reference-scaled average BE was used to verify that the two preparations were bioequivalent under fasting and fed conditions. Compared with the fasting state, the AUC0-t of the test drug, the AUC0-t, and AUC0-∞ of the reference drug were higher in the fed state. CONCLUSION: The test drug and the reference drug were BE and well tolerated in Chinese healthy subjects under both fasting and fed conditions. Food-intake may cause a significant difference in the main PK parameters of the two preparations.

Metabolomic Detection Between Pancreatic Cancer and Liver Metastasis Nude Mouse Models Constructed by Using the PANC1-KAI1/CD82 Cell Line.

Background: Pancreatic cancer (PC) has a poor prognosis and is prone to liver metastasis. The KAI1/CD82 gene inhibits PC metastasis. This study aimed to explore differential metabolites and enrich the pathways in serum samples between PC and liver metastasis nude mouse models stably expressing KAI1/CD82. Methods: KAI1/CD82-PLV-EF1α-MCS-IRES-Puro vector and PANC1 cell line stably expressing KAI1/CD82 were constructed for the first time. This cell line was used to construct 3 PC nude mouse models and 3 liver metastasis nude mouse models. The different metabolites and Kyoto encyclopedia of genes and genomes (KEGG) and human metabolome database (HMDB) enrichment pathways were analyzed using the serum samples of the 2 groups of nude mouse models on the basis of untargeted ultra-performance liquid chromatography-tandem mass spectrometry platform. Results: KAI1/CD82-PLV-EF1α-MCS-IRES-Puro vector and PANC1 cell line stably expressing KAI1/CD82 were constructed successfully, and all nude mouse models survived and developed cancers. Among the 1233 metabolites detected, 18 metabolites (9 upregulated and 9 downregulated) showed differences. In agreement with the literature data, the most significant differences between both groups were found in the levels of bile acids (taurocholic acid, chenodeoxycholic acid), glycine, prostaglandin E2, vitamin D, guanosine monophosphate, and inosine. Bile recreation, primary bile acid biosynthesis, and purine metabolism KEGG pathways and a series of HMDB pathways (P < .05) contained differential metabolites that may be associated with liver metastasis from PC. However, the importance of these metabolites on PC liver metastases remains to be elucidated. Conclusions: Our findings suggested that the metabolomic approach may be a useful method to detect potential biomarkers in PC.

A novel voltammetric approach to the detection of primary bile acids in serum samples.

An innovative voltammetric approach to the detection of cholic and chenodeoxycholic acids is presented. These two primary bile acids are important biomarkers of liver function in humans and are involved in many physiological processes in the human body. Herein we describe a way to reproducibly convert the hard-to-detect bile acid molecule into an easily detectable derivative in situ using 0.1 M HClO4 in acetonitrile (water content 0.55%). Under these conditions the bile acids are dehydrated and the resulting alkenes can be subsequently oxidized electrochemically on polished boron-doped diamond electrode under unchanged conditions at approximately +1.2 V vs. Ag/AgNO3 in acetonitrile. After optimization, differential pulse voltammetry provides competitive limits of detection of 0.5 µM and 1.0 µM for cholic and chenodeoxycholic acid, respectively, with a linear course of calibration dependency to the minimum of 80 µM. The method was applied for detection of cholic and chenodeoxycholic acids in artificial and human serum samples using single solid phase extraction on C-18 cartridge for preliminary separation of the analytes. High recoveries of 80-90% were consistently obtained by the proposed voltammetric method and reference HPLC with fluorescence detection for human serum samples, confirming good selectivity for real-life samples.

Dynamic Metabolomics Study of the Bile Acid Pathway During Perioperative Primary Hepatic Carcinoma Following Liver Transplantation.

BACKGROUND There are many situations of abnormal metabolism influencing liver graft function. This study aims to provide data for the development of liver function recovery after liver transplantation by dynamically analyzing metabolites of bile acids pathway in serum. MATERIAL AND METHODS A comprehensive metabolomics profiling of serum of 9 liver transplantation patients before transplantation, on the 1st, 3rd, and 7th days after liver transplantation, and healthy individuals were performed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Multivariate data and dynamic analysis were used to search for biomarkers between the metabolomics profiles present in perioperative liver transplantation and normal controls. RESULTS Thirty-three differential endogenous metabolites were screened by the threshold of variable importance in the projection (VIP) from an orthogonal partial least square discriminant analysis (OPLS-DA) greater than 1.0, q-value <0.05, and fold change (FC) ≤0.8 or ≥1.2 between the preoperative group and the normal controls in negative mode. The metabolite intensities of taurocholic acid, taurochenodeoxycholic acid, chenodeoxycholic acid glycine conjugate, and glycocholic acid pre-transplantation were significantly higher than those of normal controls. The average metabolite intensities of taurocholic acid and taurochenodesoxycholic acid on the first day after liver transplantation were lower than those observed pre-transplantation. The average metabolite intensities on day 3 after liver transplantation showed a sudden increase and then decreased after 7 postoperative days. The average metabolite intensities of glycocholic acid and chenodeoxycholic acid glycine conjugate showed an increasing trend on the 1st, 3rd, and 7th days after liver transplantation. CONCLUSIONS Use of taurocholic acid and taurochenodeoxycholic acid-related bile secretion, liver regeneration, and de novo bile acid synthesis may help clinical evaluation and provide data for the development of liver function recovery after liver transplantation.

Circulating primary bile acid is correlated with structural remodeling in atrial fibrillation.

BACKGROUND: Circulating primary bile acid was involved in the regulation of cardiac ionic channel currents and ventricular myocyte apoptosis, but it was unknown whether or not it played a role in structural remodeling of AF. This study was aimed to testify the hypothesis that elevated chenodeoxycholic acid (CDCA) concentration correlated with left atrial low voltage area (LVA) and could induce apoptosis of atrial myocytes in AF. METHODS AND RESULTS: Serum concentrations of 12 types of bile acids were determined in patients with paroxysmal (n = 21), persistent AF (n = 20), and type A pre-excitation and paroxysmal supraventricular tachycardia (PSVT) (n = 19) and were correlated with LVA in AF, which was obtained by electroanatomical mapping during ablation. Additionally, the impact of CDCA incubation on apoptosis of mouse atrial myocytes was evaluated. Serum levels of CDCA and cholic acid were significantly higher in AF than in PSVT. CDCA serum concentration was significantly higher in persistent AF than in paroxysmal AF. CDCA serum level was positively correlated with the size (r = 0.78, P < 0.05) and proportion of LVA (r = 0.89, P < 0.05) in AF patients. CDCA (75 µM, 100 µM) promoted atrial myocyte apoptosis in a concentration-dependent manner. CONCLUSIONS: The higher circulating level of CDCA in AF than in PSVT, positive correlation of CDCA with LVA in AF, and incubation dose-dependent increase of mouse atrial myocyte apoptosis indicated that CDCA might play a significant role in the progress of structural remodeling of AF.

Oral gavage of nano-encapsulated conjugated acrylic acid-bile acid formulation in type 1 diabetes altered pharmacological profile of bile acids, and improved glycaemia and suppressed inflammation.

BACKGROUND: Ursodeoxycholic acid (UDCA) is a secondary hydrophilic bile acid, metabolised in the gut, by microbiota. UDCA is currently prescribed for primary biliary cirrhosis, and of recently has shown ß-cell protective effects, which suggests potential antidiabetic effects. Thus, this study aimed to design targeted-delivery microcapsules for oral uptake of UDCA and test its effects in type 1 diabetes (T1D). METHODS: UDCA microcapsules were produced using alginate-NM30 matrix. Three equal groups of mice (6-7 mice per group) were gavaged daily UDCA powder, empty microcapsules and UDCA microcapsules for 7 days, then T1D was induced by alloxan injection and treatments continued until mice had to be euthanised due to weight loss > 10% or severe symptoms develop. Plasma, tissues, and faeces were collected and analysed for bile acids' concentrations. RESULTS: UDCA microcapsules brought about reduction in elevated blood glucose, reduced inflammation and altered concentrations of the primary bile acid chenodeoxycholic acid and the secondary bile acid lithocholic acid, without affecting survival rate of mice. CONCLUSION: The findings suggest that UDCA exerted direct protective effects on pancreatic ß-cells and this is likely to be associated with alterations of concentrations of primary and secondary bile acids in plasma and tissues. Three equal groups of mice were gavaged daily UDCA (ursodeoxycholic acid) powder, empty microcapsules and UDCA microcapsules for 7 days, then T1D was induced and treatments continued until mice had to be euthanised. UDCA microcapsules brought about reduction in elevated blood glucose, reduced inflammation and altered concentrations of the primary bile acid chenodeoxycholic acid and the secondary bile acid lithocholic acid, without affecting survival rate of mice. The findings suggest that UDCA exerted direct protective effects on pancreatic ß-cells and this is likely to be associated with alterations of concentrations of primary and secondary bile acids in plasma and tissues.

Ratio of Conjugated Chenodeoxycholic to Muricholic Acids is Associated with Severity of Nonalcoholic Steatohepatitis.

OBJECTIVE: Bile acids (BAs) are important molecules in the progression of nonalcoholic fatty liver disease. This study aimed to investigate BA profile alterations in Chinese nonalcoholic steatohepatitis (NASH) patients. METHODS: BA profiles in serum and liver tissues were determined by ultraperformance liquid chromatography coupled to tandem mass spectrometry in patients from two different clinical centers. RESULTS: A total of 134 participants were enrolled in this study to serve as the training (n = 87) and validation (n = 47) cohorts. The ratio of circulating conjugated chenodeoxycholic acids to muricholic acids (P = 0.001) was elevated from healthy controls to non-NASH individuals to NASH individuals in a stepwise manner in the training cohort and was positively associated with the histological severity of NASH: steatosis (R2 = 0.12), lobular inflammation (R2 = 0.12), ballooning (R2 = 0.11), and fibrosis stage (R2 = 0.18). The ratio was elevated in the validation cohort of NASH patients (P < 0.001), and it was able to predict NASH (area under the receiver operating characteristic curve: 75%) and significant fibrosis (area under the receiver operating characteristic curve: 71%) in these two cohorts. Moreover, this elevated ratio and impaired farnesoid X receptor signaling were found in the NASH liver. CONCLUSIONS: Altered BA profile in NASH is closely associated with the severity of liver lesions, and it has the potential for predicting NASH development.

An ultra-performance liquid chromatography-tandem mass spectrometry method for the determination of obeticholic acid in rat plasma and its application in preclinical pharmacokinetic studies.

Currently, ursodeoxycholic acid (UDCA) is the only clear medical treatment for primary biliary cholangitis (PBC). However, approximately 40% of patients are not sensitive to UDCA. In recent years, obeticholic acid (OCA) combined with UDCA has been used in the PBC patients who were not sensitive to UDCA, or as monotherapy for PBC adult patients who are intolerant to UDCA. OBJECTIVE: To develop and validate a specific, sensitive and reliable tandem mass spectrometry (UPLC-MS/MS) method for the determination of obeticholic acid (OCA) in rat plasma. METHODS: Plasma samples were treated with liquid-liquid extraction. Diazepam was selected as the internal standard (IS). Chromatographic separation was achieved by an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 µm) and a mobile phase consisting of acetonitrile and ultrapure water (containing 0.1% formic acid). The analyte was detected in positive ion mode by electrospray ionization mass spectrometry (ESI-MS). Multiple reaction monitoring (MRM) methods were used to detect specific precursor and product ions. The target ion pair of OCA was 419.38 → 401.22, and the IS was 285.05 → 193.02. RESULTS: The linear range of OCA in rat plasma was 0.05-50 µg/mL (R2 = 0.992); the recovery rate was 91.34%-97.37%. This assay showed good intra- and inter-day precision and accuracy. No significant matrix effects in this study. CONCLUSION: A specific, sensitive and reliable quantitative analysis method was established to detect OCA after oral/intravenous administration in rat plasma via UPLC-MS/MS. It was appropriate for preclinical pharmacokinetic studies of OCA.

Hepatic uptake of bile acids in man. Fasting and postprandial concentrations of individual bile acids in portal venous and systemic blood serum.

This investigation was undertaken in order to (a) characterize the postprandial inflow of individual bile acids to the liver and (b) determine if peripheral venous bile acid levels always adequately reflect the portal venous concentration, or if saturation of hepatic bile acid uptake can occur under physiological conditions. In five patients with uncomplicated cholesterol gallstone disease, the umbilical cord was cannulated during cholecystectomy, and a catheter was left in the left portal branch for 5 to 7 d. The serum concentrations of cholic acid, chenodeoxycholic acid, and deoxycholic acid in portal venous and systemic circulation were then determined at intervals of 15 to 30 min before and after a standardized meal. A highly accurate and specific gas chromatographic/mass spectrometric technique was used. The sum of the fasting concentrations of the three bile acids averaged 14.04+/-4.13 mumol/liter in portal venous serum, and 2.44+/-0.31 mumol/liter in peripheral venous serum. The estimated hepatic fractional uptake of cholic acid was approximately 90%, and those of chenodeoxycholic acid and deoxycholic acid were 70-80%. This resulted in an enrichment of systemic bile acids in the dihydroxy bile acid species. In response to a standardized meal, portal venous bile acid concentrations increased two- to sixfold, with a peak seen 15-60 min after the meal. The maximum postprandial portal venous bile acid concentration averaged 43.04+/-6.12 mumol/liter, and the corresponding concentration in peripheral serum was 5.22+/-0.74 mumol/liter. The estimated fractional uptakes of the individual bile acids were not affected by the increased inflow to the liver. The peripheral venous concentrations of individual as well as total bile acids were well correlated with those in portal venous serum. The results (a) give a quantitation of postprandial bile acid inflow to the liver and (b) indicate that the hepatic uptake system for bile acids in healthy man cannot be saturated during maximal inflow of endogenous bile acids. Measurement of peripheral serum bile acids can thus give important information on the status of the enterohepatic circulation.

Reduced activity of 11 beta-hydroxysteroid dehydrogenase in patients with cholestasis.

Enhanced renal sodium retention and potassium loss in patients with cirrhosis is due to activation of mineralocorticoid receptors (MRs). Increased aldosterone concentrations, however, do not entirely explain the activation of MR in cirrhosis. Here, we hypothesize that cortisol activates MRs in patients with cholestasis. We present evidence that access of cortisol to MRs is a result of bile acid-mediated inhibition of 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2), an MR-protecting enzyme that converts cortisol to cortisone. Twelve patients with biliary obstruction and high plasma bile acid levels were studied before and after removal of the obstruction. The urinary ratio of (tetrahydrocortisol + 5 alpha-tetrahydrocortisol)/tetrahydrocortisone, a measure of 11 beta-HSD2 activity, decreased from a median of 1.91 during biliary obstruction to 0.78 at 4 and 8 weeks after removal of the obstruction and normalization of plasma bile acid concentrations. In order to demonstrate that bile acids facilitate access of cortisol to the MR by inhibiting 11 beta-HSD2, an MR translocation assay was performed in HEK-293 cells transfected with human 11 beta-HSD2 and tagged MR. Increasing concentrations of chenodeoxycholic acid led to cortisol-induced nuclear translocation of MR. In conclusion, 11 beta-HSD2 activity is reduced in cholestasis, which results in MR activation by cortisol.

Bioavailability and hypoglycemic activity of the semisynthetic bile acid salt, sodium 3alpha,7alpha-dihydroxy-12-oxo-5beta-cholanate, in healthy and diabetic rats.

Previous studies in our laboratory have shown that the semisynthetic bile acid derivative, sodium 3alpha,7alpha-dihydroxy-12-oxo-5beta-cholanate (MKC), has hypoglycemic activity. The aim of this study was to investigate the relationship between the pharmacokinetics and hypoglycemic activity of MKC in healthy and diabetic rats. Groups of healthy and alloxan-induced diabetic rats were dosed intravenously (i.v.) and orally with MKC (4 mg/kg). Blood samples were taken before administration of the dose and at 20, 40, 60, 80, 120, 150, 180, 210 and 240 minutes post-dose. MKC serum concentration was measured by HPLC, and pharmacokinetic parameters determined using the WinNonlin program. The absolute bioavailability of MKC was found to be low in healthy and diabetic rats (29 and 23% respectively) and was not significantly different between the two groups. Mean residence time (MRT), volume of distribution (Vd) and half-life (t1/2) of MKC after oral administration were significantly lower in diabetic than in healthy rats (21, 31 and 29% respectively). After the i.v. dose, the change in blood glucose concentration was not significant in either healthy or diabetic rats. After the oral dose, the decrease in blood glucose concentration was significant, reaching a maximum decrease from baseline of 24% in healthy rats and 15% in diabetic rats. The results suggest that a first-pass effect is crucial for the hypoglycemic activity of MKC, indicating that a metabolite of MKC and/or interference with metabolism and glucose transport is responsible.

Lowered fasting chenodeoxycholic acid correlated with the decrease of fibroblast growth factor 19 in Chinese subjects with impaired fasting glucose.

The gut-derived hormone Fibroblast growth factor 19 (FGF19) could regulate glucose metabolism and is induced by bile acids (BAs) through activating Farnesoid X Receptor (FXR). FGF19 was found to decrease in subjects with isolated-impaired fasting glucose (I-IFG) and type 2 diabetes mellitus (T2DM). However, the reason for the change of FGF19 in subjects with different glucometabolic status remained unclear. Here we measured six BAs including chenodeoxycholic acid (CDCA), cholic acid, deoxycholic acid, their glycine conjugates and FGF19 levels during oral glucose tolerance test (OGTT) in normal glucose tolerance (NGT), isolated-impaired glucose tolerance, I-IFG, combined glucose intolerance (CGI) and T2DM subjects. After OGTT, serum FGF19 peaked at 120 min in all subjects. Glycine conjugated BAs peaked at 30 min, while free BAs did not elevated significantly. Consistent with the decrease trend in FGF19 levels, fasting serum CDCA levels in subjects with I-IFG, CGI and T2DM were significantly lower than NGT subjects (P < 0.05). Fasting serum CDCA was independently associated with FGF19. CDCA strongly upregulated FGF19 mRNA levels in LS174T cells in a dose- and time-dependent manner. These results suggest that the decrease of FGF19 in subjects with I-IFG was at least partially due to their decrease of CDCA acting via FXR.

Molecular adsorbent recirculating system (MARS) application in liver failure: clinical and hemodepurative results in 22 patients.

PURPOSE: Acute liver failure (ALF) and acute on chronic liver failure (ACLF) still show a poor prognosis. MARS was used in 22 patients with ALF or ACLF to prolong patient survival for liver function recovery or as a bridge to transplantation. DESIGN: Evaluation of depurative efficiency, biocompatibility, hemodynamics, encephalopathy (HE) and clinical outcome. PROCEDURES: During 71 five-hour sessions we evaluated (0', 60', 120', 180', 240', 300'): bilirubin, ammonia, cholic acid (CCA), chenodeoxycholic acid (CCDCA), leukocytes, platelets, hemoglobin and mean arterial pressure (MAP). Serum creatinine, electrolytes, cardiac output, cardiac index (bioimpedence) and HE (West Haven Criteria score) were evaluated at 0' and 300'. STATISTICAL METHODS AND OUTCOME MEASURES: Student's t-test for pre- vs. end-session values was used. For bilirubin and ammonia the correlation test was made between pre- and end-session values and between pre-session values and removal rates (RRS). MAIN FINDINGS: Survival was 90.9% at 7 days, 40.9% at 30 days. Pre- vs. end-session: bilirubin from 37.2 +/- 12.5 mg/dL to 24.9 +/- 8.9 mg/dL (p < 0.01), ammonia from 88.0 +/- 60.4 micromol/L to 43.6 +/- 32.9 micromol/L (p < 0.01), CCA from 42.8 +/- 21.0 micromol/L 18.2 +/- 9.8 micromol/L (p < 0.01), CCDCA from 26.3 +/- 6.3 micromol/L to 15.7+/-7.6 micromol/L (p<0.01). The correlation test between pre-session values of bilirubin and ammonia vs. RR S was respectively 0.32 (p = 0.01) and 0.30 (p = 0.04). Leukocytes, platelets and hemoglobin remained stable. MAP increased from 82.0 +/- 12.0 mmHg to 87.0 +/- 13.0 mmHg (p < 0.05), West Haven Criteria score decreased from 2.7 +/- 0.7 to 0.7 +/- 0.7 (p < 0.001). CONCLUSION: MARS treatment led in all patients to an improvement of clinical, hemodynamic and neurological conditions, with significant reduction in the hepatic toxins blood level. Treatment biocompatibility and tolerance were satisfactory.