Effect of endoplasmic reticulum stress on endothelial ischemia-reperfusion injury in humans.

Endoplasmic reticulum stress contributes to ischemia-reperfusion (I/R) injury in rodent and cell models. However, the contribution of endoplasmic reticulum stress in the pathogenesis of endothelial I/R injury in humans is unknown. We tested the hypothesis that compared with placebo, inhibition of endoplasmic reticulum stress via ingestion of tauroursodeoxycholic acid would prevent the attenuation of endothelium-dependent vasodilation following I/R injury. Twelve young adults (6 women) were studied following ingestion of a placebo or 1,500 mg tauroursodeoxycholic acid (TUDCA). Endothelium-dependent vasodilation was assessed via brachial artery flow-mediated dilation (duplex ultrasonography) before and after I/R injury, which was induced by 20 min of arm ischemia followed by 20 min of reperfusion. Endothelium-independent vasodilation (glyceryl trinitrate-mediated vasodilation) was also assessed after I/R injury. Compared with placebo, TUDCA ingestion increased circulating plasma concentrations by 145 ± 90 ng/ml and increased concentrations of the taurine unconjugated form, ursodeoxycholic acid, by 560 ± 156 ng/ml (both P < 0.01). Ischemia-reperfusion injury attenuated endothelium-dependent vasodilation, an effect that did not differ between placebo (pre-I/R, 5.0 ± 2.1% vs. post-I/R, 3.5 ± 2.2%) and TUDCA (pre-I/R, 5.6 ± 2.1% vs. post-I/R, 3.9 ± 2.1%; P = 0.8) conditions. Similarly, endothelium-independent vasodilation did not differ between conditions (placebo, 19.6 ± 4.8% vs. TUDCA, 19.7 ± 6.1%; P = 0.9). Taken together, endoplasmic reticulum stress does not appear to contribute to endothelial I/R injury in healthy young adults.

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.

Metabolite reanalysis revealed potential biomarkers for COVID-19: a potential link with immune response.

Aim: To understand the pathological progress of COVID-19 and to explore the potential biomarkers. Background: The COVID-19 pandemic is ongoing. There is metabolomics research about COVID-19 indicating the rich information of metabolomics is worthy of further data mining. Methods: We applied bioinformatics technology to reanalyze the published metabolomics data of COVID-19. Results: Benzoate, ß-alanine and 4-chlorobenzoic acid were first reported to be used as potential biomarkers to distinguish COVID-19 patients from healthy individuals; taurochenodeoxycholic acid 3-sulfate, glucuronate and N,N,N-trimethyl-alanylproline betaine TMAP are the top classifiers in the receiver operating characteristic curve of COVID-severe and COVID-nonsevere patients. Conclusion: These unique metabolites suggest an underlying immunoregulatory treatment strategy for COVID-19.

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.

Use of a Bile Salt Export Pump Knockdown Rat Susceptibility Model to Interrogate Mechanism of Drug-Induced Liver Toxicity.

Inhibition of the bile salt export pump (BSEP) may be associated with clinical drug-induced liver injury, but is poorly predicted by preclinical animal models. Here we present the development of a novel rat model using siRNA knockdown (KD) of Bsep that displayed differentially enhanced hepatotoxicity to 8 Bsep inhibitors and not to 3 Bsep noninhibitors when administered at maximally tolerated doses for 7 days. Bsep KD alone resulted in 3- and 4.5-fold increases in liver and plasma levels, respectively, of the sum of the 3 most prevalent taurine conjugated bile acids (T3-BA), approximately 90% decrease in plasma and liver glycocholic acid, and a distinct bile acid regulating gene expression pattern, without resulting in hepatotoxicity. Among the Bsep inhibitors, only asunaprevir and TAK-875 resulted in serum transaminase and total bilirubin increases associated with increases in plasma T3-BA that were enhanced by Bsep KD. Benzbromarone, lopinavir, and simeprevir caused smaller increases in plasma T3-BA, but did not result in hepatotoxicity in Bsep KD rats. Bosentan, cyclosporine A, and ritonavir, however, showed no enhancement of T3-BA in plasma in Bsep KD rats, as well as Bsep noninhibitors acetaminophen, MK-0974, or clarithromycin. T3-BA findings were further strengthened through monitoring TCA-d4 converted from cholic acid-d4 overcoming interanimal variability in endogenous bile acids. Bsep KD also altered liver and/or plasma levels of asunaprevir, TAK-875, TAK-875 acyl-glucuronide, benzbromarone, and bosentan. The Bsep KD rat model has revealed differences in the effects on bile acid homeostasis among Bsep inhibitors that can best be monitored using measures of T3-BA and TCA-d4 in plasma. However, the phenotype caused by Bsep inhibition is complex due to the involvement of several compensatory mechanisms.

A new oral formulation for the release of sodium butyrate in the ileo-cecal region and colon.

AIM: To develop a new formulation with hydroxy propyl methyl cellulose and Shellac coating for extended and selective delivery of butyrate in the ileo-caecal region and colon. METHODS: One-gram sodium butyrate coated tablets containing 13C-butyrate were orally administered to 12 healthy subjects and 12 Crohn's disease patients and the rate of 13C-butyrate absorption was evaluated by 13CO2 breath test analysis for eight hours. Tauroursodeoxycholic acid (500 mg) was co-administered as a biomarker of oro-ileal transit time to determine also the site of release and absorption of butyrate by the time of its serum maximum concentration. RESULTS: The coated formulation delayed the 13C-butyrate release by 2-3 h with respect to the uncoated tablets. Sodium butyrate was delivered in the intestine of all subjects and a more variable transit time was found in Crohn's disease patients than in healthy subjects. The variability of the peak 13CO2 in the kinetic release of butyrate was explained by the inter-subject variability in transit time. However, the coating chosen ensured an efficient release of the active compound even in patients with a short transit time. CONCLUSION: Simultaneous evaluation of breath 13CO2 and tauroursodeoxycholic acid concentration-time curves has shown that the new oral formulation consistently releases sodium butyrate in the ileo-cecal region and colon both in healthy subjects and Crohn's disease patients with variable intestinal transit time. This formulation may be of therapeutic value in inflammatory bowel disease patients due to the appropriate release of the active compound.

High metabolic function of primary human and porcine hepatocytes in a polyurethane foam/spheroid culture system in plasma from patients with fulminant hepatic failure.

It has been demonstrated that plasma from patients with fulminant hepatic failure (FHF) interferes extensively with cellular function. We placed primary human and primary porcine hepatocytes in a polyurethane foam (PUF)/spheroid culture system and compared the metabolic functions in the plasma of patients with FHF in a 24-h stationary culture to those in a monolayer culture. The PUF/spheroid culture system using primary human and primary porcine hepatocytes significantly decreased ammonia content during 28-day culture. Fisher's ratio significantly increased at culture days 3 and 7. Tauroursodeoxycholic acid significantly increased and glycochenodeoxycholic acid and taurochenodeoxycholic acid decreased in the FHF patients' plasma at culture day 3. During at least a 24-h culture in the FHF patients' plasma, metabolic functions of primary human and primary porcine hepatocytes were almost identical. The present results indicate that the PUF/spheroid culture system using primary human or primary porcine hepatocytes demonstrated more advantageous metabolic functions in the plasma from patients with FHF than the monolayer culture.

Tauroursodeoxycholate prevents taurocholate induced cholestasis.

The nature of transport pathway(s) for the biliary excretion of taurocholate and tauroursodeoxycholate was examined by comparing the biliary transport maximum (Tm) value for taurocholate during the infusion of taurocholate alone with that of taurocholate combined with tauroursodeoxycholate. The combined infusion of tauroursodeoxycholate resulted in an appreciable excretion of tauroursodeoxycholate while the excretion rate of taurocholate was not reduced in comparison with the Tm value of taurocholate alone. Furthermore, the Tm state of taurocholate was maintained for a much longer period with the simultaneous infusion of tauroursodeoxycholate than by the infusion of taurocholate alone. The cholestasis usually produced by the excess infusion of taurocholate was also prevented when tauroursodeoxycholate was simultaneously infused. Since plasma taurocholate concentration was not significantly different from the two rat groups, the results suggest the presence of the facilitative interaction of tauroursodeoxycholate with the taurocholate excretion.

Conjugated bile acid and cholesterol levels in pregnant Africans and Caucasians.

Pregnancy serum glycochenodeoxycholic acid (GCDA) and taurochenodeoxycholic acid (TCDA) were simultaneously assayed by solid-phase fluoroimmunoassay (FIA) for apparently healthy Nigerians and caucasians domiciled in Nigeria and London respectively. Serum cholesterol values were also obtained for the same gestational period, that is, the second and third trimesters. The bile acid and cholesterol values were compared with those of their respective non-pregnant counterparts. Although there was a considerable fluctuation in the mean bile acid concentrations throughout this period for both pregnant groups, the mean bile acid value for each group was, however, within the normal range. The mean bile acid concentration for the pregnant African was significantly lower than that of her Caucasian counterpart, a notable factor in the assessment of hepatobiliary function for the African during pregnancy. Furthermore, a progressive rise in serum cholesterol during pregnancy, previously observed by other workers, was confirmed for both groups of women. But the increase in cholesterol concentration, which was greater in the Caucasian than the African, did not, however, appear to have affected the bile acid concentration during pregnancy.

Vertical sleeve gastrectomy reduces hepatic steatosis while increasing serum bile acids in a weight-loss-independent manner.

OBJECTIVE: Our objective was to investigate the role of bile acids in hepatic steatosis reduction after vertical sleeve gastrectomy (VSG). DESIGN AND METHODS: High fat diet (HFD)-induced obese C57Bl/6 mice were randomized to VSG, Sham operation (Sham), Sham operation with pair feeding to VSG (Sham-PF), or nonsurgical controls (Naïve). All mice were on HFD until sacrifice. Mice were observed postsurgery and data for body weight, body composition, metabolic parameters, serum bile acid level and composition were collected. Further hepatic gene expression by mRNA-seq and RT-PCR analysis was assessed. RESULTS: VSG and Sham-PF mice lost equal weight postsurgery while VSG mice had the lowest hepatic triglyceride content at sacrifice. The VSG mice had elevated serum bile acid levels that positively correlated with maximal weight loss. Serum bile composition in the VSG group had increased cholic and tauroursodeoxycholic acid. These bile acid composition changes in VSG mice explained observed downregulation of hepatic lipogenic and bile acid synthetic genes. CONCLUSION: VSG in obese mice results in greater hepatic steatosis reduction than seen with caloric restriction alone. VSG surgery increases serum bile acids that correlate with weight lost postsurgery and changes serum bile composition that could explain suppression of hepatic genes responsible for lipogenesis.

A surgical model in male obese rats uncovers protective effects of bile acids post-bariatric surgery.

Bariatric surgery elevates serum bile acids. Conjugated bile acid administration, such as tauroursodeoxycholic acid (TUDCA), improves insulin sensitivity, whereas short-circuiting bile acid circulation through ileal interposition surgery in rats raises TUDCA levels. We hypothesized that bariatric surgery outcomes could be recapitulated by short circuiting the normal enterohepatic bile circulation. We established a model wherein male obese rats underwent either bile diversion (BD) or Sham (SH) surgery. The BD group had a catheter inserted into the common bile duct and its distal end anchored into the middistal jejunum for 4-5 weeks. Glucose tolerance, insulin and glucagon-like peptide-1 (GLP-1) response, hepatic steatosis, and endoplasmic reticulum (ER) stress were measured. Rats post-BD lost significantly more weight than the SH rats. BD rats gained less fat mass after surgery. BD rats had improved glucose tolerance, increased higher postprandial glucagon-like peptide-1 response and serum bile acids but less liver steatosis. Serum bile acid levels including TUDCA concentrations were higher in BD compared to SH pair-fed rats. Fecal bile acid levels were not different. Liver ER stress (C/EBP homologous protein mRNA and pJNK protein) was decreased in BD rats. Bile acid gavage (TUDCA/ursodeoxycholic acid [UDCA]) in diet-induced obese rats, elevated serum TUDCA and concomitantly reduced hepatic steatosis and ER stress (C/EBP homologous protein mRNA). These data demonstrate the ability of alterations in bile acids to recapitulate important metabolic improvements seen after bariatric surgery. Further, our work establishes a model for focused study of bile acids in the context of bariatric surgery that may lead to the identification of therapeutics for metabolic disease.

Fasting and postprandial serum concentrations of glycine- and taurine-conjugated bile acids in Crohn's disease.

Fasting and postprandial serum concentrations of glycine and taurine conjugates of cholic, chenodeoxycholic, and deoxycholic acid were measured with a high-pressure liquid chromatographic-enzymatic assay in 17 patients with ileal Crohn's disease and in 17 controls. The postprandial concentrations of the taurine-conjugated bile acids in the patients were significantly lower than in the controls, whereas the concentrations of the glycine conjugates were not significantly different. The total glycine to taurine ratios of serum bile acids were significantly higher in the patients (means, 2.9 fasting and 4.8 postprandial) than in the controls (1.9 and 2.6). Of the patients, 65% had a postprandial total G/T ratio of serum bile acids which was above the control interval.

Saturation of hepatic transport of taurocholate in rats in vivo.

A single intravenous injection of [14C]taurocholate was followed up in blood and bile of rats submitted to steady intravenous infusions of taurocholate (TC) at rates of 0.0, 0.5, 1.0, and 1.5 mumol.min-1.100 g body wt-1 for at least 30 min. The transport rate constants and the amounts of TC in different compartments were estimated by weighted least-squares adjustment of a six-compartment model to the experimental data (3 compartments for TC distribution in blood, 2 compartments for liver, and 1 compartment for sinusoidal blood space). The saturation of the TC excretion rate was reached at 0.8 mumol.min-1.100 g body wt-1. It was characterized by a decrease of both the uptake and excretion rate constants, by an increase of the ratio of the amounts of TC in the two intrahepatic compartments (H'/H), and by an intrahepatic TC concentration of approximately 2 mM. When tauroursodeoxycholate (TUDC) was infused at a rate of 0.5 mumol.min-1.100 g body wt-1 together with TC at a rate of 1.5 mumol.min-1.100 g body wt-1, the TC excretion rate increased to 1.2 mumol.min-1.100 g body wt-1, and the excretion rate constant and H'/H decreased toward control values. These results support the hypothesis that the saturation of the transport of TC is due to TC hepatotoxicity and can be reduced by TUDC. Michaelis-Menten parameters, derived from saturation curves for both uptake and excretion steps, closely matched earlier results, thus confirming the good descriptive capacity of the model.

Diagnostic value of serum primary bile acids in detecting bile acid malabsorption.

Serum cholic and chenodeoxycholic acid conjugates were measured in fasting conditions and after meals in 14 patients with bile acid malabsorption due to ileal resection. Mean serum fasting levels of both primary bile acids did not differ from the controls. After meals, serum cholic acid peaks were lower in patients with ileal resection than in control subjects (p less than 0.001), while chenodeoxycholic acid peaks were reduced in colectomised patients (p less than 0.01). In the sera from patients with ileal resection, the glycine/glycine + taurine ratio for cholic and chenodeoxycholic acid increased (p less than 0.001) from morning to evening, and glycine/glycine + taurine ratio for chenodeoxycholic acid was significantly (p less than 0.01) different from the controls in the sera collected in the evening. The results are consistent with the concept of a better intestinal conservation of chenyl, mainly of the glycine conjugated from, than of cholylconjugates, in patients with ileal resection; this is probably because of passive absorption in the intestine. The postprandial peaks of serum cholic acid conjugates may therefore be regarded as a test of ileal dysfunction, while peaks of chenodeoxycholic acid conjugates suggest colonic impairment.

Hepatic uptake and intestinal absorption of bile acids in the rabbit.

The existence of transporters for bile acids (BA) in liver and intestine has been well documented, but information is still needed as to their respective transport capacity. In the present investigation, we compared the hepatic and intestinal transport rates for BA, using perfused livers and intestines. The livers and intestines were separately perfused and dose-response curves (0.25-10 mM) for tauroursodeoxycholate, taurocholate and taurodeoxycholate were obtained. The intestinal and mesenteric concentration and bile acid pattern were also evaluated in six non-fasting rabbits. Taurocholic, tauroursodeoxycholic and taurodeoxycholic acid ileal absorption showed saturation kinetics in the intestine as in the liver; the maximal uptake velocity for each bile acid in the liver was tenfold higher than the respective maximal transport velocity in the intestine; the Km values obtained in the liver were of the same order of magnitude, i.e. in the millimolar range. Taurocholic, tauroursodeoxycholic and taurodeoxycholic acid transport differences in the liver paralleled those in the intestine. Although the intestine was not homogeneously filled, the bile acid concentration in the ileal content fell into the range of the Km for the three studied bile acids, while the portal blood total bile acid concentration was inferior to the observed Kms of liver uptake. Therefore, both the hepatic and intestinal systems do not operate at their maximal transport rates at the prevailing concentrations in portal blood and luminal content, and the hepatic transport occurs at its highest efficiency (below the Km values) in physiological conditions.

Daily determination of individual serum bile acids allows early detection of hepatic allograft dysfunction.

Acute graft rejection is still a major cause of morbidity after orthotopic liver transplantation, and its diagnosis necessitates an invasive liver biopsy. Our aim has been to determine whether changes in individual serum bile acid levels after engraftment are sensitive, specific and reliable indicators of graft function and whether these changes can antedate other biochemical indicators of hepatic allograft rejection. Individual bile acids in 200 serum samples taken serially from eight adult liver transplant patients were measured. Patients with biopsy-confirmed graft dysfunction due to rejection or nonrejection causes (n = 6 episodes) had significantly higher serum concentrations of glycocholate plus glycochenodeoxycholate and taurocholate/taurochenodeoxycholate ratios than did noncomplicated grafts (n = 3). These changes antedated any other conventional biochemical parameters by at least 48 hr and were 100% sensitive and specific. None of the conventional liver function tests could match this. Acute rejection episodes (n = 3) were then compared with nonrejection causes of graft dysfunction (n = 3). In acute rejection we noted a significant increase in the concentration of glycodeoxycholate plus deoxycholate and a significant decrease in the cholate/chenodeoxycholate ratio compared with that in nonrejection graft malfunction. Both of these changes antedated any other biochemical parameters by 24 hr. In conclusion, individual serum bile acid assays, after orthotopic liver transplantation, can detect graft dysfunction resulting from any cause at an earlier time than routine biochemical tests, and they are sensitive, specific and reliable for early detection of graft dysfunction. In addition, acute rejection can be distinguished from other causes of graft dysfunction.

Comparative analysis of conjugated bile acids in human serum using high-performance liquid chromatography and capillary electrophoresis.

This paper describes the analysis of conjugated bile acids in human serum using reversed-phase high-performance liquid chromatography (HPLC) and micellar electrokinetic capillary electrophoresis (CE). Samples of healthy subjects and patients with different hepatic diseases were pretreated with a simple preparation procedure using a solid-phase extraction technique. The optimal analytical conditions of both chromatographic methods were investigated for the convenience and reliability for routine analysis. Both HPLC and CE methods were found to be reliable and compatible. The recoveries of nine bile acid conjugates using both methods were generally >85% and reproducibility >90%. The day-to-day variation of retention time was <5% for HPLC, while the variation of migration time for CE was <3%. Although the detection limit of the HPLC method (1 nmol/ml) was five times more sensitive than that of the CE method, the CE method was considered to be more time and cost effective.