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Lee C.-S.,National Taiwan University Hospital | Lee C.-S.,Taipei Tzu Chi Hospital | Kimura A.,Kurume University | Wu J.-F.,National Taiwan University Hospital | And 7 more authors.
Journal of Lipid Research | Year: 2017

Tetrahydroxy bile acids (THBAs) are hydrophilic and are present at minimal or undetectable levels in healthy human adults, but are present at high levels in bile salt export pump (abcb11)-knockout mice. The roles of THBAs in human cholestatic diseases are unclear. We aimed to investigate the presence of THBAs in patients with infantile intrahepatic cholestasis and its correlation with outcome. Urinary bile acids (BAs) were analyzed by GC-MS. Data were compared between good (n = 21) (disease-free before 1 year old) and poor prognosis groups (n = 19). Good prognosis patients had a higher urinary THBA proportion than poor prognosis patients [25.89% (3.45-76.73%) vs. 1.93% (0.05-48.90%)]. A urinary THBA proportion >7.23% predicted good prognosis with high sensitivity (95.24%), specificity (84.21%), and area under the curve (0.91) (P < 0.0001). A THBA proportion ?7.23% was an independent factor for decreased transplantfree survival (hazard ratio = 7.16, confidence interval: 1.24- 41.31, P = 0.028). Patients with a confirmed ABCB11 or tight junction protein 2 gene mutation (n = 7) had a minimally detectable THBA proportion (0.23-2.99% of total BAs). Three patients with an ATP8B1 mutation had an elevated THBA proportion (7.51-37.26%). In conclusion, in addition to disease entity as a major determinant of outcome, a high THBA level was associated with good outcome in the infantile intrahepatic cholestasis patients.


PubMed | Buddhist Tzu Chi Medical Foundation, Kurume University, Junshin Clinic Bile Acid Institute and National Taiwan University Hospital
Type: | Journal: Journal of lipid research | Year: 2017

Tetrahydroxy bile acids (THBAs) are hydrophilic and are present at minimal or undetectable levels in healthy human adults but are present at high levels in bile salt export pump (abcb11)-knockout mice. The roles of THBAs in human cholestatic diseases are unclear. We aimed to investigate the presence of THBAs in patients with infantile intrahepatic cholestasis and its correlation with outcome. Urinary bile acids were analysed by GC-MS. Data were compared between good (N=21) (disease-free before 1 year old) and poor prognosis groups (N=19). Good prognosis patients had a higher urinary THBA proportion than poor prognosis patients (25.89% (3.45-76.73) vs. 1.93% (0.05-48.90)). A urinary THBA proportion >7.23% predicted good prognosis with high sensitivity (95.24%), specificity (84.21%) and area under the curve (0.91) (p<0.0001). A THBA proportion 7.23% was an independent factor for decreased transplant-free survival (hazard ratio=7.16, confidence interval: 1.24-41.31, p=0.028). Patients with a confirmed ABCB11 or TJP2 mutation (N=7) had a minimally detectable THBA proportion (0.23-2.99% of total bile acids). Three patients with an ATP8B1 mutation had an elevated THBA proportion (7.51-37.26%). In conclusion, in addition to disease entity as a major determinant of outcome, a high THBA level was associated with good outcome in the infantile intrahepatic cholestasis patients.


PubMed | Virginia Commonwealth University, Nihon University, Health Sciences University of Hokkaido, Junshin Clinic Bile Acid Institute and 3 more.
Type: Journal Article | Journal: Clinical and translational gastroenterology | Year: 2016

Rifaximin has clinical benefits in minimal hepatic encephalopathy (MHE) but the mechanism of action is unclear. The antibiotic-dependent and -independent effects of rifaximin need to be elucidated in the setting of MHE-associated microbiota. To assess the action of rifaximin on intestinal barrier, inflammatory milieu and ammonia generation independent of microbiota using rifaximin.Four germ-free (GF) mice groups were used (1) GF, (2) GF+rifaximin, (3) Humanized with stools from an MHE patient, and (4) Humanized+rifaximin. Mice were followed for 30 days while rifaximin was administered in chow at 100mg/kg from days 16-30. We tested for ammonia generation (small-intestinal glutaminase, serum ammonia, and cecal glutamine/amino-acid moieties), systemic inflammation (serum IL-1, IL-6), intestinal barrier (FITC-dextran, large-/small-intestinal expression of IL-1, IL-6, MCP-1, e-cadherin and zonulin) along with microbiota composition (colonic and fecal multi-tagged sequencing) and function (endotoxemia, fecal bile acid deconjugation and de-hydroxylation).All mice survived until day 30. In the GF setting, rifaximin decreased intestinal ammonia generation (lower serum ammonia, increased small-intestinal glutaminase, and cecal glutamine content) without changing inflammation or intestinal barrier function. Humanized microbiota increased systemic/intestinal inflammation and endotoxemia without hyperammonemia. Rifaximin therapy significantly ameliorated these inflammatory cytokines. Rifaximin also favorably impacted microbiota function (reduced endotoxin and decreased deconjugation and formation of potentially toxic secondary bile acids), but not microbial composition in humanized mice.Rifaximin beneficially alters intestinal ammonia generation by regulating intestinal glutaminase expression independent of gut microbiota. MHE-associated fecal colonization results in intestinal and systemic inflammation in GF mice, which is also ameliorated with rifaximin.


Muto A.,Junshin Clinic Bile Acid Institute | Takei H.,Junshin Clinic Bile Acid Institute | Unno A.,Junshin Clinic Bile Acid Institute | Murai T.,Health Sciences University of Hokkaido | And 12 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2012

The synthesis of bile salts from cholesterol is a complex biochemical pathway involving at least 16 enzymes. Most inborn errors of bile acid biosynthesis result in excessive formation of intermediates and/or their metabolites that accumulate in blood and are excreted in part in urine. Early detection is important as oral therapy with bile acids results in improvement. In the past, these intermediates in bile acid biosynthesis have been detected in neonatal blood or urine by screening with FAB-MS followed by detailed characterization using GC-MS. Both methods have proved difficult to automate, and currently most laboratories screen candidate samples using LC-MS/MS. Here, we describe a new, simple and sensitive analytical method for the identification and characterization of 39 conjugated and unconjugated bile acids, including Δ 4-3-oxo- and Δ 4,6-3-oxo-bile acids (markers for Δ 4-3-oxo-steroid 5β-reductase deficiency), using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In this procedure a concentrated, desalted urinary sample (diluted with ethanol) is injected directly into the LC-ESI-MS/MS, operated with ESI and in the negative ion mode; quantification is obtained by selected reaction monitoring (SRM). To evaluate the performance of our new method, we compared it to a validated method using GC-MS, in the analysis of urine from two patients with genetically confirmed Δ 4-3-oxo-steroid 5β-reductase deficiency as well as a third patient with an elevated concentration of abnormal conjugated and unconjugated Δ 4-3-oxo-bile acids. The Δ 4-3-oxo-bile acids concentration recovered in three patients with 5β-reductase deficiency were 48.8, 58.9, and 49.4μmol/mmol creatinine, respectively by LC-ESI-MS/MS. © 2012 Elsevier B.V..


Murai T.,Health Sciences University of Hokkaido | Oda K.,Health Sciences University of Hokkaido | Toyo T.,Health Sciences University of Hokkaido | Nittono H.,Junshin Clinic Bile Acid Institute | And 4 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2013

A method for the determination of conjugated and unconjugated 3β-hydroxy-Δ5-bile acids and related bile acids in human urine and serum has been developed using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. Calibration curves for the bile acids were linear over the range of 10-2000pmol/mL, and the detection limit was less than 4pmol/mL for all bile acids using selected reaction monitoring analysis. The bile acids in urine and serum samples from two patients with severe cholestatic liver disease were measured by this analytical method. Glycine-conjugated 3β-hydroxy-Δ5-bile acid 3-sulfates were determined to be the major bile acids in the urine and serum from patients with a 3β-hydroxy-Δ5-C27-steriod dehydrogenase/isomerase deficiency or dysfunction. © 2013 Elsevier B.V.


Kakiyama G.,Virginia Commonwealth University | Pandak W.M.,Virginia Commonwealth University | Gillevet P.M.,George Mason University | Hylemon P.B.,Virginia Commonwealth University | And 13 more authors.
Journal of Hepatology | Year: 2013

Background & Aims: The 7α-dehydroxylation of primary bile acids (BAs), chenodeoxycholic (CDCA) and cholic acid (CA) into the secondary BAs, lithocholic (LCA) and deoxycholic acid (DCA), is a key function of the gut microbiota. We aimed at studying the linkage between fecal BAs and gut microbiota in cirrhosis since this could help understand cirrhosis progression. Methods: Fecal microbiota were analyzed by culture-independent multitagged-pyrosequencing, fecal BAs using HPLC and serum BAs using LC-MS in controls, early (Child A) and advanced cirrhotics (Child B/C). A subgroup of early cirrhotics underwent BA and microbiota analysis before/after eight weeks of rifaximin. Results: Cross-sectional: 47 cirrhotics (24 advanced) and 14 controls were included. In feces, advanced cirrhotics had the lowest total, secondary, secondary/primary BA ratios, and the highest primary BAs compared to early cirrhotics and controls. Secondary fecal BAs were detectable in all controls but in a significantly lower proportion of cirrhotics (p <0.002). Serum primary BAs were higher in advanced cirrhotics compared to the rest. Cirrhotics, compared to controls, had a higher Enterobacteriaceae (potentially pathogenic) but lower Lachonospiraceae, Ruminococcaceae and Blautia (7α-dehydroxylating bacteria) abundance. CDCA was positively correlated with Enterobacteriaceae (r = 0.57, p <0.008) while Ruminococcaceae were positively correlated with DCA (r = 0.4, p <0.05). A positive correlation between Ruminococcaceae and DCA/CA (r = 0.82, p <0.012) and Blautia with LCA/CDCA (r = 0.61, p <0.03) was also seen. Prospective study: post-rifaximin, six early cirrhotics had reduction in Veillonellaceae and in secondary/primary BA ratios. Conclusions: Cirrhosis, especially advanced disease, is associated with a decreased conversion of primary to secondary fecal BAs, which is linked to abundance of key gut microbiome taxa. © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.


Kakiyama G.,Virginia Commonwealth University | Hylemon P.B.,Virginia Commonwealth University | Zhou H.,Virginia Commonwealth University | Pandak W.M.,Virginia Commonwealth University | And 12 more authors.
American Journal of Physiology - Gastrointestinal and Liver Physiology | Year: 2014

Alcohol abuse with/without cirrhosis is associated with an impaired gut barrier and inflammation. Gut microbiota can transform primary bile acids (BA) to secondary BAs, which can adversely impact the gut barrier. The purpose of this study was to define the effect of active alcohol intake on fecal BA levels and ileal and colonic inflammation in cirrhosis. Five age-matched groups {two noncirrhotic (control and drinkers) and three cirrhotic [nondrinkers/nonalcoholics (NAlc), abstinent alcoholic for >3 mo (AbsAlc), currently drinking (CurrAlc)]} were included. Fecal and serum BA analysis, serum endotoxin, and stool microbiota using pyrosequencing were performed. A subgroup of controls, NAlc, and CurrAlc underwent ileal and sigmoid colonic biopsies on which mRNA expression of TNF-α, IL-1β, IL-6, and cyclooxygenase-2 (Cox-2) were performed. One hundred three patients (19 healthy, 6 noncirrhotic drinkers, 10 CurrAlc, 38 AbsAlc, and 30 NAlc, age 56 yr, median MELD: 10.5) were included. Five each of healthy, CurrAlc, and NAlc underwent ileal/colonic biopsies. Endotoxin, serum-conjugated DCA and stool total BAs, and secondary-to-primary BA ratios were highest in current drinkers. On biopsies, a significantly higher mRNA expression of TNF-α, IL-1β, IL-6, and Cox-2 in colon but not ileum was seen in CurrAlc compared with NAlc and controls. Active alcohol use in cirrhosis is associated with a significant increase in the secondary BA formation compared with abstinent alcoholic cirrhotics and nonalcoholic cirrhotics. This increase in secondary BAs is associated with a significant increase in expression of inflammatory cytokines in colonic mucosa but not ileal mucosa, which may contribute to alcohol-induced gut barrier injury. © 2014 the American Physiological Society.


PubMed | Lund University, Junshin Clinic Bile Acid Institute and Nihon University
Type: Journal Article | Journal: Cell stem cell | Year: 2016

During development, hematopoietic stem cells (HSCs) undergo a rapid expansion in the fetal liver (FL) before settling in the adult bone marrow. We recently reported that proliferating adult HSCs are vulnerable to ER stress caused by accumulation of mis-folded proteins. Here, we find that FL-HSCs, despite an increased protein synthesis rate and a requirement for protein folding, do not upregulate ER chaperones. Instead, bile acids (BAs), secreted from maternal and fetal liver, coordinate to serve as chemical chaperones. Taurocholic acid, the major BA in FL, supports growth of HSCs in vitro by inhibiting protein aggregation. In vivo, reducing BA levels leads to ER stress elevation and accumulation of aggregated proteins and significantly decreases the number of FL-HSCs. Taken together, these findings reveal that BA alleviation of ER stress is a mechanism required for HSC expansion during fetal hematopoiesis.


PubMed | Juntendo University, Junshin Clinic Bile Acid Institute, Nihon University, Jichi Medical University and Hokkaido University
Type: | Journal: Clinica chimica acta; international journal of clinical chemistry | Year: 2015

The primary bile acids found in meconium vary with the gestational age of the fetus and the intestinal location of the meconium. We determined the composition of bile acids in samples that were collected from the gallbladder and intestine.The bile-acid profiles of intestinal contents and the gallbladder were obtained from nine fetuses who died from abortion or respiratory failure within 72 h after birth. Intestinal content samples were collected from seven intestinal locations. The bile-acid profiles of meconium were also obtained from seven full-term live births for comparison. The profiles were analyzed using liquid chromatography-tandem mass spectrometry.The bile acids in meconium collected from stillborn and live births were mainly chenodeoxycholic acid and cholic acid, conjugated with taurine, glycine, and sulfate. The same bile acids were found in the gallbladder, except that sulfate was not found.Sulfate-conjugated bile acid is found in urine, but rarely in stool. In this study, the gallbladder bile acid contained no sulfate conjugates, but these were present in intestinal contents and meconium. These results indicate that sulfate-conjugated bile acids are not excreted into the intestine through the biliary tract but originate from swallowed amniotic fluid that contains fetal urine.


Kakiyama G.,Virginia Commonwealth University | Muto A.,Junshin Clinic Bile Acid Institute | Takei H.,Junshin Clinic Bile Acid Institute | Nittono H.,Junshin Clinic Bile Acid Institute | And 5 more authors.
Journal of Lipid Research | Year: 2014

We have developed a simple and accurate HPLC method for measurement of fecal bile acids using phenacyl derivatives of unconjugated bile acids, and applied it to the measurement of fecal bile acids in cirrhotic patients. The HPLC method has the following steps: 1 ) lyophilization of the stool sample; 2 ) reconstitution in buffer and enzymatic deconjugation using cholylglycine hydrolase/sulfatase; 3 ) incubation with 0.1 N NaOH in 50% isopropanol at 60°C to hydrolyze esterified bile acids; 4 ) extraction of bile acids from particulate material using 0.1 N NaOH; 5 ) isolation of deconjugated bile acids by solid phase extraction; 6 ) formation of phenacyl esters by derivatization using phenacyl bromide; and 7 ) HPLC separation measuring eluted peaks at 254 nm. The method was validated by showing that results obtained by HPLC agreed with those obtained by LC-MS/ MS and GC-MS. We then applied the method to measuring total fecal bile acid (concentration) and bile acid profile in samples from 38 patients with cirrhosis (17 early, 21 advanced) and 10 healthy subjects. Bile acid concentrations were significantly lower in patients with advanced cirrhosis, suggesting impaired bile acid synthesis.-Kakiyama, G., A. Muto, H. Takei, H. Nittono, T. Murai, T. Kurosawa, A. F. Hofmann, W. M. Pandak, and J. S. Bajaj. A simple and accurate HPLC method for fecal bile acid profile in healthy and cirrhotic subjects: validation by GC-MS and LC-MS. J. Lipid Res. 2014. 55: 978-990. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

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