Hajime Takei

Modulation of the fecal bile acid profile by gut microbiota in cirrhosis.

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.

Colonic inflammation and secondary bile acids in alcoholic cirrhosis

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.

Neonatal Cholestatic Liver Disease in an Asian Patient With a Homozygous Mutation in the Oxysterol 7α-hydroxylase Gene

We diagnosed a Taiwanese infant with oxysterol 7a-hydroxylase deficiency resulting from a homozygous mutation in the corresponding gene. We know of only 1 report of this inborn error of bile acid synthesis, by Setchell et al (1). The clinical and laboratory findings in our patient resembled those in patients with other inborn errors of bile acid synthesis. However, this rare form is difficult to treat with bile acid therapy because of its rapid progression to cirrhosis at an early stage. We present the clinical features, diagnostic evaluation, and treatment of our patient.

A simple and accurate HPLC method for fecal bile acid profile in healthy and cirrhotic subjects: validation by GC-MS and LC-MS.

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.

Oral bile acid treatment in two Japanese patients with Zellweger syndrome

*Department of Pediatrics and Child Health, Kurume University School of Medicine, Kurume; †Junshin Clinic, Yokohama; ‡Tokyo Health Service Association, Tokyo; §Department of Pediatrics, University of Occupational and Environmental Health, Kitakyushu; ¶Department of Pediatrics and Neonatal Medicine, Kumamoto City Hospital, Kumamoto; and Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Japan

SRD5B1 gene analysis needed for the accurate diagnosis of primary 3‐oxo‐Δ4‐steroid 5β‐reductase deficiency

Background and Aim:  We encounter hyper‐3‐oxo‐Δ4 bile aciduria in patients with severe cholestatic liver disease or fulminant liver failure during the neonatal period. However, simply by bile acid analysis, it is difficult to distinguish hyper‐3‐oxo‐Δ4 bile aciduria from primary 3‐oxo‐Δ4‐steroid 5β‐reductase deficiency.

Detection of Δ4-3-oxo-steroid 5β-reductase deficiency by LC-ESI-MS/MS measurement of urinary bile acids.

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 49.4 μmol/mmol creatinine, respectively by LC-ESI-MS/MS.

Rifaximin Exerts Beneficial Effects Independent of its Ability to Alter Microbiota Composition.

Objectives: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.Methods: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 100 mg/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-1β, IL-6), intestinal barrier (FITC-dextran, large-/small-intestinal expression of IL-1β, 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).Results: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.Conclusions: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.

Successful heterozygous living donor liver transplantation for an oxysterol 7α-hydroxylase deficiency in a Japanese patient.

Only 2 patients with an oxysterol 7α‐hydroxylase deficiency caused by mutations of the cytochrome P450 7B1 (CYP7B1) gene have been reported; for both, the outcome was fatal. We describe the clinical and laboratory features, the hepatic and renal histological findings, and the results of bile acid and CYP7B1 gene analyses for a third patient. This Japanese infant presented with progressive cholestatic liver disease and underwent successful heterozygous living donor liver transplantation. Sources of relevant data included medical records, hepatic and renal histopathological findings, gas chromatography/mass spectrometry analyses of bile acids in serum and urine samples, and analyses of the CYP7B1 gene in the DNA of peripheral blood lymphocytes. Large excesses of 3β‐hydroxy‐5‐cholen‐24‐oic acid were detected in the patients serum and urine. Cirrhosis and polycystic changes in the kidneys were documented. The demonstration of compound heterozygous mutations (R112X/R417C) of the CYP7B1 gene led to the diagnosis of an oxysterol 7α‐hydroxylase deficiency. After liver transplantation with an allograft from a heterozygous living donor (the patients mother), the features of decompensated hepatocellular failure abated, and the renal abnormalities were resolved. In conclusion, we report the first Japanese patient with an oxysterol 7α‐hydroxylase deficiency associated with compound heterozygous mutations of the CYP7B1 gene; in this patient, liver transplantation with an allograft from a parental donor was effective. Liver Transpl 17:1059–1065, 2011.

Bile Acids Protect Expanding Hematopoietic Stem Cells from Unfolded Protein Stress in Fetal Liver.

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.