Continuum of Host-Gut Microbial Co-metabolism: Host CYP3A4/3A7 are Responsible for Tertiary Oxidations of Deoxycholate Species.

The gut microbiota modifies endogenous primary bile acids (BAs) to produce exogenous secondary BAs, which may be further metabolized by cytochrome P450 enzymes (P450s). Our primary aim was to examine how the host adapts to the stress of microbe-derived secondary BAs by P450-mediated oxidative modifications on the steroid nucleus. Five unconjugated tri-hydroxyl BAs that were structurally and/or biologically associated with deoxycholate (DCA) were determined in human biologic samples by liquid chromatography-tandem mass spectrometry in combination with enzyme-digestion techniques. They were identified as DCA-19-ol, DCA-6ß-ol, DCA-5ß-ol, DCA-6α-ol, DCA-1ß-ol, and DCA-4ß-ol based on matching in-laboratory synthesized standards. Metabolic inhibition assays in human liver microsomes and recombinant P450 assays revealed that CYP3A4 and CYP3A7 were responsible for the regioselective oxidations of both DCA and its conjugated forms, glycodeoxycholate (GDCA) and taurodeoxycholate (TDCA). The modification of secondary BAs to tertiary BAs defines a host liver (primary BAs)-gut microbiota (secondary BAs)-host liver (tertiary BAs) axis. The regioselective oxidations of DCA, GDCA, and TDCA by CYP3A4 and CYP3A7 may help eliminate host-toxic DCA species. The 19- and 4ß-hydroxylation of DCA species demonstrated outstanding CYP3A7 selectivity and may be useful as indicators of CYP3A7 activity.

Reversing the Cytotoxicity of Bile Acids by Supramolecular Encapsulation.

Supramolecular encapsulation has been developed into a powerful tool in clearance of toxic substances and hazardous waste from living body and external environments. Herein we tested the special efficacy of tyramine-modified ß-cyclodextrin (1) in inhibiting and reversing of the inherent cytotoxicity of deoxycholic acid (DCA). The decarboxylation from tyrosine to tyramine in 1 is crucial to the mutual electrostatic communication, ultimately leading to great enhancement in binding affinity and molecular selectivity toward bile acids. As a result, the DCA-mediated cytotoxicity could be largely eliminated by the biocompatible 1. Meanwhile, the excess DCA could be rapidly excreted by 1 via rat urinary clearance, thus facilitating the decrease of DCA concentration in blood. This study presents a proof of principle that the supramolecular encapsulation with functional cyclodextrin derivatives can efficiently modulate the cell progression and remove the cytotoxic DCA, which provides a practical approach to prevent or treat bile acid-involved diseases.

Role of glucuronidation for hepatic detoxification and urinary elimination of toxic bile acids during biliary obstruction.

Biliary obstruction, a severe cholestatic condition, results in a huge accumulation of toxic bile acids (BA) in the liver. Glucuronidation, a conjugation reaction, is thought to protect the liver by both reducing hepatic BA toxicity and increasing their urinary elimination. The present study evaluates the contribution of each process in the overall BA detoxification by glucuronidation. Glucuronide (G), glycine, taurine conjugates, and unconjugated BAs were quantified in pre- and post-biliary stenting urine samples from 12 patients with biliary obstruction, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The same LC-MS/MS procedure was used to quantify intra- and extracellular BA-G in Hepatoma HepG2 cells. Bile acid-induced toxicity in HepG2 cells was evaluated using MTS reduction, caspase-3 and flow cytometry assays. When compared to post-treatment samples, pre-stenting urines were enriched in glucuronide-, taurine- and glycine-conjugated BAs. Biliary stenting increased the relative BA-G abundance in the urinary BA pool, and reduced the proportion of taurine- and glycine-conjugates. Lithocholic, deoxycholic and chenodeoxycholic acids were the most cytotoxic and pro-apoptotic/necrotic BAs for HepG2 cells. Other species, such as the cholic, hyocholic and hyodeoxycholic acids were nontoxic. All BA-G assayed were less toxic and displayed lower pro-apoptotic/necrotic effects than their unconjugated precursors, even if they were able to penetrate into HepG2 cells. Under severe cholestatic conditions, urinary excretion favors the elimination of amidated BAs, while glucuronidation allows the conversion of cytotoxic BAs into nontoxic derivatives.

Development of an enzyme-linked immunosorbent assay for fecal bile acid.

It has been suggested that the bile acids in the feces act as a promoter of colon cancer. Among the bile acids, deoxycholic acid (DCA), which is one kind of the secondary bile acid, is said to have strong influence. DCA/cholic acid (CA) ratio in feces is also said to have a diagnostic significance in colon cancer. With this in mind, we created a CA and DCA's monoclonal antibody (MoAb) to measure them through the enzyme linked immunosorbent assay (ELISA) method. Using these MoAb, we were able to measure CA and DCA concentrations with low cross-reaction to other bile acids compared with the method with polyclonal antibody (PoAb). We measured CA and DCA concentrations and calculated the DCA/CA ratios in healthy subjects and patients with colon cancer. All subjects had been screened for colon cancer. We then compared the healthy subjects, the cancer patients before surgery and the same cancer patients after surgery. Cancer patients after surgery had significantly low DCA/CA ratios compared to before surgery, whereas there was no significant difference between healthy subjects and the pre-operative colon cancer patients.

Fetal bile acid metabolism: analysis of urinary 3beta-monohydroxy-delta(5) bile acid in preterm infants.

To elucidate the urinary concentration of total bile acids after birth and the profile of the usual and unusual urinary bile acids, especially 3beta-hydroxy-5-cholen-24-oic acid (Delta(5)-3beta-ol), we measured the concentrations of 13 bile acids in the urine from preterm infants vs. full-term controls by gas chromatography-mass spectrometry. The urinary concentration of total bile acids in early preterm infants below 32 weeks of gestational age significantly exceeded that of the late preterm and full-term infants (p < 0.0005). The major urinary bile acids in early preterm infants were cholic acid, 1beta,3alpha,7alpha,12alpha-tetrahydroxy-5beta-cholan-24-oic acid and Delta(5)-3beta-ol. In conclusion, the high urinary concentrations of total bile acids in preterm infants may be due to an overproduction, or more likely to a low hepatic bile acid clearance. An alternative fetal pathway, the acidic pathway, may be a major route of bile acid biosynthesis in preterm infants.

Stimulation of bile acid 6 alpha-hydroxylation by rifampin.

BACKGROUND: Rifampin was shown to relieve pruritus in cholestatic liver diseases. There has been much speculation about the origin of pruritus, but it has not yet been comprehensively explained. The role of bile acids in producing pruritus is obscure and still under debate. Since rifampin both inhibits the uptake of bile acids into the hepatocyte and strongly induces mixed-function oxidases in the liver, the beneficial effects of this drug might be a consequence of altered bile acid metabolism. METHODS: We investigated the influence of rifampin on urinary bile acid excretion with special respect to glucuronide and sulphate conjugates in 14 healthy volunteers before and after administration of rifampin, 600 mg x 7 days, using each subject as his or her own control. RESULTS: Bile acid glucuronide excretion increased from 0.55 to 1.19 mumol/24 h. This was in particular due to a significant increase of the urinary excretion of the 6 alpha-hydroxylated hyocholic and hyodeoxycholic acids, the relative amounts of which accounted for about two thirds of the urinary bile acid excretion. Excretion of sulphates, however, decreased from 1.40 to 0.86 mumol/24 h due to a significantly reduced excretion of lithocholic acid sulphate. No changes in the excretion rates of other primary and secondary bile acids and no changes in their conjugation patterns were observed. CONCLUSIONS: The results provide evidence that rifampin induces 6 alpha-hydroxylation of bile acids. The products are subsequently glucuronidated at the 6 alpha-hydroxy group, thus stimulating renal excretion of potentially toxic bile acids.

Metabolism and time-course excretion of murideoxycholic acid, a 6 beta-hydroxylated bile acid, in humans.

The metabolism and time-courses of urinary and fecal excretions of murideoxycholic acid (MDCA; 3 alpha,6 beta-dihydroxy-5 beta-cholanoic acid), a 6 beta-hydroxylated bile acid, was investigated in man. The study was carried out in two groups of subjects. Six cholecystectomized patients fitted with a cystic duct drain ingested 100 mg of a tracer dose of 3H-MDCA. Time-course of radioactivity in plasma was then followed for an 8-h period. Biliary, urinary and fecal excretions of radioactivity were measured for a 5-day period and excreted MDCA metabolites were identified. Five lithiasic patients with intact enterohepatic circulation ingested 500 mg of the same tracer dose of 3H-MDCA. Radioactivity in plasma was followed for a 49-h period and urinary and fecal excretions of radioactivity were measured daily for 7 days. In the first group, the excretion of the radioactivity by the three routes (bile+urine+feces) reached 97.8 +/- 1.5% of the ingested dose but dropped to 75 +/- 8.3% (urine+feces) in patients in the second group. In cholecystectomized patients, the estimation of intestinal MDCA absorption was dependent on cystic duct drain flow rate and gave values ranging from 20% to 87%. The biological half-life of MDCA in lithiasic patients averaged 3.4 +/- 0.7 days. Radioactivity appeared in the plasma in the first hour and reached a maximum 6 and 3 h after the beginning of the experiment in group I and II respectively. In the second group, another peak of radioactivity in plasma was observed just after breakfast.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for bile acid glucosides as normal constituents in human urine.

A glucosyltransferase catalysing formation of bile acid glucosides was recently isolated from human liver microsomes. In order to investigate the potential occurrence of such bile acid derivatives in vivo, a method was devised for their isolation and purification from urine. Conditions were established with the aid of glucosides of radiolabelled, unconjugated glycine and taurine conjugated bile acids prepared enzymatically using human liver microsomes. Analysis by gas chromatography and mass spectrometry of methyl ester trimethylsilyl ether derivatives indicated the excretion of glucosides of nonamidated hyodeoxycholic, chenodeoxycholic, deoxycholic, ursodeoxycholic and cholic acids and of glycine and taurine conjugated chenodeoxycholic and cholic acids. Additional compounds were present giving mass spectral fragmentation patterns typical of di- and trihydroxy bile acid glycosides. Semiquantitative estimates indicated a total daily excretion of about 1 mumol.

Intestinal absorption, excretion, and biotransformation of hyodeoxycholic acid in man.

Five patients fitted with a biliary T-tube after cholecystectomy were given orally a tracer dose of [14C]hyodeoxycholic acid and 500 mg of the same unlabeled acid. Intestinal absorption and biotransformation, liver metabolism, bile secretion, fecal and urinary excretions of this acid or of its metabolites were studied. Hyodeoxycholic acid was well absorbed by the human intestine. It was not subjected to intestinal transformations and, particularly, did not produce a significant amount of lithocholic acid, which does not support the existence of intestinal bacterial 6 alpha-dehydroxylases. The percentage of hyodeoxycholic acid and of its metabolites recovered in bile varied from 11.5 to 31%. Two major metabolites were isolated from bile: glycohyodeoxycholic acid and hyodeoxycholic acid glucuronide. Analysis of urinary bile acids showed that a large proportion (30-84%) of the administered hyodeoxycholic acid was excreted by the kidney as a glucuronide. The large extent of both glucuronidation and urinary excretion of hyodeoxycholic acid is a unique example of bile acid metabolism and excretion in man.

Hydroxylation of cholic, chenodeoxycholic, and deoxycholic acids in patients with intrahepatic cholestasis.

The metabolism of 14C-labeled chenodeoxycholic, cholic, and deoxycholic acids was studied in patients with intrahepatic cholestasis. Radioactively labeled metabolites were isolated from urine and were identified by gas-liquid chromatography-mass spectrometry. About 5% of the radioactivity was recovered in urine after administration of labeled chenodeoxycholic acid to a patient with mild intrahepatic cholestasis. In urine collected 0-24 hr after the injection, 20% of the radioactivity appeared in the combined glycine and taurine conjugate fractions, and the predominant metabolite in these fractions was identified as hyocholic acid. Eighty percent of the activity was eluted in the sulfate fraction presumably representing mainly sulfated chenodeoxycholic acid conjugates. Twenty percent of the radioactivity was recovered in urine following administration of labeled cholic acid to a patient with biliary cirrhosis and severe cholestasis. In urine collected on the fifth day, half of this radioactivity appeared in the glycine and taurine conjugate fractions, and 10% of this activity was present as tetrahydroxycholanoates. The major metabolites in this fraction were 3 alpha, 6 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta- and 1 xi, 3 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta-cholanoic acids. The former compound constituted about 50% of the tetrahydroxycholanoates. Three additional minor tetrahydroxy bile acids were present, one of which was tentatively identified as 6 beta-hydroxycholic acid. About 5% of the radioactivity appeared in urine after oral administration of labeled deoxycholic acid to a patient with mild intrahepatic cholestasis. Twenty-two percent of the activity appeared in the glycine and taurine conjugate fractions isolated from urine collected on the second day after the administration. About 75% of this activity was associated with trihydroxycholanoates. The main metabolite was 1 beta-hydroxydeoxycholic acid with small amounts of, tentatively, 6 alpha-hydroxydeoxycholic acid.

Urinary bile acid excretion in correlation to liver histopathology in cystic fibrosis.

Urinary bile acid excretion and liver morphology were compared in 25 patients with cystic fibrosis (CF). None showed clinical signs of liver disease. Most of the patients had normal liver function tests. Bile acids were determined in 24-h samples by a modification of the method of Almé. All patients had increased urinary excretion of trihydroxy bile acids, mainly cholic, 3 beta, 7 beta, 12 alpha- and 3 alpha, 7 beta, 12 alpha-trihydroxy-5 beta-cholanoic acids. Lithocholic acid excretion was lower in CF than in normal children. The urinary excretion of 3 beta-hydroxy-5-cholenoic acid was not increased in CF. In three patients with cirrhosis the urinary excretion of chenodeoxycholic acid was increased. The ratio of cholic to 3 beta-hydroxy-5-cholenoic acids was increased in all but three patients, and the ratio of chenodeoxycholic to 3 beta-hydroxy-5-cholenoic acids was increased in those with cirrhosis. These ratios differed more between cirrhotic and non-cirrhotic CF patients in this series than the ratio of cholic to chenodeoxycholic acids.

Urinary bile acids during development of recurrent cholestasis of pregnancy.

The pregnancies of two patients with mild intrahepatic cholestasis of pregnancy (RCP) were followed with detailed analyses of bile acids in urine. About twenty-five different bile acids were determined by GC/MS following separation according to mode of conjugation. The results were collated with the clinical course of the disease. The first detectable change in bile acid excretion was the appearance of tetrahydroxylated bile acids at about the 30th gestational week. Somewhat later and concomitant with the rise in urinary oestriol, the total bile acid excretion started to increase. In one of the patients, who had a maximum total excretion of 84 mumol/24 h, deoxycholic acid was a major constituent, comprising about 40% of the total. The same patient had only slightly elevated levels of tetrahydroxylated bile acids and serum amino-transferases. The possible effect of low-fat diet on these results is discussed. Monohydroxylated bile acids were present throughout the pregnancies in small amounts and their role as aetiological factors is discussed. The care of RCP patients is outlined, and the need for simple, specific and quantitative methods for following the course of RCP is pointed out.

Sulfation and renal excretion of bile salts in patients with cirrhosis of the liver.

Renal excretion of bile salts was studied in 17 patients with cirrhosis of the liver. The average quantity of bile salts in urine was 10.2 plus or minus 8.3 mg per 24 hr, 56% of which were sulfated. Of the individual urinary bile salts, 24% oithocholate were sulfated. In contrast, neither sulfated nor nonsulfated bile salts could be detected in urine from 2 normal subjects. Kinetics of bile salt metabolism was measured in 2 of the cirrhotic patients after oral administration of [14C] cholate and [3H] chenodeoxycholate. Approximately 3 to 12% of bile salts synthesized in liver were excreted in urine. Most urinary bile salts (76 to 80%) were sulfated, whereas only 4 to 5% of serum bile salts and 7 to 10% of biliary bile salts were sulfated. Renal clearance of cholate was more than 3 times greater than the clearance of chenodeosycholate or deoxycholate. Renal clearance of sulfated bile salts was 20 to 200 times greaterthan the clearance of the corresponding nonsulfated bile salts.

Sulfated and nonsulfated bile acids in urine, serum, and bile of patients with hepatobiliary diseases.

Large amounts of bile acid sulfate were found in the urine of patients with hepatobiliary diseases. In patients with acute hepatitis, daily excretion of bile acid into urine was 68.24 plus or minus 51.80 mumoles per day, and the percentage of sulfated bile acid was 83.4 plus or minus 16.7%. In patients with chronic hepatitis and cirrhosis, a slight increase of urinary bile acid was observed (2.89 plus or minus 2.69 and 5.27 plus or minus 4.28 mumoles per day, respectively), and the percentage of sulfated bile acid was 73.9 plus or minus 28.6 and 44.6 plus or minus 30.4%, respectively. In patients with obstructive jaundice, a moderate increase of urinary bile acid was found (32.62 plus or minus 18.35 mumoles per day), and the percentage of sulfated bile acid was 58.3 plus or minus 22.6%. In patients with hepatobiliary diseases, the elevation of both levels of sulfated and nonsulfated bile acids in serum was observed. The percentage of sulfated bile acid was 9% in normal serum, and varied from zero to 82.8% in pathological sera. A remarkable increase of sulfated bile acid was found in patients with obstructive juandice and acute hepatitis, while a slight elevation was found in patients with chronic hepatitis and cirrhosis. Sulfated bile acid in bile was nonexistent or below 0.5% of total bile acid. According to these findings, the increased bile acid in serum of patients with hepatobiliary diseases might be more easily excreted into the urine as sulfated bile acid.