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.

Determination of 3ß-hydroxy-Δ5-bile acids and related compounds in biological fluids of patients with cholestasis by liquid chromatography-tandem mass spectrometry.

A method for the determination of conjugated and unconjugated 3ß-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 2000 pmol/mL, and the detection limit was less than 4 pmol/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 3ß-hydroxy-Δ5-bile acid 3-sulfates were determined to be the major bile acids in the urine and serum from patients with a 3ß-hydroxy-Δ5-C27-steriod dehydrogenase/isomerase deficiency or dysfunction.

Measurement of transport activities of 3ß-hydroxy-Δ(5)-bile acids in bile salt export pump and multidrug resistance-associated proteins using LC-MS/MS.

A method has been developed for the measurement of transport activities in membrane vesicles obtained from Sf9 cells for 3ß-hydroxy-Δ(5)-bile acids by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. Calibration curves for the bile acids were linear over the range of 10 to 2000 pmol/mL, and the detection limit was less than 1 pmol/mL for 3ß-hydroxy-Δ(5)-bile acids using selected reaction monitoring analysis. The analytical method was applied to measurements of transport activities in membrane vesicles obtained from human multidrug resistance-associated protein 2-, 3-, and human bile salt export pump-expressing Sf9 cells for conjugated 3ß-hydroxy-Δ(5)-bile acids. The present study demonstrated that human multidrug resistance-associated protein 3 vesicles accepted conjugated 3ß-hydroxy-Δ(5)-bile acids along with common bile acids such as glycocholic acid and taurolithocholic acid 3-sulfate.

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.

Neonatal cholestasis with increased 3ß-monohydroxy-Δ5 bile acids in serum and urine: not necessarily primary oxysterol 7α hydroxylase deficiency.

BACKGROUND: Inborn errors of bile acid synthesis are rare genetic disorders that can present with cholestatic liver disease. Recently we encountered 3 infants with neonatal cholestasis and excessive 3ß-monohydroxy-Δ5-C24 bile acids in serum and urine. We investigated whether identification of 3ß-hydroxy-5-cholestenoic acid and 27-hydroxycholesterol in serum and urine of cholestatic patients is necessary for diagnosis of primary oxysterol 7α-hydroxylase deficiency. METHODS: These 3 patients initially led us to suspected oxysterol 7α-hydroxylase deficiency. However, sequence analysis of genomic DNA resulted in diagnosis of 2 patients with oxysterol 7α-hydroxylase deficiency and 1 patient with 3ß-hydroxy-Δ5-C27-steroid dehydrogenase/isomerase deficiency. We examined identification of 3ß-hydroxy-5-cholestenoic acid and 27-hydroxycholesterol by gas chromatography-mass spectrometry after diagnosis. RESULTS: Interestingly, we detected a peak for 3ß-hydroxy-5-cholestenoic acid in serum and 27-hydroxycholesterol of the neutral sterol in urine from 2 patients who were diagnosed with primary oxysterol 7α-hydroxylase deficiency. CONCLUSION: In evaluating infants with cholestasis and excessive 3ß-monohydroxy-Δ5-C24 bile acids in infancy, one needs to conduct C24 bile acid analysis serially. Results can guide performance and interpretation of genomic DNA analysis. Moreover, identification of 3ß-hydroxy-5-cholestenoic acid in serum and 27-hydroxycholesterol in urine is highly important for diagnosis of oxysterol 7α-hydroxylase deficiency as is genomic DNA analysis.

Detection of Δ4-3-oxo-steroid 5ß-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 5ß-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 5ß-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 5ß-reductase deficiency were 48.8, 58.9, and 49.4 µmol/mmol creatinine, respectively by LC-ESI-MS/MS.

Characterization of urinary bile acids in a pediatric BRIC-1 patient: effect of rifampicin treatment.

BACKGROUND: Benign recurrent intrahepatic cholestasis type 1 (BRIC-1), a rare autosomal recessive disorder characterized by recurrent episodes of jaundice and pruritus, is caused by mutations in the ATP8B1 gene. Rifampicin has been reported to be an effective treatment of jaundice and pruritus in patients with BRIC. Proposed mechanisms of effect for rifampicin include enhancement of multidrug-resistance protein 2 expression, activation of the enzymes of uridine diphosphate glucuronosyltransferase 1A1 and cytochrome P450 3A4, and stimulation of 6α-hydroxylation of bile acids. METHODS: To confirm the diagnosis of BRIC-1 and demonstrate the effect of rifampicin treatment on bile acid metabolism, we analyzed the patient's ATP8B1 gene and bile acids in urine. RESULTS: We detected 2 heterozygous mutations in the ATP8B1 gene, and increasing amounts of unusual bile acids such as 1ß-hydroxylated cholic acid, 2ß-hydroxylated cholic acid, 4ß-hydroxylated cholic acid, 6α-hydroxylated cholic acid, and hyocholic acid in urine during rifampicin treatment. CONCLUSIONS: We diagnosed a jaundiced pediatric patient with BRIC-1 caused by 2 novel mutations (1226delA/2210delA) in the ATP8B1 gene. Rifampicin was effective in treating cholestasis. Results of urinary bile acid analyses during rifampicin treatment in this patient, suggested that rifampicin might stimulate 1ß-, 2ß-, and 4ß-hydroxylation of bile acids in addition to 6α-hydroxylation.

Potential corticoid metabolites: chemical synthesis of 3- and 21-monosulfates and their double-conjugates of tetrahydrocorticosteroids in the 5alpha- and 5beta-series.

Here, we describe the chemical synthesis of the complete sets of 18 novel 3- and 21-monosulfates and their double-conjugated form of tetrahydrocortisol (THF), tetrahydro-11-deoxycortisol (THS), and tetrahydrocortisone (THE) in the 5alpha- and 5beta-series. The principal reactions involved are: (1) selective protection of a specific hydroxy group in substrates; (2) catalytic hydrogenation at C-5 of Delta(4)-3-ketosteroids with 10% Pd(OH)(2)/C to yield 3-oxo-5beta-steroids and reductive allomerization with 10% Pd/C to yield 3-oxo-5alpha-isomers; (3) reduction of the resulting 3-oxo-5beta- and 3-oxo-5alpha-steroids to the corresponding 3alpha-hydroxy-compounds with Zn(BH(4))(2) and K-Selectride((R)), respectively; and (4) sulfation of hydroxy groups at C-3 and/or C-21 in the tetrahydrocorticosteroid derivatives with sulfur trioxide-triethylamine complex.

Chemical synthesis of 3beta-sulfooxy-7beta-hydroxy-24-nor-5-cholenoic acid: an internal standard for mass spectrometric analysis of the abnormal delta5-bile acids occurring in Niemann-Pick disease.

In Niemann-Pick disease, type C1, increased amounts of 3beta,7beta-dihydroxy-5-cholenoic acid are reported to be present in urinary bile acids. The compound occurs as a tri-conjugate, sulfated at C-3, N-acetylglucosamidated at C-7, and N-acylamidated with taurine or glycine at C-24. For sensitive LC-MS/MS analysis of this bile acid, a suitable internal standard is needed. We report here the synthesis of a satisfactory internal standard, 3beta-sulfooxy-7beta-hydroxy-24-nor-5-cholenoic acid (as the disodium salt). The key reactions involved were (1) the so-called "second order" Beckmann rearrangement (one-carbon degradation at C-24) of hyodeoxycholic acid (HDCA) 3,6-diformate with sodium nitrite in a mixture of trifluoroacetic anhydride and trifluoroacetic acid, (2) simultaneous inversion at C-3 and elimination at C-6 of the ditosylate derivatives of the resulting 3alpha,6alpha-dihydroxy-24-nor-5beta-cholanoic acid with potassium acetate in aqueous N,N-dimethylformamide, and (3) regioselective sulfation at C-3 of an intermediary 3beta,7beta-dihydroxy-24-nor-Delta(5) derivative using sulfur trioxide-trimethylamine complex. Overall yield of the desired compound was 1.8% in 12 steps from HDCA.

A new, major C27 biliary bile acid in the red-winged tinamou (Rhynchotus rufescens):25R-1beta, 3alpha,7alpha-trihydroxy-5beta-cholestan-27-oic acid.

The chemical structures of the three major bile acids present in the gallbladder bile of the Red-winged tinamou (Rhynchotus rufescens), an early evolving, ground-living bird related to ratites, were determined. Bile acids were isolated by preparative reversed-phase HPLC. Two of the compounds were identified as the taurine N-acylamidates of 25R-3alpha,7alpha-dihydroxy-5beta-cholestan-27-oic acid (constituting 22% of biliary bile acids) and 25R-3alpha,7alpha,12alpha-trihydroxy-5beta-cholestan-27-oic acid (constituting 51%). The remaining compound, constituting 21% of biliary bile acids, was an unknown C27 bile acid. Its structure was elucidated by LC/ESI-MS/MS and NMR and shown to be the taurine conjugate of 25R-1beta, 3alpha, 7alpha-trihydroxy-5beta-cholestan-27-oic acid, a C27 trihydroxy bile acid not previously reported. Although C27 bile acids with a 1beta-hydroxyl group have been identified as trace bile acids in the alligator, this is the first report of a major biliary C27 bile acid possessing a 1beta-hydroxyl group.

A facile synthesis of C-24 and C-25 oxysterols by in situ generated ethyl(trifluoromethyl)dioxirane.

Experiments were performed to compare the regioselective hydroxylation of the isopropyl C-H bond at C-25 in 5alpha-cholestan-3beta-yl acetate by in situ generated dimethyldioxirane, methyl(trifluoromethyl)dioxirane, hexafluoro(dimethyl)dioxirane or ethyl(trifluoromethyl)dioxirane (ETDO). The dioxiranes were generated from the corresponding ketones and potassium peroxymonosulfate in aq. NaHCO(3), pH 7.5-8.0. Of the four dioxiranes examined, partially fluorinated, sterically bulky ETDO displayed the highest reactivity and regioselectivity. Using in situ generated ETDO, a facile, synthesis was developed for two naturally occurring oxysterols, i.e., 25-hydroxycholesterol, as well as its 3-sulfate (overall yield of the sulfate, 24%) and 24-oxocholesterol (16%), starting from cholesterol.