Bariatric surgery reveals a gut-restricted TGR5 agonist with anti-diabetic effects.

Bariatric surgery, the most effective treatment for obesity and type 2 diabetes, is associated with increased levels of the incretin hormone glucagon-like peptide-1 (GLP-1) and changes in levels of circulating bile acids. The levels of individual bile acids in the gastrointestinal (GI) tract after surgery have, however, remained largely unstudied. Using ultra-high performance liquid chromatography-mass spectrometry-based quantification, we observed an increase in an endogenous bile acid, cholic acid-7-sulfate (CA7S), in the GI tract of both mice and humans after sleeve gastrectomy. We show that CA7S is a Takeda G-protein receptor 5 (TGR5) agonist that increases Tgr5 expression and induces GLP-1 secretion. Furthermore, CA7S administration increases glucose tolerance in insulin-resistant mice in a TGR5-dependent manner. CA7S remains gut restricted, minimizing off-target effects previously observed for TGR5 agonists absorbed into the circulation. By studying changes in individual metabolites after surgery, the present study has revealed a naturally occurring TGR5 agonist that exerts systemic glucoregulatory effects while remaining confined to the gut.

Transcriptome analysis of Clarias magur brain and gonads suggests neuro-endocrine inhibition of milt release from captive GnRH-induced males.

Transcriptome analysis of Clarias magur brain and gonads at preparatory, mature, 6 and 16 h post-GnRH injection (hpi) stages yielded 9.5 GB data with 39,738 contigs. Sequences of 45 reproductive genes were identified for the first time in C. magur along with unique and differentially expressed genes. The expression of 20 genes was validated by qRT-PCR. Upregulation of Cyp11A1, Cyp17A1 and FTZF1 genes in the 16hpi testis accompanied by the 17ß-HSD3 expression indicates testosterone (T) synthesis in response to LH surge, while reduced expression of CYP11B1 suggests a high T: 11-KT ratio. It is evident by the gene expression analysis that the inhibitory neurotransmitter GABA, altered T: 11-KT, increased testicular bile acids, and oxytocin-like neuropeptide in the male brain, appear to be involved in arresting the pulsatile motion of testicular smooth muscles. The work generates important leads for an effective induced breeding strategy for silurid catfish.

Global chemical effects of the microbiome include new bile-acid conjugations.

A mosaic of cross-phylum chemical interactions occurs between all metazoans and their microbiomes. A number of molecular families that are known to be produced by the microbiome have a marked effect on the balance between health and disease1-9. Considering the diversity of the human microbiome (which numbers over 40,000 operational taxonomic units10), the effect of the microbiome on the chemistry of an entire animal remains underexplored. Here we use mass spectrometry informatics and data visualization approaches11-13 to provide an assessment of the effects of the microbiome on the chemistry of an entire mammal by comparing metabolomics data from germ-free and specific-pathogen-free mice. We found that the microbiota affects the chemistry of all organs. This included the amino acid conjugations of host bile acids that were used to produce phenylalanocholic acid, tyrosocholic acid and leucocholic acid, which have not previously been characterized despite extensive research on bile-acid chemistry14. These bile-acid conjugates were also found in humans, and were enriched in patients with inflammatory bowel disease or cystic fibrosis. These compounds agonized the farnesoid X receptor in vitro, and mice gavaged with the compounds showed reduced expression of bile-acid synthesis genes in vivo. Further studies are required to confirm whether these compounds have a physiological role in the host, and whether they contribute to gut diseases that are associated with microbiome dysbiosis.

Conversion of chenodeoxycholic acid to cholic acid by human CYP8B1.

The human cytochrome P450 enzyme CYP8B1 is a crucial regulator of the balance of cholic acid (CA) and chenodeoxycholic acid (CDCA) in the liver. It was previously shown to catalyze the conversion of 7α-hydroxycholest-4-en-3-one, a CDCA precursor, to 7α,12α-dihydroxycholest-4-en-3-one, which is an intermediate of CA biosynthesis. In this study we demonstrate that CYP8B1 can also convert CDCA itself to CA. We also show that five derivatives of luciferin are metabolized by CYP8B1 and established a rapid and convenient inhibitor test system. In this way we were able to identify four new CYP8B1 inhibitors, which are aminobenzotriazole, exemestane, ketoconazole and letrozole.

Bile acid analysis in human disorders of bile acid biosynthesis.

Bile acids facilitate the absorption of lipids in the gut, but are also needed to maintain cholesterol homeostasis, induce bile flow, excrete toxic substances and regulate energy metabolism by acting as signaling molecules. Bile acid biosynthesis is a complex process distributed across many cellular organelles and requires at least 17 enzymes in addition to different metabolite transport proteins to synthesize the two primary bile acids, cholic acid and chenodeoxycholic acid. Disorders of bile acid synthesis can present from the neonatal period to adulthood and have very diverse clinical symptoms ranging from cholestatic liver disease to neuropsychiatric symptoms and spastic paraplegias. This review describes the different bile acid synthesis pathways followed by a summary of the current knowledge on hereditary disorders of human bile acid biosynthesis with a special focus on diagnostic bile acid profiling using mass spectrometry.

Cytochrome P450s in the synthesis of cholesterol and bile acids--from mouse models to human diseases.

The present review describes the transgenic mouse models that have been designed to evaluate the functions of the cytochrome P450s involved in cholesterol and bile acid synthesis, as well as their link with disease. The knockout of cholesterogenic Cyp51 is embrionally lethal, with symptoms of Antley-Bixler syndrome occurring in mice, whereas the evidence for this association is conflicting in humans. Disruption of Cyp7a1 from classic bile acid synthesis in mice leads to either increased postnatal death or a milder phenotype with elevated serum cholesterol. The latter is similar to the case in humans, where CYP7A1 mutations associate with high plasma low-density lipoprotein and hepatic cholesterol content, as well as deficient bile acid excretion. Disruption of Cyp8b1 from an alternative bile acid pathway results in the absence of cholic acid and a reduced absorption of dietary lipids; however, the human CYP8B1 polymorphism fails to explain differences in bile acid composition. Unexpectedly, apparently normal Cyp27a1(-/-) mice still synthesize bile acids that originate from the compensatory pathway. In humans, CYP27A1 mutations cause cerebrotendinous xanthomatosis, suggesting that only mice can compensate for the loss of alternative bile acid synthesis. In line with this, Cyp7b1 knockouts are also apparently normal, whereas human CYP7B1 mutations lead to a congenital bile acid synthesis defect in children or spastic paraplegia in adults. Mouse knockouts of the brain-specific Cyp46a1 have reduced brain cholesterol excretion, whereas, in humans, CYP46A1 polymorphisms associate with cognitive impairment. At present, cytochrome P450 family 39 is poorly characterized. Despite important physiological differences between humans and mice, mouse models prove to be an invaluable tool for understanding the multifactorial facets of cholesterol and bile acid-related disorders.

Bioproduction of bile acids and the glycine conjugates by Penicillium fungus.

It has been found that the Penicillium endophytic filamentous fungus with the young stems of Scurrula atroprupurea (Loranthaceae) produces cholic acid, deoxycholic acid and the glycine conjugates.

Biosynthesis of bile acids in a variety of marine bacterial taxa.

Several marine bacterial strains, which were isolated from seawater off the island Dokdo, Korea, were screened to find new bioactive compounds such as antibiotics. Among them, Donghaeana dokdonensis strain DSW-6 was found to produce antibacterial agents, and the agents were then purified and analyzed by LC-MS/MS and 1D- and 2D-NMR spectrometries. The bioactive compounds were successfully identified as cholic acid and glycine-conjugated glycocholic acid, the 7alpha-dehydroxylated derivatives (deoxycholic acid and glycodeoxycholic acid) of which were also detected in relatively small amounts. Other masine isolates, taxonomically different from DSW-6, were also able to produce the compounds in a quite different production ratio from DSW-6. As far as we are aware of, these bile acids are produced by specific members of the genus Streptomyces and Myroides, and thought to be general secondary metabolites produced by a variety of bacterial taxa that are widely distributed in the sea.

Fish oil increases bile acid synthesis in male patients with hypertriglyceridemia.

Fibrates are drugs of choice in patients with hypertriglyceridemia (HTG), but may increase the risk for gallstones by decreasing bile acid synthesis. Fish oil might be a therapeutic alternative, but its effect on bile acid metabolism in humans is unknown. We compared the effects of triglyceride-lowering therapy by fish oil or bezafibrate on cholesterol synthesis and bile acid metabolism in HTG. Cholesterol synthesis, bile acid pool sizes, and synthesis rates were compared between 9 male HTG patients and 10 normolipidemic controls matched for age, sex, and BMI. Effects of bezafibrate or fish oil were studied only in HTG patients in a randomized crossover trial. Patients had 14-fold higher serum triglyceride concentrations and greater cholesterol synthesis, as indicated by a 107% higher ratio of serum lathosterol to cholesterol (P < 0.01) than controls. The groups did not differ in bile acid metabolism. Both bezafibrate and fish oil reduced serum TG concentration (-68 and -51% vs. baseline, respectively). Compared with baseline, bezafibrate therapy was associated with reduced cholesterol synthesis (-25%, P = 0.009) without changes in bile acid synthesis rate and pool size. In contrast, fish oil increased bile acid synthesis (+31% vs. baseline, P = 0.07 and +53% vs. bezafibrate, P = 0.02) and altered bile acid distribution, as reflected by an increased ratio of the cholic acid (CA) synthesis rate to the chenodeoxycholic acid (CDCA) synthesis rate (+35% vs baseline, P = 0.05 and + 32% vs bezafibrate, P = 0.07) without effects on bile acid pool size or cholesterol synthesis. In conclusion, cholesterol synthesis is greater in HTG patients than in controls, whereas bile acid synthesis does not differ. Bezafibrate and fish oil have similar triglyceride-lowering capacities, but distinct effects on cholesterol synthesis. Bile acid synthesis is increased by fish oil, but not by bezafibrate therapy.

Bile acids are new products of a marine bacterium, Myroides sp. strain SM1.

Strain SM1 was isolated as a biosurfactant-producing microorganism from seawater and presumptively identified as Myroides sp., based on morphology, biochemical characteristics and 16S rDNA sequence. The strain produced surface-active compounds in marine broth, which were purified, using emulsification activity for n-hexadecane as an indicator. The purified compounds were identified by thin-layer chromatography, (1)H- and (13)C-NMR spectra and fast atom bombardment mass spectrometry as cholic acid, deoxycholic acid and their glycine conjugates. Type strains of the genus Myroides, M. odoratus JCM7458 and M. odoramitimus JCM7460, also produced these compounds. Myroides sp. strain SM1 possessed a biosynthetic route to cholic acid from cholesterol. Thus, bile acids were found as new products of prokaryotic cells, genus Myroides.

Gene structure of pig sterol 12alpha-hydroxylase (CYP8B1) and expression in fetal liver: comparison with expression of taurochenodeoxycholic acid 6alpha-hydroxylase (CYP4A21).

Cholic acid is the major trihydroxy bile acid formed in most mammals. The domestic pig (Sus scrofa) is an exception. The bile of adult pig is devoid of cholic acid whereas hyocholic acid is found in amounts equal to that of cholic acid in humans. The pathway leading to formation of hyocholic acid is believed to be species-specific and to have evolved in the pig to compensate for a nonexistent or deficient cholic acid biosynthesis. However, a high level of cholic acid has recently been found in the bile of fetal pig. Here we describe that a gene encoding the key enzyme in cholic acid biosynthesis, the sterol 12alpha-hydroxylase (CYP8B1), is in fact present in the pig genome. The deduced amino acid sequence shows 81% identity to the human and rabbit orthologues. CYP8B1 mRNA is expressed at significant levels in fetal pig liver. Both CYP8B1 and the key enzyme in hyocholic acid formation, taurochenodeoxycholic acid 6alpha-hydroxylase (CYP4A21), were found to be expressed in pig liver in a developmental-dependent but opposite fashion.

Enterohepatic circulation of bile salts in farnesoid X receptor-deficient mice: efficient intestinal bile salt absorption in the absence of ileal bile acid-binding protein.

The bile salt-activated farnesoid X receptor (FXR; NR1H4) controls expression of several genes considered crucial in maintenance of bile salt homeostasis. We evaluated the physiological consequences of FXR deficiency on bile formation and on the kinetics of the enterohepatic circulation of cholate, the major bile salt species in mice. The pool size, fractional turnover rate, synthesis rate, and intestinal absorption of cholate were determined by stable isotope dilution and were related to expression of relevant transporters in the livers and intestines of FXR-deficient (Fxr-/-) mice. Fxr-/- mice showed only mildly elevated plasma bile salt concentrations associated with a 2.4-fold higher biliary bile salt output, whereas hepatic mRNA levels of the bile salt export pump were decreased. Cholate pool size and total bile salt pool size were increased by 67 and 39%, respectively, in Fxr-/- mice compared with wild-type mice. The cholate synthesis rate was increased by 85% in Fxr-/- mice, coinciding with a 2.5-fold increase in cholesterol 7alpha-hydroxylase (Cyp7a1) and unchanged sterol 12alpha-hydroxylase (Cyp8b1) expression in the liver. Despite a complete absence of ileal bile acid-binding protein mRNA and protein, the fractional turnover rate and cycling time of the cholate pool were not affected. The calculated amount of cholate reabsorbed from the intestine per day was approximately 2-fold higher in Fxr-/- mice than in wild-type mice. Thus, the absence of FXR in mice is associated with defective feedback inhibition of hepatic cholate synthesis, which leads to enlargement of the circulating cholate pool with an unaltered fractional turnover rate. The absence of ileal bile acid-binding protein does not negatively interfere with the enterohepatic circulation of cholate in mice.

Cyclosporin a and enterohepatic circulation of bile salts in rats: decreased cholate synthesis but increased intestinal reabsorption.

Cyclosporin A (CsA) has been shown to inhibit synthesis and hepatobiliary transport of bile salts. However, effects of CsA on the enterohepatic circulation of bile salts in vivo are largely unknown. We characterized the effects of CsA on the enterohepatic circulation of cholate, with respect to synthesis rate, pool size, cycling time, intestinal absorption, and the expression of relevant transporters in liver and intestine in rats. CsA (1 mg. 100 g(-1). day(-1) s.c.) or its solvent was administered daily to male rats for 10 days. Cholate synthesis rate and pool size were determined by a 2H4-cholate dilution technique. Bile and feces were collected for determination of cholate and total bile salts, respectively. Cycling time and intestinal absorption of cholate were calculated. The mRNA levels and corresponding transporter protein levels in liver and intestine were assessed by real-time polymerase chain reaction and Western analysis, respectively. CsA treatment decreased cholate synthesis rate by 71%, but did not affect pool size or cycling time. CsA reduced the amount of cholate lost per enterohepatic cycle by approximately 70%. Protein levels of the apical sodium-dependent bile salt transporter (Asbt) were 2-fold increased in distal ileum of CsA-treated rats, due to post-transcriptional events. In conclusion, chronic CsA treatment markedly reduces cholate synthesis rate in rats, but does not affect cholate pool size or cycling time. Our results strongly suggest that CsA enhances efficacy of intestinal cholate reabsorption through increased Asbt protein expression in the distal ileum, which contributes to maintenance of cholate pool size in CsA-treated rats.

Conversion of 7 alpha-hydroxycholesterol to bile acid in human subjects: is there an alternate pathway favoring cholic acid synthesis?

Despite the fact that most human subjects synthesize about twice as much cholic acid as chenodeoxycholic acid, available evidence suggests that 7 alpha-hydroxycholesterol, the first intermediate in the major pathway for bile acid synthesis, is converted about equally to these two bile acids. Synthesis through the main alternate pathway can not explain this discrepancy because 27-hydroxycholesterol, the first intermediate in that pathway, is converted preferentially to chenodeoxycholic acid. To examine the validity of these contradictory observations, we administered (24-(14)C)-cholic acid and (24-(14)C)-chenodeoxycholic acid together with (7 beta-(3)H)-7 alpha-hydroxycholesterol on one occasion and (22,23-(3)H)-27-hydroxycholesterol on a separate occasion to eight normal human subjects. Synthesis of the two primary bile acids was determined by means of standard isotope dilution kinetics of the carbon 14-specific activities of biliary bile acids. Conversion of (7 beta-(3)H)-7 alpha-hydroxycholesterol and (22,23-(3)H)-27-hydroxycholesterol to bile acid was calculated from the tritium/carbon 14 ratio in cholic and chenodeoxycholic acid. For synthesis, the mean +/- SEM cholic/chenodeoxycholic ratio was 1.82 +/- 0.26. For apparent conversion of (7 beta-(3)H)-7 alpha-hydroxycholesterol to bile acid, the mean +/- SEM cholic/ chenodeoxycholic ratio was 1.02 +/- 0.09, whereas for (22,23(3)H)-27-hydroxycholesterol, the mean +/- SEM cholic/chenodeoxycholic ratio was 0.38 +/- 0.03. These data imply that, on average, more than 40% of cholic acid in these subjects was synthesized through a pathway that bypassed initial 7 alpha-hydroxylation. However, consideration of all potential candidates for such a pathway raises doubts that any of them contributes substantially to bile acid synthesis.

Colonic transit influences deoxycholic acid kinetics.

BACKGROUND & AIMS: Prolonged large bowel transit, and an increase in the proportion of deoxycholic acid (DCA), have been implicated in the pathogenesis of cholesterol gallstones-including those developing in acromegalics treated with octreotide. However, there are few data on the effects of intestinal transit on bile acid kinetics. METHODS: We therefore measured the kinetics of DCA and cholic acid (CA) using stable isotopes, serum sampling, and mass spectrometry. The results were related to mouth-to-caecum (MCTT) and large bowel transit times (LBTTs) in 4 groups of 8 individuals: (1) non-acromegalic controls, (2) acromegalics untreated with octreotide, (3) acromegalics on long-term octreotide, and (4) patients with constipation. Paired, before and during octreotide, studies were performed in 5 acromegalics. RESULTS: In the unpaired and paired studies, octreotide significantly prolonged MCTT and LBTT. In the paired studies, the octreotide-induced prolongation of LBTT caused an increase in the DCA input rate (6.4 +/- 2.8 to 12 +/- 2.6 micromol. kg. d, P < 0.05) and pool size (18 +/- 12 to 40 +/- 13 micromol/kg, P < 0.05), and a decrease in CA pool size (45 +/- 15 to 25 +/- 11 micromol/kg, P < 0.05). Furthermore, during octreotide treatment, the mean conversion of 13C-CA to 13C-DCA (micromoles) was greater (P < 0.05) on study days 3, 4, and 5. There were also positive linear relationships between LBTT and DCA input rate (r = 0.78), pool size (r = 0.82, P < 0.001), and a weak (r = -0.49) negative linear relationship between LBTT and CA pool size (P < 0.01). CONCLUSIONS: These data support the hypothesis that, by increasing DCA formation and absorption, prolongation of large bowel transit is a pathogenic factor in the formation of octreotide-induced gallstones.

Expression of sterol 12alpha-hydroxylase alters bile acid pool composition in primary rat hepatocytes and in vivo.

BACKGROUND & AIMS: The rate of 12alpha-hydroxylation of bile acid intermediates is believed to determine the ratio of cholic acid (CA) to chenodeoxycholic acid (CDCA) biosynthesis and the overall hydrophobicity of the bile acid pool. The aim of this study was to determine the effects of the level of expression of sterol 12alpha-hydroxylase (CYP8b1) and cholesterol 7alpha-hydroxylase (CYP7a1) on rates of CA biosynthesis and bile acid pool composition. METHODS: Expression of CYP8b1 and CYP7a1 was accomplished through infection of primary rat hepatocytes (PRH) or intact male SD rats with replication-defective recombinant adenoviruses encoding either CYP8b1 or CYP7a1. RESULTS: Increased expression of CYP7a1 over basal levels in PRH dramatically increased bile acid biosynthesis (586% +/- 82%, P < 0.001) but did not alter the ratio of CA to CDCA. Conversely, increased expression of CYP8b1 in vitro had no significant effect on the rates of total bile acid synthesis but significantly increased (4.1-fold) the rates of CA biosynthesis, resulting in an increase in the CA-CDCA ratio from 1:6.6 to 2.8:1. In whole rats, increased CYP8b1 expression over basal levels markedly increased the CA in the bile acid pool from 36% +/- 3.4% to 50% +/- 2.9% in 5 days. CDCA and its muricholic acid derivatives decreased from 64% +/- 3.4% to 50% +/- 2.9%. CONCLUSIONS: Increased expression of CYP8b1 led to a marked increase in CA biosynthesis both in PRH and in whole animals. CYP8b1 is capable of 12alpha-hydroxylating bile acid intermediates from both the classic and acidic pathways.

Bile acid synthesis in cultured human hepatocytes: support for an alternative biosynthetic pathway to cholic acid.

The biosynthesis of bile acids by primary cultures of normal human hepatocytes has been investigated. A general and sensitive method for the isolation and analysis of sterols and bile acids was used, based on anion exchange chromatography and gas chromatography-mass spectrometry (GC/MS). Following incubation for 5 days, 8 oxysterols and 8 C(27)- or C(24)-bile acids were identified in media and cells. Cholic and chenodeoxycholic acids conjugated with glycine or taurine were by far the major steroids found, accounting for 70% and 24% of the total, respectively, being consistent with bile acid synthesis in human liver. Small amounts of sulfated 3beta-hydroxy-5-cholenoic acid and 3beta,7alpha-dihydroxy-5beta-cholanoic acid were also detected. Nine steroids were potential bile acid precursors (2% of total), the major precursors being 7alpha, 12alpha-dihydroxy-3-oxo-4-cholenoic acid and its 5beta-reduced form. These 2 and 5 other intermediates formed a complete metabolic sequence from cholesterol to cholic acid (CA). This starts with 7alpha-hydroxylation of cholesterol, followed by oxidation to 7alpha-hydroxy-4-cholesten-3-one and 12alpha-hydroxylation. Notably, 27-hydroxylation of the product 7alpha, 12alpha-dihydroxy-4-cholesten-3-one and further oxidation and cleavage of the side chain precede A-ring reduction. A-Ring reduction may also occur before side-chain cleavage, but after 27-hydroxylation, yielding 3alpha,7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid as an intermediate. The amounts of the intermediates increased in parallel to those of CA during 4 days of incubation. Suppressing 27-hydroxylation with cyclosporin A (CsA) resulted in a 10-fold accumulation of 7alpha, 12alpha-dihydroxy-4-cholesten-3-one and a decrease of the production of CA and its acidic precursors. These results suggest that the observed intermediates reflect an alternative biosynthetic pathway to CA, which may be quantitatively significant in the cells.

Alpha 1-fetoprotein transcription factor is required for the expression of sterol 12alpha -hydroxylase, the specific enzyme for cholic acid synthesis. Potential role in the bile acid-mediated regulation of gene transcription.

Cholesterol conversion to bile acids occurs via the "classic" (neutral) or the "alternative" (acidic) bile acid biosynthesis pathways. Sterol 12alpha-hydroxylase/CYP8b1 is the specific enzyme required for cholic acid synthesis. The levels of this enzyme determine the ratio of cholic acid to chenodeoxycholic acid and thus the hydrophobicity of the circulating bile acid pool. Expression of the 12alpha-hydroxylase gene is tightly down-regulated by hydrophobic bile acids. In this study, we report the characterization of two DNA elements that are required for both the 12alpha-hydroxylase promoter activity and bile acid-mediated regulation. Mutation of these elements suppresses 12alpha-hydroxylase promoter activity. Mutations of any other part of the promoter do not alter substantially the promoter activity or alter regulation by bile acids relative to the wild type promoter. These two DNA elements bind alpha(1)-fetoprotein transcription factor (FTF), a member of the nuclear receptor family. We also show that overexpression of FTF in a non-liver cell line activates the sterol 12alpha-hydroxylase promoter. These studies demonstrate the crucial role of FTF for the expression and regulation of a critical gene in the bile acid biosynthetic pathways.

Regulation of 25- and 27-hydroxylation side chain cleavage pathways for cholic acid biosynthesis in humans, rabbits, and mice. Assay of enzyme activities by high-resolution gas chromatography;-mass spectrometry.

In classic cholic acid biosynthesis, a series of ring modifications of cholesterol precede side chain cleavage and yield 5beta-cholestane-3alpha, 7alpha, 12alpha-triol. Side chain reactions of the triol then proceed either by the mitochondrial 27-hydroxylation pathway or by the microsomal 25-hydroxylation pathway. We have developed specific and precise assay methods to measure the activities of key enzymes in both pathways, 5beta-cholestane-3alpha, 7alpha, 12alpha-triol 25- and 27-hydroxylases and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases. The extracts from either the mitochondrial or microsomal incubation mixtures were purified by means of a disposable silica cartridge column, derivatized into trimethylsilyl ethers, and quantified by gas chromatography;-mass spectrometry with selected-ion monitoring in a high resolution mode. Compared with the addition of substrates in acetone, those in 2-hydroxypropyl-beta-cyclodextrin increased mitochondrial triol 27-hydroxylase activity 132% but decreased activities of the enzymes in microsomal 25-hydroxylation pathway (triol 25-hydroxylase and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases) 13;-60% in human liver. The enzyme activities in both pathways were generally 2- to 4-times higher in mouse and rabbit livers compared with human liver. In all species, microsomal triol 25-hydroxylase activities were 4- to 11-times larger than mitochondrial triol 27-hydroxylase activities but the activities of tetrol 24S-hydroxylase were similar to triol 27-hydroxylase activities in our assay conditions. The regulation of both pathways in rabbit liver was studied after bile acid synthesis was perturbed. Cholesterol feeding up-regulated enzyme activities involved in both 25- (64;-142%) and 27- (77%) hydroxylation pathways, while bile drainage up-regulated only the enzymes in the 25-hydroxylation pathway (178;-371%). Using these new assays, we demonstrated that the 25- and 27-hydroxylation pathways for cholic acid biosynthesis are more active in mouse and rabbit than human livers and are separately regulated in rabbit liver.