Anodic electrochemical oxidation of cholic acid

Regioselectivity in the anodic electrochemical oxidation of cholic acid with different anodes is described. The oxidation with PbO(2) anode affords the dehydrocholic acid in quantitative yield after 22 h. 3alpha,12alpha-Dihydroxy-7-oxo-5beta-cholan-24-oic acid (59%) and 3alpha-hydroxy-7,12-dioxo-5beta-cholan-24-oic acid (51%) are obtained stopping the reaction at lower time. The rate of the OH-oxidation is C7 > C12 > C3. The electro-oxidation with platinum foil anode gives selectively the 7-ketocholic acid in 40% yield. On the other hand, the graphite plate anode, varying the reaction conditions, produces selectively the dehydrocholic acid in quantitative yield or the 3alpha,12alpha-dihydroxy-7-oxo-5beta-cholan-24-oic acid (96%) while the 3alpha,7alpha-dihydroxy-12-oxo-5beta-cholan-24-oic acid (34%) is obtained together with the other oxo acids.     

Novel 18beta-glycyrrhetinic acid analogues as potent and selective inhibitors of 11beta-hydroxysteroid dehydrogenases

Extensive structural modifications to the 18beta-glycyrrhetinic acid template are described and their effects on the SAR of the 11beta-hydroxysteroid dehydrogenase isozymes type 1 and 2 from the rat are investigated. Isoform selective inhibitors have been discovered and compound 7 N-(2-hydroxyethyl)-3beta-hydroxy-11-oxo-18beta-olean-12-en-30-oic acid amide is highlighted as a very potent selective inhibitor of 11beta-hydroxysteroid dehydrogenase 2 with an IC(50) = 4pM.     

A novel 18 beta-glycyrrhetinic acid analogue as a potent and selective inhibitor of 11 beta-hydroxysteroid dehydrogenase 2

Using 18beta-glycyrrhetinic acid as a template, the synthesis of a series of secondary amides at the 30-position is described and the effects of these modifications on the SAR of the 11beta-hydroxysteroid dehydrogenase isozymes type 1 and 2 from the rat are investigated. An isoform selective inhibitor has been discovered and compound 5, N-(2-hydroxyethyl)-3beta-hydroxy-11-oxo-18beta-olean-12-en-30-oic acid amide, is highlighted as a very potent and selective inhibitor of 11beta-hydroxysteroid dehydrogenase 2 with an IC(50)=4 pM.     

Steroid catechol degradation: disecoandrostane intermediates accumulated by Pseudomonas transposon mutant strains

Eleven transposon mutant strains affected in bile acid catabolism were each found to form yellow, muconic-like intermediates from bile acids. To characterize these unstable intermediates, media from the growth of one of these mutants with deoxycholic acid was treated with ammonia, then the crude product was methylated with diazomethane. Four compounds were subsequently isolated; spectral evidence suggested that they were methyl 12 alpha-hydroxy-3-oxo-23,24-dinorchola-1,4-dien-22-oate, methyl 4-aza-12 beta-hydroxy-9(10)-secoandrosta-1,3,5-triene-9,17-dione-3-carboxyl ate, 4-aza-9 alpha, 12 beta-dihydroxy-9(10)-secoandrosta-1,3,5-trien-17-one-3- methyl carboxylate and 4 alpha-[3'-propionic acid]-5-amino-7 beta-hydroxy-7 alpha beta-methyl- 3a alpha, 4,7,7a-tetrahydro-1-indanone-delta-lactam. It is proposed that the mutants are blocked in the utilization of such muconic-like compounds as the 3,12 beta-dihydroxy-5,9,17-trioxo-4(5),9(10)- disecoandrostal (10),2-dien-4-oic acid formed from deoxycholic acid. A further mutant was examined, which converted deoxycholic acid to 12 alpha-hydroxyandrosta-1,4-dien-3,17-dione, but accumulated yellow products from steroids which lacked a 12 alpha-hydroxy function, such as chenodeoxycholic acid. The products from the latter acid were treated as above; spectral evidence suggested that the two compounds isolated were methyl 4-aza-7-hydroxy-9(10)-secoandrosta-1,3,5- triene-9,17-dione-3-carboxylate and 4 alpha-[1'alpha-hydroxy-3'-propionic acid]-5-amino-7a beta-methyl-3a alpha,4,7,7a-tetrahydro-1-indanone-delta-lactam.     

Bile acid induction of 7 alpha- and 7 beta-hydroxysteroid dehydrogenases in Clostridium limosum

When grown in the presence of bile acids, two strains of Clostridium limosum were found to contain significant amounts of NADP-dependent 7 alpha/7 beta-hydroxysteroid dehydrogenase and NAD-dependent 7 alpha-hydroxysteroid dehydrogenase which were active against conjugated and unconjugated bile acids. No measurable activity could be found when deoxycholic acid (3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oic acid) was used as substrate. No 7 beta-hydroxysteroid dehydrogenase activity and only a trace of 7 alpha-hydroxysteroid dehydrogenase activity could be demonstrated when bile acid was deleted from the growth medium. If bile acid was added after the time of inoculation, the amounts of 7 alpha/7 beta-hydroxysteroid dehydrogenase were greatly reduced. Enzyme enhancement was blocked by addition of rifampicin. The 7 alpha/7 beta-hydroxysteroid dehydrogenase components had pH optima of approximately 10.5. Both the 7 alpha/7 beta-hydroxysteroid dehydrogenase activities were heat-labile, with the 7 beta-component being the more stable of the two. When ranked according to the level of enzymes induced, the order in increasing bile acid induction power on an equimolar scale (0.4 mM) was: 7-ketodeoxycholic acid, cholic acid, chenodeoxycholic acid, and deoxycholic acid. Both 7-ketolithocholic acid and ursodeoxycholic acid were ineffective as enzyme inducers. Optimal induction was achieved with high concentrations of cholic acid (5 mM) and a harvest time of 24 hr. Addition of ursodeoxycholic acid to medium containing optimal concentrations of deoxycholic acid suppressed enzyme induction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concurrent occurrence of 3 beta,12 alpha-dihydroxy-5-cholenoic acid associated with 3 beta-hydroxy-5-cholenoic acid and their preferential urinary excretion in liver diseases

3 beta-Hydroxy-(delta 5-3 beta-ol), 3 beta,12 alpha-dihydroxy-(delta 5-3 beta,12 alpha-ol), 3 beta,7 alpha-dihydroxy-(delta 5-3 beta,7 alpha-ol) and 3 beta,7 beta-dihydroxy-(delta 5-3 beta,7 beta-ol) 5-cholenoic acids were identified in patients with liver diseases by gas-liquid chromatography-mass spectrometry (GLC-MS). Of these unusual 3 beta-hydroxy-5-en-metabolites, delta 5-3 beta-ol and delta 5-3 beta,12 alpha-ol were found as major components in the urine of patients with liver diseases (cholestasis, liver cirrhosis, chronic hepatitis, acute hepatitis). Other 3 beta-dihydroxy-5-en-metabolites, delta 5-3 beta,7 alpha-ol and delta 5-3 beta,7 beta-ol, were found as minor components in the urine. The levels of delta 5-3 beta-ol and delta 5-3 beta,12 alpha-ol in urine were correlated with their levels in serum, with total bile acids in the urine, and with liver function, implying that the degree of their increment correlated well with the severity of liver diseases. The most abundant amounts of delta 5-3 beta-ol and delta 5-3 beta,12 alpha-ol were found in the urine as sulfate conjugates in comparison with bile, portal and hepatic venous sera, and liver tissue of the patients. The biliary excretion and hepatic extraction of these 3 beta-hydroxy-5-en-unsaturated bile acids were more impaired and inefficient than those of cholic and chenodeoxycholic acids.     

3 beta-Hydroxysteroid dehydrogenase of Ruminococcus sp. from human intestinal bacteria

Ruminococcus sp. PO1-3 obtained from human intestinal flora is able to reduce dehydrocholate as well as 3-ketoglycyrrhetinate. From this bacterium dehydrocholate- and 3-ketoglycyrrhetinate-reducing activities were purified one thousand-fold together with 3-ketocholanate-reducing and 3-beta-hydroxyglycyrrhetinate (glycyrrhetic acid) oxidizing activities by means of Matrex Red A, Sephadex G-200 and Octyl-Sepharose column chromatography. The purified enzyme catalyzed the reduction of dehydrocholic acid to 3 beta-hydroxy-7,12-diketocholanic acid and of 3-ketocholanic acid to 3 beta-hydroxycholanic acid. Studies on substrate specificity revealed that the enzyme had absolute specificity for the beta-configuration of a hydroxyl group at the 3 position of bile acid and steroids having no double bond in the A/B ring. This enzyme was neither beta-hydroxysteroid dehydrogenase [EC 1.1.1.51] nor 3 beta-hydroxy-delta 5-steroid dehydrogenase [EC 1.1.1.145], but a novel type of enzyme, defined as 3 beta-hydroxysteroid dehydrogenase.     

Bioconversion of 3beta-hydroxy-5-cholenoic acid into chenodeoxycholic acid by rat brain enzyme systems

We have previously demonstrated that the rat brain contains three unconjugated bile acids, and chenodeoxycholic acid (CDCA) is the most abundantly present in a tight protein binding form. The ratio of CDCA to the other acids in rat brain tissue was significantly higher than the ratio in the peripheral blood, indicating a contribution from either a specific uptake mechanism or a biosynthetic pathway for CDCA in rat brain. In this study, we have demonstrated the existence of an enzymatic activity that converts 3beta-hydroxy-5-cholenoic acid into CDCA in rat brain tissue. To distinguish marked compounds from endogenous related compounds, 18O-labeled 3beta-hydroxy-5-cholenoic acid, 3beta,7alpha-dihydroxy-5-cholenoic acid, and 7alpha-hydroxy-3-oxo-4-cholenoic acid were synthesized as substrates for in vitro incubation studies. The results clearly suggest that 3beta-hydroxy-5-cholenoic acid was converted to 3beta,7alpha-dihydroxy-5-cholenoic acid by microsomal enzymes. The 7alpha-hydroxy-3-oxo-4-cholenoic acid was produced from 3beta,7alpha-dihydroxy-5-cholenoic acid by the action of microsomal enzymes, and Delta4-3-oxo acid was converted to CDCA by cytosolic enzymes. These findings indicate the presence of an enzymatic activity that converts 3beta-hydroxy-5-cholenoic acid into CDCA in rat brain tissue. Furthermore, this synthetic pathway for CDCA may relate to the function of 24S-hydroxycholesterol, which plays an important role in cholesterol homeostasis in the body.     

The degradation of cholic acid by Pseudomonas sp. N.C.I.B. 10590 under anaerobic conditions.

The bacterial degradation of cholic acid under anaerobic conditions by Pseudomonas sp. N.C.I.B. 10590 was studied. The major unsaturated neutral compound was identified as 12 beta-hydroxyandrosta-4,6-diene-3,17-dione, and the major unsaturated acidic metabolite was identified as 12 alpha-hydroxy-3-oxochola-4,6-dien-24-oic acid. Eight minor unsaturated metabolites were isolated and evidence is given for the following structures: 12 alpha-hydroxyandrosta-4,6-diene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-4,6-dien-3-one, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-1,4,6-trien-3-one, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-1,4,6-trien-3-one, 12 alpha-hydroxyandrosta-1,4-diene-3,17-dione, 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione, 3,12-dioxochola-4,6-dien-24-oic acid and 12 alpha-hydroxy-3-oxopregna-4,6-diene-20-carboxylic acid. In addition, a major saturated neutral compound was isolated and identified as 3 beta,12 beta-dihydroxy-5 beta-androstan-17-one, and the only saturated acidic metabolite was 7 alpha,12 alpha-dihydroxy-3-oxo-5 beta-cholan-24-oic acid. Nine minor saturated neutral compounds were also isolated, and evidence is presented for the following structures: 12 beta-hydroxy-5 beta-androstane-3,17-dione, 12 alpha-hydroxy-5 beta-androstane-3,17-dione, 3 beta,12 alpha-dihydroxy-5 beta-androstan-17-one, 3 alpha,12 beta-androstan-17-one, 3 alpha,12 alpha-dihydroxy-5 beta-androstan-17-one, 5 beta-androstane-3 beta,12 beta,17 beta-triol, 5 beta-androstane-3 beta,12 alpha,17 beta-triol, 5 beta-androstane-3 alpha,12 beta,17 beta-triol and 5 beta-androstane-3 alpha,12 alpha,17 beta-triol. The induction of 7 alpha-dehydroxylase and 12 alpha-dehydroxylase enzymes is discussed, together with the significance of dehydrogenation and ring fission under anaerobic conditions.     

Chemical synthesis of the 3-sulfooxy-7-N-acetylglucosaminyl-24-amidated conjugates of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, and related compounds: unusual, major metabolites of bile acid in a patient with Niemann-Pick disease type C1

The chemical synthesis of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, triply conjugated by sulfuric acid at C-3, by N-acetylglucosamine (GlcNAc) at C-7, and by glycine or taurine at C-24, is described. These are unusual, major metabolites of bile acid found to be excreted in the urine of a patient with Niemann-Pick disease type C1. Analogous double-conjugates of 3beta-hydroxy-7-oxo-5-cholen-24-oic acid were also prepared. The principal reactions involved were: (1) beta-d-N-acetylglucosaminidation at C-7 of methyl 3beta-tert-butyldimethylsilyloxy (TBDMSi)-7beta-hydroxy-5-cholen-24-oate with 2-acetamido-1alpha-chloro-1,2-dideoxy-3,4,6-tri-O-acetyl-d-glucopyranose in the presence of CdCO(3) in boiling toluene; (2) sulfation at C-3 of the resulting 3beta-TBDMSi-7beta-GlcNAc with sulfur trioxide-trimethylamine complex in pyridine; and (3) direct amidation at C-24 of the 3beta-sulfooxy-7beta-GlcNAc conjugate with glycine methyl ester hydrochloride (or taurine) using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride as a coupling agent in DMF. The structures of the multi-conjugated bile acids were characterized by liquid chromatography-mass spectrometry with an electrospray ionization probe under the positive and negative ionization modes.     

Enzymatic reduction of dehydrocholic acid to 12-ketochenodeoxycholic acid with NADH regeneration

12-Ketochenodeoxycholic acid, an essential intermediate in the synthesis of chenodeoxycholic acid, has been enzymatically prepared from dehydrocholic acid. The specific reduction of dehydrocholic with NADH was catalysed by 3α-hydroxysteroid dehydrogenase (3α-hydroxysteroid: NAD(P)⁺ oxidoreductase, EC 1.1.1.50) and 7α-hydroxysteroid dehydrogenase (7α-hydroxysteroid:NAD⁺ 7-oxidoreductase, EC 1.1.1.159). Cofactor regeneration was obtained through the formate dehydrogenase (formate:NAD⁺ oxidoreductase, EC 1.2.1.2) catalysed oxidation of formate. Complete transformation of dehydrocholic acid to the 12-keto derivative was achieved with a coenzyme turnover number up to 1200. No steroid by-products were detected by high performance liquid chromatography and thin layer chromatography. The process yielded 9 g product l^?1 in 66–84 h. The high purity of the enzymatically prepared 12-ketochenodeoxycholic acid should drastically reduce the formation of the toxic by-product lithocholic acid, which occurs in the synthesis of chenodeoxycholic acid when using chemical methods alone.     

12 beta-dehydrogenation of bile acids by Clostridium paraputrificum, C. tertium, and C. difficile and epimerization at carbon-12 of deoxycholic acid by cocultivation with 12 alpha-dehydrogenating Eubacterium lentum

12 beta-Hydroxysteroid dehydrogenating activities were detected in 13 strains of Clostridium paraputrificum, 1 strain of C. tertium, and 1 strain of C. difficile, together with a 3 alpha- and 3 beta-hydroxysteroid dehydrogenase system in many strains. Redox reactions a C-12 of disubstituted and trisubstituted bile acids were performed unspecifically by representative strains of C. paraputrificum. 3 alpha,12 beta-, 3 beta,12 beta-Dihydroxy-, 3 alpha, 7 alpha, 12 beta-trihydroxy-, and 3-keto,12 beta-hydroxy-5 beta-cholanoic acids, so far not known as bacterial bile acid metabolites, were identified. Epimerization of the 12 alpha-hydroxyl group of deoxycholate via the 12-keto intermediate was achieved by cocultivation of C. paraputrificum and Eubacterium lentum, elaborating a 12 alpha-hydroxysteroid dehydrogenase only. In addition, epimerization at C-12 was demonstrated with mixed human fecal cultures.     

12 beta-dehydrogenation of bile acids by Clostridium paraputrificum, C. tertium, and C. difficile and epimerization at carbon-12 of deoxycholic acid by cocultivation with 12 alpha-dehydrogenating Eubacterium lentum.

12 beta-Hydroxysteroid dehydrogenating activities were detected in 13 strains of Clostridium paraputrificum, 1 strain of C. tertium, and 1 strain of C. difficile, together with a 3 alpha- and 3 beta-hydroxysteroid dehydrogenase system in many strains. Redox reactions a C-12 of disubstituted and trisubstituted bile acids were performed unspecifically by representative strains of C. paraputrificum. 3 alpha,12 beta-, 3 beta,12 beta-Dihydroxy-, 3 alpha, 7 alpha, 12 beta-trihydroxy-, and 3-keto,12 beta-hydroxy-5 beta-cholanoic acids, so far not known as bacterial bile acid metabolites, were identified. Epimerization of the 12 alpha-hydroxyl group of deoxycholate via the 12-keto intermediate was achieved by cocultivation of C. paraputrificum and Eubacterium lentum, elaborating a 12 alpha-hydroxysteroid dehydrogenase only. In addition, epimerization at C-12 was demonstrated with mixed human fecal cultures.     

Potential bile acid metabolites. 9. 3,12-Dihydroxy- and 12 beta-hydroxy-5 alpha-cholanoic acids

Syntheses of the heretofore unreported 3 alpha, 12 beta-, 3 beta, 12 beta-dihydroxy-, and 12 beta-hydroxy-5 alpha-cholanic acids of the 5 alpha-series, their methyl esters, and some related derivatives are described. In addition, allodeoxycholic (3 alpha, 12 alpha-dihydroxy) acid was prepared by a new route. The principal reactions involved were the stereoselective reduction of C-12 ketones with an amino-borane reagent and of a C-3 ketone with K-Selectride, and inversion of a 3 beta-tosylate derivative with N,N-dimethylformamide.     

Purification and properties of testicular 3 beta-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase

Through the treatment of rat testicular microsomes with sodium cholate, 3 beta-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase (abbreviated as the 3 beta-hydroxysteroid dehydrogenase and isomerase, respectively) were solubilized, and then purified by DEAE and hydroxylapatite column chromatographies. The findings were as follows: With this purification procedure, the 3 beta-hydroxysteroid dehydrogenase activity could not be separated from the isomerase. For 3-oxo-4-ene-steroid formation from 3 beta-hydroxy-5-ene-steroids, NAD+ was required as a cofactor. While the 3 beta-hydroxysteroid dehydrogenase required NAD+, the isomerase also required NAD+ or its reduced form, in contrast to the microbial enzyme. On treatment of the purified enzyme with 5'-p-fluorosulfonyl-benzoyladenosine (FSBA), both enzyme activities were markedly reduced. The enzyme, affinity labeled with [adenine-8-14C]FSBA, showed a mol. wt of 46.8 K. During 4-androstenedione production from DHA, 5-androstenedione was detected as an intermediate.     

Oleanane-type triterpenes of Embelia schimperi leaves

An investigation of an ethyl acetate extract of Embelia schimperi leaves has led to the isolation of 10 oleanane-type triterpenes characterized as 3beta,16alpha-di-O-acetyl-13beta, 28-epoxyoleanane (1), 3beta-acetyl-16-oxo-13beta, 28-epoxyoleanane (2), 3beta-acetyl-16alpha-hydroxy-13beta, 28-epoxyoleanane (3), 3beta-acetyl-16alpha-hydroxyoleanane-13beta, 28-olide (4), 3beta-acetyl-28-hydroxy-16-oxo-12-oleanene (5), 3beta, 28-di-O-acetyl-16alpha-hydroxy-12-oleanene (6), 3beta-acetyl-11alpha, 28-dihydroxy-16-oxo-12-oleanene (7), 3beta, 11alpha, 16alpha, 28-tetrahydroxy-12-oleanene (8), 3beta-acetyl-16alpha, 28alpha-dihydroxy-13beta, 28-oxydooleanane (9) and 3beta, 28alpha-dihydroxy-16-oxo-13beta, 28-oxydooleanane (10). The known compounds isolated from the same extract included 3beta, 16alpha-dihydroxy-13beta, 28-epoxyoleanane (protoprimulagenin A) (11), 3beta-hydroxy-16-oxo-13beta, 28-epoxyoxyoleanane (aegicerin) (12), 3, 16-dioxo-13beta, 28-epoxyoleanane (embilionone) (13), 3beta, 28-dihydroxy-16-oxo-12-oleanene (schimperinone) (14), taraxerone (15), taraxerol (16) and stigmasterol (17). Structure elucidations were carried out spectroscopically.     

Potential bile acid metabolites. XVIII. Synthesis of stereoisomeric 3,6,12 alpha-trihydroxy-5 beta-cholanoic acids.

Two new 6-hydroxylated bile acids, 3 beta, 6 alpha, 12 alpha- and 3 beta, 6 beta, 12 alpha-trihydroxy-5 beta-cholanoic acids, were synthesized from deoxycholic acid. In addition, their C-3 epimers, 3 alpha, 6 alpha, 12 alpha- and 3 alpha, 6 beta, 12 alpha-trihydroxy acids, were prepared by a new route. The principal reactions used were 1) 6 beta-hydroxylation of 3-methoxy-3,5-dienes with m-chloroperbenzoic acid in aqueous dioxane; 2) catalytic hydrogenation of the resulting 6 beta-hydroxy-3-oxo-4-enes to the 6 beta-hydroxy-3-oxo-5 beta compounds with palladium on calcium carbonate catalyst in ethanol; and 3) stereoselective reduction of appropriate 3-oxo derivatives with potassium tri-sec-butylborohydride and tert-butylamine-borane complex. The thin-layer chromatographic, gas-liquid chromatographic, and high performance liquid chromatographic mobilities, and 1H- and 13C-nuclear magnetic resonance spectroscopic data of the four stereoisomers are presented. With this work all the 6-hydroxylated derivatives of lithocholic, deoxycholic, chenodeoxycholic, ursodeoxycholic, and cholic acids in the 5 beta series are now known and have been synthesized.     

Occurrence of 3 beta-hydroxy-5-cholestenoic acid, 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid as normal constituents in human blood

Three unconjugated C27 bile acids were found in plasma from healthy humans. They were isolated by liquid-solid extraction and anion-exchange chromatography and were identified by gas-liquid chromatography-mass spectrometry, microchemical reactions, and ultraviolet spectroscopy as 3 beta-hydroxy-5-cholestenoic, 3 beta,7 alpha-dihydroxy-5-cholestenoic, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acids. Their levels often exceeded those of the unconjugated C24 bile acids and the variations between individuals were smaller than for the C24 acids. The concentrations in plasma from 11 healthy subjects were 67.2 +/- 27.9 ng/ml (mean +/- SD) for 3 beta-hydroxy-5-cholestenoic acid, 38.9 +/- 25.6 ng/ml for 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 81.7 +/- 27.9 ng/ml for 7 alpha-hydroxy-3-oxo-4-cholestenoic acid. The levels of the individual acids were positively correlated to each other and not to the levels of the C24 acids. The cholestenoic acids were below the detection limit (20-50 ng/ml) in bile and C27 bile acids present in bile were not detected in plasma.     

Identification of new C27 and C24 bile acids in the bile of Alligator mississippiensis

Biliary bile acids of Alligator mississippiensis were analyzed by gas-liquid chromatography-mass spectrometry after fractionation by silica gel column chromatography. It was shown that the alligator bile contained 12 C27 bile acids and 8 C24 bile acids. In addition to the C27 bile acids, such as 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid, 3 alpha,7 alpha,12 alpha-trihydroxy-5 alpha-cholestanoic acid, 3 alpha,7 alpha-dihydroxy-5 beta-cholestanoic acid, 3 alpha,12 alpha-dihydroxy-5 beta-cholestanoic acid, 7 alpha,12 alpha-dihydroxy-3-oxo-5 beta-cholestanoic acid, and 3 alpha,12 alpha-dihydroxy-7-oxo-5 beta-cholestanoic acid, identified previously in the bile of A. mississippiensis, 3 alpha,7 beta-dihydroxy-5 beta-cholestanoic acid, 3 alpha,7 beta,12 alpha-trihydroxy-5 beta-cholestanoic acid, 7 beta,12 alpha-dihydroxy-3-oxo-5 beta-cholestanoic acid, 3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestanoic acid, 3 alpha,7 alpha,12 alpha,26-tetrahydroxy-5 beta-cholestanoic acid, and 1 beta,3 alpha,7 alpha,12 alpha-tetrahydroxy-5 beta-cholestanoic acid were newly identified. And in addition to the C24 bile acids, such as chenodeoxycholic acid, ursodeoxycholic acid, cholic acid, and allocholic acid, identified previously, deoxycholic acid, 3 alpha,7 alpha-dihydroxy-5 beta-chol-22-enoic acid, 3 alpha,7 alpha,12 alpha-trihydroxy-5 alpha-chol-22-enoic acid, and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-chol-22-enoic acid were newly identified.     

Partial purification and characterization of an NAD-dependent 3 beta-hydroxysteroid dehydrogenase from Clostridium innocuum

In nine strains of Clostridium innocuum, 3 beta-hydroxysteroid-dehydrogenating activities were detected. 3 beta, 7 alpha, 12 alpha-Trihydroxy- and 3 beta-hydroxy-12-keto-5 beta-cholanoic acids were identified as reduction products of the respective 3-keto bile acids by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry. One strain was shown to contain a NAD-dependent 3 beta-hydroxysteroid dehydrogenase. Enzyme production was constitutive in the absence of added bile acids. The specific enzyme activity was significantly reduced by growth medium supplementation with 3-keto bile acids, with trisubstituted acids being more effective than disubstituted ones. A pH optimum of 10.0 to 10.2 was found after partial purification by DEAE-cellulose chromatography. A molecular weight of about 56,000 was established. 3 beta-hydroxysteroid dehydrogenase activity was also found in the membrane fraction after solubilization with Triton X-100, suggesting that the enzyme was originally membrane bound. The enzyme reduced a 3-keto group in unconjugated and conjugated bile acids, lower Km values being demonstrated with disubstituted than with trisubstituted bile acids. Keto functions at C-7 and C-12 further reduced the Km value. The enzyme was found to be partially heat labile (86% inactivation at 50 degrees C for 10 min).