Formation of ursodeoxycholic acid from chenodeoxycholic acid by a 7 beta-hydroxysteroid dehydrogenase-elaborating Eubacterium aerofaciens strain cocultured with 7 alpha-hydroxysteroid dehydrogenase-elaborating organisms

A gram-positive, anaerobic, chain-forming, rod-shaped anaerobe (isolate G20-7) was isolated from normal human feces. This organism was identified by cellular morphology as well as fermentative and biochemical data as Eubacterium aerofaciens. When isolate G20-7 was grown in the presence of Bacteroides fragilis or Escherichia coli (or another 7 alpha-hydroxysteroid dehydrogenase producer) and chenodeoxycholic acid, ursodeoxycholic acid produced. Time course curves revealed that 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid produced by B. fragilis or E. coli or introduced into the medium as a pure substance was reduced by G20-7 specifically to ursodeoxycholic acid. The addition of glycine- and taurine-conjugated primary bile acids (chenodeoxycholic and cholic acids) and other bile acids to binary cultures of B. fragilis and G20-7 revealed that (i) both conjugates were hydrolyzed to give free bile acids, (ii) ursocholic acid (3 alpha, 7 beta, 12 alpha-trihydroxy-5 beta-cholanoic acid) was produced when conjugated (or free) cholic acid was the substrate, and (iii) the epimerization reaction was at least partially reversible. Corroborating these observations, an NADP-dependent 7 beta-hydroxysteroid dehydrogenase (reacting specifically with 7 beta-OH-groups) was demonstrated in cell-free preparations of isolate G20-7; production of the enzyme was optimal at between 12 and 18 h of growth. This enzyme, when measured in the oxidative direction, was active with ursodeoxycholic acid, ursocholic acid, and the taurine conjugate of ursodeoxycholic acid (but not with chenodeoxycholic, deoxycholic, or cholic acids) and displayed an optimal pH range of 9.8 to 10.2     

Formation of ursodeoxycholic acid from chenodeoxycholic acid by a 7 beta-hydroxysteroid dehydrogenase-elaborating Eubacterium aerofaciens strain cocultured with 7 alpha-hydroxysteroid dehydrogenase-elaborating organisms.

A gram-positive, anaerobic, chain-forming, rod-shaped anaerobe (isolate G20-7) was isolated from normal human feces. This organism was identified by cellular morphology as well as fermentative and biochemical data as Eubacterium aerofaciens. When isolate G20-7 was grown in the presence of Bacteroides fragilis or Escherichia coli (or another 7 alpha-hydroxysteroid dehydrogenase producer) and chenodeoxycholic acid, ursodeoxycholic acid produced. Time course curves revealed that 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid produced by B. fragilis or E. coli or introduced into the medium as a pure substance was reduced by G20-7 specifically to ursodeoxycholic acid. The addition of glycine- and taurine-conjugated primary bile acids (chenodeoxycholic and cholic acids) and other bile acids to binary cultures of B. fragilis and G20-7 revealed that (i) both conjugates were hydrolyzed to give free bile acids, (ii) ursocholic acid (3 alpha, 7 beta, 12 alpha-trihydroxy-5 beta-cholanoic acid) was produced when conjugated (or free) cholic acid was the substrate, and (iii) the epimerization reaction was at least partially reversible. Corroborating these observations, an NADP-dependent 7 beta-hydroxysteroid dehydrogenase (reacting specifically with 7 beta-OH-groups) was demonstrated in cell-free preparations of isolate G20-7; production of the enzyme was optimal at between 12 and 18 h of growth. This enzyme, when measured in the oxidative direction, was active with ursodeoxycholic acid, ursocholic acid, and the taurine conjugate of ursodeoxycholic acid (but not with chenodeoxycholic, deoxycholic, or cholic acids) and displayed an optimal pH range of 9.8 to 10.2     

Estimation of ursodeoxycholic acid in human and bear biles using Clostridium absonum 7 beta-hydroxysteroid dehydrogenase

Ursodeoxycholic acid was estimated in bile samples from humans and wild North American black bears using 7 beta-hydroxysteroid dehydrogenase purified from Clostridium absonum by Procion Red affinity chromatography. The percentage ursodeoxycholic acid was calculated by two methods: (a) 7 beta-hydroxyl groups were quantified using 7 beta-hydroxysteroid dehydrogenase and 3 alpha-hydroxyl groups (total bile acids) were quantified using 3 alpha-hydroxysteroid dehydrogenase. The percentage ursodeoxycholic acid was calculated on the basis of [7 beta-hydroxyl groups]/[3 alpha-hydroxyl groups] X 100. (b) Bile was hydrolyzed with sodium hydroxide and subjected to thin-layer chromatography. Bands corresponding to cholic acid, chenodeoxycholic acid plus deoxycholic acid, and ursodeoxycholic acid were identified by the use of standards and Komarowsky's spray reagent. Total bile acids and total ursodeoxycholic acid were measured by elution of silica gel in unsprayed areas corresponding to the bile acid standards and quantification of the total bile acid in each eluate. Direct comparison of these methods validated the use of 7 beta-hydroxysteroid dehydrogenase in the estimation of ursodeoxycholic acid in the biles of black bears and of patients fed ursodeoxycholic acid for cholesterol gallstone dissolution. Relative percentages of ursodeoxycholic acid were 8-24% in four bears and 22 and 27% in the patients ingesting 500 and 750 mg ursodeoxycholic acid per day for 3 months, respectively. Predictably lower values were obtained in two control subjects and one patient ingesting 750 mg chenodeoxycholic acid per day for 3 months.     

Oxidation of primary bile acids by a 7 alpha-hydroxysteroid dehydrogenase elaborating Clostridium bifermentans soil isolate

A gram-positive, rod-shaped anaerobe (strain F-6) was isolated from soil. This organism was identified by cellular morphology as well as fermentative and biochemical data as Clostridium bifermentans. Strain F-6 formed 7-ketolithocholic acid from chenodeoxycholic acid and 7-ketodeoxycholic acid from cholic acid in whole cell cultures, but did not transform deoxycholic acid, ursodeoxycholic acid, or ursocholic acid. This reaction is reversible. The structures of 7-ketolithocholic acid and 7-ketodeoxycholic acid were verified by mass spectroscopy and by thin-layer chromatography using Komarowsky's spray reagent. When incubated with the strain F-6 glycine and taurine conjugates of the primary bile acids were partially hydrolyzed and transformed to 7-keto products. Optimal yields of 7-ketolithocholic acid and 7-ketodeoxycholic acid were obtained after 78 h of incubation. Culture pH changed with time and was characterized by an initial drop (1.1 pH units) and a gradual increase back to the starting pH (7.3). Corroborating these observations, an inducible, NADP-dependent, 7 alpha-hydroxysteroid dehydrogenase was demonstrated in cell extracts of strain F-6. A trace of NAD-dependent 7 alpha-hydroxysteroid dehydrogenase was also found. A substantial increase in the specific activity of the NADP-dependent 7 alpha-hydroxysteroid dehydrogenase was observed when either 7-ketolithocholic acid, chenodeoxycholic acid, or deoxycholic acid was included in the growth medium. Optimal induction of the NADP-dependent 7 alpha-hydroxysteroid dehydrogenase was achieved with 0.3-0.4 mM 7-ketolithocholic acid. Production of the enzyme(s) was optimal at 6-8 h of growth and the 7 alpha-hydroxysteroid dehydrogenases had a pH optimum of approximately 11.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of NAD-dependent 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and of NADP-dependent 7 beta-hydroxysteroid dehydrogenase from Peptostreptococcus productus

A human fecal isolate, characterized by morphological, physiological and biochemical data as a strain of Peptostreptococcus roductus, was shown to contain NAD-dependent 3 alpha- and 3 beta-hydroxysteroid dehydrogenases and a NADP-dependent 7 beta-hydroxysteroid dehydrogenase. All enzyme activities could be demonstrated in crude extracts and in membrane fractions. The 3 alpha- and 3 beta-hydroxysteroid dehydrogenases were synthesized constitutively. Specific enzymatic activities were significantly reduced when bacteria were grown in the presence of 3-keto bile acids, while other bile acids were ineffective. For the 3 alpha (3 beta)-hydroxysteroid dehydrogenase, a pH optimum of 8.5 (9.5) and a molecular weight of 95,000 (132,000) was estimated. 3 alpha- and 3 beta-hydroxysteroid dehydrogenases were heat-sensitive (about 75% inactivation at 50 degrees C for 10 min). The 7 beta-hydroxysteroid dehydrogenase was already present in uninduced cells, but specific activity could be enhanced up to more than 2.5-fold when bacteria were grown in the presence of 7-keto bile acids. Disubstituted bile acids were more effective than trisubstituted ones, ursodeoxycholic acid was ineffective as an inducer. A pH optimum of 10.0 and a molecular weight of about 82,000 were shown for the 7 beta-hydroxysteroid dehydrogenase. The enzyme preparation reduced the 7-keto group of corresponding bile acids. Again the affinities of disubstituted bile acids for the enzyme were higher than those of the trisubstituted bile acids, but no significant differences between conjugated and free bile acids were observed. The 7 beta-hydroxysteroid dehydrogenase was heat-sensitive (72% inactivation at 50 degrees C for 10 min), but was detectable at 4 degrees C for at least 48 h.     

7 alpha-Dehydroxylation of bile acids by resting cells of an unidentified, gram-positive, nonsporeforming anaerobic bacterium

Transformation of bile acids by washed whole cells of strain HD-17, an unidentified gram-positive anaerobic bacterium isolated from human feces, was studied. 7 alpha-Dehydroxylase was produced only during adaptive growth on medium containing 7 alpha-hydroxy bile acids. Both the extent of hydroxylation and the state of conjugation of the bile acids had marked effects on the induction of the enzyme, and the order of the enzyme induction was conjugated cholic acid much greater than cholic acid greater than taurochenodeoxycholic acid greater than or equal to chenodeoxycholic acid. The addition of excess glucose to the growth medium appreciably reduced the enzyme level. The induced enzyme required strict anaerobic conditions for activity and had an optimal pH range of 6.5 to 7.5. In contrast with the induction of the enzyme, the induced enzyme showed a low degree of substrate specificity between cholic acid and chenodeoxycholic acid, with some preference for the former. In addition, the organism contained 3 alpha-, 7 alpha-, and 12 alpha-hydroxysteroid dehydrogenases, and the addition of bile acids to the medium somewhat enhanced the production of the oxidoreductases. The dehydrogenations were obviously stimulated by oxygen as a terminal electron acceptor. The organism also contained bile salt hydrolase.     

7 alpha-Dehydroxylation of bile acids by resting cells of an unidentified, gram-positive, nonsporeforming anaerobic bacterium.

Transformation of bile acids by washed whole cells of strain HD-17, an unidentified gram-positive anaerobic bacterium isolated from human feces, was studied. 7 alpha-Dehydroxylase was produced only during adaptive growth on medium containing 7 alpha-hydroxy bile acids. Both the extent of hydroxylation and the state of conjugation of the bile acids had marked effects on the induction of the enzyme, and the order of the enzyme induction was conjugated cholic acid much greater than cholic acid greater than taurochenodeoxycholic acid greater than or equal to chenodeoxycholic acid. The addition of excess glucose to the growth medium appreciably reduced the enzyme level. The induced enzyme required strict anaerobic conditions for activity and had an optimal pH range of 6.5 to 7.5. In contrast with the induction of the enzyme, the induced enzyme showed a low degree of substrate specificity between cholic acid and chenodeoxycholic acid, with some preference for the former. In addition, the organism contained 3 alpha-, 7 alpha-, and 12 alpha-hydroxysteroid dehydrogenases, and the addition of bile acids to the medium somewhat enhanced the production of the oxidoreductases. The dehydrogenations were obviously stimulated by oxygen as a terminal electron acceptor. The organism also contained bile salt hydrolase.     

Enzymes involved in the formation of 3 beta, 7 beta-dihydroxy-12-oxo-5 beta-cholanic acid from dehydrocholic acid by Ruminococcus sp. obtained from human intestine

Ruminococcus sp. PO1-3 from human intestinal flora reduced dehydrocholic acid to 3 beta-hydroxy-7,12-dioxo-5 beta-cholanic acid by means of the enzyme 3 beta-hydroxysteroid dehydrogenase (Akao, T., Akao, T., Hattori, M., Namba, T. and Kobashi, K. (1986) J. Biochem. (Tokyo) 99, 1425-1431). This bacterium and its crude extract gave rise to another product, showing a lower RF value on TLC, from dehydrocholic acid. The product was identified as 3 beta, 7 beta-dihydroxy-12-oxo-5 beta-cholanic acid. The crude extract reduced 7-ketolithocholic acid and its methyl ester, but not 6-ketolithocholic acid and 12-ketochenodeoxycholic acid, in the presence of NADPH, and oxidized ursodeoxycholic acid and beta-muricholic acid, but not cholic acid, chenodeoxycholic acid, deoxycholic acid and hydrocholic acid, in the presence of NADP+. Therefore, besides 3 beta-hydroxysteroid dehydrogenase, 7 beta-hydroxysteroid dehydrogenase was shown to be present in this bacterium. The two dehydrogenases were clearly separated from each other by butyl-Toyopearl 650 M column chromatography. From dehydrocholic acid, 7 beta-hydroxy-3,12-dioxo-5 beta-cholanic acid was produced by 7 beta-hydroxysteroid dehydrogenase and 3 beta, 7 beta-dihydroxy-12-oxo-5 beta-cholanic acid was produced by combination of two enzymes, 7 beta- and 3 beta-hydroxysteroid dehydrogenase.     

Formation of urso- and ursodeoxy-cholic acids from primary bile acids by a Clostridium limosum soil isolate

A gram-positive, rod-shaped anaerobe (isolate F-14) was isolated from soil. This organism was identified by cellular morphology as well as by fermentative and biochemical data as Clostridium limosum. Isolate F-14 formed ursocholic acid (UC) and 7-ketodeoxycholic acid (7-KDC) from cholic acid (CA), and ursodeoxycholic acid (UDC) and 7-ketolithocholic acid (7-KLC) from chenodeoxycholic acid (CDC) in whole cell cultures, but did not transform deoxycholic acid (DC). No hydrolysis or transformation occurred when either taurine- or glycine-conjugated bile acids were incubated with F-14. The type stain of Clostridium limosum (American Type Culture Collection 25620) did not transform bile acids. The structures of ursocholic, ursodeoxycholic, 7-ketodeoxycholic, and 7-ketolithocholic acids were verified by mass spectroscopy and by thin-layer chromatography using Komarowsky's spray reagent. The organism transformed cholic and chenodeoxycholic acids at concentrations of 20 mM and 1 mM, respectively; higher concentrations of bile acids inhibited growth. Optimal yields of ursocholic and ursodeoxycholic acids were obtained at 9-24 hr of incubation and depended upon the substrate used. Increasing yields of 7-ketodeoxycholic and 7-ketolithocholic acids, and decreasing yields of ursocholic and ursodeoxycholic acids were observed with longer periods of incubation. Culture pH changed with time and was characterized by a small initial drop (0.2-0.4 pH units) and a subsequent increase to a pH (8.1-8.2) that was above the starting pH (7.4).(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial purification and characterization of NAD-dependent 7alpha-hydroxysteroid dehydrogenase from Bacteroides thetaiotaomicron.

A NAD-dependent 7alpha-hydroxysteroid dehydrogenase was purified 18-fold over the activity in crude cell extracts prepared from Bacteroides thetaiotaomicron NCTC 10852 using Bio-Gel A 1.5-M column chromatography. A molecular weight of 320 000 was estimated for the partially purified intact enzyme. Substrate saturation kinetics were performed using the 18-fold purified enzyme and the lowest Km values were obtained for 3alpha,7alpha-dihydroxy bile acid and bile salt substrates including chenodeoxycholic acid (Km 0.048 mM), glycochenodeoxycholic acid (Km 0.083 mM) and taurochenodeoxycholic acid (Km 0.059 mM). In contrast, 3alpha,7alpha,12alpha-trihydroxy bile acid and bile salts had higher Km values, i.e. cholic acid (Km 0.22 mM), glycoholic acid Km 0.32 mM) and taurocholic acid Km 0.26 mM). NAD had a Km value of 0.20 mM. The possible physiological significance of 7alpha-hydroxy bile acid oxidation to intestinal bacteroides strains was accessed by determining the rate of conversion of [14C]-cholic acid to 7-ketodeoxy[14C]cholic acid by whole cell suspensions under different incubation conditions. The rate of biotransformation of bile acid to keto-bile acid incubated anaerobically under N2 gas increased markedly when potential electron acceptors such as fumarate (10 mM) or menadione (4 mM) was added exogenously. These results suggest that bile acid oxidation reactions may be linked to energy-generating systems in this bacterium.     

Bile acid profiles in bile, urine, and feces of a patient with cerebrotendinous xanthomatosis

Bile acid profiles of bile, urine, and feces obtained from a patient with cerebrotendinous xanthomatosis on the same day have been analyzed by gas-liquid chromatography-mass spectrometry after fractionation into groups by mode of conjugation by an ion-exchange chromatography. The predominant biliary bile acid was cholic acid conjugated with glycine and taurine. Lesser amounts of the amino acid conjugates of chenodeoxycholic acid, ursodeoxycholic acid, 7-ketodeoxycholic acid, allocholic acid, and deoxycholic acid, and of unconjugated norcholic acid and allonorcholic acid were also present in the bile. The major fecal bile acid was 7-epicholic acid. Relatively large amounts of bile acids were excreted in the urine. Unconjugated 7-epicholic acid, norcholic acid, allonorcholic acid, and cholic acid predominated. The bile acid profiles of the patient were different from those of normal subjects and should be useful for the diagnosis.     

Effect of bile acid oxazoline derivatives on microorganisms participating in 7 alpha-hydroxyl epimerization of primary bile acids

We tested bile acid oxazoline derivatives of chenodeoxycholic (CDC-OX), 7-ketolithocholic (7-KLC-OX), ursodeoxycholic (UDC-OX), and deoxycholic (DC-OX) as inhibitors of the 7-epimerization of the primary bile acids cholic acid (CA) and CDC in cultures of four species of bacteria and the human fecal flora. The organisms tested elaborate a 7 alpha- and/or 7 beta-hydroxysteroid dehydrogenase (HSDH); they were Escherichia coli (7 alpha-HSDH), Bacteroides fragilis (7 alpha-HSDH), Clostridium absonum (7 alpha- and 7 beta-HSDH) and Eubacterium aerofaciens (7 beta-HSDH). None of the oxazolines affected 7 alpha-OH oxidation of CA or CDC by E. coli or the growth of the organism. All the oxazolines (except UDC-OX) inhibited the growth of B. fragilis and its 7 alpha-HSDH. In contrast, only DC-OX blocked 7 alpha-OH epimerization of CA by C. absonum. Surprisingly, the other three oxazolines enhanced 7 alpha-OH epimerization of CA, but not that of CDC, which was inhibited (CDC-OX greater than 7-KLC-OX much greater than UDC-OX). Enzymic data suggest that CDC-OX in the presence of CA can induce a greater level of both 7 alpha- and 7 beta-HSDH than CA or CDC-OX alone, CDC-OX being more toxic in the presence of CDC. Formation of urso-bile acid from 7-keto substrates by E. aerofaciens is totally blocked by the oxazolines (except UDC-OX). Similarly, suppression of urso-bile acid formation from primary bile acids by the human fecal flora was evident with DC-OX greater than 7-KLC-OX greater than CDC-OX much greater than UDC-OX, the last being ineffective. The inhibitory activity of the oxazolines on the 7-dehydroxylation of primary bile acids by human fecal flora followed the same order.     

NADP-dependent 3 beta-, 7 alpha- and 7 beta-hydroxysteroid dehydrogenase activities from a lecithinase-lipase-negative Clostridium species 25.11.c

A lecithinase-lipase-negative Clostridium sp. 25.11.c., not fitting in any of the species of Clostridia described so far as judged by morphological, physiological, and biochemical data, was shown to contain NADP-dependent 3 beta-, 7 alpha- and 7 beta-hydroxysteroid dehydrogenases. The three hydroxysteroid dehydrogenases could be demonstrated in the supernatant and in the membrane fraction after solubilization with Triton X-100, suggesting enzymes which were originally membrane bound. The 3 beta-hydroxysteroid dehydrogenase was synthesized constitutively, and the specific enzyme activity was significantly reduced by growth medium supplementation with 3-keto bile acids and trisubstituted bile acids. A pH optimum of 7.5 and a molecular weight of approx. 104,000 were estimated by molecular sieve chromatography. The enzyme reduced the 3-keto group of bile acids; an oxidation of a 3 beta-hydroxyl function could not be demonstrated. The lowest Km values were found for disubstituted bile acids, trisubstituted and conjugated bile acids having higher Km values. 7 alpha-Hydroxysteroid dehydrogenase, but not 7 beta-hydroxysteroid dehydrogenase, was already present in uninduced cells. The specific activities, however, were greatly enhanced when cells were grown in the presence of chenodeoxycholic acid or 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid. Ursodeoxycholic acid with its 7 beta-hydroxyl group was ineffective as an inducer. Molecular weights of approx. 82,000 and 115,000 were found for the 7 alpha-hydroxysteroid dehydrogenase and the 7 beta-hydroxysteroid dehydrogenase, respectively. In contrast to the in vivo situation, the reaction could only be demonstrated in the reductive direction in vitro. Here, the pH optimum for the overall reaction was 8.5-8.7. 3 beta-, 7 alpha- and 7 beta-hydroxysteroid dehydrogenase activities were readily demonstrated for at least 48 h when preparations were stored at 4 degrees C, but were found to be heat-sensitive.     

Synthesis of alpha,beta-unsaturated C24 bile acids

Synthesis of the alpha,beta-unsaturated analogues of cholic acid, deoxycholic acid, chenodeoxycholic acid, and ursodeoxycholic acid is described. Each common bile acid was converted to the corresponding C22 aldehyde which was then converted to the delta 22 bile acid by Wittig reaction with methyl (triphenylphosphoranylidene)acetate. The synthetic unsaturated bile acids were characterized by thin-layer chromatography, gas-liquid chromatography, and mass spectrometry.     

Bile salts of the green turtle Chelonia mydas (L.)

1. Bile salts of the green turtle Chelonia mydas (L.) were analysed as completely as possible. 2. They consist of taurine conjugates of 3 alpha, 7 alpha, 12 alpha, 22 xi-tetrahydroxy-5 beta-cholestan-26-oic acid (tetrahydroxysterocholanic acid) and 3 alpha 12 alpha, 22 xi-trihydroxy-5 beta-cholestan-26-oic acid, with minor amounts of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5beta-cholan-24-oic acid (cholic acid), 3alpha, 12 alpha-dihydroxy-5beta-cholan-24-oic acid (deoxycholic acid) and possibly other bile acids. 3. Cholic acid and deoxycholic acid represent the first known examples of bile acids common to chelonians and other animal forms: they may indicate independent evolution in chelonians to C24 bile acids. 4. The discovery of a 7-deoxy C27 bile acid is the first evidence that C27 bile acids or their conjugates have an enterohepatic circulation.     

Aminopeptidase M from human liver. II. Kinetic analysis of inhibition of the enzyme by bile acids

The mechanism of inhibition of aminopeptidase M by bile acids was analyzed by application of the specific velocity plot that was introduced by Baici [Eur. J. Biochem. 119, 9-14 (1981)]. Kinetic studies with three bile acids (cholic acid, deoxycholic acid, and chenodeoxycholic acid) and three substrates (Leu-Met, Leu-Gly, and Leu-pNA) showed that the inhibition constants Ki for the bile acids were appreciably different from each other, but that the Ki for each was not affected by the substrates used, being 0.89-1.03 mM for cholic acid, 0.42-0.66 mM for deoxycholic acid, and 0.24-0.31 mM for chenodeoxycholic acid. The values of the kinetic coefficient alpha [(apparent Ks in the presence of inhibitor)/Ks] for cholic acid with Leu-Met and Leu-Gly were 9.0 and 2.5, respectively. These values were very similar to those for chenodeoxycholic acid (7.0 and 2.7) but smaller than those for deoxycholic acid (21 and 11). The values of the other kinetic coefficient beta [(apparent kp in the presence of inhibitor)/kp] were 0 except in the case of the combinations of Leu-Gly with cholic acid (0.33) and Leu-Gly with chenodeoxycholic acid (0.13). On the basis of these kinetic parameters, the inhibitions by bile acids were classified into 4 types: competitive-noncompetitive linear mixed type (1 less than alpha less than infinity, beta = 0), noncompetitive-uncompetitive linear mixed type (0 less than alpha less than 1, beta = 0), pure noncompetitive type (alpha = 1, beta = 0), and hyperbolic mixed type (1 less than alpha less than infinity, 0 less than beta less than 1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Bile acids and bile alcohols in a child with hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency: effects of chenodeoxycholic acid treatment

Duodenal bile, urine, plasma, and feces from a child with hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency were analyzed by fast atom bombardment mass spectrometry and gas chromatography-mass spectrometry to investigate the formation and excretion of abnormal bile acids and bile alcohols. The biliary bile salts consisted of glycocholic acid (25%) and of sulfated and glycine conjugated di- and trihydroxycholenoic acids (55%), two C27 bile acids, and eleven sulfated bile alcohols (mainly tetrols, 20%), all having 3 beta,7 alpha-dihydroxy-delta 5 or 3 beta,7 alpha,12 alpha-trihydroxy-delta 5 ring structures. In plasma, sulfated cholenoic acids constituted 65% and unconjugated 3 beta,7 alpha-dihydroxy-5-cholestenoic acid 25% of the total level, 71 micrograms/ml. The urinary excretion of the former was 30.4 mg/day and that of unsaturated bile alcohol sulfates, mainly pentols, 7 mg/day. The predominant bile acid in feces was an unconjugated epimer of 3 beta,7 alpha,12 alpha-trihydroxy-5-cholenoic acid, and small amounts of cholic acid were present. The minimum total excretion was 11.3 mg/day. Treatment with chenodeoxycholic acid resulted in marked clinical improvement and normalized liver function tests. Further studies are needed to define the mechanism of action. Plasma bile acids decreased to 1.6 micrograms/ml and urinary excretion to 3.4 mg/day. Chenodeoxycholic and ursodeoxycholic acids became predominant in all samples. The fecal excretion of unsaturated cholenoic acid sulfates increased to 40 mg/day compared to 89 mg/day of saturated bile acids. The results provide further support for a defective hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency, and indicate that the 3 beta-hydroxy-delta 5 bile acids are formed via 7 alpha-hydroxycholesterol. The formation of glycocholic acid may be due to an incomplete enzyme defect or to transformation of the 3 beta-hydroxy-delta 5 structure by bacterial and hepatic enzymes during an enterohepatic circulation.     

Lyophilized Clostridium perfringens 3 alpha- and Clostridium bifermentans 7 alpha-hydroxysteroid dehydrogenases: two new stable enzyme preparations for routine bile acid analysis

Preparations of 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) from Clostridium perfringens were successfully lyophilized into a stable powder form. Purification of the enzyme was achieved using triazine dye affinity chromatography. C. perfringens 3 alpha-hydroxysteroid dehydrogenase was purified 24-fold using Reactive Red 120 (Procion Red) -cross-linked agarose (70% yield). Quantitative measurement of bile acids with the purified enzymes, 3 alpha-hydroxysteroid dehydrogenase and 7 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.159) from Clostridium bifermentans (strain F-6), was achieved spectrophotometrically. Standard curves with chenodeoxycholic acid (CDC) and cholic acid were linear within a concentration range of 20-100 microM. Analysis of mixtures of ursodeoxycholic acid and CDC showed the additive nature of the 3 alpha-hydroxysteroid dehydrogenase and showed also that 7 alpha-hydroxyl groups were independently quantified by the 7 alpha-hydroxysteroid dehydrogenase. Bile acids in Folch extracts of human bile samples were measured using purified preparations of Pseudomonas testosteroni 3 alpha-hydroxysteroid dehydrogenase, C. perfringens 3 alpha-hydroxysteroid dehydrogenase, Escherichia coli 7 alpha-hydroxysteroid dehydrogenase and C. bifermentans (strain F-6) 7 alpha-hydroxysteroid dehydrogenase. Statistical comparison validated the use of C. perfringens 3 alpha- and C. bifermentans 7 alpha-hydroxysteroid dehydrogenases for the quantification of bile acids in bile.     

Mechanism of intestinal 7 alpha-dehydroxylation of cholic acid: evidence that allo-deoxycholic acid is an inducible side-product

We previously reported that the 7 alpha-dehydroxylation of cholic acid appears to be carried out by a multi-step pathway in intestinal anaerobic bacteria both in vitro and in vivo. The pathway is hypothesized to involve an initial oxidation of the 3 alpha-hydroxy group and the introduction of a double bond at C4-C5 generating a 3-oxo-4-cholenoic bile acid intermediate. The loss of water generates a 3-oxo-4,6-choldienoic bile acid which is reduced (three steps) yielding deoxycholic acid. We synthesized, in radiolabel, the following putative bile acid intermediates of this pathway 7 alpha,12 alpha-dihydroxy-3-oxo-4-cholenoic acid, 7 alpha,12 alpha-dihydroxy-3-oxo-5 beta-cholanoic acid, 12 alpha-dihydroxy-3-oxo-4,6-choldienoic acid, and 12 alpha-hydroxy-3-oxo-4-cholenoic acid and showed that they could be converted to 3 alpha,12 alpha-dihydroxy-5 beta-cholanoic acid (deoxycholic acid) by whole cells or cell extracts of Eubacterium sp. VPI 12708. During studies of this pathway, we discovered the accumulation of two unidentified bile acid intermediates formed from cholic acid. These bile acids were purified by thin-layer chromatography and identified by gas-liquid chromatography-mass spectrometry as 12 alpha-hydroxy-3-oxo-5 alpha-cholanoic acid and 3 alpha,12 alpha-dihydroxy-5 alpha-cholanoic (allo-deoxycholic acid). Allo-deoxycholic acid was formed only in cell extracts prepared from bacteria induced by cholic acid, suggesting that their formation may be a branch of the cholic acid 7 alpha-dehydroxylation pathway in this bacterium.     

Effects of bile acids on rat hepatic microsomal type I 11β-hydroxysteroid dehydrogenase

The aim of this study was to investigate the effect of various bile acids on hepatic type I 11β-hydroxysteroid dehydrogenase (11β-HSD1) activity in vitro. The rat liver microsome fraction was prepared and 11β-HSD1 activity was assayed using cortisol and corticosterone as substrates for the enzyme reaction. The substrate and various concentrations of bile acids were added to the assay mixture. After incubation, the products were extracted and analyzed using high-performance liquid chromatography. All bile acids tested except deoxycholic acid and 7-keto bile acids inhibited the 11β-HSD1 enzyme reaction to some degree. Ursodeoxycholic acid inhibited the activity less than cholic, chenodeoxycholic, and lithocholic acids. Deoxycholic acid and 7-keto bile acids did not inhibit, but enhanced the enzyme activity. Inhibitions of dehydrogenation by corticosterone were weaker than those by cortisol. Kinetic analysis revealed that the inhibition of 11β-HSD1 was competitive. The inhibition of 11β-HSD1 by bile acids depended on the three-dimensional structural difference in the steroid rings and the presence of the 7α-hydroxy molecule of the bile acids was important for the inhibition of rat hepatic 11β-HSD1 enzyme activity. These results suggest that bile acid administration might modulate 11β-HSD1 enzyme activity.