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 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)     

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.     

7alpha-Dehydroxylation of cholic acid and chenodeoxycholic acid by Clostridium leptum.

The rate of 7alpha-dehydroxylation of primary bile acids was quantitatively measured radiochromatographically in anaerobically washed whole cell suspensions of Clostridium leptum. The pH optimum for the 7alpha-dehydroxylation of both cholic and chenodeoxycholic acid was 6.5-7.0. Substrate saturation curves were observed for the 7alpha-dehydroxylation of cholic and chenodeoxycholic acid. However, cholic acid whole cell K0.5 (0.37 micron) and V (0.20 mumol hr-1mg protein-1) values differed significantly from chenodeoxycholic acid whole cell K0.5 (0.18 micron) and V (0.50 mumol-1 hr-1 mg protein-1). 7alpha-Dehydroxylation activity was not detected using glycine and taurine-conjugated primary bile acids, ursodeoxycholic acid, cholic acid methyl ester, or hyocholic acid as substrates. Substrate competition experiments showed that cholic acid 7 alpha-dehydroxylation was reduced by increasing concentrations of chendeoxycholic acid; however, chenodeoxycholic acid 7alpha-dehydroxylation activity was unaffected by increasing concentrations of cholic acid. A 10-fold increase in cholic and 7alpha-dehydroxylation activity occurred during the transition from logarithmic to stationary phase growth whether cells were cultured in the presence or absence of sodium cholate. In the same culture, a similar increase in chenodeoxycholic acid 7alpha-dehydroxylation was detected only in cells cultured in the presence of sodium cholate. These results indicate the possible existence of two independent systems for 7alpha-dehydroxylation in C. Leptum.     

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.     

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)     

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.     

Ursodeoxycholic acid, chenodeoxycholic acid, and 7-ketolithocholic acid are primary bile acids of the guinea pig

Guinea pig gallbladder bile contains chenodeoxycholic acid (62 +/- 5%), ursodeoxycholic acid (8 +/- 5%), and 7-ketolithocholic acid (30 +/- 5%). All three bile acids became labeled to the same specific activity within 30 min after [3H]cholesterol was injected into bile fistula guinea pigs. When a mixture of [3H]ursodeoxycholic acid and [14C]chenodeoxycholic acid was infused into another bile fistula guinea pig, little 3H could be detected in either chenodeoxycholic acid or 7-ketolithocholic acid. But, 14C was efficiently incorporated into ursodeoxycholic and 7-ketolithocholic acids. Monohydroxylated bile acids make up 51% and ursodeoxycholic acid 38% of fecal bile acids. After 3 weeks of antibiotic therapy, lithocholic acid was reduced to 6% of the total, but ursodeoxycholic acid (5-11%) and 7-ketolithocholic (15-21%) acid persisted in bile. Lathosterol constituted 19% of skin sterols and was detected in the feces of an antibiotic-fed animal. After one bile fistula guinea pig suffered a partial biliary obstruction, ursodeoxycholic and 7-ketolithocholic acids increased to 46% and 22% of total bile acids, respectively. These results demonstrate that chenodeoxycholic acid, ursodeoxycholic acid, and 7-ketolithocholic acid can all be made in the liver of the guinea pig.     

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)     

7-Methyl bile acids: 7 beta-methyl-cholic acid inhibits bacterial 7-dehydroxylation of cholic acid and chenodeoxycholic acid in the hamster

The effect of dietary 7 beta-methyl-cholic acid [0.075% in rodent chow (6.4 mg/animal per day)] on cholesterol and bile acid metabolism was studied and compared with that of cholic acid in the hamster. Following oral administration of 7 beta-methyl-cholic acid for 3 weeks, the glycine-conjugated bile acid analog became a major constituent of gallbladder bile. Biliary cholic acid concentration decreased significantly, while that of chenodeoxycholic acid remained unchanged. Serum and liver cholesterol levels were increased by dietary 7 beta-methyl-cholic acid and by cholic acid. Hepatic microsomal HMG-CoA reductase activity was inhibited (30% of the control value) by both bile acids; cholesterol 7 alpha-hydroxylase activity was not affected. In chow controls and cholic acid-fed animals, bacterial 7-dehydroxylation of [14C]chenodeoxycholic acid and [14C]cholic acid was nearly complete. In contrast, dietary 7 beta-methyl-cholic acid effectively prevented the 7-dehydroxylation of the two primary bile acids. These results show that dietary 7 beta-methyl-cholic acid is preserved in the enterohepatic circulation and has an effect on serum and liver cholesterol concentrations similar to those produced by the naturally occurring cholic acid. 7 beta-Methyl-cholic acid is an efficient inhibitor of the bacterial 7-dehydroxylation of the primary bile acids in the hamster.     

Characterization of serum and urinary bile acids in patients with primary biliary cirrhosis by gas-liquid chromatography-mass spectrometry: effect of ursodeoxycholic acid treatment

We have studied the effect of ursodeoxycholic acid on the serum and urinary bile acids in seven patients with moderate to severe primary biliary cirrhosis. Bile acids were characterized by gas-liquid chromatography-mass spectrometry and quantified by capillary gas-liquid chromatography. Serum bile acids were elevated 26-fold over control values, with 2.2 times more cholic acid than chenodeoxycholic acid. Urinary bile acid output was elevated 22-fold over control values with a cholic acid:chenodeoxycholic acid ratio of 1.6. In addition, lithocholic acid, deoxycholic acid, ursodeoxycholic acid, 1 beta-hydroxycholic acid, 1 beta-hydroxydeoxycholic acid, and hyocholic acid were identified in both serum and urine; the proportions of the 1- and 6-hydroxylated bile acids were much higher in urine than in serum of the patients (32.1% versus 4.2%). Three months of placebo administration did not change the serum and urinary bile acid composition. In contrast, ursodeoxycholic acid feeding (12-15 mg/kg body weight per day) for 6 months resulted in a 25% decline in the total serum bile acid concentration from the pretreatment values. The proportion of ursodeoxycholic acid increased from 2.1 to 41.2% of total bile acids, so that total fasting serum endogenous bile acid levels decreased 62.4%. Ursodeoxycholic acid feeding substantially increased urinary bile acid output, with ursodeoxycholic acid comprising 58.1%. The proportion of 1- and 6- hydroxylated endogenous bile acids was reduced by 45.5% from pretreatment levels and approximately 4.5% of the urinary bile acids were omega-muricholic acid, 1 beta-hydroxyursodeoxycholic acid, and 21-hydroxyursodeoxycholic acid. These results demonstrate significant changes in the serum and urinary bile acid pattern in primary biliary cirrhosis during ursodeoxycholic acid treatment. The beneficial effect of ursodeoxycholic acid may be due to reduction of the hydroxylated derivatives of endogenous bile acids together with the appearance of hydroxylated derivatives of ursodeoxycholic acid or it may be due to displacement of the more hydrophobic endogenous bile acids by the hydrophilic ursodeoxycholic acid.     

Feeding natural hydrophilic bile acids inhibits intestinal cholesterol absorption: studies in the gallstone-susceptible mouse

We explored the influence of the hydrophilic-hydrophobic balance of a series of natural bile acids on cholesterol absorption in the mouse. Male C57L/J mice were fed standard chow or chow supplemented with 0.5% cholic; chenodeoxycholic; deoxycholic; dehydrocholic; hyocholic; hyodeoxycholic; alpha-, beta-, or omega-muricholic; ursocholic; or ursodeoxycholic acids for 7 days. Biliary bile salts were measured by reverse-phase HPLC, and hydrophobicity indices were estimated by Heuman's method. Cholesterol absorption efficiency was determined by a plasma dual-isotope ratio method. In mice fed chow, natural proportions of tauro-beta-muricholate (42 +/- 6%) and taurocholate (50 +/- 7%) with a hydrophobicity index of -0.35 +/- 0.04 produced cholesterol absorption of 37 +/- 5%. Because bacterial and especially hepatic biotransformations of specific bile acids occurred, hydrophobicity indices of the resultant bile salt pools differed from fed bile acids. We observed a significant positive correlation between hydrophobicity indices of the bile salt pool and percent cholesterol absorption. The principal mechanism whereby hydrophilic bile acids inhibit cholesterol absorption appears to be diminution of intraluminal micellar cholesterol solubilization. Gene expression of intestinal sterol efflux transporters Abcg5 and Abcg8 was upregulated by feeding cholic acid but not by hydrophilic beta-muricholic acid nor by hydrophobic deoxycholic acid. We conclude that the hydrophobicity of the bile salt pool predicts the effects of individual fed bile acids on intestinal cholesterol absorption. Natural alpha- and beta-muricholic acids are the most powerful inhibitors of cholesterol absorption in mice and might act as potent cholesterol-lowering agents for prevention of cholesterol deposition diseases in humans.     

Bile acid synthesis in cell culture

Confluent cultures of Hep G2 cells were found to synthesize chenodeoxycholic and cholic acids continually. Chenodeoxycholic acid was synthesized at the rate of 58 +/- 8.6 micrograms/96 h, a rate more than 7-fold greater than that for cholic acid. Addition of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol but not the -3 alpha, 7 alpha-diol was followed by an increase in cholic acid synthesis, thus indicating a relatively low 12 alpha-hydroxylase activity. Endogenous synthesis of monohydroxy bile acid ester sulfates was found, with maximum rates of 135 and 74 micrograms/96 h for lithocholic and 3 alpha-hydroxy-5-cholenoic acids, respectively. Incubation of Hep G2 cells in medium containing 25% D2O permitted a comparison of the precursor/product relationship of cholesterol with 3 beta-hydroxy-5-cholenoic acid. The pattern of incorporation of deuterium was in accordance with that expected, thus allowing the conclusion that this monohydroxy bile acid is derived from cholesterol and should be considered together with chenodeoxycholic and cholic acids as a primary bile acid.     

Clostridium scindens baiCD and baiH genes encode stereo-specific 7alpha/7beta-hydroxy-3-oxo-delta4-cholenoic acid oxidoreductases

Secondary bile acids, formed by intestinal bacteria, are suggested to play a significant role in cancers of the gastrointestinal tract in humans. Bile acid 7alpha/beta-dehydroxylation is carried out by a few species of intestinal clostridia which harbor a multi-gene bile acid inducible (bai) operon. Several genes encoding enzymes in this pathway have been cloned and characterized. However, no gene product(s) has yet been assigned to the production of 3-oxo-Delta4-cholenoic acid intermediates of cholic acid (CA), chenodeoxycholic acid (CDCA) or ursodeoxycholic acid (UDCA). We previously reported that the baiH gene encodes an NADH:flavin oxidoreductase (NADH:FOR); however, the role of this protein in bile acid 7-dehydroxylation is unclear. Homology searches and secondary structural alignments suggest this protein to be similar to flavoproteins which reduce alpha/beta-unsaturated carbonyl compounds. The baiH gene product was expressed in Escherichia coli, purified and discovered to be a stereo-specific NAD(H)-dependent 7beta-hydroxy-3-oxo-Delta4-cholenoic acid oxidoreductase. Additionally, high sequence similarity between the baiH and baiCD gene products suggests the baiCD gene may encode a 3-oxo-Delta4-cholenoic acid oxidoreductase specific for CDCA and CA. We tested this hypothesis using cell extracts prepared from E. coli overexpressing the baiCD gene and discovered that it encodes a stereo-specific NAD(H)-dependent 7alpha-hydroxy-3-oxo-Delta4-cholenoic acid oxidoreductase.     

Difference between cholic acid and chenodeoxycholic acid in dependence upon cholesterol of hepatic and plasmatic sources as the precursor in rats

Some difference in functional pool of cholesterol acting as the precursor of bile acids is pointed out between cholic acid and chenodeoxycholic acid. In order to elucidate this problem further, some experiments were performed with rats equilibrated with [7(n)-3H, 4-(14)C] cholesterol by subcutaneous implantation. The bile duct was cannulated in one series of experiments and ligated in another. After the operation 14C-specific radioactivity of serum cholesterol fell, but reached practically a new equilibrium within three days. 14C-Specific radioactivity of serum cholesterol as well as of biliary bile acids in bile-fistula rats and urinary bile acids in bile duct-ligated rats was determined during a three days-period in the new equilibrated state. The results were as follows: (1) 14C-Specific radioactivity of cholic acid and chenodeoxycholic acid in bile was lower than that of serum cholesterol, and 14C-specific radioactivity of cholic acid was clearly lower than that of chenodeoxycholic acid. (2) 14C-Specific radioactivity of cholic acid and beta-muricholic acid in urine was lower than that of serum cholesterol, and 14C-specific radioactivity of cholic acid was lower than that of beta-muricholic acid. (3) Biliary as well as urinary beta-muricholic acid lost tritium label at 7-position entirely during the course of formation from [7(n)-3H, 4-(14)C]cholesterol.     

7alpha- and 12alpha-Hydroxysteroid dehydrogenases from Acinetobacter calcoaceticus lwoffii: a new integrated chemo-enzymatic route to ursodeoxycholic acid

We report the very efficient biotransformation of cholic acid to 7-keto- and 7,12-diketocholic acids with Acinetobacter calcoaceticus lwoffii. The enzymes responsible of the biotransformation (i.e. 7alpha- and 12alpha-hydroxysteroid dehydrogenases) are partially purified and employed in a new chemo-enzymatic synthesis of ursodeoxycholic acid starting from cholic acid. The first step is the 12alpha-HSDH-mediated total oxidation of sodium cholate followed by the Wolf-Kishner reduction of the carbonyl group to chenodeoxycholic acid. This acid is then quantitatively oxidized with 7alpha-HSDH to 7-ketochenodeoxycholic acid, that was chemically reduced to ursodeoxycholic acid (70% overall yield).     

Evidence for bile acid glucosides as normal constituents in human urine

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

Calcium binding by bile acids: in vitro studies using a calcium ion electrode

In this study, we compared in vitro calcium binding by the taurine and glycine conjugates of the major bile acids in human bile: cholic (CA), chenodeoxycholic (CDCA) and deoxycholic (DCA) acids, together with the cholelitholytic bile acids ursodeoxycholic (UDCA) and ursocholic (UCA) acids. At physiological total calcium (CaTOT) (1-15 mM) and bile acid (BA) (10-50 mM) concentrations, all the bile acids caused concentration-dependent falls in [Ca2+], suggesting calcium binding. Except for glycine-conjugated CDCA, all the other calcium-bile acid complexes were soluble in 150 mM NaCl. The calcium binding affinities followed the pattern: dihydroxy (CDCA, UDCA and DCA) greater than trihydroxy (CA and UCA) bile acids, and glycine conjugates greater than taurine conjugates. The glycine conjugate of UDCA, which increases during UDCA treatment, had the highest calcium binding affinity. Ten-20 mM phospholipid modestly increased calcium binding by CA conjugates, but not by CDCA, UDCA, and DCA conjugates. Phospholipid also prevented the precipitation of glyco-CDCA in the presence of calcium. Bile acid-calcium biding was pH-independent over the range 6.5-8.5. The different calcium binding affinities of the major biliary bile acids may partly explain their varying effects on biliary calcium secretion. The results also suggest that neither precipitation of calcium-bile acid complexes nor impaired calcium binding by bile acids is important in the pathogenesis of human calcium gallstone formation.     

Formation of ursodeoxycholic acid from chenodeoxycholic acid in the human colon: studies of the role of 7-ketolithocholic acid as an intermediate

The formation of ursodeoxycholic acid from chenodeoxycholic acid and the role of 7-ketolithocholic acid as an intermediate in this biotransformation were studied in vitro in fecal incubations as well as in vivo in the human colon. [24-14C]-Labeled 7-ketolithocholic and chenodeoxycholic acids were studied at various concentrations, and the biotransformation products were analyzed by thin-layer chromatography, gas-liquid chromatography, and mass spectrometry. There was rapid colonic conversion of 7-ketolithocholic acid to ursodeoxycholic acid and, to a lesser extent, to chenodeoxycholic acid. The reduction of 7-ketolithocholic to ursodeoxycholic acid proceeded significantly faster anaerobically and at acid pH than under aerobic and alkaline conditions. When chenodeoxycholic acid was incubated in vitro or instilled into the colon, various amounts of 7-ketolithocholic and ursodeoxycholic acids were formed. The formation of 7-ketolithocholic acid was favored by alkaline conditions. Isotope dilution studies, in which trace amounts of labeled 7-ketolithocholic acid were incubated with unlabeled chenodeoxycholic acid, indicate 7-ketolithocholic acid to be the major intermediate in the intestinal bacterial conversion of chenodeoxycholic to ursodeoxycholic acid.     

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.