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)     

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)     

NAD- and NADP-dependent 7alpha-hydroxysteroid dehydrogenases from bacteroides fragilis.

Twenty strains of Bacteroides fragilis were screened for hydroxysteroid oxidoreductase activity in cell-free preparations. Eighteen strains were shown to contain NAD-dependent 7alpha-hydroxysteroid dehydrogenase. Sixteen of the strains containing the NAD-dependent enzyme also contained NADP-depedent 7alpha-hydroxysteroid dehydrogenase, but invariably in lesser amounts. A strain particulary rich in both 7alpha-hydroxysteroid dehydrogenase activities was selected for further study. Measurement of activity as a function of pH revealed a fairly sharp optimal activity range of 9.5--10.0 for the NAD-dependent enzyme and a broad flat optimal range of 7.0--9.0 for the NADP-dependent enzyme. Michaelis constants for trihydroxy-bile acids for the NAD-dependent enzyme were in the range of 0.32--0.34 mM, whereas dihydroxy-bile acids gave a Km of 0.1 mM. Thin-layer chromatography studies on the oxidation product of 3alpha, 7alpha-dihydroxy-5beta-cholanoic acid (chenodeoxycholic acid) by the dehydrogenase revealed a band corresponding to that of synthetic 3alpha-hydroxy, 7-keto-5beta-cholanoic acid. Similarly the oxidation product of chenodeoxycholic acid by both 7alpha-hydroxysteroid dehydrogenase and commercially available 3alpha-hy-droxysteroid dehydrogenase revealed a band corresponding to that of synthetic 3,7-diketo-5beta-cholanoic acid. Neither of these two oxidation products could be distinguished from those by the Escherichia coli dehydrogenase oxidation previously reported. Disc-gel electrophoresis of a cell-free lyophilized preparation indicated one active band for NAD-dependent activity of mobility similar to that for the NADP-dependent E. coli enzyme. The NADP-dependent dehydrogenase was unstable and rapidly lost activity after polyacylamide disc-gel electrophoresis, ultracentrifugation, freezing on refrigeration at 4 degrees C. No 3 alpha- or 12alpha-oriented oxidoreductase activity was demonstrated in any of the strains examined.     

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.     

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     

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 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.     

3alpha-, 7alpha- and 12alpha-hydroxysteroid dehydrogenase activities from Clostridium perfringens.

25 strains of Clostridium perfringens were screened for hydroxysteroid dehydrogenase activity; 19 contained NADP-dependent 3alpha-hydroxysteroid dehydrogenase and eight contained NAD-dependent 12alpha-hydroxysteroid dehydrogenase active against conjugated and unconjugated bile salts. All strains containing 12alpha-hydroxysteroid dehydrogenase also contained 3alpha-hydroxysteroid dehydrogenase although 12alpha-hydroxysteroid dehydrogenase was invariably in lesser quantity than the 3alpha-hydroxysteroid dehydrogenase. In addition, 7alpha-hydroxysteroid dehydrogenase activity was evident only when 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholanoate was substrate but notably absent when 3alpha, 7alpha-dihydroxy-5beta-cholanoate was substrate. The oxidation product 12alpha-hydroxy-3, 7-diketo-5beta-cholanoate is rapidly further degraded to an unknown compound devoid of either 3alpha- or 7alpha-OH groups. Group specificity of these enzymes was confirmed by thin-layer chromatography studies of the oxidation products. These enzyme systems appear to be constitutive rather than inducible. In contrast to C. perfringens. Clostridium paraputrificum (five strains tested) contained no measurable hydroxysteroid dehydrogenase activity. pH studies of the C. perfringens enzymes revealed a sharp pH optimum at pH 11.3 and 10.5 for the 3alpha-OH- and 12alpha-OH-oriented activities, respectively. Kinetic studies gave Km estimates of approx. 5 X 10(-5) and 8 X 10(-4) M with 3alpha, 7a-dihydroxy-5beta-cholanoate and 3alpha, 12alpha-dihydroxy-5beta-cholanoate as substrates for two respective enzymes. 3alpha-hydroxysteroid dehydrogenase was active against 3alpha-OH-containing steroids such as androsterone regardless of the sterochemistry of the 5H (Both A/B cis and A/B trans steroides were substrates). There was no activity against 3beta-OH-containing steroids. The 3alpha- and 12alpha-hydroxysteroid dehydrogenase activities, although differing in cofactor requirements cannot be distinguished by their appearance in the growth curve, their mobility on disc gel electrophoresis, elution volume on passage through Sephadex G-200 or heat inactivation studies.     

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.     

Sex differences in gallbladder bile acid composition and hepatic steroid 12 alpha-hydroxylase activity in hamsters

The gallbladder bile acid composition and the activity of the hepatic steroid 12 alpha-hydroxylase were determined in male and female hamsters. Cholic acid, chenodeoxycholic acid, and deoxycholic acid were the major bile acids in both sexes; in addition, 7-ketodeoxycholic acid and lithocholic acid were present. A sex-linked difference in the ratio of cholic acid (plus its metabolites) to chenodeoxycholic acid (plus its metabolite) was observed. The ratio was 1.93 +/- 0.39 in males and 2.74 +/- 0.54 in females. Another sex-linked difference was found in the activity of the 12 alpha-hydroxylase. The extent of the 12 alpha-hydroxylation of 7 alpha-hydroxycholest-4-en-3-one to yield 7 alpha, 12 alpha-dihydroxycholest-4-en-3-one was about two times greater in the microsomal suspension obtained from the liver of female hamsters than in that of male hamsters. A positive correlation between the 12 alpha-hydroxylase activity and the ratio of cholic acid/chenodeoxycholic acid was also observed. These results strongly support the proposal that the activity of the 12 alpha-hydroxylase is the major factor in determining the relative proportion of cholic acid and chenodeoxycholic acid formed from cholesterol in the liver.     

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.     

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.     

Reduction of 7-ketolithocholic acid to chenodeoxycholic acid by rat liver preparations in vitro

The formation of chenodeoxycholic acid via 7-ketolithocholic acid by rat liver preparations was examined in vitro. Results showed that a rat liver preparation reduced 7-ketolithocholic acid mainly to chenodeoxycholic acid and to ursodeoxycholic acid in a smaller amount, and that the reductase required NADPH but not NADH as coenzyme and was mainly localized in the microsomes.     

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.     

Enzyme histochemical observations on the segmentation of the proximal tubules in the kidney of the female rat.

The segmentation of the proximal tubules in the kidney of the female rat was studied by means of enzyme histochemical reactions and the results compared with those observed in male and recently described by Jacobsen and J0rgensen (1973 a). Reactions were performed for the following soluble, coezyme-dependent oxido-reductases: glucose 6-phosphate dehydrogenase, alpha-glycerophosphate dehydrogenase, 3 alpha-hydroxysteroid dehydrogenase, NAD-as well as NADP-dependent isocitrate dehydrogenases, NAD-dependent malate dehydrogenase, NADP-dependent, decarboxylating malate dehydrogenase, uridine diphosphate glucose dehydrogenase. Measures were taken to reduce enzyme diffusion and eliminate interference from tissue tetrazolium reductases. Furthermore, reactions were performed for a number of less soluble or insoluble enzymes: glucose 6-phosphatase, mitochondrial alpha-glycerophosphate dehydrogenase, beta-hydroxybutyrate dehydrogenase, succinate dehydrogenase and tetrazolium reductases. In the proximal tubules of the female rat all enzymes studied--except beta-hydroxybutyrate dehydrogenase--showed segmental differences, most of them clearly revealing three segments. Sex differences were found concerning all enzymes except uridine diphosphate glucose dehydrogenase and NADP-dependent isocitrate dehydrogenase. The most pronounced sex-related differences were seen in the third segment in which part the male rat showed highest activity in respect to tetrazolium reductases, NAD-dependent isocitrate dehydrogenase, succinate dehydrogenase, beta-hydroxybutyrate dehydrogenase, 3 alpha-hydroxysteroid dehydrogenase and glucose 6-phosphate dehydrogenase and the female in respect to glucose 6-phosphatase, alpha-glycerophosphate dehydrogenases, and NADP-dependent, decarboxylating malate dehydrogenase. A few of the enzymes exhibited minor sex differences in the first two segments.     

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.     

Oryzalexin S,a Novel Stemarane-type Diterpene Rice Phytoalexin

TTUR 2-2, an alkalophilic Bacillus strain isolated from soil, grew well in media containing cholic acid (CA) at 5% or higher and efficiently converted 7α- and 12α-hydroxyl groups of CA to keto groups, with the conversion rate for both hydroxyl groups reaching 100% by 72 hours of cultivation. The strain also converted a 3α-hydroxyl group to a keto group, but the conversion rate was about 5% at 72 hours. The strain neither affected any other part of the CA molecule, nor oxidized 7β- or 12 β -hydroxyl groups.

By NTG mutagenesis, the following mutants were acquired; (1) converting only the 7α- and 12α-hydroxyl groups, (2) converting only the 12α-hydroxyl group, and (3) converting only the 7α-hydroxyl group. These mutants selectively produce 12-ketochenodeoxycholic acid (12KCDCA), 7-ketodeoxycholic acid (7KDOCA), and 7,12-diketolithocholic acid (7,12DKLCA), from CA; and 7-ketolithocholic acid (7KLCA) from cheno-deoxycholic acid (CDCA), respectively, at high yields, close to 100%.     

Enzymatic synthesis of [4R-2H]NAD (P)H and [4S-2H]NAD(P)H and determination of the stereospecificity of 7 alpha- and 12 alpha hydroxysteroid dehydrogenase

The stereospecifically labeled coenzymes [4R-2H]NADH, [4R-2H]NADPH and [4S-2H]NAD(P)H were synthesized enzymatically in high yield and high isotopic purity (greater than or equal to 95%) with 2HCOO2H/formate dehydrogenase, (CH3)2C2HOH/alchol dehydrogenase from Thermoanaerobium brockii and [1-2H]glucose/glucose dehydrogenase, respectively. This set of deuterated coenzymes was used to determine the stereospecificity of the previously unstudied 7 alpha-hydroxysteroid dehydrogenase from Escherichia coli (NAD-dependent) and 12 alpha-hydroxysteroid dehydrogenase from Clostridium group P (NADP-dependent). H-NMR and EI-MS of the nicotinamide moiety after enzymatic oxidation of deuterated NAD(P)H with dehydrocholic acid as substrate showed that both dehydrogenases are B-sterospecific.