7-ketodeoxycholic Acid Research Articles

Metabolomics analysis of urine from rats administered with long-term, low-dose acrylamide by ultra-performance liquid chromatography-mass spectrometry

1. To study the toxic effect of chronic exposure to acrylamide (AA) at low-dose levels, we applied metabolomics approach based on ultra-performance liquid chromatography/mass spectrometry (UPLC–MS). A total of 40 male Wistar rats were randomly assigned to different groups: control, low-dose AA (0.2 mg/kg.bw), middle-dose AA (1 mg/kg.bw) and high-dose AA (5 mg/kg.bw). The rats continuously received AA via drinking water for 16 weeks. Rat urine samples were collected at different time points for measurement of metabolomic profiles.2. Thirteen metabolites, including the biomarkers of AA exposure (AAMA, GAMA and iso-GAMA), were identified from the metabolomic profiles of rat urine. Compared with the control group, the treated groups showed significantly increased intensities of GAMA, AAMA, iso-GAMA, vinylacetylglycine, 1-salicylate glucuronide, PE (20:1(11Z)/14:0), cysteic acid, L-cysteine, p-cresol sulfate and 7-ketodeoxycholic acid, as well as decreased intensities of 3-acetamidobutanal, 2-indolecarboxylic acid and kynurenic acid in rat urine. Notably, three new candidate biomarkers (p-cresol sulfate, 7-ketodeoxycholic acid and 1-salicylate glucuronide) in rat urine exposed to AA have been found in this study.3. The results indicate exposure to AA disrupts the metabolism of lipids and amino acids, induces oxidative stress.

Synthesis of a Radiolabeled Zwitterionic Detergent and Its Use in Protein Purification

Radiolabeling permits the detection of traceamounts of zwitterionic detergent remaining in extracted hydrophobic or membrane proteins. To develop a sensitive and specific assay for its presence, the commonly used zwitterionic membrane protein detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) was synthesized in a tritiated form. Synthesis via 7-ketodeoxycholic acid gave [7-3H]Chaps in 53% yield with a specific activity of 0.85 mCi/mmol. A novel solvent extraction system for cholic acid obviated the need for chromatographic isolation of this intermediate. The protocol can be readily modified to yield [7-3H]Chaps of higher specific activity. [7-3H]Chaps was used to monitor the efficiency of various strategies for detergent removal from concentrated bacterial culture supernatants containing 0.2% (w/v) Chaps. Dialysis removed 95% of Chaps and the addition of detergent-affinity beads to the dialysis buffer resulted in 97% removal of Chaps. Gel-filtration chromatography removed 99.9% of Chaps, while a detergent-affinity bead chromatography column removed 99.99%. Overall, gel-filtration chromatography was the most convenient and economical method for the one-step removal of the zwittergent from complex biological mixtures.

Absence of Cholic Acid 7α-Dehydroxylase Activity in the Strains of Lactobacillus and Bifidobacterium

To investigate the presence of 7α-dehydroxylase activity on bile acids in the bacterial strains of fermented milk products, 46 strains of Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus gasseri, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Lactococcus lactis spp. lactis, and Streptococcus salivarius spp. thermophilus were tested for their ability to produce deoxycholic acid from cholic acid. The production of deoxycholic acid was quantitatively measured by radio-chromatographic analysis in anaerobically prepared washed whole resting cells and by HPLC analysis in growing cultures. Resting whole cells from a positive control strain, Eubacterium lentum-like strain c-25, converted 81.7% of .2mM cholic acid to deoxycholic acid and 3.7% to 7-keto-deoxycholic acid, when the cell suspension was incubated anaerobically at a concentration of 2mg of protein/ml for 4h at pH 7.3. However, none of the test strains investigated in this study was able to transform cholic acid under the same conditions. In growing cultures, 91.5% of 150µg/ml of cholic acid was transformed to deoxycholic acid and 1.1% to 7-ketodeoxycholic acid by E. lentum-like c-25 after a 7-d anaerobic incubation. None of the test strains showed production of either deoxycholic acid or 7-ketodeoxycholic acid as growing cultures.

Oxidation of 3-, 7-, and 12-hydroxyl Groups of Cholic Acid by an AlkalophilicBacillussp.

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%.

Identification of short side chain bile acids in urine of patients with cerebrotendinous xanthomatosis

Urine from patients with cerebrotendinous xanthomatosis (CTX) was found to contain a number of minor bile acids along with three major bile acids, 7-epicholic acid, norcholic acid, and cholic acid. The following minor bile acids were identified by combined gas-liquid chromatography-mass spectrometry: 7-ketobisnordeoxycholic acid; 12-ketobisnorchenodeoxycholic acid; 7-ketonordeoxycholic acid; 12-ketochenodeoxycholic acid; 7-ketodeoxycholic acid; 12-ketochendeoxycholic acid; bisnorcholic acid; allonorcholic acid; allocholic acid; 1 beta-hydroxybisnorcholic acid; 1 beta-hydroxynorcholic acid; 1 beta-hydroxycholic acid; 2 beta-hydroxybisnorcholic acid; 2 beta-hydroxy-norcholic acid; 2 beta-hydroxycholic acid. The presence of C22 and C23 bile acids in urine of the CTX patients suggests that bile alcohols having a hydroxyl group at C22 or C23 in the side chain may be further degraded to these bile acids.

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)

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.

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)

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)

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.

Hydrolysis of bile acid conjugates by Clostridium bifermentans

Two strains of Clostridium bifermentans have been investigated for their ability to hydrolyse bile acid conjugates under conditions suited to further transformation of the free acids liberated. In batch fermentation at 0.5 g/l substrate concentration, growing cells effected the near-quantitative hydrolysis of glycodeoxycholate, taurodeoxycholate and taurocholate within 48 h; glycocholate was 88% hydrolysed. At substrate concentration greater than 1.0 g/l however, taurine conjugates were less well hydrolysed. Further transformation of the liberated cholic acid to deoxycholic acid and/or 7-ketodeoxycholic acid was achieved, but quantitative conversion was not observed.

7 ?-Dehydroxylation of cholic acid by Clostridium bifermentans

Statistically designed experiments were used to identify variables important in the 7α-dehydroxylation of cholic acid to deoxycholic acid by strains of Clostridium bifermentans in pH-controlled anaerobic fermentation. Deoxycholic acid yields were highest in the presence of 10% CO2 and near pH 7 but were largely unaffected by the strain of organism used, time of bile acid substrate addition, mode of gas delivery, presence of thioglycollate, or the use of OH− ion or HCO 3 − ion for pH control. However, dehydroxylation was enhanced, and the redox potential remained relatively high, when temperatures were low, inoculum size small, and growth inhibitors were present. Deoxycholic acid yields of up to 40% were observed but the formation of 7-ketodeoxycholic acid side product could not be entirely prevented.

Intestinal Microflora and Bile Acids

The population levels of intestinal microflora and bile acid composition in the digestive tract were examined in rats fed bile acids to determine the relationships between gastrointestinal microflora and the host. The population level of Bacteroides was increased in the ceca of rats fed cholic acid or deoxycholic acid. In the ileum, the concentration of conjugated bile acid in rats fed cholesterol, cholic acid, hyodeoxycholic acid or lithocholic acid was higher than that in control rats, and was very low in ceca and feces of all the rats. The concentration of total free bile acid was much higher in the ceca than in the ilea of rats fed hyodeoxycholic acid or lithocholic acid. Cholic acid and deoxycholic acid were found in the ilea, ceca and feces of the cholic acid-fed rats. In the deoxycholic acid-fed rats, cholic acid was localized in the ileum. 7-Ketodeoxycholic acid was also found in the ceca of the cholic acid-fed rats. 12-Ketolithocholic acid was found in the feces of rats fed cholic acid or deoxycholic acid. 3-Ketocholanic acid was found in some samples from the lithocholic acid-fed rats. Therefore, some kinds of bile acids influence the population levels of gastrointestinal microflora and bile acid composition in the intestine.

Quantitative analysis of bile acids in serum and bile, using gas -liquid chromatography

The results of a gas—liquid chromatographical analysis of individual bile acids in serum and bile are presented. The analysis of bile acids in serum involved enzymatic hydrolysis prior to protein precipitation, purification according to Roovers et al. [2], diethyl ether extraction, preparation of trifluoroacetate methylester derivatives and gas—liquid chromatographical analysis with 1% OV-210 on Gaschrom Q as stationary phase. Protein precipitation and special purification steps were omitted in the routine analysis of bile acids in bile. With 7-ketodeoxycholic acid as internal standard adequate separation of the individual bile acids-cholic acid (chenodeoxycholic acid, deoxycholic acid and lithocholic acid) was obtained. There was a linear correlation between the dose injected and the peak height response. Recovery studies with unconjugated and conjugated bile acids, added to serum, water and bile, revealed good reproducibility. The only significant loss, mainly of cholic and lithocholic acid, occurred during the hydrolysis step. Special attention was paid to purification and diethyl ether extraction to minimize loss of bile acids during these steps. Recovery of conjugated bile acids, after correction for loss in preparation of trifluoroacetate methylester derivatives and in the gas-chromatographic procedure, was 80–95% in serum and 83–100% in bile. Duplicate analyses in serum and bile samples proved that reproducibility was good. Numerous estimates were made in serum and bile from fasting normal persons in order to establish valid and reliable normal values.

INFLUENCE OF INTESTINAL MICROORGANISMS ON THE METABOLISM OF BILE ACIDS IN MICE

In order to investigate the metabolism of bile acids of the mouse by intestinal microorganisms, isolation of the organisms responsible for the transformation of bile acids in the intestinal tract of conventional mice and their activity in vivo in mono- or polycontaminated exgermfree mice were made.Bacteroides, Lactobacillus, Enterococcus and Clostridium perfringens isolated from the intestines of conventional mice converted conjugated bile acids into free acids both in vitro and in vivo, but E. coli, Staphylococcus and Proteus did not. These microorganisms capable of converting conjugates distributed in the whole intestines, also in the cecum and upper small intestine. Among the resident intestinal microorganisms of mice, only E. coli had the ability of converting cholic acid into 7-ketodeoxycholic acid. The role of exogenic microorganisms in the conversion of free bile acids were also investigated compared with resident intestinal flora. Many species of such exogenic bacteria as Shigella, Salmonella and El Tor vibrio had no effect on the conversion of cholic acid. Some of pathogenic E. coli converted cholic acid and some did not.

Influence of E. coli infection on turnover and metabolism of cholic acid in germ-free rats

The fecal excretion of cholic acid and its metabolites has been determined in normal and germ-free rats and in rats monoinfected with Escherichia coli. The mean daily excretion was 5.1, 1.9, and 2.2 mg. in the respective groups. The mean values for the amount of cholic acid and its metabolites in the intestinal tract were 14.6, 18.4, and 20.8 mg., respectively. 7-Ketodeoxycholic acid and 3α,7β,12α-trihydroxycholanic acid are formed from cholic acid in E. coli monoinfected rats. 7-Ketodeoxycholic acid is formed by microbial enzymes and is then slowly reduced by liver enzymes to cholic acid and 3α,7β,12α-trihydroxycholanic acid.