Transformation Of Bile Acids Research Articles

Transformation of bile acids by Clostridium perfringens.

Thirty-five strains of Clostridium perfringens were examined for their ability to transform bile acids, both in growing cultures and by washed whole cells. All of the strains oxidized the 3 alpha-hydroxy group to an oxo group, and all except three converted the same alpha-hydroxy group into a beta-configuration. The oxidative 3 alpha-dehydrogenation was barely detectable under anaerobic cultural conditions but was clearly demonstrated in an aerated system using washed whole cells, with a pH optimum between 7.0 and 9.0. The epimerizing reaction amounting to 10 to 20% conversion was observed in anaerobic cultures and also with resting cells, irrespective of oxygen supply. Both reactions were carried out with seven conventional 3 alpha-hydroxy bile acids, thus producing a series of 3-oxo and 3 beta-hydroxy derivatives that could be examined for gas-liquid chromatographic and mass spectrometric behavior. No evidence for the occurrence of 7 alpha- and 12 alpha-hydroxysteroid dehydrogenase activities among the test strains was found. A highly potent deconjugating hydrolase was elaborated by all of the strains.

550 THE EXTENT OF MICROBIAL DEGRADATION OF BILE ACIDS: AN IMPORTANT FACTOR MODULATING THEIR ABSORPTION

Experimental work has shown that less polar bile acids, although better absorbed passively, have a reduced affinity for the ileal transport system and are more extensively adsorbed to residues and microbes. This study examines the fecal excretion of bile acids and the % adsorbed in relationship with the qualitative pattern in 2 categories of patients with an intact ileum. The contaminated small bowel syndrome was diagnosed in 4 cases of chronic idiopathic intestinal pseudoobstruction with fat (19.3 ± 6.8g/24h) and bile acid (693 85mg/m2/24h) malabsorption. Primary bile acids were essentially absent. The sum of deoxycholic, lithocholic, 3-ketocholanoic and 3-betacholanoic acid accounted for 80.3±6.2% of the total. Only a small % (16.7±5.4) of bile acids could be recovered in the aqueous phase. A marked reduction (P < .001) of the anaerobic flora in a fresh stool obtained from 6 CF children on triple I.V. therapy was associated with a reduction in the concentration of secondary forms (P<.005) and with a 5-fold increase (10.2 vs 52.7) in the % of bile acids in the aqueous phase when compared to 7 studied off antibiotics. In 4 CF patients studied both on oral cloxacillin and during I.V. therapy, there was no change in steatorrhea (15.9±5.7 vs 18.8±7.8g/24h) but a large decrease in bile acid loss (751±182 vs 366±93). This study suggests that the extent of microbial transformations of bile acids in the GI tract plays a role on their absorption.

Transformation of bile acids by mixed microbial cultures from human feces and bile acid transforming activities of isolated bacterial strains.

Microbiol transformation of cholic acid and chenodeoxycholic acid by anaerobic mixed cultures of human fecal microorganisms was investigated, and the results were examined in relation to the bile acid transforming activities of 75 bacterial strains isolated from the same fecal cultures. The reactions involved in the mixed cultures were dehydrogenation and dehydroxylation of the 7 alpha-hydroxy group in both primary bile acids and epimerization of the 3 alpha-hydroxy group in all metabolic bile acids. Extensive epimerization of the 7 alpha-hydroxy group of chenodeoxycholic acid yielding ursodeoxycholic acid was also demonstrated by certain fecal samples. 7 alpha-Dehydrogenase activity was widespread among the fecal isolates (88% of 16 facultative anaerobes and 51% of 59 obligate anaerobes), and 7 alpha-dehydroxylase activity was revealed in one of the isolates, and unidentified gram-positive nonsporeforming anaerobic bacterium. 3 alpha-Epimerization was effected by seven strains assigned to Eubacterium lentum, which were also active for 3 alpha- and 7 alpha-dehydrogenations. No microorganism accounting for 7 alpha-epimerization was recovered among the isolates. Splitting of conjugated bile acid was demonstrated by the majority of obligate anaerobes but the activity was rare among facultative anaerobes.

Measurement of fecal bile acid excretion in gnotobiotic rats: Comparison of gas-liquid chromatography and [4- 14c] cholesterol isotopic equilibrium

Gas-liquid chromatography (G.L.C.) and the method of [4(-14C)] cholesterol isotopic equilibrium (C.I.E.) were used to determine the fecal bile acid excretion in gnotobiotic rats. The same samples were submitted to both methods. In these conditions, it was observed that the fecal bile acid excretions determined by G.L.C. were 38% of lower than when determined by C.I.E. In thin-layer chromatographic analyses (T.L.C.) of the bile acid extracts obtained from rats in which a [4(-14C)] cholesterol isotopic equilibrium was established, 33 to 35% of the radioactivity of this fraction was not observed in the rat primary bile acids. No bile acids could be observed in G.L.C. made with eluates obtained from the T.L.C. areas containing this radioactivity. It therefore appears that the difference observed in the results obtained by G.L.C. and C.I.E. is due to the fact that chemical species which are not measured by the former method can be determined by the latter one. T.L.C. analyses of bile acid extracts from axenic rats in which either a [26(-14C)] cholesterol or a [2,4(-3H)] cholic acid and [24(-14C)] chenodeoxycholic acid equilibrium were established, lead to the conclusion that the chemical composition of these undetermined substances is complex: part of these substances comes from the transformation of bile acids; another part is made of molecules which maintain the 26(-14C) of cholesterol.

464 MICROBIAL BILE ACID TRANSFORMATION AND THE RECTAL MICROFLORA IN CONTROL AND CF CHILDREN

The possibility that the large amounts of bile acids found in CF stools could be related to an abnormal fecal flora giving rise to altered microbial bile acid transformation was examined in 7 CF off antibiotics (CF1), in 10 on oral cloxacillin (CF2) and in 6 on I.V. cloxacillin, gentamycin and carbenicillin (CF3). Their fecal flora and bile acids were also compared to those of 7 control children. The concentration (mg/g wet stool) of bile acids in CF1 (5.5 ± 1.0) decreased to (2.2 ± .4) in CF2 and to (1.0 ± .4) in CF3. A larger % of fecal bile acids in CF1 were deconjugated and had undergone either dehydroxylation or dehydrogenation as compared to CF2 and CF3. Furthermore,the conjugated bile acid fraction in CF1 showed a lower glycine/taurine ratio (3.6) than in CF2 (8.4) and CF3 (5.3) and a higher % of secondary forms. The anaerobic flora (log cts/g wet stool) was strikingly reduced in the CF3 group (4.3 ± 1.0) as compared to CF1 (9.1 ± .1) and CF2 (9.6 ± .2). In this latter group, the total flora was larger and this was mainly accounted for by an increase in aerobic gram negatives. Both the total flora and the anaerobic flora were decreased in CF1 when compared to controls. This was largely due to a diminution of total anaerobes. The close relationship between the anaerobic flora, bile acid concentration and microbial transformation observed in the CF groups was also present when CF1 children were compared to controls.

The degradation of cholic acid and deoxycholic acid by Bacteroides species under strict anaerobic conditions.

Microbial transformation of bile acids at the 3a-, 7aand 12a-hydroxyl positions, under anaerobic conditions, was first reported in Alcaligenes fueculis (Hughes & Schmidt, 1942). Other gut bacteria have also been shown to contain hydroxy steroid dehydrogenases (Aries & Hill, 1970). The most common of the three reactions, 7a-hydroxy steroid dehydrogenation, has been reported in many strains of Escherichiu coli (Haslewood et ul., 1973) and Bacteroidesfragilis (Macdonald et ul., 1975). Furthermore 3a-, 7aand 12a-hydroxy steroid dehydrogenase activity has been shown with cell-free preparations of Clostridium perfringens (Macdonald et a / . , 1976). Strains of Clostridium pnraprrtrific~im isolated from human faeces desaturate the bile acid nucleus to form 4-en-3-one (pH optimum 7.5) and 1,4-dien-3-one (pH optimum 8.5) products (Aries et a/ . , 197 1 ), and strains of Bucteroidesfragilis and Bacteroides thetaiotaomicron exhibit both 3aand 7a-dehydroxylase activity (Edenharder et ul., 1976). To date no evidence has been presented for side-chain cleavage by any bacterium indigenous to the human intestinal tract or for nuclear dehydrogenation in Bacteroides spp. This communication reports, in addition to side-chain cleavage of cholic acid and deoxycholic acid, both dehydroxylase and dehydrogenase activity in two Bacteroides spp. The nuclear dehydrogenation reactions carried out by the human intestinal flora have been implicated in the aetiology of colon cancer (Hill, 1974), but have been little studied because of the complex nutritional requirements of these bacteria. Bucteroides spp. capable of metabolizing sodium cholate and deoxycholate have been isolated from human faecal material. The isolates designated Bacteroides frugilis XF23 and Bucteroides thetaiotaomicron E59 have the ability to dehydroxylate cholic acid and oxidize both bile acids during anaerobic growth to yield metabolites with the I ,4-dien-3one structure. Anaerobic metabolism of bile acids by Bacteroides spp. was carried out in modified Todd-Hewitt broth ( I 5g/l) as basal medium containing (g/l): L-cysteine hydrochloride, 0.01 ; 1 M-NaOH, 0.01 ml; Na2S,9H,0, 1 .O; Na2C03, 8.0; MgS04,7H20, 0.1 ; bile salt, 1 .O; the final pH was 7.0 and it was dispensed to fill screw-cap bottles. The medium was autoclaved and on cooling was transferred to an anaerobic glove bag under an atmosphere of CO2/N2 (1 :9). The medium was gassed for lOmin with the above gas phase, and during this procedure the medium was inoculated aseptically with Bucteroides spp. The cultures were incubated at 37°C for 6 weeks. After acidification to pH4.0, the steroids were extracted into redistilled ethyl acetate, the solvent being removed by evaporation. Steroid products were separated by preparative t.1.c. on silica-gel GF254 plates and those with the 1,4-dien-3-one structure were detected under U.V. light; other products were detected by their colour with anisaldehyde reagent (Kritchevsky et a/., 1963). Products were identified with reference to U.V. and i.r. spectra and t.1.c. and g.1.c. mobilities of known standards (Barnes et ul., 1976). Retention times for g.1.c. were measured relative to 5a-cholestane and t.1.c. analysis was performed on Kieselgel GFZS4 in the solvent systems methanol/methylene chloride ( I : 19, v/v) and trimethylpentane/ethyl acetate/acetic acid (5 : 5 : I , by vol.). Mobilities on t.1.c. were measured relative to androsta-l,4-diene-3,17-dione. The results show (Fig. 1) that both Bucteroides spp. have the ability to dehydroxylate, dehydrogenate and carry out side-chain cleavage of cholic acid (I), yielding three acidic and three neutral products, namely: 3a,l2a-dihydroxy-5~-choIanic acid (11); 12ahydroxy-3-oxo-5~-cho1-24-oic acid (111); 12a-hydroxy-3-oxopregna-1,4-dien-20-carboxylic acid (IV); 12~-hydroxyandrosta-4-ene-3,17-dione (V) ; 128-hydroxyandrosta1,4-diene-3,17-dione (VT) and 12a-hydroxyandrosta-1,4-diene-3,17-dione (Vll). Both species also are able to dehydrogenate and carry out side-chain cleavage of deoxy-

Reduced microbial transformation of bile acids in cystic fibrosis.

The microbial transformation of bile acids by incubates of stool homogenates from children with cystic fibrosis is decreased.

Microbiological Transformation of Bile Acids

Publisher Summary This chapter discusses microbiological transformation of bile acids. There is an intimate relationship between bile acid metabolism in vivo and intestinal microorganisms. The conjugated primary bile acids are attacked by intestinal microorganisms in the lower small intestine and cecum and transformed into various metabolites that are absorbed and further transformed by liver enzymes prior to re-excretion into the bile. The liver enzymes mainly catalyze the following reactions: (1) conjugation of free bile acids with taurine or glycine, (2) reduction of keto groups to alcohols, and (3) hydroxylation of the cholane nucleus. Thus, the final composition of the bile acids in normal bile is the result of an intimate interaction between the liver and intestinal microorganisms. The bile-acid transformations in the intestine can be classified as follows: (1) hydrolysis of the conjugated bile acids; (2) elimination of hydroxyl groups, mainly at the C-7 position of the cholane nucleus; (3) oxidation of the hydroxyl groups at C-3, C-7, and C-12 to oxo groups; (4) reduction of oxo groups to both α- and β-hydroxyl groups; and (5) other miscellaneous reactions.

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.

The indigenous flora of the gastrointestinal tract.

The bacterial species that are most abundant in the gastrointestinal tract under normal conditions are anaerobic and have exacting growth requirements. As they display only a narrow range of biochemical activities, it is probable that the chemical transformation of metabolites in the gastrointestinal tract is less pronounced when these organisms predominate than when others gain the upper hand under pathologic conditions. In other words, the composition of the gastrointestinal flora determines the nature of the bioactive substances that are produced through metabolic transformation of amino acids, bile acids and other metabolites. Such biochemical activities may be as important as the orthodox pathologic lesions caused by pathogens.