Deoxycholic Acid Methyl Ester — a Novel Bacterial Metabolite of Cholic Acid

System. App!. Microbio!. 6, 18-22 (1985)

Deoxycholic Acid Methyl Ester - a Novel Bacterial Metabolite of Cholic Acid RUDOLF EDENHARDERl and RAINER HAMMANN2 1 2

Hygiene-Institut der Universitat Mainz, 6500 Mainz, Federal Republic of Germany Becton Dickinson GmbH, 6900 Heidelberg 1

Received November 20, 1984

Summary Methyl deoxycholate was identified as a novel bacterial metabolite of cholate, produced anaerobically by strains of most saccharolytic Bacteroides species (B. distasonis, B. eggerthii, B. fragi/is, B. incommunis, B. thetaiotaomicron, B. uniform is, B. variabi/is, B. vu/gatus, and unnamed species). It was also detected with a few strains of Eubacterium and Lactobacillus species. Among 2476 freshly isolated human fecal cultures the frequency of carboxyl group esterification was comparable with that of 7a-dehydroxylation (92 versus 102 cultures). Both activities were, however, lost for unknown reasons after serial transfers. Two of ten mixed fecal cultures tested esterified 3a,7a- or 3a,12a-dihydroxy bile acids at C-24, but not cholate when grown anaerobically.

Key words: Bacteroides - Eubacterium - Lactobacillus - Bile acids - 7a-dehydroxylation - C-24 methyl esterification - Mixed fecal cultures

Introduction Anaerobic intestinal bacteria split conjugated bile acids and metabolize free bile acids, primarily by dehydroxylation, oxidation, and epimerization of hydroxyl groups, mainly at C-7. While these reactions and the reduction of carbonyl moieties to a- or ~-hydroxyl functions are common transformations, other pathways seem to occur less frequently. Such pathways include generation of unsaturated bile acids, degradation of the side chain and conversion to nonsteroidal compounds, formation of Sa- from S~-bile acids, esterification of the 3-hydroxyl group with fatty acids and of the carboxyl function at C-24 with ethanol by mixed fecal and pure cultures of the rat, and hydrolytic cleavage of sulfate esters. Several reviews on these topics have recently been published (Hayakawa, 1973; Lewis and Gorbach, 1972; Macdonald et al., 1983; Midtvedt, 1974). In our own investigations, isolation of the predominant human fecal flora of a large sample pool and screening of the isolates for their cholate transforming abilities led to the discovery of bacterial methyl deoxycholate formation. In this paper, we report on this new bile acid biotransformation and its frequency among intestinal anaerobes and compare it to the ability of the cultures to perform 7a-dehydroxylation. Furthermore the esterifica-

tion of dihydroxy bile acids by mixed fecal cultures is described.

Materials and Methods Bacteriology Anaerobic bacteria, media and manipulation of anaerobes. All bacterial strains tested in this study were isolated from human feces in our laboratory by a combination procedure in the absence of air. Stools were homogenized and fecal dilutions were prepared in a sterile hood under a continous stream of oxygen free CO 2 (0 2 < 5 ppm; Messer Griesheim, Dusseldorf) from a multiple gas-supply head (Holdeman and Moore, 1977). All other isolation procedures were performed in an anaerobic glovebox similar to that described by Aranki et al. (1969). The dilution medium was brain heart infusion, pH 7.4, supplemented with 500 mg cysteine hydrochloride, 300 mg sodium formaldehydsulfoxylate, and 1 ml 0.1 % resazurin solution per liter. Fecal bacteria were isolated on Schaedler-agar, pH 7.6, to which 2 g placenta powder, 1 ml silicon anti foam emulsion and 332 mg PdCl2 were added per liter. A cooked meat medium, supplemented with Schaedler-broth and 1 ml 0.1 % resazurin solution per liter, pH 7.6, containing 0.25 mM potassium cholate, was used for transformation experiments. Stock cultures were

Methyl Deoxycholate Formation by Intestinal Anaerobes maintained in the same medium without cholate. All media used were prereduced and anaerobically sterilized. For screening experiments, the transformation medium was inoculated with sing,~ Ie colonies from agar plates within the glove-box. This culture, incubated for 24 h after tubidity had reached its maximum, was used for the identification of isolates as well as for the determination of the cholate transformation pattern. The isolates were subcultured and identified according to standard methods (Hamman and Werner, 1980; Holdeman and Moore, 1977). Analysis of bile acids. Extraction of bile acids from bacterial suspensions, gas-liquid chromatography (GLC), and combined GLC-MS (mass spectrometry) were performed as described earlier (Edenharder and Knaflic, 1981; Edenharder and Slemr, 1981). Briefly, the supernatants of centrifuged cultures were extracted with ether and the bacterial sediments with acetone. The bile acids were analyzed as TFA-HFIP (trifluoroacetyl-hexafluoroisopropyl) derivatives on 3 % QF-1 at 230°C. Chemicals. Trifluoroacetic anhydride and hexafluoroisopropanol were purchased from Merck, Darmstadt. Chenodeoxycholic acid was obtained from Calbiochem, Frankfurt, methyl deoxycholate from Roth, Karlsruhe, while 3a,7a-dihydroxy-5~­ chol-ll-enoic acid, prepared according to Cowen et al. (1976) was available in our laboratory from previous work.

Results Methyl deoxycholate formation by human intestinal anaerobes

2476 Isolates of the predominant human intestinal bacteria, freshly isolated from the feces of 51 persons, were screened for their cholate transforming capacities. Transformation products were identified by their GLC properties and by comparing mass spectra with those of authentic reference compounds. While 74 of the 2476 isolates dehydroxylated cholate at C-7, resulting in the formation of deoxycholate, 28 isolates generated a pattern of transformation products of the type shown in Fig. 1. The ketonic bile acids 3a,12a-dihydroxy-7-keto- and 3-

o

10

20

30

min

Fig. 1. Bile acids obtained by transformation 6f cholate by Bacteroides vulgatus K 24/18. Analysis by GLC as TFA-HFIP derivatives as described in Materials and Methods. Structures were verified by GLC-MS (mass spectrometry); spectra were identical with those published earlier (Edenharder and Slemr, 1981). (a), Deoxycholic acid; (b), deoxycholic acid methyl ester; (c), cholic acid; (d), 3a,12a-dihydroxy-7-keto-5~-cholanoic acid; (e), 3keto,7a,12a-dihydroxy-5~-cholanoic acid.

19

keto,7a,12a-dihydroxy-5~-cholanoic acids as well as deoxycholate are known bacterial transformation products. Compound (b), however, was neither 3~-hydroxy12-keto-5~-cholanoate as first expected nor 3~,7a,12a­ trihydroxy-5~-cholanoate, but methyl deoxycholate as demonstrated by GLC-MS. In a total of 22 random samples analyzed using this method, the two corresponding peaks were identified as deoxycholic acid and methyl deoxycholate. This simple bile acid derivative was detected here for the first time as a bacterial metabolite of cholate. As can be seen in Table 1,33 strains produced deoxycholate and 27 strains produced methyl deoxycholate. Amounts of these metabolites produced were less than 35 % of total bile acids present (absolute concentration range: - 100 Ilg/ ml). However, 23 cultures generated deoxycholate in higher amounts, but only two cultures produced methyl deoxycholate. Formation of methyl deoxycholate without the concomitant formation of deoxycholate was observed in 64 other cultures. Many but not all isolates simultaneously produced reduction- and oxidation products of cholate, the 7a-hydroxyl function being most frequently oxidized. Monohydroxy-diketo-cholanoic acids were also formed but in trace or minor amounts only. When the 166 bacterial isolates able to 7a-dehydroxylate cholate were identified, 85 isolates were found to be pure cultures. The remaining isolates were either binary or ternary mixed cultures or could not be completely identified. Table 1 shows the species designation of the pure cultures. The number of mixed cultures containing strains of the listed species is in parentheses. Species detected in mixed cultures only are not presented. Most strains, the 7a-dehydroxylating ones as well as those with the additional ability to produce methyl deoxycholate, belonged to different species of the genus Bacteroides, especially B. vulgatus, and B. thetaiotaomicron, which were the species with the highest number of isolates. In general, a correlation seems to exist between the detection frequency of 7adehydroxylating and 24-carboxyl group esterifying bacteria and the isolation frequency, at least'within the genus Bacteroides. In the genus Lactobacillus only a few strains were detected with both activities. The active strain of the genus Fusobacterium and all active strains of the genus Eubacterium produced deoxycholate exclusively. In this respect it is noteworthy that 13 of the 16 active strains of Eubacterium (Lachnospira multiparus included) were isolated from the feces of a single person, patient P 22 (Table 2). The 166 cultures able to transform cholate into methyl deoxycholate or deoxycholate were isolated from the feces of 28 people out of the 51 people investigated. These 28 people could further be divided into two groups: one group, consisting of 17 donors, provided less than 5 isolates per donor (most frequently 1-2) and a second group of 11 donors, provided more than 5 isolates per person, resulting in a total of 122 cultures. The isolates from the second group consisted of isolates that generally dehydroxylated cholate at C-7 only (P 22-K 11), and those that were able to both dehydroxylate at C-7 and esterify the carboxyl group at C-24 (K 16-K 29). No intermediary group could be detected.

20

R. Edenharder and R. Hammann

Table 1. Transformation of cholate into deoxycholate and methyl deoxycholate by human intestinal anaerobes

Number of strains producing 3a,12a' 3a,12a-Me' Total number of isolates tested

Species

Bacteroides distasonis B. eggerthii B. fragilis B. incommunis B. thetaiotaomicron B. uniformis B. variabilis B. vulgatus B. "3452 A" B. "ssp a" B. "T-4-l" Bacteroides, not fitting in known species Eubacterium aerofaciens E. fissicatena E. recta Ie Eubacterium, not fitting in known species Lachnospira multiparus Fusobacterium russii Lactobacillus fermentum Lactobacillus, not fitting in known species

3a,12a' and 3a,12a-Me'

in the percentual range indicated 3-35%

-70%

99 42 122 19 205 116 49 321 46 26 21

3 (1)

2 (1)

141 2 2 8

1 (2)

2 (1) (1) 5 (5) 1 1 9 (1) 5 (1) 2 1

39 12 1 56

2

65

1

-100%

1 (1)

-70%

3-70%

2 2 4 1 4 2

(1)

2 1 (1) 3 1 2 1 (1)

1

5 (1)

(1) (1) (3) (6)

6 (5) 1 (2) 1 1

(1) 2

1 1

3-35%

1

1 (5)

1 (1)

1 (1)

1 (1)

4 6

1

1 (1)

3a,12a, deoxycholic acid; 3a,12a-Me, deoxycholic acid methyl ester. , The number of strains, being part of binary or ternary mixed culures, which converted cholate into the indicated products, is given in parentheses. Tests in which the transformation products were present in amounts below 3% were disregarded. Table 2. Origin and distribution of bacterial isolates able to transform cholate into deoxycholate respectively methyl deoxycholate

Label of person

Total number of isolates

p 22 K 5 K 6 K 9 K 10 Kll K 16 K 23 K24 K27 K29

52 40 28 39 39 37 47 78 24 35 40

Number of isolates producing 3a,12a' 3a,12a-Me' 3a,12a + 3a,12a-Me' 22 (8) 8 5 7 (1) 5 5

2 1

2

1

2 (1)

1 1

1

2 3 2 1 1

(8) (12) (1) (2)

1 1 6 1 2

(1) (1)

(2) (1)

3a,12a, deoxycholic acid; 3a,12a-Me, deoxycholic acid methyl ester. , The number of binary and ternary mixed cultures which produced the indicated compounds from cholate is given in parentheses.

Esterification of deoxycholate and of other bile acids All strains lost the ability to form methyl deoxycholate after a few serial transfers. Only four strains, one of Eubacterium aerofaciens and three of Lachnospira mul-

tiparus, continuously and abundantly dehydroxylated at C-7, but were unable to esterify the 24-carboxyl function. In order to compare cholate transformation patterns of individual isolates of the predominant fecal flora with their corresponding 10-8 and 10-9 dilutions, two samples were taken from each dilution originating from 10 persons. In general only ketonic bile acid derivatives of cholate were produced by such fecal dilutions, but dilutions of subject K 16 generated between 70-80 % 7a-dehydroxylated bile acids of total bile acids present: deoxycholate was the main product, while methyl deoxycholate amounted to about 10 % of total bile acids present. A survey of cholate transformation by mixed fecal cultures, dilution 10-\ showed that samples from 8 out of 10 persons intensively dehydroxylated at C-7, but did not esterify at C-24. Two samples of each of the remaining two persons produced deoxycholate and methyl deoxycholate, the latter amounting to about 5-15 % of total bile acids present. Neither methyl cholate nor methyl esters of ketonic bile acid transformation products were detected. Stool suspensions of the two persons were also able to esterify deoxycholate, chenodeoxycholate and 3a,7a-dihydroxy-5 ~-chol-l1, 12-enoate.

Methyl Deoxycholate Formation by Intestinal Anaerobes Discussion Though, in vivo, 7a-dehydroxylation is quantitatively the most important microbial transformation of primary bile acids, it has up to now been thought to be a rare feature among intestinal anaerobes. This has been concluded from the in vitro 7a-dehydroxylation activity demonstrated in only a few strains of "lactobacilli", Bacteroides, Clostridium, and Eubacterium commonly with low yields (Hayakawa, 1973; Lewis and Gorbach, 1972; Macdonald et al., 1983; Midtvedt, 1974). In contrast with these findings Hill (1976) asserted that 7a-dehydroxylating abilities were widespread among intestinal anaerobes including Bacteroides. The results presented here support this point of view. The screening technique employed here - first checking for cholate transformation, then identifying isolates - may explain the detection of 7a-dehydroxylation in relatively numerous strains of various genera of intestinal anaerobes especially Bacteroides. On the other hand, the loss of 7a-dehydroxylase activity by nearly all strains after a few serial transfers may explain the well known difficulty to detect such an activity in pure cultures although 7a-dehydroxylating bacteria may occur in high numbers, at least in the feces of some people. Surprisingly, many strains which were able to reduce cholate to deoxycholate in our experiments were also able to convert it into methyl deoxycholate. This esterification process, the mechanistic details of which are unknown, represents a novel biosynthetic reaction. It is probably not identical with the recently detected esterification of lithocholate by rat intestinal microflora or by pure cultures of Bacteroides thetaiotaomicron, Citrobacter species, and Peptostreptococcus productus (Kelsey and Sexton, 1976; Kelsey and Thompson, 1976). Esterification of lithocholate required anaerobic growth conditions as well as the presence of ethanol, while during the methyl deoxycholate formation reported here no methanol had been added to the medium. Transfer of a metabolically produced C1-unit, possibly a methyl or methoxy group, to the carboxyl function of suitable bile acids seems to be more plausible than bacterial bile acid esterification with methanol. Such an analogized mechanism would require metabolic formation of methanol by the bacteria listed in Table 1, which is conceivable but unknown. The frequency of bacterial methyl deoxycholate formation from cholate was comparable with the frequency of 7 a-dehydroxylation. Ninety two cultures, corresponding to 3.7 % of total isolates, were able to esterify the carboxyl group at C-24, while 74 cultures (3.0 %) produced deoxycholate only. A further parallel was that both abilities, i. e. features of the original isolates, were lost by pure strains for unknown reasons. In mixed fecal cultures, however, bile acid esterification seems to be much rarer than 7a-dehydroxylation. With stool suspensions carboxyl group esterification was demonstrated with some dihydroxy bile acids but not with cholate. These findings were confirmed by the results of the original screening experiments: methyl cholate was generated microbially but infrequently and in traces only, while the formation of methyl esters of ketonic bile acids was never detected.

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Esterification of dihydroxy bile acids - deoxycholic, chenodeoxycholic, and 3a, 7 a-dihydroxy-5~-chol-11, 12enoic acids - but not of cholic acid and its oxidation products by pure or mixed fecal cultures suggests a relation to bacterial growth inhibition by these acids. While concentrations of about 5-25 mM of these trisubstituted bile acids are necessary to be inhibitory for many intestinal anaerobes, concentrations as low as 0.1-2 mM of 3a,7aor 3a,12a-dihydroxy bile acids inhibited growth (Binder et al., 1975; Kurzbach, 1980). The major determinant to explain these differences is thought to be the concentration ratio of undissociated bile acid/dissociated bile acid (Binder et al., 1975). This ratio is pH dependent and determined by the pK. value for a given structure. Therefore any change in the molecular structure which affects solubility should result in modified bacterial growth inhibition. Indeed, dissimilar solution and membrane phenomena of chenodeoxycholic and ursodeoxycholic acids, differing only in 7-hydroxyl group orientation, could be explained by pH-solubility relations (lgimi and Carey, 1980). The bacterial carboxyl group esterification of dihydroxy bile acids mentioned above, which reduces the bile acid concentration by the generation of less soluble compounds, diminishes or even abolishes growth inhibition and should therefore be a protective mechanism for these bacteria. From this work we suggest that so far unknown fecal bile acid methyl esters may be detected, at least in specific individuals, if appropriate analytical methods are applied avoiding saponification and analysis of bile acids as methyl ester derivatives. If bile acids shall be analyzed as methyl esters new separation techniques must be introduced (Setchell et al., 1983). Acknowledgements: The authors thank Mrs. C. Bohres and Mr. j. Fontaine, Organisch-Chemisches Institut der Universitiit Mainz, for performing the mass spectrometric analyses. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg. Identification of bacteria was performed by one of us (R. H.) at the Institut fur Medizinische Mikrobiologie und Immunologie der Universitiit Bonn.

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anaerobic bacteria from human gingiva and mouse cecum by means of a simplified glove box procedure. Appl. Microbiol. 17,568-576 (1969) Binder, H. j., Filburn, B., Floch, M.: Bile acid inhibition of intestinal anaerobic organisms. Amer. J. Clin. Nutrit. 28, 119-125 (1975) Cowen, A. E., Hofmann, A. F., Hachey, D. L., Thomas, P. j., Belobaba, D. T. E., Klein, P. D., Tokes, L.: Synthesis of 11, 12-2H 2- and 11,12- 3H r labeled chenodeoxycholic and lithocholic acids. J. Lipid Res. 17, 231-237 (1976) Edenharder, R., Knaflic, T.: Epimerization of chenodeoxycholic acid by human intestinal lecithinase-lipase-negative Clostridia. J. Lipid Res. 22, 652-658 (1981) Edenharder, R., Slemr, j.: Gas chromatographic and mass spectrometric analysis of bile acids as trifluoroacetyl-hexa-

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fluoroisopropyl and heptafluorobutyryl derivatives. ]. Chromatogr. 222, 1-12 (1981 ) Hammann, R., Werner, H. : Fermentation products (using g.!'c.) in the differentiation of non-sporing anaerobic bacteria. In: Goodfellow, M., Board, R. G. (eds.), Microbiological classification and identification, pp. 257-271. London-New York, Academic Press, Inc. 1980 Hayakawa, S.: Microbiological transformation of bile acids. In: R. Paoletti and D. Kritchevsky (eds.), Advanc. Lipid Res., vol. XI, pp. 143-192. New York-London, Academic Press 1973 Hill, M. J.: Fecal steroids in the etiology of large bowel cancer. In: Nair, P. P., Kritchevsky, D. (eds.), The bile acids, vol. 3, pp. 169-200. New York-London, Plenum Press 1976 Holdeman, L. V., Moore, W. E. c.: Anaerobe laboratory manual, 4 th ed. Blacksburg, Virginia Polytechnic Institute and State University 1977 Igimi, H., Carey, M. c.: pH-Solubility relations of chenodeoxycholic and ursodeoxycholic acids: physical-chemical basis for dissimilar solution and membrane phenomena. ]. Lipid Res. 21, 72-90 (1980)

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Dr. Rudolf Edenharder, Hygiene-Institut def Univ., Hochhaus am Augustusplatz, 0-6500 Mainz