Secondary metabolite; microbial metabolite; secondary bile acid; endogenous metabolite

In vitro activity. [1]
The in vitro transformations of unconjugated bile acids by monocultures of E. lentum R1; strain R3, and strain M67 and by binary cultures of E. lentum with either strain R3 or M67 are listed in Table 2. Both binary cultures converted P-MCA into ω-MCA for 59 and 71%, respectively, after 72 h of incubation (mean of four determinations, varying between 50 and 90%). This transformation proceeded in two steps: first E. lentum Rl oxidized P-MCA to 3a,7p-dihydroxy-6-oxo-5p-cholanoic acid; the identity of this metabolite was confirmed by combined gas chromatography-mass spectrome try. Either of the two Fusobacterium sp. could then reduce the 6-oxo-group to a 6a-hydroxyl group, yielding ω-MCA. The latter conversion was confirmed by the appearance of ω-MCA in monocultures of strain R3 or M67 admixed with the 6-oxo derivative of 1-MCA. In contrast, aMCA and ω-MCA were not transformed by the isolates. Hyocholic acid was partially converted by E. lentum Rl into 3a,6a-dihydroxy-7-oxo-5Pcholanoic acid as shown by the mobility of the compound on gas-liquid chromatography and by comparison of the data from the mass spectrometry with those of the reference compound. CA was oxidized by E. lentum Rl to 3a,12a-dihydroxy-7-oxo-5p-cholanoic acid (50 to 60%) and to small amounts of the 12-oxo derivative. Strain R3 oxidized CA to the 12-oxo derivative. E. lentum Rl oxidized 40% of CDCA to 3a-hydroxy-7-oxo-5,B-cholanoic acid. The 7,B-hydroxyl of ursodeoxycholic acid, however, was not oxidized. None of the isolates transformed HDCA, although the 61-isomer MCA was oxidized to the 6-oxo derivative by either E. lentum Rl or strain M67. This 6-oxo group was reduced back to the 61-OH group of MCA by E. lentum Rl. In contrast, strains R3 and M67 reduced the 6-oxo group to a 6a-OH group to yield HDCA. This caused isomerization of MCA to HDCA in both binary cultures of E. lentum Rl with either strain R3 or M67. Lithocholic acid was not transformed by any of the three isolates. E. lentum Rl deconjugated tauro- and glycoCA, tauro- and glyco-CDCA, and tauro-j-MCA for 60 to 90%. The unidentified compound A, a derivative of tauro-1-MCA found in the feces of gnotobiotic rats monoassociated with E. lentum Rl (see below), was not detected in vitro. Strain M67 deconjugated both tauro- and glvco-CDCA for more than 90%; deconjugation of tauro- and glyco-CA was more irregular and varied between 50 and 75%, whereas tauro-1-MCA was not deconjugated.

In vivo activity. [1]
E. lentum Rl was easily established as a monoassociate in germfree rats; in contrast, strains M67 and R3 could not be established in gnotobiotic rats unless the oxidation-reduction potential in the intestinal tract was lowered by association with Clostridium sp. C18. In gnotobiotic rats associated with E. lentum Rl and Clostridium sp. C18, more than 80% of the fecal bile acids were unconjugated and more than 50% of CA was converted into the 7-keto derivative of CA; small amounts of the 3-keto and 12-keto derivatives were also formed. The amounts of these oxo derivatives were not markedly influenced by Clostridium sp. C18, strain M67, or strain R3. 3a,71-dihydroxy-6-oxo-513-cholanoic acid, and this reaction occurred independently of the presence or absence of strain M67 or R3 (Fig. 1). In animals associated with E. lentum Rl and Clostridium sp. C18 plus strain M67 or R3, 26% (group with M67) and 27% (group with R3) of ,BMCA was converted into ω-MCA. In rats associated with the four strains, nearly 70% of P-MCA was converted into ω-MCA. Feces from gnotobiotic rats associated with E. lentum Rl or E. lentum Rl plus Clostridium sp. C18 also contained an unidentified bile acid, termed compound A. This compound was not found in germfree rats. Furthermore, compound A was only detected in the unconjugated fraction or after enzymatic deconjugation of the sample; it was destroyed upon alkaline deconjugation with 20% KOH in ethylene glycol. In gasliquid chromatography its retention times relative to those of the same derivative of 23- nordeoxycholic acid were 1.11 and 1.04 as methyl ester trimethylsilyl ether on 1% QF-1 and 3% OV-225 and 3.06 and 3.35 as methyl ester acetate on 3% OV-1 and 3% OV-17, respectively. The mass spectrum of the trimethylsilyl ether showed intense peaks at mle 285 and 195, characteristic of a 6t,713-bis-trimethylsiloxy structure. The compound further gave peaks at mle 636, 546, and 456, suggesting a trihydroxycholanoate structure with one double bond. The presence of an ABCD-ring fragment ion at mle 253 and the absence of a definite side chain ion peak at mle 115 indicated that the unsaturation was in the side chain. The compound gave 1BMCA upon catalytic hydrogenation of the methyl ester acetate for 18 h at room temperature in 5 ml of 90% ethanol, using 0.5 mg of palladium oxide. These results indicated that compound A was a conjugated derivative of 1-MCA with a double bond in the side chain; the exact position of the double bond could not be derived from our data.

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Three anaerobic bacteria, isolated from the ceca of rats and mice, converted, through a concerted mechanism, beta-muricholic acid, the predominant bile acid in germfree rats, into omega-muricholic acid. One isolate was a Eubacterium lentum strain; the second and third isolates were tentatively identified as atypical Fusobacterium sp. strains. The conversion of beta-muricholic acid into omega-muricholic acid proceeded in two steps: E. lentum oxidized the 6 beta-hydroxyl group of beta-muricholic acid to a 6-oxo group, which was reduced by either of the two other species to a 6 alpha-hydroxyl group, yielding omega-muricholic acid. This transformation occurred both in vitro and in gnotobiotic rats. Monoassociation of germfree rats with the E. lentum strain gave rise to an unidentified fecal bile acid, probably a derivative of beta-muricholic acid having a double bond in the side chain [1].

Bile acid analysis. [1]
The procedure used to determine bile acids in feces and intestinal contents from germfree and gnotobiotic rats has been described elsewhere. In vitro transformations were studied in cultures incubated for 3 or 4 days with the substrates under investigation. To study transformations of unconjugated bile acids, 3 ml of culture medium was mixed with 1 ml of methanol and 0.5 ml of internal standard solution containing 50 ,ug of 23-nordeoxycholic acid. This mixture was acidified to pH <2 with HCI and extracted three times with 5 ml of diethyl ether; the ether extract was evaporated and the bile acids were derivatized to methyl ester trimethylsilyl ethers and methyl ester acetates for gas-liquid chromatography. In vitro hydrolysis of conjugated bile acids was determined by comparing the amount of free bile acid in the grown culture to that of the conjugate in an uninoculated control tube. To analyze the conjugated bile acids, 3 ml of medium was mixed with 0.5 ml of internal standard solution, evaporated, and subjected to alkaline deconjugation with 20% (wt/vol) KOH-ethylene glycol; the hydrolysate was then acidified and further treated as for unconjugated bile acids. Cultures with tauro-p-MCA were enzymatically hydrolyzed since feces of gnotobiotic rats contained an unknown metabolite of tauro-1-MCA that could only be detected after enzymatic deconjugation by the cholylglycine hydrolase procedure of Nair and Garcia. Identification of bile acids was carried out by gasliquid chromatography and mass spectrometry. Methyl ester acetates were chromatographed on columns of 3% OV-1 and 3% OV-17; trimethylsilyl ethers were chromatographed on columns of 1% QF-1 and 3% OV-225. When required for the identification of certain peaks, mass spectra were run via a gas chromatography-mass spectrometry combination technique and were compared with those of reference compounds.

Isolation and identification procedures. [1]
Strains Rl and R3 were isolated from the cecal contents of a conventional rat. The cecum was transferred into the ananerobic chamber, and its contents were suspended in 10 ml of medium A. Aliquots (10 ,ul) of serial 10-fold dilutions of this suspension were streaked onto medium A agar plates. After 3 to 5 days of incubation in the anaerobic isolator, single colonies and mixtures of different types of colonies were subcultured for 5 days in 10 ml of medium A containing 500 ,ug of 13-MCA. None of 250 pure cultures converted 1-MCA into ω-MCA. One of the mixed cultures produced ω-MCA when incubated with P-MCA and contained no more than three or four morphologically different microorganisms. Strains Rl and R3 were isolated from this simplified culture on medium A agar plates. Strain M67 was isolated on medium B agar plates streaked with a loopful of cecal contents from a gnotobiotic mouse associated with a microflora composed exclusively of anaerobic species; this flora originated from the cecum of antibiotic-treated conventional mice and had been provided by D. Van der Waaij. Strain C18 is an atypical Clostridium species of group II (gelatin liquefied, spores subterminal) that had previously been isolated from the cecal conterits of a conventional rat. This strain allows the establishment of fastidious anaerobes in gnotobiotic animals, since it lowers considerably the oxidation-reduction potential of the cecum. Clostridium sp. C18 shows no activity on bile acids in vivo. The isolates were tentatively identified according to the methods described by Holdeman et al., with the following modifications: (i) the basal medium for all tests was medium A for strains R3 and M67; (ii) medium C was used for strain Rl, except for the fermentation of carbohydrates, for which medium A was used; (iii) nitrate reduction was investigated by adding 1% KNO3 to the basal media; (iv) medium A or C agar plates admixed with 10% horse blood were used to study colonial appearance, Gram reaction (KopeloffWs modification), and hemolysis; (v) supplemented brain heart infusion agar slants were used for catalase detection and triple sugar iron agar slants or SIM medium for H2S detection; (vi) motility and indole production were investigated in semisolid medium A or C supplemented with 0.2% DL-tryptophan. (iii) Inoculation of animals. The culture tubes were sealed by heat, and the surface was sterilized with 2% peracetic acid before introduction into the germfree isolators. Strains were administered via the anal route, using a syringe fitted with a soft plastic cannula. The germfree rats were first associated with strain Rl. Subsequent introduction of strain R3 or M67 was unsuccessful. These strains became established only after the previous inoculation of Clostridium sp. C18.

[1]. Eyssen H, De Pauw G, Stragier J, Verhulst A. Cooperative formation of omega-muricholic acid by intestinal microorganisms. Appl Environ Microbiol. 1983 Jan;45(1):141-7.

Omega-muricholic acid is a member of the class of muricholic acids in which the hydroxy groups at positions 6 and 7 have alpha and beta configuration, respectively.

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Until recently, it was uncertain whether ω-MCA was exclusively a tertiary bile acid, i.e., a secondary bile acid that had been converted by the hepatocyte into a chemical species different from primary bile acids. Madsen et al. assumed that ω-MCA could be a tertiary bile acid since they observed that rat hepatocytes can 7p-hydroxylate the secondary bile acid HDCA into ω-MCA. The isolation by Sacquet et al.of a rat intestinal Clostridium sp. which converts 1-MCA into ω-MCA shows that this substance can be considered as a secondary bile acid, since rat hepatocytes were not required for its formation. This is supported by the in vitro conversion of ,B-MCA into ω-MCA by the concerted activity of the bacteria described in the present study. Several findings on bile acid metabolism in rats have pointed to an apparent relationship between the relative amounts of ω-MCA and HDCA, both secondary bile acids arising from 1-MCA. Wostmann et al. have shown that a steam-sterilized, lactose-containing diet fed to conventional rats increases the fecal w-MCAI HDCA ratio; these authors observed a similar shift in this ratio in antibiotic-treated conventional rats. Sacquet et al. observed a significant increase in the ratio when the bile of conventional rats was surgically shunted to the urinary bladder. In addition, Brydon et al. reported an increase of the ratio of 3-MCA + ω-MCA/HDCA in the colon of conventional rats when they were changed from a pelleted to a low-fiber diet. Presumably, these treatments have a notable effect on the intestinal microflora and hence on the microbial bile acid transformations in the intestinal tract. To clearly understand the above-mentioned observations, further investigations are required to elucidate the pathway(s) leading to HDCA and the potential involvement of w-MCA in its formation. The genus Eubacterium comprises several steroid-inverting species, possessing bile acid deconjugase, 3a- or 12a-hydroxysteroid dehydrogenase or both, bile acid 7a-dehydroxylase, corticoid 16a-dehydroxylase, corticoid 21-dehydroxylase and A5-sterol hydrogenase activity. The present E. lentum isolate showed bile acid deconjugase and bile acid hydroxysteroid dehydrogenase activity; bile acid dehydroxylase activity was not observed. Under the conditions of our study, E. lentum Rl developed bile acid 6p-, 7a-, and 12adehydrogenase activities in vitro; 3a-dehydrogenase activity was only observed in vivo. It remains to be established whether the production of 6,- and 7a-hydroxysteroid dehydrogenase activities is specific for E. lentum Rl or whether these characteristics also occur in other E. lentum strains. It also remains to be established whether the absence of 3a-hydroxysteroid dehydrogenase activity might be because our studies were carried out under strict anaerobic conditions with living cultures. In studies with cell-free preparations, Macdonald et al. observed bile salt 3a-dehydrogenase activity in 15 of 32 strains of E. lentum and related organisms. The bacterial formation of a 6-oxo derivative of P-MCA has not been reported heretofore. However, the Clostridium sp. isolated by Sacquet et al. might invert the 6p3-hydroxyl group of P-MCA by means of the same intermediate, since the closely related isomerization reaction of the 6,-hydroxyl group of MCA proceeded through the corresponding 6-oxo intermediate. In fact, the identity of the 6-oxo derivative of P-MCA has only recently been established since feces of germfree rats fed a cholesterol diet contain substantial amounts of this, then primary, bile acid. The in vitro formation of the compound by E. lentum Rl shows that this bile acid can also be a secondary one. Current investigations of the distribution of 6p-hydroxysteroid dehydrogenase among a series of 33 E. lentum strains indicate that this activity is not a common feature of this species since it was found only in strain Rl (Eyssen et al., unpublished data). The association of germfree rats with the E. lentum isolate also gave rise to a monounsaturated bile acid, closely related to ,-muricholate. The data from mass spectrometry indicate that the double bond is in the side chain. Hence, this compound might be identical to the unidentified chemical species which has been found by several authors in the bile of conventional rats. That this bile acid was not found in germ-free rats or in the E. lentum Rl culture media admixed with conjugated ,-MCA suggests that it could be a tertiary bile acid; however, more investigations are required before definite conclusions can be drawn.[1]

C24H40O5

408.57140827179

408.287

6830-03-1

5283851

White to off-white solid powder

1.2±0.1 g/cm3

565.7±40.0 °C at 760 mmHg

310.0±23.8 °C

0.0±3.5 mmHg at 25°C

1.558

Rat intestinal microflora

3.82

4

5

4

29

637

11

C[C@H](CCC(=O)O)[C@H]1CC[C@@H]2[C@@]1(CC[C@H]3[C@H]2[C@H]([C@@H]([C@H]4[C@@]3(CC[C@H](C4)O)C)O)O)C

DKPMWHFRUGMUKF-NTPBNISXSA-N

InChI=1S/C24H40O5/c1-13(4-7-19(26)27)15-5-6-16-20-17(9-11-23(15,16)2)24(3)10-8-14(25)12-18(24)21(28)22(20)29/h13-18,20-22,25,28-29H,4-12H2,1-3H3,(H,26,27)/t13-,14-,15-,16+,17+,18+,20+,21-,22-,23-,24-/m1/s1

(4R)-4-[(3R,5R,6R,7R,8S,9S,10R,13R,14S,17R)-3,6,7-trihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoic acid

omega-muricholic acid; 6830-03-1; (3a,5b,6a,7b)-3,6,7-trihydroxy-Cholan-24-oic acid; omega-Muricholate; omega-MCA; 3a,6a,7b-Trihydroxy-5b-cholanoic acid; (4R)-4-[(3R,5R,6R,7R,8S,9S,10R,13R,14S,17R)-3,6,7-trihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoic acid; 3alpha,6alpha,7beta-Trihydroxy-5beta-cholan-24-oic Acid;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~20 mg/mL (~49 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)