Oxidoreduction of different hydroxyl groups in bile acids during their enterohepatic circulation in man

The extent of oxidoreduction of the 3 alpha-, 7 alpha- and 12 alpha-hydroxyl groups in bile acids during the enterohepatic circulation in man was studied with the use of [3 beta-3H]-labeled deoxycholic acid and cholic acid, [7 beta-3H]-labeled cholic acid, and [12 beta-3H]-labeled deoxycholic acid and cholic acid. Each [3H]-labeled bile acid was given per os to healthy volunteers, together with the corresponding [24-14C]-labeled bile acid. The rate of oxidoreduction was calculated from the decrease in the ratio between 3H and 14C in the respective bile acid isolated from duodenal contents collected at different time intervals after administration of the labeled bile acids. The mean fractional conversion rate was found to be 0.29 day-1 for the 3 alpha-hydroxyl group in deoxycholic acid (n = 2), 0.18 day-1 for the 12 alpha-hydroxyl group in deoxycholic acid (n = 6), 0.09 day-1 for the 3 alpha-hydroxyl group in cholic acid (n = 3), 0.05 day-1 for the 7 alpha-hydroxyl group in cholic acid (n = 2), and 0.03 day-1 for the 12 alpha-hydroxyl group in cholic acid (n = 2). The extent of oxidoreduction of the 12 alpha-hydroxyl group in [12 beta-3H]-labeled deoxycholic acid given to two patients operated with subtotal colectomy and ileostomy was markedly reduced (less than 20% of normal).(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of intestinal 7 alpha-dehydroxylation of cholic acid: evidence that allo-deoxycholic acid is an inducible side-product

We previously reported that the 7 alpha-dehydroxylation of cholic acid appears to be carried out by a multi-step pathway in intestinal anaerobic bacteria both in vitro and in vivo. The pathway is hypothesized to involve an initial oxidation of the 3 alpha-hydroxy group and the introduction of a double bond at C4-C5 generating a 3-oxo-4-cholenoic bile acid intermediate. The loss of water generates a 3-oxo-4,6-choldienoic bile acid which is reduced (three steps) yielding deoxycholic acid. We synthesized, in radiolabel, the following putative bile acid intermediates of this pathway 7 alpha,12 alpha-dihydroxy-3-oxo-4-cholenoic acid, 7 alpha,12 alpha-dihydroxy-3-oxo-5 beta-cholanoic acid, 12 alpha-dihydroxy-3-oxo-4,6-choldienoic acid, and 12 alpha-hydroxy-3-oxo-4-cholenoic acid and showed that they could be converted to 3 alpha,12 alpha-dihydroxy-5 beta-cholanoic acid (deoxycholic acid) by whole cells or cell extracts of Eubacterium sp. VPI 12708. During studies of this pathway, we discovered the accumulation of two unidentified bile acid intermediates formed from cholic acid. These bile acids were purified by thin-layer chromatography and identified by gas-liquid chromatography-mass spectrometry as 12 alpha-hydroxy-3-oxo-5 alpha-cholanoic acid and 3 alpha,12 alpha-dihydroxy-5 alpha-cholanoic (allo-deoxycholic acid). Allo-deoxycholic acid was formed only in cell extracts prepared from bacteria induced by cholic acid, suggesting that their formation may be a branch of the cholic acid 7 alpha-dehydroxylation pathway in this bacterium.     

Evidence for a lack of regulatory importance of the 12 alpha-hydroxylase in formation of bile acids in man: an in vivo study

The possibility that the 12 alpha-hydroxylase involved in formation of bile acids is of regulatory importance for the ratio between cholic acid and chenodeoxycholic acid in bile was studied with an in vivo technique. [4-14C]7 alpha-Hydroxy-4-cholesten-3-one and [6 beta-3H]7 alpha, 12 alpha-dihydroxy-4-cholesten-3-one were synthesized, and a mixture of these two bile acid intermediates was administered intravenously in five healthy subjects and in one patient with severe liver cirrhosis. The patient with liver cirrhosis was included in the study because of a considerable reduction in biosynthesis of cholic acid. Since the [4-14C]-labeled steroid is an intermediate just proximal to and since the [6 beta-3H]-labeled steroid is an intermediate just distal to the 12 alpha-hydroxylase step, the 3H/14C ratio in the cholic acid formed should reflect the relative 12 alpha-hydroxylase activity. The 3H/14C ratio varied between 1.8 and 3.9 in the cholic acid isolated from the healthy subjects and was 3.6 in the cholic acid isolated from the patient with liver cirrhosis. The ratio between cholic acid and chenodeoxycholic acid varied between 0.6 and 3.9 in the bile from the control subjects and was only 0.4 in the bile from patients with liver cirrhosis. There was no correlation between the 3H/14C ratios and the ratios between cholic acid and chenodeoxycholic acid in bile.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of cholic acid via 3 alpha, 7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid in the dog.

The proposed cholic precursor, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-[3H]cholestan-26-oic acid, and [14C]cholesterol were infused intravenously at a constant rate into two dogs for 25 days. If the specific activities of trihydroxy[3H]cholestanoic acid and [3H]cholic acid will be equal after an isotopic steady-state is achieved. The specific activities of [14C]deoxycholic acid (formed from [14C]cholic acid) isolated in the stool of these two dogs were equal the last four days of the infusion indicating that labeled deoxycholic acid (and presumably labeled cholic acid) was in an isotopic steady-state. However, the specific activities of trihydroxy[3H]cholestanoic acid were 3.3 and 5.7 times greater than the specific activities of [3H]cholic acid, respectively. These data suggest that either an alternate route of cholic acid synthesis exists exclusive of trihydroxycholestanoic acid or that an isotopic steady state of trihydroxycholestanoic acid cannot be reached during an infusion of labeled trihydroxycholestanoic acid.     

Formation of C21 bile acids from plant sterols in the rat

Formation of bile acids from sitosterol in bile-fistulated female Wistar rats was studied with use of 4-14C-labeled sitosterol and sitosterol labeled with 3H in specific positions. The major part (about 75%) of the 14C radioactivity recovered as bile acids in bile after intravenous administration of [4-14C]sitosterol was found to be considerably more polar than cholic acid, and only trace amounts of radioactivity had chromatographic properties similar to those of cholic acid and chenodeoxycholic acid. It was shown that polar metabolites were formed by intermediate oxidation of the 3 beta-hydroxyl group (loss of 3H from 3 alpha-3H-labeled sitosterol) and that the most polar fraction did not contain a hydroxyl group at C7 (retention of 3H in 7 alpha,7 beta-3H2-labeled sitosterol). Furthermore, the polar metabolites had lost at least the terminal 6 or 7 carbon atoms of the side chain (loss of 3H from 22,23-3H2- and 24,28-3H2-labeled sitosterol). Experiments with 3H-labeled 7 alpha-hydroxysitosterol and 4-14C-labeled 26-hydroxysitosterol showed that none of these compounds was an efficient precursor to the polar metabolites. By analysis of purified most polar products of [4-14C] sitosterol by radio-gas chromatography and the same products of 7 alpha,7 beta-[2H2]sitosterol by combined gas chromatography-mass spectrometry, two major metabolites could be identified as C21 bile acids. One metabolite had three hydroxyl groups (3 alpha, 15, and unknown), and one had two hydroxyl groups (3 alpha, 15) and one keto group. Considerably less C21 bile acids were formed from [4-14C]sitosterol in male than in female Wistar rats. The C21 bile acids formed in male rats did not contain a 15-hydroxyl group. Conversion of a [4-14C]sitosterol into C21 bile acids did also occur in adrenalectomized and ovariectomized rats, indicating that endocrine tissues are not involved. Experiments with isolated perfused liver gave direct evidence that the overall conversion of sitosterol into C21 bile acids occurs in this organ. Intravenously injected 7 alpha,7 beta-3H-labeled campesterol gave a product pattern identical to that of 4-14C-labeled sitosterol. Possible mechanisms for hepatic conversion of sitosterol and campesterol into C21 bile acids are discussed.     

Quantitative aspects of the conversion of 5 beta-cholestane intermediates to bile acids in man.

The in vivo conversion of several 5 beta-cholestane intermediates to primary bile acids was investigated in three patients with total biliary diversion. The following compounds were administered intravenously: 5 beta-[G-3H]-cholestane-3 alpha, 7 alpha-diol, 5 beta-[G-3H]cholestane-3 alpha, 7alpha, 26-triol, and 5 beta-[24-14C]cholestane-3 alpha, 7 alpha-25-triol. Bile was then collected quantitatively at frequent intervals for the next 21 to 28 h. The administered 5 beta-[G-3H]cholestane-3alpha, 7alpha, 26-triol was found to be efficiently converted to cholic and chenodeoxycholic acids in two patients; 61 and 75% of the administered label was found in primary bile acids. The proportion of labeled cholic to chenodeoxycholic acid was 1.20 and 1.02 in the bile of these patients, indicating that the C-26 triol was efficiently converted to cholic acid. The ratio of cholic to chenodeoxycholic acid (mass) in the bile of these patients was 1.23 and 2.32. The 5 beta-cholestane-3alpha, 7alpha-diol intermediate was also efficiently converted (71%) to both primary bile acids. The cholic to chenodeoxycholic acid ratios by mass and label were similar (2.97 versus 2.23). By contrast, the 5beta-cholestane-3alpha, 7alpha, 25-triol was poorly converted to bile acids in three patients. Following the administration of this compound almost all of the administered radioactivity found in the bile acid fraction was in cholic acid (5 to 19%) and very little (less than 5%) was found in chenodeoxycholic acid. These findings indicate that ring hydroxylation at position 12 is not materially hindered by the presence of a hydroxyl group on the side chain at C-26 in patients with biliary diversion. The labeled C-26-triol which was efficiently converted to both primary bile acids in a proportion similar to that which was observed for the bile acids synthesized by the liver suggests that this 5beta-cholestane derivative may be a major intermediate in the synthesis of both cholic and chenodeoxycholic acids.     

Evidence for a multigene family involved in bile acid 7-dehydroxylation in Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 is a human intestinal isolate which has an inducible bile acid 7-dehydroxylation activity. At least two cholic acid-induced polypeptides, with molecular masses of 27,000 and 45,000 daltons, respectively, coelute with bile acid 7-dehydroxylation activity. The 45,000-dalton polypeptide appears to be encoded by a cholic acid-induced mRNA species of greater than 6 kilobases, which suggests that the gene coding for this polypeptide is part of a larger operon. A gene has been cloned which flanks the gene encoding the 45,000-dalton polypeptide, in the upstream (5') direction. This gene appears to encode a second 27,000-dalton polypeptide. The gene bears striking homology at both the nucleotide (80%) and deduced amino acid sequence (89%) levels with the gene which encodes the 27,000-dalton polypeptide that has been shown previously to be involved in the bile acid 7-dehydroxylation reaction sequence. The implications of this homology and the possible function(s) of the two homologous genes in bile acid 7-dehydroxylation are discussed. Evidence is presented which suggests that the two homologous genes involved in bile acid 7-dehydroxylation may be part of a larger multigene family in Eubacterium sp. strain VPI 12708.     

Assessment of fecal bacteria with bile acid 7 alpha-dehydroxylating activity for the presence of bai-like genes.

Eubacterium sp. strain VPI 12708 has several bile acid-inducible (bai) genes which encode enzymes in the bile acid 7 alpha-dehydroxylation (7 alpha DeOH) pathway. Twelve 7 alpha DeOH-positive intestinal bacterial strains were assayed for 7 alpha DeOH activity, and 13 strains were tested for hybridization with bai genes. Cholic acid 7 alpha DeOH activity varied greatly (> 100-fold) among these strains. Southern blot experiments showed that DNA prepared from 7 of 13 strains hybridized with at least one of the bai genes from Eubacterium sp. strain VPI 12708.     

Bile acid induction specificity of 7α-dehydroxylase activity in an intestinal Eubacterium species

The addition of cholic acid to growing cultures of $\text{Eubacterium}$ species V.P.I. 12708 caused a 25 and 46-fold increase in 7α-dehydroxylation activity using cell extracts or whole cell suspensions, respectively. Bile acid conversion rates using either [¹⁴C]-cholic acid or [¹⁴C]-chenodeoxycholic acid as substrates increased at approximately the same rate when either cholic or chenodeoxycholic acid was added to growing cultures as inducer. The induction of 7α-dehydroxylase activity was highly specific requiring a free C-24-carboxyl group and an unhindered 7α-hydroxy group on the B ring of the steroid nucleus. Unexpectedly, cholic acid also rapidly induced NADH:flavin oxidoreductase activity in growing cultures of this bacterium.     

Difference between cholic acid and chenodeoxycholic acid in dependence upon cholesterol of hepatic and plasmatic sources as the precursor in rats

Some difference in functional pool of cholesterol acting as the precursor of bile acids is pointed out between cholic acid and chenodeoxycholic acid. In order to elucidate this problem further, some experiments were performed with rats equilibrated with [7(n)-3H, 4-(14)C] cholesterol by subcutaneous implantation. The bile duct was cannulated in one series of experiments and ligated in another. After the operation 14C-specific radioactivity of serum cholesterol fell, but reached practically a new equilibrium within three days. 14C-Specific radioactivity of serum cholesterol as well as of biliary bile acids in bile-fistula rats and urinary bile acids in bile duct-ligated rats was determined during a three days-period in the new equilibrated state. The results were as follows: (1) 14C-Specific radioactivity of cholic acid and chenodeoxycholic acid in bile was lower than that of serum cholesterol, and 14C-specific radioactivity of cholic acid was clearly lower than that of chenodeoxycholic acid. (2) 14C-Specific radioactivity of cholic acid and beta-muricholic acid in urine was lower than that of serum cholesterol, and 14C-specific radioactivity of cholic acid was lower than that of beta-muricholic acid. (3) Biliary as well as urinary beta-muricholic acid lost tritium label at 7-position entirely during the course of formation from [7(n)-3H, 4-(14)C]cholesterol.     

Metabolism of bile acid oxazoline derivatives by hepatocyte monolayer cultures and intestinal anaerobic bacteria

Certain bile acid oxazoline derivatives (100 microM), but not corresponding unconjugated bile acids (100 microM), were found to inhibit the growth of Eubacterium sp. V.P.I. 12708. The growth inhibition was correlated with the polarity of the steroid portion of the bile acid oxazoline. Primary cultures of adult rat hepatocyte monolayer cultures converted [7 epsilon-14C]methylchenooxazoline3 into MeOH-H2O soluble derivatives. Certain intestinal bacteria were capable of metabolizing [17 epsilon-14C]methylchenooxazoline as well as the MeOH-soluble hepatocyte derivative(s). These results suggest that bile acid oxazoline derivatives may undergo hepatic, as well as bacterial metabolism during enterohepatic circulation.     

Suitability of primary monolayer cultures of adult rat hepatocytes for studies of cholesterol and bile acid metabolism

Monolayer cultures of hepatocytes isolated from cholestyramine-fed rats and incubated in serum-free medium converted exogenous [4-14C]cholesterol into bile acids at a 3-fold greater rate than did cultures of hepatocytes prepared from untreated rats. Cholic acid and beta-muricholic acid identified and quantitated by gas-liquid chromatography and thin-layer chromatography were synthesized by cultured cells for at least 96 h following plating. The calculated synthesis rate of total bile acids by hepatocytes prepared from cholestyramine-fed animals was approximately 0.058 micrograms/mg protein/h. beta-Muricholic acid was synthesized at approximately a 3-fold greater rate than cholic acid in these cultures. Cultured hepatocytes rapidly converted the following intermediates of the bile acid pathway; 7 alpha-hydroxy[7 beta-3H]cholesterol, 7 alpha-hydroxy-4-[6 beta-3H] cholesten-3-one, and 5 beta-[7 beta-3H]cholestane-3 alpha, 7 alpha, 12 alpha-triol into bile acids. [24-14C]Chenodeoxycholic acid and [3H]ursodeoxycholic acid were rapidly biotransformed to beta-muricholic acid. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase activity measured in microsomes of cultured hepatocytes decreased during the initial 48 h following plating, but remained relatively constant for the next 72 h. In contrast, cholesterol 7 alpha-hydroxylase activity appeared to decrease during the first 48 h, followed by an increase over the next 48 h. Despite the apparent changes in enzyme activity in vitro, the rate of bile acid synthesis by whole cells during this time period remained constant. It is concluded that primary monolayer cultures of rat hepatocytes can serve as a useful model for studying the interrelationship between cholesterol and bile acid metabolism.     

Metabolism of bile alcohols in the perfused rabbit liver.

The mechanism and sequence of side chain hydroxylation of cholesterol in bile acid synthesis was studied in the isolated perfused rabbit liver. A comparison was made between the importance of 26- and 25-hydroxylation in cholic acid biosynthesis in the rabbit. The formation of [G-3H]cholic acid was observed when the liver was perfused with 5beta-[G-3H]cholestane-3alpha, 7alpha-diol, 5beta-[G-3H]cholestane-3alpha, 7alpha-12alpha-triol, and 5beta-[G-3H]cholestane-3alpha, 7alpha, 26-triol. No [G-3H]chenodeoxycholic acid was detected in the bile. These findings indicate that potential precursors of chenodeoxycholic acid were hydroxylated at position 12alpha either subsequent to or before hydroxylation of the cholesterol side chain. In addition, no other intermediates (tetrahydroxy or pentahydroxy bile alcohols) were found in the bile when these compounds were perfused in the liver. Bile acid precursors were detected in bile when the rabbit liver was perfused with 5beta-[24-14C]cholestane-3alpha, 7alpha, 25-triol. The 5beta-[24-14C]cholestane-3alpha, 7alpha, 25-triol was hydroxylated in the liver at the 12alpha position to yield the corresponding 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol. The tetrol was further metabolized to a series of pentols (5beta-cholestane-3alpha, 7alpha, 12alpha, 22, 25-pentol; 5beta-cholestane-3alpha, 7alpha, 12alpha, 23, 25-pentol; 5beta-cholestane-3alpha, 7alpha, 12alpha, 24, 25-pentol; and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25, 26-pentol). The major bile acid obtained from the perfusion of the 5beta-cholestane-3alpha, 7alpha, 25-triol was cholic acid. The experiments indicated that in the rabbit liver 12alpha-hydroxylation can occur after hydroxylation of the cholesterol side chain at either C-25 (5 beta-cholestane-3alpha, 7alpha, 25-triol) or C-26 (5beta-cholestane-3alpha, 7alpha-26-triol). Apparently, the rabbit can form cholic acid via the classical 26-hydroxylation pathway as well as via 25-hydroxylated intermediates.     

Biosynthesis of a novel bile acid nucleotide and mechanism of 7 alpha-dehydroxylation by an intestinal Eubacterium species

Eubacterium species V.P.I. 12708 has inducible bile acid 7-dehydroxylase activity that can use either 7 alpha or 7 beta bile acids as substrates. Cell extracts prepared from bacteria grown in the presence of cholic acid catalyzed the rapid conversion of free bile acids into a highly polar bile acid metabolite (HPBA). This conjugation activity co-eluted with bile acid 7-dehydroxylase activity on high performance gel filtration chromatography (GFC). The HPBA was purified by a combination of high performance GFC and reverse-phase high performance liquid chromatography (HPLC). The intact HPBA eluted earlier from reverse-phase HPLC than deoxycholyl-CoA and had a Mr of 1102 by Bio-Gel P-2 (GFC). The HPBA had an absorption peak at 255 nm and was sensitive to treatment with phosphodiesterase I or nucleotide pyrophosphatase. The HPBA has a free phosphate as shown by an increase in elution volume on reverse-phase HPLC following treatment with alkaline phosphatase. Treatment of the purified HPBA with nucleotide pyrophosphate plus alkaline phosphatase yielded adenosine, whereas, treatment with nucleotide pyrophosphatase alone generated 5',3'-ADP. A bile acid metabolite was also generated by nucleotide pyrophosphatase treatment. The bile acid metabolite had different chromatographic properties (HPLC and TLC) than the corresponding free bile acid. Gas liquid chromatography-mass spectrometry showed the bile acid metabolite to be 12 alpha-hydroxy-3-oxo-4-cholenoic acid. We hypothesize that the HPBA is an intermediate in 7-dehydroxylation and consists of this compound linked at the C-24 with an anhydride bond to the beta phosphate (5') of ADP-3'-phosphate. These results suggest a novel mechanism of bile acid 7 alpha/7 beta-dehydroxylation in Eubacterium sp. V.P.I. 12708.     

Side chain conjugation prevents bacterial 7-dehydroxylation of bile acids

The effect of side chain conjugation on 7-dehydroxylation of bile acids has been investigated. C24-bile acids and their glycine and taurine conjugates and keto bile acids were incubated with pure strains of Eubacterium sp. VPI 12708. Bile acids of the 5 alpha- or 5 beta-series with a free terminal carboxyl group and a 3 alpha, 7 alpha-dihydroxy system were very effectively 7 alpha-dehydroxylated, whereas 7 beta-hydroxy bile acids resisted 7-dehydroxylation. Oxo bile acids were metabolized at the oxygen function also. Glycine- and taurine-conjugated bile acids were neither deamidated nor 7-dehydroxylated by the bacteria. Thus, side chain conjugation prevents 7-dehydroxylation of bile acids by Eubacterium sp. VPI 12708.     

In vitro formation of bile acids from di- and trihydroxy-5 beta-cholestanoic acid in human liver peroxisomes

The conversion of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-[3H]cholestanoic acid into cholic acid and 3 alpha,7 alpha-dihydroxy-5 beta-[3H]cholestanoic acid into chenodeoxycholic acid has been studied in subcellular fractions of human liver. The products were separated from the substrates by high-pressure liquid chromatography and identified by combined gas chromatography-mass spectrometry. The highest rates of conversion were found in the light mitochondrial fraction. This fraction also contained the highest amount of the marker enzymes for peroxisomes. The maximal rates of cholic acid and chenodeoxycholic acid formation were 1.3 and 1.8 nmol/mg protein per h, respectively. The presence of KCN in the incubation medium stimulated the formation of bile acids. Peroxisomes were prepared from the light mitochondrial fraction by sucrose-gradient centrifugation. By use of different marker enzymes, it was confirmed that the major part of the activity for cholic acid formation in the light mitochondrial fraction was located in the peroxisomes. It is concluded that liver peroxisomes are important for the oxidative cleavage of the C27 steroid side chain in bile acid formation in man.     

Determination of deoxycholic acid pool size and input rate using [24-13C]deoxycholic acid and serum sampling

We have developed an isotope dilution method for determination of deoxycholic acid pool size and input rate which employs oral administration of 50 mg of [24-13C]deoxycholic acid and serum sampling. The method has been validated by classical isotope dilution technique using [24-14C]deoxycholic acid and bile sampling in five patients with colonic adenomas. Excellent agreement between pool sizes and input rates determined with 13C/12C isotope ratio measurements in serum and 14C measurements in bile was obtained when isotope ratios were measured in the conjugated fraction of deoxycholic acid in serum. We conclude that pool size and input rate of deoxycholic acid can accurately be determined by blood sampling after oral administration of [24-13C]deoxycholic acid, therewith eliminating the use of radioactive tracers and the need for bile sampling.     

The bile acid-inducible baiB gene from Eubacterium sp. strain VPI 12708 encodes a bile acid-coenzyme A ligase.

The baiB gene from Eubacterium sp. strain VPI 12708 was previously cloned, sequenced, and shown to be part of a large bile acid-inducible operon encoding polypeptides believed to be involved in bile acid 7 alpha-dehydroxylation. In the present study, the baiB gene was subcloned and expressed in Escherichia coli and shown to encode a bile acid-coenzyme A (CoA) ligase. This ligase required a C-24 bile acid with a free carboxyl group, ATP, Mg2+, and CoA for synthesis of the final bile acid-CoA conjugate. Product analysis by reverse-phase high-performance liquid chromatography revealed final reaction products that comigrated with cholyl-CoA and AMP. A putative bile acid-AMP intermediate was detected when CoA was omitted from the reaction mixture. The bile acid-CoA ligase has amino acid sequence similarity to several other polypeptides involved in the ATP-dependent linking of AMP or CoA to cyclic carboxylated compounds. The bile acid-CoA ligation is believed to be the initial step in the bile acid 7 alpha-dehydroxylation pathway in Eubacterium sp. strain VPI 12708.