Novel bile acid biosynthetic pathways are enriched in the microbiome of centenarians.

Centenarians have a decreased susceptibility to ageing-associated illnesses, chronic inflammation and infectious diseases1-3. Here we show that centenarians have a distinct gut microbiome that is enriched in microorganisms that are capable of generating unique secondary bile acids, including various isoforms of lithocholic acid (LCA): iso-, 3-oxo-, allo-, 3-oxoallo- and isoallolithocholic acid. Among these bile acids, the biosynthetic pathway for isoalloLCA had not been described previously. By screening 68 bacterial isolates from the faecal microbiota of a centenarian, we identified Odoribacteraceae strains as effective producers of isoalloLCA both in vitro and in vivo. Furthermore, we found that the enzymes 5α-reductase (5AR) and 3ß-hydroxysteroid dehydrogenase (3ß-HSDH) were responsible for the production of isoalloLCA. IsoalloLCA exerted potent antimicrobial effects against Gram-positive (but not Gram-negative) multidrug-resistant pathogens, including Clostridioides difficile and Enterococcus faecium. These findings suggest that the metabolism of specific bile acids may be involved in reducing the risk of infection with pathobionts, thereby potentially contributing to the maintenance of intestinal homeostasis.

Enhanced activity and substrate tolerance of 7α-hydroxysteroid dehydrogenase by directed evolution for 7-ketolithocholic acid production.

7-Ketolithocholic acid (7-KLCA) is an important intermediate for the synthesis of ursodeoxycholic acid (UDCA). UDCA is the main effective component of bear bile powder that is used in traditional Chinese medicine for the treatment of human cholesterol gallstones. 7α-Hydroxysteroid dehydrogenase (7α-HSDH) is the key enzyme used in the industrial production of 7-KLCA. Unfortunately, the natural 7α-HSDHs reported have difficulty meeting the requirements of industrial application, due to their poor activities and strong substrate inhibition. In this study, a directed evolution strategy combined with high-throughput screening was applied to improve the catalytic efficiency and tolerance of high substrate concentrations of NADP+-dependent 7α-HSDH from Clostridium absonum. Compared with the wild type, the best mutant (7α-3) showed 5.5-fold higher specific activity and exhibited 10-fold higher and 14-fold higher catalytic efficiency toward chenodeoxycholic acid (CDCA) and NADP+, respectively. Moreover, 7α-3 also displayed significantly enhanced tolerance in the presence of high concentrations of substrate compared to the wild type. Owing to its improved catalytic efficiency and enhanced substrate tolerance, 7α-3 could efficiently biosynthesize 7-KLCA with a substrate loading of 100 mM, resulting in 99% yield of 7-KLCA at 2 h, in contrast to only 85% yield of 7-KLCA achieved for the wild type at 16 h.

[Effects of intestinal flora on the expression of cytochrome P450 3A in the liver].

Living organisms eliminate foreign low-antigenic substances, such as drugs and environmental pollutants, by detoxification mediated by metabolizing cytochrome P450 (CYP). We have examined the possible regulation of CYP expression by enteric bacteria. Cyp mRNA expression levels, Cyp3a protein expression level, and the activity of Cyp3a in hepatic microsomal fractions were compared in germ-free (GF) and specific pathogen-free (SPF) mice. We evaluated hepatic Cyp3a11 mRNA expression levels and Cyp3a metabolic activity in GF and SPF mice after five days of antibiotic administration. The fecal levels of lithocholic acid (LCA)-producing bacteria and hepatic taurolithocholic acid (TLCA) were also measured. Cyp mRNA expression levels, Cyp3a protein expression level, and the activity of Cyp3a in SPF mice were higher than those in GF mice, indicating that enteric bacteria increases hepatic Cyp3a expression. The effects of enteric bacteria-reducing antibiotics on Cyp3a expression were examined. We observed that decreasing enteric bacteria with antibiotics in SPF mice caused a significant decrease in the hepatic Cyp3a11 mRNA expression, TLCA, and fecal LCA-producing bacteria compared to the group that did not receive antibiotics. No change in Cyp3a11 expression was observed in GF mice that were treated with antibiotics. Administration of LCA to GF mice showed an increase in Cyp3a11 expression similar to that of SPF mice. The enzymes of the enteric bacteria are believed to metabolize and detoxify drugs by either reduction or hydrolysis. The results of this study indicate that changes in enteric bacteria may alter the expression and activity of hepatic drug metabolizing enzymes and pharmacokinetics. Therefore, enteric bacteria should be closely monitored to ensure the safe use of drugs.

Antibiotics suppress Cyp3a in the mouse liver by reducing lithocholic acid-producing intestinal flora.

We previously demonstrated that ciprofloxacin (CPX), a new quinolone antibiotic, suppresses Cyp3a in the mouse liver by reducing the hepatic level of lithocholic acid (LCA) produced by intestinal flora. The present study investigated the possibility that other antibiotics with antibacterial activity against LCA-producing bacteria also cause a decrease in the LCA level in the liver, leading to reduced expression of Cyp3a11. While the mRNA expression of Cyp3a11 in the liver was significantly reduced when SPF mice were administered antibiotics such as ampicillin, CPX, levofloxacin, or a combination of vancomycin and imipenem, no significant changes were observed after antibiotic treatment of GF mice lacking intestinal flora. LCA-producing bacteria in the feces as well as the hepatic level of the taurine conjugate of LCA were significantly reduced in the antibiotic-treated SPF mice, suggesting that the decrease in Cyp3a11 expression can be attributed to the reduction in LCA-producing intestinal flora following antibiotic administration. These results suggest that the administration of antibiotics with activity against LCA-producing bacteria can also cause a decrease in the LCA level in humans, which may lower CYP3A4 expression. The intestinal flora are reported to be altered not only by drugs, such as antibiotics, but also by stress, disease, and age. The findings of the present study suggest that these changes in intestinal flora could modify CYP expression and contribute to the individual differences in pharmacokinetics.

Tauroursodeoxycholate increases rat liver ursodeoxycholate levels and limits lithocholate formation better than ursodeoxycholate.

BACKGROUND & AIMS: To explain the greater hepatoprotective effect of tauroursodeoxycholic acid vs. ursodeoxycholic acid, the absorption, hepatic enrichment, and biotransformation of these bile acids (250 mg/day) were compared in rats. METHODS: Bile acids were determined in intestinal contents, feces, urine, plasma, and liver by gas chromatography-mass spectrometry. RESULTS: The concentration of ursodeoxycholate in the liver of animals administered tauroursodeoxycholic acid (175 +/- 29 nmol/g) was greater (P < 0.05) than in animals administered ursodeoxycholic acid (79 +/- 19 nmol/g). Hepatic lithocholate was substantially higher after ursodeoxycholic acid administration (21 +/- 10 nmol/g) than after tauroursodeoxycholic acid administration (12 +/- 1 nmol/g). A concomitant reduction in the proportion of hydrophobic bile acids occurred that was greatest during tauroursodeoxycholic acid administration. In the intestinal tract, the mass of ursodeoxycholate and its specific metabolites was greater in rats administered tauroursodeoxycholic acid (27.2 mg) than those administered ursodeoxycholic acid (13.2 mg). In feces, the proportion of lithocholate was 21.9% +/- 4.9% and 5.4% +/- 4.0% after ursodeoxycholic acid and tauroursodeoxycholic acid administration, respectively. CONCLUSIONS: Compared with ursodeoxycholic acid, tauroursodeoxycholic acid induces a greater decrease in the percent composition of more hydrophobic bile acids within the pool, limits lithocholate formation, and increases hepatic ursodeoxycholate concentration. These differences are explained by increased hepatic extraction and reduced intestinal biotransformation and not by enhanced absorption of the amidated species.

Formation, absorption, and biotransformation of delta 6-lithocholenic acid in humans.

delta 6-Lithocholenic acid was identified in small amounts in fecal samples in vitro after incubation with ursodeoxycholic acid and in vivo in controls and after chenodeoxycholic and ursodeoxycholic acid ingestion. Fourteen to 45.0% of delta 6-[24-14C]lithocholenic acid was biotransformed in vitro in feces within 30 s. After colonic instillation of delta 6-[24-14C]lithocholenic acid, 50% of the radioactivity appeared in bile acids, most of it in lithocholic acid, within 3 h. Jejunal perfusions with delta 6-[24-14C]lithocholenic acid showed 33-92% absorption. One hour after jejunal instillation of 1 mmol, 4.4-27.5% of the biliary radioactivity was found in ursodeoxycholic, chenodeoxycholic, lithocholic, and 7-ketolithocholic acids. A sulfated glycine conjugate of delta 6-lithocholenic acid was identified in bile. One hour after intravenous injection of delta 6-[24-14C]lithocholenic acid, 40.1-42.6% of biliary radioactivity appeared in 7-ketolithocholic, chenodeoxycholic, lithocholic/isolithocholic, and ursodeoxycholic acids. The studies show that delta 6-lithocholenic acid is 1) formed in colonic lumen from chenodeoxycholic and ursodeoxycholic acids, 2) well absorbed in small intestine, and 3) biotransformed in both the colonic lumen and liver. The studies also identified delta 6-lithocholenic acid as a new intermediate in formation of lithocholic acid. Finally, the studies showed that a small portion of delta 6-lithocholenic acid is excreted as a sulfated glycine conjugate in bile.

Faecal steroids and colorectal cancer: the effect of lactulose on faecal bacterial metabolism in a continuous culture model of the large intestine.

The effect of lactulose on intestinal bacterial metabolism in two identical single-stage chemostats has been studied. The study was designed as a single stage crossover. Both cultures were inoculated and treated in an identical manner except that whilst one fermenter was subject to pH control the other was not and vice versa. Complex bacterial populations were maintained for 23 days and many of the metabolic functions carried out by the micro-organisms in vivo were demonstrated in vitro. The predominant organisms belonged to the genera Bacteroides, Bifidobacterium and Clostridium with abundant levels of anaerobic Gram-positive rods. The redox potential in each fermenter showed considerable variation with maximum values of below -300 eV being attained. An indication of the stability of the bacterial communities was the production of short-chain volatile fatty acids; during steady-state conditions the mean ratio of the major acids acetic, propionic and butyric being 3.90:0.69:1.00 and 3.65:0.76:1.00 in each fermenter, respectively. During steady-state conditions, 7 alpha-dehydroxylation of the primary bile acid chenodeoxycholic acid was maintained at a constant rate with lithocholic acid representing over 85% of total steroid. Addition of the soluble fibre lactulose to the cultures had a profound effect on many of the parameters tested. Without pH control the culture pH dropped to below 5.0 and this coincided with a substantial decrease in total anaerobes, especially Bacteroides spp., an increase in Lactobacillus spp. (concomitant with an increase in lactic acid), a decrease in the concentration of short-chain volatile fatty acids and a substantial decrease in 7 alpha-dehydroxylation of chenodeoxycholic acid. These results show that it is possible to maintain viable intestinal bacteria in vitro using a continuous culture chemostat and that the cultures respond to changing conditions as evinced by the inhibition of 7 alpha-dehydroxylation of chenodeoxycholic acid on addition of lactulose. This indicates that the model may be of further use in studying the modulation of secondary bile acid formation, a possible risk factor in colorectal carcinogenesis.

Metabolism of lithocholic acid in the rat: formation of lithocholic acid 3-O-glucuronide in vivo.

Milligram amounts of [3 beta-3H]lithocholic (3 alpha-hydroxy-5 beta-cholanoic) acid were administered by intravenous infusion to rats prepared with a biliary fistula. Analysis of sequential bile samples by thin-layer chromatography (TLC) demonstrated that lithocholic acid glucuronide was present in bile throughout the course of the experiments and that its secretion rate paralleled that of total isotope secretion. Initial confirmation of the identity of this metabolite was obtained by the recovery of labeled lithocholic acid after beta-glucuronidase hydrolysis of bile samples. For detailed analysis of biliary metabolites of [3H]lithocholic acid, pooled bile samples from infused rats were subjected to reversed-phase chromatography and four major labeled peaks were isolated. After complete deconjugation, the two major compounds in the combined first two peaks were identified as murideoxycholic (3 alpha, 6 beta-dihydroxy-5 beta-cholanoic) and beta-muricholic (3 alpha, 6 beta, 7 beta-trihydroxy-5 beta-cholanoic) acids and the third peak was identified as taurolithocholic acid. The major component of the fourth peak, after isolation, derivatization (to the methyl ester acetate), and purification by high pressure liquid chromatography (HPLC), was positively identified by proton nuclear magnetic resonance as lithocholic acid 3 alpha-O-(beta-D-glucuronide). These studies have shown, for the first time, that lithocholic acid glucuronide is a product of in vivo hepatic metabolism of lithocholic acid in the rat.

Reduction of 3 alpha-hydroxy-5 beta-chol-6-en-24-oic acid to lithocholic acid in rats.

After [24-14C]delta 6-lithocholic acid was injected into the cecum of rats, [14C]lithocholic acid was identified as a metabolite in feces. When the labeled delta 6-bile acid was injected intraperitoneally into bile-fistula rats, radioactivity excreted in bile was contained most abundantly in the taurine-conjugated fraction of bile acids. In the fraction, taurine conjugate of [14C]delta 6-lithocholic acid but of neither [14C]lithocholic acid nor other bile acids was found. The results showed that [24-14C]delta 6-lithocholic acid was reduced to [14C]lithocholic acid by the intestinal flora but not by the liver, which, however, was capable of conjugating delta 6-lithocholic acid with taurine.

Reduction of 3-keto-5 beta-cholanoic acid to lithocholic and isolithocholic acids by human liver cytosol in vitro.

The formation of lithocholic and isolithocholic acids from 3-keto-5 beta-cholanoic acid by human liver cytosol was examined in vitro. Liver cytosol was incubated at various pH levels with 3-keto-5 beta-cholanoic acid in a phosphate buffer containing NADPH or NADH; the products formed were analyzed by gas chromatography. Results showed that human liver cytosol reduced 3-keto-5 beta-cholanoic acid to lithocholic acid at a pH level of 7.0 or above and to isolithocholic acid at a pH level of 6.0 or below when NADPH was used as a coenzyme, and it was reduced to isolithocholic acid only when NADH was used. Furthermore, two peaks for the reducing enzymes could be clearly found by column chromatography of Affi-Gel Blue. These results indicate that human liver cytosol contains two enzymes acting on reduction of 3-keto-5 beta-cholanoic acid to lithocholic and isolithocholic acids, which are dependent on the pH level and the use of NADPH or NADH in vitro. Since the 3 beta-dehydrogenation was inhibited by the addition of pyrazole, an alcohol dehydrogenase inhibitor or ethanol, and the major peak of 3 beta-hydroxysteroid dehydrogenase coincided with the peak of alcohol dehydrogenase on Affi-Gel Blue chromatography, at least some of the cytosolic 3 beta-hydroxysteroid dehydrogenase seemed to be identical to or to have characteristics similar to alcohol dehydrogenase.

Changes in bile salt composition after cholecystectomy and ileal resection.

The effect of ileal resection and cholecystectomy on bile salt metabolism was studied in female prairie dogs 4 weeks after either a sham laparotomy, cholecystectomy, ileal resection, or cholecystectomy and ileal resection. Bile was collected from a common bile duct cannula at hourly intervals for 12 hours. Pool sizes and synthetic rates of primary and secondary bile salts were determined from washout curves. Cholate, chenodeoxycholate, deoxycholate, and lithocholate levels were determined by gas chromatography from pooled collections of bile. After cholecystectomy and ileal resection, the pool sizes of primary and secondary bile salts were significantly reduced to amounts that were much less than the pool sizes after either procedure alone. Primary bile salt synthesis was significantly increased after combined cholecystectomy and ileal resection, to the same degree as cholecystectomy alone. After the combined procedures, there was a decrease in the proportion of cholate in hepatic bile associated with an increase in chenodeoxycholate, deoxycholate, and lithocholate levels. The data suggest that after the loss of both ileum and gallbladder the bile salt pool sizes are drastically reduced, the synthesis of primary bile salts is increased, and the proportion of secondary bile salts is increased. Cholecystectomy should be avoided, if possible, in patients with ileal resection in order to conserve the bile salt pool and prevent severe fat malabsorption.

Isolation and identification of delta 6-lithocholenic acid (3 alpha-hydroxy-5 beta-6-cholen-24-oic acid) as an intestinal bacterial metabolite of chenodeoxycholic acid in man.

Fecal bacterial biotransformation studies of chenodeoxycholic acid were performed. Incubations were carried out for 30-s to 12-h time intervals. delta 6-Lithocholenic acid was isolated by thin-layer chromatography. Its structure was confirmed by gas-liquid chromatography and mass spectrometry. The proportion of chenodeoxycholic acid biotransformed to delta 6-lithocholenic acid consistently ranged from 5.5 to 14.0%.

Effect of bile acid analogs on 7 alpha-dehydroxylase activity in Eubacterium sp. V.P.I. 12708.

7 beta-Methyl-chenodeoxycholic acid (7-MeCDC, 3 alpha, 7 alpha-dihydroxy-7 beta-methyl-5 beta-cholan-24-oic acid), 7 alpha-methyl-ursodeoxycholic acid (7-MeUDC, 3 alpha, 7 beta-dihydroxy-7 alpha-methyl-5 beta-cholan-24-oic acid), 7 xi-methyl-lithocholic acid (7-MeLC, 3 alpha-hydroxy-7 xi-methyl-5 beta-cholan-24-oic acid) and ursodeoxycholylsarcosine (UDCS) were tested as inhibitors of bacterial bile acid 7 alpha-dehydroxylase activity. At a concentration of 50 microM, 7-MeCDC and 7-MeUDC inhibited enzyme activity by 66% and 12%, respectively. 7 alpha-Dehydroxylase activity was not inhibited in the presence of 7-MeLC and UDCS. None of the four bile acid analogs tested inhibited the growth of Eubacterium sp. V.P.I. 12708 at concentrations up to 100 microM.