Rapid feedback inhibition of endogenous cholic and chenodeoxycholic acid synthesis by exogenous chenodeoxycholic acid in man.

Chenodeoxycholic acid (300 mg + ¹⁴C) was administered orally to a bile fistula patient receiving a constant infusion of {³H}mevalonic acid. Suppression of endogenous cholic and chenodeoxycholic acid synthesis occurred within 2 to 4 hours and continued for the next 10 hours; synthesis returned to the baseline level after 18 hours. Incorporation of {³H}mevalonic acid into both bile acids was also greatly reduced during the first several hours after chenodeoxycholic acid, but almost recovered by 5 hours. The data suggest that multiple feedback sites are involved in the regulation of bile acid synthesis in man.     

7-Methyl bile acids: 7 beta-methyl-cholic acid inhibits bacterial 7-dehydroxylation of cholic acid and chenodeoxycholic acid in the hamster

The effect of dietary 7 beta-methyl-cholic acid [0.075% in rodent chow (6.4 mg/animal per day)] on cholesterol and bile acid metabolism was studied and compared with that of cholic acid in the hamster. Following oral administration of 7 beta-methyl-cholic acid for 3 weeks, the glycine-conjugated bile acid analog became a major constituent of gallbladder bile. Biliary cholic acid concentration decreased significantly, while that of chenodeoxycholic acid remained unchanged. Serum and liver cholesterol levels were increased by dietary 7 beta-methyl-cholic acid and by cholic acid. Hepatic microsomal HMG-CoA reductase activity was inhibited (30% of the control value) by both bile acids; cholesterol 7 alpha-hydroxylase activity was not affected. In chow controls and cholic acid-fed animals, bacterial 7-dehydroxylation of [14C]chenodeoxycholic acid and [14C]cholic acid was nearly complete. In contrast, dietary 7 beta-methyl-cholic acid effectively prevented the 7-dehydroxylation of the two primary bile acids. These results show that dietary 7 beta-methyl-cholic acid is preserved in the enterohepatic circulation and has an effect on serum and liver cholesterol concentrations similar to those produced by the naturally occurring cholic acid. 7 beta-Methyl-cholic acid is an efficient inhibitor of the bacterial 7-dehydroxylation of the primary bile acids in the hamster.     

Transformation of cholic acid by Clostridium bifermentans

Suspensions of washed, resting-stage cells of Clostridium bifermentans were incubated with cholic acid under a variety of conditions in an attempt to manipulate the competing 7α-dehydroxylation and 7α-dehydrogenation reactions. Important variables thus identified were then tested in batch fermentation. Sodium thioglycollate was observed to inhibit dehydrogenation and to delay dehydroxylation. Zinc ion inhibited both dehydrogenation and dehydroxylation. In the presence of EDTA, near quantitative 7α-dehydrogenation was achieved with no associated dehydroxylation. Finally, the presence of air in the fermenter headspace enhanced both transformations, possibly through an effect on the redox potential of the medium.     

Preparation of the 3-monosulphates of cholic acid, chenodeoxycholic acid and deoxycholic acid.

Extraction with butan-1-ol of freeze-dried microsomal fractions from livers of 3-methyl-cholarthrene-pre-treated hamsters removed about 90% of the total lipid content, but the lipid remaining proved sufficient for the cytochrome P-450 enzyme system to retain about 15-40% of its original catalytic activity for dimethylnitrosamine demethylation. Addition of butan-1-ol-extracted total phospholipid or phosphatidylcholine could not restore any activity, whereas the addition of the synthetic phospholipid dilauroyl phosphatidylcholine was able to restore almost complete activity. Synthetic dipalmitoyl or distearoyl phosphatidylcholine was ineffective in restoring the activity in this reconstituted system.     

Conversion of cholic acid and chenodeoxycholic acid into their 7-oxo derivatives by Bacteroides intestinalis AM-1 isolated from human feces

Secondary bile acid-producing bacteria were isolated from human feces to improve our appreciation of the functional diversity and redundancy of the intestinal microbiota. In total, 619 bacterial colonies were isolated using a nutrient-poor agar medium and the level of secondary bile acid formation was examined in each by a liquid culture, followed by thin-layer chromatography. Of five strains analyzed by 16S rRNA gene sequencing and biochemical testing, one was identified as Bacteroides intestinalis AM-1, which was not previously recognized as a secondary bile-acid producer. GC-MS revealed that B. intestinalis AM-1 converts cholic acid (CA) and chenodeoxycholic acid into their 7-oxo derivatives, 7-oxo-deoxycholic acid (7-oxo-DCA) and 7-oxo-lithocholic acid, respectively. Thus, B. intestinalis AM-1 possesses 7α-hydroxysteroid dehydrogenase (7α-HSDH) activity. In liquid culture, B. intestinalis AM-1 showed a relatively higher productivity of 7-oxo-DCA than Escherichia coli HB101 and Bacteroides fragilis JCM11019^T, which are known to possess 7α-HSDH activity. The level of 7α-HSDH activity was higher in B. intestinalis AM-1 than in the other two strains under the conditions tested. The 7α-HSDH activity in each of the three strains is not induced by CA; instead, it is regulated in a growth phase-dependent manner.     

Tauroconjugation of cholic acid stimulates 7 alpha-dehydroxylation by fecal bacteria.

We examined the effect of the type of cholic acid conjugation (taurine-conjugated, glycine-conjugated, or unconjugated cholic acid) on cholic acid 7 alpha-dehydroxylation by intestinal flora. Cholic acid 7 alpha-dehydroxylation in fecal cultures, in cultures of a defined limited flora consisting of a mixture of seven bacterial species isolated from the intestinal tract, and in a binary culture of a 7 alpha-dehydroxylating Clostridium species plus a cholic acid-deconjugating Bacteroides species was studied. We found that tauroconjugation of cholic acid significantly (P < 0.05) increased bacterial 7 alpha-dehydroxylation of cholic acid into deoxycholic acid from 34 to 55% in fecal cultures, from 45 to 60% in defined limited fecal cultures, and from 75 to 100% in binary cultures. Equimolar concentrations of free taurine did not stimulate 7 alpha-dehydroxylation in fecal cultures or in the defined limited flora, but free taurine did stimulate 7 alpha-dehydroxylation in the binary culture. In the binary culture of Clostridium species strain 9/1 plus Bacteroides species strain R1, the minimal flora capable of increased 7 alpha-dehydroxylation of taurocholic acid, strain R1 deconjugated taurine and rapidly reduced it to H2S. Bacteroides species strain R1 did not grow unless taurine or another appropriate reducible sulfur source was present. Clostridium species strain 9/1 did not grow or 7 alpha-dehydroxylate unless H2S or another source of reduced sulfur was present. We conclude that the increased 7 alpha-dehydroxylation of tauroconjugated cholic acid depends on the reduction of taurine to H2S, which is a necessary growth factor for the 7 alpha-dehydroxylating bacteria.     

Conversion of chenodeoxycholic acid to ursodeoxycholic acid by Clostridium absonum in culture and by immobilized cells

Abstract Volatile fatty acids (VFA) have interesting biological effects on eukaryotic cells, inducing alterations of cell shape, morphological differentiation, changes in the composition of the cytoplasmic membrane and growth inhibition. This paper describes the effects of VFA on granulocyte chemotaxis. The influences observed, usually inhibitory, were shown to depend on the concentration tested and the incubation atmosphere (anaerobic or 5% CO₂).
Because these metabolites are produced by several anaerobic bacteria both in vitro and at infected sites, one can speculate that they might act as potential 'leukotoxins' contributing to the pathogenicity of anaerobic bacteria.
We suggest that the well-known ability of anaerobes to inhibit their own phagocytosis and intracellular killing, as well as that of facultative anaerobes, might be at least partially due to the impairment of granulocyte functions induced by VFA.     

Epimerization of chenodeoxycholic acid to ursodeoxycholic acid by Clostridium baratii isolated from human feces

Several H2-producing fermentative anaerobic bacteria including Clostridium, Klebsiella and Fusobacteria degraded octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) (36 microM) to formaldehyde (HCHO) and nitrous oxide (N2O) with rates ranging from 5 to 190 nmol h(-1)g [dry weight] of cells(-1). Among these strains, C. bifermentans strain HAW-1 grew and transformed HMX rapidly with the detection of the two key intermediates the mononitroso product and methylenedinitramine. Its cellular extract alone did not seem to degrade HMX appreciably, but degraded much faster in the presence of H2, NADH or NADPH. The disappearance of HMX was concurrent with the release of nitrite without the formation of the nitroso derivative(s). Results suggest that two types of enzymes were involved in HMX metabolism: one for denitration and the second for reduction to the nitroso derivative(s).     

Bacterial 7-dehydroxylation of cholic acid and allocholic acid

An obligate anaerobic organism capable of dehydroxylating cholic acid to deoxycholic acid and allocholic acid to allodeoxycholic acid was isolated from feces of the rabbit. It was a member of the bacteroides group (Gram-variable, nonsporulating anaerobes). The growth of the organism was inhibited by neomycin, 10-20 micro g/ml. The existence of this organism affords a satisfactory explanation for the development of gallstones in the cholestanol-fed rabbit and for their absence in rabbits simultaneously treated with neomycin.     

口服柔嫩梭菌对OVA致敏小鼠气道炎症的影响

目的 在动物水平探索口服柔嫩梭菌(Clostridium leptum)对哮喘小鼠气道炎症的影响.方法 建立OVA致敏的BALB/c小鼠哮喘模型,依据检测气道炎症情况和气道反应性确定哮喘模型构建成功.30只BALB/c小鼠随机分为3组:正常对照组,安慰剂组和口服柔嫩梭菌治疗组,每组10只.通过HE染色检测小鼠肺组织病理变化,细胞计数检测肺泡灌洗液中炎性细胞(嗜酸性粒细胞、中性粒细胞、淋巴细胞、巨噬细胞)的数目,ELISA方法检测肺泡灌洗液中炎性因子(IL4、IL-5、IL-13)的表达情况,并检测各组小鼠的气道反应性.结果 验证OVA致敏哮喘小鼠模型构建成功;口服柔嫩梭菌可显著减轻OVA致敏小鼠的气道高反应性,气道炎症细胞浸润和炎性细胞因子分泌(P＜0.05).结论 口服柔嫩梭菌可显著减轻OVA致敏小鼠的气道炎症和气道高反应性,这可能为哮喘治疗提供新思路.     

Biological properties of the 2-fluoro-beta-alanine conjugates of cholic acid and chenodeoxycholic acid in the isolated perfused rat liver

The isolated perfused rat liver was used to examine the hepatic extraction, biliary secretion and effect on bile flow of the 2-fluoro-beta-alanine conjugates of cholic acid and chenodeoxycholic acid. The naturally occurring taurine and glycine conjugates of these bile acids were used for comparisons. The 2-fluoro-beta-alanine conjugates were extracted by the liver to a similar extent as the taurine and glycine conjugates. The biliary secretion rate and increase in bile flow were similar for all the cholic acid conjugates. On the other hand, the maximal biliary secretion rate of the 2-fluoro-beta-alanine conjugate of chenodeoxycholate was similar to that of the glycochenodeoxycholate, but 47% lower than that of taurochenodeoxycholate. In addition, the 2-fluoro-beta-alanine conjugate of chenodeoxycholate produced a decrease in bile flow that was comparable to that observed with the glycochenodeoxycholate (54% vs. 74%), but which was greater than that produced by the taurochenodeoxycholate (12%). In summary, these data demonstrate that the biological properties of the 2-fluoro-beta-alanine conjugates of cholic acid and chenodeoxycholic acid are not markedly different from those of the naturally occurring taurine and glycine conjugates. These data also suggest that the amino acid moiety can influence the biliary secretion and cholestatic properties of chenodeoxycholic acid conjugates.     

Reduction of 7-ketolithocholic acid to chenodeoxycholic acid by rat liver preparations in vitro

The formation of chenodeoxycholic acid via 7-ketolithocholic acid by rat liver preparations was examined in vitro. Results showed that a rat liver preparation reduced 7-ketolithocholic acid mainly to chenodeoxycholic acid and to ursodeoxycholic acid in a smaller amount, and that the reductase required NADPH but not NADH as coenzyme and was mainly localized in the microsomes.     

Potential bile acid precursors in plasma--possible indicators of biosynthetic pathways to cholic and chenodeoxycholic acids in man

The plasma concentrations of 3 beta-hydroxy-5-cholestenoic acid, 3 beta,7 alpha-dihydroxy-5-cholestenoic acid and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid have been compared with that of 7 alpha-hydroxy-4-cholesten-3-one in healthy subjects and in patients with an expected decrease or increase of the bile acid production. In controls and patients with liver disease, the level of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid was positively correlated to that of 3 beta,7 alpha-dihydroxy-5-cholestenoic acid and not to that of 7 alpha-hydroxy-4-cholesten-3-one. In patients with stimulated bile acid formation the levels of the acids were not correlated to each other but there was a significant positive correlation between the levels of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid and 7 alpha-hydroxy-4-cholesten-3-one. These findings indicate that the precursor of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid differs depending on the activity of cholesterol 7 alpha-hydroxylase. Since the activity of this enzyme is reflected by the level of 7 alpha-hydroxy-4-cholesten-3-one in plasma the findings are compatible with a formation of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid from 3 beta,7 alpha-dihydroxy-5-cholestenoic acid when the rate of bile acid formation is normal or reduced and from 7 alpha-hydroxy-4-cholesten-3-one under conditions of increased bile acid synthesis. In support of this interpretation, 7 alpha,26-dihydroxy-4-cholesten-3-one was identified at elevated levels in plasma from patients with ileal resection or treated with cholestyramine. The levels of 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one were also higher than normal in these patients. Based on these findings and previous knowledge, a model is proposed for the biosynthesis of bile acids in man. Under normal conditions, two major pathways, one "neutral" and one "acidic" or "26-oxygenated", lead to the formation of cholic acid and chenodeoxycholic acid, respectively. These pathways are separately regulated. When the activity of cholesterol 7 alpha-hydroxylase is high, the "neutral" pathway is most important whereas the reverse is true when cholesterol 7 alpha-hydroxylase activity is low. In cases with enhanced activity of cholesterol 7 alpha-hydroxylase, the "neutral" pathway is connected to the "acidic" pathway via 7 alpha,26-dihydroxy-4-cholesten-3-one, whereas a flow from the acidic pathway to cholic acid appears to be of minor importance.     

A study on the mechanism of the epimerization at C-3 of chenodeoxycholic acid by Clostridium perfringens.

The mechanism of 3-hydroxy epimerization of chenodeoxycholic acid by Clostridium perfringens was investigated in 3 alpha, 7 alpha-dihydroxy-[2,2,4,4-2H4]-, 3 alpha, 7 alpha-dihydroxy-[3 beta-2H]- and 3 beta, 7 alpha-dihydroxy-[3 alpha-2H]-5 beta-cholanoic acid transformations. Our findings rule out a dehydration-rehydration pathway and agree with a redox mechanism involving 3-oxochenodeoxycholic acid as intermediate.     

Synthesis and cytotoxicity of A-homo-lactam derivatives of cholic acid and 7-deoxycholic acid

Using cholic acid and deoxycholic acid as starting materials, a series of 3-aza-A-homo-4-one bile acid and 7-deoxycholic acid derivatives were synthesized by the esterification, oxidation, reduction, oximation and Beckman rearrangement etc. The cytotoxicity of the synthesized compounds against MGC 7901 (human ventriculi carcinoma cell line), hela (human cervical carcinoma cell line), SMMC 7404 (human liver carcinoma cell line) were investigated. The results showed that bile acid and 7-deoxycholic-acid derivatives with 3-aza-A-homo-4-one configuration bearing a 6-hydroximino or 12-hydroximino group displayed a distinct cytotoxicity to Hela tumor cell line. In particular, the IC₅₀ values of the compounds 6 and 13 were 14.3 and 24.3  μmol/L against Hela human tumor cell line respectively. The information obtained from the studies may be useful for the design of novel chemotherapeutic drugs.