Cytosolic ferritin and lipid-associated ferritin are metabolically different in guinea-pig livers.

1. Using a human hepatoma (Hep G2) cell line that continually synthesizes 3 beta-hydroxy-5-cholenoic acid, lithocholic acid, chenodeoxycholic acid and cholic acid we have determined the metabolism and biological effects of 26-hydroxycholesterol and 7 alpha-hydroxycholesterol. 2. Addition of 26-hydroxycholesterol to the medium (6 microM) downregulated cholesterol and chenodeoxycholic acid synthesis. 3. The predominant metabolite of 26-hydroxycholesterol was 3 beta-hydroxy-5-cholenoic acid. 4. Cholesterol synthesis was not affected by the addition of 7 alpha-hydroxycholesterol (6 and 12 microM). The predominant metabolite of 7 alpha-hydroxycholesterol was chenodeoxycholic acid. 5. In Hep G2 cells 7 alpha-hydroxylation of 26-hydroxycholesterol is not well expressed.     

Bile acid synthesis: down-regulation by monohydroxy bile acids

The regulation of bile acid synthesis was studied in rabbits after interruption of the enterohepatic circulation by choledochoureteral anastomosis. Total daily bile acid output was 772 +/- 130 (SD) mumol/24 h, of which greater than 95% was glycocholic acid. Administration of deoxycholic or cholic acid or their conjugates (300-800 mumol) or gall-bladder bile failed to down-regulate endogenous bile acid synthesis. In contrast, chenodeoxycholic acid administration did down-regulate bile acid synthesis, but this effect was related to the formation and excretion of lithocholic acid. This observation was confirmed by the finding that i.v. infusion of 10-20 mumol of either lithocholic acid or 3 beta-hydroxy-5-cholenoic acid significantly reduced cholic acid synthesis. Thus monohydroxy bile acids, derived from either hepatic or intestinal sources, participate in the down-regulation of bile acid synthesis.     

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.     

Metabolism of potential precursors of chenodeoxycholic acid in cerebrotendinous xanthomatosis (CTX).

In patients with cerebrotendinous xanthomatosis (CTX), diminished cholic acid production is associated with incomplete oxidation of the cholesterol side chain and the excretion of C(25)-hydroxy bile alcohols. The aims of this investigation were 1) to provide quantitative information on the pool size and production rate of chenodeoxycholic acid by the isotope dilution technique; and 2) to investigate the possible existence of a block in chenodeoxycholic acid synthesis and explain the absence of chenodeoxycholic acid precursors in CTX. After the injection of [24-(14)C]chenodeoxycholic acid, measurements of chenodeoxycholic acid pool size and production rate in a CTX subject were, respectively, 1/20 and 1/6 as great as controls. Further, three potential precursors of chenodeoxycholic acid, namely [G-(3)H]7alpha-hydroxy-4-cholesten-3-one, [G-(3)H]5beta-cholestane-3alpha,7alpha,25-triol, and [G-(3)H]5beta-cholestane-3alpha,7alpha,26-triol, were administered to the CTX and control subjects and the specific activity curves of [G-(3)H]cholic acid and [G-(3)H]chenodeoxycholic acid were constructed and compared. In the control subjects, the two bile acids decayed exponentially, but in the CTX patient maximum specific activities were abnormally delayed, indicating the hindered transformation of precursor into bile acid. These results show that chenodeoxycholic acid synthesis is small in CTX and that the conversion of 7alpha-hydroxy-4-cholesten-3-one, 5beta-cholestane-3alpha,7alpha,25-triol, and 5beta-cholestane-3alpha,7alpha,26-triol to both chenodeoxycholic acid and cholic acid were similarly impaired.     

Effect of bile duct ligation on bile acid and cholesterol metabolism in rats

The effects of bile duct ligation on bile acid and cholesterol metabolism were examined in male Wistar strain rats. Quantitative and qualitative changes of bile acids and cholesterol in serum and urine occurred; beta-muricholic acid predominantly increased in serum and urine and the ratio of urinary cholic acid and beta-muricholic acid changed from about 5:3 on day 1 to about 1:8 on day 5 under biliary obstruction. The form of the increased urinary bile acids was mainly taurine-conjugated and partly sulfated. Under conditions of bile duct ligation on day 5, 14C-labeled 3 beta-hydroxy-5-cholenoic, lithocholic, and chenodeoxycholic acids were intragastrically administered to the rats after pretreatment with antibiotics and the metabolites of these three acids were investigated. 3 beta-Hydroxy-5-cholenoic acid was most efficiently converted to beta-muricholic acid. The present study strongly suggested the presence of an alternative metabolic pathway induced by bile duct ligation, which caused the change in composition of urinary bile acids, and especially the marked increase in beta-muricholic acid formation. A possible alternative pathway for bile acid biosynthesis under biliary obstruction in rats is postulated.     

Bile acids and bile alcohols in a child with hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency: effects of chenodeoxycholic acid treatment

Duodenal bile, urine, plasma, and feces from a child with hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency were analyzed by fast atom bombardment mass spectrometry and gas chromatography-mass spectrometry to investigate the formation and excretion of abnormal bile acids and bile alcohols. The biliary bile salts consisted of glycocholic acid (25%) and of sulfated and glycine conjugated di- and trihydroxycholenoic acids (55%), two C27 bile acids, and eleven sulfated bile alcohols (mainly tetrols, 20%), all having 3 beta,7 alpha-dihydroxy-delta 5 or 3 beta,7 alpha,12 alpha-trihydroxy-delta 5 ring structures. In plasma, sulfated cholenoic acids constituted 65% and unconjugated 3 beta,7 alpha-dihydroxy-5-cholestenoic acid 25% of the total level, 71 micrograms/ml. The urinary excretion of the former was 30.4 mg/day and that of unsaturated bile alcohol sulfates, mainly pentols, 7 mg/day. The predominant bile acid in feces was an unconjugated epimer of 3 beta,7 alpha,12 alpha-trihydroxy-5-cholenoic acid, and small amounts of cholic acid were present. The minimum total excretion was 11.3 mg/day. Treatment with chenodeoxycholic acid resulted in marked clinical improvement and normalized liver function tests. Further studies are needed to define the mechanism of action. Plasma bile acids decreased to 1.6 micrograms/ml and urinary excretion to 3.4 mg/day. Chenodeoxycholic and ursodeoxycholic acids became predominant in all samples. The fecal excretion of unsaturated cholenoic acid sulfates increased to 40 mg/day compared to 89 mg/day of saturated bile acids. The results provide further support for a defective hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency, and indicate that the 3 beta-hydroxy-delta 5 bile acids are formed via 7 alpha-hydroxycholesterol. The formation of glycocholic acid may be due to an incomplete enzyme defect or to transformation of the 3 beta-hydroxy-delta 5 structure by bacterial and hepatic enzymes during an enterohepatic circulation.     

HepG2. A human hepatoblastoma cell line exhibiting defects in bile acid synthesis and conjugation

We used capillary gas chromatography/mass spectrometry to demonstrate that a cell line derived from a well differentiated human hepatoblastoma, HepG2, synthesized and secreted the following bile acids (ng/10(7) cells/h): chenodeoxycholic acid (131.4), cholic acid (3.3), 3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acid (DHCA; 4.5), and 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26-oic acid (THCA; 32.0). Deuterium from [7 beta-2H]7 alpha-hydroxycholesterol, which was added to the media, was incorporated into newly synthesized chenodeoxycholic acid, DHCA, and THCA, but not into cholic acid. Since THCA is a known precursor of cholic acid, these data suggest that HepG2 is specifically deficient in the side chain cleavage that transforms THCA into cholic acid. Greater than 90% of the bile acids synthesized and secreted by HepG2 were unconjugated. Conjugation could not be stimulated by the addition of glycine or taurine to the media. Approximately 30% of newly synthesized DHCA and THCA were sulfated. Chenodeoxycholic acid and cholic acid were not appreciably sulfated. In summary, cultured HepG2 cells synthesize bile acid, but in a pattern distinct from that of adult human liver. This cell line may be a model for studying pathways of human bile acid synthesis, conjugation, and sulfation.     

Bile acid metabolism in early life: studies of amniotic fluid

Bile acid metabolism of the human fetus was examined in early gestation (weeks 13-19) and compared with the full-term fetus from the analysis of amniotic fluid collected from healthy pregnant women. Total individual bile acids were determined by gas-liquid chromatography-mass spectrometry after solvolysis and hydrolysis of bile acid conjugates. Additionally, bile acids were separated according to their mode of conjugation by lipophilic anion exchange chromatography. Qualitatively the bile acid profiles of amniotic fluid in early gestation were similar and markedly different from those of full-term fetuses. Chenodeoxycholic acid was the major bile acid identified in early gestation and concentrations exceeded those of cholic acid, but by full term this relationship was reversed. Over 50 bile acids were identified in the amniotic fluids, these included C-1, C-4, and C-6 hydroxylated species and reflected primary hepatic synthesis by the fetus. At full term, 7 alpha,12 alpha-dihydroxy-3-oxo-4-cholenoic acid was one of the major bile acids identified in amniotic fluid. The monohydroxy bile acids lithocholic and 3 beta-hydroxy-5-cholenoic acids were present in significant proportions during early gestation, but by full term these accounted for only a few percent of the total bile acids. Quantitatively the total bile acid concentration of amniotic fluid was less than 4 mumol/l. The majority of bile acids were found to be glyco-, tauro-, and sulfate-conjugates. The more hydrophobic bile acids tended to be preferentially sulfated. These data indicate that significant and major changes in bile acid metabolism take place between early and late gestation in the human fetus.     

Bile acid synthesis in cultured human hepatoblastoma cells.

Biosynthetic pathways to bile acids have been studied in HepG2 cells, a well-differentiated human hepatoblastoma cell line. Cholesterol metabolites, in total 29, were isolated from culture media and cells by liquid-solid extraction and anion-exchange chromatography and were identified by gas-liquid chromatography-mass spectrometry. The production rates/concentrations of cholic acid (CA) and chenodeoxycholic acid (CDCA) in media from control cells were 71 and 74 ng/10(7) cells/h, respectively. Major bile acid precursors were 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA), 7 alpha, 12 alpha-dihydroxy-3-oxo-4-cholestenoic acid, 7 alpha-hydroxy-3-oxo-4-cholenoic acid, and 7 alpha, 12 alpha-dihydroxy-3-oxo-5 beta-cholanoic acid, their concentrations being 60, 30, 23, and 10 ng/10(7) cells/h, respectively. These and nine other isolated intermediates formed essentially complete metabolic sequences from cholesterol to CA and CDCA. The remaining steroids were metabolites of the intermediates or autooxidation products of cholesterol. These findings and the observed effect of dexamethasone on production rates suggest that in HepG2 cells the major biosynthetic pathways to primary bile acids start with 7 alpha-hydroxylation of cholesterol and oxidation to 7 alpha-hydroxy-4-cholesten-3-one followed by hydroxylation at either the 26 or 12 alpha position. CDCA is formed by the sequence of 26-hydroxylation, oxidation, and degradation of the side chain and A-ring reduction. CA is formed by the sequence of 12 alpha-hydroxylation, 26-hydroxylation, oxidation, and degradation of the side chain and reduction of the A-ring. An alternative pathway to CA included A-ring reduction of the intermediate 7 alpha, 12 alpha-dihydroxy-3-oxo-4-cholestenoic acid to form THCA prior to side chain cleavage. These pathways are not limited to HepG2 cells but may also be important in humans.     

The bile acid composition of gastric contents from neonates with high intestinal obstruction.

The study was designed to identify 'atypical' bile acids in gastric contents from three neonates with high intestinal obstruction on the basis that this was likely to represent a rich source of primary bile acids. Cholic acid was the major component, and related 'atypical' bile acids included its C-3 and C-7 oxidation products, its 3 beta-epimer and 2 beta- and 6 alpha-hydroxylation products. Allocholic acid was the only 5 alpha-cholanic acid derivative identified. 7 alpha, 12 alpha-Dihydroxy-3-oxochol-4-en-24-oic acid was found in all three specimens and might be an intermediate in a biosynthetic pathway from cholesterol to cholic acid in which side-chain oxidation precedes at least some of the nuclear changes. Side-chain-hydroxylated derivatives of trihydroxycoprostanic acid were also detected and these may represent intermediates in biosynthetic pathways from cholesterol to cholic acid via 5 beta-cholestan-3 alpha, 7 alpha, 12 alpha-triol. The most abundant bile acid of this type was (25 epsilon)-3 alpha, 7 alpha, 12 alpha, 25-tetrahydroxy-5 beta-cholestan-26-oic acid, which suggested that C-25 hydroxylation may be an important step in the shortening of the C8 side chain of the cholestane triol to the C5 side chain of cholic acid in the neonatal period. Bile acids lacking a substituent at C-12 included chenodeoxycholic acid, its C-3 and C-7 oxidation products, its 3 beta-epimer and its 6 alpha-hydroxylation product (hyocholic acid).     

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.     

Clostridium scindens baiCD and baiH genes encode stereo-specific 7alpha/7beta-hydroxy-3-oxo-delta4-cholenoic acid oxidoreductases

Secondary bile acids, formed by intestinal bacteria, are suggested to play a significant role in cancers of the gastrointestinal tract in humans. Bile acid 7alpha/beta-dehydroxylation is carried out by a few species of intestinal clostridia which harbor a multi-gene bile acid inducible (bai) operon. Several genes encoding enzymes in this pathway have been cloned and characterized. However, no gene product(s) has yet been assigned to the production of 3-oxo-Delta4-cholenoic acid intermediates of cholic acid (CA), chenodeoxycholic acid (CDCA) or ursodeoxycholic acid (UDCA). We previously reported that the baiH gene encodes an NADH:flavin oxidoreductase (NADH:FOR); however, the role of this protein in bile acid 7-dehydroxylation is unclear. Homology searches and secondary structural alignments suggest this protein to be similar to flavoproteins which reduce alpha/beta-unsaturated carbonyl compounds. The baiH gene product was expressed in Escherichia coli, purified and discovered to be a stereo-specific NAD(H)-dependent 7beta-hydroxy-3-oxo-Delta4-cholenoic acid oxidoreductase. Additionally, high sequence similarity between the baiH and baiCD gene products suggests the baiCD gene may encode a 3-oxo-Delta4-cholenoic acid oxidoreductase specific for CDCA and CA. We tested this hypothesis using cell extracts prepared from E. coli overexpressing the baiCD gene and discovered that it encodes a stereo-specific NAD(H)-dependent 7alpha-hydroxy-3-oxo-Delta4-cholenoic acid oxidoreductase.     

Cholic acid synthesis from 26-hydroxycholesterol and 3-hydroxy-5-cholestenoic acid in the rabbit

Intravenous administration of 26-hydroxycholesterol to the rabbit with a bile fistula yielded cholic acid in proportions (84 and 86%) not significantly different from that derived from cholesterol. By contrast, the naturally occurring C27 bile acid 3 beta-hydroxy-5-cholestenoic acid yielded not more than 8% cholic acid. Thus initial 26-hydroxylation of cholesterol followed by 7-alpha-hydroxylation can provide sufficient amounts of cholic acid to be considered a quantitatively significant pathway for bile acid synthesis, and in addition it is the only pathway that can be the source of the circulating levels of C24 and C27 monohydroxy bile acids.     

Similarity of unusual bile acids in human umbilical cord blood and amniotic fluid from newborns and in sera and urine from adult patients with cholestatic liver diseases

Unusual bile acids in umbilical cord blood and amniotic fluid of term newborns and in sera and urine from adult patients with cholestatic liver diseases were analyzed by use of gas-liquid chromatography-mass spectrometry. These bile acids were compared in order to elucidate possible similarities of bile acid metabolism between fetal and cholestatic liver. In both umbilical cord blood and amniotic fluid, 14 unusual bile acids were found in addition to normal bile acids (cholic, chenodeoxycholic, deoxycholic, and lithocholic acids), and 15, excluding ursodeoxycholic acid, were found in sera and urine from patients with cholestatic liver diseases. Of the unusual bile acids detected, 12 were common to both samples. Six unusual bile acids, 3 beta-hydroxy- and 3 beta,12 alpha-dihydroxy-5-cholenoic acids, 3 alpha,6 alpha,7 alpha-trihydroxy-5 beta-cholanoic acid, 1 beta,3 alpha,12 alpha-trihydroxy-1 beta,3 alpha,7 alpha-trihydroxy-, and 1 beta,3 alpha,7 alpha,12 alpha-tetrahydroxy-5 beta-cholanoic acids were more abundant than others. They could be classified into three groups, i.e., unsaturated, 6-hydroxylated, and 1 beta-hydroxylated bile acids. 1 beta-Hydroxylated bile acids, which were not found in serum specimens, were detected in sera from umbilical cord blood and from patients with cholestatic liver diseases. The presence of these unusual bile acids suggested similarities between the altered metabolic states of the two groups examined.     

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.     

Formation of ursodeoxycholic acid from chenodeoxycholic acid by a 7 beta-hydroxysteroid dehydrogenase-elaborating Eubacterium aerofaciens strain cocultured with 7 alpha-hydroxysteroid dehydrogenase-elaborating organisms.

A gram-positive, anaerobic, chain-forming, rod-shaped anaerobe (isolate G20-7) was isolated from normal human feces. This organism was identified by cellular morphology as well as fermentative and biochemical data as Eubacterium aerofaciens. When isolate G20-7 was grown in the presence of Bacteroides fragilis or Escherichia coli (or another 7 alpha-hydroxysteroid dehydrogenase producer) and chenodeoxycholic acid, ursodeoxycholic acid produced. Time course curves revealed that 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid produced by B. fragilis or E. coli or introduced into the medium as a pure substance was reduced by G20-7 specifically to ursodeoxycholic acid. The addition of glycine- and taurine-conjugated primary bile acids (chenodeoxycholic and cholic acids) and other bile acids to binary cultures of B. fragilis and G20-7 revealed that (i) both conjugates were hydrolyzed to give free bile acids, (ii) ursocholic acid (3 alpha, 7 beta, 12 alpha-trihydroxy-5 beta-cholanoic acid) was produced when conjugated (or free) cholic acid was the substrate, and (iii) the epimerization reaction was at least partially reversible. Corroborating these observations, an NADP-dependent 7 beta-hydroxysteroid dehydrogenase (reacting specifically with 7 beta-OH-groups) was demonstrated in cell-free preparations of isolate G20-7; production of the enzyme was optimal at between 12 and 18 h of growth. This enzyme, when measured in the oxidative direction, was active with ursodeoxycholic acid, ursocholic acid, and the taurine conjugate of ursodeoxycholic acid (but not with chenodeoxycholic, deoxycholic, or cholic acids) and displayed an optimal pH range of 9.8 to 10.2     

Formation of ursodeoxycholic acid from chenodeoxycholic acid by a 7 beta-hydroxysteroid dehydrogenase-elaborating Eubacterium aerofaciens strain cocultured with 7 alpha-hydroxysteroid dehydrogenase-elaborating organisms

A gram-positive, anaerobic, chain-forming, rod-shaped anaerobe (isolate G20-7) was isolated from normal human feces. This organism was identified by cellular morphology as well as fermentative and biochemical data as Eubacterium aerofaciens. When isolate G20-7 was grown in the presence of Bacteroides fragilis or Escherichia coli (or another 7 alpha-hydroxysteroid dehydrogenase producer) and chenodeoxycholic acid, ursodeoxycholic acid produced. Time course curves revealed that 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid produced by B. fragilis or E. coli or introduced into the medium as a pure substance was reduced by G20-7 specifically to ursodeoxycholic acid. The addition of glycine- and taurine-conjugated primary bile acids (chenodeoxycholic and cholic acids) and other bile acids to binary cultures of B. fragilis and G20-7 revealed that (i) both conjugates were hydrolyzed to give free bile acids, (ii) ursocholic acid (3 alpha, 7 beta, 12 alpha-trihydroxy-5 beta-cholanoic acid) was produced when conjugated (or free) cholic acid was the substrate, and (iii) the epimerization reaction was at least partially reversible. Corroborating these observations, an NADP-dependent 7 beta-hydroxysteroid dehydrogenase (reacting specifically with 7 beta-OH-groups) was demonstrated in cell-free preparations of isolate G20-7; production of the enzyme was optimal at between 12 and 18 h of growth. This enzyme, when measured in the oxidative direction, was active with ursodeoxycholic acid, ursocholic acid, and the taurine conjugate of ursodeoxycholic acid (but not with chenodeoxycholic, deoxycholic, or cholic acids) and displayed an optimal pH range of 9.8 to 10.2     

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.     

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.     

The effect of the hypocholesteremic drug, AY 9944 on the synthesis of bile salts in rat liver

1. The compound trans-1,4 bis-(2-dichlorobenzylaminomethyl)cyclohexane dihydrochloride (AY9944) blocks cholesterol synthesis at a late stage. This leads to a decrease in cholesterol and accumulation of cholesta-5,7-diene-3-beta-ol (7-dehydrocholesterol) in tissues and plasma. 2. The effect of AY9944 on bile salt synthesis in rat liver was studied. The synthesis of conjugated cholic and chenodeoxycholic acids was measured in hepatocytes isolated from rats 2 h, 24 h and 48 h after administration of a single oral dose of AY9944. Production of the two bile salts was inhibited by 70-80% in hepatocytes from AY9944-treated as compared to untreated animals. 3. When AY9944 was added to the incubation medium in vitro of hepatocytes prepared from untreated rats the synthesis of conjugated cholic and chenodeoxycholic acids was not inhibited during the first hour of incubation, probably because of the presence of endogenous cholesterol. However when hepatocytes from untreated rats were incubated with AY9944 for periods of 2 h or longer, bile salt production was decreased markedly. 4. Bile salt synthesis is stimulated when rats are subjected to total biliary drainage for 24 h. The effect of AY9944 on this stimulation was studied. The content of conjugated cholic and chenodeoxycholic acid in the bile was measured as an indicator of bile salt synthesis. 5. In control animals the rate of secretion of biliary bile salts began to increase after about 24 h of total biliary drainage and reached a maximum after approximately 36 h. A single oral dose of AY9944 given 2 h after the start of total biliary drainage delayed and reduced this response. 6. The results show that inhibition of cholesterol synthesis by AY9944 resulting in the replacement of cholesterol by 7-dehydrocholesterol decreases but does not completely prevent bile salt synthesis.