Identification of mono- and dihydroxy bile acids in human feces by gas-liquid chromatography and mass spectrometry

The mono- and disubstituted cholanoic acids present in human feces have been investigated. Extracts of feces were fractionated on silicic acid column and individual bile acids were isolated by preparative thin-layer chromatography. The isolated compounds were studied by gas-liquid chromatography of the methyl esters, partial trimethylsilyl ethers, oxidation products, and trifluoroacetates. The probable structures deduced were confirmed by gas chromatography-mass spectrometry and by comparisons with authentic compounds. The following derivatives of 5 Beta-cholanoic acid not previously isolated from human feces were identified: 3,12-diketo, 3-keto-12alpha-hydroxy, 3alpha,12 Beta-dihydroxy, 3 Beta,12 Beta-dihydroxy, 3-keto-7alpha-hydroxy, 3alpha-hydroxy-7-keto, 3 Beta,7alpha-dihydroxy, 3alpha,7alpha-dihydroxy, and 3alpha,7 Beta-dihydroxy. The presence of 3-keto-, 3 Beta-hydroxy-, 3alpha-hydroxy-, 3 Beta-hydroxy-12-keto-, 3alpha-hydroxy-12-keto-, 3 Beta,12alpha-dihydroxy-, and 3alpha,12alpha-dihydroxy-5 Beta-cholanoic acids was confirmed. Evidence was obtained for the presence of two bile acids having at least one hydroxyl group at a carbon atom other than C(3), C(7), or C(12).     

Identification of new C27 and C24 bile acids in the bile of Alligator mississippiensis

Biliary bile acids of Alligator mississippiensis were analyzed by gas-liquid chromatography-mass spectrometry after fractionation by silica gel column chromatography. It was shown that the alligator bile contained 12 C27 bile acids and 8 C24 bile acids. In addition to the C27 bile acids, such as 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid, 3 alpha,7 alpha,12 alpha-trihydroxy-5 alpha-cholestanoic acid, 3 alpha,7 alpha-dihydroxy-5 beta-cholestanoic acid, 3 alpha,12 alpha-dihydroxy-5 beta-cholestanoic acid, 7 alpha,12 alpha-dihydroxy-3-oxo-5 beta-cholestanoic acid, and 3 alpha,12 alpha-dihydroxy-7-oxo-5 beta-cholestanoic acid, identified previously in the bile of A. mississippiensis, 3 alpha,7 beta-dihydroxy-5 beta-cholestanoic acid, 3 alpha,7 beta,12 alpha-trihydroxy-5 beta-cholestanoic acid, 7 beta,12 alpha-dihydroxy-3-oxo-5 beta-cholestanoic acid, 3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestanoic acid, 3 alpha,7 alpha,12 alpha,26-tetrahydroxy-5 beta-cholestanoic acid, and 1 beta,3 alpha,7 alpha,12 alpha-tetrahydroxy-5 beta-cholestanoic acid were newly identified. And in addition to the C24 bile acids, such as chenodeoxycholic acid, ursodeoxycholic acid, cholic acid, and allocholic acid, identified previously, deoxycholic acid, 3 alpha,7 alpha-dihydroxy-5 beta-chol-22-enoic acid, 3 alpha,7 alpha,12 alpha-trihydroxy-5 alpha-chol-22-enoic acid, and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-chol-22-enoic acid were newly identified.     

Ketonic bile acids in urine of infants during the neonatal period

Ketonic bile acids have been found to be quantitatively important in urine of healthy infants during the neonatal period. In order to determine their structures, the bile acids in urine from 11 healthy infants were analyzed by gas-liquid chromatography-mass spectrometry (GLC-MS) and three samples with particularly high levels of ketonic bile acids were selected for detailed studies by ion exchange chromatography, fast atom bombardment mass spectrometry, microchemical reactions, and GLC-MS. The major ketonic bile acid was identified as 7 alpha, 12 alpha-dihydroxy-3-oxo-5 beta-chol-1-enoic acid, not previously described as a naturally occurring bile acid. The positional isomer 7 alpha, 12 alpha-dihydroxy-3-oxo-4-cholenoic acid, recently described as a major urinary bile acid in infants with severe liver diseases, was also excreted by most infants. Three acids related to cholic acid were identified: 7 alpha, 12 alpha-dihydroxy-3-oxo-, 3 alpha, 12 alpha-dihydroxy-7-oxo-, and 3 alpha, 7 alpha-dihydroxy-12-oxo-5 beta-cholanoic acids. Five bile acids having one oxo and three hydroxy groups were also present. Based on mass spectra and biological considerations two of these were tentatively given the structures 1 beta, 7 alpha, 12 alpha-trihydroxy-3-oxo- and 1 beta, 3 alpha, 12 alpha-trihydroxy-7-oxo-5 beta-cholanoic acids. Some of the others had a hydroxy group at C-4 or C-2. The levels of ketonic bile acids were higher on the third than on the first day of life, and lower after 1 month. The formation and excretion especially of 3-oxo bile acids is proposed to result from changes of the redox state in the liver in connection with birth.     

Synthesis of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acid from 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid: configuration in the bile of Alligator mississippiensis.

Synthesis of 25R- and 25S-diastereoisomers of 3 alpha,7 alpha-dihydroxy-5 beta-cholestan-26-oic acid from 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid is described. The 25S-diastereoisomer of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan- 26-oic acid was obtained by vigorous hydrolysis of the bile of Alligator mississippiensis followed by repeated crystallization of the hydrolysate, and the 25R-diastereoisomer was isolated by hydrolysis of the bile salts in bile of A mississippiensis with rat feces. Acetylation of the 25R- or 25S-diastereoisomer of methyl 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid under controlled conditions yielded the corresponding 3 alpha,7 alpha-diacetate in approximately 70% yield. The diacetate was quantitatively oxidized to methyl 3 alpha,7 alpha-diacetoxy-12-oxo-5 beta-cholestan-26-oate, which was converted into the 12-tosylhydrazone in approximately 58% yield. Reduction of the tosylhydrazone with sodium borohydride in acetic acid yielded the 25R- or the 25S-diastereoisomer of 3 alpha,7 alpha-dihydroxy-5 beta-cholestan-26-oic acid as the major product. Purification via column chromatography yielded the pure diastereoisomers in approximately 25% overall yield. The two diastereoisomers were resolved on thin-layer chromatography and high-performance liquid chromatography. When the bile of A mississippiensis was hydrolyzed with rat fecal bacteria, the 3 alpha,7 alpha-dihydroxy-5 beta-cholestan-26-oic acid isolated via chromatographic purification was shown to be the 25R-diastereoisomer.     

Isocholic acid formation from 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid with human liver enzyme

The formation of isocholic acid from 7 alpha, 12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid by human liver preparations was examined in vitro. Liver preparations were incubated with 7 alpha, 12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid at pH 7.4 in a phosphate buffer containing NADPH or NADH. The products formed were analyzed by gas chromatography and gas chromatography/mass spectrometry. Results showed that 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid was reduced mainly to isocholic acid and to cholic acid in a smaller amount in the presence of NADPH, while it was reduced only to cholic acid in the presence of NADH. The reducing enzyme participating in the formation of isocholic acid was localized largely in the cytosol and had more specificity to the unconjugated form as substrate than to the conjugated forms. 3-Keto bile acid analogues, 3-keto-5 beta-cholanoic and 7 alpha-hydroxy-3-keto-5 beta-cholanoic acids were not reduced to the corresponding iso-bile acids by the cytosol in the same conditions used in the isocholic acid formation and the activity of the enzyme catalyzing the reduction of 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid to isocholic acid was not inhibited by the addition of 3-keto-5 beta-cholanoic acid or 7 alpha-hydroxy-3-keto-5 beta-cholanoic acid to the reaction mixture. Furthermore, on column chromatography of Affi-Gel Blue, the peak of the enzyme catalyzing the reduction of 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid to isocholic acid was clearly distinguished from that of the enzyme catalyzing the reduction of 3-keto-5 beta-cholanoic acid to isolithocholic acid and that of alcohol dehydrogenase. These results indicate that this enzyme catalyzing the reduction of 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid to isocholic acid is different from the enzyme(s) catalyzing the reduction 3-keto-5 beta-cholanoic and 7 alpha-hydroxy-3-keto-5 beta-cholanoic acids to the corresponding iso-bile acids and from alcohol dehydrogenase, and has a stereospecific character for 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid.     

Synthesis of potential cholelitholytic agents: 3 alpha,7 alpha,12 alpha-trihydroxy-7 beta-methyl-5 beta-cholanoic acid, 3 alpha,7 beta,12 alpha-trihydroxy-7 alpha-methyl-5 beta-cholanoic acid, and 3 alpha,12 alpha-dihydroxy-7 xi-methyl-5 beta-cholanoic acid

This report describes the chemical synthesis of six new bile acid analogs, namely, 3 alpha,7 alpha,12 alpha-trihydroxy-7 beta-methyl-5 beta-cholanoic acid (7 beta-methyl-cholic acid), 3 alpha,7 beta,12 alpha-trihydroxy-7 alpha-methyl-5 beta-cholanoic acid (7 alpha-methyl-ursocholic acid), 3 alpha,12 alpha-dihydroxy-7 xi-methyl-5 beta-cholanoic acid (7 xi-methyl-deoxycholic acid), 3 alpha,12 alpha-dihydroxy-7-methyl-5 beta-chol-7-en-24-oic acid, 3 alpha,12 alpha-dihydroxy-7-methyl-5 beta-chol-6-en-24-oic acid, and 3 alpha,12 alpha-dihydroxy-7-methylene-5 beta-cholan-24-oic acid. The carboxyl group of the starting material 3 alpha,12 alpha-dihydroxy-7-oxo-5 beta-cholanoic acid was protected by conversion to its oxazoline derivative. A Grignard reaction of the bile acid oxazoline with CH3MgI followed by acid hydrolysis gave two epimeric trihydroxy-7-methyl-cholanoic acids and three dehydration products. The latter were purified by silica gel column chromatography and silica gel-AgNO3 column chromatography of their methyl ester derivatives. Catalytic hydrogenation of 3 alpha,12 alpha-dihydroxy-7-methyl-5 beta-chol-6-en-24-oic acid and 3 alpha,12 alpha-dihydroxy-7-methylene-5 beta-cholan-24-oic acid gave 3 alpha,12 alpha-dihydroxy-7 xi-methyl-5 beta-cholanoic acid. The configuration of the 7-methyl groups and the position of the double bonds were assigned by proton nuclear magnetic resonance spectroscopy and the chromatographic and mass spectrometric properties of the new compounds. These compounds were synthesized for the purpose of exploring new and potentially more effective cholelitholytic agents. The hydrophilic bile acids 7 beta-methyl-cholic acid and 7 alpha-methyl-ursocholic acid are of particular interest because they should be resistant to bacterial 7-dehydroxylation.     

The effect of cholesterol feeding on gallbladder bile acids of the rabbit. Evidence that lithocholic acid is a primary bile acid in the rabbit.

The bile acid in gallbladder bile of rabbits fed a normal diet or one containing 2% (w/w) cholesterol have been determined by gas chromatography-mass spectrometry. The predominant bile acids in normally fed rabbits were 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholan-24-oic acid (cholic acid), 3 alpha, 12 alpha-dihydroxy-5 alpha-cholan-24-oic acid (allodeoxycholic acid) and 3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oic acid (deoxycholic acid) with very much smaller amounts of 3 alpha-hydroxy-5 beta-cholan-24-oic acid (lithocholic acid) and 3 alpha, 12 beta-dihydroxy-5 beta-cholan-24-oic acid. In the cholesterol-fed animals the lithocholate became a predominant bile acid. Sulphated bile acids accounted for less than 1% of the total bile acids. It is proposed that lithocholic acid may be a primary bile acid in the cholesterol-fed rabbit, formed by an alternative pathway of biosynthesis involving hepatic mitochondria.     

Identification of bile acids in the serum and urine in cholestasis. Evidence for 6alpha-hydroxylation of bile acids in man.

In this qualitative study of the pattern of bile acid excretion in cholestasis, methods are described for the isolation of bile acids from large volumes of urine and plasma. The bile acids were subjected to a group separation and identified by combined gas chromatography-mass spectrometry. The techniques were developed to allow identification of the minor components of the bile acid mixture. Four bile acids that have not previously been described in human urine and plasma were detected, namely 3beta, 7alpha-dihydroxy-5beta-cholan-24-oic acid, 3alpha, 6alpha-dihydroxy-5beta-cholan-24-oic acid (hyodeoxycholic acid), 3alpha, 6alpha, 7alpha-trihydroxy-5beta-cholan-24-oic acid (hyocholic acid) and 3alpha, 7beta, 12alpha-trihydroxy-5beta-cholan-24-oic acid. In addition three C27 steroids were found; 26-hydroxycholesterol and a trihydroxy cholestane, probably 5 beta-cholestane-3alpha, 7alpha, 26-triol were found in the sulphate fraction of plasma and urine. In the plasma sample, a sulphate conjugate of 24-hydroxycholesterol was found. The presence of these compounds probably reflects the existence of further pathways for bile acid metabolism. It is not yet known whether this is a consequence of the cholestasis or whether they are also present in normal man, at much lower concentrations.     

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.     

Metabolism of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid by rat liver in vivo and in vitro.

The metabolism of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid was studied in the bile fistula rats and in preparations from rat liver homogenates. In the bile fistula rats, the main products were chenodeoxycholic acid, alpha-muricholic acid, and beta-muricholic acid. Only small amounts of cholic acid were formed. Incubations of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid with microsomes and NADPH yielded as the main product 3 alpha, 6 beta, 7 alpha-trihydroxy-5 beta-cholestanoic acid. The formation of small amounts of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid was shown. The major product in incubations of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid with microsomes and the 100,000 g supernatant fluid fortified with ATP was identified as 3 alpha, 7 alpha, 24 xi-trihydroxy-5 beta-cholestanoic acid. This compound was converted into chenodeoxycholic acid and its metabolites in the bile fistula rat.     

Dehydroxylation of cholic acid at C12 and epimerization at C5 and C7 by Bacteroides species

Freshly isolated cultures (2060) of human intestinal bacteria of the predominant flora, among them 1029 strains of saccharolytic Bacteroides species, were tested for cholic acid transformation. Eight Bacteroides strains reduced cholate to chenodeoxycholate, while 73 strains dehydroxylated at C7, producing deoxycholate. Concurrent oxidation of hydroxyl groups, mainly at C7, was seen with many strains. No strain was able to dehydroxylate simultaneously at C7 and C12. One isolate, identified as a mixed culture of Bacteroides fragilis and B. uniformis, epimerized cholic acid at C5 and simultaneously epimerized, oxidized and dehydroxylated at C7. The following transformation products were identified: 3 alpha,7 alpha,12 alpha-trihydroxy-5 alpha-cholanoic acid, 3 alpha,7 beta,12 alpha-trihydroxy-5 beta-cholanoic acid (ursocholic acid), 3 alpha,12 alpha-dihydroxy-7-keto-5 beta-cholanoic acid, 3 alpha,12 alpha-dihydroxy-5 alpha-cholanoic acid and a 3 alpha,12 alpha-dihydroxy-5 alpha-cholenoic acid. Dehydroxylating and epimerizing abilities were detected when fresh isolates were tested first for cholate transformation. They were no longer recognizable after some serial transfers. Dehydroxylation at C12 of cholate could not be demonstrated with mixed fecal cultures. The possible intermediate, however, 3 alpha,7 alpha-dihydroxy-5 beta-chol-11-enoate, was abundantly hydrogenated by stool suspensions.     

Bile salts of the green turtle Chelonia mydas (L.)

1. Bile salts of the green turtle Chelonia mydas (L.) were analysed as completely as possible. 2. They consist of taurine conjugates of 3 alpha, 7 alpha, 12 alpha, 22 xi-tetrahydroxy-5 beta-cholestan-26-oic acid (tetrahydroxysterocholanic acid) and 3 alpha 12 alpha, 22 xi-trihydroxy-5 beta-cholestan-26-oic acid, with minor amounts of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5beta-cholan-24-oic acid (cholic acid), 3alpha, 12 alpha-dihydroxy-5beta-cholan-24-oic acid (deoxycholic acid) and possibly other bile acids. 3. Cholic acid and deoxycholic acid represent the first known examples of bile acids common to chelonians and other animal forms: they may indicate independent evolution in chelonians to C24 bile acids. 4. The discovery of a 7-deoxy C27 bile acid is the first evidence that C27 bile acids or their conjugates have an enterohepatic circulation.     

Bile acids of snakes of the subfamily Viperinae and the biosynthesis of C-23-hydroxylated bile acids in liver homogenate fractions from the adder, Vipera berus (Linn.).

1. Analysis of bile salts of four snakes of the subfamily Viperinae showed that their bile acids consisted mainly of C-23-hydroxylated bile acids. 2. Incubations of 14C-labelled sodium cholate (3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholan-24-oate) and deoxycholate (3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oate) with whole and fractionated adder liver homogenates were carried out in the presence of molecular oxygen and NADPH or an NADPH-generating system. The formation of C-23-hydroxylated bile acids, namely bitocholic acid (3 alpha, 12 alpha, 23xi-trihydroxy-5 beta-cholan-24-oic acid) and 3 alpha, 7 alpha, 12 alpha, 23 xi-tetrahydroxy-cholanic acid (3 alpha, 7 alpha, 12 alpha, 23 xi-tetrahydroxy-5 beta-cholan-24-oic acid), was observed mainly in the microsomal fraction and partly in the mitochondrial fraction. 3. Biosynthetic pathways of C-23-hydroxylated bile acids are discussed.     

Bile salts of the coelacanth, Latimeria chalumnae

Bile salts of the coelacanth, Latimeria chalumnae, Smith, have been analyzed and shown to have three bile alcohols, latimerol, 5 alpha-cyprinol, and 5 alpha-cholestane-3 beta, 7 alpha,-12 alpha,25,26-pentol, two C24 bile acids, chenodeoxycholic acid and cholic acid, one C26 bile acid, probably 3 beta, 7 alpha, 12 alpha-trihydroxy-27-nor-5 alpha-cholestan-26-oic acid, and two C27 bile acids, 3 alpha,7 alpha,12 alpha-trihydroxy-5 alpha-cholestan-26-oic acid and 3 beta,7 alpha,12 alpha-trihydroxy-5 alpha-cholestan-26-oic acid as determined by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry.     

Identification of 3 alpha,4 beta,7 alpha-trihydroxy-5 beta-cholanoic acid in human bile: reflection of a new pathway in bile acid metabolism in humans

The chemical synthesis, nuclear magnetic resonance, and mass spectrometric characteristics of the first C-4 hydroxylated bile acid analogues are described. The data definitively confirm, for the first time, the identity of 3 alpha,4 beta,7 alpha-trihydroxy-5 beta-cholanoic acid in human fetal gallbladder bile. In addition, 3 alpha,4 beta,7 alpha-12 alpha-tetrahydroxy-5 beta-cholanoic was identified in the feces from healthy newborn infants many days after birth, indicating a hepatic origin for C-4 hydroxylation of bile acids. To our knowledge bile acids hydroxylated at the C-4 position of the steroid nucleus have never been previously recognized in any mammalian species. The finding of this novel bile acid which accounts for 5-15% of the total biliary bile acids in early gestation indicates that C-4 hydroxylation is a unique and important metabolic pathway in early human development.     

Synthesis of coenzyme A esters of 3alpha,7alpha,12alpha-trihydroxy- and 3alpha,7alpha-dihydroxy-24-oxo-5beta-cholestan-26-oic acids for the study of beta-oxidation in bile acid biosynthesis

3alpha,7alpha,12alpha-Trihydroxy- and 3alpha,7alpha-dihydroxy-24-oxo-5beta-cholestan-26-oyl CoAs were chemically synthesized by the conventional method for the study of side chain cleavage in bile acid biosynthesis. 3alpha,7alpha,12alpha-Triformyloxy- and 3alpha,7alpha-diformyloxy-5beta-cholan-24-als were initially subjected to the Reformatsky reaction with methyl alpha-bromopropionate, and the products were then converted into methyl 3alpha,7alpha,12alpha-triformyloxy- and 3alpha,7alpha-diformyloxy-24-oxo-5beta-cholestan-26-oates. Protection by acetalization of the 24-oxo-group of these methyl esters with ethylene glycol, followed by alkaline hydrolysis, gave 3alpha,7alpha,12alpha-trihydroxy- and 3alpha,7alpha-dihydroxy-24,24-ethylenedioxy-5beta-cholestan-26-oic acids. These acids were condensed with coenzyme A by a mixed anhydride method, and the resulting CoA esters were treated with 4M-hydrocholic acid to remove the protecting group to give 24-oxo-5beta-cholestanoic acid CoA esters. The chromatographic behaviors of these CoA esters were also investigated.     

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.     

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.     

Synthesis of 24-nor-5 beta-cholan-23-oic acid derivatives: a convenient and efficient one-carbon degradation of the side chain of natural bile acids

An efficient procedure for obtaining nor-bile acids from natural (C24) bile acids is described. Treatment of formylated bile acids with sodium nitrite in a mixture of trifluoroacetic anhydride with trifluoroacetic acid gives, through a "second order" Beckmann rearrangement, 24-nor-23-nitriles. These compounds, on alkaline hydrolysis, afford the corresponding nor-bile acids in high yields. The sequence was successfully applied to the synthesis of 3 alpha-hydroxy-24-nor-5 beta-cholan-23-oic (norlithocholic) acid, 3 alpha,6 alpha- (norhyodeoxycholic), 3 alpha,7 alpha- (norchenodeoxycholic), 3 alpha,7 beta- (norursodeoxycholic), and 3 alpha,12 alpha-dihydroxy-24-nor-5 beta-cholan-23-oic (nordeoxycholic) acids, as well as 3 alpha,7 alpha,12 alpha-trihydroxy-24-nor-5 beta-cholan-23-oic (norcholic) acid. 13C-NMR spectra of their methyl esters are reported. The procedure provides a more rapid alternative to the Barbier-Wieland degradation for shortening by one methylene group the side chain of natural (C24) bile acids.     

The catabolism of cholesterol in vitro. Formation of 3α,7α,12α-trihydroxy-5β-cholestanoic acid from cholesterol by rat liver

1. Both 25-d- and 25-l-3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid were isolated from the gall-bladder bile of Crocodylus niloticus. 2. The catabolism of cholesterol to 25-d- and 25-l-3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid respectively was studied by using a rat liver preparation in vitro. The results show that rat liver can metabolize cholesterol to both forms of 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid. However, a preference was noted for the formation from [4-(14)C]cholesterol of 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid (25-d), which was isolated from the incubations with a specific radioactivity about four times that of 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid (25-l). 3. The results indicate that 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid is a normal intermediate in the biosynthesis of bile acids from cholesterol in the rat.