Synthesis of four diastereoisomers at carbons 24 and 25 of 3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid, intermediates of bile acid biosynthesis

The synthesis of four stereoisomers at C-24 and C-25 of 3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid is described. Pyridium chlorochromate oxidation of 3 alpha,7 alpha,12 alpha-triacetoxy-5 beta-cholan-24-ol (II) prepared from cholic acid (I) afforded 3 alpha,7 alpha,12 alpha-triacetoxy-5 beta-cholan-24-al (III) which was converted to a mixture of the four stereoisomers (IV-VII) by a Reformatsky reaction with ethyl DL-alpha-bromopropionate followed by alkaline hydrolysis. Separation of these isomers (IV-VII) was achieved by silica gel column chromatography, and subsequent reversed-phase partition column chromatography. The configurations at C-24 were elucidated by conversion of each isomer into (24R)- or (24S)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24-tetrol (XII or XI) by Kolbe electric coupling, the C-24 configurations of which were determined by modified Horeau's method and 13C-nuclear magnetic resonance spectroscopy. The stereochemistries at C-25 were deduced by comparison of IV-VII with the products of the hydroboration followed by oxidation with alkaline hydrogen peroxide of (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-en-26-oic acid (XIII).     

Configuration at C-25 in 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26-oic acid by X-ray crystallography

The two diastereoisomers at carbon-25 of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26-oic acid, a key intermediate in the biosynthetic pathway of cholic acid, were obtained in pure form by a combination of fractional crystallization and thin-layer chromatography. The configuration at C-25 of these two isomers was established by X-ray crystallography as 25S for one diastereoisomer (mp 199-201 degrees C) and 25R for the other (mp 180-182 degrees C). These findings permit us to determine, unequivocally, the configuration of this naturally occurring C27-bile acid in man and other animals and to establish the stereospecificity of the microsomal and mitochondrial omega-hydroxylation pathway for the side-chain oxidation of cholesterol to bile acids.     

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.     

Cleavage of the taurine conjugate of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid by rat fecal bacteria.

This paper describes a method for the hydrolysis of the taurine conjugates of the 25R and the 25S diastereoisomers of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid (THCA) with retention of original configuration of C-25. Rat fecal suspensions were incubated with the taurine conjugate of THCA for 5 and 60% of the free THCA was recovered. When bile from Alligator mississippiensis, which contains mostly the taurine conjugate of THCA, was analyzed by this method, THCA was obtained with the 25R configuration.     

The metabolism of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestan-26-oic acid into cholic: an enzyme assay using homogenates of human liver.

An enzyme assay was developed to measure the conversion of the bile acid precursor, 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestan-26-oic acid (THCA), into cholic acid using homogenates of human liver biopsies. The average rate of metabolism of THCA into cholic acid was found to be 3.9 +/- 0.5 (+/- 1 SD) pmoles of cholic acid formed/mg liver/minute in twelve normal liver biopsies. This assay system can be used to determine if the syndrome of neonatal cholestasis associated with a metabolic block in the conversion of THCA into cholic acid is transmitted as a genetic trait.     

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.     

Occurrence of both (25R)- and (25S)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acids in urine from an infant with Zellweger's syndrome

Urine from a patient with Zellweger's syndrome was examined for bile acids after fractionation into three groups according to mode of conjugation. 3 alpha,7 alpha,12 alpha-Trihydroxy-5 beta-cholestanoic acid was the predominant bile acid of the unconjugated and glycine-conjugated bile acid fractions. Smaller amounts of cholic acid and 1 beta-, 6 alpha-, 24-, and 26-hydroxylated derivatives of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid were found in both fractions in similar proportions. The bile acid spectrum of the taurine-conjugated bile acid fraction was different from those of the other two fractions in the occurrence of two new compounds as the major constituents. These compounds were tentatively identified as two epimers at C-23 of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestano-26,23-lactone, which were probably artifacts formed from the corresponding tetrahydroxycholestanoic acids during the procedures for extraction after hydrolysis. High-performance liquid chromatographic analysis revealed that 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid excreted into the urine as the unconjugated form consisted of a mixture of (25R)- and (25S)-isomers in the ratio of about 7:3.     

Improved synthesis of 5 beta-cholestan-26-oic acids.

An improved method for the synthesis of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acid and 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26-oic acid is described. The method involves an Arndt-Eistert rearrangement of the corresponding diazoketone obtained by the action of diazoethane on 3 alpha, 7 alpha-diformyloxy-5 beta-cholane-24-carboxylic or 3 alpha, 7 alpha, 12 alpha-triformyloxy-5 beta-cholane-24-carboxylic acid chloride. The products are obtained in good yield and no isomeric 27-nor- 24-methyl acid contaminants are formed as encountered in the commonly employed Kolbe synthesis.     

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

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

Structural and biosynthetic studies of a principal bile alcohol, 27-nor-5beta-cholestane-3alpha,7alpha,12alpha,24,25-pentol, in human urine

The stereochemistry at C-24 and C-25 of 27-nor-5beta-cholestane-3alpha,7alpha,12alpha,24 ,25-pentol, a principal bile alcohol in human urine, and its biosynthesis are studied. Four stereoisomers of the C(26)-24,25-pentols were synthesized by reduction with LiAlH(4) of the corresponding epoxides prepared from (24S)- or (24R)-27-nor-5beta-cholest-25-ene-3alpha, 7alpha,12alpha,24-tetrol. The stereochemistries at C-25 were deduced by comparison of the C(26)-24,25-pentols with the oxidation products of (24Z)-27-nor-5beta-cholest-24-ene-3alpha,7alpha, 12alpha-triol with osmium tetraoxide. On the basis of this assignment, the principal bile alcohol excreted into human and rat urine was determined to be (24S,25R)-27-nor-5beta-cholestane-3alpha,7alpha, 12alpha,24,25-pentol, accompanied by a lesser amount of (24R, 25R)-isomer. To elucidate the biosynthesis of the C(26)-24,25-pentol, a putative intermediate, 3alpha,7alpha, 12alpha-trihydroxy-27-nor-5beta-cholestan-24-one, derived from 3alpha,7alpha, 12alpha-trihydroxy-24-oxo-5beta-cholestanoic acid by decarboxylation during the side-chain oxidation of 3alpha,7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid, was incubated with rat liver homogenates. The 24-oxo-bile alcohol could be efficiently reduced to yield mainly (24R)-27-nor-5beta-cholestane-3alpha,7alpha, 12alpha,24-tetrol. If a 25R-hydroxylation of the latter steroid occurs, it should lead to formation of (24S,25R)-C(26)-24,25-pentol. Now it has appeared that a major bile alcohol excreted into human urine is (24S,25R)-27-nor-5beta-cholestane-3alpha,7alpha, 12alpha, 24, 25-pentol, which might be derived from 3alpha,7alpha, 12alpha-trihydroxy-27-nor-5beta-cholestan-24-one via (24R)-27-nor-5beta-cholestane-3alpha, 7alpha,12alpha,24-tetrol.     

Synthesis of 3 alpha,7 alpha,12 alpha,25-tetrahydroxy-5 beta-cholestan-24-one, an intermediate in the 25-hydroxylation pathway of cholic acid biosynthesis from cholesterol

This paper describes the chemical synthesis of 3 alpha,7 alpha,12 alpha,25-tetrahydroxy-5 beta-cholestan-24-one via selective oxidation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha, 24 xi,25-pentol with silver carbonate on celite. The structure of this 24-keto bile alcohol was confirmed by gas-liquid chromatography and mass spectrometry. Synthesis of this compound via pyridinium chlorochromate oxidation of the triacetoxy derivative of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24 xi,25-pentol followed by saponification further established its structure. 3 alpha,7 alpha,12 alpha,25-Tetrahydroxy-5 beta-cholestan-24-one was required for the in vivo and in vitro studies of side-chain oxidation and cleavage in the 25-hydroxylation pathway of cholic acid biosynthesis.     

Stereoselective synthesis of (24R and 24S) 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentols and (25R and 25S) 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentols using a modified osmium-catalyzed Sharpless asymmetric dihydroxylation process.

Described herein are the stereoselective syntheses of the (24R, 24S) and (25R, 25S) isomers of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentols and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentols by using a modified osmium-catalyzed Sharpless asymmetric dihydroxylation process. Also presented herein are the results of lanthanide-induced CD Cotton effect measurements and 1H- and 13C-nuclear magnetic resonance studies of (24R, 24S) and (25R, 25S)-5 beta-cholestanepentols and their derivatives. These compounds were required to study the biosynthesis of cholic acid from cholesterol.     

C26-analogs of naturally occurring bile alcohols-II. Preparation of 24-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,23-tetrols (23R and 23S) and 24-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,26-tetrols (25R and 25S) by a hydroboration procedure

A convenient procedure for the synthesis of 24-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,23-tetrol (23R and 23S) and 24-nor-5 beta-cholestane-3 alpha,7 alpha,12alpha,26-tetrol (25R and 25S) starting from 24-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol was developed. Dehydration of 24-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha, 25-tetrol with glacial acetic acid and acetic anhydride yielded a mixture of 24-nor-5 beta-cholest-23-ene-3 alpha,7 alpha,12 alpha-triol and the corresponding delta 25 compound. Hydroboration and oxidation of the mixture of unsaturated nor-triols resulted in the formation of 24-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,23-tetrols (23R and 23S) and 24-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,26-tetrols (25R and 25S). In addition, smaller amounts of 24-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,22 xi-tetrol and 24-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol were also obtained. The C26 bile alcohols epimeric at C-23 and C-25 were resolved by analytical and preparative TLC and characterized by gas-liquid chromatography and mass spectrometry. Provisional assignment of the configurations of the C-23 and C-25 hydroxyl groups were made on the basis of molecular rotation differences. These C26 alcohols will be used to test the stereospecificity of the hepatic enzymes that promote oxidation of the cholesterol side chain.     

Reverse cross-coupling in the synthesis 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid.

The present report describes the characterization of (24R and 24S)-27-nor-24-methyl-3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acids obtained in considerable amounts during the synthesis of (25RS)-3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acid by the electrolytic coupling of chenodeoxycholic acid and the half ester of methylsuccinic acid. The mixture of 24R and 24S diastereomers was resolved by analytical and preparative thin-layer chromatography and characterized by gas-liquid chromatography, proton magnetic resonance, and molecular rotation differences. For reference, the model compound, 27-nor-3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acid, was synthesized by electrolytic coupling of chenodeoxycholic acid and the half ester of succinic acid.     

Absolute configuration at C-24 of 5 beta-ranol, a principal bile alcohol of the bullfrog

The stereochemistry of the hydroxyl group at C-24 in 5 beta-ranol (27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,26-pentol) a principal bile alcohol of the bullfrog which is structurally related to the major human urinary bile alcohol, 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentol, is described. Two isomers (IIIa and IIIb) at C-24 of 27-nor-5 beta-cholest-25-ene-3 alpha,7 alpha,12 alpha, 24-tetrol were synthesized from cholic acid (I) by the conversion to 3 alpha, 7 alpha, 12 alpha-triacetoxy-5 beta-cholan-24-al (II) followed by a Grignard reaction with vinylmagnesium bromide. The absolute configurations at C-24 of the unsaturated tetrols (IIIa and IIIb) were elucidated as S and R, respectively, by means of the difference of the reactivity to Sharpless oxidation, a stereoselective epoxidation. Catalytic hydrogenation of each delta 25-tetrol (IIIa or IIIb) gave (24R)- or (24S)-27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha, 24-tetrol (IVa or IVb). The configurations at C-24 of two isomeric 3 alpha,7 alpha,12 alpha,24-tetrahydroxy-27-nor-5 beta-cholestan-26-oic acids (Va and Vb) were determined as S and R, respectively, by means of their conversion into the saturated tetrols (IVa and IVb) of known absolute configurations by a Kolbe electrolytic coupling with acetic acid. The lithium aluminum hydride reduction product of the 24R-acid (Vb) was identical with the naturally occurring 5 beta-ranol, hence 5 beta-ranol has the 24R configuration.     

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.     

Synthesis of C-22, C-23-3H-labeled 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestane

This report describes an efficient synthesis of C-22, C-23-(3)H-labeled 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestane. - Somanathan, R., and S. Krisans. Synthesis of C-22, C-23-(3)H-labeled 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestane.     

Synthesis and structure of 26 (or 27)-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24S,25 xi-pentol isolated from the urine and feces of a patient with sitosterolemia and xanthomatosis

The urine and feces of a patient with the rare inherited lipid storage disease, sitosterolemia and xanthomatosis, were analyzed. Substantial quantities of C26-bile alcohol, 26 (or 27)-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24S,25 xi-pentol along with 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24-tetrol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24R,25-pentol, and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol were found. The structure of the C26-bile alcohol was confirmed by direct comparison (gas-liquid chromatography-mass spectrometry and thin-layer chromatography) with a standard sample synthesized from cholic acid. The configurational assignment at C-24 was determined by lanthanide-induced circular dichroism Cotton effect measurements. The increased excretion of these C26- and C27-bile alcohols suggests an abnormality of bile acid biosynthesis in this disease.     

Improved synthesis of 5 beta-cholestane-3 alpha,7 alpha,12 alpha, 26(27)-tetrol isomers from cholic acid

Synthesis of a mixture of the 25(R) and 25(S) isomers of 5 beta-cholestane-3 alpha,7 alpha,12 alpha, 26(27)-tetrol from cholic acid in four steps, including a Wittig reaction, is described.     

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).