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.     

7β,12β-dihydroxy-5β-cholan-24-oic acid as an internal standard for quantitative determination of bile acids by gas chromatography

In order to find an artificial internal standard compound for quantitative determination of bile acids by gas chromatography, 7α,12α-,7α, 12β-, 7β,12α- and 7β,12β-dihydroxy-5β-cholan-24-oic acids were chemically synthesized with cholic acid (1) as the first starting material. The gas chromatographie retention time of 7β,12β-dihydroxy-5β-cholan-24-oic acid (ββ-isomer) was more different from that of natural bile acids than the other isomers. Moreover, ββ-isomer was extracted in the same fraction as the bile acids from urine, and no urinary substance had the same retention time as ββ-isomer. No artifact was produced from ββ-isomer during the analysis procedure. It was concluded that the ββ-isomer is an internal standard compound with certain advantages for the quantitative determination of bile acids in urine by gas chromatography, irrespective of the recovery rate during the analysis procedure.     

Role of endogenous and exogenous cholesterol in liver as the precursor for bile acids in rats

There is considerable evidence suggesting that compartmentalized functional pools of cholesterol in the liver contribute differently to the formation of bile acids as the precursor. The present paper deals with the incorporation of [1-¹⁴C]acetate and of [1,2-³H]cholesterol carried on lipoproteins (LDL and HDL) into biliary bile acids in perfused rat livers and bile-fistula rats. The results showed that endogenous cholesterol synthesized newly from [1-¹⁴C]acetate in the liver was incorporated into both cholic acid and chenodeoxycholic acid in a similar way, while exogenous lipoprotein-[1,2-³H]cholesterol delivered to hepatocytes from hepatic circulation was incorporated into chenodeoxycholic acid at a higher rate.     

Identification of (22R)-3 alpha,7 alpha,12 alpha,22- and (23R)-3 alpha,7 alpha,12 alpha,23-tetrahydroxy-5 beta-cholestanoic acids in urine from a patient with Zellweger's syndrome

The nature of two novel C27 bile acids present as the taurine conjugates in urine from a patient with Zellweger's syndrome was studied. Bile acids conjugated with taurine were isolated from unconjugated and glycine-conjugated bile acids by means of ion-exchange chromatography. After alkaline hydrolysis of the taurine conjugates, the hydrolysate was acidified and extracted with ether; the extract was again subjected to ion-exchange chromatography to separate neutral from acidic compounds. The neutral fraction, which consisted mainly of two steroidal lactones, was treated with lithium aluminum hydride, and the reduction products were identified as (22R)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,22,26-pentol and (23R)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,26-pentol by direct comparison of their gas-liquid chromatographic behaviors and mass spectral data with those of chemically synthesized authentic samples. Thus, the chemical structure of two native bile acids present in urine from a patient with Zellweger's syndrome should be formulated as (22R)-3 alpha,7 alpha,12 alpha,22-tetrahydroxy-5 beta-cholestanoic acid and (23R)-3 alpha,7 alpha,12 alpha,12 alpha,23-tetrahydroxy-5 beta-cholestanoic acid, respectively.     

Synthesis, intestinal absorption and metabolism of sarcosine conjugated ursodeoxycholic acid

Sarcosine conjugated ursodeoxycholic acid (SUDC) was synthesized and its intestinal absorption and metabolism were studied in rat and hamster. Intestinal absorption study using bile fistula rat shows that more than 90% of SUDC administered intraduodenally was excreted in the bile within 24 hr. No change of the administered bile acid was seen during the absorption from the intestine, the passage of the liver, and the excretion into the bile. When [24-14C]SUDC and [11,12-3H2]-ursodeoxycholic acid were administered orally to a hamster, more than 95% of both the administered 14C and 3H were recovered from the feces within 6 days. Most (77%) of the fecal 14C-labeled compound was SUDC, whereas 95% of the fecal 3H-labeled compound was unconjugated lithocholic acid. These results indicate that SUDC, unlike taurine or glycine conjugated bile acid, resists bacterial deconjugation and 7-dehydroxylation.     

Methoxylation of methyl 3α,7α-dihydroxychol-4-en-24-oate and its 3β-epimer. —a contribution to chenodeoxycholic acid biogenesis

By the conventional methods of gas liquid chromatography (GLC) as well as mass spectrometry, 3β,7α-dihydroxychol-5-en-24-oic acid (Δ⁵-acid), a key intermediate of chenodeoxycholic acid biogenesis and its metabolic by-product, 3α,7α-dihydroxychol-4-en-24-oic acid (Δ⁴-acid) have not yet been identified as such probably due to thermal decomposition. However, taking advantage of the observation that they are readily methoxylated in methanoi containing a trace of acids, their individual methoxy-compounds were easily prepared and proved to be useful for their identification, even though they are present in minimal amounts as was the case with the human or hen bile. The present paper reported physical as well as spectral properties of the methoxy-compounds derived from methyl 3α,7α-dihydroxychol-4-en-24-oate, compared with those of its 3β-epimer     

Methoxylation of 3β,7α-dihydroxychol-5-en-24-oic acid,a key intermediate of chenodeoxycholic acid biogenesis,compared with that of its 7β-epimer

The conventional methods of gas liquid chromatography or mass spectrometry failed to be useful for the identification of the biliary 3β, 7α-hydroxychol--en-24-oic acid, a key intermediate of chenodeoxycholic acid biogenesis. It has been preliminarily reported that this acid in human bile was successfully identified by gas chromatography-mass spectrometry, after the methoxylation of its allyl alcohol group. Physical as well as spectral properties of the methoxylation products derived from the acid were reported, compared with those from its 7β-epimer.     

Persistent enhancement of bile acid synthesis in guinea pigs following stimulation of cholesterol catabolism in neonatal life

Cholesterol catabolism to bile acids was stimulated in neonatal guinea pigs by feeding 1,11% cholestyramine (CT)-containing diet for 8 weeks. The animals were then switched to standard laboratory diet for an additional 4 weeks. At the end of the laboratory diet period: a) CT-pre-treated guinea pigs continued to excrete significantly higher (p<0.05) amounts of bile acids, b) the activity of hepatic 7α-hydroxylase was significantly elevated (p<0.01) in CT-pre-treated animals, and c) isolated hepatocytes from CT-pre-treated guinea pigs secreted significantly higher (p<0.05) amounts of bile acid when compared to controls during a 4-hour incubation. These data provide biochemical support for our contention that stimulation of cholesterol catabolism during neonatal life can have effects that persist into adult life.     

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.     

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.     

Influence of dietary bile acids on formation of bile acids in rat

Various taurine-conjugated bile acids were fed to rats at the 1%-level in the diet for 3 or 7 days and the effect on several hydroxylations involved in the biosynthesis and metabolism of bile acids was studied. The hydroxylations studied were all catalyzed by the microsomal fraction of liver homogenate fortified with NADPH. The 7α-hydroxylation of cholesterol was inhibited by feeding taurocholic acid, taurocheno-deoxycholic acid and taurodeoxycholic acid for 3 as well as 7 days. No marked inhibition was obtained with taurohyodeoxycholic acid or taurolithocholic acid. The 12α-hydroxylation of 7α-hydroxy-4-cholesten-3-one was inhibited after 3 as well as 7 days by all bile acids except taurohyodeoxycholic acid. With this acid a marked stimulation of 12α-hydroxylation was observed. The effects of the different bile acids on the 7α-hydroxylation of taurodeoxycholic acid were not very marked. The 6β-hydroxylation of lithocholie acid and taurochenodeoxycholic acid was stimulated by taurocholic acid and taurodeoxycholic acid. The reaction was inhibited by taurochenodeoxycholic acid, at least after 7 days. Taurohyodeoxycholic acid inhibited the 6β-hydroxylation slightly and taurolithocholic acid had no effect. The results were discussed in the light of present knowledge concerning mechanisms of regulation of formation and metabolism of bile acids and it was suggested that the mechanisms may be more complex than previously thought.     

Synthesis of 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol and 5beta-cholestane-3alpha, 7alpha, 245, 25-pentol.

This paper describes syntheses of 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol and 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol which give higher yields than previously published methods. In addition, 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol was synthesized by a different procedure, namely via performic acid oxidation of the correspinding unsaturated triol, which gave a lower yield but avoided the formation of 5beta-cholestane-3alpha, 7alpha, 12alpha, 25, 26-pentol, which normally tends to contaminate the final product. Structures were confirmed by gas-liquid chromatography, infrared-, proton magnetic resonance- and mass spectrometry, 5beta-Cholestane-3alpha, 7alpha, 12alpha, 25-tetrol and 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol were required for in vivo and in vitro studies of the (hypothetical) 25-hydroxylation pathway of cholic acid biosynthesis.     

Transformation of 23,24-bisnorchol-4-en-3-one-22-ol by Rhizopus arrhizus

The transformation of 23,24-bisnorchol-4-en-3-one-22-ol into 6β,11α,22-trihydroxy-23,24-bisnorchol-4-en-3-one by the fungus Rhizopus arrhizus has been shown to be dependent on the composition of the culture medium, with respect to yield of the triol. The transformation of the 22-alcohol to 6β,11α-dihydroxy-pregn-4-ene-3,20-dione is also reported.     

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.     

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.     

New bile alcohols--synthesis of 5beta-cholestane-3alpha, 7alpha, 25-triol and 5beta-cholestane-3alpha, 7alpha, 25-24 (14C)-triol.

5beta-Cholestane-3alpha, 7alpha, 25-triol and 5beta-cholestane-3alpha, 7alpha, 25-24(14-C)-triol were synthesized from 3alpha, 7alpha-dihydroxy-5beta-cholanoic acid (chenodeoxycholic acid). Chenodeoxycholic acid was converted to the diformoxy derivative (II) using formic acid. Reaction of II with thionyl chloride yielded the acid chloride which was treated with diazomethane (CH-2-N-2 or 14-CH-2-N-2) to produce 3alpha, 7alpha-diformoxy-24-oxo-25-diazo-25-homocholane (III, A or B). 25-Homochenodeoxycholic acid (IV, A or B) was formed from III by means of the Wolff rearrangement of the Arndt-Eistert synthesis. The methyl ester of V (A or B) was treated with methyl magnesium iodidi in ether to provide the desired triol, VI (A and B). The triol was identified by mass spectrometry and elemental analysis and was characterized by thin-layer and gas-liquid chromatography. The 3alpha, 7alpha, 25-triol is of possible significance as an intermediate in the pathway of bile acid formation from cholesterol.     

Identification of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25,26-hexol in human urine

A new bile alcohol, 5 beta-cholestanehexol, was identified in the urine of healthy humans as the glucuronide. The bile alcohol glucuronide fraction was isolated by an ion exchange chromatography on piperidinohydroxypropyl Sephadex LH-20. After enzymatic hydrolysis, the bile alcohols were converted into trimethylsilyl ether derivatives and analyzed by a combination of gas-liquid chromatography and mass spectrometry. The major bile alcohol was 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentol. As minor constituents the following C26 and C27 bile alcohols were identified: 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25,26-hexol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,26-pentol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol. In addition to these bile alcohols, a new bile alcohol was identified as a sixth component of the urinary bile alcohols. The structure was assigned as (24S)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25,26-hexol by the direct comparison of mass spectral data and chromatographic properties with synthetic standard. The average daily excretion of the new bile alcohol was 28.6 micrograms and 3.0% of the total bile alcohols. The presence of 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentol and 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25,26-hexol suggests that 26-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentol is most likely for the biosynthesis of this new bile alcohol.     

Identification of new bile alcohols, 5 beta-cholestane-3 alpha,7 alpha,24,26-tetrol, 5 beta-cholestane-3 alpha,7 alpha,25,26-tetrol, and 5 beta-cholestane-3 alpha,7 alpha,26,27-tetrol in human gallbladder bile

The nature of cholestanetetrols present as the glucurono-conjugates in human gallbladder bile was studied. Glucurono-conjugated bile alcohols were isolated by ion exchange chromatography and, after enzymatic hydrolysis, were fractionated by reversed phase partition chromatography to give a fraction containing tetrahydroxy bile alcohols which was analyzed by gas-liquid chromatography and mass spectrometry. Along with the three previously identified bile alcohols, 5 alpha- and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha,24-tetrols, and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha,26-tetrol, three new cholestanetetrols, possessing two hydroxyl groups in the ring system and two in the side chain, were detected in the tetrahydroxy bile alcohol fraction. These new bile alcohols were identified as 5 beta-cholestane-3 alpha, 7 alpha,24,26-tetrol, 5 beta-cholestane-3 alpha, 7 alpha,25,26-tetrol, and 5 beta-cholestane-3 alpha, 7 alpha,26,27-tetrol by direct comparison of their gas-liquid chromatographic behaviors and mass spectral data with those of authentic standards prepared from chenodeoxycholic acid by partial synthesis.     

Metabolism of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 26-tetrol and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 25-tetrol into cholic acid in normal human subjects.

Side chain oxidation and cleavage of precursors in cholic acid synthesis is thought to involve initial hydroxylation at either position 25 or 26 of the side chain. Therefore, the conversion of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 26-tetrol and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 25-tetrol into cholic acid was studied in normal subjects after single intravenous injections of these labeled alcohols. Eighty-six percent and 82% of 5 beta-cholestane, 3 alpha, 7 alpha, 12 alpha, 26-tetrol was converted into cholic acid in two subjects, respectively. However, only 14 and 16% of the injected 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 25-tetrol was converted into cholic acid in two subjects, respectively. Thus, this study indicates that 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 25-tetrol is an inefficient substrate for cholic acid biosynthesis in man and that the major route of cholic acid synthesis probably involves the 26-hydroxylated intermediate.     

Bile acids. LXIV. Synthesis of 5α-cholestane-3α,7α,25-triol and esters of new 5α-bile acids

Interest in the structural requirements of a sterol or bile acid for maximal activity by an hepatic microsomal steroid 12α-hydroxylase prompted the preparation of 5α-cholestane-3α, 7α, 25-triol and 5α-analogs of 3α, 7α-dihydroxy-5β-cholane-24-carboxylic acid. Methyl 3α, 7α-dihydroxy-5β-cholane-24-carboxylate derived from methyl chenodeoxycholate via the Arndt-Eistert reaction was allomerized with Raney nickel in boiling p-cymene to provide a number of products of which methyl 3,7-dioxo-5β- and 5α-cholane-24-carboxylates, methyl 3-oxo-7α-hydroxy-5β-and 5α-cholane-24-carboxylates, were identified. Reduction with K-Selectride of methyl 3-oxo-7α-hydroxy-5β-cholane-24-carboxylate, provided a high yield of methyl 3α, 7α-dihydroxy-5α-cholane-24-carboxylate. Treatment of this ester with an excess of methyl magnesium iodide afforded 5α-cholestane-3α, 7α, 25-triol. The products were characterized by thin-layer and gas liquid chromatography, proton resonance, infrared and mass spectrometry.