Bile acid synthesis. Metabolism of 3 beta-hydroxy-5-cholenoic acid to chenodeoxycholic acid

Metabolism of 3 beta-hydroxy-5-cholenoic acid to chenodeoxycholic acid has been found to occur in rabbits and humans, species that cannot 7 alpha-hydroxylate lithocholic acid. This novel pathway for chenodeoxycholic acid synthesis from 3 beta-hydroxy-5-cholenoic acid led to a reinvestigation of the pathway for chenodeoxycholic acid from 3 beta-hydroxy-5-cholenoic acid in the hamster. Simultaneous infusion of equimolar [1,2-3H]lithocholic acid and 3 beta-hydroxy-5-[14C]cholenoic acid indicated that the 14C enrichment of chenodeoxycholic acid was much greater than that of lithocholic acid. Thus, in all these species, a novel 7 alpha-hydroxylation pathway exists that prevents the deleterious biologic effects of 3 beta-hydroxy-5-cholenoic acid.     

Solvolysis of chenodeoxycholic acid sulfates

Chemical solvolysls of chenodeoxycholic acid sulfates was studied using 4 published methods. Quantitative recovery of chenodeoxycholic acid from the 3-sulfate was obtained with each method. However, only 2 methods yielded chenodeoxycholic acid after solvolysis of the 7-sulfate. In each instance a compound resembling lithocholic acid by GLC but identifiable as a derivative of chenodeoxycholic acid by mass spectrometry was obtained and represents a product formed during solvolysis. Failure to obtain adequate solvolysis of chenodeoxycholic acid 7-sulfate can lead to false identification of monohydroxy bile acids and apparent absence of the 7-sulfate and disulfate esters.     

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

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

Bioconversion of 3beta-hydroxy-5-cholenoic acid into chenodeoxycholic acid by rat brain enzyme systems

We have previously demonstrated that the rat brain contains three unconjugated bile acids, and chenodeoxycholic acid (CDCA) is the most abundantly present in a tight protein binding form. The ratio of CDCA to the other acids in rat brain tissue was significantly higher than the ratio in the peripheral blood, indicating a contribution from either a specific uptake mechanism or a biosynthetic pathway for CDCA in rat brain. In this study, we have demonstrated the existence of an enzymatic activity that converts 3beta-hydroxy-5-cholenoic acid into CDCA in rat brain tissue. To distinguish marked compounds from endogenous related compounds, 18O-labeled 3beta-hydroxy-5-cholenoic acid, 3beta,7alpha-dihydroxy-5-cholenoic acid, and 7alpha-hydroxy-3-oxo-4-cholenoic acid were synthesized as substrates for in vitro incubation studies. The results clearly suggest that 3beta-hydroxy-5-cholenoic acid was converted to 3beta,7alpha-dihydroxy-5-cholenoic acid by microsomal enzymes. The 7alpha-hydroxy-3-oxo-4-cholenoic acid was produced from 3beta,7alpha-dihydroxy-5-cholenoic acid by the action of microsomal enzymes, and Delta4-3-oxo acid was converted to CDCA by cytosolic enzymes. These findings indicate the presence of an enzymatic activity that converts 3beta-hydroxy-5-cholenoic acid into CDCA in rat brain tissue. Furthermore, this synthetic pathway for CDCA may relate to the function of 24S-hydroxycholesterol, which plays an important role in cholesterol homeostasis in the body.     

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

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

Bioconversion of acid-hydrolyzed poplar hemicellulose to acetic acid byClostridium thermoaceticum

Summary Clostridium thermoaceticum was used to ferment carbohydrate released from pretreated oat splet xylan and hemicellulose isolated from hybrid poplar. Hydrolysis with dilute sulfuric acid (2.5% (v/v) for oat spelt xylan and 4.0% (v/v) for poplar hemicellulose) at 100°C for 60 min was found to release the highest concentration of fermentable substrate.C. thermoaceticum, when grown in non-pH controlled batch culture at 55°C under a headspace of 100% CO₂, typically produced 14gl^–1 acetic acid during a 48 h fermentation in medium containing 2% xylose. In fed-batch fermentations this organism was able to produce 42gl^–1 acetic acid after 116h when the concentration of xylose was maintained at approximately 2% and the pH was controlled at 7.0.     

Involvement of 3-dehydroecdysone in the 3-epimerization of ecdysone.

The epimerization of ecdysone to 3-epiecdysone has been investigated in a dialysed cytosolic enzyme preparation from midgut of sixth instar Spodoptera littoralis larvae, with particular emphasis on establishing the intermediacy of 3-dehydroecdysone. Incubation of ecdysone with the dialysed cytosolic preparation furnished 3-dehydroecdysone as the only detectable product, the reaction being oxygen-dependent. The enzyme preparation catalysed reduction of 3-dehydroecdysone to 3-epiecdysone and ecdysone in the presence of NADH or NADPH. Whereas formation of 3-epiecdysone greatly predominated over that of ecdysone in the presence of NADPH, the converse applied when the cofactor was NADH. 3-Epiecdysone incubated with the enzyme preparation in the presence of various cofactors was not metabolized, indicating the irreversibility of the reduction of 3-dehydroecdysone to 3-epiecdysone and, hence, of the 3-epimerization process. The foregoing results, together with comparison of the metabolism of 3-dehydro[3H]ecdysone and [3H]ecdysone by the enzyme preparation in the presence of unlabelled ecdysone and NADPH, support the intermediacy of 3-dehydroecdysone in the 3-epimerization of ecdysone.     

Effects of extractive fermentation on butyric acid production byClostridium acetobutylicum

Summary The addition of an oleyl alcohol extractant to a batch fermentation of glucose byClostridium acetobutylicum resulted in a concentration profile that was distinctly different from the non-extractive control fermentation. The concentration of butyric acid increased and subsequently decreased in the control fermentation. The concentration of butyric acid increased but did not subsequently decrease in the oleyl alcohol extractive fermentation. The production of butyric acid was found to have been prolonged into the solventogenic phase in the oleyl alcohol extractive fermentation. Butyric acid was continually replenished from glucose while it was being converted to butanol. Supplementation of exogenous acetic and butyric acids, the metabolic uncoupler carbonyl cyanide 3-chlorophenylhydrazone, or decanol to the oleyl alcohol extractive fermentation helped to reinstate the normal butyric acid concentration profile. These findings are discussed with respect to the effects of these additives on the pH ofC. acetobutylicum and its importance with regard to the production of butyric acid.     

Transformation of chenodeoxycholic acid by thermophilic Geobacillus stearothermophilus

We performed a series of experiments with Geobacillus stearothermophilus, a thermophile isolated from oil-contaminated soil in the Kuwaiti desert. The organism has a good potential for the transformation of a broad spectrum of organic molecules such as steroids, amino acids, and aromatic hydrocarbons. In the present study, we tested its potential for the transformation of a bile component, chenodeoxycholic acid (CDCA). Five transformed products, namely, cholic acid, methylcholate, methylchenodeoxycholate, 3α-hydroxy-7-oxo-5β-cholanic acid, and 7α-hydroxy-3-oxo-5β-cholanic acid, were the major transformation products catalyzed by G. stearothermophilus. Under aerobic conditions, no evidence of side chain degradation, ring cleavage, or dehydrogenation was found among the metabolites of CDCA. CDCA transformation by a thermophile is reported for the first time.     

Biotransformation of linoleic acid and bile acids by Eubacterium lentum.

Eubacterium lentum is a gram-positive, nonsporeforming, nonmotile, asaccharolytic anaerobe. In the present investigations, 3 E. lentum strains (group E) isolated from rat feces were compared with 30 E. lentum strains (groups A, B, C, and D) previously studied by Macdonald et al. (I. A. Macdonald, J. F. Jellet, D. E. Mahony, and L. V. Holdeman, Appl. Environ. Microbiol. 37:992-1000, 1979). All strains alkalized (pH 8 to 8.5) arginine-containing (2 to 15 mg/ml) culture media, and growth of the majority of the strains was stimulated by arginine. All strains converted linoleic acid into transvaccenic acid by shifting the 12,13-cis double bond of linoleic acid into an 11,12-trans(?) double bond followed by biohydrogenation of the 9,10-cis double bond. Hence, biohydrogenation of linoleic acid is a new general characteristic of E. lentum. The 33 strains were also studied for bile acid deconjugase and hydroxysteroid dehydrogenase (HSDH) activities. The 6 strains in group D were steroid inactive; the 27 strains in groups A, B, C, and E were steroid active. The steroid-active group contained bile acid deconjugase-producing strains (groups C and E, plus strain 116 in group A) and nondeconjugating strains. All nondeconjugating strains of groups A and B developed 7 alpha- and 12 alpha-HSDH activities and contained 3 alpha-HSDH-positive strains and 3 alpha-HSDH-negative strains. Deconjugating strains varied in HSDH activities.     

Formation of chenodeoxycholic acid from 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid by rat liver peroxisomes

The oxidation of the side chain of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA) into chenodeoxycholic acid has been studied in subcellular fractions of rat liver. The product was separated from the substrate by high pressure liquid chromatography and identified by gas-liquid chromatography-mass spectrometry. The highest specific rate of conversion was found in the heavy (M) and the light (L) mitochondrial fractions with the highest enrichment in the L fraction. Washing the M fraction reduced the side chain cleavage activity by 90%. The peroxisomal marker enzyme urate oxidase was reduced to the same extent. The activity found in the M fraction may thus be due to peroxisomal contamination. After centrifugation of the L fraction on a Nycodenz density gradient, the highest specific activity for side chain cleavage of DHCA (31 nmol X mg-1 X h-1) was found in the fraction with the highest peroxisomal marker enzyme activity. This fraction also catalyzed conversion of 3 alpha,7 alpha,12 alpha-5 beta-cholestanoic acid (THCA) into cholic acid at the highest rate (32 nmol X mg-1 X h-1). The peroxisomal oxidation of DHCA into chenodeoxycholic acid required the presence of ATP, CoA, Mg2+, and NAD in the incubation medium. The reaction was not inhibited by KCN. It is concluded that rat liver peroxisomes contain enzymes able to catalyze the cleavage of the side chain of both DHCA and THCA. The enzymes involved are similar to, but not necessarily identical to, those involved in the peroxisomal beta-oxidation of fatty acids.     

Effect of controlled substrate feeding on butyric acid production byClostridium tyrobutyricum

Summary Production of butyric acid from wheat flour hydrolysate was studied withClostridium tyrobutyricum. The mode of substrate supply was found a key parameter for fermentation performance as large improvements were obtained by feeding with a non-limiting supply of substrate. With this procedure, increases in product concentration and productivity but also in selectivity and yield for butyrate were obtained. Substrate feeding controlled by the rate of gas production was found preferable to constant rate feeding for reason of convenience and flexibility. In these conditions, a butyrate concentration of 62.8 gl^–1 was obtained with a productivity of 1.25 gl^–1 h^–1, a selectivity of 91.5% and a yield of 0.45 g per g of glucose.     

Structural requirements of the ASBT by 3D-QSAR analysis using aminopyridine conjugates of chenodeoxycholic acid

The human apical sodium-dependent bile acid transporter (ASBT) is a validated drug target and can be employed to increase oral bioavailability of various drug conjugates. The aim of the present study was to investigate the chemical space around the 24-position of bile acids that influences both inhibition and uptake by the transporter. A series of 27 aminopyridine and aminophenol conjugates of glutamyl-chenodeoxycholate were synthesized and their ASBT inhibition and transport kinetics (parametrized as K(i), K(t), and J(max)) measured using stably transfected ASBT-MDCK cells. All conjugates were potent ASBT inhibitors. Monoanionic conjugates exhibited higher inhibition potency than neutral conjugates. However, neutral conjugates and chloro-substituted monoanionic conjugates were not substrates, or at least not apparent substrates. Kinetic analysis of substrates indicated that similar values for K(i) and K(t) implicate substrate binding to ASBT as the rate-limiting step. Using 3D-QSAR, four inhibition models and one transport efficiency model were developed. Steric fields dominated in CoMFA models, whereas hydrophobic fields dominated CoMSIA models. The inhibition models showed that a hydrophobic or bulky substitute on the 2 or 6 position of a 3-aminopyridine ring enhanced activity, while a hydrophobic group on the 5 position was detrimental. Overall, steric and hydrophobic features around the 24 position of the sterol nucleus strongly influenced bile acid conjugate interaction with ASBT. The relative location of the pyridine nitrogen and substituent groups also modulated binding.     

Rifampicin-induced CYP3A4 activation in CTX patients cannot replace chenodeoxycholic acid treatment

Cerebrotendinous xanthomatosis (CTX) is a rare neurodegenerative disorder with cholestanol accumulation resulting from mutations in the sterol 27-hydroxylase gene (CYP27A). Conventional treatment includes chenodeoxycholic acid and HMG-CoA reductase inhibitors. Mice with disrupted Cyp27A (Cyp27 KO) do not show elevated cholestanol levels nor develop CTX manifestations. This phenomenon was proposed to be due to murine CYP3A overexpression leading to an alternative pathway for degradation of bile alcohols including cholestanol. Our objective was to examine the influence of CYP3A4 induction on cholestanol elimination in CTX patients. Rifampicin (600 mg/day x 7 days), known to induce the PXR, and thereby to increase CYP3A activity, was used. The degree of CYP3A4 induction was assessed by comparing midazolam pharmacokinetics before and after rifampicin treatment. Cholestanol levels and cholestanol/cholesterol ratios were assayed during the experimental period and compared to a 3 weeks period without treatment. The results show that despite 60% increase in CYP3A4 activity following rifampicin treatment, there is no significant change in cholestanol levels. We conclude that up-regulated expression of CYP3A affects cholestanol elimination in mice differently as compared to its effect in CTX patients. Therefore, CYP3A4 inducers cannot replace chenodeoxycholic acid for the treatment of CTX.     

Influence of acetic and butyric acid addition on polysaccharide formation byClostridium acetobutylicum

Summary The production of granulose (an intracellular reserve polygranule), capsule and exopolysaccharide was investigated in a synthetic medium in which the oxido-reduction level was modified by the addition of acetic or butyric acid. After addition of the acids, granulose synthesis increased from 150 to 300 mg glucose equivalents ·1^–1 and capsular synthesis decreased by 25%. Exopolysaccharide production was unchanged under these conditions. A hypothesis that attributes a role to the polymer in the oxido-reduction sequences is discussed.     

Production of extracellular 5-aminolevulinic acid byClostridium thermoaceticum grown in minimal medium

Summary A growth associated formation of extracellular 5-aminolevulinic acid (ALA) was found in the homoacetogenesis of glucose byClostridium thermoaceticum grown in minimal defined medium. The growth and ALA production was enhanced by L-cysteine HCl both in complex medium (UM) and minimal defined medium (MDM). The amount of ALA produced extracellularly in MDM wasca. 15 mg/L after 90-h anaerobic cultivation (cell-mass: 1.5 g/l; glucose consumed: 20 g/l).     

Purification and properties of 3 alpha-hydroxyglycyrrhetinate dehydrogenase of Clostridium innocuum from human intestine

3 alpha-Hydroxyglycyrrhetinate dehydrogenase of Clostridium innocuum, isolated from human intestinal bacteria, was capable of converting 3-ketoglycyrrhetic acid to 3 alpha-hydroxyglycyrrhetic acid. The enzyme was purified to homogeneity by means of butyl-Toyopearl 650M, Sephadex G-150, Matrex Red A, Toyopearl HW-55S, and isoelectric focusing column chromatographies. The purified enzyme showed a specific activity of 156 mumol/min.mg toward 3 alpha-hydroxyglycyrrhetic acid, and showed a single band on SDS-polyacrylamide gel electrophoresis. The apparent molecular weight was 53,000, as estimated by gel filtration, and 30,000, as judged by SDS-polyacrylamide gel electrophoresis. Its isoelectric point was 5.2. The enzyme showed absolute specificity for the 3 alpha-hydroxyl and 3-ketonic groups of 18 alpha- or 18 beta-glycyrrhetic acid and required NADP+ and NADPH as cosubstrates. The enzyme did not act on any 3 alpha-hydroxyl or 3-ketonic group of steroids or bile acids. The enzyme is a novel type of enzyme, defined as 3 alpha-hydroxy-glycyrrhetinate dehydrogenase, being quite different from 3 alpha-hydroxysteroid dehydrogenase [EC 1.1.1.50].     

7alpha-Dehydroxylation of cholic acid and chenodeoxycholic acid by Clostridium leptum.

The rate of 7alpha-dehydroxylation of primary bile acids was quantitatively measured radiochromatographically in anaerobically washed whole cell suspensions of Clostridium leptum. The pH optimum for the 7alpha-dehydroxylation of both cholic and chenodeoxycholic acid was 6.5-7.0. Substrate saturation curves were observed for the 7alpha-dehydroxylation of cholic and chenodeoxycholic acid. However, cholic acid whole cell K0.5 (0.37 micron) and V (0.20 mumol hr-1mg protein-1) values differed significantly from chenodeoxycholic acid whole cell K0.5 (0.18 micron) and V (0.50 mumol-1 hr-1 mg protein-1). 7alpha-Dehydroxylation activity was not detected using glycine and taurine-conjugated primary bile acids, ursodeoxycholic acid, cholic acid methyl ester, or hyocholic acid as substrates. Substrate competition experiments showed that cholic acid 7 alpha-dehydroxylation was reduced by increasing concentrations of chendeoxycholic acid; however, chenodeoxycholic acid 7alpha-dehydroxylation activity was unaffected by increasing concentrations of cholic acid. A 10-fold increase in cholic and 7alpha-dehydroxylation activity occurred during the transition from logarithmic to stationary phase growth whether cells were cultured in the presence or absence of sodium cholate. In the same culture, a similar increase in chenodeoxycholic acid 7alpha-dehydroxylation was detected only in cells cultured in the presence of sodium cholate. These results indicate the possible existence of two independent systems for 7alpha-dehydroxylation in C. Leptum.     

Microbial production of chenodeoxycholic acid precursor, 12-ketochenodeoxycholic acid,from dehydrocholic acid

Summary Two kinds of bacteria (DC33 and DC1115) were isolated from soil as biotransformers of dehydrocholic acid to 12-ketochenodeoxycholic acid, and identified to be Brevibacterium fuscum and Lactobacillus xylosus, respectively. Dehydrocholic acid was converted via 7,12-diketolithocholic acid to 12-ketochenodeoxycholic acid by both strains, and the product and the intermediate were isolated and chemically identified. By using a jar fermentor, 12-ketochenodeoxycholic acid was produced with a more than 50% yield after 52 h by Brevibacterium fuscum with aerobic growth and anaerobic conversion, and after 24 h by Lactobacillus xylosus under anaerobic conditions, respectively.