Δ22-β-Muricholic acid in monoassociated rats and conventional ratsacid in monoassociated rats and conventional rats

Bile acids were analyzed in the bile, small and large intestines, and feces of germ-free rats after a single inoculation with one of six intestinal bacteria that had been originally isolated from human feces.Bacteroides vulgatus andBifidobacterium longum preferentially deconjugated tauro-β-muricholic acid and taurocholic acid, respectively.Clostridium ramosum, Peptostreptococcus productus andLactobacillus gasseri deconjugated both bile acids, butEscherichia coli did not deconjugate either one. Rats inoculated with bacteria that deconjugated tauro-β-muricholic acid produced Δ²²-β-Muricholic acid in the feces. In contrast, Δ²²-cholic acid could not be detected in rats inoculated with bacteria that deconjugated taurocholic acid.     

Deconjugation of bile acids by human intestinal bacteria implanted in germ-free rats

Fecal bile acids in germ-free rats were analyzed after inoculation withBacteroides vulgatus, Bifidobacterium longum, Escherichia coli orClostridium ramosum. B. vulgatus preferentially deconjugated tauro-β-muricholic acid andB. longum taurocholic acid.C. ramosum deconjugated both bile acids, butE. coli deconjugated neither. 7α-Dehydroxylation of bile acids was negligible even after 18 days of inoculation, but a small amount of 7-oxo-bile acid, less than 5%, was formed. Fecal excretion of bile acids increased after inoculation withB. vulgatus, B. longum andC. ramosum, but not withE. coli.     

Deoxycholic acid formation in gnotobiotic mice associated with human intestinal bacteria

In humans and animals, intestinal flora is indispensable for bile acid transformation. The goal of our study was to establish gnotobiotic mice with intestinal bacteria of human origin in order to examine the role of intestinal bacteria in the transformation of bile acids in vivo using the technique of gnotobiology. Eight strains of bile acid-deconjugating bacteria were isolated from ex-germ-free mice inoculated with a human fecal dilution of 10⁻⁶, and five strains of 7α-dehydroxylating bacteria were isolated from the intestine of limited human flora mice inoculated only with clostridia. The results of biochemical tests and 16S rDNA sequence analysis showed that seven out of eight bile acid-deconjugating strains belong to a bacteroides cluster (Bacteroides vulgatus, B. distasonis, and B. uniformis), and one strain had high similarity with Bilophila wadsworthia. All five strains that converted cholic acid to deoxycholic acid had greatest similarity with Clostridium hylemonae. A combination of 10 isolated strains converted taurocholic acid into deoxycholic acid both in vitro and in the mouse intestine. These results indicate that the predominant bacteria, mainly Bacteroides, in human feces comprise one of the main bacterial groups for the deconjugation of bile acids, and clostridia may play an important role in 7α-dehydroxylation of free-form primary bile acids in the intestine although these strains are not predominant. The gnotobiotic mouse with bacteria of human origin could be a useful model in studies of bile acid metabolism by human intestinal bacteria in vivo.     

Composition of cecal bile acids in ex-germfree mice inoculated with human intestinal bacteria

Germfree (GF) mice were orally inoculated with human fecal suspension or various components of human fecal microbiota. Three weeks after the inoculation, cecal bile acid composition of these mice was examined. More than 80% of total bile acids was deconjugated in the cecal contents of ex-GF mice associated with human fecal dilutions of 10⁻² or 10⁻⁶, or anaerobic growth from a dilution of 10⁻⁶. In these ex-GF mice, deoxycholic acid accounted for about 20% of total bile acids. In the cecal contents of ex-GF mice associated only with clostridia, unconjugated bile acids made up less than 40% of total bile acids, about half of those in other ex-GF groups. However, the percentage of deoxycholic acid in these mice was the same as that in the other groups. These results indicate that dominant anaerobic bacterial combination is efficient for deconjugation of primary bile acids, and that clostridia in the human feces may play an important role in 7α-dehydroxylation of unconjugated primary bile acids in the intestine.     

Fecal steroids in diarrhea: IV. Cholera

Fecal bile acid and neutral sterol patterns were studied in eight healthy adult volunteers who were challenged withVibrio cholerae classical Ogawa 395 strain in the course of vaccine development studies. Bacterial 7α-dehydroxylation of cholic and chenodeoxycholic acids was not altered during experimentally induced cholera diarrhea, despite the fact that fecal weight in g/day (wet wt) was increased greatly during diarrhea (1913±390 vs 161±11 in controls, p<0.005). Consistent with the findings on bile acids, no significant changes in the production of coprostanol, epicoprostanol, or coprostanone were observed although the percentage of unmodified cholesterol was increased during the diarrheal episode (20.7±3.3% vs 11.9±2.3, p<0.02). Total concentrations of both bile acids and cholesterol in mg/g of feces (wet wt) were decreased considerably as a result of diarrhea). However, total bile acid and neutral steroid excretions in mg/kg/day in subjects with and without diarrhea do not appear to be different. Intestinal transit times, measured in eight subjects by the use of carmine red dye, were found to be shortened in diarrhea (5.8±1.1 hr vs 23.4±4.1 hr in controls, p<0.001). The results from this study are similar to those observed in experimentally induced travellers' diarrhea associated with toxigenicEscherichia coli, but they are in striking contrast to the changes in gastrointestinal steroid metabolism observed in acute shigello sis, an invasive intestinal infection.     

Fatty acid composition of the cellular slime mold Polysphondylium pallidum

The cellular slime mold Polysphondylium pallidum was grown upon Escherichia coli B/r, and the fatty acid compositions of total lipids obtained from vegetative amebae and aggregation-competent cells were compared. Fatty acids isolated from vegetative cells included C-17 and C-19 cyclopropane fatty acids and also straight-chain, saturated fatty acids. The cyclopropane fatty acids were derived from the ingested bacteria. Development of amebae to aggregation-competent cells was accompanied by a substantial decrease in saturated cyclopropane fatty acids and a concomitant increase in unsaturated fatty acids and unsaturated cyclopropane fatty acids, mostly as 18∶3 (5,9,12). We report here the fatty acid composition and identify the occurrence of Δ5 desaturation of cyclopropane fatty acids, namely, 9,10-methylene 5-hexadecenoic acid and 11,12-methylene 5-octadecenoic acid. These fatty acids have not been reported previously in the related species Dictyostelium discoideum, which also feeds on E. coli B/r and has Δ5-desaturation activity.     

Effect of chitosan feeding on intestinal bile acid metabolism in rats

The effect of chitosan feeding (for 21 days) on intestinal bile acids was studied in male rats. Serum cholesterol levels in rats fed a commercial diet low in cholesterol were decreased by chitosan supplementation. Chitosan inhibited the transformation of cholesterol to coprostanol without causing a qualitative change in fecal excretion of these neutral sterols. Increased fiber consumption did not increase fecal excretion of bile acids, but caused a marked change in fecal bile acid composition. Litcholic acid increased sigificantly, deoxycholic acid increased to a leasser extent, whereas hyodeoxycholic acid and the 6β-isomer and 5-epimeric 3α-hydroxy-6-keto-cholanoic acid(s) decreased. The pH in the cecum and colon became elevated by chitosan feeding which affected the conversion of primary bile acids to secondary bile acids in the large intestine. In the cecum, chitosan feeding increased the concentration of α-,β-, and ω-muricholic acids, and lithocholic acid. However, the levels of hyodeoxycholic acid and its 6β-isomer, of monohydroxy-monoketo-cholanoic acids, and of 3α, 6ξ, 7ξ-trihydroxy-cholanoic acid decreased. The data suggest that chitosan feeding affects the metabolism of intestinal bile acids in rats.     

Rapid hydrolysis of bile acid conjugates using microwaves: Retention of absolute stereochemistry in the hydrolysis of (25R) 3α,7α,12α-trihydroxy-5β-cholestan-26-oyltaurine

In recent years, defects of bile acid synthesis caused by disorders of peroxisome biogenesis have led to increased interest in C₂₇ bile acids. In humans, while the majority of bile acids are C₂₄ carboxylic acids, the presence of increased concentrations of C₂₇ bile acids and their metabolites in hereditary diseases associated with peroxisomal dysfunction can serve as a useful marker for the intensity of the metabolic disorder. Our present studies describe an efficient method for the rapid hydrolysis of C₂₇ and C₂₄ bile acid conjugates using a commercial microwave oven. The advantages of this method include freedom from racemization, minimal activation, mild reaction conditions, and the highly stereocontrolled nature of the reaction, thus allowing for free bile acid recovery in high yield. For example, when (25R) 3α,7α,12α-trihydroxy-5β-cholestan-26-oyl taurine, a major compound present in the bile of Alligator mississippiensis, was deconjugated with 4% NaOH/diethylene glycol or 1 M LiOH/propylene glycol in the microwave oven for 4–6 min, 3α,7α,12α-trihydroxy-5β-cholestan-26-oic acid (THCA) was obtained in 81% yield with retention of configuration at C-25. It is suggested that present studies will be helpful in delineating the absolute stereochemistry of 3α,7α,12α-trihydroxy-5β-cholestanoyl-CoA oxidase, the peroxisomal enzyme that catalyzes the first step in the oxidation of THCA.     

Biohydrogenation of cholesterol as an index of bacterial 7α-dehydroxylase activity

Fecal steroid compositions of 82 human subjects of various ages and diets and gastrointestinal status were examined by gas liquid chromatography. Progressive increases in bacterial activities on both bile acids and neutral sterols were observed with the advance of age in infants and children. The patterns in the 4-year-olds approached those observed in adults. Bacterial activites on fecal steroids were found to be decreased in adult subjects with acute shigellosis and in those challenged by castor oil. In contrast, no significant changes in fecal steroid profiles were observed in the subjects with traveller's diarrhea assoicated with toxigenicEscherichia coli. The effects of diarrhea on fecal steroids of infants under 1^1/2 years were less consistent than those of adults. However, a close relationship was observed between the degree of 7α-dehydroxylation of cholic acid (expressed as the ratio of deoxycholic to the sum of deoxycholic and cholic acids) and the percentage of cholesterol in the feces (r= 0.921, p<0.001). The correlation between the production of lithocholic acid and the percentage cholesterol was also good (r=−0.739, p<0.001). Analysis of neutral steroids may be a good index of intraluminal bile acid metabolism.     

Effects of different levels of dietarytrans-octadecenoate on steroid metabolism in rats

Rats were fed semipurified diets containing olive oil or partially hydrogenated corn oil at the 5 or 20% level for ca. 30 days. These fat diets contained the same amount of octadecenoate but differed in the geometry with respect to each fat level. Contents oft-18∶1 were 26% and 41% of total fatty acids, respectively. The linoleic acid content was also made equivalent (3.8 energy %). After feeding on cholesterol-free diets, rats ontrans fat, compared to those oncis fat, showed: (a) no changes in serum cholesterol and apolipoprotein levels, (b) no effects on the bile flow and concentrations of biliary cholesterol or bile acids, (c) a trend toward increased fecal excretion of neutral and acidic steroids, (d) a lesser extent of transformation of cholesterol to coprostanol in the gut, and (e) no changes in the composition of biliary and fecal bile acids. Observations (c) and (d) were more marked with a hightrans fat regimen. These observations, except for serum apolipoproteins and fecal steroid excretion, were practically reproducible even when rats were fed cholesterol-enriched diets. A preliminary part of this study was presented at the 74th annual AOCS meeting, Chicago, 1983.     

Identification and Functional Expression of a Δ9-Fatty Acid Desaturase from <Emphasis Type="Italic">Psychrobacter urativorans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The Δ9-fatty acid desaturase is a key enzyme in the synthesis of unsaturated fatty acids. The fatty acid composition of membrane phospholipids in Psychrobacter urativorans is characterized by a high degree of desaturation at Δ9 position. Based on CODEHOP-mediated PCR strategy, a novel gene designated as PuFAD9, putatively encoding a Δ9-fatty acid desaturase (PuFAD9), was isolated from P. urativorans. The gene consists of 1,455 bp and codes for 484 amino acids. Analysis of the amino acid sequence reveals three histidine clusters and a hydropathy profile, typical for membrane-bound desaturases. Activity of the PuFAD9 protein, recombinantly expressed in Escherichia coli was confirmed by GC-MS analysis of the cellular fatty acid composition. It was found that the ratio between palmitoleic and palmitic acid in E. coli cells heterologously expressing the PuFAD9 gene was significantly affected by IPTG induction and the growth temperature.     

Inhibition and induction of bile acid synthesis by ketoconazole effects on bile formation in the rat

The effects of ketoconazole, an antimycotic agent, and metyrapone, an inhibitor of mixed function oxidases, on bile acid synthesis were compared in the rat bothin vitro andin vivo. In rat liver microsomes, ketoconazole was much more potent than metyrapone in inhibiting the activity of cholesterol 7α-hydroxylase, the rate-limiting enzyme in the synthesis of bile acids. The I₅₀ values were 0.42 μM and 0.91 mM for ketoconazole and metyrapone, respectively. Intraduodenal administration of ketoconazole caused a rapid, dose-dependent reduction of bile acid synthesis in eight-day bile diverted rats. A single dose of 50 mg/kg reduced bile acid synthesis to 5% of control value; the same dose of metyrapone caused a reduction to only 85%. Inhibition of bile acid synthesis by ketoconazole was followed by a marked overshoot. At 28 hr after injection of 50 mg/kg of the drug, formation of bile acids was stimulated maximally by 45% compared to control value and remained elevated for more than 20 hr thereafter. Synthesis of all primary bile acids was affected to the same extent. Cholesterol 7α-hydroxylase activity in livers of ketoconazole treated (30 mg/kg) rats with an intact enterohepatic circulation was increased by 70% at 16 hr after i.p. injection of the drug. During the very large decrease of biliary bile acid output with ketoconazole, bile flow rate was relatively increased, due to stimulation of the bile acid-independent fraction of bile flow. The latter effect can probably be explained as caused by biliary secretion of osmotically active metabolites of ketoconazole.     

Escherichia coli sepsis increases hepatic apolipoprotein B secretion by inhibiting degradation

Sepsis leads to hypertriglyceridemia in both humans and animals. Previously, we reported that plasma very low density lipoprotein apolipoprotein (apo) B and hepatic production of apoB increased during Escherichia coli sepsis. The present experiments were undertaken to determine whether the altered hepatic secretion of apoB was associated with an increase in synthesis or a decrease in degradation rate. Sepsis was induced in male, Lewis rats (225–275 g) by intravenous injection of 3.8×10⁸ live E. coli colonies/100 g body. Twenty-four hours later rats were sacrificed, and primary hepatocytes were prepared and incubated overnight with ³⁵S-methionine. Hepatocytes from E. coli-treated rats secreted twice as much apoB-48 and total apoB than the hepatocytes from control rats. Escherichia coli sepsis increased celular triglyceride mass by 86%, which was due to a stimulation in triglyceride synthesis from newly synthesized fatty acids, measured by ³H₂O incorporation into triglycerides. The apoB synthesis rate, apoB mRNA levels, and apoB mRNA editing were not altered during E. coli sepsis. The pulse-chase experiments showed that the rate of apoB degradation decreased in E. coli-treated rats. These findings demonstrate that the secretion of apoB is regulated posttranslationally during E. coli sepsis by decreasing the degradation of newly synthesized apoB, which contributes to the development of hypertriglyceridemia.     

Preparation, characterization, and surface and biological properties of N-stearoyl amino acids

Several lipoamino acids were synthesized, in which n-octadecanoic acid (stearic acid) was coupled with the α-amino group of an amino acid. The products were characterized and their identities confirmed by advanced analytical techniques like Fourier transform infrared ¹H nuclear magnentic resonance spectroscopy, and differential scanning, calorimetry. Their surface properties, such as critical micelle concentration (CMC) and foaming properties, biodegradability, and antimicrobial activity were also evaluated. The N-stearoyl amino acids (NSA) had low CMC values, and some of them showed good foaming properties. They were screened for antimicrobial activity against the gram-positive bacteria Staphylococcus aureus, Micrococcus luteus, and Bacillus cerceus, the gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and the yeast Candida albicans. All the compounds inhibited at least one of these organisms. N-Stearoyl proline was the most effective, the order of antimicrobial activity being aromatic NSA> acidic NSA>basic NSA. However, the effective inhibition by all the compounds indicates the desirability of more thorough investigation and suggests that some of these compounds may have potential utility as biostatic additives in commercial products. All NSA are highly biodegradable and can readily be removed under conditions of normal secondary sewage treatment.     

Effect of dietary bile acids,cholesterol, and β-sitosterol upon formation of coprostanol and 7-dehydroxylation of bile acids by rat

During studies of sterol metabolism in the rat, the fecal neutral sterol fraction was analyzed by a combination of thin layer chromatography and gas liquid chromatography. On a stock diet of rat chow supplemented with 5% corn oil, the rats excreted 14.5 mg/day of total neutral sterols. Coprostanol comprised 35% (5 mg/day) of this fraction. When the diet was supplemented with 0.5% sodium taurochenodeoxycholate, the amount of coprostanol in the feces remained the same as in the controls (3.2 mg/day, 32%). The addition of 0.5% sodium taurocholate to the diet resulted in a fivefold reduction of coprostanol formation (0.6 mg/day, 8%). When 1.2% cholesterol was added to the stock diet, the amount of coprostanol present in the feces decreased to an average of 11% compared to controls, but the absolute amount formed was greater (35 mg/day). On a diet enriched with 0.8% β-sitosterol, the rats, on the average, converted 23% of the cholesterol to coprostanol. Feeding diets enriched with sodium taurochenodeoxycholate and sodium taurocholate reduced the 7-dehydroxylation of primary bile acids in the feces by 28% and 42%, respectively. The conversion of primary bile acids to secondary bile acids in the feces of control, cholesterol, and β-sitosterol fed rats was the same (64%).     

The cholesterol-lowering effect of guar gum is not the result of a simple diversion of bile acids toward fecal excretion

The effects of partially hydrolyzed, nonviscous, guar gum (PHGG) on cholesterol metabolism and digestive balance have been compared with those of native guar gum (GUAR) in rats adapted to 0.4% cholesterol diets. Both types of guar gum elicited acidic fermentations in the large intestine, but only GUAR effectively lowered plasma cholesterol (P<0.001), chiefly in the triglyceride-rich lipoprotein fraction. The biliary bile acid excretion was significantly enhanced in rats fed GUAR (P<0.05), as well as the intestinal and cecal bile acid pool (P<0.001). In rats fed GUAR and to a lesser extent in those fed PHGG, the fecal excretion of bile acids and neutral sterol was higher than in controls (P<0.01). The digestive balance (cholesterol intake-steroid excretion) was positive in control rats (+47 μmol/d), whereas it was negative in rats fed GUAR (−20 μmol/d), which could involve a higher rate of endogenous cholesterol synthesis. In rats fed PHGG, the steroid balance remained slightly positive. Liver 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was very low (22 pmol/min/mg protein), owing to cholesterol supplementation, in control rats or in rats fed PHGG, whereas it was markedly higher (+463%) in rats fed GUAR. In conclusion, even if PHGG does alter some parameters of the enterohepatic cycle of cholesterol and bile acids, its effects are not sufficient to elicit a significant cholesterol-lowering effect. The intestinal (ileal or cecal) reabsorption of bile acids was not reduced, but rather increased, by GUAR; nevertheless the intestinal capacities of reabsorption were overwhelmed by the enlargement of the digestive pool of bile acids. In the present model, induction of HMG-CoA reductase probably takes place in the presence of elevated portal bile acid concentrations.     

Effect of addition of amino acids on microfiltration behavior of a microorganism slurry

The effect of the addition of amino acids on the microfiltration behavior of Escherichia coli (E coli) and Corynebacterium glutamicum (C glutamicum) slurry was examined using a dead‐end microfilter. It was found that the average specific filtration resistance, α_av, of a slurry of microorganisms increased markedly by adding amino acids. Amino acid concentration ranged from 110 to 876 mol m^?3. However the same concentration of ammonium chloride did not increase α_av. The cells were found to disperse in the presence of lysine, and this caused an increase in α_av. In the case of kaolin slurry, α_av was not affected by adding amino acids. Copyright © 2004 Society of Chemical Industry     

Synthesis and application to cellulose of reactive dye precursor of anti‐bacterial N‐halamine

To achieve textile dyeing and functional finishing in one process, a bleach‐resistant reactive dye precursor to anti‐bacterial N‐halamine was synthesised by reacting a type of dichlorotriazine reactive dye with 4‐amino‐2,2,6,6‐tetramethylpiperidine. The synthesised compound, which can be transformed to an N‐halamine molecule by exposure to dilute bleach solution, was used to dye cotton fabrics. After exposure to a dilute sodium hypochlorite solution, dyed cotton fabrics showed excellent anti‐bacterial properties against Staphylococcus aureus and Escherichia coli O157:H7, facilitating a ca. 6‐log reduction in bacteria within a short period of contact. Compared with the dichlorotriazine reactive dye, the reactive dye precursor demonstrated comparable dyeing properties including exhaustion and fixation values. No differences in rub fastness, wash fastness or bleach fastness were detected between fabrics dyed with, respectively, dichlorotriazine reactive dye and the reactive dye precursor to N‐halamine.     

Separation of bile acid methyl esters by high-performance liquid chromatography

Conjugated bile acids, namely glyco- and tauro-3α,6α-dihydroxy-5β-cholanoic acid (hyodeoxycholic acid), 3α,7α-dihydroxy-5β-cholanoic acid (chenodeoxycholic acid), 3α,6α,7α-trihydroxy-5β-cholanoic acid (hyocholic acid) and 3α-hydroxy-6-oxo-5β-cholanoic acid (6-keto-litocholic acid) were isolated from pig bile, and subsequently transformed into the corresponding methyl esters. Separation of the methyl esters of the isolated bile acids by high-performance liquid chromatography (HPLC) was accomplished on a ZORBAX-CN column (Dupont, Boston, MA) withn-hexane/2-propanol/methylene chloride (89∶6∶5, by vol) as the mobile phase containing traces (≈1%) of amyl alcohol and water as moderators. HPLC analysis of the methyl esters also showed the presence of methyl 3α-hydroxy-6-oxo-5α-cholanoate, which was probably produced in the course of alkaline hydrolysis of the conjugated bile acids.     

Bile Acids Conjugation in Human Bile Is Not Random: New Insights from <Superscript>1</Superscript>H-NMR Spectroscopy at 800 MHz

Bile acids constitute a group of structurally closely related molecules and represent the most abundant constituents of human bile. Investigations of bile acids have garnered increased interest owing to their recently discovered additional biological functions including their role as signaling molecules that govern glucose, fat and energy metabolism. Recent NMR methodological developments have enabled single-step analysis of several highly abundant and common glycine- and taurine- conjugated bile acids, such as glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, and taurochenodeoxycholic acid. Investigation of these conjugated bile acids in human bile employing high field (800 MHz) ¹H-NMR spectroscopy reveals that the ratios between two glycine-conjugated bile acids and their taurine counterparts correlate positively (R ² = 0.83–0.97; p = 0.001 × 10⁻²–0.006 × 10⁻⁷) as do the ratios between a glycine-conjugated bile acid and its taurine counterpart (R ² = 0.92–0.95; p = 0.004 × 10⁻³–0.002 × 10⁻¹⁰). Using such correlations, concentration of individual bile acids in each sample could be predicted in good agreement with the experimentally determined values. These insights into the pattern of bile acid conjugation in human bile between glycine and taurine promise useful clues to the mechanism of bile acids’ biosynthesis, conjugation and enterohepatic circulation, and may improve our understanding of the role of individual conjugated bile acids in health and disease. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.