Absence of cecal secondary bile acids in gnotobiotic mice associated with two human intestinal bacteria with the ability to dehydroxylate bile acids in vitro.

Germ-free mice were orally inoculated with human intestinal 7alpha-dehydroxylating bacterial strains to evaluate their ability to transform bile acids in vivo. Three weeks after inoculation of the bacteria, cecal bile acids were examined. Among free-form bile acids, only beta-muricholic acid was detected in the cecal contents of gnotobiotic mice associated with Bacteroides distasonis strain K-5. No secondary bile acid was observed in the cecal contents of any of the gnotobiotic mice associated with 7alpha-dehydroxylating bacteria, Clostridium species strain TO-931 or Eubacterium species strain 36S.     

Pressurized water extraction of β‐glucan enriched fractions with bile acids‐binding capacities obtained from edible mushrooms

A pressurized water extraction (PWE) method was developed in order to extract β‐glucans with bile acids‐binding capacities from cultivated mushrooms (Agaricus bisporus, Lentinula edodes, and Pleurotus ostreatus) to be used as supplements to design novel foods with hypocholesterolemic properties. Extraction yields were higher in individual than sequential extractions being the optimal extraction parameters: 200°C, 5 cycles of 5 min each at 10.3 MPa. The crude polysaccharide (PSC) fractions, isolated from the PWE extracts contained mainly β‐glucans (including chitooligosaccharides deriving from chitin hydrolysis), α‐glucans, and other PSCs (hetero‐/proteo‐glucans) depending on the extraction temperature and mushroom strain considered. The observed bile acids‐binding capacities of some extracts were similar to a β‐glucan enriched fraction obtained from cereals. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:391–400, 2014     

Metabolism of Bile Salts in Mice Influences Spore Germination in Clostridium difficile

Clostridium difficile, a spore-forming bacterium, causes antibiotic-associated diarrhea. In order to produce toxins and cause disease, C. difficile spores must germinate and grow out as vegetative cells in the host. Although a few compounds capable of germinating C. difficile spores in vitro have been identified, the in vivo signal(s) to which the spores respond were not previously known. Examination of intestinal and cecal extracts from untreated and antibiotic-treated mice revealed that extracts from the antibiotic-treated mice can stimulate colony formation from spores to greater levels. Treatment of these extracts with cholestyramine, a bile salt binding resin, severely decreased the ability of the extracts to stimulate colony formation from spores. This result, along with the facts that the germination factor is small, heat-stable, and water-soluble, support the idea that bile salts stimulate germination of C. difficile spores in vivo. All extracts able to stimulate high level of colony formation from spores had a higher proportion of primary to secondary bile salts than extracts that could not. In addition, cecal flora from antibiotic-treated mice was less able to modify the germinant taurocholate relative to flora from untreated mice, indicating that the population of bile salt modifying bacteria differed between the two groups. Taken together, these data suggest that an in vivo-produced compound, likely bile salts, stimulates colony formation from C. difficile spores and that levels of this compound are influenced by the commensal gastrointestinal flora.     

The Hylemon-Björkhem pathway of bile acid 7-dehydroxylation: history,biochemistry, and microbiology

Bile acids are detergents derived from cholesterol that function to solubilize dietary lipids, remove cholesterol from the body, and act as nutrient signaling molecules in numerous tissues with functions in the liver and gut being the best understood. Studies in the early 20th century established the structures of bile acids, and by mid-century, the application of gnotobiology to bile acids allowed differentiation of host-derived “primary” bile acids from “secondary” bile acids generated by host-associated microbiota. In 1960, radiolabeling studies in rodent models led to determination of the stereochemistry of the bile acid 7-dehydration reaction. A two-step mechanism was proposed, which we have termed the Samuelsson-Bergström model, to explain the formation of deoxycholic acid. Subsequent studies with humans, rodents, and cell extracts of Clostridium scindens VPI 12708 led to the realization that bile acid 7-dehydroxylation is a result of a multi-step, bifurcating pathway that we have named the Hylemon-Björkhem pathway. Due to the importance of hydrophobic secondary bile acids and the increasing measurement of microbial bai genes encoding the enzymes that produce them in stool metagenome studies, it is important to understand their origin.     

Comparison among fecal secondary bile acid levels, fecal microbiota and Clostridium scindens cell numbers in Japanese

Bile acid 7alpha-dehydroxylation by intestinal bacteria, which converts cholic acid and chenodeoxycholic acid to deoxycholic acid (DCA) and lithocholic acid (LCA), respectively, is an important function in the human intestine. Clostridium scindens is one of the most important bacterial species for bile acid 7alpha-dehydroxylation because C. scindens has high levels of bile acid 7alpha-dehydroxylating activity. We quantified C. scindens and secondary bile acids, DCA and LCA, in fecal samples from 40 healthy Japanese and investigated their correlation. Moreover, we used terminal restriction fragment length polymorphism (T-RFLP) analysis to investigate the effect of fecal microbiota on secondary bile acid levels. There was no correlation between C. scindens and secondary bile acid in fecal samples. On the other hand, T-RFLP analysis demonstrated that fecal microbiota associated with high levels of DCA were different from those associated with low levels of DCA, and furthermore that fecal microbiota in the elderly (over 72 years) were significantly different from those in younger adults (under 55 years). These results suggest that intestinal microbiota have a stronger effect on DCA level than does the number of C. scindens cells.     

First Report about Mineral Content,Fatty Acids Composition and Biological Activities of Four Wild Edible Mushrooms

The goal of this research was a comprehensive analysis of four wild edible mushroom species, Cantharellus cinereus, Clavariadelphus pistillaris, Clitocybe nebularis and Hygrocybe punicea, which have not been analyzed so far. Extracts of different polarities have been prepared and evaluated for their antioxidant activities by DPPH, ABTS, FRAP, TRP and CUPRAC methods. For all extracts, total phenolic content was determined. Based on the analysis, it was shown that solvent type had a significant effect on the antioxidant capacities of mushroom extracts, so water extracts showed the highest activity. Furthermore, the analysis includes determination of mineral composition, fatty acid profiles and antimicrobial activity. Unsaturated fatty acids, which are very important for human health, are dominant in the studied mushroom species. Linoleic and oleic acid consist of over 50 % of the total fatty acid composition. Seventeen biologically important and toxic elements have been analyzed by ICP‐OES and ICP‐MS and results showed that the element concentrations were species‐dependent. Also, it has been found that analyzed mushrooms did not show any antimicrobial activity. Chemometric analysis was used to understand the connection between the extracts of different polarities.     

Medium-Chain Fatty Acids Enhanced the Excretion of Fecal Cholesterol and Cholic Acid in C57BL/6J Mice Fed a Cholesterol-Rich Diet

The objective of the present study was to investigate the cholesterol-reducing effect of medium-chain fatty acids (MCFAs) completed by elevated excretion of fecal neutral steroids and/or bile acids. Blood and liver lipid profiles, fecal neutral steroids, bile acids, and mRNA and protein expression of the genes relevant to cholesterol homeostasis were measured and analyzed in C57BL/6J mice fed a cholesterol-rich diet with 2% caprylic acid or capric acid for 12 weeks. Blood total cholesterol and low-density lipoprotein cholesterol (LDL-c) levels were reduced significantly as compared to diet with palmitic acid or stearic acid. Caprylic acid promoted the excretion of fecal neutral steroids, especially cholesterol. The excretion of fecal bile acids, mainly in the form of cholic acid was enhanced and accompanied by elevated expression of mRNA and the protein of hepatic cholesterol 7α-hydroxylase (CYP7A1). These results indicate that MCFAs can reduce blood cholesterol by promoting the excretion of fecal cholesterol and cholic acid.     

A simple and accurate HPLC method for fecal bile acid profile in healthy and cirrhotic subjects: validation by GC-MS and LC-MS

We have developed a simple and accurate HPLC method for measurement of fecal bile acids using phenacyl derivatives of unconjugated bile acids, and applied it to the measurement of fecal bile acids in cirrhotic patients. The HPLC method has the following steps: 1) lyophilization of the stool sample; 2) reconstitution in buffer and enzymatic deconjugation using cholylglycine hydrolase/sulfatase; 3) incubation with 0.1 N NaOH in 50% isopropanol at 60°C to hydrolyze esterified bile acids; 4) extraction of bile acids from particulate material using 0.1 N NaOH; 5) isolation of deconjugated bile acids by solid phase extraction; 6) formation of phenacyl esters by derivatization using phenacyl bromide; and 7) HPLC separation measuring eluted peaks at 254 nm. The method was validated by showing that results obtained by HPLC agreed with those obtained by LC-MS/MS and GC-MS. We then applied the method to measuring total fecal bile acid (concentration) and bile acid profile in samples from 38 patients with cirrhosis (17 early, 21 advanced) and 10 healthy subjects. Bile acid concentrations were significantly lower in patients with advanced cirrhosis, suggesting impaired bile acid synthesis.     

Bioconversion of propionic,valeric, and 4-hydroxybutyric acids into the corresponding alcohols byClostridium acetobutylicum NRRL 527

The addition of biogenic (acetate and butyrate) or abiogenic (propionate, isobutyrate, and valerate) volatile fatty acids to theClostridium acetobutylicum growth medium reduced the lag phase. Propionate and valerate were reduced to the corresponding alcohols (l-propanol andl-pentanol). Dicarboxylic acids were not metabolized, but reduced glucose utilization and solvent synthesis. A hydroxyacid, 4-hydroxybutyric acid, was quantitatively converted to 1,4-butanediol.     

Growth inhibition of Clostridium thermocellum by carboxylic acids: A mechanism based on uncoupling by weak acids

Summary The inhibition of Clostridium thermocellum strains by acetate and other organic acids (propionate, butyrate) can be explained by a model based on the chemiosmotic theory and uncoupler action. It is proposed that the charged permeant species in the process of anion exclusion is the dimer HA _- ² . Evidence for this mechanisms is provided by ³¹P-NMR studies of whole cells and cell extracts.Abbreviations CM4-Cb CM4-cellobiose - EPC⁵⁰ equipotency concentration - P _m bilayer partition coefficient - ka acid dissociation constant     

Intestinal steroids in rats are influenced by the structural parameters of pectin

We investigated the effects of pectin with different degrees of methylation (34.5, 70.8, and 92.6%, respectively) on the composition and concentration of intestinal and fecal bile acids and neutral sterols in conventional and germfree rats. Diets containing 6.5% pectin (galacturonan) were given for 3 weeks. High concentrations of free and secondary bile acids appeared in cecum and colon of conventional rats. With increasing degree of methylation, more bile acids were transported into lower parts of intestinal tract and excreted whereas the proportion of secondary bile acids decreased. In contrast, the composition of bile acids in intestinal contents and feces was relatively unchanged in germfree rats. Exclusively cholesterol was found as a neutral sterol in germfree rats. Coprostanol appeared in cecum of conventional rats and additionally coprostanone in colon. Amounts of neutral sterols increased with increasing degree of methylation of pectin. Additionally, concentrations of bile acids in plasma decreased if the pectin-containing diets were given. Besides the degree of methylation, the molecular weight of pectin used in the diets influenced concentration and composition of intestinal and fecal steroids in rats.     

Effect of bile acid deconjugation on the fecal excretion of steroids

The effect of microbiological deconjugation of bile acids on total bile acid and neutral sterol fecal excretion by adult male rats has been studied. A screening method utilizing mice allowed selection of a Clostridium perfringens type A strain, which accelerated cholesterol catabolism in mice. When this species of bacteria was associated with germfree rats, the fecal bile acids were excreted as free bile acids (deconjugated), however the quantities of bile acids excreted were not increased compared with those of germfree rats. Conventional rats excrete twice as much bile acids (all deconjugated) as do the germfree and C. perfringens-associated rats. It is, therefore, unlikely that the microbiological deconjugation of bile acids is responsible for the increased fecal excretion of bile acids seen in conventional rats. The C. perfringens-associated rats excreted identical kinds and quantities of fecal neutral sterols as did the germfree rats.     

The Effect of Dietary Prebiotics and Probiotics on Body Weight,Large Intestine Indices,and Fecal Bile Acid Profile in Wild Type and IL10?/? Mice

Previous studies have suggested roles of probiotics and prebiotics on body weight management and intestinal function. Here, the effects of a dietary prebiotic, inulin (50 mg/g diet), and probiotic, Bfidobacterium animalis subsp. lactis (Bb12) (final dose verified at 10⁵ colony forming unit (cfu)/g diet, comparable to human consumption), were determined separately and in combination in mice using cellulose-based AIN-93G diets under conditions allowed for the growth of commensal bacteria. Continuous consumption of Bb12 and/or inulin did not affect food intake or body, liver, and spleen weights of young and adult mice. Fecal bile acid profiles were determined by nanoESI-MS/MS tandem mass spectrometry. In the presence of inulin, more bacterial deconjugation of taurine from primary bile acids was observed along with an increased cecal weight. Consumption of inulin in the absence or presence of Bb12 also increased the villus cell height in the proximal colon along with a trend of higher bile acid sulfation by intestinal cells. Feeding Bb12 alone at the physiological dose did not affect bile acid deconjugation and had little effect on other intestinal indices. Although interleukin (IL)10-null mice are susceptible to enterocolitis, they maintained the same body weight as the wild type mice under our specific pathogen-free housing condition and showed no signs of inflammation. Nevertheless, they had smaller cecum suggesting a mildly compromised intestinal development even before the disease manifestation. Our results are consistent with the notion that dietary factors such as prebiotics play important roles in the growth of intestinal microbiota and may impact on the intestinal health. In addition, fecal bile acid profiling could potentially be a non-invasive tool in monitoring the intestinal environment.     

Influence of high-fat diet on host animal health via bile acid metabolism and benefits of oral-fed Streptococcus thermophilus MN-ZLW-002

In this study, C57BL/6J male mice were fed normal chow (NC; control) or a high-fat diet (HFD) for 12 weeks, and HFD mice were supplemented with oral administration of Streptococcus thermophilus MN-ZLW-002 (HFD + MN002); n=20/group. Body weight, visceral fat, blood glucose, blood lipids and liver lipid deposition increased in the HFD group, and the composition of gut microbiota, cecum short-chain fatty acids and fecal bile acids (BAs) also changed. Oral-fed MN-002 increased the relative abundances of Ruminococcaceae, Lachnospiraceae and Streptococcaceae and improved blood glucose, liver cholesterol deposition, and serum IL-10, CCL-3 and the fecal BAs composition. In conclusion, the high-fat diet changed the composition of bile acids by shaping the gut microbiota into an obese type, leading to metabolic disturbances. Streptococcus thermophilus MN-ZLW-002 regulated gut microbiota by adjusting the composition of bile acids and improved the perturbation caused by high-fat diets. However, the effect of MN002 observed in animal experiments needs to be verified by long-term clinical trials.     

Acidic extracellular pH shifts colorectal cancer cell death from apoptosis to necrosis upon exposure to propionate and acetate,major end-products of the human probiotic propionibacteria

The human probiotic Propionibacterium freudenreichii kills colorectal adenocarcinoma cells through apoptosis in vitro via its metabolites, the short chain fatty acids (SCFA), acetate and propionate. However, the precise mechanisms, the kinetics of cellular events and the impact of environmental factors such as pH remained to be specified. For the first time, this study demonstrates a major impact of a shift in extracellular pH on the mode of propionibacterial SCFA-induced cell death of HT-29 cells, in the pH range 5.5 to 7.5 prevailing within the colon. Propionibacterial SCFA triggered apoptosis in the pH range 6.0 to 7.5, a lethal process lasting more than 96 h. Indeed at pH 7.5, SCFA induced cell cycle arrest in the G2/M phase, followed by a sequence of cellular events characteristic of apoptosis. By contrast, at pH 5.5, the same SCFA triggered a more rapid and drastic lethal process in less than 24 h. This was characterised by sudden mitochondrial depolarisation, inner membrane permeabilisation, drastic depletion in ATP levels and ROS accumulation, suggesting death by necrosis. Thus, in digestive cancer prophylaxis, the observed pH-mediated switch between apoptosis and necrosis has to be taken into account in strategies involving SCFA production by propionibacteria to kill colon cancer cells. This work has been supported by a grant from CRITT santé Bretagne.     

Impact of Mushrooms’ Vegetative Places and Morphological Parts of a Fruiting Body on the Fatty Acids Profile of Wild Leccinum aurantiacum and Leccinum versipelle

The aim of this study was to indicate potential differences in composition of fatty acids between two mushroom species as well as to examine the impact of mushrooms’ vegetative places and morphological parts of a fruiting body on the fatty acids profile. The research material consisted of 72 samples of wild Leccinum aurantiacum and Leccinum versipelle in the form of caps and stipes, collected from three selected regions of Poland. Determination of the examined compounds was performed by gas chromatography (FID). Linoleic (C18 : 2), oleic (C18 : 1) and palmitic (C16:0) acids were the predominant compounds in all samples under study. The profile of fatty acids in Leccinum aurantiacum and Leccinum versipelle was varied depending on mushroom species, a region and morphological parts of a fruiting body. The high content of polyunsaturated fatty acids in polish L. aurantiacum and L. versipelle provides that the mushroom may be recommended in different types of diets.     

Changes in Colonic Bile Acid Composition following Fecal Microbiota Transplantation Are Sufficient to Control Clostridium difficile Germination and Growth

Fecal microbiota transplantation (FMT) is a highly effective therapy for recurrent Clostridium difficile infection (R-CDI), but its mechanisms remain poorly understood. Emerging evidence suggests that gut bile acids have significant influence on the physiology of C. difficile, and therefore on patient susceptibility to recurrent infection. We analyzed spore germination of 10 clinical C. difficile isolates exposed to combinations of bile acids present in patient feces before and after FMT. Bile acids at concentrations found in patients’ feces prior to FMT induced germination of C. difficile, although with variable potency across different strains. However, bile acids at concentrations found in patients after FMT did not induce germination and inhibited vegetative growth of all C. difficile strains. Sequencing of the newly identified germinant receptor in C. difficile, CspC, revealed a possible correspondence of variation in germination responses across isolates with mutations in this receptor. This may be related to interstrain variability in spore germination and vegetative growth in response to bile acids seen in this and other studies. These results support the idea that intra-colonic bile acids play a key mechanistic role in the success of FMT, and suggests that novel therapeutic alternatives for treatment of R-CDI may be developed by targeted manipulation of bile acid composition in the colon.     

Propionate Enhances Synthesis and Secretion of Bile Acids in Primary Cultured Rat Hepatocytes via Succinyl CoA

To discover the role of propionate produced by colonic bacteria, this study examined the secretion of bile acids and cholesterol 7α-hydroxylase activity in the primary cultured hepatocytes. Addition of propionate (2 mM) to the medium for 48 h caused an increase in the bile acid secretion and enzyme activity, while acetate and butyrate had no significant influence. Bile acid secretion was increased by the addition of succinyl CoA and its precursor substances (α -ketoglutarate, valine, isoleucine, and methionine), but not malate and oxaloacetate, which are the metabolites of succinyl CoA. α -Ketoglutarate and valine also increased the activity of cholesterol 7α -hydroxylase. Since cholesterol 7α -hydroxylase is a microsomal cytochrome P-450 enzyme and the formation of δ-aminolevulinate from succinyl CoA in the mitochondria is the rate-controlling step for the subsequent synthesis of heme proteins, propionate may affect bile acid synthesis via elevation of mitochondrial succinyl CoA.     

Ascaris suum infection was associated with a worm-independent reduction in microbial diversity and altered metabolic potential in the porcine gut microbiome

The effect of infection of pigs with Ascaris suum on the microbial composition in the proximal colon and fecal matter was investigated using 16S rRNA gene sequencing. The infection significantly decreased various microbial diversity indices including Chao1 richness, but the effect on Chao1 in the colon luminal contents was worm burden-independent. The abundance of 49 genera present in colon contents, such as Prevotella and Faecalibacterium, and 179 operational taxonomic units was significantly changed as a result of infection. Notably, infection was also associated with a significant shift in the metabolic potential of the proximal colon microbiome, where the relative abundance of at least 30 metabolic pathways including carbohydrate metabolism and amino acid metabolism was reduced, while the abundance of 28 pathways was increased by infection. Furthermore, the microbial co-occurrence network in infected pigs was highly modular. Two of 52 modules or subnetworks were negatively correlated with fecal butyrate concentrations (r?<??0.7; P?<?0.05) while one module with 18 members was negatively correlated with fecal acetate, propionate and total short-chain fatty acids. A partial Mantel test identified a strong positive correlation between node connectivity of the operational taxonomic units assigned to β-Proteobacteria (especially the family Alcaligenaceae) and fecal acetate and propionate levels (r?=?0.82 and 0.74, respectively), while that of the family Porphyromonadaceae was positively correlated with fecal egg counts. Overall, Ascaris infection was associated with a profound change in the gut microbiome, especially in the proximity of the initial site of larval infection, and should facilitate our understanding of the pathophysiological consequence of gastrointestinal nematode infections.