Bile Acids and Microbiome Among Individuals With Irritable Bowel Syndrome and Healthy Volunteers.

Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder. High bile acid (BA) profiles have been associated with abdominal pain symptoms, mucosal inflammation, and diarrhea in a subgroup of those with IBS. The purpose of this study was to compare: 1) fecal primary and secondary BAs in women with and without IBS; and 2) symptoms, gut microbiome, and diet between women with high and normal BAs (i.e., similar to healthy [HC] women). Women (ages 18-45) with IBS and HCs were recruited from healthcare providers or the community. Participants kept a 28-day symptom diary, completed a 3-day food journal, and collected a stool sample for microbiome analysis (16 S rRNA gene sequencing). Primary and secondary BA levels were determined by mass spectrometry. Primary BAs did not differ between IBS (n = 45) and HC (n = 28) groups; women with IBS had significantly increased conjugated secondary BAs (glycodeoxycholic acid [p = 0.006], taurodeoxycholic acid [p = 0.006], and glycolithocholic acid [p = 0.01]). Sixty percent of women with IBS had normal BAs whereas 40% had high BAs. Women with high fecal BAs were predominantly IBS-Diarrhea or IBS-Mixed and consumed less fiber and vegetable protein and more animal protein compared to women with IBS whose fecal BAs levels were comparable to HCs. Those with high conjugated secondary fecal BAs also had a greater Firmicutes/Bacteroidetes ratio, less abundance of phylum Bacteroidetes and genus Gemmiger, and more abundance of family Erysipelotrichaceae compared to IBS women with normal BAs. Determination of fecal BA levels provides additional insights into pathophysiological links between diet and microbiome in IBS.

Stress can attenuate hepatic lipid accumulation via elevation of hepatic ß-muricholic acid levels in mice with nonalcoholic steatohepatitis.

Stress can affect our body and is known to lead to some diseases. However, the influence on the development of nonalcohol fatty liver disease (NAFLD) remains unknown. This study demonstrated that chronic restraint stress attenuated hepatic lipid accumulation via elevation of hepatic ß-muricholic acid (ßMCA) levels in the development of nonalcoholic steatohepatitis (NASH) in mice. Serum cortisol and corticosterone levels, i.e., human and rodent stress markers, were correlated with serum bile acid levels in patients with NAFLD and methionine- and choline-deficient (MCD) diet-induced mice, respectively, suggesting that stress is related to bile acid (BA) homeostasis in NASH. In the mouse model, hepatic ßMCA and cholic acid (CA) levels were increased after the stress challenge. Considering that a short stress enhanced hepatic CYP7A1 protein levels in normal mice and corticosterone increased CYP7A1 protein levels in primary mouse hepatocytes, the enhanced Cyp7a1 expression was postulated to be involved in the chronic stress-increased hepatic ßMCA level. Interestingly, chronic stress decreased hepatic lipid levels in MCD-induced NASH mice. Furthermore, ßMCA suppressed lipid accumulation in mouse primary hepatocytes exposed to palmitic acid/oleic acid, but CA did not. In addition, Cyp7a1 expression seemed to be related to lipid accumulation in hepatocytes. In conclusion, chronic stress can change hepatic lipid accumulation in NASH mice, disrupting BA homeostasis via induction of hepatic Cyp7a1 expression. This study discovered a new ßMCA action in the liver, indicating the possibility that ßMCA is available for NAFLD therapy.

Modulatory Nano/Micro Effects of Diabetes Development on Pharmacology of Primary and Secondary Bile Acids Concentrations.

BACKGROUND: Recent studies have suggested that hyperglycaemia influences the bile acid profile and concentrations of secondary bile acids in the gut. INTRODUCTION: This study aimed to measure changes in the bile acid profile in the gut, tissues, and faeces in type 1 Diabetes (T1D) and Type 2 Diabetes (T2D). METHODS: T1D and T2D were established in a mouse model. Twenty-one seven-weeks old balb/c mice were randomly divided into three equal groups, healthy, T1D and T2D. Blood, tissue, urine and faeces samples were collected for bile acid measurements. RESULTS: Compared with healthy mice, T1D and T2D mice showed lower levels of the primary bile acid, chenodeoxycholic acid, in the plasma, intestine, and brain, and higher levels of the secondary bile acid, lithocholic acid, in the plasma and pancreas. Levels of the bile acid ursodeoxycholic acid were undetected in healthy mice but were found to be elevated in T1D and T2D mice. CONCLUSION: Bile acid profiles in other organs were variably influenced by T1D and T2D development, which suggests similarity in effects of T1D and T2D on the bile acid profile, but these effects were not always consistent among all organs, possibly since feedback mechanisms controlling enterohepatic recirculation and bile acid profiles and biotransformation are different in T1D and T2D.

[Qualitative and quantitative analysis of major cholic acids in Suis Fellis Pulvis].

This study is to establish a qualitative method for rapid identification of bile acids in Suis Fellis Pulvis based on UHPLC-LTQ-Orbitrap-MS technology,and an HPLC-ELSD internal standard method for the quantitative determination of two glycine-conjugated BAs in Suis Fellis Pulvis.The chromatographic separation of the UHPLC-LTQ-Orbitrap-MS qualitative analysis was achieved on a Waters Acquity UPLC HSS T_3column(2.1 mm×100 mm,1.8µm),with 0.2%formic acid aqueous solution(A)-acetonitrile(B)as mobile phase ingradient elution.Electrospray ionization(ESI)source was applied and operated in negative ion mode.Quantitative analysis was performed at 30℃on a Diamonsil-C_(18)column(4.6 mm×250 mm,5µm).The mobile phase consisted of 0.2%formic acid solution and acetonitrile with gradient elution and the flow rate was 1.0 m L·min~(-1).An ELSD was used with a nitrogen flow-rate of1.4 L·min~(-1)at a drift tube temperature of 60℃and the gain was 1.A total of 14 bile acids in Suis Fellis Pulvis were characterized based on the accurate mass measurements,fragmentation patterns,chromatographic retention times,and reference materials.For the quantitative analysis method,the glycohyodeoxycholic acid and glycochenodeoxycholic acid had good linear relationship in the range of26.52-265.20 mg·L~(-1)(r=0.999 8)and 19.84-198.40 mg·L~(-1)(r=0.999 1),respectively.The average recoveries(n=6)were104.1%and 103.1%,and the RSD were 2.0%and 2.4%.The UHPLC-LTQ-Orbitrap-MS technology provides a fast and efficient qualitative analysis method for identification of bile acids in Suis Fellis Pulvis.The HPLC-ELSD internal standard method is accurate and reliable,which has reference value for the quality control of Suis Fellis Pulvis.

Charge-tagging liquid chromatography-mass spectrometry methodology targeting oxysterol diastereoisomers.

The introduction of a hydroxy group to the cholesterol skeleton introduces not only the possibility for positional isomers but also diastereoisomers, where two or more isomers have different configurations at one or more of the stereocentres but are not mirror images. The differentiation of diastereoisomers is important as differing isomers can have differing biochemical properties and are formed via different biochemical pathways. Separation of diasterioisomers is not always easy by chromatographic methods Here we demonstrate, by application of charge-tagging and derivatisation with the Girard P reagent, the separation and detection of biologically relevant diastereoisomers using liquid chromatography - mass spectrometry with multistage fragmentation.

Development and validation of a sensitive LC-MS-MS method for the simultaneous determination of multicomponent contents in artificial Calculus Bovis.

Artificial Calculus Bovis is a major substitute in clinical treatment for Niuhuang, a widely used, efficacious but rare traditional Chinese medicine. However, its chemical structures and the physicochemical properties of its components are complicated, which causes difficulty in establishing a set of effective and comprehensive methods for its identification and quality control. In this study, a simple, sensitive and reliable liquid chromatography-tandem mass spectrometry method was successfully developed and validated for the simultaneous determination of bilirubin, taurine and major bile acids (including six unconjugated bile acids, two glycine-conjugated bile acids and three taurine-conjugated bile acids) in artificial Calculus Bovis using a Zorbax SB-C18 column with a gradient elution of methanol and 10 mmol/L ammonium acetate in aqueous solution (adjusted to pH 3.0 with formic acid). The mass spectra were obtained in the negative ion mode using dehydrocholic acid as the internal standard. The content of each analyte in artificial Calculus Bovis was determined by monitoring specific ion pairs in the selected reaction monitoring mode. All analytes demonstrated perfect linearity (r(2) > 0.994) in a wide dynamic range, and 10 batches of samples from different sources were further analyzed. This study provided a comprehensive method for the quality control of artificial Calculus Bovis.

Biosynthesis and release of pheromonal bile salts in mature male sea lamprey.

BACKGROUND: In vertebrates, bile salts are primarily synthesized in the liver and secreted into the intestine where they aid in absorption of dietary fats. Small amounts of bile salts that are not reabsorbed into enterohepatic circulation are excreted with waste. In sexually mature male sea lamprey (Petromyzon marinus L.) a bile salt is released in large amounts across gill epithelia into water where it functions as a pheromone. We postulate that the release of this pheromone is associated with a dramatic increase in its biosynthesis and transport to the gills upon sexual maturation. RESULTS: We show an 8000-fold increase in transcription of cyp7a1, a three-fold increase in transcription of cyp27a1, and a six-fold increase in transcription of cyp8b1 in the liver of mature male sea lamprey over immature male adults. LC-MS/MS data on tissue-specific distribution and release rates of bile salts from mature males show a high concentration of petromyzonol sulfate (PZS) in the liver and gills of mature males. 3-keto petromyzonol sulfate (3kPZS, known as a male sex pheromone) is the primary compound released from gills, suggesting a conversion of PZS to 3kPZS in the gill epithelium. The PZS to 3kPZS conversion is supported by greater expression of hsd3b7 in gill epithelium. High expression of sult2b1 and sult2a1 in gill epithelia of mature males, and tissue-specific expression of bile salt transporters such as bsep, slc10a1, and slc10a2, suggest additional sulfation and transport of bile salts that are dependent upon maturation state. CONCLUSIONS: This report presents a rare example where specific genes associated with biosynthesis and release of a sexual pheromone are dramatically upregulated upon sexual maturation in a vertebrate. We provide a well characterized example of a complex mechanism of bile salt biosynthesis and excretion that has likely evolved for an additional function of bile salts as a mating pheromone.

A sensitive analytical method for quantifying petromyzonol sulfate in water as a potential tool for population monitoring of the southern pouched lamprey, Geotria australis, in New Zealand streams.

The migratory southern pouched lamprey, Geotria australis, is a culturally important fish native to New Zealand. Anecdotal evidence suggests that populations of G. australis have declined from historic levels, and presently, this species is rare in many New Zealand rivers and streams. Migratory sea lamprey (Petromyzon marinus) use a pheromone mixture to locate suitable spawning sites. This mixture is comprised of three steroids: petromyzonol sulfate (PS), petromyzonamine disulfate (PADS), and petromyzosterol disulfate (PSDS). We examined the migratory pheromone mixture released by G. australis ammocetes and found that they excrete predominantly PS. PADS has been detected on some occasions in low concentrations, and PSDS either is not released, or is released in extremely low concentrations. By using a recently developed sensitive mass spectrometry method, we compared passive sampling techniques against more traditional active water sampling as methods for estimating lamprey populations in local streams. Passive sampling provided quantitative data for PS from all sites surveyed, with uptake rates of 0.3 to 45.7 pg/day observed. Conversely, active sampling returned only one positive result out of 19 samples, and with a method detection limit of 2.5 × 10(-14) M, this suggests that concentrations of PS in these streams are either extremely low or variable. The combination of passive sampling and triple quadrupole mass spectrometry is a promising tool for monitoring of G. australis in New Zealand streams.

A rapid, sensitive, and selective method for quantitation of lamprey migratory pheromones in river water.

The methodology of using fish pheromones, or chemical signatures, as a tool to monitor or manage species of fish is rapidly gaining popularity. Unequivocal detection and accurate quantitation of extremely low concentrations of these chemicals in natural waters is paramount to using this technique as a management tool. Various species of lamprey are known to produce a mixture of three important migratory pheromones; petromyzonol sulfate (PS), petromyzonamine disulfate (PADS), and petromyzosterol disulfate (PSDS), but presently there are no established robust methods for quantitation of all three pheromones. In this study, we report a new, highly sensitive and selective method for the rapid identification and quantitation of these pheromones in river water samples. The procedure is based on pre-concentration, followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. The method is fast, with unambiguous pheromone determination. Practical quantitation limits of 0.25 ng/l were achieved for PS and PADS and 2.5 ng/l for PSDS in river water, using a 200-fold pre-concentration, However, lower quantitation limits can be achieved with greater pre-concentration. The methodology can be modified easily to include other chemicals of interest. Furthermore, the pre-concentration step can be applied easily in the field, circumventing potential stability issues of these chemicals.

Multiplex quantification of lamprey specific bile acid derivatives in environmental water using UHPLC-MS/MS.

Larval and adult sea lampreys (Petromyzon marinus) release bile salts and acids into the surrounding aquatic environment. Some of these bile salts and acids, such as petromyzonol sulfate (PZS), 3-keto petromyzonol sulfate (3k PZS), petromyzonamine disulfate (PADS), petromyzosterol disulfate (PSDS), and 3-keto allocholic acid (3k ACA), may function as pheromones. To examine the release and distribution patterns of these metabolites, which this study has termed bile acid derivatives, we developed a novel UHPLC-MS/MS method that was characterized by simple sample preparation, baseline separation, and short analysis time for all studied compounds. These five analytes were separated in 7 min using a reversed-phase C18 column containing 1.7 µm particles and a gradient elution at pH 8.9. Once separated, the analytes were subjected to electrospray ionization-mass spectrometry (negative ion mode) and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) using the multiple reaction monitoring (MRM) mode. Deuterated 3k PZS ([(2)H(5)]3k PZS) was added as the internal standard (IS) to the sample prior to solid phase extraction (SPE). Among the three types of SPE sorbent tested, mixed-mode cation-exchange and reversed-phase sorbent for bases (MAX) and acids (MCX), and reversed-phase C18 sorbent (Sep-pak), the best recoveries (84.1-99.7%) were obtained with MCX cartridges. The calibration curves of all five analytes were linear between 0.15 and 1200 ng/mL, with R(2)≥0.9997. This method had a precision of relative standard deviation (RSD) ≤9.9% and an accuracy of deviation (DEV) ≥92.5%. The developed method was successfully used to quantify bile acid derivatives found in streams where lampreys spawn (SD<1.4) and water conditioned with male sea lampreys (SD<4.8). Utilizing this method provides a routine analysis of lamprey bile acid derivatives and may prove useful for sea lamprey population estimates in future studies and applications.

The role of the acquisition methods in the analysis of natural and synthetic steroids and cholic acids by gas chromatography-mass spectrometry.

An exhaustive GC-MS acquisition study was performed, for the simultaneous analysis of natural and synthetic steroids and cholic acids (in order to insert them into the last tierce of our multiresidue analysis system), such as androsterone, ß-estradiol, transdehydroandro-sterone, transdehyroandrosterone, mestranol, dihydrotestosterone, ethinylestradiol, testosterone, norethisterone, estriol, 4-androstene-3,17-dione, gestodene, levonorgestrel, etonogestrel, coprostanol, progesterone, cholesterol, medroxyprogesterone-acetate, lithocholic acid, stigmasterol, cholic acid, chenodeoxycholic acid, ß-sitosterol, ursodeoxycholic acid, 3-hydroxy-7-ketocholic acid and dehydrocholic acid, in total 26 compounds. As novelties to the field, for the trimethylsilyl (TMS) oxime ether/ester derivatives of steroids and cholic acids, at first, a tandem mass spectrometric (MS/MS), multiple reaction monitoring (MRM) type acquisition method has been developed in a single run; also for the first time, the three acquisition techniques, the full scan (FS), the selective ion monitoring (SIM), in our case the multiple ion monitoring (MIM) and the currently optimized MRM methods, have been compared; all three, in parallel, under strictly the same derivatization/instrumental conditions, both in matrix free solutions and municipal wastewater from two Hungarian wastewater treatment plants (WWTPs). Critical evaluation of the three acquisition protocols was collated on their analytical performances and validated under the same conditions. The data of six point calibration curves for FS, MIM and MRM methods, showed that both R² (0.9995, 0.9858, 0.9975) and RSD (5.3, 5.8, 5.0), for two parallel derivatizations, each injected three times, proved to be independent of the acquisition processes. Whereas, for the method limit of quantification (LOQ) and the instrument limit of quantification (ILQ) values showed considerable differences. LOQ data, were decreasing in the FS, MIM, MRM line (expressed in ng/L), for all steroids and cholic acids. The same trend was determined in terms of the ILQ values. The practical utility of the optimized acquisition techniques was confirmed by the quantitation of the steroids and cholic acids contents of wastewater samples. Results confirmed the importance of the MRM acquisition method, even in comparison to the MIM one: with particular interest in selected cases: avoiding the extreme overestimation of the ß-estradiol (156-1325%) and that of the ethinylestradiol (582-831%) concentrations in the wastewater samples.

Impact of the composition of gastric reflux bile acids on Barrett's oesophagus.

BACKGROUND: The effect of the composition of reflux bile acids, especially the ratio of hydrophobic to hydrophilic ones, on the development of Barrett's oesophagus has not been fully investigated in human studies. AIMS: To evaluate the influence of the bile acid composition of gastric juice on Barrett's oesophagus, a prospective study was designed. METHODS: Fifty patients with and 100 patients without Barrett's oesophagus were enrolled. For all enrolled patients, gastric juice was collected by the endoscopic procedure for bile acid analysis. The ratio of hydrophobic to hydrophilic bile acids (bile hydrophobicity ratio, BHR) was calculated from 6 kinds of bile acids analysed in gastric juice. The relationship between the ratio and clinico-pathological factors of Barrett's oesophagus was investigated. RESULTS: The mean of BHR of patients with Barrett's oesophagus was significantly higher than that of patients without Barrett's oesophagus (0.26 ± 0.05 vs. 0.08 ± 0.02, p<0.05). In multivariate analysis, a high BHR value was a predictor for the presence of Barrett's oesophagus (OR 5.74, p<0.001). In patients with Barrett's oesophagus, the BHR correlated with COX-2 protein expression and with accelerated cellular proliferation. CONCLUSIONS: Patients with Barrett's oesophagus had a higher BHR in the gastric juice than those without.

A validated RP-HPLC method for quantitative determination of related impurities of ursodeoxycholic acid (API) by refractive index detection.

An isocratic RP-HPLC method was developed and validated for quantitative determination of ursodeoxycholic acid (UDCA) and its related impurities. Considering the lower molecular absorptivity of UDCA, refractive index detector was used to detect the impurities on a Phenomenex Luna C(18), 150 mm × 4.6 mm, 5 µm column. The mobile phase was 0.1% acetic acid/methanol (30:70, v/v) and flow rate was 0.8 ml/min. The detector and column temperature was maintained at 40°C. The method is linear over a range of 0.25-3.5 µg/ml for all impurities and coefficient of correlation (r(2)) was ≥0.9945. The accuracy of method demonstrated at three levels in the range of 50-150% of the specification limit and recoveries were found to be in the range of 97.11-100.75%. The precision for all related impurities was below 3.5% R.S.D. The method was applied to commercial bulk drug sample for assay purpose.

In vitro evaluation of liposomes containing bio-enhancers for the oral delivery of macromolecules.

The aim of this work was to develop a new type of liposomes containing bio-enhancers for oral delivery of hydrophilic macromolecules. The study focused on EPC/cholesterol-based formulations combined with TPGS 1000 and 400, cholylsarcosine (CS), cetylpyridinium chloride (CpCl) and stearylamine (SA) covering a broad range of different types of enhancers. Most of the tested liposomal formulations and enhancers showed neither influence on cell viability in the Alamar Blue® assay nor an increase in lactate dehydrogenase LDH release. But, at a concentration of 1 mM, CpCl exhibited a strong toxicity after 2 h, TPGS 1000 reduced the cell viability at the same concentration after 8h significantly. Only one liposomal formulation with 25% CpCl led to a decrease in viability to 60.0% after 8h at a total lipid concentration of 5 mM. In the Caco-2 Transwell® model, one formulation with 5% TPGS 400 improved the permeation of FITC-dextran 70 kDa 3.34 ± 0.62-fold, one with 10% CpCl 3.69 ± 0.67 and one with 10% CS and 2.5% SA 3.41 ± 0.51-fold without influencing the TER. Liposomes with 10% SA or 25% CpCl increased the permeation of FITC-dextran 29.02 ± 5.84, respectively 39.28 ± 2.10-fold, but led also to a strong reduction in the TER. Especially, the three formulations which enhanced the permeation of FITC-dextran around 3.5-fold without showing any cell toxicity or decrease in TER should be safe and effective candidates for the development of an oral delivery system for hydrophilic macromolecules.

Anti-invasive effect of a rare mushroom, Ganoderma colossum , on human hepatoma cells.

Ganoderma colossum is a rare species of the Ganodermataceae family with biological activity but has not been widely used to date. Because of its rareness and hard availability, the literature regarding the bioactivity of G. colossum is very limited and the bioactive components in G. colossum have never been explored. In the present study, an ethanol extract was prepared from the fruiting body of a G. colossum strain (EEGC) and then fractionated by reverese-phase high-performance liquid chromatography (HPLC). The inhibitory effects and molecular mechanisms of the EEGC on the phorbol-12-myristate-13-acetate (PMA)-induced invasion of HepG2 cells were investigated. The fractions of the EEGC cannot be totally identified, but the lucidenic acids were considered as the major component. When HepG2 cells were treated with the EEGC, the PMA-induced invasion was reduced in a dose-dependent manner and the PMA-induced matrix metalloproteinase (MMP)-9 was also suppressed at the transcriptional level. The EEGC also showed an inhibitory effect on the PMA-induced phosphorylation of extracellular signal-regulated kinase (ERK1/2) and protein kinase B (Akt) in cytosol, as well as the activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) levels in the nucleus of HepG2 cells. This study provides the first evidence demonstrating that the EEGC is an effective inhibitior on the PMA-induced invasion of hepatoma cell. The EEGC could be further tested by an in vivo model to verify whether it is effective for the prevention of hepatoma invasion or metastasis.

Bile acid aspiration in suspected ventilator-associated pneumonia.

AIMS: The aims of this study were to measure the levels of bile acids in patients with suspected ventilator-associated pneumonia (VAP) and provide a possible pathway for neutrophilic inflammation to explain its proinflammatory effect on the airway. METHODS: Bile acid levels were measured by spectrophotometric enzymatic assay, and liquid chromatography mass spectrometry was used to quantify the major bile acids. Alveolar cells were grown on modified air-liquid interface culture inserts, and bile acids were then employed to stimulate the cells. Reverse transcriptase polymerase chain reaction and Western blots were used to determine the involved gene expression and protein levels. RESULTS: The mean (+/- SE) concentration of total bile acids in tracheal aspirates was 6.2 +/- 2.1 and 1.1 +/- 0.4 mumol/L/g sputum, respectively, for patients with and without VAP (p < 0.05). The interleukin (IL)-8 level was significantly higher in the VAP group (p < 0.05). The major bile acid, chenodeoxycholic acid, stimulated alveolar epithelial cells to increase IL-8 production at both the messenger RNA and protein level through p38 and c-Jun N-terminal kinase (JNK) activation. The selective p38 and JNK inhibitors, as well as dexamethasone, successfully inhibited IL-8 production. CONCLUSION: These data suggest that early intervention to prevent bile acid aspiration may reduce the intensity of neutrophilic inflammation in intubated and mechanically ventilated patients in the ICU.

Gas chromatography-mass spectrometry of the trimethylsilyl (oxime) ether/ester derivatives of cholic acids: their presence in the aquatic environment.

This paper presents a derivatization, mass fragmentation study relating to the most common six cholic acids, such as cholic, lithocholic, chenodeoxycholic, ursodeoxycholic, 3-hydroxy,7-ketocholanic and dehydrocholic acids, identified and quantified as pollutants in the aquatic environment at the first time. Derivatizations have been performed with the two-step process (1: oximation, 2: silylation) varying the time and temperature of both reactions. Optimum responses have been obtained after 30 min oximation with hydroxylamine.HCl and 90 min silylation with hexamethyldisilazane and trifluoroacetic acid at 70 degrees C. Fragmentation patterns of the trimethylsilyl (oxime) ether/ester derivatives of all six cholic acids provided the theoretically expected, fully derivatized compounds. Reproducibility/linearity of derivatives calculated on the basis of the corresponding selective fragment ions, characterized by the relative standard deviation percentages of measurements, proved to be < or =4.9 (RSD%). The practical utility of the method was shown by the identification and quantification of cholic acids as pollutants in the aquatic environment. Subsequently to a solid phase extraction study varying the pH of extractions (pH 2, pH 4 and pH 7), applying the OASIS cartridges, it has been confirmed that the recoveries for all six cholic acids are acceptable, varying between 77% and 104%, and are independent on the pH. The total cholic acid content of a Hungarian wastewater plants' influent wastewater varied between 184 microg/L and 356 microg/L, while the Danube rivers' cholic acid content was 4.1 microg/L, only.

Perinatal bile acid metabolism: bile acid analysis of meconium of preterm and full-term infants.

BACKGROUND: Our purpose was to evaluate the metabolism of bile acids in the fetus by analyzing the bile acid composition of meconium of preterm (less than 30 weeks' gestational age) and full-term infants and comparing the results with the bile acid composition of feces of preterm and full-term infants 6 days after delivery. METHODS: The concentrations of individual bile acids were determined by gas chromatography-mass spectrometry after solvolysis and hydrolysis of bile acid conjugates. RESULTS: In meconium, the main bile acids were chenodeoxycholic and hyocholic acids. The main bile acid of feces from preterm infants at 6 days of age was the same as that of meconium. We also detected large amounts of secondary bile acids, especially deoxycholic acid and ursodeoxycholic acid. The ratio of cholic acid relative to chenodeoxycholic acid in meconium of preterm and full-term infants and in feces of preterm infants was less than 1, 0.36, 0.55, and 0.55, respectively. The percentage of chenodeoxycholic acid relative to total bile acids in meconium of preterm (P < 0.05) and full-term (P < 0.01) infants was significantly higher than that in feces of 6-day-old full-term infants. CONCLUSIONS: More than half of the main pathway, at least, for bile acid synthesis in preterm infants may be the acidic pathway until the infants reach about 7 days of age.

Determination of critical micellar concentrations of cholic acid and its keto derivatives.

The critical micellar concentration (CMC) values of keto derivatives of cholic acid (3alpha,12alpha-dihydroxy-7-oxo-5beta-cholanoic acid, 3alpha,7alpha-dihydroxy-12-oxo-5beta-cholanoic acid, 12alpha-hydroxy-3,7-dioxo-5beta-cholanoic acid, 3alpha-hydroxy-7,12-dioxo-5beta-cholanoic acid, 3,7,12-triketo-5beta-cholanoic acid) and cholic acid itself, were determined. Replacement of hydroxyl groups in cholic acid molecule with keto groups yields the derivatives whose CMC values increase with increase in the number of keto groups introduced. The CMCs of derivatives with the same number of keto groups but at different positions do not differ significantly. The relationship between the number of keto groups in the molecule of cholic acid keto derivatives and CMC value can be described by the following equation: CMC=43 number of keto groups+14.667. The effect of NaCl concentration on CMC increases with increase in the number of keto groups.

Human cecal bile acids: concentration and spectrum.

To obtain information on the concentration and spectrum of bile acids in human cecal content, samples were obtained from 19 persons who had died an unnatural death from causes such as trauma, homicide, suicide, or drug overdose. Bile acid concentration was measured via an enzymatic assay for 3alpha-hydroxy bile acids; bile acid classes were determined by electrospray ionization mass spectrometry and individual bile acids by gas chromatography mass spectrometry and liquid chromatography mass spectrometry. The 3alpha-hydroxy bile acid concentration (mumol bile acid/ml cecal content) was 0.4 +/- 0.2 mM (mean +/- SD); the total 3-hydroxy bile acid concentration was 0.6 +/- 0.3 mM. The aqueous concentration of bile acids (supernatant after centrifugation) was identical, indicating that most bile acids were in solution. By liquid chromatography mass spectrometry, bile acids were mostly in unconjugated form (90 +/- 9%, mean +/- SD); sulfated, nonamidated bile acids were 7 +/- 5%, and nonsulfated amidated bile acids (glycine or taurine conjugates) were 3 +/- 7%. By gas chromatography mass spectrometry, 10 bile acids were identified: deoxycholic (34 +/- 16%), lithocholic (26 +/- 10%), and ursodeoxycholic (6 +/- 9), as well as their primary bile acid precursors cholic (6 +/- 9%) and chenodeoxycholic acid (7 +/- 8%). In addition, 3beta-hydroxy derivatives of some or all of these bile acids were present and averaged 27 +/- 18% of total bile acids, indicating that 3beta-hydroxy bile acids are normal constituents of cecal content. In the human cecum, deconjugation and dehydroxylation of bile acids are nearly complete, resulting in most bile acids being in unconjugated form at submicellar and subsecretory concentrations.