Unsaturated Bile Acids Research Articles

Metabolomic networks connect host-microbiome processes to human Clostridioides difficile infections.

Clostridioides difficile infection (CDI) accounts for a substantial proportion of deaths attributable to antibiotic-resistant bacteria in the United States. Although C. difficile can be an asymptomatic colonizer, its pathogenic potential is most commonly manifested in patients with antibiotic-modified intestinal microbiomes. In a cohort of 186 hospitalized patients, we showed that host and microbe-associated shifts in fecal metabolomes had the potential to distinguish patients with CDI from those with non-C. difficile diarrhea and C. difficile colonization. Patients with CDI exhibited a chemical signature of Stickland amino acid fermentation that was distinct from those of uncolonized controls. This signature suggested that C. difficile preferentially catabolizes branched chain amino acids during CDI. Unexpectedly, we also identified a series of noncanonical, unsaturated bile acids that were depleted in patients with CDI. These bile acids may derive from an extended host-microbiome dehydroxylation network in uninfected patients. Bile acid composition and leucine fermentation defined a prototype metabolomic model with potential to distinguish clinical CDI from asymptomatic C. difficile colonization.

Oxylipins discriminate between whole grain wheat and wheat aleurone intake: a metabolomics study on pig plasma

A pig model was used to investigate the difference in metabolic response of plasma between whole grain wheat and wheat aleurone. Six pigs were fed in a cross-over design iso dietary fiber (DF) breads prepared from whole grain wheat and wheat aleurone and with a wash-out diet based on bread produced from refined wheat flour made iso-DF by adding Vitacel. The pigs were fitted with catheters in the mesenteric artery and the portal vein, which allow studying the enrichment of nutrient in plasma after passing the gastrointestinal tract. LC–MS measurements showed the presence of oxygenated fatty acids (oxylipins) in the plasma of pigs and with discrimination between whole grain wheat versus wheat aleurone and refined flour. The oxylipin-marker of this difference was identified as a mixture of 13-hydroxy-9,11-octadecadienoic and 9-hydroxy-10,12-octadecadienoic acid (13-HODE and 9-HODE). Similar oxylipins were also found in the flour and the bread consumed by pigs. Since the germ is part of the whole grain flour, the germ is most likely responsible for the elevated level of oxylipins in plasma after whole grain wheat consumption. This finding may also point towards bioactive compounds, which can be used as novel lipid candidate biomarkers of whole grain intake versus aleurone. Principal component analysis discriminated between venous and arterial plasma samples. We identified glycine conjugated and unconjugated bile acids to be responsible for this discrimination. Moreover we discovered the existence of unsaturated bile acid in the plasma of pigs.

Measurement of Transport Activities of Bile Acids in Human Multidrug Resistance-Associated Protein 3 Using Liquid Chromatography-Tandem Mass Spectrometry

A method has been developed for the measurement of transport activities in membrane vesicles obtained from human multidrug resistance-associated protein 3-expressing Sf9 cells for 1beta-hydroxy-, 6alpha-hydroxy- and unsaturated bile acids by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. Calibration curves for the bile acids were linear over the range of 10 to 2000 pmol/mL, and the detection limit was less than 2 pmol/mL for all bile acids using selected reaction monitoring analysis. The method was applied to measurements of adenosine triphosphate-dependent transport activities of the membrane vesicles for the above-mentioned hydroxylated and unsaturated bile acids. The present study demonstrated that the human multidrug resistance-associated protein 3 vesicles accepted 1beta-, 6alpha-hydroxylated and unsaturated bile acids along with common bile acids, such as glycocholic acid and taurolithocholic acid 3-sulfate. The developed method is useful for measurements of bile acid transport activities.

Cholestatic liver disease in adults may be due to an inherited defect in bile acid biosynthesis

An increasing number of treatable inborn errors of bile acid synthesis have been described, primarily in infants with severe cholestatic liver disease. The present patient, whose two older siblings had died from progressive cholestatic liver disease, developed neonatal cholestasis and rickets but recovered during the childhood years and follow-up was terminated at 12 years of age. The patient presented again at 26 years of age with jaundice and pathological liver function tests. This was normalized upon treatment with ursodeoxycholic acid. Electrospray mass spectrometry of urine showed predominance of unsaturated bile acids, characteristic of 3beta-hydroxy-Delta5-C27-steroid dehydrogenase/isomerase (HSD3B7) deficiency. The activity of HSD3B7 in cultured fibroblasts was less than 5% of normal. A single homozygous mutation was found in exon 4 leading to an amino acid exchange (S162R) and loss of enzyme activity. This case illustrates that infants with an inherited absence of HSD3B7 may survive the neonatal period of life and childhood without treatment with bile acids. A low level of sulphation of the abnormal trihydroxy bile acid formed as a result of enzyme deficiency may be of importance for survival. The possibility that liver disease presenting in the adult may be due to a mutation in the HSD3B7 gene should be considered, especially in cases with familial occurrence of liver disease and earlier periods of liver dysfunction.

Placental transport of bile acids: analysis of bile acids in maternal serum and urine, umbilical cord blood, and amniotic fluid.

To investigate the role of placental transport of bile acids in fetal bile acid metabolism, such as with regard to synthesis of the unusual bile acids (1 beta- and 6 alpha-hydroxylated and unsaturated bile acids), we measured the concentrations of bile acids in umbilical cord blood, amniotic fluid, maternal serum and maternal urine at delivery by means of gas chromatography-mass spectrometry. Serum and urine from healthy nonpregnant women were used as controls. We detected large amounts of unusual bile acids, especially hyocholic acid and 3 beta-hydroxy-delta 5 bile acids, in amniotic fluid and umbilical cord blood. The concentration of total bile acids in maternal serum was less than that of control serum and umbilical cord blood, and the concentration of total bile acids in maternal urine was higher than that of control urine and amniotic fluid. In conclusion, the fetus synthesized large amounts of unusual bile acids, and these compounds were transported from fetus to mother by placental transfer. We suggest that pregnant women may excrete large amounts of bile acids into the urine to control serum concentration of bile acids in fetus.

Unsaturated bile acids in the urine of patients with hepatocellular neoplasia

Unsaturated bile acids in the urine of patients with hepatocellular neoplasia

Expression of the bile acid-inducible NADH:flavin oxidoreductase gene of Eubacterium sp. VPI 12708 in Escherichia coli

The intestinal microorganism Eubacterium sp. VPI 12708 synthesizes a bile acid-inducible NADH:flavin oxidoreductase (NADH:FOR) which presumably functions in the 7α-dehydroxylation of cholic acid to deoxycholic acid. The baiH gene encoding NADH:FOR was subcloned into an IPTG-inducible expression vector, pBaiH2.2. Escherichia coli DH5α cells transformed with pBaiH2.2 expressed 10-fold higher levels of NADH:.FOR upon induction with IPTG than did Eubacterium sp. VPI 12708 cells induced with cholic acid. The NADH:FOR produced by E. coli DH5α(pBaiH2.2) was purified to > 95% electrophoretic homogeneity in three steps. The purified NADH:FOR was similar to that of Eubacterium sp. VPI 12708 in subunit and native M r (ca. 72 000 and 210 000, respectively), pH optimum, sensitivity to inhibitors, and electron acceptor specificity. It contained 1 mol of FAD, up to 2 mol of iron, and 1 mol of copper per mol of subunit. The enzyme reduced synthetic quinones, dyes, flavins, and O z with NADH as the electron donor, but did not reduce disulfide compounds, various unsaturated bile acids, cytochrome c, physiological quinones, or cell fractions from Eubacterium sp. VPI 12708. Addition of purified NADH:FOR to Eubacterium sp. VPI 12708 cell extracts altered the balance of oxidized and reduced bile acid intermediates produced during cholic acid 7α-dehydroxylation, suggesting that the enzyme may regulate the cellular ration of NAD to NADH.

Fetal bile acid metabolism during infancy: analysis of 1 beta-hydroxylated bile acids in urine, meconium and feces.

Fetal bile acids (1 beta-hydroxylated, 6 alpha-hydroxylated and unsaturated bile acids), especially 1 beta, 3 alpha,7 alpha, 12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid (CA-1 beta-ol), have been detected in urine and feces early in life. To investigate whether a fetal pathway of bile acid synthesis exists in infancy, we measured the concentrations of bile acids in the urine, meconium and feces from normal newborns and infants by means of gas chromatography-mass spectrometry. The mean ratio of total bile acids to creatinine in urine increased between birth and 7 days and then gradually decreased; however, the concentration of total bile acids in urine remained significantly higher than that in adult urine until 3 mo of age. The main urinary bile acid was CA-1 beta-ol, and substantial amounts of fetal bile acids were detected in urine until 3 mo of age. The ratio of cholic acid to chenodeoxycholic acid was abnormally low in meconium (mean, 0.44; range, 0.19 to 0.74), and hyocholic acid constituted 19.3% of total bile acids. The mean total bile acid content of feces decreased between birth and 7 days of age and thereafter increased. The mean percentage of fetal bile acids in feces decreased after birth, but substantial amounts were present in feces until 1 mo of age.(ABSTRACT TRUNCATED AT 250 WORDS)

The urinary bile acid excretion in healthy premature and full-term infants during the neonatal period.

The 24h urinary bile acid excretion was prospectively studied during the neonatal period in healthy, fully breastfed, premature and full-term infants. The urinary bile acids were identified by gas-liquid chromatography (GLC)-mass spectrometry and quantified by GLC. The excretion of bile acids in urine increased after birth, reaching maximum levels by the 3-4th day. Taurine conjugates predominated and the excretion of bile acid sulphates was remarkably low. Cholic acid and atypical bile acids were the main bile acids in urine during the first week. Tetrahydroxylated bile acids carrying hydroxyl groups at C-1, C-2 and C-6 were common, and also other 1- and 6-hydroxylated bile acids, including hyocholic and hyodeoxycholic acids. Three tentatively identified 4-hydroxylated bile acids, including one ketonic bile acid, were also found. Ketonic bile acids constituted an average of 16% of total urinary bile acids during the first week. Unsaturated bile acids were scantily found only during the first days. The excretion of atypical bile acids decreased to 1 month of age, parallel with the total bile acid excretion. The data support earlier hypothesis of a physiological cholestasis in the newborn. Atypical hydroxylated and ketonic bile acids, as well as cholic acid, constituted the major part of the urinary bile acids. The persistent atypical pattern of bile acids in urine during the first month of life indicates a longer period of immaturity of bile acid metabolism in healthy infants than previously described.

Taurine conjugate of 3 alpha,6 beta,7 beta-trihydroxy-5 beta,22-cholen-24-oic acid (tauro-delta 22-beta-muricholate): the major bile acid in the serum of female rats treated with alpha-naphthylisothiocyanate and its secretion by liver slices.

The taurine conjugate of 3 alpha,6 beta,7 beta-trihydroxy-5 beta,22-cholen-24-oic acid (tauro-delta 22-beta-muricholate) has been identified in the serum of female rats treated with alpha-naphthylisothiocyanate. Using a high performance liquid chromatographic/enzymatic method for measurement of bile acids, tauro-delta 22-beta-muricholate was the predominant bile acid in the serum of female Fischer 344 rats treated for 3 days with alpha-naphthylisothiocyanate. Other significant changes in concentrations of serum bile acids included increases in tauro-alpha-muricholate, tauro-beta-muricholate, taurocholate, taurochenodeoxycholate, and several unknown bile acids. The formation of tauro-delta 22-beta-muricholate was examined in vitro using liver slices from control rats and rats treated with alpha-naphthylisothiocyanate. Slices were incubated for 7 h in William's E medium containing no bile acids or 25 mumol/l of one of the following: beta-muricholate, tauro-beta-muricholate, or cholate. Tauro-delta 22-beta-muricholate was secreted by slices from control and treated rats and the rate was increased significantly by the addition of beta-muricholate (2.9-5.6-fold) but not tauro-beta-muricholate or cholate to the medium. Tauro-delta-beta-muricholate was formed by liver slices from endogenous precursors and from exogenous beta-muricholate. Pretreatment of rats with alpha-naphthylisothiocyanate did not alter total secretion rates but those of some important individual bile acids were affected. Because of the increased secretion of tauro-delta-beta-muricholate by liver slices with the addition of beta-muricholate to the medium, the liver may be the primary site of formation for this unsaturated bile acid.

A proposed nomenclature for bile acids.

A proposal is made for a system of nomenclature of the more common unconjugated and conjugated bile acids. Acceptable trivial names for bile acids are tabulated, and guidelines are proposed for using these existing trivial names as roots to create acceptable semi-systematic names for other bile acids, as well as for new natural bile acids that will be discovered in the future. The term alpha-hyocholic is recommended to replace hyocholic, and beta-hyocholic to replace omega-muricholic. The term murideoxycholic acid is recommended for 3 alpha,6 beta-dihydroxy-5 beta-cholan-24-oic acid. Proposals are also made for bile acids with epimeric hydroxy groups, for unsaturated bile acids, and for bile acids with oxo- and/or hydroxy-oxo- substituents on the nucleus and/or on the side chain. For conjugated bile acids, the term "aminoacyl amidates" is recommended to replace "amidates" for bile acids conjugated in N-acyl linkage with amino acids. Nomenclature for other types of conjugates (sulfates, glucuronides, glucosides) is included as well as abbreviations. It is recommended that the historic tradition of naming a newly discovered bile acid after the species from which it was isolated be abandoned, and that in the future such a bile acid should be named using the principles contained in this paper.

Capillary gas chromatographic analysis of oxo, oxo-hydroxy and unsaturated bile acid glycine conjugates

A method for the direct gas chromatographic analysis without prior hydrolysis has been developed for a series of glycine-conjugated bile acids having oxo or multiple functional groups, including oxo and hydroxyl groups and double bonds. The glycine conjugates without and with the hydroxyl groups were derivatized to their ethyl or methyl esters and ethyl ester-trimethylsilyl or methyl ester-dimethylethylsilyl ethers, respectively, and chromatographed on an aluminum-clad, flexible, fused-silica capillary column coated with a thin film (0.1 μm) of chemically bonded and cross-linked methyl polysiloxane. The change of methylene unit value exerted by derivatization and glycination for each compound is discussed.

Capillary gas chromatography/negative ion chemical ionization mass spectrometry for the quantification of bile acids including 1β‐hydroxylated and unsaturated bile acids in serum and urine

12 bile acids, including 1 beta-hydroxylated and unsaturated bile acids, have been quantified by capillary gas chromatography/negative ion chemical ionization mass spectrometry, using the trimethylsilyl(TMS) ether derivatives of bile acid pentafluorobenzyl(PFB) esters. The analysis time is 12 min and the minimum measurable amount is 100 fg for each bile acid. Bile acids in 200 microL of serum and 50 microL of urine from healthy human adults were measured. These small sample sizes enhance the practicality of using this method as a screening test for bile acids in the serum and urine of human infants, where small sample size is a major problem.

Determination of 3β,12α-dihydroxy-5-cholen-24-oic acid and related bile acids in human serum by gas chromatography-mass spectrometry

The glycine, taurine and sulphate cojugates of 3β,12α-dihydroxy-5-cholen-24-oic acid were synthesized as authentic samples for the analysis of this unusual bile acid. A highly sensitive and specific quantitative assay of the bile acid and related compounds has been developed by selected-ion monitoring in gas chromatography-mass spectrometry of their methyl ester-trimethylsilyl ether derivatives, using the deuterium labelled internal standards: [ 2H 6] 3β,12α-dihydroxy-5-cholen-24-oic acid, [ 2H 5] 3β-hydroxy-5-cholen-24-oic acid, [ 2H 7] cholic acid and their sulphates. Calibration curves for these bile acids were linear over the range 0.01–100 μg/ml in human serum. Recoveries of the bile acids and their conjugates ranged from 95 to 103% of the added amounts of their standard samples. The unsaturated bile acid was identified in a significant amount of 25.2 μg/ml in serum of an infant with liver disease, and its sulphate comprised 55.1% of the amount of the bile acid.

Synthesis of α, β-unsaturated C 24 bile acids

Synthesis of the α, β-unsaturated analogues of cholic acid, deoxycholic acid, chenodeoxycholic acid, and ursodeoxycholic acid is described. Each common bile acid was converted to the corresponding C 22 aldehyde which was then converted to the Δ 22 bile acid by Wittig reaction with methyl (triphenylphosphoranylidene)acetate. The synthetic unsaturated bile acids were characterized by thin-layer chromatography, gas-liquid chromatography, and mass spectrometry.

Metabolism of unsaturated bile acids and androstanes by human faecal bacteria

The metabolism of unsaturated bile acids and androstanes by mixed human faecal cultures has been studied. The reactions observed were mainly reductive. Unsaturated 4-ene-3-oxo and 1,4-diene-3-oxo bile acids were reduced in Ring A. 5β-3-Oxo bile acids were reduced to 5β-3-hydroxy bile acids. 4-Ene, 1,4-diene and 4,6-diene-3,17-dioxo-androstanes were reduced in Ring A with concomitant reduction of oxo groups to hydroxyl groups. The Gram-negative facultative anaerobic faecal bacteria are implicated in the reductive process, whilst the genus Clostridium does not appear to be important. Inclusion of menadione, a synthetic form of vitamin K., retards the reductive process.

Analysis and structure determination of unsaturated 5 beta - cholanoic acids.

Unsaturated cholanoic acids are known to arise as artifacts of chemical transformation processes and during storage and high-temperature gas liquid chromatography (GLC) of various derivatives of saturated bile acids. Nevertheless, there is evidence for their natural occurrence and isolation under conditions where artifactual formation of unsaturated bile acids would be unlikely. Since structural identification of such compounds is often complicated by a lack of knowledge of their analytical properties, a representative series of monounsaturated cholanoic acids with double bonds in rings A, B and C were prepared by POCl3 and ZnCL2 dehydration of saturated bile acids with selectively blocked hydroxyl functions. The cholenoic acids were indistinguishable from their saturated analogs by thin layer chromatography (TLC) on plain silica gel, but those compounds with sterically exposed double bonds were resolved by AgNO3-TLC, using chloroform/methanol solvent systems. The synthetic 5 beta-cholenoic acids obeyed the general rules of GLC mobility based on the overall shape of the molecule and the number and configuration of the functional groups. Constant retention factors attributable to the double bond were observed for all of the double bond types on several GLC phases, and theoretical retention times could be calculated for combinations of double bonds and functional groups not specifically represented among the synthetic standards. With gas chromatography-mass spectrometry (GC-MS), the unsaturated bile acids gave several characteristic fragments, which, in conjunction with the chromatographic properties of the parent compounds, permitted an unambiguous distinction among different unsaturated acids, and between unsaturated and saturated bile acids of the same number and configuration of functional groups. For complete structural identification of saturated and unsaturated bile acids, capillary GC-MS represents the ideal state of the art, but the less expensive combination of AgNO3-TLC and GLC also can yield much useful information concerning the structure of natural and synthetic 5 beta-cholenoic acids. This study emphasizes the need for special precautions in the isolation and derivatization of bile acids intended for studies of unsaturated components.

Resolution of saturated and unsaturated 5 beta-cholanoic acids by gas-liquid and thin-layer chromatography.

The thin-layer and gas-liquid chromatographic properties of the methyl ester acetates were determined for a series of 20 monounsaturated 5 beta-cholanoic acids representing the simple chemical and enzymic dehydration products of the common bile acids. The unsaturated acids were generally indistinguishable from their saturated analogues by thin-layer chromatography on plain silica gel, but resolution was achieved on silica gel impregnated with silver nitrate for compounds having sterically exposed double bonds. The gas-liquid chromatographic behaviour of the unsaturated bile acids on the OV-225, SE-30, and Poly-S-179 liquid phases was closely similar to that observed for the saturated bile acids. The 5 beta-cholenoic acids obeyed the general rules of chromatographic mobility based on the overall shape of the molecule and the number and configuration of functional groups, with a constant retention factor attributable to the olefinic bond. The structural information provided by the chromatographic behaviour of the standard unsaturated bile acids allows a distinction to be made among most of the isomeric 5 beta-cholenoates. A complete identification of all isomeric olefins is possible when chromatographic and mass spectrometric data are combined.

Preparation and mass spectral behaviour of some 5β-cholenoic acids

Monounsaturated 5 beta-cholanoic acids with double bonds in rings A, B, and C were prepared by POCl3 and ZnCl2 dehydration from natural bile acids with selectively blocked hydroxyl functions. The yields ranged from 15 to 100%. The products were purified by thin-layer and AgNO3 thin-layer chromatography and the structures were confirmed by nuclear magnetic resonance spectroscopy and mass spectrometry. The methyl ester acetates of the unsaturated 5 beta-cholanoic acids possessed chromatographic properties closely similar to those of the corresponding saturated bile acids. Several characteristic fragments were seen in the mass spectra which, in conjunction with the chromatographic properties, permitted an unambiguous distinction between different monounsaturated acids, and between saturated and unsaturated bile acids of the same number and configuration of functional groups. The 20 5 beta-cholenoic acids examined represent all of the simple chemical and enzymatic dehydration products of natural bile acids and can be completely identified by their combined chromatographic and mass spectral properties.

Bile acids of a 3200-year-old Egyptian mummy

The bile acids of the gall bladder and hepatic tissue of a 3200-year-old Egyptian mummy were isolated by thin-layer chromatography and identified by combined gas-liquid chromatrography and mass spectrometry. Except for complete deconjugation and extensive dehydration, the bile acids were found to correspond in their qualitative and quantitative composition to the gall bladder bile acids of modern man. The secondary bile acids constituted about 50% of the total and were identified as the normal bacterial oxidoreduction products of the primary bile acids and their dehydration products. In addition a series of unsaturated bile acids were identified, which corresponded to the dehydration products of cholic and chenodeoxycholic acids. It is suggested that both bile acid deconjugation and the limited oxidoreduction were probably brought about by the Clostridium organisms identified in the tissue. On the basis of the bile acid composition it is concluded that the ancient man metabolized cholesterol along the same pathways as modern man.