Altered metabolism of bile acids in cholestasis: determination of 1 beta- and 6 alpha-hydroxylated metabolites

Trihydroxy and tetrahydroxy bile acid metabolites substituted at the C-1 or C-6 position were studied using the urine, serum and liver tissue from sixteen patients with cholestatic liver diseases. Following extraction, isolation and hydrolysis, bile acids were converted into the dimethylethylsilyl derivatives and assayed by capillary gas chromatography-mass spectrometry. Five 1 beta-hydroxylated bile acids, viz. 1 beta,3 alpha,12 alpha-trihydroxy-, 1 beta,3 alpha,7 alpha-trihydroxy-, 1 beta,3 alpha,7 beta-trihydroxy-, 1 beta,3 alpha,7 alpha,12 alpha-tetrahydroxy-5 beta-cholanoic acids and an epimer of the first compound, and two 6 alpha-hydroxylated bile acids, viz. 3 alpha,6 alpha,7 alpha-trihydroxy-, 3 alpha,6 alpha,7 alpha,12 alpha-tetrahydroxy-5 beta-cholanoic acids, were completely or partially identified. Large amounts of 1 beta-hydroxylated and 6 alpha-hydroxylated bile acids were found in the urine, whereas only trace amounts were detected in the serum and liver tissue. These findings indicate that altered metabolism, such as 1 beta- or 6 alpha-hydroxylation of bile acids, is enhanced in cholestasis, and that the resulting hydroxylated metabolites are eliminated in the urine.     

Capillary gas chromatography/negative ion chemical ionization mass spectrometry for the quantification of bile acids including 1 beta-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 3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid and related bile acids in human serum by gas chromatography-mass spectrometry

The glycine, taurine and sulphate conjugates of 3 beta,12 alpha-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: [2H6]3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid, [2H5]3 beta-hydroxy-5-cholen-24-oic acid, [2H7]cholic acid and their sulphates. Calibration curves for these bile acids were linear over the range 0.01-100 micrograms/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 micrograms/ml in serum of an infant with liver disease, and its sulphate comprised 55.1% of the amount of the bile acid.     

Potential bile acid metabolites. 25. Synthesis and chemical properties of stereoisomeric 3alpha,7alpha,16- and 3alpha,7alpha,15-trihydroxy-5beta-cholan-24-oic acids

Epimeric 3alpha,7alpha,16- and 3alpha,7alpha,15-trihydroxy-5beta-cholan-24-oic acids and some related compounds were synthesized from chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), respectively. The key reaction involved one-step remote oxyfunctionalization of unactivated methine carbons at C-17 of CDCA and at C-14 of UDCA as their methyl ester-peracetate derivatives with dimethyldioxirane (DMDO). After dehydration of the resulting 17alpha- and 14alpha-hydroxy derivatives with POCl(3) or conc. H(2)SO(4), the respective Delta(16)- and Delta(14)-unsaturated products were subjected to hydration via hydroboration followed by oxidation to yield the 3,7,16- and 3,7,15-triketones, respectively. Stereoselective reduction of the respective triketones with tert-butylamine-borane complex afforded the epimeric 3alpha,7alpha,16- or 3alpha,7alpha,15-trihydroxy derivatives exclusively. A facile formation of the corresponding epsilon-lactones between the side chain carboxyl group at C-24 and the 16alpha- (or 16beta-) hydroxyl group in bile acids is also clarified.     

Studies on steroids. CCLIII. Capillary gas chromatographic behaviour of diethylhydrogensilyl-diethylsilylene derivatives of stereoisomeric bile acids.

The capillary gas chromatographic behaviour of diethylhydrogensilyl (DEHS) ethers and/or diethylsilylene (DES) derivatives of fifty bile acids including 4- and 6-hydroxylated compounds is described. The methylene unit (MU) values of methyl and pentafluorobenzyl esters of bile acids were determined as their trimethylsilyl (TMS), dimethylethylsilyl (DMES) ethers and DEHS-DES derivatives. The differences in methylene unit values between the corresponding TMS ethers and DMES ethers or DEHS-DES derivatives were used for estimating the number and stereochemistry of hydroxyl groups on the steroid nucleus. On treatment with the silylating agent N,O-bis (diethylhydrogensilyl)trifluoroacetamide, bile acids possessing isolated hydroxyl in addition to diaxial trans-glycol groups were easily converted into the DEHS ehters, whereas those having a vicinal glycol group except for the diaxial group were converted into cyclic DES derivatives. The mass spectrometric properties obtained with negative-ion chemical ionization detection are discussed.     

Recognition of bile acids at cyclodextrin-modified gold electrodes.

Lipoylamino-beta- and gamma-cyclodextrin (LP-beta-CD and LP-gamma-CD, respectively) were adsorbed at the surface of gold electrodes by sulfur-gold bonding. The resultant electrodes exhibited quasi-reversible voltammograms for the redox reaction of Fe(CN)6(3-/4-) in aqueous solutions, with peak-to-peak separation (deltaEp) being 85 mV at 20 mV s(-1) as a potential sweep rate. When bile acids are added to the solution, deltaEp values increased to 200-300 mV with increasing the concentration of bile acids. A Langmuir-type adsorption analyses satisfactorily afforded the binding constants (Ksurf) of the surface-confined LP-beta-CD and LP-gamma-CD with the bile acids. The obtained Ksurf values of LP-gamma-CD are 5.0-50 times larger than the corresponding binding constants of gamma-CD in homogeneous aqueous solutions. Cyclic voltammetric experiments with positively, negatively, and non-charged adamantane derivatives as well as pH titration experiments revealed that the retardation of the electrode reaction of negatively charged Fe(CN)6(3-/4-) caused by bile acids was attributable (1) to electric potential changes due to the accumulation of the negative charges at the electrode surface, and (2) to an increase in the hydrophobicity of the electrode surface due to the binding of hydrophobic bile acids to the LP-beta-CD and LP-gamma-CD membranes.     

Bile acids. LXXVI. Analyses of bile acids and sterols by high-performance liquid chromatography with a microbore column

The mobilities of several free and conjugated 5 beta-bile acids, cholesterol and analogues, and alpha, beta-unsaturated sterols and steroidal acids have been investigated with a microbore reversed-phase high-performance liquid chromatographic column (50 cm X 1 mm I.D., 12% C18) with appropriate solvent mixtures at flow-rates of 50-100 microliter/min and a UV monitor set at 193, 198, 212, or 243 nm. With a solvent mixture of 2-propanol-10 mM phosphate buffer, pH 7.0 (160:340) bile acids or their conjugates were separated in a manner similar to those by microBondapak columns (10% C18). The lower detection limit of the conjugates was 20 pmoles with the UV detector set at 193 nm, whereas the lower limit for alpha, beta-unsaturated keto sterols or steroidal acids was 5 pmoles at 243 nm. The detection limit for cholesterol with the UV monitor at 198 nm was 10 pmoles. Contributions of substituent groups of sterols to their time of elution (capacity factor) were calculated for several substituted 4-cholesten-3-ones.     

Selective oxoammonium salt oxidations of alcohols to aldehydes and aldehydes to carboxylic acids

The oxidation of alcohols to aldehydes using stoichiometric 4-acetamido-2,2,6,6-tetramethylpiperidine-1-oxoammonium tetrafluoroborate (1) in CH(2)Cl(2) at room temperature is a highly selective process favoring reaction at the carbinol center best able to accommodate a positive charge. The oxidation of aldehydes to carboxylic acids by 1 in wet acetonitrile is also selective; the rate of the process correlates with the concentration of aldehyde hydrate. A convenient and high yield method for oxidation of alcohols directly to carboxylic acids has been developed.     

杂多酸(盐)及单缺位磷钨杂多化合物在醇类氧化反应中的催化活性

在相转移条件下,研究了杂多化合物在苄醇,环己醇氧化反应中的催化活性.六种Keggin结构杂多酸的催化活性按GeMo~12(H~4GeMo~12O~40的简写,其余类推,PW~12,PMo~12,SiMo~12,GeW~12,SiW~12顺序下降,杂多酸中的质子可分别被其它阳离逐渐取代而达到酸性修饰. H~3PW~12O~40随着其质子逐步被Na^+取代,酸性下降,催化活性大大提高;杂多酸(盐)的催化活性随体系pH值的改变将发生奇妙剧烈的变化;单缺位杂多化合物显示出较饱和杂多酸(盐)更高催化活性.溶剂对催化活性有明显影响.     

Electrochemical characterization of some ferrocenylcarboxylic acids

Summary Several carboxy-substituted ferrocene compounds are prepared and investigated by cyclic voltammetry in acetonitrile solution. The half-wave potentials of most of the acids studied (E _1/2=0.34–0.58 V versus s.c.e.) are more positive than that of ferrocene (0.33 V), reflecting a diminished susceptibility to oxidation of these compounds relative to the parent metallocene. Only -ferrocenylpropionic acid (0.32₅ V) is effectively identical with the latter in its oxidation behaviour, and -ferrocenylbutyric acid (0.31 V) tends to be more readily oxidized. The results are of interest for subsequent chemical oxidation studies of ferrocenylcarboxylic acids.     

Synthesis of mono- and difluoronaphthoic acids

Aryl carboxamides are useful structural units found in several biologically active compounds. Unlike their benzoic acid counterparts, fluorinated versions of naphthoic acids are relatively unknown. In connection with a recent project, we needed viable syntheses of several mono- and difluorinated naphthoic acids. Herein we describe the synthesis of 5-, 6-, 7-, and 8-fluoro-1-naphthalenecarboxylic acids and 5,7-, 5,8-, 6,7-, and 4,5-difluoro-1-naphthalenecarboxylic acids. The 5-fluoro derivative 1was obtained from the corresponding 5-bromo compound via electrophilic fluorination of the lithio-intermediate. The rest of the monofluoro (2, 3, and 4) and the difluoro acids (5, 6, and 7) were prepared by a new, general route which entailed the elaboration of commercial fluorinated phenylacetic acids to 2-(fluoroaryl)glutaric acids with differential ester groups; selective hydrolysis to a mono acid, intramolecular Friedel-Crafts cyclization, and aromatization furnished the target structures. An alternative process to assemble a naphthalene skeleton is also presented for the difluoro acids 5 and 6. Finally, 4,5-difluoro-1-naphthalenecarboxylic acid (8) was prepared expeditiously from 1,8-diaminonaphthalene by adapting classical reactions.     

Synthesis of 6-hydroxylated bile acids and identification of 3 alpha,6 alpha,7 alpha,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid in human meconium and neonatal urine

Three 6-hydroxylated bile acids, 3 alpha,6 alpha,7 alpha,12 alpha-, 3 alpha,6 beta,7 alpha,12 alpha- and 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acids, were synthesized from methyl cholate, and a sensitive method was developed for analyzing them by gas chromatography-mass spectrometry for the stoichiometric study of fetal bile acids. 3 alpha,6 alpha,7 alpha,12 alpha-Tetrahydroxy-5 beta-cholan-24-oic acid (6 alpha-hydroxylated cholic acid) was identified from human meconium and healthy neonatal urine by comparison with the mass spectrum of the reference compound. In human meconium, 6 alpha-hydroxylated cholic and chenodeoxycholic acids were determined in 1.2% and 29.0% of the total bile acids, respectively. We discuss the significance of hydroxylation at the C-1 beta and C-6 alpha positions of bile acids and their elimination in fetal and neonatal periods.     

An efficient and practical system for the catalytic oxidation of alcohols, aldehydes, and alpha,beta-unsaturated carboxylic acids

Upon exposure to commercial bleach (approximately 5% aqueous sodium hypochlorite), nickel(II) chloride or nickel(II) acetate is transformed quantitatively into an insoluble nickel species, nickel oxide hydroxide. This material consists of high surface area nanoparticles (ca. 4 nm) and is a useful heterogeneous catalyst for the oxidation of many organic compounds. The oxidation of primary alcohols to carboxylic acids, secondary alcohols to ketones, aldehydes to carboxylic acids, and alpha, beta-unsaturated carboxylic acids to epoxy acids is demonstrated using 2.5 mol % of nickel catalyst and commercial bleach as the terminal oxidant. We demonstrate the controlled and selective oxidation of several organic substrates using this system affording 70-95% isolated yields and 90-100% purity. In most cases, the oxidations can be performed without an organic solvent, making this approach attractive as a "greener" alternative to conventional oxidations.     

Measurement of conjugated 1 beta-hydroxycholic acid in urine of newborns by specific radioimmunoassay.

Anti-tauro 1 beta-hydroxycholic acid antisera were prepared by immunizing rabbits with N-(1 beta,3 alpha,7 alpha,12 alpha-tetrahydroxy-5 beta-cholan-24-oyl)glycine bovine serum albumin conjugate. The immunoglobulin G fraction was obtained by ammonium sulfate precipitation, followed by diethylaminoethyl cellulose column chromatography. The antibody was characterized using [2-3H]tauro 1 beta-hydroxycholic acid which has a high affinity (Ka = 1.09 x 10(9) M-1) and reasonable specificity. Cross-reactivity for glyco 1 beta-hydroxycholic acid was 100% and those for other 1 beta-hydroxylated bile acids ranged from 4.32 to 29.6%. Concentrations of conjugated 1 beta-hydroxycholic acid in urine of newborns at 0-20 d after birth were determined by radioimmunoassay to be significant (0.2-11.1 micrograms/ml), exhibiting a tendency to increase during the 20 d after birth.     

Polyurethanes made from bile acids

New polyurethanes based on bile acids were synthesized from alcohol derivatives of cholic and lithocholic acids and hexamethylene diisocyanate, in an effort to improve the biocompatibility and biodegradability of polyurethanes through the use of natural compounds. The hydrogen bonding in the polymers is confirmed by IR spectral analysis. The glass transition temperatures of the polymers are in the range of 82-138 ℃ and degradation temperatures in the range of 267-298 ℃ as studied by thermal analyses. Thermogravimetric studies indicate that the comonomers are of equimolar amounts in the polyurethanes derived from both bile acids.     

Preparation of glycine-conjugated bile acids and their gas/liquid chromatographic analysis on an aluminum-clad flexible fused silica capillary column.

This paper describes a method for the direct gas/liquid chromatographic (GC) analysis of 46 glycine-conjugated bile acids, which differ from one another in the number, position and configuration of the hydroxyl groups at positions C-2, C-3, C-4, C-6, C-7 and/or C-12. Free bile acids were converted quantitatively on a micro scale to ethyl ester-trimethylsilyl (Et-TMS) and methyl ester-dimethylethylsilyl (Me-DMES) ether derivatives of the corresponding glycine conjugates. The Et-TMS and Me-DMES ethers of the glycine conjugates were chromatographed on an aluminum-clad flexible fused silica capillary column coated with a thin film (0.1 micron) of chemically bonded and cross-linked methylpolysiloxane. Relative retention time (RRT) and methylene unit (MU) values were determined for the 46 compounds and their GC behaviour was discussed. The derivatization procedure and the retention data would be useful for the direct GC identification of unknown glycine-conjugated bile acid mixtures extracted from biological samples.     

Oxathiaphospholane approach to N- and O-phosphorothioylation of amino acids

A method of highly efficient synthesis of N- and O-phosphorothioylated amino acids was developed. N- and O-(2-Thiono-1,3,2-oxathiaphospholanyl)amino acid methyl esters (3) were prepared in high yields in reaction of amino acid methyl esters with 2-chloro-1,3,2-oxathiaphospholane in pyridine in the presence of elemental sulfur. Compounds 3 were converted in high yield into the corresponding methyl or benzyl phosphorothioamides 6 and 7 by DBU-assisted treatment with methanol or benzyl alcohol. When 3-hydroxypropionitrile was used instead of methanol or benzyl alcohol, the corresponding 2-cyanoethylphosphorothioamidates 4 were obtained in high yield, from which the 2-cyanoethyl group was removed with concentrated ammonium hydroxide. The oxathiaphospholane methodology was also applied for the phosphorylation of amino acids. Thus, 2-oxo-1,3,2-oxathiaphospholane derivatives 10 were prepared by oxidation of compounds 3 with SeO(2.) Compounds 10 were transformed into the corresponding phosphate diesters or amidoesters upon treatment with 3-hydroxypropionitrile in the presence of DBU. The DBU-assisted oxathiaphospholane ring-opening process in 3 and 10 did not cause any measurable C-racemization of phosphorothioylated/phosphorylated amino acids.     

Studies on steroids. CXXVIII. Separation and determination of bile acids by high-performance liquid chromatography

A method is described for the simultaneous determination of major bile acids by high-performance liquid chromatography without prior hydrolysis. A mixture of bile acids is divided into the free, glyco- and tauro-conjugate groups by thin-layer chromatography. Separation of each group into cholate, ursodeoxycholate, chenodeoxycholate, deoxycholate and lithocholate is attained in two stages on a muBondapak C18 column; first, 0.3% ammonium carbonate-acetonitrile (9:4) is used as a mobile phase for the separation of the last three compounds. Subsequently cholate and ursodeoxycholate are resolved by chromatography in 0.3% ammonium carbonate-acetonitrile (11:4).     

Synthesis of some 1-methyl-3-alkoxy-5-arylpyrrolecarboxylic acids and derivatives

By a combination of hydrolysis, decarboxylation, and methylation diethyl 1-methyl-3-hydroxy-5-phenylpyrrole-2,4-dicarboxylate was converted into 1-methyl-3-methoxy-5-phenyl-pyrrole-2,4-dicarboxylic acid (5) and into the isomeric compounds ethyl 1-methyl-2-phenyl-4-methoxypyrrole-3-carboxylate (4a) and ethyl 1-methyl-3-methoxy-5-phenylpyrrole-2-carboxylate (9a). 1-Methyl-2-phenyl-4-methoxypyrrole-3-carboxylic acid was synthesized both by the selective decarboxylation of 5 and by the hydrolysis of 4a. Hydrolysis of 9a, however, did not give the corresponding acid, but rather an oxidation product, 1-methyl-3-methoxy-5-hydroxy-5-phenyl-3-pyrrolin-2-onc (10a). Compound 10a was shown to arise from the air oxidation of the completely decarboxylated product, 1-methyl-2-phenyl-4-methoxypyrrole. Reduction of 9a with lithium aluminum hydride gave 1-methyl-3-methoxy-5-phenylpyrrole-2-methanol, which yielded 10a upon oxidation with silver oxide.     

Pharmacophore model for bile acids recognition by the FPR receptor

Formyl-peptide receptors (FPRs) belong to the family A of the G-protein coupled receptor superfamily and include three subtypes: FPR, FPR-like-1 and FPR-like-2. They have been involved in the control of␣many inflammatory processes promoting the recruitment and infiltration of leukocytes in regions of inflammation through the molecular recognition of chemotactic factors. A large number of structurally diverse chemotypes modulate the activity of FPRs. Newly identified antagonists include bile acids deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA). The molecular recognition of these compounds at FPR receptor was computationally investigated using both ligand- and structure-based approaches. Our findings suggest that all antagonists bind at the first third of the seven helical bundles. A closer inspection of bile acid interaction reveals a number of unexploited anchor points in the binding site that may be used to aid the design of new potent and selective bile acids derivatives at FPR.