Fluorescence properties of trans-ethyl-p-(dimethylamino) cinnamate in presence of bile acid host

The knowledge of the formation of bile acid micellar aggregates is of great importance because of the biological significance of these compounds and their pharmacological applications. The intramolecular charge transfer (ICT) fluorescence property of trans-ethyl-p-(dimethylamino) cinnamate is used to study the micelles formed by aggregation of three most important bile acids, viz. cholic acid, deoxycholic acid and chenodeoxycholic acid by steady state and picosecond time-resolved fluorescence spectroscopy. The ICT fluorescence band intensity was found to increase with concomitant blue shift with the addition of bile acids. The blue shift in ICT fluorescence maxima as well as decrease in nonradiative decay constants in presence of bile acids indicate the passage of the probe towards the micro domains formed from the aggregated bile acids. Binding constant of the probe with micelles as well as critical micelle concentration and average polarity parameter of the micellar environments were obtained from the variation of fluorescence intensity on increasing concentration of bile acids in the medium.     

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

The critical micellar concentration (CMC) values of keto derivatives of cholic acid (3,12-dihydroxy-7-oxo-5β-cholanoic acid, 3,7-dihydroxy-12-oxo-5β-cholanoic acid, 12-hydroxy-3,7-dioxo-5β-cholanoic acid, 3-hydroxy-7,12-dioxo-5β-cholanoic acid, 3,7,12-triketo-5β-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.     

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.     

Potential bile acid metabolites. XV. Synthesis of 4 beta-hydroxylated bile acids; unique bile acids in human fetal bile

The 4 beta-hydroxylated derivatives of lithocholic, deoxycholic, chenodeoxycholic, and cholic acids were synthesized from their respective parent compounds. The principal reactions employed were 1) beta-face cis-dihydroxylation of delta 3 intermediates with osmium tetroxide-N-methylmorpholine N-oxide, 2) selective cathylation of vicinal 3 beta,4 beta-diols followed by oxidation of the resulting 4 beta-monocathylates, or direct selective oxidation at C-3 of 3 beta,4 beta-diols with pyridinium chlorochromate, and 3) stereoselective reduction of the 3-oxo compounds with tert-butylamine-borane complex. The results of analysis of the prepared 4 beta-hydroxylated bile acids with a diequatorial trans-glycol structure and their 3 beta-epimers by proton and carbon-13 nuclear magnetic resonance spectroscopies are briefly discussed along with the mass spectrometric properties.     

Aqueous amphiphilic drug (amitriptyline hydrochloride)-bile salt mixtures at different temperatures

The formation of mixed micelles built of 7,12-dioxolithocholic and the following hydrophobic bile acids was examined by conductometric method: cholic (C), deoxycholic (D), chenodeoxycholic (CD), 12-oxolithocholic (12-oxoL), 7-oxolithocholic (7-oxoL), ursodeoxycholic (UD) and hiodeoxycholic (HD). Interaction parameter (β) in the studied binary mixed micelles had negative value, suggesting synergism between micelle building units. Based on β value, the hydrophobic bile acids formed two groups: group I (C, D and CD) and group II (12-oxoL, 7-oxoL, UD and HD). Bile acids from group II had more negative β values than bile acids from group I. Also, bile acids from group II formed intermolecular hydrogen bonds in aggregates with both smaller (2) and higher (4) aggregation numbers, according to the analysis of their stereochemical (conformational) structures and possible structures of mixed micelles built of these bile acids and 7,12-dioxolithocholic acid. Haemolytic potential and partition coefficient of nitrazepam were higher in mixed micelles built of the more hydrophobic bile acids (C, D, CD) and 7,12-dioxolithocholic acid than in micelles built only of 7,12-dioxolithocholic acid. On the other hand, these mixed micelles still had lower values of haemolytic potential than micelles built of C, D or CD. The mixed micelles that included bile acids: 12-oxoL, 7-oxoL, UD or HD did not significantly differ from the micelles of 7,12-dioxolithocholic acid, observing the values of their haemolytic potential.     

Enzymatic fluorimetric determination of cholic acid and chenodeoxycholic acid in aqueous solutions and blood serum using a differential kinetic method

An enzymatic fluorimetric method is described for the determination of chenodeoxycholic acid and its conjugates and of cholic acid and its conjugates in aqueous solutions and serum. The method is based on the oxidation of 7 α-hydroxy bile acids by β-NAD⁺ in the presence of 7 α-hydroxysteroid dehydrogenase; the NADH produced is monitored fluorimetrically. Chenodeoxycholic acid is determined in the presence of cholic acid by a differential kinetic procedure; the sum of the two acids (primary bile acids) is determined by an equilibrium procedure, and cholic acid is calculated by difference. The r.s.d. was ca. 3% and 10% for aqueous solutions and sera, respectively. Recoveries of chenodeoxycholic acid, cholic acid and primary bile acids added to serum samples averaged 100.5, 105.1, and 102.9%, respectively. Ten samples can be analyzed per working day.     

Effects of aminotriazole treatment on biosyntheses of primary bile acids in vivo.

The influence of aminotriazole treatment on primary bile acid biosynthesis was studied in detail. After administration of aminotriazole to rats, bile was collected for 8 h. The content of chenodeoxycholic acid in the bile was increased to 144% of the control by aminotriazole treatment, but that of cholic acid was decreased to 48.4%. In another experiment, [4-14C]cholesterol was injected into rats immediately after aminotriazole treatment, and then bile was collected. The content of radioactive chenodeoxycholic acid in the bile was significantly increased to 130% of the control, but that of radioactive cholic acid was unchanged. In a similar experiment with [2-14C]mevalonate, the content of radioactive chenodeoxycholic acid in the bile was hardly changed by aminotriazole treatment, but that of radioactive cholic acid was greatly decreased to 41.2% of the control. Aminotriazole treatment did not affect the ratios of tauroconjugate to glycoconjugate of the two bile acids. Thus, aminotriazole treatment affects the syntheses of not only cholesterol (F. Hashimoto, C. Sugimoto and H. Hayashi, Chem. Pharm. Bull., 38, 2532 (1990); F. Hashimoto and H. Hayashi, Biochim. Biophys. Acta, 1086, 115 (1991)) but also primary bile acids in vivo. Namely, aminotriazole treatment activated biosynthesis of chenodeoxycholic acid from exogenous cholesterol, but did not affect that of cholic acid. Aminotriazole hardly affected the synthesis of chenodeoxycholic acid through endogenous cholesterol (from mevalonate), but inhibited that of cholic acid.     

Determination of bile acids and lysolecithin in artificial and natural gastric juice by HPLC for characterization of antacida

Work-up procedures and HPLC separation systems for determination of taurocholic, glycocholic, chenodeoxycholic, and cholic acids and lysolecithin in artificial and natural gastric juice are described. These compounds are used for testing the binding capacity of antacida to the individual analytes. Work-up is simple, no extraction or filtration being required. The optimized combinations of stationary and mobile phases allow selective and sensitive determination of the respective bile acids in gastric juice. For optimization, the capacity factors of two related bile acids were evaluated for numerous commercial stationary phases. The mechanism of retention is different for free and conjugated bile acids. Special aspects of routine analysis are discussed.     

The Effect of the Side Chain on Gelation Properties of Bile Acid Alkyl Amides

Six bile acid alkyl amide derivatives were studied with respect to their gelation properties. The derivatives were composed of three different bile acids with hexyl or cyclohexyl side chains. The gelation behaviour of all six compounds were studied for 36 solvents with varying polarities. Gelation was observed mainly in aromatic solvents, which is characteristic for bile-acid-based low molecular weight gelators. Out of 108 bile acid-solvent combinations, a total of 44 gel systems were formed, 28 of which from lithocholic acid derivatives, only two from deoxycholic acid derivatives, and 14 from cholic acid derivatives. The majority of the gel systems were formed from bile acids with hexyl side chains, contrary to the cyclohexyl group, which seems to be a poor gelation moiety. These results indicate that the spatial demand of the side chain is the key feature for the gelation properties of the bile acid amides.     

Studies on steroids. CCXXVIII Trace analysis of bile acids by gas chromatography-mass spectrometry with negative ion chemical ionization detection

A suitable derivatization method for the trace analysis of bile acids by gas chromatography (GC) in combination with negative ion chemical ionization (NICI) mass spectrometry is described. Of various derivatives for the carboxyl group, the pentafluorobenzyl (PFB) ester provided the highest value of the ratio of the negative to the positive ion current. A characteristic carboxylate anion [M - 181]- was produced as the most abundant ion by the loss of the PFB group in NICI. The PFB esters were further derivatized to the dimethylethylsilyl (DMES) ethers, whereby lithocholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid and cholic acid were distinctly separated by GC on a cross-linked methyl silicone fused-silica capillary column. The detection limit for the PFB-DMES derivatives of dihydroxylated bile acids was 2 fg when the fragment ion was monitored at m/z 563 in the NICI mode using isobutane as a reagent gas.     

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.     

FUNCTIONALIZED β-CYCLODEXTRIN POLYMERS FOR THE SORPTION OF BILE SALTS

Poly(β-cyclodextrin) (PCD) resins were prepared by a crosslinking reaction of β-cyclodextrin with different amounts of epichlorhydrin. Some hydroxyl groups of these polymers were functionalized with alkyl quaternary ammonium groups. The polymers were tested for their ability to bind several bile salts (including the sodium salts of cholic acid, glycocholic acid, and chenodeoxycholic acid), individually and competitively, from phosphate buffer solutions. In all cases, the aminated PCD resin had a higher binding capacity for bile salts. The binding of chenodeoxycholate by the resins was much more effective than that of cholate and its conjugate, which indicates the importance of the host cavity size relative to that of the guest molecules. The degree of hydrophobicity of bile acids also plays a role in their binding by PCD resins. The variable temperature studies indicate that the electrostatic interactions become weaker at higher temperatures while the hydrophobic interactions are not as much affected.     

Simultaneous determination of keto and non-keto bile acids in human serum by gas chromatography with selected ion monitoring

A reliable method for the simultaneous determination of keto and non-keto bile acids in human serum was developed. Carbonyl substituents of bile acid ethyl esters were converted into methyloxime and hydroxyl substituents into dimethylethylsilyl ethers and the products were analysed directly by capillary gas chromatography with selected ion monitoring using [2H4]chenodeoxycholic and [2H4]3 alpha-hydroxy-7-oxo-5 beta-cholanoic acids as internal standards. The bile acid peaks on the selected ion chromatogram were separated without interference from endogenous substances present in serum. Recoveries of individual keto bile acids added to serum range from 74.4 to 94.7% with a mean of 87.1%. Eight kinds of keto bile acids not previously found in sera of normal subjects, namely 3-oxo-, 3-oxo-7 alpha-hydroxy-, 3-oxo-12 alpha-hydroxy-, 3 alpha-hydroxy-7-oxo, 3 alpha-hydroxy-12-oxo-, 3-oxo-7 alpha,12 alpha-dihydroxy-, 3 alpha,7 alpha-dihydroxy-12-oxo- and 3 alpha,12 alpha-dihydroxy-7-oxo-5 beta-cholanoic acids were identified and quantified. The total concentration of keto bile acids was found to be 0.16 +/- 0.08 nmol/ml and constituted 2.9 +/- 1.5% of that of the usual non-keto bile acids in peripheral venous serum.     

Determination of some hydroxycholesterols in human serum samples

The simultaneous determination of some hydroxycholesterols in human serum samples is described. The procedure is based on hydrolysis and extraction of these compounds in serum samples, followed by removal of especially cholesterol (making use of reversed-phase high-performance liquid chromatography) and derivatization of the purified compounds to their trimethylsilyl ethers and subsequent gas chromatography using flame ionization detection. Serum levels of 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol and 26-hydroxycholesterol were determined in several groups of patients: normals, untreated patients suffering from cerebrotendinous xanthomatosis, patients suffering from cerebrotendinous xanthomatosis and treated with either chenodeoxycholic acid or cholic acid in an effective dose, patients suffering from cerebro-hepato-renal syndrome, patients suffering from hypercholesterolemia and treated with cholestyramine for prolonged periods and one patient presumed to be suffering from an inborn error of metabolism in bile acid synthesis. It can be concluded that the 7 alpha-hydroxycholesterol concentration in serum is a good parameter for establishing disorders involving the metabolic conversion of 7 alpha-hydroxycholesterol towards bile acids. In addition, 26-hydroxycholesterol levels in patients suffering from cerebrotendinous xanthomatosis are beyond detectable limits, even during treatment with bile acids in an effective dose, whereas in all other conditions this compound is substantially present.     

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.     

O-trimethylsilylquinoxalinol derivatives of aromatic alpha-keto acids. Mass spectra and quantitative gas chromatography

As an extension of earlier work on aliphatic alpha-keto acids, a method is described for the quantitative gas chromatographic determination of urinary aromatic alpha-keto acids. The keto acids are derivatized with o-phenylenediamine to yield the quinoxalinols. These compounds are chromatographed after trimethylsilylation. The aromatic keto acids are stabilized by sodium dithionite (4 mg/ml urine) and storage below 0 degrees. The final derivatives are stable for weeks at room temperature. Low resolution mass spectra are reported. The fragmentation mechanisms are elucidated by analysis of O-trimethylsilyl-(TMS)-quinoxalinois, O-(TMS-d9)-quinoxalinois and O-TMS-6(7)-chloroquinoxalinois.     

Thin-layer chromatography/liquid secondary ion mass spectrometry in the determination of major bile salts in dog bile

Positive and negative ion liquid-state secondary-ion mass spectrometry (LSIMS) was applied to several bile acids and bile salts and their spectra were measured directly from the surface of silica gel thin-layer chromatograms. Such spectra were identical to the LSIMS spectra of the pure compound at the same concentration. Three-dimensional ion images were obtained of a model mixture of cholic, chenodeoxycholic and lithocholic acids in both the positive and negative ion modes. A sample of dog bile was prepared, and the bile acids extracted from it were separated by high-performance TLC; TLC/LSIMS spectra were obtained for sodium taurocholate and sodium taurochenodeoxycholate/taurodeoxycholate, the predominant bile salts present. Quantitative estimates of the analytes were obtained by monitoring the ion intensity for the sample spot and a standard spot that had been developed in parallel on the same TLC plate. The concentration of sodium taurocholate in the bile of this particular dog was found to be 38 mg/ml.     

Differentiation of various traditional Chinese medicines derived from animal bile and gallstone: simultaneous determination of bile acids by liquid chromatography coupled with triple quadrupole mass spectrometry

Animal biles and gallstones are popularly used in traditional Chinese medicines, and bile acids are their major bioactive constituents. Some of these medicines, like cow-bezoar, are very expensive, and may be adulterated or even replaced by less expensive but similar species. Due to poor ultraviolet absorbance and structural similarity of bile acids, effective technology for species differentiation and quality control of bile-based Chinese medicines is still lacking. In this study, a rapid and reliable method was established for the simultaneous qualitative and quantitative analysis of 18 bile acids, including 6 free steroids (cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, hyodeoxycholic acid, and ursodeoxycholic acid) and their corresponding glycine conjugates and taurine conjugates, by using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). This method was used to analyze six bile-based Chinese medicines: bear bile, cattle bile, pig bile, snake bile, cow-bezoar, and artificial cow-bezoar. Samples were separated on an Atlantis dC₁₈ column and were eluted with methanol–acetonitrile–water containing ammonium acetate. The mass spectrometer was monitored in the negative electrospray ionization mode. Total ion currents of the samples were compared for species differentiation, and the contents of bile acids were determined by monitoring specific ion pairs in a selected reaction monitoring program. All 18 bile acids showed good linearity (r² > 0.993) in a wide dynamic range of up to 2000-fold, using dehydrocholic acid as the internal standard. Different animal biles could be explicitly distinguished by their major characteristic bile acids: tauroursodeoxycholic acid and taurochenodeoxycholic acid for bear bile, glycocholic acid, cholic acid and taurocholic acid for cattle bile, glycohyodeoxycholic acid and glycochenodeoxycholic acid for pig bile, and taurocholic acid for snake bile. Furthermore, cattle bile, cow-bezoar, and artificial cow-bezoar could be differentiated by the existence of hyodeoxycholic acid and the ratio of cholic acid to deoxycholic acid. This study provided bile acid profiles of bile-based Chinese medicines for the first time, which could be used for their quality control.     

Simultaneous quantification of the major bile acids in Artificial Calculus bovis by high‐performance liquid chromatography with precolumn derivatization and its application in quality control

An accurate and sensitive high‐performance liquid chromatography method coupled with ultralviolet detection and precolumn derivatization was developed for the simultaneous quantification of the major bile acids in Artificial Calculus bovis, including cholic acid, hyodeoxycholic acid, chenodeoxycholic acid, and deoxycholic acid. The extraction, derivatization, chromatographic separation, and detection parameters were fully optimized. The samples were extracted with methanol by ultrasonic extraction. Then, 2‐bromine‐4’‐nitroacetophenone and 18‐crown ether‐6 were used for derivatization. The chromatographic separation was performed on an Agilent SB‐C18 column (250 × 4.6 mm id, 5 μm) at a column temperature of 30°C and liquid flow rate of 1.0 mL/min using water and methanol as the mobile phase with a gradient elution. The detection wavelength was 263 nm. The method was extensively validated by evaluating the linearity (r² ≥ 0.9980), recovery (94.24–98.91%), limits of detection (0.25–0.31 ng) and limits of quantification (0.83–1.02 ng). Seventeen samples were analyzed using the developed and validated method. Then, the amounts of bile acids were analyzed by hierarchical agglomerative clustering analysis and principal component analysis. The results of the chemometric analysis showed that the contents of these compounds reflect the intrinsic quality of artificial Calculus bovis, and two compounds (hyodeoxycholic acid and chenodeoxycholic acid) were the most important markers for quality evaluating.