Fluorescence high-performance liquid chromatographic determination of free and conjugated bile acids in serum and bile using 1-bromoacetylpyrene as a pre-labeling reagent

A fluorescence high-performance liquid chromatographic method is described for the determination of free and conjugated bile acids in serum and bile. Free and conjugated bile acids are extracted from serum or bile using a Sep-Pak C18 cartridge and then fractionated on a piperidinohydroxypropyl Sephadex LH-20 column. Free and glycine-conjugated bile acids are labeled with 1-bromoacetylpyrene in acetonitrile using dicyclohexyl-18-crown-6-ether as catalyst. Taurine-conjugated bile acids are hydrolyzed by cholylglycine hydrolase and then derivatized by the same reagent. Derivatized bile acids are separated stepwise on a reversed-phase column (Radial Pak A) using acetonitrile-methanol-water (A) (100 : 50 : 40) and (B) (100 : 50 : 20) as mobile phase. The eluate is monitored by a fluorophotometer at 370 nm (excitation) and 440 nm (emission). Linearities of fluorescence intensities (peak heights) with the amounts of free and conjugated bile acids were obtained between 50 pmol and 200 pmol for free bile acids and between 25 pmol and 100 pmol for glycine-conjugated bile acids, respectively. Recoveries from serum and bile samples are not less than 90%. This method is sensitive, reliable and useful for the simultaneous determination of free and conjugated bile acids in serum and bile.     

Enzymatische Bestimmung von Gallensäuren in Körperflüssigkeiten und Geweben

A method for the quantitative determination of bile acids in serum, bile and liver is described. Tetran-heptylammonium salts of the bile acids are extracted from aqueous solutions by means of ethyl acetate. For the enzymatic analysis with 3 α-hydroxysteroid-NAD-oxidoreductase (E.C. 1.1.1.50) the bile acids are isolated from their tetra-n-heptylammonium compounds by cationic exchange (Dowex 50 WX 2).     

Enzymatic fluorimetric determination of the individual bile acids in blood serum

An enzymatic fluorimetric method is described for the determination of total bile acids (cholic acid and deoxycholic acid), primary bile acids (cholic and chen acids and individual bile acids in serum without prior separation of the acids. Total and primary bile acids are determined by equilibrium procedures by conver of the 3α- and 7α-hydroxy bile acids to 3-oxo and 7-oxo bile acids by α-NAD⁺, in the presence of 3α- and 7α-hydroxysteroid dehydrogenase (HSD), respectively, and measurement of the generated NADH fluorimetrically. Chenodeoxycholic acid is determined with 7α-HSD in the presence of cholic and deoxycholic acids by a differential kinetic procedure, and cholic and deoxycholic acids are calculated by difference. Interferents are removed by treatment of serum with Sachrom rein. Only 1.00 ml of serum is required. Low cost, simplicity and reliability are the main features of the method. The recovery of bile acids added to serum averaged 103% (range 83–122%). The method is suitable for routine use in small clinical laboratories.     

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.     

Determination of serum metabolic profiles of bile acids by microcolumn liquid chromatography/laser-induced fluorescence

A method for the determination of individual free and conjugated bile acids in serum using microcolumn liquid chromatography coupled with a laser-induced fluorescence detector is described. Bile acids are separated into free/glycine-conjugate and taurine-conjugate fractions using a Sep-Pak SIL cartridge. The taurine-conjugated bile acid fraction is subjected to enzymatic hydrolysis. Subsequently, free and conjugated bile acids are labeled using 4-(bromomethyl)-7-methoxycoumarin as a fluorogenic reagent, producing stable derivatives that can be excited by the 325 nm line of a He/Cd laser. Prior to their fluorimetric detection, the individual components of a bile acid serum profile are separated by reversed-phase microcolumn liquid chromatography.     

Class separation of bile lipids by thin-layer chromatography

A thin-layer chromatography technique is described that permits separation of each class of bile lipid, such as cholesterol, free (unconjugated) bile acids, glycine- and taurine-conjugated bile acids and phospholipids, in a single run. The use of silica gel G-aluminium pre-coated sheets facilitates further processing, such as the extraction in situ of each class of separated bile lipids for determination by conventional methods.     

Gas chromatographic determination of serum bile acids — Application of a novel extraction procedure

Summary The profile of serum bile acids is a result of their liver metabolism and enterohepatic circulation.In the present work size exclusion chromatography is used for extraction of serum bile acids to optimize the methodology for analyzing serum bile acids by high resolution gas chromatography.Compared to other extraction methods like adsorption-[1–3] or reversed phase chromatography [4,5], this novel technique yielded a satisfactory recovery (75–104%) with high reproducibility. Therefore a reliable determination of serum bile acids is possible.     

Chromatographic methods for the determination of toxicants in faeces.

Modern chromatographic techniques and their application in the determination of toxic compounds in faeces are reviewed. Faecal analysis may be of importance in toxicokinetic studies of xenobiotics in order to determine factors such as metabolism, body burden and major routes of elimination. Compounds of interest include various food constituents, drugs and occupational or environmental factors. Further, various mutagenic or carcinogenic compounds which are excreted by faeces have been indicated to represent risk factors for colorectal cancer. In this context, the chromatographic determination of the endogenously generated fecapentaenes and bile acids, both postulated etiological factors in colorectal carcinogenesis, is reviewed. For fecapentaene determination, several high-performance liquid chromatographic (HPLC) methods are available; however, the applicability of some of these methods is limited owing to insufficient separation of various isomeric forms or discrimination between fecapentaenes and their precursors. For the determination of bile acids in faeces, many chromatographic procedures have been reported, and the characteristics of the most relevant methods are compared and discussed. It is concluded that separation by gas chromatography (GC) in combination with mass spectrometry provides the highest selectivity and sensitivity. A relatively rapid alternative analysis for the determination of total and aqueous faecal bile acids is proposed. Further, methods for the determination of polycyclic aromatic hydrocarbons (PAHs) are reviewed. Although the use of radiolabelled PAHs in animal studies has many advantages, it cannot be applied for human biological monitoring and HPLC and GC provide sensitive alternatives. An HPLC method for the determination of non-metabolized PAHs in faeces is described.     

Spectrofluorimetric Determination of Kinetic Parameters of Bile Acids With 3α-Hydroxysteroid Dehydrogenase from Mutant Pseudomonas Testosteroni

Abstract

The spectrofluorimetric determination of kinetic parameters (K_m, V and k_obs) of conjugated bile acids with 3α-hydroxysteroid dehydrogenase from mutant Ps. Testosteroni is described. Under optimal pH conditions and pseudo- first order kinetics([β-NAD⁺)20 K_mNAD+) the kinetic parameters of bile acids in different buffers are determined. The bile acids exhibit larger kinetic differences in glycine and Tris buffer compared to those measured in pvrophosphate buffer. From these data it is concluded that under certain conditions the differential kinetic determination of one bile acid in the presence of others is possible without prior separation.     

Determination of diagnostically important free and conjugated bile acids in blood plasma by high-performance liquid chromatography

A procedure was proposed for the determination of free bile acids and their conjugates in blood plasma by the reversed-phase HPLC using the column Lichrospher 100 RP-18 (250 + 4.6 mm) with gradient elution and UV-detection at 206 nm. The procedure allowed the simultaneous determination of diagnostically important cholic acids, tauro-and glyco-cholates in blood plasma of patients with no preliminary separation of the analytes into subtypes. The bile acids and their conjugates were isolated from the sample matrix by solid phase extraction in a Sep-Pack C18 cartridge. The limits of detection were 0.11–0.15 mM for free acids and 0.015–0.025 mM for conjugates.     

Separation and Determination of Bile Acids in Human Bile by High-Performance Liquid Chromatography

Abstract

A method for simultaneous determination of major bile acids in human bile is described. The unconjugated, glycine- and taurine-conjugated bile acids are extracted with Sep-pak C¹⁸ and separated into groups by ion-exchange chromatography on a lipophilic gel. Subsequently, resolution of each group into ursodeoxycholate, cholate, chenodeoxycholate, deoxycholate and lithocholate is attained into two stages by high-performance liquid chromatography on a Radial-PAK A column. First, 0.3% ammonium phosphate (pH 7.7)/acetonitrile (19:8, v/v) is used for separation of the latter three as a mobile phase. Ursodeoxycholate and cholate are efficiently separated in 0.3% ammonium phosphate (pH 7.7)/acetonitrile (23:8, v/v). The present method is applicable to quantitation of bile acids in human bile with satisfactory accuracy and precision.     

高效液相色谱／光散射检测器测定肝病病人血清中的胆汁酸

利用反相高效液相色谱／挥发激光散射检测器发展了分析胆汁酸的新方法。采用甲醇和乙酸铵缓冲液混合溶液为冲洗体系，梯度淋洗，１０种甘氨型和牛磺型胆汁酸有２５ｍｉｎ内完成基线分离。方法简单快速，不需衍生，血清榈经预处理，平均回收率为９６．１％，在４－５０，ｇ／Ｌ浓度范围内对峰面积与浓度进行线性回归分析，回归系数０．９９１５－０．９９８２之间可用于正常人和肝硬化病人血清中胆汁酸的测定。     

在胆汁酸盐背景中n种氨羧配位剂的加质子常数及其钙配合物稳定常数

为测定在胆酸盐和脱氧胆酸盐存在下氨提配位剂的加质子常数和钙配合物稳定常数,对常规电住法做了一些修正。用所建立的方法测定了EDTA、DTPA、EGTA及NTA的有关常数及其随胆汁酸浓度的变化。     

A highly sensitive determination of individual serum bile acids using high-performance liquid chromatography with immobilized enzyme

We have developed a highly sensitive method for the simultaneous determination of individual bile acids in serum using high-performance liquid chromatography (HPLC) combined with immobilized 3 alpha-hydroxysteroid dehydrogenase. Both the HPLC column and the immobilized-enzyme column are suitable for use with alkaline solutions necessary in working with this enzyme system. With this method we were able to determine simultaneously fifteen different serum bile acids.     

Quantitative analysis of some bile acids by differential scanning calorimetry combined with TLC

An analytical method useful for the quantitative determination of some bile acids is proposed. The analysis is carried out in two steps. The first is based on the thin layer chromatographic (TLC) separation of the bile acids on alumina plates. The second is based on the application of differential scanning calorimetry (DSC), which permits the characterization and determination of the amount of compound contained in each spot. The DSC signal is proportional to the amount of sample present in the spot layer, while the various peaks and peak temperatures are used to identify the separated compound.     

Improved method for the determination of the major neutral steroids and unconjugated bile acids in human faeces using capillary gas chromatography

An improved method has been developed for the determination of the major neutral steroids (cholesterol and 5 beta-cholestan-3 beta-ol) and unconjugated bile acids (deoxycholic acid and lithocholic acid) in human faeces, using capillary gas chromatography with flame ionization detection. The freeze-dried faecal sample was subjected to a two-stage Soxhlet extraction followed by an aqueous alkali-organic solvent partition step to separate neutral steroids from bile acids. The neutral steroids were analysed as their trimethylsilyl ether derivatives on an OV-1 capillary column. The bile acids were further purified on a Sep-Pak C18 cartridge and then fractionated on a Sep-Pak SIL cartridge. Unconjugated bile acids were analysed as their methyl ester-trimethylsilyl ether derivatives also on an OV-1 capillary column. Quantitation of neutral steroids and unconjugated bile acids was achieved by reference to appropriate internal standards, added to the faecal extract immediately after the Soxhlet extraction stage. The method is being used in a study of the effect of diet on the metabolic activity of human gut flora.     

Determination of bile acids in pig liver, pig kidney and bovine liver by gas chromatography-chemical ionization tandem mass spectrometry with total ion chromatograms and extraction ion chromatograms

An effective method has been developed for quantitative determination of six bile acids including lithocholic acid (LCA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), hydodeoxycholic acid (HDCA), cholic acid (CA) and ursodeoxycholic acid (UDCA) in biological tissues including pig liver, pig kidney and bovine liver by gas chromatography-chemical ionization/tandem mass spectrometry (GC-CI/MS/MS). Camphor-10-sulphonic acid (CSA) was proposed as effective catalyst for bile acid derivatization. Reactions were accelerated ultrasonically. The effects of different catalysts and reaction times on derivatization efficiency were evaluated and optimized. Bile acids were determined as methyl ester-trimethylsilyl ether and methyl ester-acetate derivatives. The efficiency of trimethylsilylation and acetylation was evaluated. Trimethylsilylation was done with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) as the trimethylsilyl donating reagent in a ultrasonic bath for 20 min. Acetylation was done in pyridine with acetic anhydride at 40-45°C for 4 h. The former reaction was faster than the latter. Thus, trimethylsilylation was employed for the quantitative analysis. Negligible interferences from sterols in biological matrices were observed when the biological samples were treated with solid phase extraction before GC-CI/MS/MS. The linearity, reproducibility, detection limit and recovery were evaluated under the optimized conditions. Satisfactory results were obtained when bile acid derivatives of LCA, CDCA, HDCA, and UDCA were determined with total ion chromatograms (TIC) while DCA and CA were determined with extracted ion chromatograms (EIC), respectively. The detection limits (S/N=3) for six bile acids in biological tissues were ranging from 0.40 to 1.6 ng/mL and the recoveries indicated that the proposed method was feasible for the determination of trace bile acids in the biological samples studied. The experimental results for the animal tissues purchased from five different markets were compared. Interestingly, all of the six bile acids were present in pig liver while only the dihydroxy bile acids, DCA, CDCA and HDCA were found in pig kidney. In addition to DCA and CDCA, trihydroxy bile acid, CA, are the major bile acids in bovine liver.     

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.     

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).