Bile acids LIII: Application of reverse-phase high-pressure liquid chromatography to the analysis of conjugated bile acids in bile samples

The common conjugated bile acids of deproteinated bile from the human or the rat can be separated by high-pressure liquid chromatography and quantitated within 30 min with a 4-mm × 30-cm “fatty-acid analysis” column (Waters Associates) in 2-propanol/8.8 mm potassium phosphate buffer (pH 2.5) 160:340, coupled to a uv flow detector set at 193-nm wavelength. Detection limits were at least 0.1 nmol for the tauro-conjugates and 0.2 nmol for the glyco-derivatives.     

Determination of individual conjugated bile acids in human bile

A method has been developed and validated for the determination of the six major conjugated bile acids, cholesterol, and total phospholipids in bile of human subjects previously injected with 4-(14)C-cholesterol. The procedure is designed for use with 5-10 ml of duodenal or T-tube bile and eliminates difficulties associated with existing methods for bile acid determination, in particular the requirement for preliminary saponification under pressure or the use of paper chromatography. Saponification under pressure is employed only in steps where partial destruction of the steroid moiety of conjugated bile acids is not a crucial matter. A preliminary Folch extraction and washing step separated free cholesterol and phospholipids (bottom layer) from the six major conjugated bile acids (top layer). The conjugated bile acids were then fractionated cleanly by thin-layer chromatography to give four groups, the (14)C content of each of which was determined. A second aliquot of the top layer was used to determine (after deconjugation) the radioactivity ratio of deoxycholic acid to chenodeoxycholic acid for the two unresolved groups (dihydroxycholanoic acid conjugates with glycine and taurine, respectively). A third aliquot was used for determination of specific activities of the methyl esters of cholic, chenodeoxycholic, and deoxycholic acids derived from the total bile salts. Appropriate calculations yielded the concentration in bile of all six major bile acid conjugates.     

Determination of conjugated bile acids in human bile and duodenal fluid by reverse-phase high-performance liquid chromatography

A simple mehtod using reverse-phase liquid chromatography is presented for resolution and quantitation of the major conjugated bile acids of man, including the glycine and taurine conjugates of the dihydroxy bile acids, chenodeoxycholic and deoxycholic acid. Using modern, high-performance chromatographic equipment, analysis time is less than 30 minutes. The quantitative range of the method, with detection by refractive index, is 0.05 to 0.1 mumol of bile acid and the limit of detection for an injection sample is 0.01 mumol. This provides a sensitivity sufficient for analysis of dilute duodenal and gallbladder bile with minimal sample preparation.     

Glyco-7 alpha, 12 alpha-dihydroxy-5 beta-cholanic acid as internal standard for high-pressure liquid chromatographic analysis of conjugated bile acids

Glyco-7 alpha, 12 alpha-dihydroxy-5 beta-cholanic acid was tested as internal standard for high-pressure liquid chromatographic analysis of the five main glycine- and taurine-conjugated bile acids present in adult human serum and bile. When the standard is added to the samples before extraction, the recovery rate throughout the procedure is similar to that of other bile acids. For all bile acids studied, the response, relative to the internal standard, is linear at 205 nm. Baseline separation is observed between the internal standard and all other bile acids, both in artificial mixtures and extracts of biological samples. Thus, glyco-7 alpha, 12 alpha-dihydroxy-5 beta-cholanic acid is a reliable internal standard for HPLC analysis of conjugated bile acids in serum and bile.     

Bile acids: XLVIII. Separation of conjugated bile acids by high-pressure liquid chromatography

Major conjugated bile acids of human bile have been resolved by high-pressure liquid chromatography. The elutions are carried out in two stages on Corasil II or μPorasil columns; first, an alkaline solvent system (2-propanol/ethyl acetate/water/7n ammonium hydroxide, 260:600:50:3) was used for separation into groups: tauro-dihydroxy derivatives, taurocholate, glyco-dihydroxy derivatives, and glycocholate. The fraction containing glyco-dihydroxy conjugates was separated by rechromatography in acetonitrile/acetic acid, 400:10, and the fraction containing tauro-dihydroxy conjugates could be partially resolved by rechromatography in acetonitrile/acetic acid/formic acid (97%)/water, 500:10:5:10. Three samples of prepared human bile have been similarly treated.     

Determination of conjugated bile acids in human urine by high-performance liquid chromatography with chemiluminescence detection

A qualitative and quantitative analysis of the conjugated 1β- and 6α-hydroxy bile acids, including common bile acids, in human urine using high-performance liquid chromatography with chemiluminescence detection is described. After extraction of urine with C₁₈ silica cartridges, the bile acids were separated into non-conjugated, glycine, taurine and sulphate fractions by ion-exchange chromatography on a lipophilic gel. Solvolysis of the sulphate was carried out by treatment with trifluoroethanol in acetone containing hydrochloric acid, and the liberated amino acid conjugates were fractionated again. The individual bile acids were separated on a reversed-phase C₁₈ column (Bile Pak II), with detection by an immobilized 3α-hydroxysteroid dehydrogenase enzyme reactor and chemiluminescence reaction of the generated NADH using 1-methoxy-5-methylphenazinium methylsulphate—isoluminol—microperoxidase system. The assay method showed the detection limits ranging from 8 to 250 pmol for the bile acids tested. Analysis of urine samples obtained from newborns, non-pregnant women and women in late pregnancy showed a large difference in bile acid composition and conjugation mode, suggesting that bile acid metabolism is different during fetal and neonatal periods.     

Quantitation of free and conjugated bile acids in human feces using a high-pressure liquid chromatography counterion method

A method has been developed for extraction and purification of the major bile acids in human feces, and for their quantitative estimation using high-pressure liquid chromatography. Freeze-dried fecal material was extracted with alkaline ethanol and, after removal of neutral steroids, was subjected to thin-layer chromatography, followed by reversed-phase C18 silica cartridge (Sep-Pak) purification. The mixture was further separated into free, and glyco and tauro conjugates by ion-exchange chromatography. Subsequent resolution of individual bile acids was accomplished by HPLC using a counterion pairing method.     

Bile acids. LXXII. High-performance liquid chromatographic analysis of bile acid coenzyme A derivatives

The mobilities of coenzyme A and coenzyme A derivatives of cholate, chenodeoxycholate, deoxycholate, lithocholate, and their 5 alpha analogs were studied in reversed-phase high-performance liquid chromatography. With a C18 Radial-PAK A cartridge (10-micron particles) and a solvent mixture of 2-propanol/10 mM phosphate buffer (pH 7.0, 140:360), separation of the chenodeoxycholyl and deoxycholyl coenzyme A derivatives was not observed. An increase in ionic strength of the buffer to 50 mM afforded separation, which was markedly augmented with a C18 Radial-PAK A cartridge with 5-micron particles. Lowering the pH of the buffer to 5.5 did not materially change the separations regardless of the ionic strength. Quantitation was carried out to a lower level of 8.5 X 10(-12) mol.     

基于色谱技术定量分析胆汁酸的研究进展

胆汁酸以其在吸收脂肪和脂溶性维生素方面的功能而闻名。然而,胆汁酸也是调节胆固醇、葡萄糖和能量稳态的重要信号分子,并且是人体与肠道微生物代谢之间的关键中间分子。胆汁酸循环的稳态与人体代谢和免疫功能密切相关。但胆汁酸家族成员的化学结构多样,并且在复杂生物基质的样品中以较低的浓度存在。因此,精确测量这些重要代谢物意义重大,尽管酶法测定总胆汁酸在临床实验室中最为普遍,但这些方法只提供了有限的胆汁酸信息。先进的分析方法,如气相色谱和液相色谱-质谱技术,是高度信息丰富的技术,有助于识别和量化复杂生物基质中的个别胆汁酸。本文对几种利用色谱技术定量分析胆汁酸的方法进行了综述。     

Pre-column derivatization of free bile acids for high-performance liquid chromatographic and gas chromatographic—mass spectrometric analysis

Pentachlorophenyl (PCP) esters of five free bile acids (FBA) were obtained by reacting the FBA and Kovacs' complex (KC) in a 1:8 molar ratio in acetone at 65°C, and were purified by column chromatography on silica gel. The esters were crystallized from benzene—hexane, derivatized as trimethylsilyl ethers for gas chromatography on a DB-1 capillary column and for gas chromatography—mass spectrometry with a DB-5 column, and mass spectrometry (MS) in the electron-impact (EI) positive-ion mode at 70 eV. The reaction is specific for FBA even in the presence of glycine and taurine conjugates of bile acids. The PCP esters were treated with benzylamine in chloroform or methanol to produce N-benzyl derivatives of FBA. The N-benzylamides were separated by high-performance liquid chromatography (HPLC) on a 4-μm Nova-Pak C₁₈ column, studied by thermospray—LC—MS, and in the direct insertion probe—EI positive-ion mode.     

High pressure liquid chromatographic separation of the metabolites of 3-methylcholanthrene

All the known metabolites of 3-methylcholanthrene can be separated by high pressure liquid chromatography on a μBondapak C₁₈ column. The separation is accomplished with a reverse phase linear gradient of 40% acetonitrile water to 100% acetonitrile at room temperature. The addition of 0.2% acetic acid to the 40% acetonitrile chamber is required for resolution of the $\text{cis}$ 1,2-dihydrodiol.     

Ligand-exchange chromatographic resolution ofDl-amino acids in the presence of nucleic acids

Summary Nine representativeDl-amino acids were resolved using ligand-exchange chromatography whose mobile phase containe Cu (II) and ribonucleic acids. It was also found that deoxyribonucleic acids could be substituted for RNAs. In the presence of 5-GMP, 4XMP, (a mixture of 5-GMP,-AMP,-CMP, and-UMP), and oligomer of RNA,L-amino acids, which seemed to give more stable complexes, were eluted more slowly than the opposite isomers.     

Rapid determination of glycine- and taurine-conjugated bile acids in human bile by high-performance liquid chromatography

With use of an anion-exchange packing, TSK Gel IEX 540 DEAE, for high-performance liquid column chromatography, glycine- and taurine-conjugated bile acids were separated in 10 min and detected with a differential refractometer. Human bile could be analyzed after a simple pretreatment. The purity of the peaks of glycine- and taurine-conjugated bile acids in human bile was confirmed by enzymatic determination using 3α-hydroxysteroid:NAD oxidoreductase. The molar ratios of the two forms of the conjugates (glycine/taurine ratios) in bile from normal subjects and from patients suffering from various hepatobiliary diseases were measured.     

A rapid non-chromatographic analysis of individual bile acids in human bile extracts

It is postulated that the six conjugated bile acids of most common occurrence in human bile could be analyzed by three enzymic and one chemical assay without any prior chromatographic separation of the bile acids. In health, all bile acids in liver or gall bladder bile are conjugated with either glycine or taurine and have an a-hydroxyl group at the 3 position. In addition, the trihydroxy bile acid, cholic (C) has a 7α- and a 12α-hydroxy group while the dihydroxy bile acids either have a second hydroxyl group at the 7α-position (chenodeoxycholic acid, CDC) or at the 12α-position (deoxycholic acid, DC). Hydroxysteroid dehydrogenases (HSDH) specific for oxido-reductase activity at the 3α-, 7α- and 12α-positions would directly quantify these 3α-, 7α- and 12α-hydroxyl groups in a sample of bile or bile extract. Subsequent data would be used to solve three simultaneous equations yielding solutions for the overall concentrations of conjugated C, conjugated CDC and conjugated DC on the assumption that the overall concentration of lithocholic acid is negligible (< 2 %). A suitable assay for the sulphonate group containing taurine conjugates, such as that described by Christie, Macdonald & Williams, 1975, along with the total bile acid measurement would readily facilitate the estimation of the glycine/taurine ($\text{G}\text{T}$) ratio. This ratio applied to the enzymatically derived estimates for conjugated DC, CDC and C would approximate the glycodeoxycholate (GDC), glycochenodeoxycholate (GCDC), glycocholate (GC), taurodeoxycholate (TDC), taurochenodeoxycholate (TCDC) and taurocholate (TC) concentrations. Figures for these concentrations would be based on the assumption that the $\text{G}\text{T}$ ratio is approximately the same for each bile acid and that all the bile acids are conjugated.     

Determination of sulfated and nonsulfated bile acids in serum by mass fragmentography

A Sep-Pak C18 cartridge was used for purification of bile acids from serum. Three kinds of deuterium labeled internal standards were required for accurate measurement of individual sulfated and nonsulfated bile acids. These internal standards were added to the serum before its application to the cartridge. Separation of sulfated and nonsulfated bile acids was performed on piperidinohydroxypropyl Sephadex LH-20 column chromatography. The nonsulfate fraction was submitted to alkaline hydrolysis, and the sulfate fraction to solvolysis followed by alkaline hydrolysis. Each fraction was converted to the hexafluoroisopropyl-trifluoroacetyl derivatives and quantitated by mass fragmentography. The recovery of each bile acid sulfate was quite satisfactory. In fasting healthy subjects the mean of total nonsulfated bile acids in serum was 1.324 micrograms/ml, and that of total sulfated bile acids was 0.450 micrograms/ml. Sulfated lithocholic acid comprised a large part of sulfated bile acids in healthy subjects.     

Serum concentrations of glucuronidated and sulfated bile acids in children with cholestasis

Serum concentrations of nonglucuronidated-nonsulfated, glucuronidated, and sulfated bile acids in 9 control children and 16 children with cholestasis were quantitated by mass fragmentography. Total bile acid levels in control children were 19.55 +/- 2.78 mumol/liter (mean +/- SEM), and glucuronidated and sulfated bile acids comprised 2.6 +/- 0.5 and 17 +/- 3.1%, respectively. In 9 patients with congenital biliary atrasia, total bile acid levels were 167.34 +/- 11.18 mumole/liter of which 2.1 +/- 0.3% were glucuronidated and 15 +/- 1.4% were sulfated. Lithocholic and 3 beta-hydroxy-5-cholenoic acids, which have hepatotoxic effects, were presented in only small amounts in cholestatic children, and they were almost all glucuronidated or sulfated. The percentages of glucuronidated bile acids in control and cholestatic children were lower than in healthy and cholestatic adults, which may be explained by the lower activity of UDP-glucuronyltransferase in neonatal liver.     

New simplified procedures for the extraction and simultaneous high-performance liquid chromatographic analysis of retinol, tocopherols and carotenoids in human serum

A short, simple extraction procedure and a sensitive reversed-phase high-performance liquid chromatographic assay, which utilizes isocratic elution and detection either by a photodiode-array detector or by two detectors set at 300 and 450 nm, have been developed to measure retinol, tocopherols and several carotenoids in human serum simultaneously. By relying on characteristic UV—visible spectra, seventeen carotenoids, retinol and α- and γ-tocopherols were identified in concentrated serum extracts by use of the three-dimensional data mode of a photodiode-array detector. The presence of some recently reported carotenoids in human serum has been confirmed.     

High-performance liquid chromatographic determination of α-keto acids in human urine and plasma

A high-performance liquid chromatographic method has been developed for the determination of α-keto acids in human urine and plasma. These acids were prepurified using a column of hydrazide gel and derivatized with o-phenylenediamine into 2-quinoxalinol derivatives, which were extracted into ethyl acetate. The 2-quinoxialinol derivatives were separated by reversed-phase paired-ion chromatography using a 250 × 4 mm-i.d. column packed with LiChrosorb RP-8 (5 μm). This method is sensitive, selective, and reproducible. The α-keto acids in urine and plasma from normal individuals were determined.     

High-performance liquid chromatographic analysis of ammonium in human urine

A method for the quantitative determination of ammonium in human urine by high-performance liquid chromatography (HPLC) is described. After making fluorescent substances with fluorescamine, they were separated and quantified by their fluorometric intensity. The intensity (as measured by peak height) was linear between 0.5 and 5.0 micrograms, and coefficients of variation for elution time and peak height on replicate analysis of the standard were 0.15 and 4.2%, respectively. Recoveries of added ammonium were 96.5 and 97.3%, respectively, on 2.0 and 3.0 micrograms by this method. Detection limit of this method was 0.2 microgram. There was good agreement between the proposed HPLC method (X) and ion chromatographic method (Y), giving the relationships as Y = 0.956X + 0.012, r = 0.991.     

Chiral discrimination by albumin: A mechanistic study of liquid chromatographic optical resolution of nonaromatic carboxylic acids

In order to get further insight into the mechanism by which bovine serum albumin (BSA) discriminates between enantiomers of organic acids, some radioisotopically labeled, nonaromatic carboxylic acids were studied under varying mobile phase conditions. It was found for a series of N-alkanoyl-DL -[³H]leucines that the D -enantiomers were much more strongly retained and that the composition of the mobile phase could be adjusted to give very large (α > 20) enantiomeric separation factors. The elution order was consistent with what has been found earlier for other N-acyl derivatives as well as for N-arylcarbamoyl derivatives of simple aliphatic amino acids. A marked increase in the hydrophobic interaction of the D -enantiomers with the chiral phase was found upon a lowering of the mobile phase strength, conditions under which the L -form was only slightly influenced. These and other results are consistent with a chiral recognition model by which inclusion of the compound in a hydrophobic chiral cavity of BSA with simultaneous charge interaction is assumed to take place and whereby discrimination is determined by the steric bulk and orientation of the α-substituent. © 1993 Wiley-Liss, Inc.