Gas-liquid chromatographic assay of serum bile acids

A sensitive method has been established for the analysis of serum bile acids by gas-liquid chromatography (glc). Bile acids are extracted from 0.5–2 ml of serum and analysed as methyl ester trifluoroacetates following enzymatic hydrolysis of the taurine and glycine conjugates. The method as described has been used to estimate serum bile acid levels in health and disease although bile acid sulphates are not detected. Inclusion of a solvolysis procedure before enzymatic hydrolysis would allow their measurement.     

A gas-liquid-chromatographic procedure for separating a wide range of metabolites occuring in urine or tissue extracts

1. A gas–liquid-chromatographic procedure is described which permits separation and identification on the same chromatogram of a wide range of substances occurring in urine or tissue extracts. The method uses hydrogen flame ionization, which detects organic compounds whether free or conjugated with no requirement for specific reactive groups. 2. For chromatography, carboxyl groups are quantitatively converted into methyl esters or trimethylsilyl esters. Phenolic, alcoholic and potential enolic groups are converted into trimethylsilyl ethers. Separations are carried out on a 6ft. column of either 10% F-60 (a polysiloxane) or 1% F-60, temperature programming at 2°/min. being used over such part of the temperature range 30°–260° as is required. Propionyl derivatives of hydroxy compounds can also be used, but only on a non-quantitative basis. Derivatives and columns have been selected for optimum range of usefulness when large numbers of samples are examined by using automated gas chromatography. 3. The method is applicable to: fatty acids above butyric acid; di- and tri-carboxylic acids; hydroxy acids and keto acids; polyhydroxy and alicyclic compounds such as glycerol, inositol, quinic acid, shikimic acid, ascorbic acid and sugar alcohols; aromatic hydroxy and acidic compounds, both benzenoid and indolic; sesquiterpenes; steroids; glycine conjugates; mercapturic acids; glucuronides. It is not satisfactory for sulphate conjugates, iminazoles or polypeptides. 4. Methylene units provide an accurate and reproducible parameter for characterizing peak position. Methylene unit values are reported for a large variety of substances occurring in, or related to those occurring in, urine and tissue extracts. 5. The nature of derivatives was confirmed by combining gas chromatography with mass spectrometry. Combined gas chromatography–mass spectrometry gives a diagnostic tool of great power in the evaluation of metabolic patterns, and various uses are discussed.     

Capillary gas-liquid chromatography of glycine-conjugated bile acids without prior hydrolysis

A method is described for the gas-liquid chromatographic (GLC) analysis of intact glycine conjugates of the major bile acids present in human plasma. It is, therefore, now possible to analyze glycine-conjugated and unconjugated bile acids together on a single GLC column without the necessity for a hydrolytic step. A large number of derivatives of bile acid glycine conjugates were examined, but only acetate- and silyl ether-derivatives of carboxylic acid methyl esters were found initially to be suitable. It was not possible to make acetates consistently, and trimethylsilyl ethers did not allow resolution of the glycine conjugates of cholic and chenodeoxycholic acids. Dimethylethylsilyl ether methyl ester derivatives were subsequently found to give the best results. Chromatographic conditions for successful analysis of these derivatives were examined and it was found to be necessary to use wall-coated capillary columns of thin film thickness (0.12 micron) and very high carrier gas flow rates (ca. 20 ml/min hydrogen). Using acetonitrile and Bond Elut extraction, fractionation on Sep-Pak SIL cartridges, and derivatization as dimethylethylsilyl ether methyl esters, the capillary gas-liquid chromatography of intact glycine-conjugated bile acids from human plasma was demonstrated for the first time.     

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.     

Method for combined analysis of profiles of conjugated progesterone metabolites and bile acids in serum and urine of pregnant women

A method for analysis of profiles of conjugated progesterone metabolites and bile acids in 10 ml of urine and 1–4 ml of serum from pregnant women is described. Total bile acids and neutral steroids from serum and urine were extracted with octadecylsilane-bonded silica. Groups of conjugates were separated on the lipophilic ion-exchanger triethylaminohydroxypropyl Sephadex LH-20 (TEAP-LH-20). Fractions were divided for steroid or bile acid analyses. Sequences of hydrolysis/ solvolysis and separations on TEAP-LH-20 permitted separate analyses of steroid glucuronides, monosulfates and disulfates and bile acid aminoacyl amidates, sulfates, glucuronides and sulfate-glucuronides. Radiolabelled compounds were added at different steps to monitor recoveries and completeness of separation, and hydrolysis/solvolysis of conjugates was monitored by fast-atom bombardment mass spectrometry. The extraction and solvolysis of steroid disulfates in urine were studied in detail, and extraction recoveries were found to be pH-dependent. Following methylation of bile acids, all compounds were analysed by capillary gas chromatography and gas chromatography—mass spectrometry of their trimethylsilyl ether derivatives. Semiquantification of individual compounds in each profile by gas—liquid chromatography had a coefficient of variation of less than 30%. The total analysis required 3 days for serum and 4 days for urine.     

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.     

Quantitative assay of conjugated and free bile acids as heptafluorobutyrate derivatives by gas-liquid chromatography.

Quantitative analyses of individual bile acids in biological samples are limited by the lengthy multistep preparations necessary. Using heptafluorobutyric acid anhydride in pyridine as derivatizing agent, we reduced several steps to one. Bile acids and their glycine and taurine conjugates form stable heptafluorobutyrate derivatives, climinating the need for deconjugation and preparation of methyl esters. The derivatives have been characterized by mass spectrometry, and optimum reaction yields have been determined. Operating conditions for analyzing the bile acid heptafluorobutyrates by gas-liquid chromatography on various column packings were investigated, and 0.5% QF-1 or 3% OV-255 was found suitable. The bile acid derivatives were identical whether starting with the bile acid or the glycine or taurine conjugates. The procedure was applied to a quantitative analysis of artificial mixtures of bile acids and bile conjugates, and also of human bile. The results compared favorably to those obtained with a 3 alpha- and 7 alpha-hydroxysteroid dehydrogenase fluorimetric method.     

Measurement of faecal bile acid sulphates

A method is described for the measurement, by difference, of the sulphate fractions of faecal bile acids. A solvolysis step (for the deliberate hydrolysis of the bile acid sulphates) was added to the procedure of sample homogenisation, extraction, enzymatic hydrolysis and thin-layer chromatography. The bile acids were quantitated by gas—liquid chromatography of their methyl ester and trifluoroacetate methyl ester derivatives on 3% QF-1 columns. The total bile acid excretion in 15 control subjects was 603 ± 71 mg/24 h (x̄ ± S.E.M.). The major bile acid peaks (mg/24 h) were: lithocholic acid, without solvolysis 118 ± 26 and including solvolysis 175 ± 30; deoxycholic acid 60 ± 8 and 90 ± 18 and chenodeoxycholic acid 13 ± 7 and 15 ± 7. It was concluded that bile acid sulphates may form a considerable proportion of the total bile acids excreted in man.     

Separation of methylated free bile acids from their taurine and methyl glycine conjugates by thin-layer chromatography

Class separation of methylated free bile acids from bile acids conjugated with taurine and methylglycine was accomplished using a solvent system of 2,2,4-trimethylpentane-absolute ethanol 10:1 (v/v). By developing a silica thin-layer plate two times with solvent in a Brinkmann sandwich tank, the difficult resolution between methyl cholate and methyl glycolithocholate was achieved. Evidence is presented that this separation system may be useful as a preparative step in the analysis of bile acids by gas-liquid chromatography or high pressure liquid chromatography.--Bolt, M. J. G. Separation of methylated free bile acids from their taurine and methyl glycine conjugates by thin-layer chromatography.     

Development and validation of a method for measuring the glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography

We developed and validated a simple method for measuring the individual glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography with a C₁₈ reversed-phase column using an isocratic solvent system of acidified methanol—potassium phosphate. Without preliminary derivatization or purification, complete separation of the ten major conjugated bile acids present in bile could be achieved in 65 min. Total bile acid concentrations were identical when measured enzymatically and by summing the individual bile acids determined by high-performance liquid chromatography. Bile acid composition determined by gas—liquid chromatography correlated with results by high-performance liquid chromatography. Finally, measurements of individual glycine and taurine conjugates in human bile and in mixtures of bile acid standards by high-performance liquid chromatography and thin-layer chromatography gave similar results. This high-performance liquid chromatographic system permits simultaneous quantification of total and individual bile acids and their glycine and taurine conjugates in bile.     

Synthesis of new bile acid analogues and their metabolism in the hamster: 3 alpha, 6 alpha-dihydroxy-6 beta-methyl-5 beta-cholanoic acid and 3 alpha, 6 beta-dihydroxy-6 alpha-methyl-5 beta-cholanoic acid

This paper reports the chemical synthesis of two new bile acid analogues, namely, 3 alpha, 6 beta-dihydroxy-6 alpha-methyl-5 beta-cholanoic acid from 3 alpha-hydroxy-6-oxo-5 beta-cholanoic acid and describes their metabolism in the hamster. A Grignard reaction of the oxo acid with methyl magnesium iodide in tetrahydrofuran gave two epimeric dihydroxy-6-methyl-cholanoic acids which were separated as the methyl esters by silica gel column chromatography. The configuration of the 6-methyl groups was assigned by proton nuclear magnetic resonance spectroscopy and was supported by the chromatographic properties of the new compounds. The metabolism of the two new bile acid analogues was studied in the hamster. After intraduodenal administration of the 14C-labeled analogues into bile fistula hamsters, both compounds were absorbed rapidly from the intestine and secreted into bile. Intravenous infusion studies revealed that these compounds were efficiently extracted by the liver; the administered analogues became major biliary bile acids, present as either the glycine or taurine conjugates. These compounds are useful to study the effect of methyl-substituted bile acids on cholesterol and bile acid metabolism and may possibly possess cholelitholytic properties.     

Synthesis of alpha,beta-unsaturated C24 bile acids

Synthesis of the alpha,beta-unsaturated analogues of cholic acid, deoxycholic acid, chenodeoxycholic acid, and ursodeoxycholic acid is described. Each common bile acid was converted to the corresponding C22 aldehyde which was then converted to the delta 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.     

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.     

Gas chromatography-mass spectrometry of isobutyl ester trimethylsilyl ether derivatives of bile acids and application to the study of bile sterol and bile acid biosynthesis in rat liver epithelial cell lines

The derivatization of bile acids into trimethylsilyl ether isobutyl ester (IBTMS) and of neutral sterols into trimethylsilyl ether (TMS) allowed the separation on an OV-1 capillary gas chromatography column of 15 bile steroids as follows: cholesterol, 7 alpha-hydroxycholesterol, 6 beta-hydroxycholesterol, 6 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, lithocholate, deoxycholate, 25-hydroxycholesterol, chenodeoxycholate, cholate, murocholate, hyodeoxycholate, ursodeoxycholate, hyocholate, and beta-muricholate. Fragmentation data of the coupled gas chromatographic-mass spectrometric (GC-MS) analysis of these nine bile acids as IBTMS derivatives under electron impact and chemical ionizations (methane, isobutane, and ammonia) are given. The ammonia chemical ionization appears to be the best mode for compound identification and quantitation due to fragmentations into high mass ions. The comparison of methylene units of the five sterols as TMS derivatives and of each type of methyl, TMS, or isobutyl ester of the nine bile acids as TMS ethers showed that isobutyl esterification increased dramatically the retention time of the bile acids, allowing their separation after the neutral sterols. Different methods of GC-MS analysis were applied to the study of bile steroid secretion in long-term rat liver epithelial cell lines, either serum-supplemented cell lines or serum-free cell lines, growing in serum-free medium since the primary explanation or after adaptation of serum-supplemented lines to this medium. It is demonstrated for the first time that liver epithelial cell lines maintain the metabolic pathway leading from synthesized cholesterol to dioxygenated sterols and the two normal main primary bile acids of the liver, chenodeoxycholic acid and cholic acid, up to 32-47% of the in vivo daily rate, and in addition the production of alpha-muricholic acid, the bile acid marker of murine liver.     

Rapid transesterification of bilirubin glucuronides in methanol

The current studies present evidence that bilirubin conjugates derived from rat bile undergo rapid transesterification, $\text{in}$$\text{vitro}$, in solutions containing methanol. The conjugates of bilirubin and the methyl esters formed from them by exposure to methanol were isolated by thin layer chromatography. The isolates were chemically quantitated for their bilirubin and glucuronic acid composition. Characterization of the bilirubin methyl esters was performed by mass spectrometric analysis of the trimethylsilyl and phenylazo derivatives.     

Modulation of gamma-glutamyl transpeptidase activity by bile acids

The free bile acids (cholate, chenodeoxycholate, and deoxycholate) stimulate the hydrolysis and transpeptidation reactions catalyzed by gamma-glutamyl transpeptidase, while their glycine and taurine conjugates inhibit both reactions. Kinetic studies using D-gamma-glutamyl-p-nitroanilide as gamma-glutamyl donor indicate that the free bile acids decrease the Km for hydrolysis and increase the Vmax; transpeptidation is similarly activated. The conjugated bile acids increase the Km and Vmax of hydrolysis and decrease both of these for transpeptidation. This mixed type of modulation has also been shown to occur with hippurate and maleate (Thompson, G.A., and Meister, A. (1980) J. Biol. Chem. 255, 2109-2113). Glycine conjugates are substantially stronger inhibitors than the taurine conjugates. The results with free cholate indicate the presence of an activator binding domain on the enzyme with minimal overlap on the substrate binding sites. In contrast, the conjugated bile acids, like maleate and hippurate, may overlap on the substrate binding sites. The results suggest a potential feedback role for bile ductule gamma-glutamyl transpeptidase, in which free bile acids activate the enzyme to catabolize biliary glutathione and thus increase the pool of amino acid precursors required for conjugation (glycine directly and taurine through cysteine oxidation). Conjugated bile acids would have the reverse effect by inhibiting ductule gamma-glutamyl transpeptidase.     

Gas-liquid chromatographic determination of human fecal bile acids

A method for the determination of total bile acids in human feces that is suitable for routine application is described and discussed. Bile acids are extracted from freeze-dried feces with acetic acid and toluene, in the presence of the internal standard 23-nordeoxycholic acid. After saponification of the extract, bile acids and the internal standard are methylated and converted by mild chromic acid oxidation into their ketonic derivatives. The resultant mixture of a few stable compounds can be separated and measured quantitatively by gas-liquid chromatography on a methylsiloxane polymer. A reference bile acid mixture including the internal standard is also taken through the entire procedure with each series of samples. It has been demonstrated that, in spite of the omission of the usual purification steps, the method is specific for bile acids.     

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.     

Quantitative determination of bile acids in bile with reversed-phase high-performance liquid chromatography

Separation and quantitation of glycine and taurine conjugates of commonly occurring bile acids in bile, i.e. lithocholic, deoxycholic, chenodeoxycholic, ursodeoxycholic and cholic acids in their naturally occurring states have been successfully accomplished using high-performance liquid chromatography. No preliminary purification of bile acids is required except ethanol extraction of bile. A μ Bondapak C₁₈ column and acetonitrile—methanol—phosphate buffer and ultraviolet detector at 200 nm were used. Detection limit was 0.05 μg and linearity was observed in the range up to 16 μg. Bile acid composition of ten randomly chosen normal human gallbladder bile samples is given. A large difference in bile acid composition between glycine and taurine conjugates was found to be present.     

A convenient synthesis of 3-keto bile acids by selective oxidation of bile acids with silver carbonate-Celite.

A number of 3-keto bile acids were synthesized by the selective oxidation of bile acid methyl esters with silver carbonate-Celite in refluxing toluene. The pure 3-keto bile acids were isolated simply by filtering the reaction mixture and concentrating the filtrate. The relation of the bile acid structure to the oxidation rate is also discussed.