Differentiated quantification of human bile acids in serum by high-performance liquid chromatography-tandem mass spectrometry

Determination of quantitative changes in the pattern of serum bile acids is important for the monitoring of diseases affecting bile acid metabolism. A sensitive and specific high-performance liquid chromatography (HPLC)-MS/MS method was developed for the differentiated quantification of unconjugated as well as glycine- and taurine-conjugated cholic, chenodeoxycholic (CDCA), deoxycholic (DCA), ursodeoxycholic (UDCA) and lithocholic acid (LCA) in serum samples. After solid-phase extraction and reversed-phase HPLC separation, detection of the conjugated bile acids was performed using electrospray ionization (ESI)-MS/MS and selected reaction monitoring mode, whereas unconjugated bile acids were determined by ESI-MS and selected ion monitoring mode. The within-day and between-day coefficients of variation were below 7% for all bile acids and the recovery rates of the extraction procedure were between 84.9 and 105%. The developed method was applied to a group of 21 healthy volunteers and preliminary reference intervals in serum were established. In patients with drug-induced cholestasis, an elevation of primary bile acids has been shown.     

7β,12β-dihydroxy-5β-cholan-24-oic acid as an internal standard for quantitative determination of bile acids by gas chromatography

In order to find an artificial internal standard compound for quantitative determination of bile acids by gas chromatography, 7α,12α-,7α, 12β-, 7β,12α- and 7β,12β-dihydroxy-5β-cholan-24-oic acids were chemically synthesized with cholic acid (1) as the first starting material. The gas chromatographie retention time of 7β,12β-dihydroxy-5β-cholan-24-oic acid (ββ-isomer) was more different from that of natural bile acids than the other isomers. Moreover, ββ-isomer was extracted in the same fraction as the bile acids from urine, and no urinary substance had the same retention time as ββ-isomer. No artifact was produced from ββ-isomer during the analysis procedure. It was concluded that the ββ-isomer is an internal standard compound with certain advantages for the quantitative determination of bile acids in urine by gas chromatography, irrespective of the recovery rate during the analysis procedure.     

Determination of bile acids in biological fluids by liquid chromatography-electrospray tandem mass spectrometry

A simple, sensitive, and specific liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the determination of bile acids in human bile has been developed. The bile acids were extracted with a C(18) (octadecyl) reversed-phase column and identified and quantified by simultaneous monitoring of their parent and daughter ions, using the multiple reaction monitoring mode. Identification and quantification of conjugated bile acids in bile was achieved in 5 min. The detection limit was 1 ng, and the determination was linear for concentrations up to 100 ng. The percent recovery of standards made of single conjugated (glycine and taurine) bile acid or of mixture of glycine- or taurine-conjugated cholic acid, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, and lithocholic acid averaged 71.73% to 95.92%. The percent recovery of the same standard bile acids was also determined by gas chromatography-mass spectrometry (GC-MS), using the selected ion monitoring mode, and averaged 66% to 96%. A biliary bile acid profile of human gallbladder bile was obtained by LC-MS/MS and GC-MS.The results showed a good correlation between the two techniques and no significant differences between the two methods were observed. The LC-MS/MS method was also used for the analysis of serum, urine, and fecal bile acids. In conclusion, LC-MS/MS is a simple, sensitive, and rapid technique for the analysis of conjugated bile acids in bile and other biological samples. - Perwaiz, S., B. Tuchweber, D. Mignault, T. Gilat, and I. M. Yousef. Determination of bile acids in biological fluids by liquid chromatography-electrospray tandem mass spectrometry. J. Lipid Res. 2001. 42: 114;-119.     

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.     

Radioassay of bile acid coenzyme A:glycine/taurine: N-acyltransferase using an n-butanol solvent extraction procedure

A rapid, specific, and sensitive radioassay for measuring bile acid CoA:glycine/taurine: N-acyltransferase (EC 2.3.1) has been developed. In this assay, 3H-labeled amino acids (glycine or taurine) are conjugated with unlabeled bile acid CoA derivatives to form 3H-labeled bile acid amidates. Following incubation, the 3H-labeled bile acid amidate is separated from the unreacted amino acid by an n-butanol extraction method. The extraction procedure was developed by evaluating the effects of buffer concentration and pH on the recovery of radiolabeled bile acid amidate standards in the presence of human hepatic cytosol. Highest recovery (greater than 90%) of bile acid amidate standards occurred under acidic conditions (pH 2) in the presence of 1% (w/v) SDS. When the radioassay and accompanying n-butanol extraction procedure were utilized to study the amidation of glycine or taurine with cholic acid in human hepatic cytosol, a single peak of radioactivity corresponding with either authentic glycocholate or taurocholate was detected in the n-butanol phase by high-performance liquid chromatography. This assay for bile acid CoA:glycine/taurine: N-acyltransferase activity was linear with incubation time and protein concentration. This assay should be useful in the biochemical studies of this enzyme, as well as in the examination of bile acid amidation in clinical liver specimens.     

Characterization of serum and urinary bile acids in patients with primary biliary cirrhosis by gas-liquid chromatography-mass spectrometry: effect of ursodeoxycholic acid treatment

We have studied the effect of ursodeoxycholic acid on the serum and urinary bile acids in seven patients with moderate to severe primary biliary cirrhosis. Bile acids were characterized by gas-liquid chromatography-mass spectrometry and quantified by capillary gas-liquid chromatography. Serum bile acids were elevated 26-fold over control values, with 2.2 times more cholic acid than chenodeoxycholic acid. Urinary bile acid output was elevated 22-fold over control values with a cholic acid:chenodeoxycholic acid ratio of 1.6. In addition, lithocholic acid, deoxycholic acid, ursodeoxycholic acid, 1 beta-hydroxycholic acid, 1 beta-hydroxydeoxycholic acid, and hyocholic acid were identified in both serum and urine; the proportions of the 1- and 6-hydroxylated bile acids were much higher in urine than in serum of the patients (32.1% versus 4.2%). Three months of placebo administration did not change the serum and urinary bile acid composition. In contrast, ursodeoxycholic acid feeding (12-15 mg/kg body weight per day) for 6 months resulted in a 25% decline in the total serum bile acid concentration from the pretreatment values. The proportion of ursodeoxycholic acid increased from 2.1 to 41.2% of total bile acids, so that total fasting serum endogenous bile acid levels decreased 62.4%. Ursodeoxycholic acid feeding substantially increased urinary bile acid output, with ursodeoxycholic acid comprising 58.1%. The proportion of 1- and 6- hydroxylated endogenous bile acids was reduced by 45.5% from pretreatment levels and approximately 4.5% of the urinary bile acids were omega-muricholic acid, 1 beta-hydroxyursodeoxycholic acid, and 21-hydroxyursodeoxycholic acid. These results demonstrate significant changes in the serum and urinary bile acid pattern in primary biliary cirrhosis during ursodeoxycholic acid treatment. The beneficial effect of ursodeoxycholic acid may be due to reduction of the hydroxylated derivatives of endogenous bile acids together with the appearance of hydroxylated derivatives of ursodeoxycholic acid or it may be due to displacement of the more hydrophobic endogenous bile acids by the hydrophilic ursodeoxycholic acid.     

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.     

Human cecal bile acids: concentration and spectrum

To obtain information on the concentration and spectrum of bile acids in human cecal content, samples were obtained from 19 persons who had died an unnatural death from causes such as trauma, homicide, suicide, or drug overdose. Bile acid concentration was measured via an enzymatic assay for 3alpha-hydroxy bile acids; bile acid classes were determined by electrospray ionization mass spectrometry and individual bile acids by gas chromatography mass spectrometry and liquid chromatography mass spectrometry. The 3alpha-hydroxy bile acid concentration (mumol bile acid/ml cecal content) was 0.4 +/- 0.2 mM (mean +/- SD); the total 3-hydroxy bile acid concentration was 0.6 +/- 0.3 mM. The aqueous concentration of bile acids (supernatant after centrifugation) was identical, indicating that most bile acids were in solution. By liquid chromatography mass spectrometry, bile acids were mostly in unconjugated form (90 +/- 9%, mean +/- SD); sulfated, nonamidated bile acids were 7 +/- 5%, and nonsulfated amidated bile acids (glycine or taurine conjugates) were 3 +/- 7%. By gas chromatography mass spectrometry, 10 bile acids were identified: deoxycholic (34 +/- 16%), lithocholic (26 +/- 10%), and ursodeoxycholic (6 +/- 9), as well as their primary bile acid precursors cholic (6 +/- 9%) and chenodeoxycholic acid (7 +/- 8%). In addition, 3beta-hydroxy derivatives of some or all of these bile acids were present and averaged 27 +/- 18% of total bile acids, indicating that 3beta-hydroxy bile acids are normal constituents of cecal content. In the human cecum, deconjugation and dehydroxylation of bile acids are nearly complete, resulting in most bile acids being in unconjugated form at submicellar and subsecretory concentrations.     

General methods for the analysis of metabolic profiles of bile acids and related compounds in feces

A general method is described for the detailed qualitative and quantitative analysis of bile acids and related compounds from feces. The technique utilizes a novel combination of liquid-gel and liquid-solid extraction, lipophilic ion exchange chromatography, and capillary column gas-liquid chromatography coupled to mass spectrometry, which permits the detailed composition of bile acids in feces in terms of both the individual bile acids present and their mode of conjugation in the original fecal sample. The extraction, purification, and isolation procedures have been evaluated using fecal samples containing endogenous radioactive bile acid metabolites and from the addition of radiolabeled standards to fecal homogenates. The applicability of the general procedure is illustrated with examples from the analysis of bile acids and sterols in the feces collected from normal healthy subjects, patients with chronic diarrhea, and an adult female Sprague-Dawley rat. The flexibility of the method, and the general problems encountered in the extraction, purification, and isolation of bile acids and related classes of compounds from feces for subsequent analysis of gas-liquid chromatography are discussed in detail.     

Microanalysis of free and conjugated bile acids by thin-layer chromatography and in situ spectrofluorimetry

A combination of thin-layer chromatography (TLC) and in situ spectrofluorimetry for the determination of free bile acids and bile acids conjugated with glycine or taurine is described. This method makes it possible to determine bile acids concentrations as low as 0.15-0.25 nmol (0.05-0.1 microgram) in a simple and reproducible way. Moreover, information can be obtained about conjugation patterns and relative concentrations of mono-, di-, and trihydroxy bile acids as well as about the presence of abnormal bile acids. After TLC the bile acids are made visible in uv light by dipping the layer in sulfuric acid in diethyl ether and warming it under well-described conditions. The fluorescence of the bile acids on the thin layer can be measured and makes it possible to quantitate them. The method presented here is applicable to bile acid-containing extracts from serum, bile, and feces, and the results are in good agreement with those obtained by enzymatic and gas-liquid chromatographic techniques.     

Hepatic bile acid metabolism during early development revealed from the analysis of human fetal gallbladder bile

A detailed study of the qualitative and quantitative composition of bile acids in human fetal gallbladder bile is described. Bile was collected during early gestation (weeks 16-19) and analyzed by gas chromatography and mass spectrometry, fast atom bombardment ionization mass spectrometry, and high performance liquid chromatography. Bile acids were separated into different conjugate groups by chromatography on the lipophilic anion exchange gel, diethylaminohydroxypropyl Sephadex LH-20. Quantitatively more than 80% of the bile acids were secreted into bile conjugated to taurine. Unconjugated bile acids and glycine conjugates accounted for 5-10% of the total biliary bile acids. Bile acid sulfates were present only in trace amounts indicating that quantitatively sulfation is not an important pathway in bile acid metabolism during development. Total biliary bile acid concentrations were low (0.1-0.4 mM) when compared to reported values for adult bile (greater than 10 mM). Chenodeoxycholic acid was the major biliary bile acid and exceeded cholic acid concentrations by 1.43-fold indicating either a relative immaturity in 12 alpha-hydroxylase activity during early life or a dominance of alternative pathways for chenodeoxycholic acid synthesis. A relatively large proportion of the biliary bile acids comprised metabolites not found in adult bile. The presence of relatively high proportions of hyocholic acid (often greater than cholic acid) and several 1 beta-hydroxycholanoic acid isomers indicates that C-1 and C-6 hydroxylation are important pathways in bile acid synthesis during development. We describe, for the first time, evidence for the existence of a C-4 hydroxylation pathway in the metabolism of bile acids, which may be unique to early human development. Mass spectrometry was used to confirm the identification of 3 alpha,4 beta,7 alpha-trihydroxy-5 beta-cholanoic and 3 alpha,4 beta-dihydroxy-5 beta-cholanoic acids. Quantitatively, these C-4 hydroxylated bile acids accounted for 5-15% of the total biliary bile acids of the fetus, suggesting that C-4 hydroxylation is quantitatively an important pathway in the bile acid metabolism during early life.     

Extraction of bile acids from rat feces containing cholestyramine

The fecal extraction procedure described by Evrard and Janssen (1) was inadequate for the complete extraction of conjugated bile acids from feces containing the bile acid sequestrant, cholestyramine. As judged by gas-liquid chromatographic analysis, substitution of 0.5 n HCl in absolute ethanol for glacial acetic acid allowed for complete recovery (98-104%) of three different conjugated bile salts in the presence of the resin.     

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.     

Radioimmunological determination of serum 3 beta-hydroxy-5-cholenoic acid in normal subjects and patients with liver disease.

A radioimmunoassay for the determination of 3 beta-hydroxy-5-cholenoic acid in human serum has been developed, using 3 beta-hydroxy-5-cholenoyl-thyroglobulin as immunogen and 3 beta-hydroxy-5-cholenoylglycyl-125I histamine as radioactive ligand. The association constant was 6.3 X 10(8) l/mol. Cross reactivity with other bile acids of human serum was not detectable, but was 5.6% with cholesterol. Serum sample preparation included extraction of 3 beta-hydroxy-5-cholenoic acid from serum, solvolysis of sulfates, hydrolysis of conjugates, and separation from cholesterol by thin-layer chromatography. Serum concentrations of 3 beta-hydroxy-5-cholenoic acid were 0.23 +/- SD 0.12 mumol/l and 0.21 +/- SD 0.09 mumol/l in healthy males and females, respectively. In patients with primary biliary cirrhosis the serum concentration of 3 beta-hydroxy-5-cholenoic acid and the quotient 3 beta-hydroxy-5-cholenoic acid over total 3 alpha-hydroxy-bile acids (measured enzymatically) were significantly higher (P less than 0.02) than in patients with chronic active hepatitis or alcoholic cirrhosis. Analysis of 17 sera with elevated concentration of 3 beta-hydroxy-5-cholenoic acid by radioimmunoassay and capillary gas-liquid chromatography showed a close correlation (r = 0.91, slope = 0.97) between the results of the two methods.     

A one-vial method for routine extraction and quantification of free fatty acids in blood and tissue by HPLC

A one-vial procedure has been developed to quantify free fatty acids in human blood serum and rat heart tissue. To allow routine analysis the system has been constructed to allow simultaneous processing of nine samples (Praechromat). The free fatty acids are extracted with Freon 11 and then derivatized to coumarin esters prior to HPLC. The Freon 11 extraction of the free fatty acids is rapid and complete. Neither hydrolytic degradation of natural fatty acid esters nor oxidative damage of unsaturated fatty acids was observed. Fifteen free fatty acids (FFA) were routinely quantified by isocratic elution with high reproducibility (SD less than 4%) and good recovery (0.1 mM FFA: 98-100%, 0.02 mM: 91-105%). The free fatty acids could be determined in the range from 20 pmol to 20 nmol.     

Serum bile acid determination for assessment of hepatic injury in the woodchuck

A direct spectrophotometric assay for determination of the serum bile acid concentration in the woodchuck (Marmota monax) has been validated. The assay relies on the conversion of 3-hydroxy bile acids to 3-oxo bile acids by 3 alpha-hydroxysteroid dehydrogenase with concomitant reduction of NAD+ to NADH. Reduction of NAD+ is coupled via a diaphorase catalyst to the formation of a diformazan dye from nitrotetrazolium blue and the diformazan product is measured spectrophotometrically at 540 nm. Interfering endogenous dehydrogenase activity present in woodchuck sera was inactivated with sodium pyruvate. Mean recovery of seven exogenous bile acids added to woodchuck sera was 102.0 +/- 2.2%. Intra-assay precision was determined with ten replicate samples giving a mean +/- standard error of the mean of 1.94 +/- 0.12 micron/L with a coefficient of variation of 3.9%. The mean serum bile acid concentration determined in 33 clinically healthy animals was 5.52 +/- 0.81 micron/L. The serum bile acid concentration increased following surgical ligation of the bile duct from 3.78 +/- 0.58 micron/L to a maximum value of 148.0 +/- 30.7 micron/L and remained increased for the 42 day study period. In woodchucks treated with carbon tetrachloride, the serum bile acid concentration peaked at 16 hours following treatment at 72.7 +/- 29.3 micron/L, and returned to pretreatment concentration within 6 days. The serum bile acid concentration therefore appears to be a sensitive biochemical test of cholestasis and hepatocellular forms of hepatic injury and of potential value in the clinical assessment of hepatic disease associated with woodchuck hepatitis virus infection.     

Improved method for the measurement of large neutral amino acids in biological matrices

A high-performance liquid chromatographic method for measuring neutral amino acids in rat sera, brain tissues, and perfusates was developed by using o-phthalaldehyde sulfite as a pre-column derivatization reagent. With the present method, it was possible to separate the neutral amino acids within a single run in 25 min, while the acidic amino acids were eluted near or at the solvent front. The recovery was above 88.8% with a relative standard deviation (RSD) below 4.2%. The within- and between-day assay reproducibility for the determination of rat serum amino acids showed RSDs below 1.35 and 7.61%, respectively. In the present study, the neutral amino acids were assayed with high sensitivity, accuracy and good reproducibility in a relatively short time and on a small sample size.     

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.     

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.