Hyocholic acid species as novel biomarkers for metabolic disorders.

Hyocholic acid (HCA) is a major bile acid (BA) species in the BA pool of pigs, a species known for its exceptional resistance to spontaneous development of diabetic phenotypes. HCA and its derivatives are also present in human blood and urine. We investigate whether human HCA profiles can predict the development of metabolic disorders. We find in the first cohort (n = 1107) that both obesity and diabetes are associated with lower serum concentrations of HCA species. A separate cohort study (n = 91) validates this finding and further reveals that individuals with pre-diabetes are associated with lower levels of HCA species in feces. Serum HCA levels increase in the patients after gastric bypass surgery (n = 38) and can predict the remission of diabetes two years after surgery. The results are replicated in two independent, prospective cohorts (n = 132 and n = 207), where serum HCA species are found to be strong predictors for metabolic disorders in 5 and 10 years, respectively. These findings underscore the association of HCA species with diabetes, and demonstrate the feasibility of using HCA profiles to assess the future risk of developing metabolic abnormalities.

Modulatory Nano/Micro Effects of Diabetes Development on Pharmacology of Primary and Secondary Bile Acids Concentrations.

BACKGROUND: Recent studies have suggested that hyperglycaemia influences the bile acid profile and concentrations of secondary bile acids in the gut. INTRODUCTION: This study aimed to measure changes in the bile acid profile in the gut, tissues, and faeces in type 1 Diabetes (T1D) and Type 2 Diabetes (T2D). METHODS: T1D and T2D were established in a mouse model. Twenty-one seven-weeks old balb/c mice were randomly divided into three equal groups, healthy, T1D and T2D. Blood, tissue, urine and faeces samples were collected for bile acid measurements. RESULTS: Compared with healthy mice, T1D and T2D mice showed lower levels of the primary bile acid, chenodeoxycholic acid, in the plasma, intestine, and brain, and higher levels of the secondary bile acid, lithocholic acid, in the plasma and pancreas. Levels of the bile acid ursodeoxycholic acid were undetected in healthy mice but were found to be elevated in T1D and T2D mice. CONCLUSION: Bile acid profiles in other organs were variably influenced by T1D and T2D development, which suggests similarity in effects of T1D and T2D on the bile acid profile, but these effects were not always consistent among all organs, possibly since feedback mechanisms controlling enterohepatic recirculation and bile acid profiles and biotransformation are different in T1D and T2D.

Tandem mass spectrometric determination of atypical 3ß-hydroxy-Δ5-bile acids in patients with 3ß-hydroxy-Δ5-C27-steroid oxidoreductase deficiency: application to diagnosis and monitoring of bile acid therapeutic response.

BACKGROUND: 3ß-Hydroxy-Δ(5)-C27-steroid oxidoreductase (HSD3B7) deficiency, a progressive cholestatic liver disease, is the most common genetic defect in bile acid synthesis. Early diagnosis is important because patients respond to oral primary bile acid therapy, which targets the negative feedback regulation for bile acid synthesis to reduce the production of hepatotoxic 3ß-hydroxy-Δ(5)-bile acids. These atypical bile acids are highly labile and difficult to accurately measure, yet a method for accurate determination of 3ß-hydroxy-Δ(5)-bile acid sulfates is critical for dose titration and monitoring response to therapy. METHODS: We describe a electrospray ionization LC-MS/MS method for the direct measurement of atypical 3ß-hydroxy-Δ(5)-bile acid sulfates in urine from patients with HSD3B7 deficiency that overcomes the deficiencies of previously used GC-MS methods. RESULTS: Separation of sulfated 3ß-hydroxy-Δ(5)-bile acids was achieved by reversed-phase HPLC in a 12-min analytical run. The mean (SE) urinary concentration of the total 3ß-sulfated-Δ(5)-cholenoic acids in patients with HSD3B7 deficiency was 4650 (1711) µmol/L, approximately 1000-fold higher than in noncholestatic and cholestatic patients with intact primary bile acid synthesis. GC-MS was not reliable for measuring 3ß-hydroxy-Δ(5)-bile acid sulfates; however, direct analysis of urine by fast atom bombardment mass spectrometry yielded meaningful semiquantitative assessment of urinary excretion. CONCLUSIONS: The tandem mass spectrometry method described here for the measurement of 3ß-hydroxy-Δ(5)-bile acid sulfates in urine can be applied to the diagnosis and accurate monitoring of responses to primary bile acid therapy in HSD3B7 patients.

Comparison between drug screening by immunoassay and ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry in post-mortem urine.

Immunoassay is currently the most common approach for urine drug screening. However, the continuous emergence of new psychoactive substances (NPS) and their low urinary concentrations have challenged the scope and sensitivity of immunoassays. Consequently, specialized toxicology laboratories rely more and more on mass spectrometry (MS) based techniques. Ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOF-MS) is an especially attractive technique for comprehensive drug screening. The objective was to compare the performances of immunoassay and UHPLC-HR-TOF-MS in terms of scope, flexibility, sensitivity, and reliability of substance identification. A total of 279 post-mortem urine samples were analyzed using a method representative of each technique. The immunoassay method was an Emit II Plus enzyme immunoassay for the following drug groups: amphetamines, benzodiazepines, buprenorphine, cannabis, and opiates. The UHPLC-HR-TOF-MS method was a recently published method covering hundreds of drugs: conventional drugs of abuse, abused prescription drugs, and NPS of various classes. UHPLC-HR-TOF-MS produced a lower number of false positive (FP) results for the drug groups covered by immunoassay. Many of the false negative (FN, n = 40) and FP (n = 22) immunoassay results were obviously due to the higher cut-off concentrations and interfering matrix, respectively. Moreover, the wider scope of UHPLC-HR-TOF-MS allowed detection of NPS and prescription drugs. UHPLC-HR-TOF-MS gave FP results related to a few particular substances. The future option of adjusting all compound-specific reporting parameters individually would allow the method's sensitivity and specificity to be fully exploited.

Determination of 3ß-hydroxy-Δ5-bile acids and related compounds in biological fluids of patients with cholestasis by liquid chromatography-tandem mass spectrometry.

A method for the determination of conjugated and unconjugated 3ß-hydroxy-Δ5-bile acids and related bile acids in human urine and serum has been developed using high-performance liquid chromatography electrospray ionization-tandem mass spectrometry. Calibration curves for the bile acids were linear over the range of 10 2000 pmol/mL, and the detection limit was less than 4 pmol/mL for all bile acids using selected reaction monitoring analysis. The bile acids in urine and serum samples from two patients with severe cholestatic liver disease were measured by this analytical method. Glycine-conjugated 3ß-hydroxy-Δ5-bile acid 3-sulfates were determined to be the major bile acids in the urine and serum from patients with a 3ß-hydroxy-Δ5-C27-steriod dehydrogenase/isomerase deficiency or dysfunction.

Characterization of urinary bile acids in a pediatric BRIC-1 patient: effect of rifampicin treatment.

BACKGROUND: Benign recurrent intrahepatic cholestasis type 1 (BRIC-1), a rare autosomal recessive disorder characterized by recurrent episodes of jaundice and pruritus, is caused by mutations in the ATP8B1 gene. Rifampicin has been reported to be an effective treatment of jaundice and pruritus in patients with BRIC. Proposed mechanisms of effect for rifampicin include enhancement of multidrug-resistance protein 2 expression, activation of the enzymes of uridine diphosphate glucuronosyltransferase 1A1 and cytochrome P450 3A4, and stimulation of 6α-hydroxylation of bile acids. METHODS: To confirm the diagnosis of BRIC-1 and demonstrate the effect of rifampicin treatment on bile acid metabolism, we analyzed the patient's ATP8B1 gene and bile acids in urine. RESULTS: We detected 2 heterozygous mutations in the ATP8B1 gene, and increasing amounts of unusual bile acids such as 1ß-hydroxylated cholic acid, 2ß-hydroxylated cholic acid, 4ß-hydroxylated cholic acid, 6α-hydroxylated cholic acid, and hyocholic acid in urine during rifampicin treatment. CONCLUSIONS: We diagnosed a jaundiced pediatric patient with BRIC-1 caused by 2 novel mutations (1226delA/2210delA) in the ATP8B1 gene. Rifampicin was effective in treating cholestasis. Results of urinary bile acid analyses during rifampicin treatment in this patient, suggested that rifampicin might stimulate 1ß-, 2ß-, and 4ß-hydroxylation of bile acids in addition to 6α-hydroxylation.

Titration of bile acid supplements in 3beta-hydroxy-Delta 5-C27-steroid dehydrogenase/isomerase deficiency.

OBJECTIVES: 3beta-Hydroxy-Delta 5-C27-steroid dehydrogenase/isomerase deficiency is a bile acid synthesis defect responsive to primary bile acids. We reviewed its clinical features and response to treatment with a mixture of ursodeoxycholic (UDCA) and chenodeoxycholic acid (CDCA) to titrate the dose of supplements required for disease control. PATIENTS AND METHODS: We studied our patients by liquid chromatography-tandem mass spectrometry, liver function tests, and histology. After diagnosis all of the patients received a balanced mixture of UDCA/CDCA and the dose was titrated according to urinary levels of 3beta,7 alpha-dihydroxy-5-cholenoic acid (u-3beta-D-OH-5C). RESULTS: Five patients presenting with giant cell hepatitis, biliary cirrhosis, and cryptogenic cirrhosis (1 each), and picked up by neonatal screening (2 patients) were diagnosed at a median age of 2.5 years (range 0.1-5.5). Normal levels of u-3beta-D-OH-5C were achieved after 4 months (range 3-28 months) from the start of the treatment. The minimum dose of UDCA/CDCA required to maintain normal u-3beta-D-OH-5C levels was 5/5 mg x kg(-1) x day(-1). A follow-up biopsy in 2 patients showed no progression of liver disease. CONCLUSIONS: A mixture of UDCA/CDCA can effectively control 3beta-hydroxy-Delta 5-C27-steroid dehydrogenase/isomerase deficiency. Dose titration by liquid chromatography-tandem mass spectrometry warrants the maintenance of negative feedback on the abnormal synthetic pathway and avoids disease progression.

LC/ESI-tandem mass spectrometric determination of bile acid 3-sulfates in human urine 3beta-Sulfooxy-12alpha-hydroxy-5beta-cholanoic acid is an abundant nonamidated sulfate.

We developed a highly sensitive and quantitative method to detect bile acid 3-sulfates in human urine employing liquid chromatography/electrospray ionization-tandem mass spectrometry. This method allows simultaneous analysis of bile acid 3-sulfates, including nonamidated, glycine-, and taurine-conjugated bile acids, cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), and lithocholic acid (LCA), using selected reaction monitoring (SRM) analysis. The method was applied to analyze bile acid 3-sulfates in human urine from healthy volunteers. The results indicated an unknown compound with the nonamidated common bile acid 3-sulfates on the chromatogram obtained by the selected reaction monitoring analysis. By comparison of the retention behavior and MS/MS spectrum of the unknown peak with the authentic specimen, the unknown compound was identified as 3beta,12alpha-dihydroxy-5beta-cholanoic acid 3-sulfate.

Comparative study of macro- and microsurgical extrahepatic cholestasis in the rat.

The long-term (5-week) evolution of two experimental models of extrahepatic cholestasis, i.e., macrosurgical by bile duct ligation (n = 20) and microsurgical by biliary tract resection (n = 13), is studied. All cholestatic animals showed jaundice, choluria, and portosystemic collateral circulation. Macrosurgical cholestasis causes greater hepatosplenomegaly, hilar biliary pseudocysts, and ascites. Microsurgical extrahepatic cholestasis occurs with a lower degree of hepatosplenomegaly as well as with serum increase (P < 0.001) of gamma-GT and alkaline phosphatase. The bile ductular proliferation in the four hepatic lobes is very intense (P < 0.001) in both experimental models. The differences between both experimental models may be considered secondary to the increase of the predisposition to infection in rats with bile duct ligation, that complicates their evolution. The microsurgical cholestasis model could be useful in studying cholestasis secondary to biliary atresia.

Perinatal bile acid metabolism: analysis of urinary unsaturated ketonic bile acids in preterm and full-term infants.

AIM: To compare urinary concentrations of unsaturated ketonic bile acids in preterm and full-term infants. METHODS: Urinary unsaturated ketonic bile acids were determined using gas chromatography-mass spectrometry. RESULTS: Urinary concentrations of total bile acids in early preterm infants (of less than 29wk gestational age) exceeded concentrations in late preterm (between 30 and 37 wk) and full-term infants (between 38 and 41 wk; p < 0.01). The percentage of ketonic bile acids (7alpha, 12alpha-dihydroxy-3-oxo-4-cholenoic acid and 7alpha-hydroxy-3-oxo-4-cholenoic acid) among total urinary bile acids in full-term infants (20.2 +/- 14.1%) was higher than that in early preterm infants (8.94 +/- 8.1%; p < 0.05). The percentage of unsaturated bile acids (3beta-hydroxy-delta5-bile acids) among total bile acids in urine did not differ greatly between groups. CONCLUSION: The percentage of 3-oxo-delta4 bile acids among total bile acids in urine gradually increased from early to late preterm infants, while healthy full-term infants excreted large amounts of 3-oxo-delta4 bile acids in urine at delivery.

Appearance of atypical 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid in spgp knockout mice.

Bile formation and its canalicular secretion are essential functions of the mammalian liver. The sister-of-p-glycoprotein (spgp) gene was shown to encode the canalicular bile salt export protein, and mutations in spgp gene were identified as the cause of progressive familial intrahepatic cholestasis type 2. However, target inactivation of spgp gene in mice results in nonprogressive but persistent cholestasis and causes the secretion of unexpectedly large amounts of unknown tetrahydroxylated bile acid in the bile. The present study confirms the identity of this tetrahydroxylated bile acid as 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid. The data further show that in serum, liver, and urine of the spgp knockout mice, there is a significant increase in the concentration of total bile salts containing a large amount of tetrahydroxy-5 beta-cholan-24-oic acid. The increase in total bile acids was associated with up-regulation of the mRNA of cholesterol 7 alpha-hydroxylase in male mice only. It is suggested that the lower severity of the cholestasis in the spgp knockout mice may be due to the synthesis of 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid, which neutralizes in part the toxic effect of bile acids accumulated in the liver.

Fetal bile acid metabolism: analysis of urinary 3beta-monohydroxy-delta(5) bile acid in preterm infants.

To elucidate the urinary concentration of total bile acids after birth and the profile of the usual and unusual urinary bile acids, especially 3beta-hydroxy-5-cholen-24-oic acid (Delta(5)-3beta-ol), we measured the concentrations of 13 bile acids in the urine from preterm infants vs. full-term controls by gas chromatography-mass spectrometry. The urinary concentration of total bile acids in early preterm infants below 32 weeks of gestational age significantly exceeded that of the late preterm and full-term infants (p < 0.0005). The major urinary bile acids in early preterm infants were cholic acid, 1beta,3alpha,7alpha,12alpha-tetrahydroxy-5beta-cholan-24-oic acid and Delta(5)-3beta-ol. In conclusion, the high urinary concentrations of total bile acids in preterm infants may be due to an overproduction, or more likely to a low hepatic bile acid clearance. An alternative fetal pathway, the acidic pathway, may be a major route of bile acid biosynthesis in preterm infants.

Differences in the efficacy of ursodeoxycholic acid and bile acid metabolism between viral liver diseases and primary biliary cirrhosis.

AIM AND METHODS: The effects of ursodeoxycholic acid (UDCA, 600 mg/day) on liver function test values, and serum and urinary bile acids levels in hepatitis C virus-related chronic hepatitis (CH, n = 39) and liver cirrhosis (LC, n = 25), and in primary biliary cirrhosis (PBC, n = 25) were compared. RESULTS: The percentages of improvement in alanine transaminase (ALT) and gamma-glutamyl transpeptidase (gamma-GTP) in CH were almost the same in LC. The rates of improvement in ALT in PBC were negatively correlated with histological stages in the liver. Total serum bile acid levels in LC rose to the same extent as in CH, but the increases in PBC were significantly smaller at stages 3-4 than stages 1-2. The urinary levels of hydroxylated metabolites of UDCA only slightly increased in LC, but they increased significantly at PBC stages 3-4. CONCLUSIONS: The efficacy of UDCA was preserved in LC, but diminished at PBC stages 3-4. The poor enrichment of UDCA in the bile acid pool and extensive biotransformation of UDCA may cause the limited efficacy of UDCA in the cirrhotic stage of PBC.

Urinary choloyl-PABA excretion in diagnosing small intestinal bacterial overgrowth: evaluation of a new noninvasive method.

The synthetic substrate cholyl-PABA, developed by conjugating cholic acid with paraaminobenzoic acid, is hydrolyzed by the bacterial enzyme cholyl hydrolase to release free PABA. This study aimed to evaluate whether quantitating urinary excretion of PABA after oral administration of cholyl-PABA can detect small intestinal bacterial overgrowth. In the first phase, investigations were performed on 10 healthy volunteers to study the dynamics of urinary excretion of PABA and any adverse reactions after oral administration of 1.2 g of cholyl-PABA. Another 10 healthy volunteers and 25 adult patients with various gastrointestinal disorders participated in the second phase, where the urinary cholyl-PABA test was compared to the [14C]xylose breath test (XBT). The upper limit of normal levels of urinary PABA excretion at the end of 4 h was 1.1% of the administered dose of cholyl-PABA. The urinary PABA excretion after 4 hr [median (range), in percentage] in the XBT-positive group was 1.6 (0.6-35.0), which was significantly higher than those in the XBT-negative group [0.7 (0.4-1.8)] and the healthy controls [0.7 (0.2-1.1)]. The agreement between the XBT and the urinary cholyl-PABA test was 85.7% (P < 0.01). No adverse effect was noted. In conclusion, the urinary cholyl-PABA test offers a simple, safe, noninvasive, and rapid method for diagnosing small intestinal bacterial overgrowth and warrants further clinical evaluation.

A paucity of unusual trihydroxy bile acids in the urine of patients with severe liver diseases.

To clarify the relationship between the occurrence of unusual trihydroxy bile acids, namely hyocholic acid, ursocholic acid (UCA), and omega-muricholic acid (omega-MCA) in urine and liver disease severity, urinary bile acids were analyzed by gas-liquid chromatography in acute and late phases of acute hepatitis and before and after ursodeoxycholic acid (UDCA) loading in healthy adults and liver cirrhosis patients. In 11 patients with acute hepatitis, the occurrence rates and amounts of unusual trihydroxy bile acids were increased in the late (recovery) phase, as compared with those in the early phase. In 10 patients with severe acute hepatitis who had prothrombin times exceeding 16 seconds, these bile acids had completely disappeared from the urine in the early phase but reappeared in the late phase in those who had a good outcome, though never in a patient who died. After UDCA administration for a week, the amounts of unusual bile acids, especially UCA and omega-MCA, which are thought to be synthesized through 12 alpha- and 6 alpha-hydroxylations, respectively, from UDCA, were clearly increased in 10 healthy adults but only slightly changed in 10 patients with liver cirrhosis. In conclusion, hepatic hydroxylations of dihydroxy bile acids as a detoxification reaction were impaired in severe liver diseases, which may play a role in the intensification and perpetuation of hepatocellular injuries.

Perinatal bile acid metabolism: analysis of urinary bile acids in pregnant women and newborns.

The metabolism of bile acids in 30 pregnant women was evaluated by analyzing the urinary composition of bile acids during late gestation (weeks 30-41) and again in these women and their newborn infants during the first week after delivery. The levels of individual bile acids were determined by gas chromatography-mass spectrometry after solvolysis and hydrolysis of bile acid conjugates. The mean total bile acid/creatinine ratio in pregnant women decreased from 1.22 micromol/mmol creatinine at 30-32 weeks of gestation to 0.15 micromol/mmol creatinine at 6-7 days after delivery. The mean percentage of 1beta-hydroxylated bile acids peaked at 27% at 3-4 days after delivery. In newborn infants, the mean total bile acid/creatinine ratio rapidly increased from 3.39 micromol/mmol creatinine at birth to 54.33 micromol/mmol creatinine at 7 days. During this period, large amounts (40-50%) of unsaturated ketonic bile acids, especially 7alpha,12alpha-dihydroxy-3-oxo-5beta-chol-1-en-24-oic acid and 7alpha,12alpha-dihydroxy-3-oxo-4-cholen-24-oic acid, were observed in the infants' urine. These data suggest that, during the perinatal period, the formation of polyhydroxylated and unsaturated ketonic bile acids probably represents a mechanism for the excretion of bile salts, and that the metabolism of bile acids in both the mother and the infant changes significantly after birth.

Ursocholic acid, a hydrophilic bile acid, fails to improve liver function parameters in primary biliary cirrhosis: comparison with ursodeoxycholic acid.

OBJECTIVE: To compare the effect of short term feeding of ursocholic acid, a hydrophilic bile acid, as the unconjugated acid and the taurine conjugate, on clinical and biochemical features and bile acid metabolism with that of ursodeoxycholic acid in four patients with primary biliary cirrhosis. METHODS: Four patients with stage II primary biliary cirrhosis were studied. Two were fed ursocholic acid (900 mg/day), and two were given tauroursocholate (900 mg/day) in three divided doses. After 1 month, all patients were given 900 mg/day of ursodeoxycholic acid. Fasting serum, bile, and 24-hour urine levels were measured before and at the end of ursocholic acid and tauroursocholate feeding and after 1 month of ursodeoxycholic acid feeding. Clinical and biochemical symptoms were measured by routine hospital methods, and bile acids were measured by gas-liquid chromatography. RESULTS: One month of ursocholic acid or tauroursocholate feeding did not improve clinical or biochemical findings in any patient. Approximately 21-25% ursocholic acid was present in the serum and bile, with substantial metabolism to deoxycholic acid. Increased ursocholic acid was excreted in the urine. In comparison, ursodeoxycholic acid improved biochemical parameters and was 45-65% enriched in the serum and bile. CONCLUSION: Ursocholic acid as the free bile acid or as taurine conjugate, although more hydrophilic, is poorly enriched in serum and bile and is ineffective in patients with primary biliary cirrhosis.

Determination of fetal bile acids in biological fluids from neonates by gas chromatography-negative ion chemical ionization mass spectrometry.

A method has been developed for microanalysis of fetal bile acids in biological fluids from neonates by capillary gas chromatography-mass spectrometry using negative-ion chemical ionization of pentafluorobenzyl ester-dimethylethylsilyl ether derivatives of bile acids. Calibration curves for the bile acid derivatives are useful over the range 0.1-100 pg and the detection limit for bile acids was 1 fg (S/N = 5) using isobutane as a reagent gas. Recoveries of the bile acids and their glycine and taurine conjugates from bile acid-free serum and dried blood discs ranged from 92 to 101% and from 93 to 108%, respectively, of the added amounts of their standard samples. The analysis of bile acids on a dried blood disc, meconium and urine from infants, exhibited significant hydroxylation at the 1 beta-, 2 beta-, 4 beta- and 6 alpha-positions of the usual bile acids, cholic and chenodeoxycholic acids, for the urinary or fecal excretion of bile acids in the fetal and neonatal periods. The present method was applied clinically to analyze bile acids on a dried blood disc from neonatal patients with congenital biliary atresia and hyper-bile-acidemia.

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.