Takao Kurosawa

Profile of urinary bile acids in infants and children : developmental pattern of excretion of unsaturated ketonic bile acids and 7β-hydroxylated bile acids

Unusual bile acids, such as unsaturated ketonic and 7β-hydroxylated bile acids, have been detected in urine early in life. To elucidate the normal profiles of usual and unusual urinary bile acids in the neonatal and pediatric periods, we measured the concentrations of 28 kinds in urine from normal newborns, infants, and children by gas chromatography-mass spectrometry. The mean total bile acid/Cr ratio in 7-d-old infants was significantly higher than in subjects of other age groups (birth, 2-4 mo, 5-7 mo, 11-12 mo, 2-3 y, 9-14 y, and adult) (p < 0.05). Relatively large amounts of unusual bile acids were detected during infancy, especially during the period up to 1 mo of age. At that time, 1β,3α,7α,12α-tetrahydroxy-5β-cholan-24-oic, 7α,12α-dihydroxy-3-oxo-5β-chol-1-en-24-oic, and 7α,12α-dihydroxy-3-oxo-4-cholen-24-oic acids were predominant among the unusual urinary bile acids present. Moreover, the levels of 3α,7β,12α-trihydroxy-5β-cholan-24-oic acid increased significantly after 2-4 mo of age. These results indicate that bile acid synthesis and metabolism in the liver of developing infants are significantly different from that occurring in the liver of adults. Significant amounts of urinary isomerized 7β-hydroxylated bile acids were detected after late infancy, probably because of changes in the intestinal bacterial flora response to a change in nutrition. We describe, for the first time, evidence of the epimerization of the 7α-hydroxyl group of cholic acid, which may be unique to human development.

A convenient synthesis of 3β,12α-, 3β,7α-, and 3β,7β-dihydroxy-5-cholen-24-oic acids: Unusual bile acids in human biological fluids

Abstract The unusual bile acids 3β,12α- (V), 3β,7a- (XIIIa), and 3β,7β- (XIIIb) dihydroxy-5-cholen-24-oic acids were synthesized conveniently from the 3-oxo derivatives of deoxycholic (I) and lithocholic (VI) acids, respectively, to provide authentic samples for the gas chromatography-mass spectrometric determination of these bile acids in the abnormal metabolism of bile acids.

Separation of stereoisomers of C27-bile acid CoA esters by liquid chromatography and its application to the study of the stereospecificities of D- and L-bifunctional proteins in bile acid biosynthesis

Abstract The separation and quantitation analysis of the stereoisomers of 3α,7α,12α,24-tetrahydroxy- and 3α,7α,24-trihydroxy-5β-cholestan-26-oyl CoAs (VA-CoA and DVA-CoA), known as one of the intermediates for bile acid biosynthesis, were achieved by liquid chromatography. The above stereoisomers of C 27 -bile acids were effectively separated by a linear gradient solvent system of methanol/acetonitrile/sodium acetate buffer (pH 5) and acetonitrile/sodium acetate buffer (pH 5) using a reversed phase C18 column. Application for the study of the stereochemistry of the products by enzymatic reaction related to the β-oxidation of bile acids in rat liver was also demonstrated. The products (VA-CoA and DVA-CoA) from the corresponding 3α,7α,12α-trihydroxy- and 3α,7α-dihydroxy-5β-cholest-24-en-26-oyl CoAs (24 E -THCA-CoA and 24 E -DHCA-CoA) from a catalytic reaction with rat liver peroxisomal D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase or enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase were quantitatively determined. The results indicated different stereospecificities of these enzymes which might prove useful for the evaluation of their role and function.

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.

Urinary 7α-hydroxy-3-oxochol-4-en-24-oic and 3-oxochola-4,6-dien-24-oic acids in infants with cholestasis

BACKGROUND/AIMS Urinary 3-oxo-delta4 bile acids have been detected in infants who ultimately died of liver disease. We used qualitative and quantitative methods to compare urinary 3-oxo-delta4 bile acids in liver disease, determining their composition and evaluating the prognostic implication in patients of various ages with various liver diseases. METHODS Gas chromatography-mass spectrometry was used to measure 3-oxo-delta4 bile acids in the urine of patients and healthy controls. RESULTS Patients with a deficiency of 3-oxo-delta4-steroid 5beta-reductase and acute hepatic failure exhibited a significantly higher percentage of 3-oxo-delta4 bile acids in total bile acids in urine than the healthy controls or other patient groups, including those with neonatal cholestasis or biliary atresia (p<0.0001). The urinary 3-oxo-delta4 bile acids in patients with 3-oxo-delta4-steroid 5beta-reductase deficiency who had a poor prognosis were mainly 7alpha-hydroxy-3-oxochol-4-en-24-oic acid and 3-oxochola-4,6-dien-24-oic acid. CONCLUSIONS Our results indicate that an increase in the 7alpha-hydroxy-3-oxochol-4-en-24-oic acid and 3-oxochola-4,6-dien-24-oic acid in the urine of patients with hepatobiliary disease indicates a poor prognosis.

Two neonatal cholestasis patients with mutations in the SRD5B1 (AKR1D1) gene: Diagnosis and bile acid profiles during chenodeoxycholic acid treatment

Background and aimsIn two Japanese infants with neonatal cholestasis, 3-oxo-Δ4-steroid 5β-reductase deficiency was diagnosed based on mutations of the SRD5B1 gene. Unusual bile acids such as elevated 3-oxo-Δ4 bile acids were detected in their serum and urine by gas chromatography–mass spectrometry. We studied effects of oral chenodeoxycholic acid treatment.Patients and methodsSRD5B1 gene analysis used peripheral lymphocyte genomic DNA. Diagnosis and treatment of these two patients were investigated retrospectively and prospectively investigated.ResultsWith respect to SRD5B1, one patient was heterozygous (R266Q, a novel mutation) while the other was a compound heterozygote (G223E/R261C). Chenodeoxycholic acid treatment was effective in improving liver function and decreasing unusual bile acids such as 7α-hydroxy- and 7α,12α-dihydroxy-3-oxo-4-cholen-24-oic acids in serum and urine.ConclusionPrimary bile acid treatment using chenodeoxycholic acid was effective for these patients treated in early infancy before the late stage of chronic cholestatic liver dysfunction.

Analysis of stereoisomeric C27-bile acids by high performance liquid chromatography with fluorescence detection.

A method for differentially measuring the 24-hydroxylated stereoisomeric intermediates (3 alpha,7 alpha,12 alpha,24-tetrahydroxy- and 3 alpha,7 alpha,24-trihydroxy-5 beta-cholestan-26-oic acids) and related C27-bile acids in beta-oxidation of bile acid biosynthesis has been developed by high performance liquid chromatography with fluorescence detection. The method involved the derivatization of the above intermediable C27-bile acids into fluorescent esters with 3-(4-bromomethylphenyl)-7-diethylaminocoumarin, a newly synthesized labeling reagent for carboxylic acids. The fluorescent derivatives were subjected to a short silica gel column to eliminate interfering products prior to analysis by high performance liquid chromatography. The separation of the 16 kinds of bile acids containing stereoisomers was carried out using a reversed-phase Inertsil C8-column by gradient elution and detected with a fluorometer (Ex. 400 nm, Em. 475 nm). The linearity of calibration curve for each bile acid was from 0.5 to 250 pmol (r = 0.999) and the detection limits were about 15 fmol at a signal-to-noise ratio of 3. The method was applied to the determination of intermediates in beta-oxidation of bile acid biosynthesis using rat liver homogenate. The results showed that two stereoisomers of 24-hydroxylated C27-bile acids were predominantly produced, indicating the formation of the isomers by the cis-hydration with water.

Synthesis of 3α,7α,12α-trihydroxy- and 3α,7α-dihydroxy-5β-cholestan-26-oic acids by the use of β-ketosulfoxide

The biosynthetic intermediates of bile acid, 3 alpha, 7 alpha, 12 alpha-trihydroxy- and 3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acids, were synthesized by means of the thermal elimination of beta-ketosulfoxides. The alpha, beta-unsaturated ketones as key compounds of the synthesis, 3 alpha, 7 alpha, 12 alpha-trihydroxy- and 3 alpha, 7 alpha-dihydroxy-5 beta-cholest-25-en-24-ones, were effectively derived from the beta-ketosulfoxides prepared from methyl cholate or chenodeoxycholate by reaction with methylsulfinylcarbanion. These unsaturated ketones were converted into 3 alpha, 7 alpha, 12 alpha, 26-tetrahydroxy- and 3 alpha, 7 alpha, 26-trihydroxy-5 beta-cholestanes by reductive deoxygenation and hydroboration, of which stereoisomers were chromatographically separated into 25S- and 25R- isomers. The oxidation of each of the above isomeric alcohols after the protection of the hydroxyl groups on the steroidal ring and the following hydrolysis gave the title 26-carboxylic acids.

Effect of the hydroxyl group on the oxidative cleavage (β-oxidation) of steroidal side chain for bile acid biosynthesis in rat liver homogenate

Mono-, di-, tri-, and tetrahydroxy-5 beta-cholestan-26-oic acids were incubated with rat liver homogenate (800 x g supernatant and light mitochondrial fraction) to study substrate specificity in the side-chain cleavage reaction (beta-oxidation) of bile acid biosynthesis. The C27-intermediates (5 beta-cholest-24-en-26-oic acids and 24-hydroxy-5 beta-cholestan-26-oic acids) in beta-oxidation and the corresponding C24-bile acids were quantitatively determined by capillary gas chromatography. Monohydroxy-5 beta-cholestan-26-oic acid was not converted into C24-bile acid. Di- and trihydroxy-5 beta-cholestan-26-oic acids were effectively transformed into the C27-intermediates and C24-bile acids. Tetrahydroxy-5 beta-cholestan-26-oic acids were also converted into C27-intermediates and corresponding C24-bile acids. The intermediate 24-hydroxy-5 beta-cholestan-26-oic acids could not be detected in the products by incubation with the light mitochondrial fraction. The total specific activity of protein in the light mitochondrial fraction for the production of C27-intermediates and C24-bile acids was higher than that of 800 x g supernatant solution. The effects of the number and the position of hydroxyl groups on the side-chain degradation are discussed.