A convenient synthesis of 3 beta,12 alpha-, 3 beta,7 alpha-, and 3 beta,7 beta-dihydroxy-5-cholen-24-oic acids: unusual bile acids in human biological fluids.

The unusual bile acids 3 beta,12 alpha- (V), 3 beta,7 alpha- (XIIIa), and 3 beta,7 beta- (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.

An efficient synthesis of 4 beta- and 6 alpha-hydroxylated bile acids.

An efficient method for the preparation of 4 beta- and 6 alpha-hydroxylated bile acids has been developed. It involved a highly stereoselective acetoxylation at the 4 beta and 6 alpha positions of 3- and 7-oxo bile acids, respectively, with lead tetraacetate in the presence of boron trifluoride etherate in acetic acid. Reduction of the resulting alpha-acetoxy ketones with sodium borohydride or tert-butylamine borane complex, and alkaline hydrolysis, provided the desired bile acids in good yields.

Determination of 3 beta-hydroxy-5-cholen-24-oic acid and its sulfate in human serum by gas chromatography-mass spectrometry.

The glycine conjugate of 3 beta-hydroxy-5-cholen-24-oic acid and its sulfate labeled with deuterium at the C-2, -4, and -23 positions were synthesized. A highly sensitive and specific quantitative assay of the bile acid has been developed by selected ion monitoring in gas chromatography-mass spectrometry of the methyl ester trimethylsilyl ether derivatives using the deuterium labeled conjugates as internal standards. Calibration curves for the bile acid and its sulfate exhibited a linear relationship over the range of 0.01-100 micrograms/ml in human serum.

Large amounts of 1 beta-hydroxylated bile acids in urine during taurine therapy.

The management of infants with cholestasis remains a difficult challenge. On the hypothesis that taurine is effective in treating neonatal cholestasis, taurine (1 g/day, per os) was administered to 2 patients with neonatal hepatitis and the bile acids were analyzed using gas chromatography-mass spectrometry (GC-MS). The serum levels of bilirubin and bile acids were significantly decreased by taurine. Before the treatment, cholic acid (CA) and chenodeoxycholic acid (CDCA) were predominant (79.2% in both patients) in the urine. There was a significant elevation of 1 beta-hydroxylated bile acids (1 beta BA), especially 1 beta, 3 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid (CA-1 beta-ol), in urine collected during the taurine therapy, and 1 beta BA became predominant (57.7-78.3%). Therefore, increased amounts of urine 1 beta BA were excreted during taurine administration. Taurine therapy is recommended, because it might be effective for treating neonatal cholestasis.

Synthesis of 6-hydroxylated bile acids and identification of 3 alpha,6 alpha,7 alpha,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid in human meconium and neonatal urine.

Three 6-hydroxylated bile acids, 3 alpha,6 alpha,7 alpha,12 alpha-, 3 alpha,6 beta,7 alpha,12 alpha- and 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acids, were synthesized from methyl cholate, and a sensitive method was developed for analyzing them by gas chromatography-mass spectrometry for the stoichiometric study of fetal bile acids. 3 alpha,6 alpha,7 alpha,12 alpha-Tetrahydroxy-5 beta-cholan-24-oic acid (6 alpha-hydroxylated cholic acid) was identified from human meconium and healthy neonatal urine by comparison with the mass spectrum of the reference compound. In human meconium, 6 alpha-hydroxylated cholic and chenodeoxycholic acids were determined in 1.2% and 29.0% of the total bile acids, respectively. We discuss the significance of hydroxylation at the C-1 beta and C-6 alpha positions of bile acids and their elimination in fetal and neonatal periods.

Synthesis of 19-hydroxylated bile acids and identification of 3 alpha,7 alpha,12 alpha,19-tetrahydroxy-5 beta-cholan-24-oic acid in human neonatal urine.

The synthesis of 19-hydroxylated bile acids (3 alpha,19-dihydroxy-, 3 alpha,7 alpha,19-trihydroxy-, 3 alpha,12 alpha,19-trihydroxy- and 3 alpha,7 alpha,12 alpha,19-tetrahydroxy-5 beta-cholan-24-oic acids) was described. These synthesized 19-hydroxylated bile acids were used as standard samples for the analysis of bile acids in human urine by gas chromatography-mass spectrometry. 3 alpha,7 alpha,12 alpha,19-Tetrahydroxy-5 beta-cholan-24-oic acid was identified in neonatal urines (0.1-1.5 micrograms/ml and 1.5-7% of total bile acids).

1 beta-hydroxylated bile acids in the urine of healthy neonates.

In order to clarify the metabolism of bile acids in neonates, 1 beta-hydroxylated bile acids in the urine of healthy newborns were examined by gas chromatography-mass spectrometry. The results showed that the percentage of total 1 beta-hydroxylated bile acids, 3 beta, 12 alpha-dihydroxy-5-cholenoic acid and hyocholic acid in neonates was significantly higher than in older children. The ratio of 1 beta-hydroxylated bile acids to their comparable primary bile acids was also higher in neonates than in older children. These results suggest that 1 beta- and 6 alpha-hydroxylation of bile acids are the predominant pathways of bile acid metabolism in neonates.

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.

Determination of 3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid and related bile acids in human serum by gas chromatography-mass spectrometry.

The glycine, taurine and sulphate conjugates of 3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid were synthesized as authentic samples for the analysis of this unusual bile acid. A highly sensitive and specific quantitative assay of the bile acid and related compounds has been developed by selected-ion monitoring in gas chromatography-mass spectrometry of their methyl ester-trimethylsilyl ether derivatives, using the deuterium labelled internal standards: [2H6]3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid, [2H5]3 beta-hydroxy-5-cholen-24-oic acid, [2H7]cholic acid and their sulphates. Calibration curves for these bile acids were linear over the range 0.01-100 micrograms/ml in human serum. Recoveries of the bile acids and their conjugates ranged from 95 to 103% of the added amounts of their standard samples. The unsaturated bile acid was identified in a significant amount of 25.2 micrograms/ml in serum of an infant with liver disease, and its sulphate comprised 55.1% of the amount of the bile acid.

Determination of urinary 18-hydroxycortisol by isocratic normal-phase high-performance liquid chromatography.

A method for the determination of urinary 18-hydroxycortisol by high-performance liquid chromatography is described. Urinary samples were first mixed with an internal standard, 18-hydroxyprednisolone. 18-Hydroxycortisol and 18-hydroxyprednisolone, extracted by a Bond Elut column, were dehydrated by 1% (w/v) p-toluenesulphonic acid to the 11,18-epoxides. The epoxides were separated into two distinct peaks on a Resolve Silica column with a mobile phase of chloroform-methanol (100:2.5, v/v). The detection wavelength was 248 nm. The urinary 18-hydroxycortisol concentration was calculated from peak-height ratio of 11,18-epoxycortisol to 11,18-epoxyprednisolone. The linearity of the ratio was satisfactory in the range 12.5-300 ng per injection of 11,18-epoxycortisol. A specific increase of urinary 18-hydroxycortisol in patients with primary aldosteronism was demonstrated.

Abnormal low ratio of cholic acid to chenodeoxycholic acid in a cholestatic infant with severe hypoglycemia.

We report a premature infant with severe hypoglycemia (serum glucose: 6 mg/dl) and cholestasis (serum total bile acids: 211.55 mumol/L) caused by hypoplasia of the interlobular bile ducts. This patient had developed intracranial hemorrhage and sepsis while undergoing treatment for hypoglycemia. As a result of endocrine evaluation, we made a diagnosis of idiopathic panhypopituitarism, congenital absence or hypoplasia of the pituitary gland. Moreover, we found abnormal bile acid profiles: The ratio of cholic acid to chenodeoxycholic acid was abnormally low in serum (0.04) and in biliary bile (0.33). However, 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid and bile alcohols were not detected. We therefore suspected that the severe cholestasis and abnormal bile acid profiles in the serum and biliary bile in this patient were related to physiologic immaturity of the enterohepatic circulation of bile acids and immaturity of hepatic 12 alpha-hydroxylation.

Identification of short side chain bile acids in urine of patients with cerebrotendinous xanthomatosis.

Urine from patients with cerebrotendinous xanthomatosis (CTX) was found to contain a number of minor bile acids along with three major bile acids, 7-epicholic acid, norcholic acid, and cholic acid. The following minor bile acids were identified by combined gas-liquid chromatography-mass spectrometry: 7-ketobisnordeoxycholic acid; 12-ketobisnorchenodeoxycholic acid; 7-ketonordeoxycholic acid; 12-ketochenodeoxycholic acid; 7-ketodeoxycholic acid; 12-ketochendeoxycholic acid; bisnorcholic acid; allonorcholic acid; allocholic acid; 1 beta-hydroxybisnorcholic acid; 1 beta-hydroxynorcholic acid; 1 beta-hydroxycholic acid; 2 beta-hydroxybisnorcholic acid; 2 beta-hydroxy-norcholic acid; 2 beta-hydroxycholic acid. The presence of C22 and C23 bile acids in urine of the CTX patients suggests that bile alcohols having a hydroxyl group at C22 or C23 in the side chain may be further degraded to these bile acids.

Similarity of unusual bile acids in human umbilical cord blood and amniotic fluid from newborns and in sera and urine from adult patients with cholestatic liver diseases.

Unusual bile acids in umbilical cord blood and amniotic fluid of term newborns and in sera and urine from adult patients with cholestatic liver diseases were analyzed by use of gas-liquid chromatography-mass spectrometry. These bile acids were compared in order to elucidate possible similarities of bile acid metabolism between fetal and cholestatic liver. In both umbilical cord blood and amniotic fluid, 14 unusual bile acids were found in addition to normal bile acids (cholic, chenodeoxycholic, deoxycholic, and lithocholic acids), and 15, excluding ursodeoxycholic acid, were found in sera and urine from patients with cholestatic liver diseases. Of the unusual bile acids detected, 12 were common to both samples. Six unusual bile acids, 3 beta-hydroxy- and 3 beta,12 alpha-dihydroxy-5-cholenoic acids, 3 alpha,6 alpha,7 alpha-trihydroxy-5 beta-cholanoic acid, 1 beta,3 alpha,12 alpha-trihydroxy-1 beta,3 alpha,7 alpha-trihydroxy-, and 1 beta,3 alpha,7 alpha,12 alpha-tetrahydroxy-5 beta-cholanoic acids were more abundant than others. They could be classified into three groups, i.e., unsaturated, 6-hydroxylated, and 1 beta-hydroxylated bile acids. 1 beta-Hydroxylated bile acids, which were not found in serum specimens, were detected in sera from umbilical cord blood and from patients with cholestatic liver diseases. The presence of these unusual bile acids suggested similarities between the altered metabolic states of the two groups examined.

Altered metabolism of bile acids in cholestasis: determination of 1 beta- and 6 alpha-hydroxylated metabolites.

Trihydroxy and tetrahydroxy bile acid metabolites substituted at the C-1 or C-6 position were studied using the urine, serum and liver tissue from sixteen patients with cholestatic liver diseases. Following extraction, isolation and hydrolysis, bile acids were converted into the dimethylethylsilyl derivatives and assayed by capillary gas chromatography-mass spectrometry. Five 1 beta-hydroxylated bile acids, viz. 1 beta,3 alpha,12 alpha-trihydroxy-, 1 beta,3 alpha,7 alpha-trihydroxy-, 1 beta,3 alpha,7 beta-trihydroxy-, 1 beta,3 alpha,7 alpha,12 alpha-tetrahydroxy-5 beta-cholanoic acids and an epimer of the first compound, and two 6 alpha-hydroxylated bile acids, viz. 3 alpha,6 alpha,7 alpha-trihydroxy-, 3 alpha,6 alpha,7 alpha,12 alpha-tetrahydroxy-5 beta-cholanoic acids, were completely or partially identified. Large amounts of 1 beta-hydroxylated and 6 alpha-hydroxylated bile acids were found in the urine, whereas only trace amounts were detected in the serum and liver tissue. These findings indicate that altered metabolism, such as 1 beta- or 6 alpha-hydroxylation of bile acids, is enhanced in cholestasis, and that the resulting hydroxylated metabolites are eliminated in the urine.

Concurrent occurrence of 3 beta,12 alpha-dihydroxy-5-cholenoic acid associated with 3 beta-hydroxy-5-cholenoic acid and their preferential urinary excretion in liver diseases.

3 beta-Hydroxy-(delta 5-3 beta-ol), 3 beta,12 alpha-dihydroxy-(delta 5-3 beta,12 alpha-ol), 3 beta,7 alpha-dihydroxy-(delta 5-3 beta,7 alpha-ol) and 3 beta,7 beta-dihydroxy-(delta 5-3 beta,7 beta-ol) 5-cholenoic acids were identified in patients with liver diseases by gas-liquid chromatography-mass spectrometry (GLC-MS). Of these unusual 3 beta-hydroxy-5-en-metabolites, delta 5-3 beta-ol and delta 5-3 beta,12 alpha-ol were found as major components in the urine of patients with liver diseases (cholestasis, liver cirrhosis, chronic hepatitis, acute hepatitis). Other 3 beta-dihydroxy-5-en-metabolites, delta 5-3 beta,7 alpha-ol and delta 5-3 beta,7 beta-ol, were found as minor components in the urine. The levels of delta 5-3 beta-ol and delta 5-3 beta,12 alpha-ol in urine were correlated with their levels in serum, with total bile acids in the urine, and with liver function, implying that the degree of their increment correlated well with the severity of liver diseases. The most abundant amounts of delta 5-3 beta-ol and delta 5-3 beta,12 alpha-ol were found in the urine as sulfate conjugates in comparison with bile, portal and hepatic venous sera, and liver tissue of the patients. The biliary excretion and hepatic extraction of these 3 beta-hydroxy-5-en-unsaturated bile acids were more impaired and inefficient than those of cholic and chenodeoxycholic acids.

Neonatal Dubin-Johnson syndrome with severe cholestasis: effective phenobarbital therapy.

We described Dubin-Johnson syndrome (DJS) with severe cholestasis in a 20-day-old Japanese boy. Although neonatal DJS has been sporadically reported. DJS with severe cholestasis has not to our knowledge been described in the English literature. The ratio of urinary coproporphyrin isomer I to urinary total coproporphyrin in our patient was high (93%). Liver histology showed cytoplasmic pigment granules in the liver cells. Administration of phenobarbital (PB) significantly decreased the levels of bilirubin and bile acids in the serum. There was a significant elevation of 1 beta-hydroxylated bile acids in the urine. It is predicted that severe cholestasis in neonatal DJS may cause metabolic abnormalities in both bilirubin and bile acids transport.

Fetal bile acid metabolism during infancy: analysis of 1 beta-hydroxylated bile acids in urine, meconium and feces.

Fetal bile acids (1 beta-hydroxylated, 6 alpha-hydroxylated and unsaturated bile acids), especially 1 beta, 3 alpha,7 alpha, 12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid (CA-1 beta-ol), have been detected in urine and feces early in life. To investigate whether a fetal pathway of bile acid synthesis exists in infancy, we measured the concentrations of bile acids in the urine, meconium and feces from normal newborns and infants by means of gas chromatography-mass spectrometry. The mean ratio of total bile acids to creatinine in urine increased between birth and 7 days and then gradually decreased; however, the concentration of total bile acids in urine remained significantly higher than that in adult urine until 3 mo of age. The main urinary bile acid was CA-1 beta-ol, and substantial amounts of fetal bile acids were detected in urine until 3 mo of age. The ratio of cholic acid to chenodeoxycholic acid was abnormally low in meconium (mean, 0.44; range, 0.19 to 0.74), and hyocholic acid constituted 19.3% of total bile acids. The mean total bile acid content of feces decreased between birth and 7 days of age and thereafter increased. The mean percentage of fetal bile acids in feces decreased after birth, but substantial amounts were present in feces until 1 mo of age.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Unusual bile acids, such as unsaturated ketonic and 7beta-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, 1beta,3alpha,7alpha,12alpha-tetrahydroxy-5bet a-cholan-24-oic, 7alpha, 12alpha-dihydroxy-3-oxo-5beta-chol-1-en-24-oic, and 7alpha,12alpha-dihydroxy-3-oxo-4-cholen-24-oic acids were predominant among the unusual urinary bile acids present. Moreover, the levels of 3alpha,7beta,12alpha-trihydroxy-5beta-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 7beta-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 7alpha-hydroxyl group of cholic acid, which may be unique to human development.