Nobuo Yamaga

Transformation of bile acids and sterols by clostridia (fusiform bacteria) in Wistar rats

The effects on bile acid and sterol transformation of clostridia (fusiform bacteria), the dominant intestinal bacteria in rodents (ca. 1010 counts per g wet feces) were examined in Wistar rats. After inoculation of clostridia into germ-free rats and into rats previously inoculated solely with Escherichia coli, most of the endogenous bile acids were deconjugated, and cholic acid and chenodeoxycholic acid were 7α-dehydroxylated to deoxycholic acid and lithocholic acid, respectively. Tauro-β-muricholic acid, another major bile acid in rats, was deconjugated, but only part of it (ca. 30%) was transformed into hyodeoxycholic acid. Cholesterol and sitosterol were also reduced to coprostanol and sitostanol, respectively. Escherichia coli transformed neither bile acids nor sterols. These data suggest that clostridia play an imporant role in the formation of secondary bile acids and coprostanol in rats.

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

Abstract 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.

Internal standards for quantitative gas chromatography of individual bile acids after group separation of bile acids in urine.

Sodium glyco-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oate, sodium tauro-7 beta,12 beta-dihydroxy-5 beta-cholan-24-oate and disodium glyco-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oate 7 alpha-sulphate have been synthesized for the first time. These compounds, together with 7 beta,12 beta-dihydroxy-5 beta-cholan-24-oic acid, which were added to a sample prior to extraction, were found to be useful as internal standards for determination by gas chromatography of individual bile acids in each fraction after group separation of urinary bile acids.

Metabolism of 7β-Hydroxycholesterol-4-14C in rat

7β-Hydroxycholesterol-14C was administered to bile-fistula rats, whose bile was collected and analyzed. As the major metabolites of this hydroxy sterol, the following unsaturated C24-bile acids were identified: the 4 possible stereoisomers of 3,7-dihydroxychol-5-enoic acid as well as those two of 3-hydroxy-7-oxochol-5-enoic acid. In addition, cheno- and ursodeoxycholic acids were indentified as minor metabolites. The meaning of these findings was discussed.

Synthesis of deuterium-labeled 17-hydroxyprogesterone suitable as an internal standard for isotope dilution mass spectrometry

A synthesis is reported of 17-hydroxyprogesterone, labeled with four atoms of deuterium at ring C and suitable for use as an internal standard for isotope dilution mass spectrometry. Base-catalyzed equilibration of methyl 3 alpha-acetoxy-12-oxo-cholanate (III) with 2H2O, followed by reduction of the 12-oxo group by the modified Wolff-Kisher method using [2H]diethylene glycol and [2H]hydrazine hydrate afforded [11,11,12,12,23,23(-2)H]lithocholic acid (V). The Meystre-Miescher degradation of the side chain of V yielded 3 alpha-hydroxy-5 beta-[11,11,12,12(-2)H]pregnan-20-one (X). Oxidation of the 3,20-enol-diacetate of X with perbenzoic acid followed by saponification afforded 3 alpha,17-dihydroxy-5 beta-[11,11,12,12(-2)H]pregnan-20-one (XI). Oxidation of XI with N-bromoacetamide yielded 17-hydroxy-5 beta-[11,11,12,12(-2)H]pregnane-3,20-dione (XII). Bromination of XII followed by dehydrobromination yielded 17-hydroxy-[11,11,12,12(-2)H] progesterone (XIV), consisting of 0.3% 2H0-, 1.1% 2H1-, 8.6% 2H2-, 37.1% 2H3-, 52.1% 2H4-, and 0.8% 2H5-species.

Determination of 17-hydroxyprogesterone in plasma by gas chromatography—mass spectrometry with high-resolution selected-ion monitoring

A method for determining 17-hydroxyprogesterone in plasma by isotope dilution-mass spectrometry is described. For the internal standard 17-hydroxy [2H4]progesterone is used. Extraction of plasma is followed by conversion into the 3,20-dienol,17-tristrimethylsilyl ether derivative and analysis by capillary gas chromatography-mass spectrometry with selected-ion monitoring, at a resolution of 6000. The lower limit of quantitation was 1 pg, judged from a criterion of a signal-to-noise ratio of 10. The precision and accuracy of the method were satisfactory.

Bile acids of patients with renal failure receiving chronic hemodialysis.

Bile acids in serum, urine and dialysate of 8 patients with renal failure in chronic hemodialysis were analyzed by gas chromatography and gas chromatography-mass spectrometry. The following results were obtained: 1. Lithocholic acid, 3 beta-hydroxy-5-cholen-24-oic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, and cholic acid were identified in hemodialysate as well as in serum and urine. 2. The serum bile acid concentration of the patients was 2.78 +/- 0.57 micrograms/mL before hemodialysis and 1.34 +/- 0.48 micrograms/mL after a 5-h period hemodialysis with cuprophane membrane. The proportions of secondary bile acids in predialysis and postdialysis serum of patients were significantly higher than those of healthy subjects. 3. Two out of 8 patients excreted urine. But the amounts of bile acids in urine of the patients were very small compared to those of healthy subjects. 4. The amount of bile acids removed from blood by hemodialysis was 0.70 +/- 0.25 mg. In dialysate, cholic acid constituted a larger proportion of the total bile acids, and lithocholic acid a smaller proportion, when compared to those in urine of patients and healthy subjects.

Measurement of serum 17α-hydroxyprogesterone in newborn infants by stable isotope dilution - Gas chromatography/mass spectrometry

Immunochemical measurement of serum 17α-hydroxyprogesterone (17OHP), the most important parameter for diagnosis of classical 21-hydroxylase deficiency (21OHD) in newborn infants, is known to be inaccurate due to the cross-reactivity of antibodies with a large quantity of fetal adrenal steroids. The aims of this study were 1) to establish reference values for the serum 17OHP level in Japanese newborn infants using non-immunochemical stable isotope dilution —gas chromatography/mass spectrometry (SID-GC/MS) and 2) to compare the serum 17OHP levels determined by SID-GC/MS with those determined by radioimmunoassay (RIA). The first study subjects were used for determination of reference values and included 57 healthy full-term newborn infants (4–5 d of age). The second study subjects were used for comparison of SID-GC/MS with RIA and included 27 healthy full-term newborn infants (3–6 d of age) and two subjects with neonatal transient hyper 17OHPnemia; these two subjects were 16 and 27 d of age, respectively. In the first study subjects, the intra-assay coefficient of variation for SID-GC/MS was 3% (n=5), the recovery rate was 98%, the sensitivity was 0.2 ng/ml, and the range of linearity was 0.5–200 ng/ml. The reference values for the serum 17OHP level determined by SID-GC/MS ranged from 0.3–1.5 (0.6) (ng/ml) (median). In the second study subjects, the serum 17OHP levels determined by SID-GC/MS were lower in one of the 27 subjects and both of the two subjects with neonatal transient hyper 17OHPnemia compared with the levels determined by RIA. Measurement of the serum 17OHP level using SID-GC/MS may be clinically useful for definitive diagnosis of classical 21OHD in newborn infants.

Conversion of 5α-pregnane-3,20-dione, 3α-hydroxy-5α-pregnan-20-one and 3β-hydroxy-5α-pregnan-20-one toΔ16-C19 steroids by the reconstitutedΔ16-C19-steroid synthetase system

Abstract The substrate specificity of the reconstitutedΔ 16 -C 19 -steroid synthetase system, which catalyzes the formation of 5,16-androstadien-3β-ol or 4,16-androstadien-3-one from pregnenolone or progesterone, respectively, was studied. The reconstituted system consisted of a partially purified cytochrome P -450, NADPH-cytochrome P -450 reductase, cytochrome b 5 and NADH-cytochrome b 5 reductase all from pig testicular microsomes. It was found that 5α-reduced C 21 steroids such as 5α-pregnane-3,20-dione, 3α-hydroxy-5α-pregnan-20-one and 3β-hydroxy-5α-pregnan-20-one can be substrates for the enzyme system, resulting in the formation of 5α-androst-16-en-3-one, 5α-androst-16-en-3α-ol and α5-androst-16-en-3β-ol, respectively. The results suggest that 5α-reducedΔ 16 -C 19 steroids might be synthesized from pregnenolone and progesterone via 5α-reduced C 21 steroids as intermediates. The pathways would bypass 5,16-androstadien-3β-ol and 4,16-androstadien-3-one which have been assumed as obligatory intermediates in the formation of 5α-reducedΔ 16 -C 19 steroids from pregnenolone and progesterone.

Bile acids of patients with renal failure (possibility of bile acid secretion in the distal tubule)

Bile acids of patients with renal failure and of healthy subjects were analyzed by capillary gas chromatography after group separation. The amount of bile acids in the total dialysate (150 L) of the patients was smaller than that in the 24-h urine of healthy subjects. Polar bile acid sulfates constituted 17.3% and 30.9% of the total bile acids in serum and urine of healthy subjects, respectively, 26.0% in predialysis serum of patients, and only 11.3% in dialysate of patients. The amount of bile acid sulfates in the hemodialysate converted during a 24-h period dialysis, was still smaller than that in 24-h urine of healthy subjects. We propose that the distal tubule secretes bile acid sulfates.