Kyutaro Shimizu

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.

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.

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.

Cytosol estradiol receptor content in the adult rat brain after neonatal treatment with estradiol benzoate or testosterone propionate

Abstract: The effects of neonatal treatment with estrogen or androgen on the development of cytosol estradiol (E2) receptors were examined in the rat pituitary, hypothalamus and amygdala. The treatment with estradiol benzoate (EB, 100 μg) significantly reduced the E2 receptor content in all of these regions in both sexes. But the treatment with testosterone propionate (TP, 1 mg) to the female rats significantly decreased the E2 receptor content in the pituitary and hypothalamus but not in the amygdala. The TP treatment to the male rats decreased the E2 receptor content in the hypothalamus and amygdala but not in the pituitary. The decreased cytosol E2 receptors in the hypothalamus and amygdala seem to be implicated in preventing the normal sexual development.