Kaori Takanashi

Comparison of cytochrome P-450 species which catalyze the hydroxylations of the aromatic ring of estradiol and estradiol 17-sulfate

For identification of microsomal cytochrome P-450 (P-450) enzymes which catalyze 2- or 4-hydroxylations of estrogens in the rat liver, estradiol (E2) and estradiol 17-sulfate (E2-17-S) were selected as the substrates and incubated with various kinds of purified P-450 enzymes: PB-1, PB-2, PB-4 and PB-5 obtained from phenobarbital-treated male rats (Sprague-Dawley); MC-1 and MC-5 from 3-methylcholanthrene-treated male rats; and UT-1, UT-2, UT-4 and UT-5 from untreated animals. The reactions were carried out under the P-450-reconstructed system, and the resulting products were determined by HPLC using electrochemical detection. All the enzymes tested were shown to have varying degrees of catalytic activities for 2-hydroxylation of the two substrates; UT-1 and UT-2 had the highest activity. Of the induced P-450 enzymes, PB-2 and MC-1 showed fairly high catalytic activity for 4-hydroxylation of E2. The P-450 enzymes obtained from the untreated male rats, especially UT-4, showed the highest catalytic activity for 4-hydroxylation of the two substrates. From these results and also from kinetic experiments, the P-450 enzymes which catalyze 2- and 4-hydroxylations of estrogen were considered to be different species. A part of E2 was converted to such metabolites as estrone and those having a hydroxyl group at positions 6 beta, 15 alpha or 16 alpha, each production of which was estimated to be catalyzed by single or multiple P-450s.

Determination of estradiol-17-sulfate in human urine by a direct radioimmunoassay: Urinary levels throughout the menstrual cycle

The measurement of urinary estradiol-17-sulfate concentration by direct radioimmunoassay was established. The urinary estradiol-17-sulfate levels measured by the radioimmunoassay correlated well with those determined by high-performance liquid chromatography equipped with electrochemical detector. Estradiol-17-sulfate concentrations in early morning urine of six healthy adult men was 8.2 +/- 2.0 ng/mL, or 5.7 +/- 1.8 ng/mg creatinine. The urinary levels in women throughout the menstrual cycle showed a characteristic three-peak excretion pattern: the first and the second peaks appeared just after and three days before the urinary LH peak, and the third peak appeared a few days before menstruation.

Synthesis of 6- and 7-hydroxyestradiol 17-sulfates: the potential metabolites of estradiol 17-sulfate by female rat liver microsomes

The potential ring-B hydroxylated metabolites of estradiol 17-sulfate (1) by female rat liver microsomes were chemically prepared as authentic compounds. They are 6alpha- and 6beta-hydroxyestradiol 17-sulfates (7 and 9), and 7alpha- and 7beta-hydroxyestradiol 17-sulfates (12 and 16), whose synthetic procedures are described.

The physiological role of estradiol 17-sulfate during pregnancy

To clarify the physiological role of estradiol 17-sulfate (ES) during pregnancy, experiments were conducted and the following results were obtained: (1) serum or urinary ES levels rose as a function of gestational age; (2) placental microsomes showed fairly high 2- and 4-hydroxylase activity for ES; and (3) the catechol products, 2- and 4-hydroxy-ES, had a strong inhibitory effect upon the in vitro production of lipid peroxides. These results suggest that ES acts as a precursor to the catechol metabolites which maintain normal gestation. This is coincident with the negative correlation of serum levels in ES and lipid peroxides observed in late pregnancy.

Metabolism of [6,7-3H, 35S] estradiol 17-sulfate in rats

To confirm whether or not the sulfo group of estradiol 17-sulfate (ES) is removed during in vivo metabolism in rats, the doubly labeled conjugate [6,7-3H, 35S] ES was injected into rats, and its biliary and urinary metabolites were determined by reverse isotope dilution method (RIDM). In male rats, the major radioactivity was detected in biliary disulfate fraction, which was composed of mainly ES and its two minor metabolites, 2-hydroxyestradiol 17-sulfate (2-OH-ES) and 2-methoxyestradiol 17-sulfate (2-MeO-ES). In female rats, in contrast, the radioactivity was dispersed into three fractions:biliary monosulfate, biliary disulfate, and urinary monosulfate fractions (Frs.) In both monosulfate Frs., 7beta-hydroxyestradiol 17-sulfate was detected as the major metabolite followed by 6alpha-, 6beta-, and 15beta-hydroxyestradiol 17-sulfates. Like male rats, 2-OH-ES and 2-Meo-ES as the minor products were detected in biliary disulfate fraction. The isotope ratios of ES and its metabolites in both sexes were essentially the same as that of the dose except that of 6alpha-hydroxylated metabolite, which may be derived from the loss of the tritium labeled at C6. These results confirm the occurrence of the direct metabolism of ES in rats.

Comparison of ex vivo inhibitory effect between 2-hydroxyestradiol and its 17-sulfate on rat hepatic microsomal lipid peroxidation

Two endogenous antioxidants that are speculated to be defense substances against preeclampsia, 2-hydroxyestradiol (2-OH-E2) and its 17-sulfate, 2-hydroxyestradiol 17-sulfate (2-OH-E2-17-S), were administered to rats to compare their inhibitory effects on hepatic microsomal lipid peroxidation, and the lipid peroxides were determined in NADPH- and ascorbic acid-dependent systems. The two catechols showed a strong inhibitory effect on lipid peroxidation in both systems, and the effect was dose dependent. However, a large difference was observed in their inhibition patterns. After administration of 2-OH-E2, the effect appeared immediately and decreased gradually with time. In contrast, the effect of 2-OH-E2-17-S appeared some time after administration and persisted for a longer time. Both catechols also showed a striking difference in their dynamics. After administration, 2-OH-E2 was detected in the blood together with its metabolites, 2-methoxyestradiol and 2-methoxyestrone, and they disappeared immediately. In contrast, 2-OH-E2-17-S was present in the blood for a longer time together with its O-methylated product, 2-methoxyestradiol 17-sulfate, but disappeared from liver microsomes within 2 h after administration. The results imply no occurrence of a direct inhibition effect of 2-OH-E2-17-S.

On the structures of hydroxylated metabolites of estradiol 17-sulfate by rat liver microsomes

The metabolism of estradiol 17-sulfate (ES) by hepatic microsomes of female rats produced four new metabolites in addition to 2- and 4-hydroxyestradiol 17-sulfates (2- and 4-OH-ES), which were detected on an HPLC chromatogram. By comparison with synthetic specimens, three of these compounds were identified as 6alpha-, 6beta-, and 7beta-hydroxyestradiol 17-sulfates. To elucidate the structure of the remaining metabolite, a large-scale incubation of ES was carried out, followed by isolation using preparative HPLC to give the single material, which was assigned as 15beta-hydroxyestradiol 17-sulfate by instrumental analyses. On the other hand, when ES was incubated with the microsomes of male rats, 2-OH-ES was produced accompanied by two minor products: 4-OH-ES and a metabolite of unknown structure. The results show clearly that the metabolism of ES by rat hepatic microsomes is remarkably different between the sexes.

[Studies on the metabolism of conjugated drugs. III. 3-Deoxyestradiol 17-sulfate. (3). The metabolism by rat liver microsomes].

When 17 beta-hydroxyestra-1,3,5(10)-triene 17-sulfate was incubated with rat liver microsomes, the hydroxylations occurred predominantly at the position 2, followed by the 3. The hydroxylation at 4 was negligible. With proceeding the incubation time, two catechol metabolites hydroxylated at the positions 2,3 and 3,4 were produced. These catechols were confirmed as the second products derived from the above phenolic metabolites. Some kinetic parameters obtained by these hydroxylations showed that all the hydroxylases concerned are different enzymes.