Itsuo Yoshizawa

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.

Clinical analysis on steroids : XXV. Assay of estradiol 17-sulfate 2-hydroxylase activity by high-performance liquid chromatography

A simple method for the assay of the 2-hydroxylation enzyme of estradiol 17-sulfate in rat liver microsomes has been established by using reversed-phase high-performance liquid chromatography with electrochemical detection. The technique devised involves elution with 50 mM acetate buffer, pH 5.0--methanol (3:2, v/v) using an ODS SIL column and monitoring the potentials at 1.1 V vs. the silver/silver chloride reference electrode. The calibration curve of the relationship between the amounts of 2-hydroxyestradiol 17-sulfate (5-50 ng) injected and peak heights (cm) of the chromatogram was linear. By this method, some kinetic parameters of estradiol 17-sulfate 2-hydroxylase were measured.

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.

Evidence of 2-hydroxylation of estradiol-17β 17-glucuronide by male rat liver microsomes

When estradiol-17 beta 17-glucuronide was incubated with male rat liver microsomal preparations with a NADPH-generating system, 2-hydroxyestradiol-17 beta 17-glucuronide was obtained. This 2-hydroxylation was shown to occur without cleavage of the conjugate group. The result clearly indicates that estradiol-17 beta 17-glucuronide could act as substrate for rat liver microsomal 2-hydroxylase.

[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.

Studies on the Metabolism of Conjugated Drugs. II. 3-Deoxyestradiol 17-Sulfate. (2). Analytical Procedure for Determination by High-Performance Liquid Chromatography of Potential Metabolites

To investigate the metabolic pattern of such a drug having two counter functional groups in the molecule: a hydrophylic sulfate group and a hydrophobic aromatic ring, as 17-sulfate of 17 beta-hydroxyestra-1,3,5(10)-triene (3-deoxyestradiol) was incubated with rat hepatic microsomes under a reduced nicotinamide adenine dinucleotide phosphate-generating system. The metabolites assigned were mono- and dihydroxylated products hydroxylated only at the aromatic ring. The phenolic products having hydroxyl group at the position 2, 3, or 4 were detected in the male and female rats. The two catechol products having hydroxyl groups at 2, 3 and 3, 4 were detected only in the male rats. No other metabolites were formed except a phenol product hydroxylated at the position 1 in the case of the male rats. These metabolisms were confirmed to proceed without removal of the sulfate group. A simple method for the determination of these metabolites was established by using reversed-phase high-performance liquid chromatography. The devised technique involves an elution with a mixture of 0.5% NH4H2PO4 (pH 3.0) and methanol on a TSK-Gel ODS-120A column with electrochemical and/or ultraviolet detectors.