Mitsuteru Numazawa

A simple radiometric assay for estradiol 2-hydroxylase activity☆

Abstract [2- 3 H]Estradiol was synthesized from 2-iodoestradiol by reduction with sodium borotritiride in the presence of palladium chloride as a catalyst. When the labeled substrate was incubated with the rat liver and kidney microsomes, the tritium label was liberated quantitatively depending upon the 2-hydroxylase activity. Tritiated water produced in the incubation medium was recovered as a satisfactory rate by passage through a column of Amberlite XAD-2 resin without any interference due to the labeled substrate. The present method for the assay of estradiol 2-hydroxylase activity was found to be simple, reliable, and sensitive (detection limit:29 pmol of 2-hydroxyestradiol).

Picogram order enzyme immunoassay of oestradiol.

Radioinmunoassay (RIA) methods have been widely used for determination of endogenous and exogenous substances in the biological fluids. In recent years enzyme immunoassay (EIA) which possesses several advantageous features has been developed as an alternative to RIA [1]. This method, however, is still unsatisfactory in respect of the sensitivity and versatility. Although several papers dealing with EIA of steroid hormones have been published [2-5], an application of this method to human plasma has not yet been reported. The purpose of this communication is to describe the development of an enzyme immunoassay method for oestradiol at levels ranging from 50 pg using the antibody-acrylamine glass conjugate, horse radish peroxidase (HRP)-labelled oestradiol and fluorogenic substrate.

Properties of estradiol 2-hydroxylase and 2-hydroxy-3-deoxyestradiol 3-hydroxylase in rat liver☆

The properties of enzyme systems involved in the formation of catechol estrogen from estradiol and 2-hydroxy-3-deoxyestradiol in rat liver microsomes have been investigated. Molecular oxygen dissolved in the incubation medium was enough for the occurrence of 2- and 3-hydroxylations. The presence of carbon monoxide suppressed the formation of catechol estrogen from the two substrates where the CO/O2 ratios needed for 50% inhibition of the bioconversion were 12.0 and 14.1, respectively. The inhibitory effect was reversed almost completely by illumination with white light. Pretreatment with phenobarbital and 3-methylcholanthrene did not exert any significant change in the activities of 2- and 3-hydroxylases, but increased the content of cytochrome P-450 or P-448 in liver microsomes. The storage of microsomal preparation in the frozen state resulted in a marked decrease in the hydroxylase activities. These results strongly imply that a similar cytochrome P-450 system would be operative in the formation of catechol estrogen from estradiol and 2-hydroxy-3-deoxyestradiol in rat liver microsomes.

Determination of estrogen ring D glucuronides in pregnancy plasma by direct radioimmunoassay without hydrolysis

A direct radioimmunoassay method using highly specific antisera without prior deconjugation has been developed for determination of estradiol 17-glucuronide and estriol 16-glucuronide in human plasma. Antisera were elicited in the rabbit by immunization with antigens in which the steroid haptens are linked to a carrier protein through the C-2 or C-4 position. After treatment with Rivanol the protein of albumin-free antiserum was covalently bound to a p-arylamine glass bead support through the cross-linkage with glutaraldehyde. A simple and reliable assay method employing the antibody-glass preparation was established and applied to measurement of estrogen ring D glucuronide concentration in peripheral plasma throughout normal pregnancy.

Metabolism of 2-hydroxy-3-deoxyestradiol by rat liver microsomes.

Abstract The biotransformation of [6,7-3H]2-hydroxy-3-deoxyestradiol, which is a positional isomer of the naturally occurring estrogen, has been investigated with rat liver microsomes. 2-Hydroxyestradiol separated from the lipophilic fraction of the incubation mixture was unequivocally characterized by means of thin-layer chromatography, gas chromatography-mass spectrometry, and the reverse isotope dilution method. The formation of glutathione 1- and 4-thioethers of 2-hydroxyestradiol as watersoluble metabolites was also confirmed by chromatographic properties and coloration tests, followed by reverse isotope dilution analysis of the aglycone produced by desulfurization with Raney nickel. Incubation of [3-2H]2-hydroxy-3-deoxyestradiol with rat liver microsomes yielded 2-hydroxyestradiol without any retention of the label, indicating that C-3 hydroxylation proceeded through a quinoid intermediate. When a 1 to 1 mixture of 3-deuterated and nonlabeled 2-hydroxy-3-deoxyestradiols was incubated, no substantial change was seen in the deuterium content with the recovered substrate, which implied the absence of the isotope effect in C-3 hydroxylation.

Properties of enzyme systems involved in the formation of catechol estrogen glutathione conjugates in rat liver microsomes

Abstract The properties of enzyme systems involved in the formation of the glutathione 1- and 4-thioethers of catechol estrogen from estradiol, 2-hydroxy-3-deoxyestradiol, or 2-hydroxyestradiol in rat liver microsomes have been investigated. Molecular oxygen was essential and NADPH was preferable as a cofactor to obtain the maximum activity with the three substrates. The presence of carbon monoxide suppressed the formation of the thioethers where the CO O 2 ratios needed for 50 per cent inhibition of the bioconversion were 2.1 to 3.6. This inhibitory effect was reversed almost completely by illumination with white light. SKF-525A inhibited considerably the formation of the glutathione conjugates. Pretreatment with phenobarbital stimulated the formation of the thioethers from the three substrates by 100–220 per cent, whereas administration of 3-methylcholanthrene did not exert any significant influence. The storage of frozen microsomes resulted in a marked decrease in the enzyme activity: the initial activity was depressed to 50 per cent at 24 hr for catechol estrogen and at 4–5 days for phenol estrogens. The NADH-dependent enzyme activities were also inhibited by both SKF-525A and CO; the CO inhibition was reversed by light irradiation. It is evident from these data that cytochrome P-450 participated in both NADPH- and NADH-dependent formation of 2-hydroxyestradiol glutathione thioethers and two different enzyme systems are involved in this biotransformation between phenol estrogens and catechol estrogen.

Preparation of antiserum specific for oestradiol 17-glucuronide

Considerable attention has been recently focused on the physiological significance of steroid hormone conjugates. Usually the conjugated steroids are determined indirectly after hydrolysis by enzymatic and/or chemical means. Such procedures have inevitable disadvantages of lack of reliability due to incomplete hydrolysis and formation of artifacts, and in loss of information about the conjugate forms. Consequently several investigators have attempted to prepare antisteroid glucuronide sera for use in radioimmunoassays [l-5]. However, antisera so far obtained are not satisfactory in respect of specificity. We have undertaken the development of a radioimmunoassay method specific for oestrogen glucuronides without prior hydrolysis. In this communication we report the synthesis of oestradiol 17-glucuronide-bovine serum albumin (BSA) conjugate in which the hapten is linked to protein through the C-2 position and characterisation of antiserum raised against it.