Tadashi Ohkubo

Serum and urinary estrone sulfate during the menstrual cycle, measured by a direct radioimmunoassay, and fate of exogenously injected estrone sulfate

Serum and early-morning urinary levels of estrone sulfate during the menstrual cycle were measured by a direct radioimmunoassay without hydrolysis. These levels were high and showed prominent peaks [serum, 2.67 +/- 0.37 ng/ml (mean +/- SE); urine, 5.82 +/- 2.3 micrograms/l] around the day of the preovulatory estradiol-17 beta peak, and increased again during the luteal phase. Following intravenous injection of estrone sulfate, serum estrone sulfate, estrone and estradiol-17 beta were measured. The conversion of estrone sulfate to estrone and/or estradiol-17 beta was very small during their transit in the general circulation.

Determination of mitomycin C in serum by high-performance liquid chromatography with dual-electrode coulometric detection

In recent years, mitomycin C (Fig. 1) has been widely used for cancer treatment, but its clinical applications are limited owing to inherent severe toxicity. The therapeutic efficacy of mitomycin C is closely related to the drug concentration in blood and tissues, depending on the dose, route and frequency of administration. Quantitation of mitomycin C in biological fluids was first attained by the bioassay technique [ 11. Various other methods have been devised, such as enzyme immunoassay [2] and high-performance liquid chromatography (HPLC ) with UV detection [ 3-51, differential pulse polarographic [ 61 and reduction-mode electrochemical detections [ 71. However, development of a more feasible and sensitive assay method is needed for therapeutic

Prepartion and antigenic properties of estriol 16-glucuronide- and estradiol 17-glucuronide-[C-6]-bovine serum albumin conjugates

The preparation and antigenic properties of estriol 16-glucuronide-bovine serum albumin (BSA) and estradiol 17-glucuronide-BSA conjugates in which the hapten molecule is linked to the carrier protein through an (O-carboxymethyl)oxime bridge at the C-6 position on the steroid nucleus, have been described. Antibodies raised against the two immunogens in the rabbit possessed excellent specificity to estriol 16-glucuronide and estradiol 17-glucuronide, respectively, exhibiting no significant cross-reactions with estrogen glucuronides having a substituent in ring A and no cross-reactivities with free estrogens, their sulfates and related steroids.

Synthesis of 2-hydroxyestriol monoglucuronides and monosulfates.

The ring A monoglucuronides and monosulfates of 2-hydroxyestriol were synthesized from 2-hydroxyestriol 16,17-diacetate by means of the Koenigs-Knorr reaction with methyl alpha-acetobromoglucuronate and sulfation with sulfur trioxide-pyridine complex, respectively. The conjugated positions of these compounds were definitely established by conversion to 2-hydroxyestriol monomethyl ethers by methylation, then enzymatic hydrolysis. The ring D monoglucuronides and monosulfates of 2-hydroxyestriol were also prepared from 2-hydroxyestriol 2,3-dibenzyl ether by glucuronidation and sulfation in a similar fashion followed by debenzylation, respectively. The positions of conjugation were established on the basis of their 1H-nuclear magnetic resonance spectral data.

Determination of estrone sulfate in plasma by radioimmunoassay without deconjugation

A radioimmunoassay method using highly specific antiserum without prior deconjugation has been developed for determination of estrone sulfate in human plasma. Antiserum was elicited in the rabbit by immunization with antigen in which the sulfated steroid hapten is linked to a carrier protein through the C-6 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 method for measurement of estrone sulfate which involves elimination of endogenous interferences by use of Sep-pak C18 followed by radioimmunoassay employing the antibody-glass preparation, was established and applied to peripheral plasma throughout normal pregnancy.

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.