The Determination of Oxytocin Levels in Posterior Pituitary and Plasma of Rats by a Radioimmunoassay

Abstract

The method of a radioimmunoassay for oxytocin was developed. Antibody was produced in rabbits by the repeated injections of oxytocin-bovine serum albumin conjugate emulsified with Freund complete adjuvant, and ¹³¹I-oxytocin was prepared by the modified chloramine T method and purified with two successive gel filtrations through the column of Sephadex G-15. The radioimmunoassay was carried out by incubation of a mixture of antibody, ¹³¹I-oxytocin and oxytocin sample for 2 days at 4°C, using saline-0.01M phosphate buffer (pH 7.4) containing 0.25% egg albumin as a diluent buffer, followed by separation of antibody-bound hormone (B) from free (F) by the dextran coated charcoal method (or ammonium sulfate precipitation method). The radioactivity of F (or B) and total reaction mixture was estimated, and the contents of oxytocin were calculated from the percentage of B. The sensitivity was 10μμg (4μU). Crude oxytocin extracted from the posterior pituitary of cow according to Kamm's method equally cross-reacted to synthetic oxytocin used as a standard in this assay, without any interferences from any substances except oxytocin that was included in this extract. But the cross reactivity of synthetic lys-vasopressin to oxytocin was only 0.5%, and bovine serum albumin employed in the process of antibody production did not show any influences on the assay of oxytocin.
This radioimmunoassay method was applied for the determination of oxytocin contents in biological samples of rats. Pituitary samples were prepared by extracting with 0.25% acetic acid and by washing with ether. Plasma samples were prepared by extracting with cold acetone and 0.25% acetic acid, and by washing with ether. The reproductibility of oxytocin from plasma by this extraction method was 63%. The normal levels of oxytocin were ca 1.4 μg/mg in the posterior pituitary and ca 40μμg/ml in plasma, but not detectable in the anterior pituitary of rats. The oxytocin levels decreased to ca 1.1 μg/mg in the posterior pituitary and to ca 30μμg/ml in plasma 2 weeks after male and female rats were castrated. By this assay method it was confirmed that the plasma oxytocin levels during a reproductive cycle of female rats increase just before and after parturition and 1 minuite after suckling. In the determination of rat biological samples a result of bioassay by a rat uterine muscle contractian method was about 10% higher than this radioimmunoassay.