Assay of oxytocin in blood

Abstract

The antidiuretic hormone (ADH) of the neurohypophysis can be detected and assayed in blood and urine by numerous methods, and the conditions under which it is released have therefore received much attention (for review see Heller, 1951); the release of oxytocin has been less thoroughly investigated owing to the difficulty of assaying this hormone in body fluids. Methods using the uterus of the rat or guinea-pig in situ have not proved suitable for quantitative work, while assay on the fowl's blood pressure (Coon, 1939) is not sensitive enough to estimate the small amounts of oxytocin likely to be present in blood.By applying a method described by Melville (1937) for extracting ADH from blood with acid alcohol, we have obtained concentrated extracts which can be assayed for oxytocic activity on the isolated uterus of the rat, and we have used this method to determine the amount of oxytocin in the blood of rats under ether anaesthesia. METHOD Preparation ofthe extracts. Male albino rats, weighing from 260 to 360 g, were anaesthetized with ether and given heparin, 4 units per 100 g, by injection into the saphenous vein. One minute later the carotid artery on one side, or the external jugular veins on both sides were cut, and the blood was collected in a vessel containing enough heparin to give a concentration of 4 units per ml. of blood. The bloodwas mixedwith 9vol. 80 % (v/v) ethanol, and N-H2SO4 was added until the solution was blue to Congo Red. The mixture was left standing for 3 hr at room temperature with frequent shaking. The precipitate was removed by centrifuging and the supernatant concentrated in vacuo to about one-tenth its original volume, the temperature ofthe bath not exceeding 450 C. The concentrated extract was then centrifuged and the precipitate discarded. The flask was thoroughly washed out with small amounts of 80 % (v/v) ethanol, and the washings, together with the supernatant, were transferred to a clean flask and evaporated in vacuo almost to dryness. The residue was taken up in about 10 ml. distilled water and centrifuged, and the supernatant left overnight at room temperature in an open Petri dish to evaporate to dryness. The new residue was then dissolved in about 5 ml. distilled water and, in order to remove potassium, the solution was in most cases dialysed in a Cellophane membrane (Nojax casings 8/32) against running tap water; for this purpose 30 min was usually sufficient. If necessary the extract was neutralized after dialysis by the addition of N-Na2CO3. Ifit was kept for any length of time before the assay, the extract was made acid to ensure its stability.

The Effects of Nicotine, Hexamethonium and Ethanol on the Secretion of the Antidiuretic and Oxytocic Hormones of the Rat

“…Non-specific activity was present principally in those experiments in which glassware was still in use to collect blood, and in these cases the concentration of oxytocin was estimated as the difference in activity before and after treatment with thioglycollate. The validity of this procedure was established by previous work (Bisset and Walker, 1954).…”

“…Extraction and assay of oxytocin were carried out by the method of Bisset and Walker (1954). In every instance, except where the total activity was very small, extracts were treated with sodium thioglycollate (Van Dyke, Chow, Greep, and Rothen, 1942), and it was assumed that any activity persisting after this treatment was due not to oxytocin but to some nonspecific factor.…”

“…In this investigation an attempt has been made to block the antidiuretic and chloruretic actions of nicotine in the rat by means of the ganglionblocking agent hexamethonium. A preliminary account of this work has been given (Bisset and Walker, 1953).…”

THE METABOLISM OF EXOGENOUS AND ENDOGENOUS ANTIDIURETIC HORMONE IN THE KIDNEY AND LIVER IN VIVO

“…Acid Ethanol Extracts.-Procedures used by Vogt (1953) for the extraction of vasopressin from the hypothalamus and by Bisset and Walker (1954) for the extraction of oxytocic and antidiuretic activity from blood, which are based on the method of Melville (1937), were therefore modified as shown on I u p e r n a t a n t Table I. The interval between decapitation of the animal and immersion of the liver or the kidneys in ice-cold acid ethanol was approximately 3 min.…”

“…The neutralization was therefore done in two stages. First, the precipitate formed at pH 4 to 5 was removed by centrifugation and, second, the clear supernatant was then adjusted to pH 7.2 and dialysed as described by Bisset and Walker (1954). Flamephotometric control estimations showed that 30 min.…”