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.
