A Method for Assay of Ovarian Hormone in Blood and Urine and Relation of Assay to Menstrual Cycle.

Abstract

Ten cc. of blood are drawn from the Median Basilic vein into oxalate to prevent coagulation. The blood is centrifuged, and the blood plasma pipetted off. The blood corpuscles are washed with normal saline solution twice, and the washing fluid is added to the blood plasma. Since no ovarian hormone was found in the blood corpuscles they are not included in the extraction. To the blood plasma and saline is added about 5 cc. of N NaOH. This is extracted 3 times with an equal volume of ether (free of peroxides). The ether extract is put into a distilling flask and the ether boiled off under reduced pressure. The residue is taken up in normal saline. For purposes of quantitative assays, no further purification is necessary, as this extract is not so irritating as to induce sloughs in mice. Castrated white mice are used, which have been observed to be in continuous dioestrus pause, for at least 2 weeks following castration; and when used again after having been injected, at least 4 days of dioestrus are necessary. Urine: Twenty-four hour urine specimens are obtained from normal non-pregnant women. Five cc. of chloroform is used as a preservative. The urine is made alkaline to phenolphthalein with NaOH, and then extracted 3 times with one-fourth pound of ether for each extraction. (When the urine is acidified before extraction with ether, toxic substances pass into the ether. In most of our experiments this acid-extract caused death to the mouse in 2 hours.) The ether extract of the alkalinized urine is then treated the same as the blood extract, and the residue taken up in normal saline. In assaying this hormone, because of the large number of assays of samples to be made, we employed a single injection instead of the 3 injections of the Allen-Doisy method or the 8 injections of the Laquer method.