Intravaginal Assay of Urinary Estrin

Abstract

Since estrin is not commonly standardized on the basis of its ability to produce behavioristic estrus, but rather by virtue of its growth-promoting influence on the vaginal epithelium, there seems but little need to saturate the entire animal to gain this end. The vaginal epithelium reacts to extremely minute quantities of estrin applied locally,∗ although Pratt and Smeltzer 1 and Powers, et al., 2 found their methods of vaginal administration only one-third to one-half as efficacious as the subcutaneous.

We have used the intravaginal method of assaying urinary estrin not only because minute amounts may be detected (even in male urine), but also to avoid the toxic effects produced by parenteral injection of crude extracts. Twenty-four-hour urines were obtained from 4 women† on days 7, 14, 21, and 28 of the menstrual cycle, extracted immediately, or stored at 0°C. Half of the 24-hour sample was evaporated in vacuo over a steam bath to a salty sludge, (a few drops of capryl alcohol being added to lessen foaming). The sludge was extracted with two 100 cc. portions of hot absolute C₂H₅OH and the insoluble material discarded. The alcohol was distilled off, and the residue neutralized with N/1 or stronger NaOH. A graded series of dilutions of this extract was prepared with distilled water so that the 0.02 cc. used in testing represented original urinary volumes of from 0.05 to 1.0 cc. 0.01 cc. of each dilution was introduced twice (24-hour interval) into the vaginae of 3 full-grown rats ovariectomized at least 10 days previously. The minimal effective urinary equivalent was considered the smallest amount causing cornification in the vaginae of at least 2 of the 3 rats, 24 hours after the second dose. After an interval of 5 days or more, the rats were used again.