Concentration and Purification of the Gonadotropic Substance in Urine of Ovariectomized and Post-Menopausal Women. ∗

Abstract

While attempting to concentrate the gonadotropic activity of the urine of post-menopausal (or castrate) women, it has been found that the active material may be practically quantitatively concentrated into relatively non-toxic extracts by the use of tannic acid. The method developed for the precipitation and partial purification of the gonadotropic material is as follows.

The chloroform-preserved urine is chilled, siphoned from any sediment and acidified to pH 5 with acetic acid. For each liter of urine, 20 cc. of a freshly prepared aqueous 10% tannic acid solution are added. An immediate precipitate forms. After thorough mixing, the precipitate is allowed to settle in the cold room and is then collected at the centrifuge. The bulky precipitate, which contains considerable water, is extracted once with at least 5 parts of 95% ethyl alcohol followed by 3–4 extractions with 80% alcohol. Alcohol is removed by several washings with acetone and the residue is freed of acetone by reduced pressure. In addition to removing considerable pigment and other inert material, the alcohol-acetone treatment removes any estrogenic substances which may be present.

The resultant powder (100 to 200 mg. per liter of urine) contains 75–100% of the original gonadotropic activity of the urine‡ as indicated by parallel assays of the raw urine (or alcohol precipitates thereof). The activity of the dry tannate is quite stable, no loss being noted even after storage for more than a year at room temperature.

Assays are conducted by giving 20–22-day-old mice (8–12 gm. body weight) subcutaneous injections once daily for 3 days, the animals being sacrificed 72 hours after the first injection. Because with minimal doses ovarian weight alone is an unreliable criterion, the mouse unit has been tentatively defined as that amount which, when administered in the above manner, will cause vaginal canalization, at least a 200% increase in the weight of the uterus drained of fluid (controls 4–7 mg.), and a slight increase in ovarian weight as compared to uninjected controls.