Extraction of Gonadotropic Factors from the Blood. Improved Technic

Abstract

An improved method for gonadotropic hormone (G.H.) extraction from blood was reported from this laboratory in 1935. By means of this method, both follicle stimulating as well as luteinizing effects were obtained with 40 cc. of blood taken from normal menstruating women, from those past the physiological menopause as well as from surgical castrates. This method, which involved desiccation of the blood with sodium sulphate, was used because it enabled both G.H. and E.S. (estrogenic substance) determinations to be performed on the same specimen. By the present method the deleterious effect of alcohol upon the G.H. factors is avoided, giving a more complete G.H. extraction without impairing the E.S. yield. Moreover, the extracts obtained by this method are non-toxic, in sharp contrast with extracts by the sodium sulphate method which caused the death of many animals because small amounts of sodium sulphate could not be removed. Forty cc. of freshly drawn blood is run into 150 cc. of cold acetone. A fine precipitate forms which settles to the bottom. The blood-acetone mixture is shaken in a mechanical shaker for 20 minutes, centrifuged and the supernatant acetone poured off. This is repeated twice more with 125 cc. of fresh cold acetone. The acetone fractions are united, evaporated and utilized for estrogenic hormone determinations. The acetone precipitate is dried under a fan, powdered and then extracted with 100 cc. of water, the mixture being acidified with dilute HC1 to pH 4.8 (Brom-cresolgreen). This mixture is stirred by hand for 10 minutes, centrifuged, and the supernatant aqueous extract decanted. This aqueous extract is filtered through a single layer of gauze to remove suspended particles. To the aqueous extract, 400 cc. of cold acetone are added, a fine buff colored precipitate settling out.