Further Purification of the Adrenal Cortical Hormone. ∗

In studying the problem of further purification of the adrenal cortical hormone we have used the following 3 types of fractionation procedure on the active material obtained from whole beef adrenal glands by the usual permutit fractionation : (1) distribution between an immiscible solvent and aqueous alkali, (2) distribution between an immiscible solvent and aqueous acid, and (3) fractionation with organic solvent mixtures. The starting material and the various fractions obtained were assayed on adrenalectomized dogs by the technique previously described. 1

Four hundred and seventy mg. of alcohol-soluble fraction obtained from 4000 gm. of beef adrenal glands and containing 8000 D. U. (dog units) were dissolved in ether and washed with 0.05 N NaOH. The aqueous alkaline solution was washed with fresh ether and the ether solutions combined. The ether-soluble fraction (200 mg.) was transferred to water (70 mg. water-soluble) for assay and was found to contain less than 500 D. U. The alkaline washings were adjusted to pH 5.6 and on assay contained between 500 and 1000 D. U. In this manipulation, therefore, about 6500 D. U. were apparently destroyed.

The above procedure was repeated on another aliquot of 470 mg. containing 8000 D. U. except that 0.05 N HCl was used instead of alkali. The ether soluble fraction (350 mg.) contained between 500 and 1000 D. U., while the aqueous acid washings after adjustment to pH 5.2 assayed between 6000 and 7000 D. U. The cortical hormone can apparently be transferred to aqueous acid without destruction. This fractionation step is being used in further studies.

One of the most potent fractions thus far obtained was prepared by hexane fractionation. An aliquot of 470 mg. containing 8000 D. U. was dissolved in 10 cc. of absolute ethyl alcohol and precipitated by the gradual addition of 9 volumes of hexane.