J. J. Pfiffner

On the Cortical Hormone of the Adrenal Gland.

The crude lipid extract of beef adrenal cortex 1 and the aqueous extract 2 obtained from the active lipid fraction contain, with the cortical hormone, small quantities of adrenalin. The adrenalin content of the aqueous extract is sufficiently low to permit the demonstration of its efficacy in maintaining indefinitely the lives of adrenalectomized cats. 2 , 3 A simple method for the separation of adrenalin from the cortical hormone has been found. 4 The 70% alcohol soluble fraction obtained by our previously described method 2 is transferred to 95% alcohol and filtered through permutit. An extract with an adrenalin concentration of less than 1:2,000,000 (bio-assay-blood pressure) and a tissue equivalent of 30 gm. of cortex per cubic centimeter can be prepared by filtering through permutit twice using 20 gm. per kilo of tissue on the first filtration and 10 gm. per kilo on the second. The active material remaining in the permutit filter after each filtration is washed out with alcohol. The active fraction is transferred to water and the extract clarified by Seitz filtration. Besides adrenalin the permutit removes most of the contaminating pigment substances along with other inert material. Intravenous injections of this type of extract have been used successfully in the crises of Addisons disease. 5 Fractionation with permutit has made possible the preparation of active extracts from whole beef adrenal glands thereby doing away with the expense of dissection. These extracts have been found to be just as active in restoring prostrate adrenalectomized cats to apparently normal health as extracts prepared from dissected adrenal cortex. Whole adrenal gland extract (1 cc. equivalent to 50 gm. of whole gland) has an adrenalin content of approximately 1:2,500,000. This extract has been found suitable for subcutaneous, intraperitoneal and intravenous use. The solid content ranges in different batches from 0.3 to 0.4%.

Vitamin C and the Adrenal Gland in the Dog.

Conclusion No evidence has been obtained that the adrenal gland, as an organ in the dog, is concerned with either the synthesis or the metabolism of vitamin C. If the adrenals do play any role in the metabolism of this compound the action is conferred by the adrenal cortical hormone. Harris and collaborators 1 , 2 have shown that dogs do not require vitamin C in their diets for adequate nutrition. Others have kept dogs upon synthetic rations, presumably devoid of this vitamin, for periods of 1-4 years without symptoms of scurvy. Tissues from such animals, notably the adrenals and liver, show by chemical and biological tests the presence of large amounts of ascorbic acid. Similar tissues from guinea pigs kept upon scorbutic diets reveal a marked diminution in the content of ascorbic acid as scurvy develops. The early observation 3 that the cortex of the adrenal gland contained large amounts of “hexuronic acid”, later shown to be vitamin C 4 , 5 , 6 has led to speculation upon the relationship of this vitamin to the functions of the adrenal cortex. The belief that the medulla 7 did not contain “hexuronic acid” supported the idea of a functional significance for this uneven distribution. It has since been demonstrated that other animal tissues contain appreciable amounts of ascorbic acid, e. g., liver, 8 corpora lutea, 9 , 10 pituitary 11 thymus. 12 Huszák 13 has shown that the adrenal medulla contains as much vitamin C as does the cortex. Such a wide distribution is hardly indicative of a specific relationship between vitamin C and the functions of the adrenal cortex. In those species that do not develop scurvy one may reasonably consider any tissue containing much vitamin C as having a possible rôle in protecting the animal from this nutritional disorder. The question arises, is the adrenal gland in the dog concerned specifically with the synthesis and metabolic functioning of vitamin C and the consequent protection of the animal from scurvy?

A Quantitative Study of Adrenal Cortical Hormone Extraction.

It was demonstrated 1 , 2 that cortical hormone can be prepared from whole beef adrenal glands by essentially the same methods of extraction used in the preparation of cortical hormone from dissected cortex. 3 , 4 The elaboration of a biological method of assay based on the minimum maintenance requirement of the adrenalec-tomized dog 5 provided a reliable means of comparing the potency of whole adrenal extract with that prepared from dissected cortex. It has been shown that cortex extract (1 cc. represents 30 gm. beef adrenal cortex) contains 4 to 10 dog units (D. U.) per cc, the potency varying with different batches. 5 Extracts prepared from whole glands are many times as potent as those prepared from dissected cortex (on an equivalent weight basis). Whole gland extract (1 cc. represents 40 gm. whole beef adrenal gland) contains 40 to 80 D. U. per cc. The following is a summarized comparison of whole gland and dissected cortex extract: Whole gland extracts of approximately equal potency but lower solid content can be prepared by using the same fractionation procedure as previously described but decreasing by 50% the thoroughness of extraction of the respective fractions. In this simplified technique the glands are extracted once with alcohol for 48 hours, the benzene soluble fraction is extracted twice with acetone, the acetone soluble fraction is distributed twice between 70% alcohol and petroleum ether and the alcohol soluble fraction is filtered only once through permutit. The adrenalin concentration is less than 1:2,000,000 (blood pressure-dog). Typical assay data follow:

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.