Gordon L. Farrell

INDOLE COMPOUNDS: ISOLATION FROM PINEAL TISSUE.

Five indole compounds have been isolated from bovine pineal tissue and characterized as 5-methoxytryptophol, N-acetyl-5-methoxytryptamine (melatonin), 5-hydroxytryptophol, 5-methoxyindole-3-acetic acid, and 5-hydroxyindole-3-acetic acid. Pineal hydroxyindole-O-methyltransferase, with S-adenosylmethionine, converts 5-hydroxytryptophol to 5-methoxytryptophol.

Increased Aldosterone Secretion in Response to Blood Loss

A marked increase in the adrenal secretion of the potent electrolyte-active steroid, aldosterone, was observed in the dog in response to loss of blood. The proportionate increase in adrenal output of alclostcrone exceeded that of hydrocortisone. The physiologic mechanism by which the increased aldosterone release is mediated remains obscure. The stimulus to increased aldosterone secretion was not a change in the serum electrolytes. Expansion of the volume of the vascular system by the infusion of large amounts of plasma substitutes failed to uniformly prevent the increased aldosterone output.

Isolation and identification of aldosterone from adrenal venous blood.

Summary A highly potent sodium-retaining steroid has been isolated from adrenal venous blood and demonstrated to be identical with aldosterone with regard to: 1) infra-red spectrum, 2) chromatographic behavior of the parent compound and 2 derivatives, 3) biological activity in inducing sodium retention in adrenalectomized rats, 4) ultraviolet absorption and 5) sulfuric acid chromogen.

Isolation of a potent sodium-retaining substance from adrenal venous blood of the dog.

Summary A substance has been isolated from the adrenal venous blood of dogs which is approximately 25 times as potent as DOCA in producing sodium retention in adrenalec-tomized rats. It has not been established with certainty that this substance is pure. The material is slightly less polar than cortisone, exhibits an absorption maximum at 240 m/x, does not react with the phenylhydrazine reagent described by Porter and Silber, and does react with a phenylhydrazine reagent developed in this laboratory. The substance shows absorption maxima at 285 and 390 m/x when mixed with concentrated sulfuric acid.

Alterations in electrolyte intake and adrenal steroid secretion.

Severe limitation of sodium intake in the dog leads to an approximate twofold increase in adrenal secretory rate of aldosterone, as determined by isolation of the steroid from adrenal venous blood. The rate of secretion of hydrocortisone remains unchanged. The administration of large amounts of potassium appears to be ineffective in altering adrenal secretion of aldosterone or of hydrocortisone. It is postulated that the increased urinary aldosterone associated with increased potassium intake described by others may be due to altered renal handling of the steroid.

Steroids in Adrenal Venous Blood of the Dog.

Summary Adrenal venous blood of the dog has been quantitatively analyzed by paper chromatography for steroid components. Fourteen fractions have been isolated; 3 have been identified as 17-hydroxycorticosterone, corticosterone and 11-desoxy-17-hydroxycorticosterone; 7 of the unidentified fractions exhibit chemical reactions characteristic of adrenocorticosteroids. Addendum. In a subsequent experiment conducted in collaboraton with Dr. Hans Hirschmann a sample containing Fractions 7 and 8 was acetylated and chromatographed on paper 901/2 hours, employing the system propylene glycol-hexane described by Savard. Five distinct zones were found by examining the strip under ultraviolet light. These subfractions (numbered 1-5 in order of their position on the chromatogram, starting at the origin) were eluted with methanol and were examined spectroscopically in this solvent and after reaction with phenylhydrazine-sulfuric acid reagent. Subfractions 1, 2, and 3 absorbed maximally near 240 mμ; subfraction 4, near 235 mμ, while subfraction 5 showed only a shoulder in this region. With phenylhydrazine reagent subfractions 1 and 5 absorbed maximally at 390-400 mμ, whereas subfractions 2, 3, and 4 gave products which did not exhibit absorption peaks in this region. Each subfraction was treated with acetylcholinesterase in glycylglycine buffer at pH 7.5 for 4 hours at 30°C, and assayed for sodium-retaining activity by the method described in an earlier communication (5). All subfractions were inactive except subfraction 4 which possessed high sodium-retaining activity. No claim of homogeneity is made for any of these subfractions, but it would appear that the sodium-retaining activity and the chromogenicity in the phenylhydrazine reagent shown by our previously described Fraction 8 are not due to the same substance.

Isolation of desoxycorticosterone from adrenal venous blood of the dog; effect of hypophysectomy and ACTH.

Summary A biologically active compound has been isolated from adrenal venous blood of the dog. It is in all probability 11-desoxy-corticosterone. Its concentration in adrenal venous blood has been shown to be markedly increased over that in arterial blood, indicating that it is secreted by the adrenal. The rate of secretion of the isolated substance has been found to decrease following acute hypophysec-tomy and to increase following intravenous ACTH injection.

Decline of corticosteroid secretion following hypophysectomy.

Summary The rates of secretion of 17-hydroxycorticosterone and corticosterone prior to and following hypophysectomy have been determined. The rates of secretion of these steroids decline after hypophysectomy, reaching a low and apparently constant level after 3 hours.

Secretion of 17-Hydroxycorticosterone by Adrenal of Hypophysectomized Dog Effect of ACTH.

Summary The rate of secretion of 17-hydroxycorticosterone by the adrenal of the dog as determined by chromatographic analysis of adrenal venous blood reached a low and apparently constant level within 4 hours after hypophysectomy. Two minutes after intravenous ACTH injection in these animals; the rate of secretion was significantly increased.

Steroids in adrenal venous blood of the dog: venous-arterial differences across the adrenal.

Summary Concentrations of steroids in arterial and venous blood of the adrenal have been determined. Those steroids whose concentrations in the blood are increased by passage through the gland are considered to be adrenal secretory products. Three steroids are consistently of adrenal origin: 17-hydrox-ycorticosterone, Fraction 8, and corticoster-one. In 2 of 3 experiments 11-desoxy-17-hy-droxycorticosterone was contributed to the blood by the adrenal gland. A steroid less polar than corticosterone (Fraction 13) also appeared to be an adrenal secretory product in 2 of 3 experiments. Five substances (Fractions 2, 5, 77 9, and 10) are clearly not secreted by the adrenal gland, since they were found in the same concentrations in arterial blood as in adrenal venous blood. The status of 4 substances (Fractions 1, 3, 4, and 14) as adrenal secretory products has not been established. 17-Hydroxycorticosterone and corticosterone, when added to arterial blood, are recovered without substantial alteration in the pattern of steroids present in such blood.