ISOLATION OF PREGNANE-3-ALPHA, 17-ALPHA,21- TRIOL-20-ONE (TETRAHYDRO COMPOUND S) FROM THE URINE OF A WOMAN WITH METASTATIC ADRENOCORTICAL CARCINOMA*
TO THE EDITOR:
Pregnane-3-alpha,17-alpha,21-triol-20-one (tetrahydro compound S) has been isolated from the urine of a 48-year-old woman with metastases from a previously removed adrenocortical carcinoma. This case has been previously described in considerable detail (1). The patient showed marked hirsutism, acne, a low pitched voice, an enlarged clitoris, atrophy of the breasts, and marked muscular weakness, associated with abnormalities of electrolyte metabolism at times similar to those found in Cushing’s syn- drome, but she had periods of hypoglycemia (prior to, and after develop- ment of recognizable liver metastases). The urine for chromatography was collected one month before her death. At approximately this time, the total neutral 17-ketosteroid excretion varied between 162 and 320 mg. per twenty-four hours. Dehydroisoandrosterone excretion was 27 mg. (method of Allen et al. (2)) and urinary estrogens were greater than 400 and less than 660 mouse units per twenty-four hours. Neutral lipid-soluble reducing sub- stances found in the extract subjected to paper chromatography totaled 64 mg. per twenty-four hours (high normal by the method used is less than 20 mg.). A semiquantitative estimate indicated that the 24-hour urine col- lection contained approximately 12 mg. of tetrahydro compound S (ex- tractable by the method employed).
The urine was adjusted to pH 4.5 with acetic acid and sodium acetate and incubated for forty-eight hours with beta-glucuronidase (100 units per cc.). After acidification to pH 1 with hydrochloric acid, the urine was extracted with equal volumes of chloroform on an oscillating shaker for one hour. Paper chromatography was performed by the method of Burton, Zaffaroni and Keutmann (3). Chromatography, using 1.1 per cent of the 24-hour urine extract, showed a large blue tetrazolium-reacting zone in the posi- tion where tetrahydro compound S is found. Eluates of this zone from multiple chroma- tograms run at the same time were examined in various solvent systems and the material was shown to have the same running rates as the sample of pregnane-3-alpha, 17-alpha, 21-triol-20-one (tetrahydro compound S) kindly supplied by Dr. Thomas F. Gallagher. The material, after acetylation, was further purified by chromatography on silica gel and shown to have absorption spectra in sulfuric acid, according to the method of Zaffaroni (4), duplicating that of the standard. The isolated material was subjected to infrared
* This investigation was supported in part by research grant H-922 from the National Heart Institute, U. S. Public Health Service, and by a grant from the American Heart Association.
spectrometry through the courtesy of Dr. Thomas F. Gallagher at the Sloan-Kettering Institute for Cancer Research, Memorial Center, New York, and was found to be identical with the standard. There were several other smaller alpha-ketolic zones on the chromatograms. These have not been identified.
Comment : Reichstein and von Euw (5) and others have isolated 17-alpha- hydroxy-11-desoxycorticosterone (compound S) from animal adrenals. More recently, compound S has been identified in blood from the adrenal vein of dogs (6). We have demonstrated that pregnane-3-alpha, 17-alpha, 21-triol-20-one (tetrahydro compound S) is the major alpha-ketolic steroid metabolite of 17-alpha-hydroxy-11-desoxycorticosterone (compound S) found in the urine after administration of this compound to individuals with markedly reduced adrenocortical function (7). It has not been found following the administration of hydrocortisone, cortisone, corticosterone, 11-dehydrocorticosterone, or 11-desoxycorticosterone. It, therefore, seems probable that the adrenocortical tumor tissue secreted the parent com- pound. The possibility that compound S may be found in the urine is being explored.
J. C. TOUCHSTONE, PH.D. E. M. RICHARDSON, PH.D. HELEN BULASCHENKO, M.S. IRMGARD LANDOLT, B.S. F. C. DOHAN, M.D.
The Endocrine Section of the William Pepper Laboratory of Clinical Medicine, University of Pennsylvania, Philadelphia, Pa.
REFERENCES
1. DOHAN, F. C .; ROSE, E .; EIDMAN, J. W .; RICHARDSON, E. M., and ZINTEL, H .: Increased urinary estrogen excretion associated with adrenal tumors: report of four cases, J. Clin. Endocrinol. & Metab. 13: 415 (Apr.) 1953.
2. ALLEN, W. M .; HAYWARD, S. J., and PINTO, A .: A color test for dehydroisoandroster- one and closely related steroids, of use in the diagnosis of adrenocortical tumors, J. Clin. Endocrinol. 10: 54 (Jan.) 1950.
3. BURTON, R. B .; ZAFFARONI, A., and KEUTMANN, E. H .: Paper chromatography of steroids. II. Corticosteroids and related compounds, J. Biol. Chem. 188: 763 (Feb.) 1951.
4. ZAFFARONI, A .: Micromethods for the analysis of adrenocortical steroids in Recent Progress in Hormone Research, edited by G. Pincus. New York, Academic Press Inc., 1953, vol. 8, p. 51.
5. REICHSTEIN, T., and VON Euw, J .: Ueber Bestandteile der Nebennierenrinde: Isolierung der Substanzen Q (Desoxy-corticosteron) and R sowie weiterer Stoffe, Helvet. chim. acta 21: 1197, 1938.
6. FARRELL, G. L., and LAMUS, B .: Steroids in adrenal venous blood of dogs, Proc. Soc. Exper. Biol. & Med. 84: 89 (May ) 1953.
7. RICHARDSON, E. M .; TOUCHSTONE, J. C., and DOHAN, F. C .: Urinary alpha-ketolic steroid metabolites of cortical hormones administered to subjects with adrenal cortical insufficiency, (to be published).
URINARY CORTICOIDS IN PHEOCHROMOCYTOMA
TO THE EDITOR:
The view that adrenaline is a physiologic stimulant of adrenocortical secretion by reason of a specific effect on the secretion of ACTH has come under recent question. In animal experiments (1, 2) large doses of adren- aline provoke increased cortical secretion by pituitary stimulation but such stimulation seems to be nonspecific in studies on human subjects (3, 4).
From the clinical aspect, attention has been drawn to the association of adrenocortical adenoma with functioning pheochromocytoma (5) as indirect evidence of adrenocortical stimulation by medullary hypersecre- tion. On the other hand, with one reported exception (6), patients with functioning pheochromocytoma do not exhibit the clinical signs of hyper- corticoidism. A further test of the clinical association between adrenal medullary and cortical functions would be the measurement of urinary corticoids in patients with pheochromocytoma.
In this regard, Sprague and Mason (7) have found increased outputs of “free” formaldehydogenic corticoids (FFC) and 17-ketosteroids in 2 of 14 patients with pheochromocytoma. This report extends these observations by data from 3 adult female patients. The measurements made were FFC (pH 1 extractable) and TFC (pH 1 extractable after glucuronidase hydrol- ysis), using the methods of Corcoran and Page (8) and of Dustan, Mason and Corcoran, Procedure 1 (9). Diagnoses were made from clinical signs and symptoms, pharmacologic tests, presence of increased urinary catechol amines and demonstration of such in the tumors found at opera- tion, both colorimetrically (method of von Euler and Hellner (10)) and by bioassay.
The data are summarized in Table 1. They show that outputs of FFC were somewhat increased in 1 patient preoperatively, but were normal in 2, whereas in all 3 the outputs of TFC were within or below the normal range.
These observations confirm the impression that adrenocortical hyper- function is not a significant aspect of pheochromocytoma. The increased outputs of FFC without associated increases in TFC found in one of the subjects recalls the similar pattern found in some patients with essential hypertension (9) and may represent an abnormality of excretion in some patients with hypertensive disease.
The data indicate that prolonged and severe adrenomedullary hyper-