K. WOLFE,LOUISF. FIESER AND H. B. FRIEDGOOD
[CONTRIBUTION FROM TEE CHEMICAL LABORATORY, HARVARD UNIVERSITY, AND BENTBRIGHAM HOSPITAL]
Vol. 63 THE
Nature of the Androgens in Female Adrenal Tumor Urine1 BY JOHN R. WOLFE,LOUISF. FIESERAND H. B. FRIEDGOOD One of the most striking observations concern- limitation and offer promise of being capable of ing the hormone content of different urines is that elaboration to a point where all substances excreted females suffering from adrenal virilism excrete in significant amounts can be characterized indiexcessive amounts of male hormones and that an vidually. For the improvement and extension of increased output of oestrogenic hormones is as- existing methods, particularly as applied to pathosociated with adrenal feminism in males.2 The logical urines, further information is required on very high levels of hormone excretion occurring the composition of the steroid mixtures and on the in cases of adrenal cortical tumor are not paralleled efficacy and validity of procedures for the hyin other pathological conditions, and the recogni- drolysis and extraction of urine and for the fraction of a highly excessive hormone output is thus tionation of the extracted material. The present of definite diagnostic value and constitutes one investigation of the nature of the androgens of of the most prominent of present clinical appli- adrenal tumor urine was undertaken partly with cations of hormone assay.3 The problem of this objective in view and partly in the hope of determining urinary steroids and interpreting gaining a further insight into intermediary steroid the results is complicated by the appearance in metabolism. the urine of several steroids associated with each The neutral ketonic fraction obtained by reof the two sex groups and by the fact that these moval of the phenolic oestrogens with alkali and substances are not themselves true hormones separation of the ketones from the non-ketonic possessing characteristic physiological functions material with Girard’s reagent may be designated but are excretory transformation products of the “neutral 17-ketosteroid fraction” or, since i t glandular hormones. A knowledge of the output includes all of the urinary steroids which possess of steroids in the urine nevertheless can be of androgenic activity, the “androgen fraction.” distinct value when it provides an accurate if The androgen fraction from the urine of normal indirect index of the activity of the glands pro- males has been shown to contain androsterone4s5 ducing the true hormones. Since both the gonads (I, rather strongly androgenic), 3a-hydroxyaetioand the adrenals appear t o be involved in the pro- cholanone-176 (11, inactive) , and dehydroisoanduction of hormones which are excreted in altered form in the urine, data concerning the output of excretory products can be translated in terms of the activity of a given gland only if determination is made of individual urinary steroids whose point of origin in the body is known or can be H O ,,bid HO,A A v ‘ safely inferred. Certain of the urinary steroids retain the biI I1 ological properties of glandular hormones, Androsterone 3a-Hydroxyaetiocholanone-17 although in diminished potency, while others are wholly devoid of biological activity. Since the activity or lack of activity is purely incidental and has no physiological significance, bio-assays for androgenic and oestrogenic activity can provide only a very incomplete and unsatisfactory indication of glandular functioning. Methods of deterI11 mination based upon specific chemical or physical Dehydroisoandrosterone properties are not subject to any such inevitable (1) This work was supported b y grants from the Milton Fund of Harvard University and the National Advisory Cancer Council. (2) Burrows, Cook, Roe and Warren, Biochcm. J., 31, 950 (1937). (3) R. K. Callow, Proc. Roy. SOC.Mcd., 31, 841 (1938).
(4) Butenandt and Tscherning, Z. angcw. Chem., 44,905 (1931). (5) Butenand t. Dannenbaum, Hanisch and Kudszus, 2. physiol. Chcm., 237, 57 (1935). (8) N. H. Callow, Biochcm. J . , 33, 559 (1939).
ANDROGENS IN FEMALE ADRENAL TUMOR URINE
drosterone7*8 (111, moderately androgenic). adrenals,14 but the nature of the precursor is still The amounts of these steroids which have been obscure. The suggestion that the substance is a isolated from normal male urine in an identi- reduction product of a A4-3-ketosteroidls lacks fiable form are in the order of 0.5-1.5 mg. any foundation of analogye and seems to us unper liter of urine6l9; still other substances may be likely; the process would require the migration present in small amounts but have not been of the double bond a t 4,5, presumably after reidentified. The same three Steroids have been duction of the carbonyl group, away from its isolated from the urine of normal females1° in position of conjugation with the oxygen atom. amounts comparable with the yields from normal The adrenal origin of dehydroisoandrosterone male urine.ll That the substances can be pro- i's further indicated by Callow's1Gisolation of an duced elsewhere than in the genital organs has abnormally large amount of this steroid from the been established by the isolation of androsterone, urine of a young girl having an adrenal tumor 3a-hydroxyaetiocholanone-17, and dehydroiso- and showing symptoms of virilism. Butler and androsterone from the urine of eunuchs9 and from Marrian investigated the neutral steroids from the urine of ovariectomized women. l 2 There the unhydrolyzed urine of women suffering from is little doubt that the adrenal gland is responsible adrenal tumors17 and from adrenal hyperplasia1*J9 for the production of these steroids in castrates and encountered no dehydroisoandrosterone but and, since in the case of ovariectomized women isolated four other crystalline products. One of the output is only slightly below normal, i t may these proved to be 3a-hydroxyaetiocholanone-17 be inferred that in the female organism the (11), a steroid of normal female urine, and the adrenal gland probably is the sole source of the others were characterized as isoandrosterone components of the androgen fraction. Since (IV), pregnane-3a,17,20-triol (V), and an isomer castrated males excrete somewhat less androster- which was assigned tentatively the probone and 3a-hydroxyaetiocholanone-17than do able structure of pregnane-3/3,17,20 - triol (VI). normals, a part of the production of these two CHI I substances in males may be attributed to the 0 CHOH testes. The evident precursor in the genital gland is testosterone, from which the two Cs-epimers are derivable by a process of partial reduction and disproportionation. Indeed administration of testosterone to males results in an increased outHO H put of androsterone and 3a-hydroxyaetiocholaHO/ H n0ne-17,~J~By analogy i t would appear that Isoandrosterone Pregnane-3a, 17,20-triol the androsterone and 3a-hydroxyaetiocholanoneCHa 17 of adrenal origin are end-products of the LHOH metabolism of a,Bunsaturated ketones of the cortical steroid group. The dehydroisoandrosterone found in the urine of males and females alike may all come from the
(7) Butenandt and Tscherning, 2. physiol. Chcm., 229, 107 (1934). (8) Nomenclature: Fieser, "The Chemistry of Natural Products Related to Phenanthrene," 2nd edition, p. 233, Reinhold Publishing Corp., New York, N . Y..1937. The substance is also referred to in the literature as dehydroandrosterone (Butenandt) and Iransdehydroandrosterone (Ruzicku). The former name seems incorrect because the compound is the dehydro derivative not of androsterone but of its isomer (Cx.OH.8). The latter name to be strictly descriptive should be changed to dehydrofransandrosterone. (9) N. H. Callow and R . K. Callow, Riochcm. J . , 84, 276 (1940). (10) (a) N . H. Callow and R. K.Callow, ibrd., 82, 1759 (1938); (b) 33, 931 (1Y39). (11) According to a preliminary report by Pearlman, J . Biol. Chem., 136, 807 (1'540), normnl female urine also contains small amounts of isonndrosterone. (12) Hirschmann, (a) ibid., 180, 421 (1939); (b) 186, 483 (1940). (13) Dorfman, Cook and Hamilton, ibid., 180, 286 (1939).
H d vHv Pregnane-3&17,20-triol
Burrows, Cook, Roe and Warren2 processed the acid-hydrolyzed urine of a male patient with a malignant tumor of the adrenal gland and (14) N. H. Cnllow, R. K. Callow, Emmens and Stroud, J . Endocrinology, 1, 76 (lY3Y). (15) Marker, ' h i s J O U R N A L , 60, 1725 (1038). (10) R. K. Callow, J . SOC.Chcm. fnd., 66, 1030 (1036); Crooke and I