Although 9a-fluorohydrocortisone was more effective than the other steroids in depressing body weight, it was poorly effective in pro- moting urinary loss of water, Na, and K; rea- son for excessive body weight loss is unknown. 41 U was the only steroid that promoted body weight gain in intact or adrenalectomized rats; it has weak physiological activity favorable for the adrenalectomized rat (unpublished).
Experiments dealing with effects of 11-oxy steroids on adrenal weight demonstrated that each compound caused some adrenal atrophy, but only hydrocortisone acetate and predniso- lone were significantly effective on both actual and relative weight bases. These 2 steroids are then presumably the most effective ones of the 10 used in inhibiting endogenous pro- duction of ACTH and adrenal cortical hor- mones.
Summary. In the force-fed intact rat, treatment with small daily doses of cortisone or hydrocortisone or their acetates and 41 ana- logues had slight, if any effects on serum Na and K but augmented urinary excretion of water (urine volume), Na and K, suppressed body weight gain, and caused reduction in adrenal weight. Hydrocortisone acetate and prednisolone were most effective of 10 ster- oids tested in causing adrenal atrophy. Treat-
ment with 9a-fluorohydrocortisone resulted in marked increase in serum Na, severe decrease in serum potassium, marked loss in body weight, no change in urinary sodium, and mild increases in urinary volume and K. Corticos- terone, 11-dehydrocorticosterone, or 41,4. pregnadiene-17, 20, 21-triol-3, 11-dione (41 U), in the doses employed, failed to affect water and electrolyte balance. These 3 com- pounds tended to suppress adrenal weight and, excepting A1U, caused slight reduction in body weight gain. In general, renal responses were similar to those found previously in adrenalectomized rats.
1. Chart, J. J., Hetzel, N., Gaunt, R., PROC. Soc. EXP. BIOL. AND MED., 1956, v91, 73.
2. Ingle, D. J., Oberle, E. A., Am. J. Physiol., 1946, v147, 222.
3. Fisher, R. A., Statistical Methods for Research Workers, 1950, Hafner Pub. Co., N. Y.
4. Sala, G., Leutcher, J. A., Endocrinol., 1954, v55, 516.
5. Ingle, D. J., Prestud, M. C., Rice, K. L., ibid., 1950, v46, 510.
6. Leith, W., Beck, J. C., J. Clin. Endocrinol., 1957, v17, 280.
7. Swingle, W. W., Baker, C., Eisler, M., Le Brie, S. J., Brannick, L., Endocrinol., 1955, v57, 220.
8. Ingle, D. J., Sheppard, R., Oberle, E. A., Kui- zenga, M. H., ibid., 1946, v39, 52.
Received June 24, 1958. P.S.E.B.M., 1958, v99.
Excretion of Pregnan-3a, 17a, 21-Triol-20-One (Tetrahydro S) by Patients with Adrenocortical Carcinoma .* (24324)
MORTIMER B. LIPSETT AND BARBARA DAMAST . (Introduced by Roy Hertz) Dept. of Health, P.H.S., National Cancer Inst., Endocrinology Branch, Bethesda, Md.
An increased excretion of Porter-Silber chromogens in urine has generally implied the presence of increased amounts of tetrahydro- cortisone (tetrahydro E) and tetrahydrohy- drocortisone (tetrahydro F), the reduced products of cortisone and hydrocortisone re- spectively. However, in 2 patients with adrenocortical carcinoma, tetrahydro S has been shown to comprise the major part of the * We wish to thank Dr. Hildegarde Wilson of the Nat. Inst. of Arthritis and Metabolic Diseases for generous advice.
urinary neutral reducing lipids and exceed ex- cretion of tetrahydro E and tetrahydro F(1). In a patient with the hypertensive variant of congenital adrenal hyperplasia, tetrahydro S was the major component of the Porter-Silber chromogens(2). It was isolated in increased amount from urine of 2 patients with adreno- cortical carcinoma (3). Since excretion of tetrahydro S probably accurately reflects se- cretion of 4-pregnen-17a, 21-diol-3, 20-dione (Compound S), determination of its urinary excretion provides data regarding the secre-
| Patient | Porter-Sil- ber chromo- gens | Tetra- hydro S | 17-keto- steroids* | Dehydro- epiandro- steronet |
|---|---|---|---|---|
| mg/24 hr | ||||
| V.G. | 89 | 13 | 134 | 36 |
| J.B. | 50 | 15 | 152 | 75 |
| V.F. | 72 | 44 | 70 | 29 |
| R.S. | 61 | 11# | 57 | 19 |
| G.T. | 20 | 7 | 23 | 3 |
| T.R. | 6 | .4 | 130 | 47 |
| G.H.§ | .6 | .3 | 107 | 34 |
| J.H. | 5 | .7 | 4.5 | .6 |
| M.S. | 5 | .3 | 44 | 17 |
| Normal | & 7 | .9 | 12 | |
* Modification of Callow’s method.
t Paper chromatography and micro Zimmerman.
# Excretion of dihydro S-3 mg/24 hr.
§ 15-mo-old boy.
tion of one of the probable intermediates in synthesis of hydrocortisone. Since one of our patients with adrenocortical carcinoma excreted 60 mg of Porter-Silber chromogens daily and was without clinical evidence of Cushing’s syndrome we searched for tetra- hydro S in his urine and subsequently in the urine of 8 other patients with adrenocortical carcinoma. This resulted in the finding of increased urinary excretion of tetrahydro S in 5 patients and the presumptive identifica- tion of pregnan-17a, 21-diol-3, 20-dione (di- hydro S) in one patient.
Methods. Most urines examined had been stored at -4℃ up to 2 years. Aliquots of 24-hour urines were hydrolyzed with beef liver beta glucuronidase (Ketodase®) at pH 5.0 for 5 days. They were then continuously extracted with ether at pH 1 for 48 hours, and for 24 hours at a concentration of sulfuric acid of 1 normal. After washing with 1N sodium hydroxide and water, aliquots of the crude material in ethanol were chromatogrammed in the Bush B5 system, a toluene-ethylene gly- col system, or in the E4 system of Eberlein and Bongiovanni (4). This last proved most useful as blue tetrazolium-reacting steroids more polar than tetrahydro S have low Rf’s. Tetrahydro S moved 19 to 23 cm in 16 hours at room temperature. The steroids were eluted with cold methanol and determined quantita- tively with blue tetrazolium(5). Recovery experiments with tetrahydro S from paper averaged between 85 and 110% Tetrahydro
S was identified by its mobility in the 3 sol- vent systems, its reaction with blue tetrazo- lium and phenylhydrazine and infra-red spectroscopy.+ Dihydro S was presumptively identified in urine of R. S. by its mobility in 2 solvent systems, and comparison of its ace- tate with authentic dihydro S acetate in the E1 system (4) and its reaction with blue tetra- zolium and phenylhydrazine.
Results. In Table I, data are presented comparing 24-hour excretion of tetrahydro S in 9 patients with proven adrenocortical car- cinoma with excretion of Porter-Silber chro- mogens, 17-ketosteroids, and dehydroepian- drosterone. Whenever excretion of Porter- Silber chromogen was elevated, tetrahydro S was likewise increased. However, when 17- ketosteroids alone were elevated, excretion of tetrahydro S was low.
Discussion. Of the steps leading to synthe- sis of hydrocortisone, 11-beta hydroxylation is generally considered the final one. Excretion of tetrahydro S undoubtedly closely corre- sponds to production of Compound S. Thus, these results suggest that when an adrenocortical carcinoma produces excessive amounts of hydrocortisone, 11-beta hydroxy- lation of Compound S may be the rate limit- ing step in the series of reactions leading to synthesis of hydrocortisone. Whether this is peculiar to adrenocortical carcinoma or is a general consequence of the high rate of pro- duction of hydrocortisone is unanswered by these data. Evidence to support the first al- ternative is the demonstration that a patient with an adrenocortical adenoma excreted only small amounts of tetrahydro S in spite of an excretion of tetrahydro E and tetrahydro F as great as in patients with adrenal carcinoma and Cushing’s syndrome(1). It is apparent, of course, that in the 4 patients without ele- vated urinary Porter-Silber chromogens, only small amounts of tetrahydro S were detected.
Hydroxylation at C17 was not similarly im- peded as no alpha-ketols less polar than di- hydro S were seen in any of the chromato- grams. This would exclude the presence of
t We wish to thank Dr. Thomas F. Gallagher of Sloan-Kettering Institute for the infra-red spectrom- etry.
4-pregnen-21-ol-3, 20-dione (tetrahydrol DOC) in amounts greater than 500 µg per 24 hours. The inconsistency between high ex- cretion of Porter-Silber chromogens and ab- sence of Cushing’s syndrome in patient R.S. could not be attributed to excretion of tetra- hydro S and dihydro S as this patient also ex- creted amounts of tetrahydro E and tetra- hydro F which were well above normal. The occurrence of dihydro S was unexpected. To our knowledge, this compound has not previ- ously been found in urine. As it has been shown that reduction of the C3-keto group is the rate-limiting step in metabolism of the 3-keto, 44 group(6), no dihydro S should be excreted. It was noted in only one patient, R.S., who had extensive hepatic metastases at the time. Whether or not this affected the further metabolism of dihydro S is conjec- tural.
Summary. Tetrahydro S was excreted in amounts ranging from 7 to 44 mg daily in 5
patients with adrenocortical carcinoma exhib- iting excessive production of Porter-Silber chromogens. Dihydro S was presumptively identified in the urine of one of these patients. The amount of tetrahydro S in 4 patients with adrenocortical carcinoma with normal levels of Porter-Silber chromogens was likewise normal.
1. Touchstone, J. C., Bulaschenko, H., Richardson, E. M., Dchan, F. C., J. Clin. Endocrinol. and Metab., 1957, v17, 250.
2. Eberlein, W. R., Bongiovanni, A. M., ibid., 1955, v15, 1531.
3. Rcsselet, J. P., Overland, L., Jailer, J. W., Lieber- man, S., Helvet. Chim. Acta, 1954, v37, 1933.
4. Eberlein, W. R., Bongiovanni, A. M., Arch. Biochem. and Biophys., 1955, v59, 90.
5. Nowaczynski, W., Goldner, M., Genest, J., J. Lab. and Clin. Med., 1955, v45, 818.
6. Tomkins, G. M., Rec. Prog. Horm. Res., 1956, v12, 125.
Received June 26, 1958.
P.S.E.B.M., 1958, v99.
Immunological Tolerance to Male Skin Isografts in Female Mice .* (24325)
T. MARIANI,+ C. MARTINEZ,+ J. M. SMITH AND R. A. GOODS Departments of Physiology and Pediatrics, University of Minnesota Medical School, Minneapolis.
Eichwald and Silmser(1) reported among certain members of inbred strains of mice a histo-incompatibility determined by the sex. This observation has been confirmed by Prehn and Main(2) and Short and Sobey (3) for some strains of mice but not for others. In these studies, isografts from male to female mice of either the A or C57 BL strain are often unsuccessful. When skin isotransplants are made from female to male, male to male or fe- male to female, skin grafts are regularly suc- cessful and resemble autografts. The basis for rejection of male skin isograft by the fe-
male of the same inbred strain has been inter- preted by Eichwald et al.(4) and Snell(5) as the function of an immune response due to a histocompatability gene located on the Y- chromosome. According to this theory the female (XX), which lacks the donor’s isoan- tigen originating from a gene residing on the Y-chromosome, is capable of an immune re- sponse and rejects the skin isotransplant. However, other possible explanations for fail- ure of male skin isotransplants to survive on females have been considered. For example, endocrinological influences, by mechanisms not yet understood, might account for the re- sults observed(4). Reasoning that rejection of male skin by females of the same strain is an immunological phenomenon based on the same principles as those responsible for skin homograft rejection, it seemed possible that the effects of the so-called Y-antigen might be
* Aided by grants from U.S.P.H.S. and Minn. Di- vision of Am. Cancer Soc.
t National Science Faculty Fellow.
# William O’Brien Cancer Research Professor Univ. of Minnesota.
§ American Legion Memorial Heart Research, Prof. of Pediatrics.