Metabolism of testoterone and related steroids in metastatic interstitial cell carcinoma of the testis. M B Lipsett, … , C W Bardin, L M Fishman J Clin Invest. 1966;45(11):1700-1709. https://doi.org/10.1172/JCI105476. Research Article Find the latest version: https://jci.me/105476/pdf Journal of Clinical Investigation Vol. 45, No. 11, 1966 Metabolism of Testosterone and Related Steroids in Metastatic Interstitial Cell Carcinoma of the Testis * M. B. LIPSETT,t G. A. SARFATY, H. WILSON, C. WAYNE BARDIN, AND L. M. FISHMAN (From the Endocrinology Branch, National Cancer Institute, Bethesda, Md.) Interstitial cell carcinoma of the testis is a singu- production rate has been shown to be a conse- larly rare steroid-producing cancer. Of the seven quence of metabolism of dehydroepiandrosterone reported cases (1-7), urinary 17-ketosteroid (17- sulfate. KS) excretion was high in the four cases in which it was measured. Abelson, Bulaschenko, Trom- Methods mer, and Valdes-Dapena (7) fractionated the uri- Routine methods were used to analyze the following: nary 17-ketosteriods and corticoids in one recently urinary 17-KS (8), urinary 17-hydroxycorticoids (9), reported case. There is, however, no comprehen- plasma Silber-Porter chromogens (10), and plasma tes- tosterone (11). sive study of either the production of androgens Gas-liquid chromatography. We carried out gas-liquid or related steroids by this tumor. We have had chromatography (GLC) in a Glowell Chromolab gas the opportunity to study a patient with metastatic chromatograph utilizing a 'Sr ionization detector oper- interstitial cell carcinoma, and we have examined ating at 1,050 v. The coiled glass columns, 6 feet by i inch several related steroids. From i.d., were packed with 2% SE-30 on chromosorb W. the androgens and Trimethylsilyl derivatives were prepared as noted previ- these data, we have concluded that interstitial cell ously (12). carcinoma resembles virilizing adrenal carcinoma in Fractionation of urinary steroids. Urinary steroid sul- its pattern of biosynthesis of C-19 steroids and 21- fates and glucuronides were separated by thin layer deoxypregnane analogues. The high testosterone 1 chromatography in system TN (13) after an ether- ethanol extraction (14). Glucuronides and sulfates were * Submitted for publication April 6, 1966; accepted hydrolyzed separately either with 8-glucuronidase (Keto- July 27, 1966. dase) or by solvolysis (15). In the subsequent dis- Presented in part at the 47th Meeting of the Endo- cussion, steroid sulfates are defined as those steroids crine Society, New York, 1965. having the mobility of steroid monosulfates in TN and t Address requests for reprints to Dr. Mortimer B. hydrolyzed by solvolysis. Glucuronides are defined as Lipsett, Endocrinology Branch, National Cancer Insti- those steroids having the mobility of steroid glucuronides tute, Bethesda, Md. 20014. in TN and released by j8-glucuronidase. Further steps 1 The chemical names for the steroids used in this in purification were chromatography on the silica-alumina paper are as follows: 17,8-hydroxy-androst-4-en-3-one column (16) and group fractionation with Girard re- (testosterone) ; 17a-hydroxy-androst-4-en-3-one (epites- agent T and digitonin. For isolation of individual me- tosterone); 3a-hydroxy-5,B-androstan-17-one (etiocholano- tabolites, the resulting ketonic and nonketonic alpha lone); 3a-hydroxy-5a-androstan-17-one (androsterone); and beta fractions of differing polarity were then chro- 3a,11,8-dihydroxy-51-androstan-3-one (11-hydroxyetiocho- matographed in the appropriate systems listed below. lanolone); 3a-hydroxy-58-androstan-3,11-dione (ll-keto- Paper chromatography systems (after Bush) were as etiocholanolone) ; 3a,11'B-dihydroxy-Sa-androstan-17-one follows: Al-ligroin: methanol: H20 (100: 90: 10); B1 (1l-hydroxy-androsterone); 3,B-hydroxy-androst-5-en-3- -ligroin: toluene: methanol: H20 (75:25:70:30); B1 one (dehydroepiandrosterone, DHA); androst-4,en-3,17- -ligroin: toluene: methanol: H20 (50:50: 70: 30); B4 dione (androstenedione); 3fi,16a-dihydroxy-5-androstan- 17-one (16-hydroxy DHA); androst-5-en-3,8,17P-diol (an- 3,20-dione (cortisol) ; 3ac,11P,17a,21 -tetrahydroxy-5#9- drostenediol); pregn-5-en-3p8,20a-diol (pregnenediol); pregnan-20-one (tetrahydrocortisol); 3a,17a,21-trihy- pregn-5-en-3fi,17a,20a-triol (pregnenetriol) ; 5,6-pregnan- droxy-5fi-pregnan-11,20-dione (tetrahydrocortisone) ; 3a, 3a,20a-diol (pregnanediol); 5P-pregnan-3a,17a,20a-triol 11P,17a,21-tetrahydroxy-5a-pregnan-20-one (allotetrahy- (pregnanetriol); 3a,17a-dihydroxy-5p8-pregnan-20-one drocortisol) ; 3a,17a,21 -trihydroxy-5Sf-pregnan-20-one (17-hydroxypregnanolone); 3a,17a,20a-trihydroxy-59- (tetrahydro substance S); 3-hydroxyestratrien-1,3,5 (10)- pregnan-l1-one (11-ketopregnanetriol); 5a-androst-16-en- 17-one (estrone); 1,3,5 (10-estratriene-3,17P-diol (estra- 3cr-ol (androstenol); llB,17a,21-trihydroxy-pregn-4-en- diol); 1,3,5 (10)-estratriene-3,16a,17P-triol (estriol). 1700 ANDROGEN METABOLISM IN TESTICULAR INTERSTITIAL CELL CARCINOMA 1,701 NFON-KETONIC A (Mg. per Day) SULFATES 5-1PTROLt - F TRIOL 3.7 1.2 I~~~~~ 0.2 0.4 0.6 0.8 1.0 02 0.4 0.6 0.8 1.0 R T VS CHOLEcSTANE n eisll Testic. Carc FIG. 1. GAS-LIQUID CHROMATOGRAMS OF NONKETONIC-3P8-HYDROXYSTEROIDS. Glu- curonides and sulfates were separated before hydrolysis, and each column fraction was treated with Girard's reagent T and digitonin. 5-A'diol = 5-androstenediol; 5-P'diol = 5-pregnenediol; and 5-P'triol = 5-pregnenetriol. -toluene: methanol: H20 (100: 50: 50) ; and C-toluene- Estrogens. The three classical urinary estrogens were ethyl acetate: methanol: Hs0 (90: 10: 50: 50). isolated from the phenolic fraction as suggested by Engel Thin layer silica gel systems were as follows: T4-ben- (18). Tritiated estrone, estradiol, and estriol 2 were zene: ethyl acetate (40: 60); T6-benzene: ethyl acetate added to the phenolic extract to correct for losses oc- (60: 40); TE-benzene: ethanol (90: 10); and TN- curring after P-glucuronidase hydrolysis. We separated ethyl acetate: ethanol: ammonia (50: 50: 10). estriol from estrone and estradiol by partitioning between 3a-Hydroxyketonic steroids. Etiocholanolone and an- petroleum -ether-benzene and water. Estrone and estra- drosterone were quantified as the trimethylsilyl ethers by diol were chromatographed on thin layer silica gel plates GLC of the appropriate fractions after chromatography in TE, and estriol was chromatographed in 100%o ethyl in Al and T6. The 11-oxo-17-ketosteroids were measured acetate. All samples were acetylated and chromato- as the free steroids by GLC after chromatography in B1 graphed on thin layer silica gel. Chloroform was used to and T4. develop the chromatograms. After saponification, paper 33a-Hydroxy nonketonic steroids. Androstenol was chromatography was performed as follows: estrone, Bi; measured by GLC of the least polar nonketonic alpha estradiol, Bi; and estriol, C. Subsequent thin layer fraction (17). Pregnanediol and pregnanetriol were iso- chromatography of estrone and estradiol in T6 and es- lated from appropriate nonketonic alpha fraction by chro- triol in T4 was performed. Gas-liquid chromatography matography in T4 and estimated by GLC as the free ster- of these fractions resulted in single clean peaks for es- oids. We could not identify li-ketopregnanetriol by trone and estriol. Estradiol could- not be identified. GLC after chromatography in T4. 16-Hydroxy DHA. Since this steroid has been found AY-3fi-Hydroxy nonketonic steroids. Androstenediol, only rarely in urine, its identity was assured by the cor- pregnenediol, and pregnenetriol were measured by GLC respondence of the isolated material with authentic stand- directly after column chromatography and Girard and ard3 in paper, thin layer, and gas-liquid chromatography digitonin fractionation. Figure 1 is an example of the as the free steroid and the acetate. The color changes chromatograms obtained from two such fractions. The produced by the Allen reagent and the sulfuric acid clearly defined peaks, well separated from contaminants, spectra were identical for the presumptive and authentic permitted accurate measurements. 16-hydroxy DHA. Cortisol metabolites. Cortisol and its metabolites, tetrahydrobortisol, tetrahydrocortisone, and allotetrahy- 2 We wish to thank Dr. Mortimer Levitz, New York drocortisol, were measured together as phenylhydrazine University Medical School, -for a gift of estriol-'H, chromogens after silica-alumina column fractionation and 3We wish to thank Dr. D. Fukushima, Institute for thin layer chromatography in TE. Tetrahydro sub- Steroid Research, New York, and Dr. A. Colas, Uni- stance was sought in another column fraction after pa- versity of Oregon Medical School, Portlan4 for gifts of per chromatography in B4. 16-hydroxy DHA. - 1702 LIPSETT, SARFATY, WILSON, BARDIN, AND FISHMAN obtained at 50, 60, and 70 minutes of the infusion. The plasma was separated, and 400 dpm of testosterone-14C and 50 #g of testosterone were added. Testosterone ex- tracted from the plasma was purified through five chro- STANDARD 16a -OH-DH'A matography systems. The specific activities and the 2 ug 'H/f'C ratios agreed within 5% during the last three chromatographies, in which the testosterone was mea- sured successively as the acetate, the free alcohol, and androstenedione. We determined the production rates of DHA and DHA sulfate with 20.45 Ac of DHA sulfate-8H and 2.77 juc of DHA-14C injected intravenously over a 5-minute pe- riod. Urine was collected for 3 days, processed as de- scribed above, and the specific activity of the DHA sul- fate and DHA glucuronide was measured. 16a-OH-DHA FROM Specific activities of DHA, 16-hydroxy DHA, and GLUCURONIDE FRACTION OF URINE EXTRACT androstenediol. The appropriate fractions containing J.AT. a DHA, 16-hydroxy DHA, and androstenediol were chro- matographed on 45-cm thin layer silica gel plates in T4, and they were then chromatographed in systems Al, B1, and Bi, respectively. The DHA was chromatographed on T6, and the 16-hydroxy DHA and androstenediol were chromatographed in T4.