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Biosynthesis of steroid hormones in human endocrine tissue and in the rat testis Ford, Henry Crawford 1969

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BIOSYNTHESIS OF STEROID HORMONES IN HUMAN ENDOCRINE TISSUE AND IN THE RAT TESTIS by Henry Crawford Ford B.A., Wesleyan University, 1954 M.D., University of Pennsylvania, 1958 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Biochemistry We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1969 In presenting t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree tha permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s re p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of B vo cA/te-wus^vi The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada Date f^rewvVer Z H ; i ABSTRACT This thesis reports the results of studies on steroid metabolism i n a subject with an adrenocortical carcinoma and hypoglycemia, i n the testes obtained from a subject with v i r i l i z i n g male pseudohermaphroditism and i n the testes of the normal rat. Incubations of cell-free homogenates of an adrenocortical carcin-oma from a 51 year old female with severe hypoglycemia were performed using ^H-pregnenolone and '^C-progesterone as substrates. Transformation 3 of H-pregnenolone to progesterone, dehydroepiandrosterone and andros-tenedione was observed; no metabolites of ^ AC-progesterone were detected. The excretion rate i n urine of 3°<- ,17,21-trihydroxy-5,s -pregnan-20-one, a metabolite of cortexolone, was elevated which suggests that a defect i n 11 £ -hydroxylase activity was present. The excretion rates i n urine of tot a l 17-ketosteroids, 17-hydroxycorticoids and 17-ketogenic steroids were elevated; the excretion rates of testosterone, dehydroepiandrosterone, preg-nandiol, pregnanetriol and free Cortisol were not elevated. The etiology of the hypoglycemia that may accompany some adrenocortical tumors remains unknown. I t was not possible to relate the results of the investigations of steroid metabolism reported herein to the hypoglycemia that was present. Steroid biosynthesis i n v i t r o was investigated i n testes obtained during puberty from a 14- year old subject with v i r i l i z i n g male pseudo-hermaphroditism. Cell-free homogenates of gonadal tissue e f f i c i e n t l y metabolized %-pregnenolone, -progesterone and 14c -androstenedione to testosterone; formation of estrone and estradiol-17,S was not detected. 16* -Hvdroxyprogesterorie was formed from both %-pregnenolone and ^ C-progesterone. The results are similar to those of others who have i i investigated the steroidogenic capacity of gonadal tissue i n patients with male pseudohermaphroditism and feminization at puberty. A defect i n the formation of progesterone from pregnenolone has been suggested to explain the results of a previous study i n which the gonadal tissue obtained from a patient with v i r i l i z i n g male pseudohermaphroditism was 3 incubated with H-pregnenolone as substrate. In the investigations r e -ported herein, transformationsof %-pregnenolone to testosterone and and-rostenedione occurred both v i a 17-hydroxypregnenolone and dehydroepiand-rosterone and v i a progesterone and 17-hydroxyprogesterone. The f a i l u r e of patients with v i r i l i z i n g male pseudohermaphroditism to masculinize during embryonic development contrasts with the v i r i l i z a t i o n that occurs during puberty. A biochemical abnormality may exert a transient effect during embryonic development. Al t e r n a t i v e l y , the s e n s i t i v i t y to andro-genic hormones may be subnormal i n ce r t a i n tissues and normal i n other tissues of patients with v i r i l i z i n g male pseudohermaphroditism. The biosynthesis of testosterone from progesterone and pregnenolone was investigated i n the rat t e s t i s . Time studies were performed using c e l l - f r e e homogenates and %-progesterone and -17-hydroxyprogesterone i n combination as substrates. I t was demonstrated that the side-chain cleavage of 17-hydroxyprogesterone i s the r a t e - l i m i t i n g reaction i n the biosynthesis of testosterone from progesterone and the evidence suggested that 17-hydroxyprogesterone was present as a bound intermediate (at least i n part). The progesterone 17-hydroxylase and the 17-hydroxyprogesterone side, chain cleavage enzyme of the rat t e s t i s can be s o l u b i l i z e d by treatment of l y o p h i l i z e d microsomes with Triton N-101. Both enzymes displayed maxi-mal a c t i v i t y at pH 6.8 and at 37°. Progesterone rather than pregnenolone i s the preferred substrate for the 176* -hydroxylase. Either NADH or i i i NADPH can serve as the reductant for active 17-hydroxylation of proges-terone and for side-chain cleavage of 17-hydroxyprogesterone. The solu-ble fraction contains NADPH dehydrogenase, non-heme iron protein and cy-tochrome P-450. The presence of these compounds i n association with the 17«. -hydroxylase and the side-chain cleavage enzyme ac t i v i t i e s suggests that these reactions are catalyzed by elaborate enzymatic systems analo-gous to those required for 11£ -hydroxylation and cholesterol side-chain cleavage i n adrenal mitochondria. i v TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS i v LIST OF TABLES i x LIST OF FIGURES xiv GLOSSARY x v i i ACKNOWLEDGEMENTS xx GENERAL INTRODUCTION 1 GENERAL METHODS 7 (a) Chromatography 7 (i) S i l i c a gel column chromatography 7 ( i i ) Paper chromatography 7 ( i i i ) Thin layer chromatography 8 (b) Detection and quantitation of steroids 8 (c) Criteria employed to assess the purity of compounds and methods used to calculate the minimal per cent conversion of substrate to product 10 (i) Crystallizations 10 ( i i ) Paper chromatography 10 (d) Assay of radioactivity 11 (i) Liquid s c i n t i l l a t i o n spectrometry 11 ( i i ) Gas flow detection 11 (e) Separation by solvent partition 12 (i) Hexane-aqueous methanol partition 12 ( i i ) Partition into neutral, "estrone-estradiol" and " e s t r i o l " fractions 12 V Page (f) Reactions 1 3 (i) Acetylation 1 3 ( i i ) Girard 1 3 (g) Purification of unlabeled steroids added as carriers 14 (h) Solvent purification 14 PART I STUDIES ON A SUBJECT WITH ADRENOCORTICAL NEOPLASM AND ! HYPOGLYCEMIA: STEROIDS EXCRETED IN THE URINE AND STER-OID METABOLISM BY THE TUMOR IN VITRO 16 INTRODUCTION 16 (a) Metabolism of adrenal steroids i n normal subjects and i n patients with adrenocortical neoplasms 16 (b) Hypoglycemia associated with extra-pancreatic neo-plasms that are not of adrenal origin 17 (i) Enhanced glucose disappearance due to the presence of excessive insulin or insulin-l i k e material i n the blood 17 ( i i ) Excessive glucose consumption by the tumor IB ( i i i ) Deficient gluconeogenesis due to a specific effect of tryptophan metabolites IS (iv) Other hypotheses 19 (c) Hypoglycemia associated with adrenocortical tumors 19 MATERIALS AND METHODS 21 (a) Patient 21 '. (b) Tumor 22 (c) Incubation conditions 22 (d) Extraction, resolution and purification procedures 23 (e) Assay of steroids in urine 24 RESULTS. 25 DISCUSSION 26 SUMMARY AND CONCLUSIONS 30 v i Page PART II STEROID BIOSYNTHESIS IN VITRO BY THE GONADS OF A PATIENT WITH VIRILIZING MALE PSEUDO-HERMAPHRODITISM 58 INTRODUCTION 58 (a) General considerations and classification 58 (b) Steroid biosynthesis by the normal testis 59 (c) Studies i n v i t r o of gonads obtained from patients with male pseudohermaphroditism 59 MATERIALS AND METHODS 6 0 (a) Patient 6 0 (b) Gonads 61 (c) Incubation conditions 61 (d) Extraction, resolution and purification procedures 62 RESULTS 64 (a) Substrate "^C-androstenedione incubation 64 (b) Substrate "^C-progesterone incubation 64 (c) Substrate %-pregnenolone incubation 65 (d) Substrate "^C-mevalonate incubations 65 DISCUSSION 65 SUMMARY AND CONCLUSIONS 73 PART III STUDIES ON STEROID BIOSYNTHESIS IN VITRO BY THE RAT TESTIS 109 PRELIMINARY EXPERIMENTS AND TIME STUDIES 109 (a) Introduction 109 (b) Materials and methods 110 (i) Rats 110 ( i i ) Incubations with quartered testes 110 ( i i i ) Incubations with cell-free homogenates 113 (iv) Time studies U 4 v i i Page (c) Results 117 (d) Discussion 120 PREPARATION AND PROPERTIES OF A SOLUBLE SYSTEM OBTAINED FROM RAT TESTICULAR MICROSOMES THAT CATALYZES THE TRANS-FORMATION OF PROGESTERONE TO 17-HYDROXYPROGESTERONE AND ANDROGENS ' 137 (a) Introduction 137 (b) Materials and methods 137 (i) Rats 137 ( i i ) Tissue preparation 138 ( i i i ) Radioactive substrates 139 (iv) Incubation conditions 140 (v) Extraction, resolution and purification procedures ' 140 (vi) Column chromatography 142 (vii) Other procedures 143 (c) Results 144 (i) Preparation of a soluble fraction con-taining 17** -hydroxylase and side-chain cleavage enzyme act i v i t y 144 ( i i ) Other enzymatic a c t i v i t i e s i n the soluble fraction 147 ( i i i ) Effects of changes of temperature and pH on the 17<* -hydroxylase and side-chain cleavage enzyme a c t i v i t i e s of the soluble fraction 148 (iv) Resolution and purification of components of the soluble fraction 148 (d) Discussion 152 STUDIES ON THE PYRIDINE NUCLEOTIDE COENZYME SPECIFICITIES OF THE PROGESTERONE 17-HYDROXYLASE, 17-HYDROXYPROGESTERONE SIDE-CHAIN CLEAVAGE ENZYME AND 17/8 -HYDROXYSTEROID DEHYDROGENASE OF THE RAT TESTIS 175 v i i i Page (a) Introduction 175 (b) Materials and methods 176 (i) Rats 176 ( i i ) Tissue preparation 176 ( i i i ) Radioactive substrates 176 (iv) Incubation conditions 177 (v) Extraction, resolution and purification procedures 178 (vi) Chromatography of pyridine nucleotide coenzymes 179 (vii) Other procedures 179 (c) Results 180 (d) Discussion 184 SUMMARY AND CONCLUSIONS 191 BIBLIOGRAPHY 198 i x LIST OF TABLES Table Page I Data concerning previously reported cases of adrenocortical tumor and hypoglycemia 32 II Excretion rates of 17-hydroxysteroids i n the urine of patients with adrenocortical tumors and hypo-glycemia 33 I I I Excretion rates of 17-ketosteroids i n the urine of patients with adrenocortical tumors and hypo-glycemia 34 IV Excretion rates of steroids in the urine of patients with adrenocortical tumors and hypoglycemia 35 V Studies on the etiology of the hypoglycemia i n patients with adrenocortical tumors 36 VI Substrate "^C-progesterone incubation: column absorption chromatography of the 90% aqueous methanol fraction 37 VII Substrate -^C-progesterone incubation: further paper chromatography of eluates containing carrier andros-tenedione, 17-hydroxyprogesterone and testosterone acetate 38 VIII Substrate %-pregnenolone incubation: column ab-sorption chromatography of the aqueous methanol fraction 39 IX Substrate -'H-pregnenolone incubation: further paper chromatography of eluates containing testosterone acetate, 17-hydroxyprogesterone, Cortisol and pro-gesterone 40 X Substrate %-pregnenolone incubation: thin layer chromatography 41 XI Substrate %-pregnenolone incubation: paper chroma-tography of compounds previously separated by thin layer chromatography 42 XII Substrate %-pregnenolone incubation: c r y s t a l l i -zations of androstenedione 43 XIII Substrate %-pregnenolone incubation: c r y s t a l l i -zations of testosterone acetate 44 X Page XIV Substrate 3H-pregnenolone incubation: c r y s t a l l i -zations of dehydroepiandrosterone 45 XV Substrate %-pregnenolone incubation: c r y s t a l l i -zations of 17-hydroxyprogesterone and progesterone 46 XVI Substrate %-pregnenolone incubation: c r y s t a l l i -zations of Cortisol, aldosterone and cortisone 47 XVII Results of incubations of tumor tissue with sub-strate •^•C-progesterone and substrate 3H-preg-nenolone 48 XVIII Steroids excreted i n the urine 49 XLX Products i d e n t i f i e d following incubations of gonads from patients with male pseudohermaphro-dit i s m and feminization with radioactive pro-gesterone as substrate 74 XX Products i d e n t i f i e d following incubations of gonads from patients with male pseudohermaphroditism and feminization with substrate pregnenolone 75 XXI Products i d e n t i f i e d following incubations of gonads from patients with male pseudohermaphroditism and feminization with radioactive testosterone arid radioactive androstenedione as substrates 76 XXII Products i d e n t i f i e d following incubations of gonads from patients with male pseudohermaphroditism and feminization with radioactive dehydroepiandrosterone and radioactive dehydroepiandrosterone sulfate as substrates 77 XXIII Products i d e n t i f i e d following incubations of gonads from patients with male pseudohermaphroditism and feminization with radioactive acetate, radioactive 17-hydroxyprogesterone and radioactive 17-hydroxy-pregnenolone as substrates 78 XXIV Results of incubations of gonads from patients with v i r i l i z i n g male pseudohermaphroditism with radioactive pregnenolone and radioactive 17-hydroxypregnenolone as substrates 79 XXV Per cent of substrate r a d i o a c t i v i t y present i n fractions following extraction, p a r t i t i o n and separation prodedures 80 XXVI. Substrate •^C-androstenedione incubation: c r y s t a l l i -zations of testosterone 81 x i XXVII Substrate "^C-androstenedione incubation: crys-tallizations of estradiol-17/9 XXVIII Substrate •^C-androstenedione incubation: crys-tallizations of estrone XXIX Substrate -^C-progesterone incubation: c r y s t a l l i -zations of 16<* -hydroxyprogesterone XXX Substrate "^C-progesterone incubation: c r y s t a l l i -zations of androstenedione XXXI Substrate "^C-progesterone incubation: c r y s t a l l i -zations of testosterone acetate XXXII Substrate "^C-progesterone incubation: c r y s t a l l i -zations of 17-hydroxyprogesterone XXXIII Substrate -^C-progesterone incubation: c r y s t a l l i -zations of estrone and estradiol-17/3 XXXIV Substrate %-pregnenolone incubation: further paper chromatography of eluates containing carrier steroids XXXV Substrate %-pregnenolone incubation: c r y s t a l l i -zations of 17-hydroxypregnenolone XXXVI Substrate %-pregnenolone incubation: c r y s t a l l i -zations of 16**- -hydroxyprogesterone XXXVII Substrate %-pregnenolone incubation: c r y s t a l l i -zations of dehydroepiandrosterone XXXVIII Substrate ^H-pregnenolone incubation: c r y s t a l l i -zations of androstenedione 3 XXXIX Substrate ^H-pregnenolone incubation: c r y s t a l l i -zations of testosterone acetate XL Substrate ^H-pregnenolone incubation: c r y s t a l l i -zations of 17-hydroxyprogesterone XLI Substrate ^H-pregnenolone incubation: c r y s t a l l i -zations of progesterone XLII Substrate %-pregnenolone incubation: c r y s t a l l i -zations of estradiol-17/9 Page 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 x i i Substrate -^-pregnenolone incubation: crystal-lizations of estrone Results of incubations of testes from a patient with v i r i l i z i n g male pseudohermaphroditism Results of incubations of preparations of rat testes with -^C-progesterone as substrate Incubations with %-progesterone and "^C-17-hydroxyprogesterone i n combination as sub-strates: results of time studies Incubations with %-progesterone and "^ fG-17-hydroxyprogesterone i n combination as sub-strates: results of time studies Distribution of radioactivity following incu-bations of preparations of rat testes Results of incubations of supernatants obtain-ed following high speed centrifugation of Triton N-101-treated lyophilized microsomes Distribution of radioactivity following incu-bations of fractions obtained after centrifug-ation (105,000 x gf 2 hours) of lyophilized microsomes treated with 0.2% Triton N-101 Distribution of radioactivity following incu-bations of the soluble fraction- obtained after treatment of lyophilized microsomes with 0.2% Triton N-101 and ultrasound Studies on the effects of the composition of the incubation medium on progesterone 17-hydroxylation, 17-hydroxyprogesterone side-chain cleavage and androstenedione 17-oxo reduction The per cent of substrate radioactivity i n isolated compounds formed during incubations of the soluble fraction with radioactive ster-oids Distribution of radioactivity following incu-bations of the precipitate obtained by t%Ofo saturation of the soluble fraction with am-monium sulfate x i i i Specific activity of the progesterone 17-hydroxylas e Non-heme ironeand protein content of wash-ed and unwashed mitochondria and microsomes Effects of pyridine nucleotide coenzymes on the distribution of radioactivity following incubations of lyophilized microsomes with •^C-androstenedione as substrate Effects of NADH and NADPH on the distribution of radioactivity following incubations of microsomes with -%-progesterone as substrate Effects of NADH and NADPH on the distribution of radioactivity following incubations of lyophilized microsomes and soluble fractions with ^H-progesterone as substrate Effects of NADH and NADPH concentrations on the distribution of radioactivity following incubations of lyophilized microsomes with 3H -progesterone as substrate xiv LIST OF FIGURES Figure Page 1. General outline of steroid biosynthetic pathways in the adrenal 3 2. Metabolism of Cortisol 4 3. Metabolism of androgens 5 4. Biosynthesis of androgens 6 5. Substrate ^"C-progesterone incubation: flow sheet for extraction, 90% aqueous methanol-hexane pa r t i t -ion, column adsorption chromatography and i n i t i a l paper chromatography of fractions 5-7 of the column eluate 50 6. Substrate •^fC-progesterone incubation: resolution and purification of progesterone 51 7. Substrate "^C-progesterone incubation: paper chroma-tography of selected eluates prior to and following the addition of unlabeled carrier androstenedione, testosterone and 17-hydroxyprogesterone 52 8. Substrate ^C-progesterone incubation: paper chroma-tography of fractions 8 and 9 from the column chroma-togram 53 3 9. Substrate H-pregnenolone incubation: flow sheet for extraction, 90$ aqueous methanol-hexane partition, column absorption chromatography, and i n i t i a l paper chromatography of fractions 5-7 of the column eluate 54 3 10. Substrate ^H-pregnenolone incubation: paper chroma-tography of eluates containing pregnenolone and androstenedione 55 11. Substrate %-pregnenolone incubation: paper chroma-tography of fractions 8 and 9 of the column chroma-togram 56 12. Substrate ^H-pregnenolone incubation: paper chroma-tography of eluates containing carrier testosterone and dehydroepiandrosterone 57 13. Substrate "^C-androstenedione incubation: paper chromatography of the neutral fraction 1D0 14. Substrate -^-C-androstenedione incubation: paper chromatography of androstenedione 101 XV Page 15. Substrate -progesterone incubation: paper chromatography of the neutral fraction 102 16. Substrate -^C-progesterone incubation: paper chroma-tography of testosterone acetate and 17-hydroxypro-gesterone 103 17. Substrate ^ H-pregnenolone incubation: paper chroma-tography of the neutral fraction 104 18. Substrate ^ H-pregnenolone incubation: paper chroma-tography of the eluate of the origin area of the neutral fraction paper chromatogram 105 19. Substrate ^ H-pregnenolone incubation: paper chroma-tography of testosterone-17-hydroxyprogesterone eluate 106 20. Substrate %-pregnenolone incubation: paper chroma-tography of pregnenolone-androstenedione eluates 107 21. Substrate •^C-mevalonate incubations: paper chroma-tography of neutral fractions 108 22. Incubations of cell-free homogenates of rat testes with 3H_progesterone and l^C-17-hydroxyprogesterone i n combination as substrates: results of time studies 134 23. Incubations of cell-free homogenates of rat testes with ^H-progesterone and ^C-17-hydroxyprogesterone i n combination as substrates: results of time studies 135 24. Incubations of cell-free homogenates of rat testes with 3H-progesterone and 14C-17-hydroxyprogesterone i n combination as substrates: results of time studies 136 25. The effect of pH on the activities of the 17* -hydroxylase and the side-chain cleavage enzyme in the soluble fraction 166 26. The effect of temperature on the activities of the 17<sc -hydroxylase and the side-chain cleavage enzyme in the soluble fraction 167 27. Absorbance spectrum of the precipitate obtained by 1+Ofo saturation of the soluble fraction with ammon-ium sulfate 168 x v i Page 28. Sephadex G-200 column chromatography I69 29. ' Ultraviolet spectrum of Triton N-101 170 30. Sephadex G-200 column chromatography 171 31. DEAE-Sephadex A-25 column chromatography 172 32. DEAE-Sephadex A-25 column chromatography 173 33. Sephadex G-200 column chromatography 174 34. Column ion exchange chromatography of NADH and NADPH 197 x v i i GLOSSARY (a) Steroids Aldosterone, 11/8, 21-dihydroxy-18-oxo-pregn-4-ene-3, 20-dione. Androstenediol, androst-5-ene-3,r3 , 17/3 - d i o l . Androstenediol sulfate, 3/3 -yl-suLfate-androst-5-en-17/3 - o l . Androstenedione, androst^4-ene-3,17-dione. 5/S -Androsterone, see etiocholanolone. Androsterone, 3 ^ -hydroxy-^**- -androstan-17-one. Cholesterol, cholest-5-en-3/S ~ o l . Cholesterol sulfate, cholest-S-en-Sy 8 -yl-sulfate. Cortexolone, 17 ,21-dihydroxypregn-4-ene-3,20-dione. Cortexolone acetate, 21-acetoxy-17 -hydroxypregn-4-ene-3,20-dione. Corticosterone, 11(3 ,21-dihydroxypregn-4-ener3,20-dione. Cortisol, llyS ,17,21-trihydroxypregn-4-ene-3,20-dione. Cortisone, 17,21-dihydroxypregn-4-ene-3,11,20-trione. Dehydroepiandrosterone, 3 / -hydroxyandrost-5-en-17-one. Dehydroepiandrosterone sulfate, 3p -yl-sulfate-androst-5-en-17-one. De oxycorticosterone, 21-hydroxypr egn-4-ene-3,20-dione. Deoxycorticosterone acetate, 21-acetoxypregn-4-ene-3,20-dione. 11-Deoxycortisol, see cortexolone. DHEA, see dehydroepiandrosterone. Equilinin, 3-hydroxyestra-l,3,5(lO), 6,8(9)-pentaen-17-one. Estradiol-17 /3 , estra-l ,3^5(lO)-triene-3,17^ - d i o l . Estrone, 3-hydroxyestra-l,3,5(lO)-trien-17-one. Etiocholanolone, 3-hydroxy - 5p -androstan-17-one. x v i i i 6 p -Hydroxyandrostenedione, 6 f -hydroxyandrost-4-ene-3,17-dione. 11^ -Hydroxyandrostenedione, 11^ -hydroxyancb?ost-4-ene-3,17-dione. 16 ^  -Hydroxyandrostenedione, 16 <<• -hydroxyandrost-4-ene-3,17-dione. 19-Hydroxyandrostenedione, 19-hydroxyandrost-4-ene-3,17-dione. :l6«C-Hydroxypregnenolone, 3 p , 16 -dihydrcex:ypregn-5-en-20-one. 17-Hydroxypregnenolone, 3 p, 17 -dihydroxypregn-5-en-20-6ne. 16*6 -Hydroxyprogesterone, 16«< -hydroxypregn-4-ene-3,20-dione. 17-Hydroxyprogesterone, 17 -hydroxypregn-4-ene-3,20-dione. 6p -Hydroxytestosterone, 6^ ,17/?-dihydroxyandrost-4-en-3-one. 19-Hydroxytestosterone, 17/3 ,19-dihydroxyandrost-4-en-3-one. Pregnanediol,5^ -pregnane-3 °<- ,20*--diol. Pregnanetriol, 5J5 -pregnane-3^ , 17, 20<*-triol. Pregnenolone, 3/3 -hydroxypregn-5-en-20-one. Pregnenolone sulfate, 3fi -yl-sulfate-pregn-5-en-20-one. Progesterone, pregn-4-ene-3,20-dione. Testosterone, 17/5 -hydroxyandrost-4-en-3-one. Testosterone acetate, 17^ -acetoxyandrost-4-en-3-one. Tetrahydrocortisol, 3"*- fliP ,17,21-tetrahydroxy-5p -pregnan-20-one. Tetrahydrocortisone, 3*- ,17,21-trihydroxy-5/S -pregnane-11,20-dione. THE, see tetrahydrocortisone. THF, see tetrahydrocortisol. THS, 3* ,17,21-trihydroxy-5/8 -pregnan-20-one. xix (b) Solvents and reagents Girard's reagent T, hydrazino-carbonyl-methyl-trimethyl ammonium chloride. Ligroin, light petroleum ether (b.p. 80-100°). NAD , oxidized nicotinamide-adenine dinucleotide. NADH, reduced NAD . NADP , oxidized nicotinamide-adenine dinucleotide phosphate. NADPH, reduced NADP . Py* pyridine. (c) Paper chromatographic solvent systems BI, Bush BI. B5, Bush B5. BA, Bush A. B/F, benzene-formamide. L/PG, ligroin-propylene glycol. T/PG> toluene--propylene glycol. (d) Miscellaneous Ac, acetyl group, b.p., boiling point, cpm, counts per minute. dpm, disintegrations per minute. S.A., specific activity, v;, vol; volume, w, wt; weight. XX ACKNOWLEDGEMENTS I am deeply grateful to Dr. V.J. 0 ,Donnell for his guidance during the course of the investigations reported herein and for his assistance i n the preparation of this manuscript. I wish to thank Mrs. LaVerne Shortt for her conscientious typing. I would l i k e to acknowledge the Medical Research Council of Canada and the United States Public Health Service for financial support i n the form of fellowships awarded to me during these studies. The re-search was supported by grants to Dr. V.J. O'Donnell from the Medical Research Council of Canada and from the National Cancer Institute of Canada. 1 GENERAL INTRODUCTION This thesis reports the results of studies on steroid metabolism i n a subject with adrenal cortical carcinoma and hypoglycemia (Part I ) , i n the testes obtained from a subject with v i r i l i z i n g male pseudoherma-phroditism (Part II) and i n the testis of the normal rat (Part I I I ) . The investigations involving the two abnormal human subjects were undertaken to extend the meager knowledge that i s currently available regarding ster-oid metabolism i n the rare disorders that were present and to search for biochemical explanations for the c l i n i c a l manifestations that the patients exhibited. Furthermore, abnormal tissue may display distortions of the normal metabolic pathways that exaggerate subtle relationships that are d i f f i c u l t to observe i n normal tissues. Both the anabolism and catabol-ism of steroid hormones were studied i n the patient with adrenal cortical carcinoma and hypoglycemia; the investigations encompassed the steroid bio-synthetic pathways of the adrenal (Fig. 1) as well as the metabolism of Cortisol and related compounds (Fig. 2) and the metabolism of androgens (Fig. 3). The biosynthesis of androgens (Fig. 4) and the formation of estrogens by aromatization of ring A of androstenedione and testosterone were investigated i n the testes obtained from the patient with v i r i l i z i n g male pseudohermaphroditism. The formation of testosterone from progesterone (Fig. 4) consti-tutes the segment of the pathway of androgen biosynthesis that i s the principal subject of the studies reported i n Part III of the thesis. The results of preliminary experiments on the metabolism of radioactive pro-gesterone and 17-hydroxyprogesterone by the rat testis i n vi t r o were used 2 to design further investigations of the enzyme systems that catalyze the 17-hydroxylation of progesterone, the side-chain cleavage of 17-hydroxy-progesterone and the reduction of the 17-oxo group of androstenedione. CORTISOL ESTRONE Fig. 2. Metabolism of Cortisol Itfl-HYDROXY- Hj8-HYDROXY-5/9- DIHYDROCORTISOL DIHYDROCORTISONE M-KETO- ll-KETO-5/3-ANDROSTERONE ANDROSTERONE I I ANDROSTERONE ANDROSTERONE CORTOL 0-CORTOL CORTOLONE /3-CORTOLONE F i g . 3. Metabolism of androgens A N D R O S T A N E - 3 a , 17/3-DIOL 5 / 3 - A N D R O S T A N E - 3 a , l 7 / J - DIOL 6 Fig. 4. Biosynthesis of androgens P R O G E S T E R O N E P R E G N E N O L O N E 1 7 - H Y D R O X Y P R O G E S T E R O N E I 7 - H Y D R 0 X Y P R E G N E N 0 L 0 N E T E S T O S T E R O N E A N D R O S T E N E D I O L 7 GENERAL METHODS (a) Chromatography (i) S i l i c a gel column chromatography: S i l i c a gel (Davidson, grade 923) was dried i n an oven at 90° for 2 hours and added to the column as a slurry i n hexane. Hexane was then run through the column for 1 hour. The experimental sample was dried i n an evacuated dessicator overnight at room temperature and taken up i n a minimal volume of a hexane-benzene solution (1:1, by vol) and add-ed to the column. Stepwise elution was then performed using hexane-ben-zene (100 ml minus the volume used to transfer the sample), benzene with increasing proportions of ethyl acetate, ethyl acetate with increasing proportions of methanol and, f i n a l l y , methanol alone. The volume of elu-ent of each composition was 100 ml. Anhydrous hexane, ethyl acetate and benzene were used. A glass tube of 1 cm internal diameter was used and the height of the s i l i c a gel column was 18-21 cm. ( i i ) Paper chromatography: Whatman no. 1 chromatography paper was washed with metha-nol and dried prior to use. The following solvent systems were used: l i -groin-propylene glycol ( l ) j benzene-formamide ( l ) ; toluene-propylene gly-col (2); ligroin-methanol-water (5:4:1, by vol), Bush A (3); benzene-metha-nol-water (2:1:1, by vol), Bush B5 (3); and toluene-ligroin-methanol-water. (5*5:7:3, by vol), Bush BI (3). Paper strips were impregnated with a 40-50$ methanol solution of formamide or propylene glycol prior to the appli-cation of the sample. Following chromatography tfietchromatograms were 8 dried for 12-24 hours at room temperature. Chromatography using the Bush systems was initiated after 6-18 hours equilibration. A l l paper chroma-tography was performed at room temperature in a descending direction. Following chromatography, the compounds were eluted i n the following man-ner: a rectangular portion of the paper that contained the material to be eluted was suspended over a collecting vessel and methanol (15 ml) was allowed to flow slowly on to the upper end of the paper. Large zones were cut into smaller pieces prior to elution. ( i i i ) Thin layer chromatography: Glass plates coated with s i l i c a gel G (according to Stahl) and Eastman Kodak chromatogram sheets (type K301R2) were employed for thin layer chromatography. Radelin GS-115 phosphor was extracted with metha-nol i n a Soxlet apparatus and dried. To prepare the chromatographic plates, 50 g of s i l i c a gel G, 25 mg of Radelin GS-115 phosphor and 100 ml of dis-t i l l e d water were mixed and the slurry spread on 20 x 20 cm glass plates with a Desaga apparatus. The plates were then dried at 1 0 0 ° and stored i n a dessicator cabinet. Prior to sample application the chromatogram sheets and the coated glass plates were washed with methanol and dried. Following chromatography on coated glass plates, the s i l i c a ;gel G that contained the steroids was scraped from the plates and leached with metha-nol ( 3 x 5 ml). The chromatogram sheets were cut and eluted i n the same way that was used for paper chromatograms. (b) Detection and quantitation of steroids Steroids containing the £>^ -3-ketone grouping were detected i n paper and thin layer chromatograms by viewing the chromatogram under short wave ultraviolet light (Chromato-Vue, model C-3, Ultra-violet Products 9 Inc., San Gabriel, California). A -Steroids were detected by treatment of the paper or thin layer chromatogram with an ethanol solution of phos-phomolybdic acid (4). In some cases, a narrow strip cut from the experi-mental chromatogram was used; otherwise, chromatograms containing stan-dard A compounds were run simultaneously and the positions of the ex-perimental steroids were estimated from the locations of the correspond-ing standards as ascertained by treatment with an ethanol solution of phosphomolybdic acid. Absorbance was measured with a Beckman DU spectrophotometer using matched quartz cells of 1 cm light path. Steroids were dissolved i n metha-nol and were read against a blank prepared by elution of a zone of size equal to that containing the steroid. The blank str i p of paper and the experimental chromatogram were prepared and run together. The values used as molar extinction coefficients (at 240 nm wave-length) were as follows: 20«.-acetoxypregn-4-en-3-one, 16,000; andros-tenedione, 16,000; aldosterone, 15,800; cortexolone, 16,800; cortexolone acetate, 16,000; Cortisol, 15,900; cortisone, 15,900; corticosterone, 16,500; deoxycorticosterone, 18,000; deoxycorticosterone acetate, 18,000; 20*< -hydroxypregn-4-en-3-one, 16,000; 20? -hydroxypregn-4-en-3-one, 16,308; l6<-hydroxyprogesterone, 16,900; 17-hydroxyprogesterons, 15,600; proges-terone, 16,400; testosterone, 16,000; and testosterone acetate, 16,100. 10 (c) Criteria employed to assess the purity of compounds and methodssused  to calculate the minimal per cent conversion of substrate to product (i) Crystallizations: The weights of compounds containing the -ketone grouping were assessed i n the crystalline and mother liquor fractions both by weighing after drying and by measurement of the absorbance of a methanol solution i n the region of 240 nm. The specific a c t i v i t i e s of the fractions were calculated using the weights obtained by both proced-ures and both results were used to assess the purity of the fractions. When i t appeared that a compound was radiochemically homogeneous, the re-sults attained from the procedure for weight determination that caused the lesser variation i n the calculated specific a c t i v i t i e s of crystals and mother liquors were employed to calculate the per cent minimal con-version of substrate to product. The specific a c t i v i t y of a compound was considered to be constant i f the values for the crystals and the mother liquors i n two successive crystallizations did not differ by more than five per cent. In calculating the per cent minimal conversion of substrate to product, the average values of the specific a c t i v i t i e s of the crystals from the f i n a l two crystallizations were multiplied by the amounts of carrier steroid added i n i t i a l l y . When the f i n a l product was an acetyl derivative, the per cent minimal conversion was calculated i n terms of the free steroid. ( i i ) Paper chromatography: In most cases, chromatography of the steroids containing the &^-3-ketone grouping was repeated u n t i l constant specific ac t i v i t i e s 11 were attained. The specific a c t i v i t y of a compound was considered to be constant i f the values for the specific activities of the compound i n the eluates of two successive chromatograms did not d i f f e r by more than five per cent. (d) Assay of radioactivity (i) Liquid s c i n t i l l a t i o n spectrometry: A Nuclear-Chicago liquid s c i n t i l l a t i o n spectrometer (model 725) was employed for assay of radioactivity by li q u i d s c i n t i l -lation spectrometry. Steroid samples i n methanol solution were pipet-ted into counting v i a l s and the solvent evaporated. Each residuum was then dissolved i n 5 ml of toluene containing 2,5-diphenyloxazole (0.4%), 1,4-bis ^ 2-(5~phenoxyloxazolyl)j -••benzene (0.01%) and absolute ethanol (2%). The counting efficiency was 25% for \ and 85% for -^C i n samples containing a single isotope. In samples containing both isotopes, the efficiency was 20% for tritium and 40$ for •^C. In samples containing both 3H and "^C, the dpm for each isotope was calculated according to the simultaneous equation method of Okita and coworkers (5). ( i i ) Gas flow detection: A Nuclear-Chicago gas flow detector (model D47) operating in the Geiger region was used without a window. Copper planchets were cleaned with concentrated sulfuric acid saturated with sodium dichromate. After rinsing with d i s t i l l e d water, the planchets were wiped with ace-tone and dried. A 0.1 or 0.2 ml portion of a methanol solution of the steroid was pipetted on to the planchet and dried i n a i r at room tempera-ture. The gas flow detector was operated at an efficiency of approxi-mately 45% for and approximately 20% for % . 12 (e) Separation by solvent partition (i) Hexane-aqueous methanol partition: The steroid sample ms partitioned between equal v o l -umes (100 ml) of hexane and aqueous methanol (70 or 90%). Following equilibration, the aqueous methanol fraction was removed and the hydro-carbon phase was extracted with aqueous methanol (2 x 100 ml or 2 x 50 ml). The aqueous methanol extracts were evaporated under reduced pres-sure at 40°, absolute ethanol was added, and the extracts were dried by azeotropic d i s t i l l a t i o n . ( i i ) Partition into neutral, "estrone-estradiol" and " e s t r i o l " fractions (6): The dried steroid mixture was transferred to a separa-tory funnel i n absolute ethanol (2.5 ml). Benzene (50 ml) and lig r o i n (50 ml) were added and two extractions with 50 ml of d i s t i l l e d water were made. The combined water extract was extracted with ether (3 x 100 ml) to yield the " e s t r i o l " fraction. The benzene-ligroin fraction was then extracted with 0.04 N sodium hydroxide (2 x 50 ml) and d i s t i l l e d water (2 x 10 ml). The alkaline and aqueous extracts were combined and brought to pH 8.5 using 6 N hydrochloric acid and then extracted with ether (3 x 100 ml) to yield the "estrone-estradiol" fraction. The re-maining benzene-ligroin mixture constituted the neutral fraction. The solvents were removed from the neutral, "estrone-estradiol" and "es t r i o l " fractions under reduced pressure at 40°, absolute ethanol was added and the fractions were dried by azeotropic d i s t i l l a t i o n . 13 (f) Reactions (i) Acetylation: The dry substrate was treated with anhydrous pyridine (0.3 nil) and acetic anhydride (0.15 ml) overnight at room temperature i n a stoppered test tube. Following the reaction period, several drops of methanol were added to the reaction mixture and the solvents were e-vaporated under a gentle stream of nitrogen at 40°. Several additional drops of methanol were then added and evaporated i n a similar manner. ( i i ) Girard (7): Girard's T reagent (8) was crystallized from absolute ethanol prior to use. The steroid mixture was dried over calcium chlor-ide i n an evacuated dessicator; 0.5 ml of glacial acetic acid and 100 mg of Girard's T reagent were added. The tube was loosely stoppered with a cork wrapped i n t i n f o i l and placed i n an o i l bath at 95° for 20 minutes. The tube was removed from the o i l bath and 15 ml of ice water were added and the reaction mixture was immediately transferred to a small separa-tory funnel. Sufficient 10$ sodium hydroxide was added to neutralize nine-tenths of the acetic acid. Three extractions with 20 ml portions of ether were made. The ether extracts were combined and washed once with 10 ml of ice water. This aqueous wash was combined with the rest of the aque-ous ketonic fraction. The ether was washed with 10 ml of 2.5% sodium carbonate and three 10 ml portions of d i s t i l l e d water. The ether con-tained the non-ketonic fraction. The aqueous ketonic fraction was a c i d i -f i e d with 3 ml of concentrated hydrochloric acid, allowed to stand at room temperature for 2 hours and then extracted with ether (3 x 10 ml). 1U This ether extract was washed with 2.5$ sodium carbonate (1 x 10 ml) and d i s t i l l e d water (3 x 10 ml). This second ether extract contained the ketonic fraction. The ketonic and non-ketonic fractions were evaporated under reduced pressure at 40°, absolute ethanol was added and the f r a c t -ions were dried by azeotropic d i s t i l l a t i o n . (g) Purification of unlabeled steroids added as carriers Unless otherwise stated, unlabeled steroids added as carriers were purified by a series of crystallizations and their purity was as-sessed by melting point determinations using a Kofler hot stage appara-tus and polarized light. (h) Solvent purification Solvents (AR grade) were purified as follows: acetone was reflux-ed for 1 hour with potassium permanganate and potassium carbonate and then d i s t i l l e d twice. Anhydrous benzene was prepared by d i s t i l l a t i o n from calcium hydride under anhydrous conditions. Chloroform was shaken with concentrated sulfuric acid, washed with d i s t i l l e d water, dried over calcium chloride, f i l t e r e d and d i s t i l l e d . Absolute ethanol was refluxed for 2 hours with zinc and potassium hydroxide under anhydrous conditions and then d i s t i l l e d twice under anhydrous conditions. Ether was washed with a dilute, weakly acidic solution of ferrous sulfate (3 x one-tenth vol), washed to neutrality with d i s t i l l e d water and d i s t i l l e d . Anhydrous ethyl acetate was prepared by drying i t over calcium chloride for 2 hours, followed by f i l t r a t i o n and d i s t i l l a t i o n under anhydrous conditions. An-hydrous hexane was prepared by d i s t i l l a t i o n from calcium hydride under anhydrous conditions. Methylene chloride was washed with d i s t i l l e d water, 15 dried over calcium chloride, f i l t e r e d and d i s t i l l e d . Pyridine was reflux-ed for 1 hour with barium oxide under anhydrous conditions, d i s t i l l e d under anhydrous conditions and stored over potassium hydroxide pellets. The following solvents were r e d i s t i l l e d prior to use: acetic an-hydride, benzene, ethyl acetate, hexane, methanol, l i g r o i n , propylene glycol and toluene. 26 PART I STUDIES ON A SUBJECT WITH ADRENOCORTICAL NEOPLASM AND HYPOGLY-CEMIA: STEROIDS EXCRETED IN THE URINE AND STEROID METABOLISM BY THE TUMOR IN VITRO INTRODUCTION (a) Metabolism of adrenal steroids i n normal subjects and i n patients  with adrenocortical neoplasms A general outline of the steroid biosynthetic pathways i n the adrenal i s given i n Fig. 1 (9,10,11,12). At present, l i t t l e i s known regarding which routes i n this complex network of steroid transformat-ions are preferentially u t i l i z e d under any given set of circumstances. Evidence was obtained i n incubations of human adrenal c o r t i c a l tissue from patients with mammary carcinoma that Cortisol formation proceeded from pregnenolone via 17whydroxypregnenolone rather than via progester-one (13). Most studies i n vitro of adrenal steroid biosynthesis have been performed on tissue from animals other than man or on glands obtain-ed from diseased human subjects. Studies i n vitro using human adrenal tissue obtained shortly after the accidental death of a male subject have been reported (14). Substrate -^C-pregnenolone was transformed to progesterone (51.2%), corticosterone (11.9%), Cortisol (10.3%), andros-tenedione (2.25%), 3/5 ,11/ ,17* ,21-tetrahydroxy-5* -pregnan-20-one (1%) and 17-hydroxyprogesterone (0.04%). Cortexolone, testosterone, 11^ -hy-droxyandrostenedione and dehydroepiandrosterone were not detected. The metabolic pathways for Cortisol are shown i n Fig. 2 and the pathways of androgen biosynthesis and metabolism are depicted i n Figs. 3 and 4. 17 The steroid biosynthetic capabilities of tumors of the adrenal cortex and the metabolism of steroids i n patients harboring these tumors have been assessed by assay of metabolites i n the urine, by measurement of the steroid content of neoplastic tissue or venous blood, and by i n -cubations of portions of the tumor (15-52). Considerable variation i n steroid metabolism has been observed and may reflect, i n part, the d i f -ferent c l i n i c a l manifestations which accompany adrenocortical neoplasia, such as v i r i l i s m , feminization, Cushing's syndrome and derangements of f l u i d and electrolyte metabolism. Even among subjects with similar c l i n i -cal manifestations, marked qualitative and quantitative differences i n steroids excreted via the kidney and i n steroid transformations i n vitro by the tumor tissue have been reported. (b) Hypoglycemia associated with extra-pancreatic neoplasms that are not  of adrenal origin A number of hypotheses have been advanced to explain the hypogly-cemia that occurs i n some patients with extra-pancreatic neoplasms (53-59). (i) Enhanced glucose disappearance due to the presence of excessive insulin or insulin-like material i n the blood: Increased insulin or insulin-like a c t i v i t y i n the circulation could be the result of secretion of insulin or insulin-like material by the tumor, pancreatic stimulation by the tumor, supression of physiologic insulin antagonists, or delayed degradation of insulin. Insulin or i n -sulin-like a c t i v i t y has been found i n increased amounts i n the tumor or serum of some patients with non-islet c e l l extra-pancreatic tumors and hypoglycemia (60-65). In other cases, normal or decreased insulin and 18 insulin-like a c t i v i t y have been reported (66-70). In one subject (61) pancreatectomy did not ameliorate the hypoglycemia which suggests that the extra-pancreatic tumor did not stimulate pancreatic insulin secretion. ( i i ) Excessive glucose consumption by the tumor: Evidence for enhanced glucose u t i l i z a t i o n by the tumor includes the pattern of changes i n concentrations of blood glucose, phosphate, and free fatty acids after glucose administration or epinephrine injection; high plasma lactate concentration; large differences i n arterial-venous glucose concentrations across the tumor i n vivo and excessive glucose up-take by the tumor tissue i n vitro (53,55,71,72). Others (60,73) have found only small differences between a r t e r i a l and venous blood glucose concentrations i n arteries and veins contiguous with the tumor and only moderate glucose consumption by the tumor tissue i n vitro (61,74). ( i i i ) Deficient gluconeogenesis due to a specific effect of trypto-phan metabolites: Certain metabolites of tryptophan cause inhibition of phos-phoenolpyruvate carboxykinase i n vitro (75). Increases i n the concentra-tions of several indole compounds i n the blood and urine were observed dur-ing episodes of severe hypoglycemia i n a group of patients with neoplasms of extra-pancreatic origin, but not i n patients with local or metastatic pancreatic i s l e t - c e l l carcinomas (76,77). On the other hand, normal con-centrations of tryptophan metabolites i n blood and urine during hypogly-cemic episodes have been found i n other patients with tumors of extra-pancreatic origin (53,77). 19 (iv) Other hypotheses: Other hypotheses advanced to explain the hypoglycemia i n cer-tain patients with extra-pancreatic neoplasms include hepatic insuf-ficiency from metastases, blockage of sympathetic innervation of the l i v e r , presence of atypical metastatic pancreatic i s l e t - c e l l tissue and torsion of the pancreatico-duodenal vein. None of these hypotheses has received substantial experimental support, (c) Hypoglycemia associated with adrenocortical tumors Nineteen cases of hypoglycemia associated with adrenocortical neoplasms have been reported (15,16,40,77-89). General information con-cerning each case i s l i s t e d i n Table I. The age of the patients has ranged from 12 to 64 yearsj there have been 12 females and 7 males. The tumors have been large and about two-thirds have involved the l e f t adrenal. One adenoma and one sarcoma have been reported, the remainder have been carcinomas. V i r i l i s m , gynecomastia, hypertension, acne and hypokalemic alkalosis were present i n some patientsj i n seven patients there was no evidence of excessive secretion of steroid hormones. The rates of steroid excretion via the kidney i n patients with hypoglycemia and adrenocortical tumor are listed i n Tables I I to IV. With one exception, the excretion rates of 17-ketosteroids and 17-hydroxy-steroids were elevated. In the patientsof Scholz and coworkers (89), the 17-ketosteroid excretion rate was elevated on one occasion and normal when measured again and the excretion rate of 17-ketogenic steroids was normal. The excretion rate of dehydroepiandrosterone was elevated i n a l l four patients i n whom i t was measured. Gynecomastia was present i n the 20 patient of Dohan and coworkers (15) and the excretion rate of estrogens was elevated when measured by bioassay of the "phenolic" fraction ob-tained by alkaline partition of a carbon tetrachloride extract of acid-hydrolyzed urine. The aldosterone excretion rate was normal i n one sub-ject and elevated i n another. The excretion rate of 3<* ,17,20-trihydroxy-5P -pregnan-20-one (THS) was elevated i n a l l three patients i n whom i t was measured. The excretion rate of free Cortisol was elevated i n two patients neither of whom had Cushing!s syndrome. The excretion rates of the following steroids were measured i n one subject and found to be ele-vated: 16°C-hydroxypregnenolone, pregn-5-ene-3 £ ,16* , 20<* - t r i o l , preg-nandiol, free corticosterone and pregnanetriol plus 17-hydroxypregneno-lone measured together. The excretion rate of pregnanetriol was normal i n one patient and elevated i n another. Investigations of the steroids present i n the urine of patients with adrenocortical tumors and hypogly-cemia have not provided an explanation for the hypoglycemia. The results of studies on the etiology of the hypoglycemia i n patients with adrenocortical neoplasia are given i n Table V. Assay of insulin-like activity i n extracts of the tumor have given normal or nega-tive results i n four cases. Extracts of the tumor removed from the patient described by Kuhnlein and Meythaler (84) caused hypoglycemic convulsions when injected into micej control experiments were not reported. Wikman and McCracken (88) reported "significant" insulin-like a c t i v i t y in acid-alcohol extracts of tumor tissue when tested by rat hemidiaphragm and epididymal fat pad technics. Serum insulin-like a c t i v i t y has been normal or diminished i n a l l five of the subjects i n whom i t has been measured. 21 Ifymontt and coworkers (87) found no arteriovenous difference i n blood glucose concentration across the tumor at the time of surgery and they suggested that the tumor did not consume glucose at an excessive rate. Williams and coworkers (16) were unable to demonstrate glucose-6-phos-phatase a c t i v i t y i n tumor tissue of one patient. The authors postulated that excessive consumption of glucose by the tumor occurred and that an enzymatic defect i n the carcinoma prevented mobilization of the stored glycogen. The normal concentrations of tryptophan metabolites i n the blood and urine of two patients (77,89), suggests that products of tryp-tophan degradation were not the cause of the hypoglycemia (76). The investigation reported i n this part of the thesis concerns studies of steroid biosynthetic pathways i n vitro i n an adrenocortical carcinoma removed from a patient with severe hypoglycemia. Results of urinary steroid assays are also presented. The metabolism of steroids by the tumor tissue of a patient with adrenocortical neoplasia and hy-poglycemia has not been previously assessed i n vi t r o . MATERIALS AND METHODS (a) Patient The patient was a 51 year old white female. There were no s t i g -mata of excessive production of glucocorticoids or sex steroids. Fast-ing hypoglycemia was present; blood glucose concentrations as low as 30 mg per 100 ml were observed. Following removal of the primary tumor the hypoglycemia was ameliorated, but i t recurred prior to death when wide-spread metastases were present. 22 (b) Tumor1 The carcinoma consisted of a spherical bosselated mass 13 cm i n diameter, weighing 1285 g. On section, the tissue was pink, dark red and golden. An area of necrosis and cavitation was presentj. Microscopic examination showed that the tumor consisted of large multangular cells with prominent cytoplasmic walls, finely granular eosinophilic cytoplasm and central irregular nuclei. The nuclei varied i n size and contained small nucleoli. Occasional mitotic a c t i v i t y was observed. The cells resembled adrenocortical c e l l s . (c) Incubation conditions 70c -%-Pregnenolone (The Radiochemical Centre, Amersham, England) was not further purified prior to use; 9.056 x 10^ cpm and 4.6 p-g were added to each of two incubation flasks. 4-^fC-Progesterone (The Radio-chemical Centre, Amersham, England) was purified by thin layer chroma-2 , N tography using the solvent system ethyl acetate-benzene (1:1, by vol;* 2.528 x 10^ cpm and 50.2 \~g were added to each of three incubation flasks. The radioactive substrates were transferred to the reaction flasks i n each instance i n a methanol solution, the solvent was evapor-ated and the substrates were redissolved in 0.2 ml of propylene glycol immediately prior to the addition of the cell-free homogenate. The tumor was stored at -19° following excision. Seven days later, i t was partially thawed and a section was taken for incubation. A f i f t y gram portion of tumor tissue was cut into small pieces and am indebted to Dr. G.B. E l l i o t for the macroscopic and micro-scopic description of the tumor. 2see General Methods section. 23 homogenized at pH 7.4 in Krebs-Ringer phosphate buffer (90), i n which Na"^  and K + were interchanged, containing glucose (0.01 M) and nicot ina-mide (0.04 M); a Potter-Elvehjem-type homogenizer was used (91). The homogenate was centrifuged (1500 x _g, 10 minutes, 4 ° ) and the super-natant (150 ml) was divided equally among f ive incubation f lasks . In-cubations were performed in a gas phase of 95% oxygen-5% carbon dioxide for 3 hours at 37° using a Dubnoff metabolic shaking incubator, (d) Extract ion, resolution and puri f icat ion procedures Data concerning the extraction, resolution and pur i f icat ion pro-cedures employed are summarized i n F igs . 5 to 8 and Tables VI and VII 14 for the experiment using C-progesterone as substrate and in F i g s . 9 to 12 and Tables VIII to XVI for the experiment using ^H-pregnenolone a s substrate. At the end of the incubation period the contents of the i n -cubation f lasks containing "^C-progesterone were combined and diluted to 200 ml with d i s t i l l e d water. The contents of the f lasks containing % -pregnenolone were treated in a similar manner. Extraction with ethyl acetate (4 x 200 ml) was then performed; the combined incubation medium from the experiment using "^C-progesterone was also extracted with methy-lene chloride (4 x 200 ml) and the ethyl acetate and methylene chloride extracts were combined. The extracts were evaporated under reduced pres-sure at 4 0 ° , absolute ethanol was added and the extracts were dried by azeotropic d i s t i l l a t i o n . The organic extract from the incubations with "^•C-progesterone and the organic extract from the incubations with 3 H -prejgnenolone were each partitioned between hexane and 90% aqueous metha-2 no l . The combined aqueous methanol extract of each radioactive substrate 24 experiment was then subjected to column partition chromatography using 2 s i l i c a gel. The compositions of the eluents used for stepwise elution are given i n Tables VI and VIII. In each isotope experiment, eluates from the s i l i c a gel columns were combined and subjected to paper chroma-tography^ i n the solvent systems ligroin-propylene glycol, toluene-propylene glycol and Bush B5. Thin layer chromatography2 on coated glass plates i n the solvent system ethyl acetate-chloroform-water (90:10:1, by vol) (92) was also used i n the experiment with %-pregnenolone as sub-strate. Unlabeled steroids (100-200 p-g) were added as carriers at d i f -ferent stages of the experiments (Figs. V to XVI and Tables IX and X). Paper chromatography of the steroids containing a £^-3-ketone system 2 was repeated u n t i l constant specific a c t i v i t y was obtained or u n t i l negligible radioactivity remained with the carrier steroid. In instances where constant specific a c t i v i t y was observed for A^-3-ketosteroids and after several paper chromatograms of the steroids, more purified car-2 r i e r compounds were added and crystallizations were performed u n t i l radio-chemical homogeneity was accomplished or u n t i l the crystals contained neg-l i g i b l e radioactivity. 2 Radioactivity was measured with a li q u i d s c i n t i l l a t i o n spectro-meter and with a gas flow detector, (e) Assay of steroids i n urine Testosterone i n urine was measured by a modification of the meth-od of Ismail and Harkness (93). Paper chromatography in the Bush A so l -2 vent system using Whatman no. 1 paper and thin layer chromatography i n the solvent system ethyl acetate-benzene (1:3, by vol) were employed 25 instead of the single chromatography on Whatman no. 42 paper as described in the original procedure. Quantitative estimation of testosterone was was performed by measuring the absorbance at 595 nm following treatment of the f i n a l eluate with a 0.5$ solution of v a n i l l i n i n ethanol-sulfuric acid (93). An Allen correction (94) using the absorbances at 510 and 680 nm was applied to the absorbance at 595 nm. Absorbance was measured i n a Unicam SP 800 spectrophotometer with quartz cuvettes of 1 cm opti-c a l path. The rates of excretion i n urine of 17-ketosteroids (95-97), de-hydroepiandrosterone (98-101), 17-ketogenic steroids (96,97,102), and pregnanediol (103) were measured by Bio-Science Laboratories, Van Nuys, California. The rates of excretion i n urine of 17-hydroxycorticosteroids (104), pregnanetriol (105) and free Cortisol (106) were measured by Bio-medical Assay Laboratories, Worcester, Massachusetts. The excretion rate i n urine of 3C* ,17,21-trihydroxy-5/B -pregnan-20-one was measured by both commercial laboratories using the same method (107). RESULTS The results of incubations of the tumor tissue with radioactive progesterone and pregnenolone are given i n Table XVII. No radioactive steroids were identified as metabolites of ^ C-progesterone. Following 3 incubation with H-pregnenolone, radioactive androstenedione (0.05 per cent minimal conversion), dehydroepiandrosterone (0.20 per cent minimal conversion) and progesterone (1.0 per cent minimal conversion) were id e n t i -f i e d . 26 The excretion rates i n urine of individual steroids and steroid fractions are given i n Table XVIII. No testosterone was found i n two 24 hour urine specimens. Carrier testosterone (10 p*g) was added to a portion of a third specimen prior to analysis and a value of 11.1 p.g was obtained. Assays of a number of steroids and steroid fractions were performed on one 24 hour urine specimen by two commercial laboratories. The excretion rates of t o t a l 17-ketosteroids, 17-hydroxycorticosteroids and 17-ketogenic ster-oids were elevated; the excretion rates of dehydroepiandrosterone, preg-nanetriol, pregnandiol and free Cortisol were within the normal range. The excretion rate of 3 o C, lY^l-trihydroxy-S/s -pregnan-20-one i n the same urine specimen was found to be elevated i n two determinations by both com-mercial laboratories. An elevated excretion rate of tot a l 17-ketosteroids and 17-ketogenic steroids was also observed i n two other 24 hour urine specimens. DISCUSSION Incubations of portions of the tumor with H-pregnenolone and "^C-progesterone resulted i n minimal transformations of the radioactive sub-strates to identifiable metabolites. A large percentage of both radio-active substrates was recovered unchanged and three radioactive meta-bolites of %-pregnenolone, namely, progesterone, androstenedione and dehydroepiandrosterone, were found i n low yield. In view of the normal or elevated rates of excretion i n urine of several steroids and steroid fractions that were observed prior to removal of the tumor, the failure to demonstrate active steroid biosynthesis in vitro may be attributed to some aspect of the experimental procedure rather than to the absence i n 27 the tumor of the requisite enzymatic machinery. Of the many causes that could be invoked to explain the low enzymatic ac t i v i t y that was observed i n v i t r o , one or more of the following are the most l i k e l y : inactive por-tions of the tumor may have been selected for incubation or the quantity of tissue that was incubated may have been too small to transform measure-able amounts of the radioactive substrates. The tumor was large ( 1 , 2 8 5 g) and contained areas of necrosis and the rate of steroid biosynthesis per gram of tissue was undoubtedly low. The tumor was frozen for one week between excision and incubation and during that time interval dam-age to enzymes may have occurred. Deficiencies of essential coenzymes during the incubation period may have caused low enzymatic activity; i n -cubations were performed i n a Krebs-Ringer phosphate buffer containing only nicotinamide and glucose. 3 lit The minimal metabolism of H-pregnenolone and C-progesterone by the tumor i n vitro may not have reflected quantitatively the i n vivo act i v i t y of the tumor. In contrast to the results of the experiments i n  vi t r o , the investigations of the steroids i n the urine of the patient prior to removal of the tumor provided a means of studying the steroid biosyn-thetic capabilities of the tumor. The predominant pathway of Cortisol biosynthesis from progesterone i n the normal adrenal cortex procedes via successive hydroxylations of progesterone at positions C 1 7 , C 2 1 , and C l l (9-12''and Fig. l ) . Studies i n patients with deficiencies i n hydroxylase act i v i t y and observations following the administration of steroids to normal subjects have shown that the major metabolites in urine of proges-terone, 17-hydroxyprogesterone and cortexolone are pregnanediol, 28 pregnanetriol and 3°<-, 17,21-trihydroxy~5/^ -pregnan-20-one, respectively (108). The elevated rates of excretion i n urine of 17-hydroxycorticoids and 17-ketogenic steroids by the patient reported herein and the normal excretion rates of pregnanediol and pregnanetriol suggest that l / t o C- and N 21-hydroxylase ac t i v i t i e s were normal. This conclusion i s i n accord with the results of studies of steroids i n the urine of other patients with adrenocortical neoplasms and hypoglycemia (Tables I I to IV). Williams and coworkers (16) found the pregnanediol excretion rate to be 10 mg/24 hours i n one female patient; the elevated value may have reflected,in;-pairt^ the production of progesterone by the ovaries. The elevated rate of ex-cretion i n urine of 3 1* ,17,21-trihydroxy-5/B -pregnan-20-one i n the patient reported herein suggests that a defect i n llj& -hydroxylase activity was present. 3*,17,21-Trihydroxy-5/ -pregnan-20-one, the major ketol meta-bolite of cortexolone (109), i s normally found i n very small quantities i n the urine (38) and in adrenal vein blood (110). The rate of excret-ion i n urine of 3* ,17,21-trihydroxy-5r3 -pregnan-20-one has been elevated i n a l l patients with an adrenocortical tumor and hypoglycemia i n whom i t has been measured (Table I I ) . It i s doubtful that cortexolone functions as a hypoglycemic agent i n these patients because the rate of excretion of 3 < ,17,21-trihydroxy-5^ -pregnan-20-one may also be elevated i n patients with adrenocortical tumors without hypoglycemia (16,37,38). The 17-hydroxycorticosteroid fraction of steroids consists of compounds with the dihydroxyacetone side-chain at C17; the 17-ketogenic fraction includes steroids with an oxo or a hydroxyl group at C20 and a 29 hydroxyl group at C17. The marked elevation i n the rate of excretion i n urine of 17-ketogenic steroids accompanied by a lesser elevation i n the excretion rate of 17-hydroxycorticosteroids suggests that unidentified 20-oxygenated 17-hydroxysteroids lacking the dihydroxyacetone side-chain configuration were excreted i n abnormally large quantities. The rates of excretion i n urine of t o t a l 17-ketosteroids and of dehydroepiandrosterone have been elevated in many patients with adreno-cortical carcinoma (17,18,111) and have been elevated i n a l l patients with an adrenocortical neoplasm and hypoglycemia i n whom they have been measur-ed (Table I I I ) . Eymontt and coworkers (87) have suggested that i n some patients the hypoglycemia might be caused by excessive production by the tumor of anabolic steroids that stimulate amino acid incorporation into protein, thus making the amino acids unavailable for gluconeogenesis. I t has been reported that hypoglycemia may follow testosterone administration (112). The excretion rate of t o t a l 17-ketosteroids was s l i g h t l y above the normal range i n the patient reported herein and the excretion rates of testosterone and of dehydroepiandrosterone were very low. In view of these findings, i t i s unlikely that dehydroepiandrosterone or testosterone were responsible for the hypoglycemia that was present i n the patient re-ported herein; however, i t i s possible that the tumor produced other ster-oids with potent anabolic a c t i v i t y (Figs. 3 and 4). The causes of the hypoglycemia that occurs i n some patients with extra-pancreatic neoplasms are not understood; more than one mechanism may coexist i n the same patient and the causative factors may differ a-mong patients (113). Patients with adrenocortical tumors and hypogly-cemia are a rarity and few detailed investigations have been performed. 30 Studies of steroid metabolism i n vivo and i n v i t r o i n patients with ad-renocortical neoplasms and hypoglycemia have yielded results that have also been observed i n patients with adrenocortical tumors without hypo-glycemia and no distinctive abnormality of steroid biochemistry has been observed i n the patients with low blood glucose concentrations. It i s possible, however, that the hypoglycemia may be caused by an unrecogniz-ed steroid produced by some adrenocortical tumors. Steroid metabolism has not been studied extensively i n patients with hypoglycemia and extra-pancreatic tumors that arise from organs and tissues other than the ad-renal. It may be that the etiology of the low blood glucose concentration i n patients with extra-pancreatic neoplasms i s unrelated to the organ from which the tumor originates. SUMMARY AND CONCLUSIONS (i) Incubations of portions of an adrenocortical tumor from a patient with severe hypoglycemia were performed using %-pregnenolone and ^ "C-progesterone as substrates. Transformation of %-pregnenolone to pro-gesterone, dehydroepiandrosterone and androstenedione was observed; no metabolism of "^"C-progesterone was detected. ( i i ) The excretion rate i n urine of 3oL,17,21-trihydroxy-5/S -pregnan-20-one was elevated which suggests that a defect i n 11/J -hydroxylase activ-i t y was present. The excretion rates i n urine of t o t a l 17-ketosteroids, 17-hydroxycorticosteroids and 17-ketogenic steroids were elevated; the excretion rates of testosterone, dehydroepiandrosterone, pregnanetriol, pregnanediol and free Cortisol were not elevated. 31 ( i i i ) The etiology of the hypoglycemia that may accompany adrenocortical neoplasms i n some patients remains unknown. It was not possible to relate the results of the investigations of steroid metabolism reported herein to the hypoglycemia that was present. TABLE I > Data concerning previously reported cases of adrenocortical tumor and hypoglycemia Authors Age Sex Signs of steroid Tumor hormone excess Size Side Histology Anderson(78) 33 M None 0.4 kg L Carcinoma Lawrence (79) 24 F Hypertension, acne "Grapefruit" L Carcinoma Broster & Patterson(80) 14 F V i r i l i s m 2.98 kg* L Carcinoma S t a f f i e r i et al{8l) Thannhauser (8"2) 25 M Gynecomastia 20x11x9 cm R Sarcoma •- ........ -Case # 1 F Carcinoma Case # 2 M Carcinoma Case # 3 F Carcinoma Dohan et a l . (15) 48 F Virilism,hypokalemic alkalosis,' 2.2 kg L Carcinoma Schamaun et al(83) hypertension, gynecomastia 37 M None 1.7 kg R Adenoma Askanazy et aL(85) 26 F None 2.2 kg L Carcinoma Kuhnlein & Meythaler(84) 45 M None 2.6 kg R Carcinoma Boss (86) 64 M None "Grapefruit" L Carcinoma Williams et al.(l6) Case f l 24 F Viril i s m , acne, hypertension, hypokalemic alkalosis 2.0 kg L Carcinoma Case # 2 48 M Hypertension 1.1 kg L Carcinoma Eymontt et a l . (87) 19 F Viril i s m , acne, hypertension 1.4 kg L Carcinoma Wikman & McCracken (88) 59 M None 2.4 kg L Carcinoma Scholz et al.(89) 16 F None 1.8 kg a L Carcinoma Cara (40 F~ 12 F Hypertension, v i r i l i s m 0.7 kg . R Carcinoma Silverstein et al.(77) 25 F height of tumor plus kidney. TABLE I I Excretion rates of 17-hydroxysteroids i n the urine of patients with adrenocortical tu-mors and hypoglycemiaa>B mg/24 hrs p-g/24 hrs Authors Total THS THE THF P» t r i o l Free F Gara (40) 31° 7.2 2.4 Willaims et a l . (16) Case Wl 162C Increased 0.9 367 (7-16) (trace) (oa-1.9) (9-82) Case # 2 > 4 C O C 193 16.8 6.3 (1.6) (2.8) Eymontt et a l . (87) 35.7-48.9° 2,199 Scholz et a l . (89) 12.2d P ' t r i o l , pregnanetriol; F, Cortisol. See Glossary for other abbreviations and for systemic equivalents of the t r i v i a l names used. ^Normal values given by the authors are shown i n parentheses. °17-Hydroxycorticosteroids. 17-Ketogenic steroids. TABLE I I I Excretion rates of 17-ketosteroids i n the urine of patients with adrenocortical tumors and hypoglycemia 3 1* Authors mg/24 hrs Total D A E ll-0xy Broster & Patterson (80) 1,980 Increased Dohan et a l . (15) 80 25-27 Thannhauser (82) Case # 1 Case # 2 Cara (40) 43 1.3 7.6 (0.1-0.3) Williams et a l . (16) Case F " l Case # 2 56.8 (4-14) 130.1 19.9 (1-1.4) 80.8 9.1 (2^) 15.2 17.0 (4-6) 11.3 4.8 ( O i A-0.9) 13.9 Eymontt et a l . (87) 85.8-23.6 43% c (16-20) 16% c (38-50) 41$ c (30-47) Shamaun et a l . (83) 33-41 Kuhnlein & Meythaler (84) 166 Scholz et a l . (89) 7-31 S t a f f i e r i et a l . (81) 23.2 aD, dehydroepiandrosterone; A, androstenedione; E, etiocholanolone; 11-Oxy, 11-oxy-genated 17-ketosteroids. ^Normal values given by the authors are shown i n parentheses. value i s given as per cent of t o t a l 17-ketosteroids excreted per 24 hours. 35 TABLE 17 Excretion rates of steroids i n the urine of patients with adrenocortical tumors and hypoglycemia Authors Steroids 0 Dohan et a l (15) Cara (40) Williams et a l . (16) Case F l Case # 2 Eymontt et al.(87) Estrogens increased 16-Hydroxypregnenolone, 8.7 mg/24 hrs Pregn-5-ene-3/5 ,3i><>c ,20*--triol, 11 mg/24 hrs Aldosterone, 5.8 hg/24 hrs (4-19) Pregnanediol, 10 mg/24 hrs (0.2-0.7) Free cbrticosterone, 33.6 p.g/24 hrs (2-9) Pregnanetriol •+• 17-hydroxypregnenolone 10.3 mg/24 hrs (1-8) Aldosterone, 50 f*g/24 hrs formal values given by the authors are shown i n parentheses TABLE V Studies on the etiology of the hypoglycemia i n patients with adrenocortical tumors Authors Insulin-like activity Other studies Tumor Fasting plasma Askanazy et al.(85) Negative Boss (86) Negative Williams et al.(l6) Case Wl Negative Case # 2 Kuhnlein and Meythaler(84) Positive 0 Eymontt et al.(87) 3 5 0 ^ g 3 Wikman & McCracken (88) Scholz et al.(89) Silverstein et al.(77) "Significant" Normal Normal 180K u/ml b Normal 2^U/ml No glucose-6-phosphatase i n tumor No arteriovenous difference i n glu-ccose concentration across tumor Normal concentrations of tryptophan metabolites i n blood and urine Normal concentrations of tryptophan metabolites i n blood and urine ON fValue i s within the normal range for muscle, l i v e r and kidney. bNormal range 60-250 y*. U/ml. cSee Text for details. TABLE VI Subs t ra te ^ C - p r o g e s t e r o n e i n c u b a t i o n : column a d s o r p t i o n chromatography o f the 90$ aqueous methanol f r a c t i o n E l u e n t : compos i t ion E l u a t e F r a c t i o n — —- • — r number • ( v / v ) Dry weight Rad ioac t i v i t jr (mg) (cpm) 1 Hexane-benzene (1:1) 89.92 707,500 2 Benzene 0.38 3 E t h y l acetate-benzene (1:99) 4.90 3,450 4 M .i i« (2:98) 17.96 5 » " 1 1 (4:96) 6.68 6 " " " (10:90) 5.45 4,886,500 7 " » " (20:80) 5.12 8 » " " (40:60) 8.04 9 E t h y l ace ta te 6.64 608,900 10 Methano l : e t h y l acetate(2:98) 66.04 57,100 11 " (5:95) 98.70 31,850 12 " " " (10:90) 13.23 42,850 426,300 13 Methano l 49.65 T o t a l 371.71 6,764,450 a S e e Text f o r d e t a i l s . ^ F r a c t i o n s 2,3 and 4j 5,6 and 7j 8 and 9 were combined p r i o r t o assay of r a d i o a c t i v i t y . 38 TABLE VII Substrate -progesterone incubation: further paper chromatography of eluates containing carrier androstenedione, 17-hydroxyprogesterone and testosterone acetate a „ , ™ , , . Contents of eluate Compound Chromatographic solvent system Radioactivity Weight Specific (cpm) (|^ g) act i v i t y (cpm/Kg) Androstenedione L/PG L/PG L/PG L/PG L/PG L/PG 17-Hydroxyprogest-erone T/PG T/PG Testosterone acetate L/PG L/PG L/PG L/PG L/PG 23,840 93.8 254 16,610 79.1 -210: 11,790 78.9 150 8,100 60.3 133 6,860 57.2 120 4,870 49.9 98 2,660 42.0 63 28,960 66.2 437 20,025 58.2 353 8,950 50.1 175 62,240 9,930 58.0 171 7,540 47.6 158 5,020 38.8 129 3,850 35.9 107 3,235 26.4 122b aSee Fig. 7 for prior paper chromatography. "The f i n a l testosterone acetate eluate was subjected to crystallization following the addition of unlabeled testosterone acetate; the crystals contained negligible radioactivity. TABIE VIII Substrate -'H-pregnenolone incubation: column adsorption chromatography of the aqueous methanol fraction a Fraction number Eluent• composition (v/v) Dry weight (mg) Eluate Radioa ct ivity* 5 (cpm) 1 2 3 4 5 6 7 8 9 10 11 12 13 Hexane-benzene (1:1) Benzene Ethyl acetate-benzene (1:99) » " » (2:98) it II II (4.96) " " " (10:90) " " " (20:80) " " » (40:60) Ethyl acetate Methanol-ethyl acetate(2:98) n .. (5.95) " " " (10:90) Methanol 41.13 1.59 12.21 9.99 3.55 0.75 1.15 2.61 1.46 49.38 56.70 6.01 55.94 468,900 56,350 . 163,350 Hi5S?;500 2,987,400 370,050 194,800 169,920 687,500 Total 242.47 16,685,770 kSee Text for details. fractions 2 and 3; 5,6 and 7; 8 and 9 were combined prior to assay of radioactivity. 40 TABLE IX Substrate -^-pregnenolone incubation: further paper chromatography of eluates containing testosterone acetate, 17-hydroxyprogesterone, Cortisol and progesterone Contents of eluate Compound Chromatographic solvent system Radioactivity Weight ( V g ) Specific activity cpm c p n y V g Testosterone acetate a L/PG L/PG L/PG L/PG L/PG 150,500 68,075 43,650 29,006 17,250 12,110 230 209 189 183 143 104 654 326 231 159 121 ll£> 17-Hydroxypro-gesteronea T/PG T/PG 32,200 13,425 8,280 69.0 41.2 201° Cortisol** B5 49,050 32,720 99.0 63.0 4 9 5 e 519 f Progesterone L/PG L/PG L/PG L/PG dpm 1,099,950 737,360 655,263 418,263 261 193 180 113 dpm/M.g 4,214 3,821 3,650 3,705c aSee Fig. 12, footnote f. bSee Table XIII. °See Table XV. See Fig. 11, footnote g. ®See Table XVI. fSee Fig. 9, footnote f. TABLE X Substrate %-pregnenolone incubation: Thin layer chromatography Distance from origin (cm) Compound or zone i Contents of combined methanol extract Radioactivity Weight Specific activity (cpm) (Kg) (cpm/Kg) 0-1 Origin 47,920 1-2.9 51,270 2.9-3.9 Aldosterone*5 34,640 94.7 366 3.9-5.6 120,440 5.6-6.5 Corticosterone b 213,790 84.1 2,542 6.5-7.3 Cortisone 0 190,850 64.8 2,945 7.3-8.1 Cortexolone^ 80,990 8.1-9.1 86,060 83.1 1,036 9.1-10 38,970 10-12 17-HydroxypregnenoloneD 39,900 12-15 3,490 15-17 Solvent front 450 Total 908,770 aThe source of the sample subjected to thin layer chromatography i s depicted i n Pig. 11, foot-note e. bSee Table XI. 42 TABLE XI Substrate ^H-pregnenolone incubation: paper chromatography of compounds previously separated by thin layer chromatography3. Compound Chromatographic solvent system Aldosterone • B5 B5 B5 Corticosterone T/PG T/PG T/PG Cortisone B5 B5 Cortexolone T/PG T/PGC T/PG T/PG 17-IHydroxypregnenolone T/PG Contents of eluate ' Radioactivity Weight Specific (cpm) (H-g) a c t i v i t y (cpm/Kg) 34,640 94.7 366 8,000 65.0 123 6,960 48.4 144 v 6,100 38.3 159 b 213,790 84.1 2,542 25,800 45.0 573 12,890 33.4 386 7,285 25.7 283 190,850 64.8 2,945 6,000 20.0 300, 4,221 13.6 310 b 86,060 83.1 1,036 3,620 32.0 113 7,830 27.0 290 4,025 17.5 230 1,760 9.7 181 39,900 8,510 aSee Table X. bSee Table XVI. cOverflow and eluate containing carrier cortexolone from the f i r s t chroma-togram were combined prior to the second chromatogram. TABLE XII Substrate %-pregnenolone incubation: c r y s t a l l i z a t i o n s of androstenedione 3 Radioactivity Weight Spe c i f i c a c t i v i t y Fraction (cpm) (mg) (cpm/mg) Balance 1 5 UV C Balance 1 3 UVC Pool 19,475 10.13 1,923 F i r s t crystals 5,100 8.03 7.75 635 658 F i r s t mother liquor 9,030 2.12 2.08 4,259 4,341 Second crystals 2,470 6.76 6.74 365 396 Second mother l i q u o r 1,600 1.33 0.95 1,416 1,684 Third crystals 1,810 5.60 5.79 323 313 Third mother liquor 876 1.01 0.73 867 1,200 Fourth crystals 1,170 3.67 3.42 319 342 Fourth mother liquor 705 1.91 1.50 369 470 F i f t h crystals 745 2.32 2.24 321 333 F i f t h mother liquor 373 1.18 0.90 316 414 S i x t h crystals 385 1.14 1.01 337 381 Sixth mother liquor 255 0.80 0.65 319 392 ^See F i g . 10, footnote b. ^Weight of steroid measured by weighing on a balance. h e i g h t of steroid calculated from the absorbance of u l t r a v i o l e t l i g h t i n the region of 240 nm. TABLE XIII Substrate ^-pregnenolone incubation: crystallizations of testosterone acetate a> b,c Radioactivity Weight Specific activity Fraction (cpm) (rag) (cpm/mg) Balance UV Balance UV Pool 12,110 10.15 1,183 F i r s t crystals 970 6.49 6.51 149 149 First mother liquor 9,560 2.74 2.67 3,489 3,581 Second crystals 338 3.39 3 .40 100 99 Second mother liquor 65$ 2.72 3.53 242 259 Third crystals 105 0.97 0.94 108 112 Third mother liquor 220 2.14 2.20 103 100 Pool d 758 4.35 174 First crystals 305 3.03 2.96 101 103 F i r s t mother liquor 488 1.09 0.83 448 588 Second crystals 130 1.90 1.93 68 67 Second mother liquor 133 0.86 0.71 155 187 Third crystals 55 0.92 0.93 60 59 Third mother liquor 34 0.69 0.56 49 61 fSee Table XII for explanation of abbreviations. height and specific activities calculated for free testosterone. °See Table IX. ^Second and third mother liquors and third crystals combined. TAB IE XIV Substrate %-pregnenolone incubation: crystal l izat ions of dehydroepiandrosteronea Fraction Radioactivity (cpm) Weight 0 (mg) Specif ic ac t i v i t y (cpm/mg) Pool 213,600 15.19 14,062 F i r s t crystals F i rs t mother l iquor 58,475 102,425 13.91 2.15 4,204 47,640 Second crystals Second mother l iquor 22,600 35,450 10.48 3.23 2,156 10,975 Third crystals Third mother l iquor 15,460 4,660 8.63 1.83 1,791 2,546 Fourth crystals Fourth mother liquor 10,725 3,075 5.97 1.75 1,796 1,757 F i f th crystals F i f th mother l iquor 720 2,490 4.10 1.41 1,756 1,766 a See Table XII for explanation of abbreviations. b See F i g . 12, footnote e. cWeight determined by weighing on analyt ical balance. 46 TABLE XV Substrate H-pregnenolone incubation: crystallizations of 17-hydroxypro-gesterone and progesterone3. Compound and Radioactivity Weight (mg) Specific a c t i v i t y fraction Balance UV Balance UV cpm/mg 17-Hydroxyprogesterone cpm Pool 8,280 10.15 816 F i r s t crystals 2,310 6.75 7.13 342 324 F i r s t mother liquor 4,380 3.59 3.51 1,220 ' 1,248 Second crystals 710 3.84 3.98 185 178 Second mother liquor 938 2.78 2.70 337 347 Third crystals 700 3.21 3.25 218 215 Third mother liquor 246 0.46 0.35 535 703 Fourth crystals 443 1.93 2.02 230 219 Fourth mother liquor 203 0.93 , 0.55 218 369 F i f t h crystals 269 1.24 1.33 217 202 F i f t h mother liquor 234 0.84 0.44 279 532 Progesterone 0 dpm dpm/mg Pool 418,263 15.23 27,463 F i r s t crystals 208,030 8.97 23,192 F i r s t mother liquor 206,409 6.50 31,775 Second crystals 185,470 7.81 23,747 Second mother liquor 21,850 0.59 37,034 Third crystals 146,757 6.12 23,980 Third mother liquor 28,535 1.16 24,599 3. See Table XII for explanation of abbreviations. bSee Table IX. TABLE XVI Substrate %-pregnenolone incubation: crystallizations of Cortisol, aldosterone and cortisone 3 Radioactivity Weight Specific a c t i v i t y Compound Fraction (cpm) (mg) (cpm/mg) Balance UV Balance UV Co r t i s o l b Pool 32,720 15.00 2,181 Fir s t crystals First mother liquor 4,000 29,730 13.93 2.38 13.40 2.10 287 L2,492 299 34,157 Second crystals Second mother liquor 250 4,030 10.59 2.98 9.81 2.78 24 1,352 25 1,450 Aldosterone 0 Pool 6,100 12.00 508 First crystals F i r s t mother liquor 10 Lost 10.49 1.58 10.80 1.41 < 1 < 1 Cortisone 0 Pool 4,221 10.00 422 Firs t crystals First mother liquor 75 2,223 4.64 4.30 5.25 5.84 16 517 14 381 aSee Table XII for explanation of abbreviations. bSee Table IX. cSee Table XI. 48 TABLE XVII Results of incubations of tumor tiss u e with sub-s t r a t e -^C-progesterone and substrate 3H-pregnenolone Steroids i s o l a t e d Per cent of substrate r a d i o a c t i v i t y -Progesterone -^-Pregnenolone 17-Hydroxypregnenolone ~ 0 Progesterone 1.0 17-Hydroxyprogesterone ~ 0 ~ 0 Androstenedione ~ 0 0.05 Testosterone ~ 0 . ~ 0 Dehydroepiandrosterone 0.2 C o r t i s o l 0 ~ 0 Cortisone ~ 0 ^ 0 Cortexolone ~ 0 *~ 0 Corticosterone -~ 0 ~ 0 Aldosterone ~ 0 ~ 0 TABLE XVIII Steroids excreted i n the u r i n e a * D Urine mg/24 hrs M-g/24 hrs specimen " 17-KS DHEA 17-0HS 17-KGS P ' t r i o l P ' d i o l THS Free F 2 3 20° (6-15) 16° < l c 2 5 . 6 ° (3-9) IT 6 2 c (3-15) 62° 5 8 c 3 . 1 Q (<4) 4.4 « 7) 2 5 . 4 d 15.5? 1 4 . 0 ° ( < D 1 4 . 5 C 5 9 . 0 U (20-90) < 1 < 1 a17-KS, t o t a l 1 7-ketosteroids; 17-0HS, t o t a l 17-hydroxysteroids; 17-KGS, t o t a l 17-ketogenic s t e r -oids; P.Jtriol, pregnanetriol; P ' d i o l , pregnanediol: F, Cortisol; T, testosterone. ^Normal values shown i n parentheses. °Assay performed by Bioscience Laboratories (See Text). Assay performed by Biomedical Assay Laboratories (See Text). 50 Fig. 5 . Substrate -^C-progesterone incubation: flow sheet for ex-traction, 90$ aqueous methanol-hexane partition, column adsorption chromatography and i n i t i a l paper chromatography of fractions 5 -7 of the column eluate Incubation mixture. ( 7 , 5 8 4 , 0 0 0 cpm, 151 Kg progesterone) Ethyl acetate 4-methylene chloride extraction iCombined extract ( 6 , 6 8 7 , 5 0 0 cpm) Hexane-90% aqueous methanol partition Hexane ( 17 ,900 cpm) f Aqueous methanol ( 6 , 9 8 8 , 5 0 0 cpm) Column adsorption chromatography3. I1 Fractions 5 - 7 Combined ( 4 , 8 8 8 , 5 0 0 cpm) i 0 - 4 cm ( 245 ,200 cpm) Fractions 8,9 Combined (608,000 cpm) L/PG, 57 hrs Testosterone 0 4—14 cm ( 54 ,075 cpm) ^ Other fractions (1,269,050 cpm) Androstenedione(  Progesterone c 14 -44 cnr ( 149 ,900 cpm) Overflow 6 ( 4 , 1 3 9 , 7 5 0 cpm) aSee Table VI. bSee Fig. 8 . cLocations of standards chromatographed simultaneously. ^Eluates combined; see Fig. 7 , footnote a. eSee Fig. 6 . 51 F i g . 6. Substrate -^C- progesterone incubation: resolution and p u r i -f i c a t i o n of progesterone Androstenedione 0-6 cmc (107,300 cpm) 0-13 cm (555,725 cpm)c I 0-12 cm (178,000 cpm)c Overflow 3 (4,139,750 cpm) L/PG, 4 hrs Progesterone (4,221,000 cpm) L/PG, 4 hrs / 1 Progesterone (3,313,250 cpm, 64.3 Kg, S.A. 51,528 cpm/w-g) L/PG, 4 hrs P _ Progesterone (2,751,500 cpm, 54,7 ug, S.A. 50,302 cpm/ug) f 17-42 cm (70,200 cpm) 20-40 cm (181,550 cpm) — r 20-4.0 cm (49,625 cpm) aSee F i g . 5, footnote e. Location of standard androstenedione chromatographed simultaneously. cEluates combined (See F i g . 7, footnote a ) . 52 Fig. 7. Substrate ^ C-progesterone incubation: paper chromatography of selected eluates prior to and following the addition of unlabeled carrier androstenedione, testosterone and 17-hydroxyprogesterone Combined eluate a (1,045,000 cpm) L/PG, 15 hrs Testosterone Androstenedione r I Origin 1-6 cm (451,590 cpm) (258,610 cpm) Combined (710,200 cpm) Carrier testosterone (100 M-g) and carrier 17-hydroxyproges-terone (100 v*- g) added T/PG, 4 hrs T 6-25 cm (192,060 cpm) Progesterone 25-40 cm -T-& Overflow (136,050 cpm) L/PG, 15 hrs Androstenedione (23,840 cpm, 93.8 Kg, S.A. 254 cpm/ M.g) Origin 4 (269,150 cpm) Testosterone, 17-hydroxypro-gesterone (118,100 cpm) H 18-40 cm (187,000 cpm) Ac20,py T/PG, 4 hrs 17-Hydroxypr ogesterone (28,960 cpm, 66.2 Kg, S.A. 437 cpm/»^g) 1 Testosterone acetate 0 (62,240 cpm) fsee Fig. 5, footnote d,5.and Fig. 6, footnote c. "Locations of standards chromatographed simultaneously. °See Table VII., See Fig. 8, footnote b. F i g . 8. Substrate ^-progesterone incubation: paper chromatography of fractions 8 and 9 from the column chromatogram3. C o r t i s o l OCortisone 0 Fractions 8 and 9 from column chromatogram combined and o r i g i n eluate from paper chroma-togram added D (877,150 cpm) T/PG, 33 hrs C o r t i -Cortexolone 0 costerone 0  1 "1 P i f F Origin 1-6 cm 6-19 cm 19-25 cm 25^ -33 cm 33-40 cm Overflow (153,040 cpm) (106,075 cpm) (17,210 cpm) (11,735 cpm) (7,680 cpm) (5,930 cpm) (477,000 cpm) B5, 5 hrs C o r t i s o l 0 Cortisone 0 T/PG, 12 hrs r 1 0-9 cm 9-16 cm 16-32 cm 22-42 cm (15,960 cpm) (10,023 cpm) (13,686 cpm) (51,110 cpm) 27-40 cm Overflow (50,860 cpm) (308,325 cpm) T/PG, 4 hrs ir 1 0-20 cm 20-41 cm (64,850 cpm) (244,600 cpm) aSee Table VI. bSee F i g . 7, footnote d. Locations of standards chromatographed simultaneously. 54 Fig. 9. Substrate %-pregnenolone incubation: flow sheet for extraction, 90% aqueous methanol-hexane partition, column adsorption chromatography, and i n i t i a l paper chromatography of fractions 5-7 of the column eluate Incubation mixture (18,112,000 cpm, 9.2 ^ g pregnenolone) Efcnylaacetate extraction Extract (17,230,000 cpm) 400 M-g testosterone added Hexane-90$ aqueous methanol partition r Hexane (419,620 cpm) Aqueous methanol (14 ,490,000 cpm) Carrier Cortisol 200 (utg added Column adsorption chromatographya r » Fractions 5-7 ^Combined", cfrV (11 ,587,500 cpm) L/PG, 24 hrs DHEAC f Fractions 8 & 9 Combined15 (2 ,987,400 cpm) Androstenedione0 Pregnenolone0 Jgne I Other fractions (2,110,870 cpm) Progesterone* Origin 15-14 cmd 14-24 cme 24I4O cm 0verflow f (172,300 cpm) (295,800 cpm) (8,822,000 cpm) (672,000 cpm) (1,091,000 cpm) aSee Table VIII. bSee Fig. 11. °Locations of standards chromatographed simultaneously. dSee Fig. 12, footnote d. |See Fig. 10. See Table IX. 55 Fig. 10. Substrate -'H-pregnenolone incubation: paper chromatography of eluates containing pregnenolone and androstenedione 1 0-15 cm (748,600 cpm) : Jj 0-14 cm (22,830 cpm) Pregnenolone •+• androstenedione3 (8,822,000 cpm) 200 M.g carrier androstenedione added Ac20„ py L/PG, 17 hrs r-—: 1 r Androstenedione 25-40 cm Overflow (202,250 cpm, 176 Kg, (135,250 cpm) Pregnenolone S.A. 1,150 cpm/(Ag) acetate (6,978,000 cpm) Ac20,py L/PG, 16 hrs Androstenedione (23,450 cpm, 118 K g , SI A. 199 cpm/Kg) 1 1 22i40 cm Overflow (8,925 cpm) (73,325 cpm) L/PG, 16 hrs 1 b Androstened ione (19,475 cpm, 101 K g , S.A. 193 cpm/Kg) See Fig. 9. 'See Table XII. Fig. 11. Substrate %-pregnenolone incubation: paper chromatography of fractions 8 and 9 of column chromatogram3 Fractions 8 and 9 of column chromatogram combined (2,987,400 cpm) Cortisol 17-Hydroxypregnen-. olone b Origin (715,000 cpm) 1 2-10 cm (729,300 cpm) DHEA T/PG, 5 hrs T/PG, 16 hrs I Testosterone0 (700,875 cpm, 307 Kg, S.A. 2,283 cpm/Kg) | Origin (104,500 cpm) J Cortisol (161,300 cpm, 182, M-g, S.A. 886 cpm/Kg) B5, 3 hrs Cdrtisonec 3-9 cme (52,400 cpm) 0-4 cm (74,230 cpm) 17-Hydroxy-pregnenolone" 9-40 cme -t-Overflow (571,500 cpm) Testosterone* 4-7 cmd (75,980 cpm) j 22^42 cm (643,000 cpm) L/PG, 48 hrs f -7-12 cm (36,510 cpm) DHEA 12-16 cm1" (57,300 cpm) fl 0-6 cm (43,950 cpm) Cortisol^ (49,050 cpm, 99 wg, S.A. 495 cpm/Kg) 1 10^40 cme (52,750 cpm) } • 16-40 cm -+• Overflow (243,475 cpm) fsee Table 8 and Fig. 9, footnote b. Eluates combined, see Table X. Locations of standards chromatographed simultaneously. %ee Fig. 12, footnote d. °See Fig. 12, footnote a. gSee Table IX. dSee Fig. 12, footnote b. Fig. 12. Substrate -'H-pregnenolone incubation: paper chromatography of eluates containing carrier testosterone and dehydroepiandrosterone 0-3 cm (187,920 cpm) 1 Origin (16,730 cpm) Testosterone9, (700,875 cpm, 307 Kg, S.A. 2,283 cpm/Kg) L/PG, 48 hrs Testosterone 7-12 cm (229,700 cpm, 250 Kg, (22,150 cpm) S.A. 918 cpm/Kg) Eluate of testosterone zone addedb 119 Kg Carrier 17-hydroxy-progesterone added DHEA T 12-16 cm (10,500 cpm) 1 16-42 cm -t-Overflow (6,250 cpm) Eluates of DHEA zones addedd L/PG, 18 hrs DHEA Ac20,py L/PG, 4 hrs (213,600 cpm) 17-Hydroxy-progesterone* (32,200 cpm) ~ 1 3-22 cm (24,480 cpm) 1 7 Testosterone acetate-1-(150,500 cpm, 230 Kg, S.A. 654 cpm/Kg) r 30-40 cm (59,125 c] fsee Fig. 11, footnote c. £See Fig. 11, footnote d. Location of standard dehydroepiandrosterone chromatographed simultaneously. dSee Fig. 9, footnote d and Fig. U, footnote f. |See Table XIV. See Table IX. 58 PART II STEROID BIOSYNTHESIS IN VITRO BY THE GONADS OF A PATIENT WITH VIRILIZING MALE PSEUDOHERMAPHRODITISM INTRODUCTION (a) General considerations and classification Male pseudohermaphroditism is defined as that condition in which a genetic male with gonads containing only testicular tissue shows phenotypic evidence of incomplete differentiation and development of the external genitalia (114,115). The appearance of the external genitalia varies widely among patients because of different degrees of labio-scro-t a l fusion and penile development. Wilkens (115) has divided male pseudo-hermaphroditism into two major groups: (i) those with external genitalia that simulate the male or are ambiguous and (ii) those with external geni-talia that resemble the female. In the Latter group, feminization usually occurs at puberty and the syndrome has been termed "male pseudohermaphro-ditism with testicular feminization" (116). Subjects in whom the external genitalia resemble the male or are ambiguous have been designated as pre-senting an "incomplete" form of testicular feminization (117); this term is misleading because some patients with ambiguous genitalia will v i r i l -ize at puberty. In this part of the thesis, patients with external geni-talia resembling the female who undergo feminization or in whom only mini-mal changes occur at puberty wil l be designated as exhibiting male pseudo-hermaphroditism with feminization; patients who viri l i z e at puberty will be designated as exhibiting virilizing male pseudohermaphroditism. 59 (b) Steroid biosynthesis by the normal testis Knowledge of the steroid biosynthetic capabilities of the testis (Fig. 4) has accumulated from a large number of experiments i n vivo and i n -vitro (118). However, l i t t l e i s known regarding steroid bio-synthesis i n vitro by the normal human testis ; most incubation studies have been performed on testes obtained from animals or diseased humans. In one instance (119), fresh testicular tissue from a 16 year old ac-cident victim was incubated with •^C-pregnenolone as substrate. The main products formed were testosterone and 17-hydroxyprogesterone. Other metabolites included 17,20<*-dihydroxypregn-4-en-3-one and i t s 20,8 epimer; 17-hydroxypregnenolone/ estra-l,3,5(10)-triene-3,6<*- ,17/? - t r i o l ; 3 , 1 7 ^ -dihydroxyestra-l,3,5(lO)-trien-6-one; 6/5 -hydroxyandrostenedione; JL6*<*-hydroxyandrostenedione, androstenedione; pregn-5-ene-3/ff , 20/9 - d i o l ; and dehydroepiandrosterone. (c) Studies i n vit r o of gonads obtained from patients with male pseudo- hermaphroditism Results of studies i n vitro of gonads obtained from patients with the syndrome of male pseudohermaphroditism and feminization are l i s t -ed i n Tables XIX to XXIII. In one patient (120) both mascullnization and feminization occurred at puberty. A number of different substrates have been employed; however, no unusual products or metabolic pathways have been discovered. Furthermore, convincing evidence for deficient androgen production or excessive estrogen production has not been reported. JL6oc-Hydroxyprogesterone may be a prominent metabolite of substrate progesterone (Table XIX). Green and coworkers (114) have reported studies i n vitro of gonads obtained from two siblings with v i r i l i z i n g male pseudohermaphroditism 60 (Table XXIV). In both cases transformation of -%-17<-hydroxypregnenolone to radioactive androstenedione, testosterone and dehydroepiandrosterone occurred and no radioactivity was found i n the "phenolic" fractions ( a l -kaline washes of ethyl acetate extracts of the incubation media). In one experiment i n which %-pregnenolone was used as substrate, t r i t i a t e d and-rostenedione, testosterone and dehydroepiandrosterone were formed, but radioactive progesterone and 17-hydroxyprogesterone were not detected. The authors suggested that androgen synthesis proceeded from pregnenolone via 17-hydroxypregnenolone and dehydroepiandrosterone rather than via progesterone and 17-hydroxyprogesterone. In this section of the thesis, the results of incubations of gonads obtained from a patient with v i r i l i z i n g male pseudohermaphroditism with radioactive mevalonate, pregnenolone, progesterone and androstenedione are reported. MATERIALS AND METHODS (a) Patient The patient was 14 years of age. At a previous laparotomy no internal genitalia were found. The vagina ended i n a blind pouch, the labia had a scrotal appearance, the c l i t o r i s was markedly" enlarged and inguinal gonads were present. There was no inguinal, a x i l l a r y or f a c i a l hair, nor was there breast development. Leukocytic sex chromatin was ab-sent. The immediate cause for excision of the gonads was deepening of the voice and masculinization of body build. 61 (b) Gonads1 p The two gonads were collected i n ice. Microscopic examination of a small wedge from each testis showed i n t e r s t i t i a l c e l l hyperplasia and absence of spermatogenesis. Following the removal of extraneous tissue, the right gonad weighed 8,01 g and the l e f t gonad weighed 5.60 g. (c) Incubation conditions 7 * -^-Pregnenolone ( 3 5 . 4 3 x 10° cpm, 18 Kg), 4-14C-androstene-dione (10,80 x 10^ cpm, 108 Kg) and 4-^C-progesterone ( 5 . 5 7 x 10° cpm, 106 ^g) were each added to a different reaction flask. 2-^C-D, L-Meva-lonic acid, lactone form, (0 .05 mCi ,5.85 ms) was added to two other re-action flasks. The "^-progesterone (The Radiochemical Centre, Amersham, England) was purified prior to use by Celite column partition chromato-graphy using an iso-octane-90% aqueous methanol solvent system. The androstenedione (New England Nuclear Corporation, Boston, Mass.) was sub-jected to paper partition chromatography i n the ligroin-propylene glycol solvent system prior to use. The 3H-pregnenolone (The Radiochemical Centre, Amersham, England) and the •^••C-mevalonic acid (The Radiochemical Centre, Amersham, England) were not further purified prior to use. The radio-active substrates were transferred to the reaction flasks i n each instance i n a methanol solution, the solvent was evaporated and the substrates were redissolved i n 0.2 ml of propylene glycol. . The gonadal tissue was homogenized at pH 7 . 4 i n Krebs-Ringer •*The cooperation of Dr. F.E. Bryans i n obtaining the tissue i s gratefully acknowledged. 2 Microscopic examination performed by Dr. A.A. Syed. 62 phosphate buffer, i n which Na and K were interchanged, containing glucose (0.01 M) and nicotinamide (0.04 M); a Potter-Elvehjem-type homo-genizer was used. The homogenate was centriguged (650 x _y 20 minutes, 4°) and the supernatant (110 ml) was divided equally among five incubation flasks. Each flask contained NADP"** (14.6 mg), glucose-6-phosphate (26.0 mg) and glucose-6-phosphate dehydrogenase (41.7 Kornberg units). Pergonal^ i n an amount equivalent to 500 units of follicle-stimulating hormone was added to one of the incubation flasks containing ^C-mevalonic acid as substrate. Incubations were begun approximately 4 hours after excision of the testes and were performed for 2 hours at 37° with a gas phase of 95% oxygen-5% carbon dioxide using a Dubnoff metabolic shaking incubator, (d) Extraction, resolution and purification procedures: Data concerning the extraction, resolution and purification procedures employed i n the five incubation experiments are summarized i n Tables XXV to XLIII and i n Figs. 13 to 21. At the end of the incubation period, the contents of each incubation flask were extracted with ethyl acetate (5 x 100 ml). The incubation media from the experiments with 3 1A H-pregnenolone and "^'C-mevaIonic acid were each further extracted with chloroform (4 x 100 ml). The organic extracts from each incubation were evaporated under reduced pressure at 40°, absolute ethanol was added, and the extracts were dried by azeotropic d i s t i l l a t i o n . The combined organic extract from each incubation was then partitioned^ between hexane and 90% aqueous methanol (70$ aqueous methanol was used for the combined organic extract from the incubation with -^C-androstenedione as substrate). 3 A^ g i f t from Cutter Laboratories, Berkeley, California that i s gratefully acknowledged. •^See General Methods section. 63 The aqueous methanol fraction of each incubation was separated into "estrone-estradiol", " e s t r i o l " and neutral fractions 4. The " e s t r i o l " fractions were not processed further. The "estrone-estradiol" fractions were separated into ketonic and non-ketonic portions 4 and estrone and es-tradiol-17/8 were purified by a series of crystallizations. The neutral fractions were further resolved by paper chromato-graphy4 i n the solvent systems ligroin-propylene glycol, toluene-propy-lene glycol, benzene-formamide and Bush A. Chromatography of steroids containing the A.4-3-ketone grouping was usually repeated i n the last system used u n t i l constant specific a c t i v i t y was attained 4. Milligram quantities of unlabeled carrier were then added to the eluates and crys-tallizations were performed u n t i l radiochemical homogeneity was accomplish-ed 4 or u n t i l the crystals contained negligible radioactivity. In a l l experiments unlabeled estrone and estradiol-17,* (9-11 mg of each) were added immediately prior to hexane-aqueous methanol parti-tion. The amounts of neutral steroid carriers added for crystallizations are given as the weights of the pools in Tables XXVI, XXLX to XXXII and XXXV to XLI. In the incubations with -^C-androstenedione as substrate, no neutral steroid carriers^ - were added u n t i l immediately prior to crys-t a l l i z a t i o n . In the experiment with ^4C-progesterone as substrate, un-labeled androstenedione (200 ^g) and testosterone (200 v*g) were added prior to hexane-aqueous methanol partition and 16* -hydroxyprogesterone and 17-hydroxyprogesterone were added immediately prior to crystallization. In the other experiments 200 Kg of each of the following unlabeled ster-oids were added prior to hexane-aqueous methanol partition: progesterone, 64 16*. -hydroxyprogesterone, 17-hydroxyprogesterone, 17-hydroxypregnenolone, dehydroepiandrosterone, testosterone and androstenedione. Pregnenolone (200 * A g ) was also added i n the experiments with -^-C-mevaIonic acid as substrate. Radioactivity was measured with either a liqu i d s c i n t i l l a t i o n spectrometer^ or a gas flow detector^. RESULTS The results of incubations of the cell-free homogenates of the testes from a patient with v i r i l i z i n g male pseudohermaphroditism with androstenedione, 14C -progesterone and %-pregnenolone are given i n Table XLTV. The biosynthesis of conjugated steroids was not investigated. (a) Substrate ^ C-androstenedione incubation The per cent minimal conversion of substrate "^C-androstenedione radioactivity to testosterone was 16.4 which represents 17.7 h*g of sub-strate converted. No demonstrable biosynthesis of estrone or estradiol-17/9 from ^(^androstenedione occurred (Tables XXVII and XXVIII). The specific activity of the '^C-androstenedione recovered following paper chromatography (Fig. 14) differed very l i t t l e from that of the substrate. (b) Substrate ^ P-progesterone incubation Similar quantities of substrate 14c -progesterone were trans-formed to testosterone (13.7 v - g ) and androstenedione (11.6 w.g), 3.6 u»g were transformed to 16»C -hydroxyprogesterone and 3.0 ^  g to 17-hydroxy-progesterone. Synthesis of estrone and estradiol-17fl could not be demon-strated (Table XXXIII). 6 5 Only 2 . 6 % of the o r i g i n a l C-progesterone r a d i o a c t i v i t y was recovered unchanged. Most of the i n i t i a l substrate r a d i o a c t i v i t y was ac-counted f o r i n other fractions (Fig. 1 5 ) . (c) Substrate ^H-pregnenolone incubation Testosterone ( 3 . 9 4 y-g) was the major radioactive metabolite re-3 covered following incubation of the tissue with substrate H-pregnenolone. The formation of radioactive androstenedione ( 1 . 8 4 Kg), 16«* -hydroxypro-gesterone ( 0 . 0 2 Kg), 17-hydroxyprogesterone ( 0 . 0 1 Kg), 17-hydroxypreg-nenolone ( 0 . 0 0 2 v*-g) and dehydroepiandrosterone ( 0.36 yxg) was also demon-3 strated. The transformation of H-pregnenolone to radioactive progesterone (Table XLI), estrone (Table XLIII) and estradiol - 1 7 / 3 (Table XLIl) was not observed. (d) Substrate -^•C-mevalonate incubations Transformation of -^C-mevalonate to steroids was not observed following incubations performed i n the presence or absence of Perganol (Table XXV and F i g . 2 1 ) . DISCUSSION I t i s established that the gonads of patients with male pseudo-hermaphroditism and feminization ( 1 1 7 , 1 2 0 - 1 2 5 , 1 2 7-131 , 1 3 3 - 1 3 6 ) or v i r i l i -zation ( 1 1 4 ) are capable of transforming a v a r i e t y of radioactive sub-strates to testosterone i n v i t r o (Tables XIX-XXIV). The results reported herein u t i l i z i n g the gonads of a patient with v i r i l i z i n g male pseudo-hermaphroditism are i n accord with these findings; r e l a t i v e l y large amounts of substrate progesterone, pregnenolone and androstenedione were con-verted to testosterone (Table XLIV). Green et a l . (114), who have re-ported studies i n v i t r o on the gonads of two patients with v i r i l i z i n g 66 male pseudohermaphroditism, were unable to detect radioactive progesterone or 17-hydroxyprogesterone following incubations of the tissue with sub-strate ^H-pregnenolone and %-17-hydroxypregnenolone. I t was suggested (114) that a defect existed i n the 3 B -ol-dehydrogenase and &5-isomerase that u t i l i z e pregnenolone or 17-hydroxypregnenolone as substrates and, therefore, testosterone was formed via 17-hydroxypregnenolone and dehydro-epiandrosterone (Fig. 4). An alternate explanation of the findings of Green et a l . (114) i s that radioactive progesterone and 17-hydroxypro-gesterone were formed and then rapidly metabolized. The p l a u s i b i l i t y of this explanation i s enhanced by the results reported herein. In the ex-periment using substrate •^'C-progesterone (Table XLIV), the per cent of i n i t i a l substrate radioactivity recovered as progesterone (2.6$) and 17-hydroxyprogesterone (2.8$) was considerably less than that recovered as androstenedione (11.1$) and testosterone (12 .9$). These results cannot be explained by losses of radioactive progesterone and 17-hydroxyproges-terone incurred during the extraction, resolution and purification pro-cedures (Table XXV, Figs. 15 and 16). Fallowing the incubation with ^E-pregnenolone as substrate (Table XLIV), 10.2 per cent of the substrate radioactivity was recovered as androstenedione and 21.9 per cent as tes-tosterone. In contrast, the recovery of radioactive 17-hydroxypregneno-lone (0.01$) and 17-hydroxyprogesterone (0.07$) was low. Radioactive progesterone was not demonstrated; however, 0.10$ of the substrate radio-activity was recovered as 160C,-hydroxyprogesterone. The results of the experiments with substrate "^"C-progesterone and %-pregnenolone show that progesterone, 17-hydroxyprogesterone and 17-hydroxypregnenolone were eff i c i e n t l y metabolized by the tissue and 67 the data are i n accord with the concept that the metabolic a c t i v i t y of a compound cannot necessarily be inferred from i t s r e l a t i v e abundance, par-t i c u l a r l y when the compound i s an intermediate i n a complex metabolic path-way. The results suggest that the tissue was capable of transforming pregnenolone to testosterone both v i a 17-hydroxypregnenolone and dehydro-epiandrosterone and v i a progesterone, 17-hydroxyprogesterone and andros-tenedione (Fig. 4). The data do not permit assessment of the r e l a t i v e importance of the two major biosynthetic pathways because the sizes of the endogenous pools of the precursors and intermediates under i n v e s t i -gation are unknown and because of interconnections between the pathways. The conversion of substrate progesterone to 16<* -hydroxypro-gesterone has been demonstrated i n v i t r o using human ovarian (137,138), placental (139), adrenal (140,141,142,143,144) and t e s t i c u l a r (123-125, 128,131,145-147) tissue as w e l l as the hog adrenal (148) and the r a t t e s t i s (149). 1J6°V -Hydroxyprogesterone was a prominent metabolite of progesterone i n incubations with the testes of some patients with male pseudohermaphroditism and feminization (Table XLX); t h i s compound has not been reported as a product of incubations of gonadal tissue from these patients with substrate pregnenolone (Table XX) nor has i t been observed previously i n incubations of the gonads from patients with v i r i -l i z i n g male pseudohermaphroditism with pregnenolone as substrate (Table XXIV). The r o l e of 16°<- -hydroxyprogesterone i n the t e s t i s i s unknown. Adadevoil and Engel have suggested (144) that l£>< -hydroxylation of pro-gesterone i n the adrenal represents a regulatory process which serves to withdraw substrate that would otherwise undergo 17-hydroxylation and 21-hydroxylation. Calvin and Lieberman (150) ad^iinistered radioactive 68 16<*• -hydroxyprogesterone to a normal male subject and found 3°^hydroxy-17-iso-pregnan-20-one as a metabolite i n the urine; the evidence suggested a A"^ intermediate. The conversion of substrate mevalonate to steroids i n vitro hf, preparations of endocrine tissues that form steroid hormones has been ob-served by some investigators (151-153); however, the extent of conversion has been low and others have been unable to demonstrate transformation of substrate mevalonate (154-156). Salokangas and coworkers (157) showed that the failure to obtain significant conversions of substrate mevalonate in rat testis preparations involving unbroken cells was due to the ina-b i l i t y of mevalonate to penetrate intact c e l l s . These workers also pos-tulated that the rapid destruction of adenosine triphosphate by a micro-somal adenosine triphosphatase was responsible for the i n a b i l i t y of c e l l -free homogenates to metabolize substrate mevalonate to squalene. Nightin-gale et a l . (158) confirmed the presence of an adenosine triphosphatase i n cell-free homogenates of rat testes and also reported that the microsomal fraction contained a pyrophosphate pyrophosphohydrolase that catalyzed the hydrolysis of pyrophosphorylated intermediates of sterol biosynthesis. The conversion of substrate mevalonate to squalene was enhanced by the presence of an adenosine triphosphate-generating system (pyruvate kinase, phosphoenolpyruvate and adenosine diphosphate). In the experiments re-ported i n this section of the thesis, an adenosine triphosphate-generating system was not included i n the incubation media and the failure to demon-strate transformation of substrate •^C-mevalonate to steroids may be due to the presence of adenosine triphosphatase and pyrophosphate pyrophos-phohydrolase a c t i v i t y i n the cell-free homogenate. The etiology of the syndrome of male pseudohermaphroditism i s 69 not understood. The following hypotheses have been suggested: deficient androgen synthesis by the test is (159), excessive peripheral conversion of androgens to estrogens, rapid metabolism of circulat ing androgens to b io log ica l ly inactive compounds, inactivation of androgens by antagonists (160) and diminished target-organ sensi t iv i ty to androgens (161,162). The cause of male pseudohermaphroditism must exert i t s influence during the f i r s t trimester of embryonic development because the di f ferent iat ion of the external genital ia i s complete by the twelfth week of gestation (163). Experiments on animals have shown that the development of the male ex-ternal genital ia is dependent on the presence of the f e t a l t e s t i s ; cas-t rat ion of the male fetus in utero leads to female urogenital sinus de-velopment (164). Androgenic steroids do not appear to inhib i t the de-velopment of the Mullerian system, hence the absence of a uterus i n patients with male pseudohermaphroditism may be explained by the pre-sence of the Mullerian inhibitory factor (165) that prevents the formation of the uterus in the male. The results of a number of studies provide substantial evidence that male pseudohermaphroditism i s usually not associated with deficient androgen production or excessive estrogen production. Testosterone con-centrations i n peripheral venous plasma prior to castration have f r e -quently been found to be in the range of normal males (120,121,133, 166-169); lower values were observed postoperatively when plasma tes -tosterone concentrations were measured preoperatively and following gonadectomy (121,130,133,166). Elevated peripheral venous plasma con-centrations of dehydroepiandrosterone and dehydroepiandrosterone s u l -fate have also been reported (117,133); these values declined following 70 castration. The value for the plasma testosterone concentration was re-ported to be i n the range of normal females i n one subject (117) and be-tween the upper l i m i t for normal females and the lower li m i t for normal males i n another subject (130). Two groups of investigators have report-ed that the values for the blood production rate of testosterone were i n the range of normal males i n patients with male pseudohermaphroditism (130,168,169) and elevated secretion rates of dehydroepiandrosterone and i t s sulfate have been observed (166). The rate of testosterone excretion v i a the kidney was reported to be i n the range for normal males (114,120, 133,170) and normal females (114,129,171). In one study (117), dehydro-epiandrosterone and androstenedione were isolated from the gonadal tissue; testosterone and estrogens were not found. The values for the excretion rate of estrogens via the kidney prior to castration have usually been i n the overlapping region of low normal for nonpregnant females and high nor-mal for males and postoperative values were frequently lower than preop-erative values (121,123,128,129,132,134,167,172-176). Estrone and estra-diol-17/5 concentrations i n peripheral venous plasma were observed to be in range of normal nonpregnant females (167) or males .'(121); i n the l a t -ter study, no decrease occurred following castration. Studies of gonadal vein blood obtained at the time of castration have revealed the presence of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, estrone, estradiol-17£ , testosterone and androsterone sulfate (117,120, 129,1309133166,167); i n several of these studies concentrations of the steroids under'investigation have been considerably higher i n gonadal vein plasma than i n plasma obtained from a peripheral vein of the same subject. 71 Other studies have provided evidence that excessive peripheral transformation of testosterone to estrogens or unusually rapid metabolism of testosterone does not occur i n subjects with the syndrome of male pseudo-hermaphroditism. The metabolic clearance rate and the h a l f - l i f e of testos-terone in plasma were reported to be normal (166-169). Following admini-stration of "^C-testosterone to one patient (167), the pattern of radio-active metabolites excreted in the urine was similar to that of normal subjects; very l i t t l e radioactivity was found i n the phenolic fraction. In another subject (129) no increase occurred i n the excretion rate of estrogens in urine during the administration of large doses of testoster-one. At present, the evidence favors the hypothesis that a defect i n target-organ sensitivity to androgens exists i n patients with male pseudohermaphroditism and feminization. Wilkins (162) observed a lack of v i r i l i z a t i o n in patients treated with large amounts of testosterone and this finding has been confirmed (129) . French and coworkers (167) administered large doses of testosterone to a subject under metabolic balance conditions and reported no change i n the excretion rates of n i t -rogen, phosphorus and c i t r i c acid; the excretion rates of these substances normally decrease during testosterone administration (167, 176-181). Investigators in search of an explanation for a target-organ defect in patients with the syndrome of male pseudohermaphroditism and feminization have studied the metabolism of testosterone in peripheral tissues of these patients. It has been reported that 17/8 -hydroxy-5* -androstan-3-one i s the only metabolite present i n prostatic nuclei f o l -lowing intravenous administration of radioactive testosterone to rats (182) 72 and after incubation of rat prostatic tissue with testosterone (183). Furthermore, 17 £ -hydroxy-5<* -androstane steroids have been observed to be more potent androgens in certain tests than testosterone i t s e l f (184, 185). These findings have led to the suggestion (182,183,186) that 17/8 -hydroxy-^* -androstan-3-one may be the active form of androgens i n prostatic nuclei. Mauvais-Jarvis et a l . (187) rubbed labeled testosterone on ab-dominal and thoracic skin of patients with male pseudohermaphroditism and feminization and noted diminished excretion of 5* -metabolites i n the urine of these patients when compared to normal subjects. Northcutt and cowork-ers (188) incubated portions of abdominal skin, vas deferens and epididy-mus and pubic hair f o l l i c l e s obtained from normal subjects and from patients with male pseudohermaphroditism and feminization with radioactive testos-terone as substrate and observed that the tissues from the patients trans-formed less testosterone to 17fi -hydroxy-5«< -androstan-3-one than did cor-responding tissue from the normal subjects. Both groups of investigators suggested that the failure of patients with the syndrome of male pseudo-hermaphroditism and feminization to respond to testosterone may be due to a defect i n testosterone 5 0 t -reductase activity i n the target organs. Patients with v i r i l i z i n g male pseudohermaphroditism masculinize at puberty and the results of the experiments reported i n this part of the thesis show that the testes removed during puberty from a patient with v i r i l i z i n g male pseudohermaphroditism produce substantial amounts of and-rogens in vitro from substrate H-C-progesterone and substrate %-preg-nenolone. The data suggest that these patients are able to respond to androgens at the time of puberty. A l l studies on patients with the syn-drome of male pseudohermaphroditism aimed at elucidating the cause of 73 this disorder have been performed many years after the i n i t i a l etiologic factor has exerted its effect and the biochemical data obtained by these studies may not apply to the conditions present during early embryonic development. SUMMARY AND CONCLUSIONS (i) Steroid biosynthesis in vitro was investigated in testes obtained during puberty from a patient with virilizing male pseudohermaphroditism. 3 Cell-free homogenates of gonadal tissue efficiently metabolized -'H-preg-nenolone, "^C-progesterone and "^C-androstenedione to testosterone; for-mation of estrone and estradiol- 17i» was not demonstrated. 16<*- -Hydroxy-progesterone was formed from both %-pregnenolone and ^C-progesterone. (ii) The results are similar to those of others who have investigated the steroidogenic capacity of gonadal tissue in patients with male pseudoherma-phroditism and feminization at puberty. Green and coworkers (114), who have reported the only other detailed study of subjects with virilizing male pseudohermaphroditism, postulated that the formation of progesterone from pregnenolone was defective. In the study reported herein, transfor-mation of pregnenolone to testosterone and androstenedione occurred both via 17-hydroxypregnenolone and dehydroepiandrosterone and via progesterone and 17-hydroxyprogesterone. ( i i i ) The failure of patients with virilizing male pseudohermaphroditism to masculinize during embryonic development contrasts with the virilization that occurs during puberty. A biochemical abnormality may exert a tran-sient effect during embryonic development. Alternatively, the sensitivity to androgenic hormones may be subnormal in certain tissues and normal in other tissues of patients with virilizing male pseudohermaphroditism. TABLE XIX Products identified following incubations of gonads from patients with male pseudohermaphro-ditism and feminization with radioactive progesterone as substrate Product Reference6 A B a G D E F (p H I J c K X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X xd xd xd X X X X 17-Hydroxyprogesterone Androstenedione Testosterone Estradiol-17/9 Estrone 20* -Hydroxypregn-4-en-3-one 17,20* -Dihydroxypregn-4-en-3-one 17,20-Dihydroxypregn-4-en-3-one 16* -Hydroxyprogesterone 19-Hydroxyandrostenedione Gonads from three patients were incubated i n three separate experiments. The results were qualitatively the same i n a l l experiments, ^Gonads from two patients were incubated i n two separate experiments. The results were quali-tively the same i n both experiments. cGonads from two patients were incubated in two separate experiments. Testosterone was formed i n both experiments; the identification of androstenedione was not established i n one experiment. Identification tentative. e A , ( l 2 l ) ; B,(122); C,(l23); D,(l24); E,(l25); F, (127); G,(128); H,(117); 1,(129); J,(l30); K,(131). 75 TABLE XX Products identified following incubations of gonads from patients with male pseudohermaphroditism and feniinization with substrate pregnenolone Product Reference6 A a B c D> E pC G Progesterone X X X X x d Andros tenedione X X X X X X De hydroepiandrosterone X X X X X X Testosterone X X X X X x d X 17-Hydroxyprogesterone X X X X xd X Estrone X X Estradiol-17 £ X X X 17-Hydroxypregnenolone X X X X Androstenediol X Pregn-5-ene-3 P ,17,20 * - t r i o l X A 20* -Hydroxypregn-4-ene-3-one 19-Hydroxyandrostenedione Pregnenolone sulfate Dehydroepiandrosterone sulfate x x x aGonads from three patients were incubated i n three separate ex-periments. 17-Hydroxyprogesterone, estrone and estradiol-17^ were detected as products i n two of the incubations, the other products l i s t e d were found i n a l l three incubations. bGonads from two patients were incubated i n two separate experi-ments. The results were qualitatively the same i n botheexperi-ments. cNon-radioactive pregnenolone used as substrate, dldentification tentative. % ( U 2 2 ) ; B,(125); C,(l27); D,(l28). E,(117); F,(l32) ; G,(129). 76 TABLE XXI Products identified following incubations of gonads from patients with male pseudohermaphroditism and feminization with radioactive testosterone and radioactive androstenedione as substrates Substrate Product Referencec A B O D E F G3, H Testosterone Estradiol-17/S Estrone Androstenedione 19-Hydroxytestosterone 6/6 -Hydroxytestosterone x X X X X Androstenedione Testosterone 19-Hydroxytestosterone 19-Hydroxyandrostenedione 6l* -Hydroxytestosterone 6p -Hydroxyandrostenedione Estrone Estradiol-17/? Equilinin x x x x b xb x b x 1 3 5 > X" X1" x x x X X X X Gonads from two patients were incubated i n two separate experiments. The results were qualitatively the same i n both experiments, i d e n t i f i c a t i o n tentative. CA,(121); B,(l23)j C,(U24); D,(l25); E,(U27); F,(133); G,(128); H,(117). 77 TABLE XXII Products identified following incubations of gonads from patients with male pseudohermaphroditism and feminization with radioactive dehydroepiandrosterone and radioactive dehydroepiandrosterone s u l -fate as substrates Substrate Product , Reference b A a B C D E F Dehydroepiandrosterone Androstenedione x x x x x x Testosterone x x x x x x Estrone x x Estradiol-17£ x x x Androstenediol x x x 19-Hydroxyandrostenedione x DHEA-sulfate x Androstenediol sulfate x Dehydroepiandrosterone Androstenedione x sulfate DHEA x Testosterone x Androstenediol x Androstenediol sulfate x aGonads from three patients were incubated i n three separate experi-ments. Estrone was formed i n one experiment, the other products -^listed were formed i n a l l three experiments. 'A, (122); B,(125); C,(l34); D,(l27); E , ( l l 7 ) , F,(l35). 78 \ TABLE XXIII Products identified following incubations of gonads from patients with male pseudohermaphroditism and feminization with radioactive acetate, radioactive 17-hydroxyprogesterone and radioactive 17-hydroxypregnenolone as substrates j N Reference0 Substrate Product A B C I T E Acetate Pregnenolone x x Progesterone x 17-Hydroxyprogesterone x Dehydroepiandrosterone x x Androstenedione x Testosterone x x Estradiol-17p x Estrone Cholesterol x Cholesterol sulfate x 17-Hydroxy- None x progesteroneiTestosterone x 17,20ot- -Dihydroxypregn-4-en-3-one x 17,20^ -Dihydroxypregn-4-en-3-one x Androstenedione x D 17-Hydroxy- Dehydroepiandrosterone x pregnenolone Pregn-5-ene-3P ,17,20* - t r i o l x Androstenedione Testosterone x 17-Hydroxyprogesterone x Gonads from two patients were incubated i n two separate experi-ments. The results were qualitatively the same i n both experi-ments. "Identification tentative. CA,(120); B,(123); C,(l34); D,(228)5 E,(l36). TABLE XXIV Results of incubations of gonads from patients with v i r i l i z i n g male pseudo-hermaphroditism with radioactive pregnenolone and radioactive ^-hydroxy-pregnenolone as substrates Substrate Product cpm/g of tissue incubated Case l a Case 2d H-Pregneno-lone D 3H-17-Hydroxy-pregnenolone0 Androstenedione 464,348 Dehydroepiandrosterone 40,935 Testosterone 67,478 Progesterone 0 17-Hydroxyprogesterone 0 Androstenedione 202,428 Dehydroepiandrosterone 153,325 Testosterone 61,725 17-Hydroxyprogesterone 0 129,868 107,763 29,860 0 aReference ' 114. b1.0 j*Gi per incubation flask. c100 K-Ci per incubation flask. TABLE XXV Per cent of substrate radioactivity present i n fractions following extraction, partition and separation procedures3 Procedure Fraction Per cent of substrate radioactivity^ Radioactive substrate Androstene-dione Progest-erone Pregnen-olone Mevalonate (control) Mevalonate (Pergonal) Extraction of incubation medium Hexane-aqueous methanol p a r t i -tion Partition of aqu-eous methanol fraction Girard separation of the "estrone-estradiol" fraction Organic extract 96 101 86 22 22 Hexane < 1 C 1 2 £ 1 t 1 Aq. methanol 97 99 86 20 15 Neutral 96 70 65 £ 1 < 1 "Estrone-Estradiol" * 1 6 6 < 1 < 1 "Estriol" < 1 12 11 1 1 Ketonic < 1 3 3 Non-ketonic < 1 1 aSee the Text for details of the procedures TABLE XXVI Substrate -^C-androstenedione incubation: crystallizations of testosterone3-' Radioactivity Weight Specific a c t i v i t y Fraction (cpm) (mg) (cpm/mg) Balance uv Balance UV Pool 2,136,875 14.78 144,579 F i r s t crystals 1,286,500 9.69 132,767 First mother liquor 767,500 5.14 149,319 Second crystals 1,091,000 8.91 122,446 Second mother liquor 127,800 1.43 89,371 Third crystals 1,027,250 7.90 130,031 Third mother liquor 122,825 1.11 110,653 Fourth crystals 932,250 7.46 7.19 124,966 129,659 Fourth mother liquor 67,600 0.59 114,576 Fi f t h crystals 592,700 5.33 5.17 111,201 114,642 Fi f t h mother liquor 243,750 2.30 2.00 105,978 121,875 Sixth crystals 217,525 2.01 1.75 108,221 124,300 Sixth mother liquor 381,500 3.59 3.19 106,267 119,592 See Table XII for explanation of abbreviations 'See Fig. 13, footnote c. TABLE XXVII Substrate ^C-^drostenedione incubation: crystallizations of estradiol-17,5 T" 7"! Radioactivity Weight3 Specific a c t i v i t y ft""*"" (cpm) (mg) (cpm/mg) Pool 16,850 4.75 3,547 F i r s t crystals 5,350 2.60 2,058 F i r s t mother liquor 12,350 1.95 6,333 Second crystals 1,380 1.94 711 Second mother liquor 3,588 0.44 8,155 Third crystals 489 1.56 313 Third mother liquor 688 0.30 2,293 Fourth crystals 311 1.36 229 Fourth mother liquor 184 0.24 767 F i f t h crystals 194 1.19 163 F i f t h mother liquor 68 0.17 400 iiaWeight determined by weighing on an analytical balance TABLE XXVIII Substrate ^G-androstenedione incubation: crystal l izat ions of estrone Fraction Radioactivity (cpm) Weight 3 (mg) Specif ic ac t i v i t y (cpm/mg) Pool 98,350 4.10 23,988 F i r s t crystals 4,700 3.16 1,487 F i r s t mother l iquor 87,150 0.72 121,041 Second crystals 655 2.26 290 Second mother l iquor 4,065 0.87 4,672 Third crystals 314 1.67 188 Third mother l iquor 359 0.41 876 Fourth crystals 190 1.32 144 Fourth mother liquor 89 0.30 297 F i f th crystals 122 0.89 137 F i f t h mother l iquor 75 0.44 170 h e i g h t determined by weighing on an analyt ical balance TABLE XXIX-1 Substrate -^C-progesterone incubation: crystal l izat ions of 16*- -hydroxyprogesterone' Radioactivity Weight Speci f ic ac t i v i t y Fraction (cpm) (mg) (cpm/mg) Balance UV Balance UV £ool 376,000 10.38 36,224 F i r s t crystals 165,575 8.11 8.07 20,416 20,517 F i r s t mother liquor 182,230 2.21 2.09 82,457 87,191 Second crystals 111,425 5.87 5.98 19,982 18,633 Second mother liquor 54,510 2.22 2.08 24,554 26,207 Third crystals 69,800 3.82 3.78 18,272 18,466 Third mother liquor 36,670 2.10 2.10 17,462 17,462 Fourth crystals 45,390 2.35 2.53 19,315 17,941 Fourth mother liquor 20,195 1.15 1,18 17,561 17,114 a See Table XII for explanation of abbreviations. b See F i g . 15, footnote c. TABLE XXX Substrate ^C-progesterone incubation: crystallizations of candrostenedionea» Radioactivity Weight Specific a c t i v i t y Fraction (cpm) (mg) ?J (cpm/mg) Balance UV Balance UV Pool 4 6 7 , 0 0 0 1 5 . 0 0 3 1 , 1 3 3 First crystals 297,325 9 . 7 4 1 0 . 1 0 3 0 , 5 2 6 2 9 , 4 3 8 F i r s t mother liquor 173 ,050 5 .47 5 .36 3 1 , 6 3 6 3 2 , 2 8 5 Second crystals 163,525 5 .65 5 5 U 9 6 28 ,942 2 7 , 4 3 7 Second mother liquor 119,625 4 . 1 3 3 . 9 5 28,965 3 0 , 2 8 5 Third crystals 4 9 , 0 5 0 1 .72 1.80 28 ,517 2 7 , 2 5 0 Third mother liquor 109 ,400 3 .85 3 . 9 0 28,416 28 ,051 aSee Table XII for explanation of abbreviations bSee Fig. 1 5 , footnote d. TABLE XXXI Substrate -^C-progesterone incubation: crystallizations of testosterone acetate^, 0' Radioactivity Weight Specific activity Fraction (cpm) (mg) (cpm/mg) Balance UV Balance UV Pool 636,700 17.45 36,487 First crystals 349,200 11.10 11.85 31,459 29,468 First mother liquor 259,200 6.70 6.41 38,687 40,437 Second crystals 283,275 8.94 9.18 31,686 30,858 Second mother liquor 52,430 1.62 1.71 32,364 30,661 Third crystals 221,575 7.56 7.65 29,309 28,964 Third mother liquor 43,890 1.43 1.31 30,692 33,504 fSee Table XII for explanation of abbreviations. "Weights and specific activities calculated for free testosterone cSee Fig. 16, footnote b. TABLE XXXII Substrate 14c -progesterone incubation: crystallizations of 17-hydroxyprogesterone Radioactivity Weight Specific a c t i v i t y Fraction (cpm) (mg) (cpm/mg) Balance UV Balance UV Pool 219,125 15/.00 14,608 First crystals 104,125 11.04 11.80 9,432 8,824 Firs t mother liquor 127,460 4.30 4.19 29,642 30,420 Second crystals 77,110 8.72 9.01 8,843 8,558 Second mother liquor 26,240 2.42 2.12 10,843 12,377 Third crystals 50,200 5.97 6.26 8,409 8,019 Third mother liquor 20,720 2.32 2.42 8,931 8,562 Fourth crystals 31,100 3.97 4.14 7,834 7,512 Fourth mother liquor 13,655 1.76 1.83 7,758 7,462 F i f t h crystals 18,770 2.36 2.35 7,953 7,987 F i f t h mother liquor 10,675 1.32 1.24 8,087 8,609 aSee Table XII for explanation of abbreviations bSee Fig. 16, footnote c. TABLE XXXIII Substrate 14C -progesterone incubation: c r y s t a l l i z a t i o n s of estrone and estradiol -17/S Fraction Radioactivity (cpm) Weight a S p e c i f i c a c t i v i t y (mg) (cpm/mg) Estrone Pool F i r s t crystals F i r s t mother liquor Second crystals Second mother liquor E s t r a d i o l - 1 7 ^ Pool F i r s t crystals F i r s t mother liquor Second crystals Second mother l i q u o r 172,825 3 ,243 169,975 135 1,934 9 ,625 665 7 ,465 243 465 7 . 9 7 6 . 3 7 1 . 8 9 2 . 4 0 1 .64 9 . 2 1 4 . 3 0 4 . 7 8 2 . 8 7 1 .04 2 1 , 6 8 4 509 89 ,934 56 1 ,156 1 ,045 156 1 ,562 85 4447 h e i g h t determined by weighing on an a n a l y t i c a l balance. 89 TABLE XXXIV Substrate -'H-pregnenolone incubation: further paper chromatography of eluates containing carrier steroids Compound Chromatographic solvent system Contents of eluate Radioactivity (cpm) Weight ( K g ) Specific a c t i v i t y (cpm/K-g) DHEAa L/PG L/EG 1,887,000 1,276,425 921,875 Androstenedione13 L/PG L/PG L/PG 2,658,850 2,570,750 2,423,850 2,270,550 148 121 127 134 17,965 21,246 19,085 19,917 Testosterone acetate c L/PG L/PG L/PG 6,612,500 5,930,875 5,292,000 5,210,000 342 348 341 135 46,567 40,073 37,532 38,592 17-Hydroxy-progesterone T/PG T/PG 86,750 57,100 54,625 346 336 136 594 420 402 Progesterone15 L/PG L/PG L/PG L/PG 688,600 428,175 332,550 274,950 249,625 221 145 123 116 106 3,316 2,953 2,704 2,370 2,355 aSee Fig. 17, footnote c and Table XXXVII. bSee Fig. 20, footnote b and Table XXXVIII. °See Fig. 19, footnote b and Table XXXIX. dSee Fig. 19, footnote b and Table XL. eSee Fig. 17, footnote f and Table XLI. TABLE XXXV Substrate ^H-pregnenolone incubation: crystallizations of 17-hydroxypregnenolonea Fraction Radioactivity (cpm) Weight 0 (mg) Specific a c t i v i t y (cpm/mg) Pool 108,040 15.00 7,203 Fi r s t crystals 10,800 12.69 851 F i r s t mother liquor 84,560 2.43 34,798 Second crystals 3,790 10.38 365 Second mother liquor 8,425 2.08 4,050 Third crystals 2,030 7.58 268 Third mother liquor 920 1.61 571 Fourth crystals 1,260 6.16 205 Fourth mother liquor 498 1.31 380 F i f t h crystals 1,020 4.90 208 Fi f t h mother liquor 272 1.27 214 Sixth crystals 801 3.99 201 Sixth mother liquor 154 0.74 208 See Fig. 18, footnote c. "Weight determined by weighing on an analytical balance. TABLE XXXVI Substrate ^H-pregnenolone incubation: crystallizations of lo**--hydroxyprogesteronea> Radioactivity Weight Specific a c t i v i t y Fraction (cpm) (mg) (cpm/mg) i Balance UV Balance UV Pool 304,900 8.87 34,374 First crystals 61,030 6.01 5.40 10,155 11,302 F i r s t mother liquor 194,010 3.36 2.63 57,741 73,768 Second crystals 22,025 3.13 3.03 7,037 7,269 Second mother liquor 37,785 2.75 2.65 13.740 14,258 Third crystals 8,063 1.25 1.29 6,450 6,250 Third mother liquor 12,295 1.88 1.59 6,540 7,733 Fourth crystals 6,180 6.24 c 6.27 990 986 Fourth mother liquor 1,205 1.15 1.05 1,048 1,148 F i f t h crystals 3,730 3.70 3.55 1,008 1,051 F i f t h mother liquor 2,475 2.41 2.35 1,027 1,053 aSee Table XII for explanation of abbreviations. bSee Fig. 18, footnote d. c6.13 *wvg of additional unlabeled 16*> -hydroxyprogesterone added prior to fourth cry-st a l l i z a t i o n . TABLE XXXVII Substrate %-pregnenolone incubation: crystallizations of dehydroepiandrosterone3 Fraction Radioactivity Weightb Specific a c t i v i t y (cpm) (mg) (cpm/mg) Pool 921.875 20.30 45,413 F i r s t crystals 650,000 15.72 41,348 First mother liquor 258,000 4.89 52,761 Second crystals 532,000 14.30 37,203 Second mother liquor 68,500 1.02 67,157 Third crystals 456,000 13.04 34,969 Third mother liquor 46,400 1.12 41,429 Fourth crystals 437,000 12.38 35,299 Fourth mother liquor 14,600 0.41 35,561 F i f t h crystals 392,000 11.26 35,813 F i f t h mother liquor 34,000 1.01 33,663 aSee Table XXXIV. ^Weight determined by weighing on an analytical balance. TABLE XXXVIII o a Substrate •'H-pregnenolone incubation: c r y s t a l l i z a t i o n s of androstenedione ' Radioactivity Weight Specific a c t i v i t y Fraction (cpm) (mg) (cpm/mg) Balance UV Balance UV Pool 2,270,550 20.20 112,403 F i r s t crystals 1,344,000 12.67 13.15 106,077 102,205 F i r s t mother liquor 863,775 7.62 7.96 113,356 108,514 Second crystals 1,028,850 9.55 10.13 107,733 101,565 Second mother liquor 291,850 2.90 2.85 100,638 102,404 Third crystals 748,175 7.20 7.43 103,913 100,697 Third mother liquor 224,730 2.23 2.20 100,776 102,150 aSee Table XII for explanation of abbreviations bSee Table XXXIV. TABLE XXXIX Substrate %-pregnenolone incubation: crystallizations of testosterone acetate a> b, c Radioactivity Weight Specific a c t i v i t y Fraction (cpm) (mg) (cpm/mg) Balance UV Balance UV Pool 5,210,000 12.63 412,509 F i r s t crystals 3,782,700 10.15 8.63 372,680 438,320 F i r s t mother liquor 1,341,650 2.77 2 .51 484,350 534,522 Second crystals 2,885,000 8.12 8.60 355,296 335,465 Second mother liquor 666,250 1.79 1.81 372,207 368,094 Third crystals 2,350,950 6.33 6.26 371,398 375,551 Third mother liquor 634,520 1.76 1.75 360,522 362,583 aSee Table XII for explanation of abbreviations. bSee Table XXXIV. cWeights and specific activities calculated for free testosterone. TABLE XL Substrate 3H-pregnenolone incubation: c r y s t a l l i z a t i o n s of 17-hydroxyprogesterone' Radioactivity Weight S p e c i f i c a c t i v i t y Fraction (cpm) (mg) (cpm/mg) Balance UV Balance UV Pool 54,625 10.10 5,408 F i r s t crystals 14,400 6.87 7.60 2,096 1,895 F i r s t mother liq u o r 29,890 3.09 3.04 9,673 9,832 Second crystals 8,080 4.38 4.85 1,845 1,666 Second mother liquor 5,290 2.28 2.48 2,320 2,133 Third crystals 5,165 3.09 3.06 1,672 1,688 Third mother liquor 2,023 1.18 1.12 1,714 1,806 Fourth crystals 2,855 1.73 1.74 1,650 1,6U Fourth mother liquor 2,168 1.33 1.32 1,630 1,642 F i f t h crystals 1,508 0.93 0.97 1,622 1,555 F i f t h mother liquor 1,083 0.66 0.71 1,641 1,525 aSee Table XII f o r explanation of abbreviations. bSee Table XXXIV. TABLE XLI Substrate %-pregnenolone Incubation: crystal l izat ions of progesterone a ' Radioactivity Weight Specif ic ac t i v i t y Fraction (cpm) - (mg) (cpm/mg) Balance UV Balance UV Pool 249,625 10.06 24,814 F i rs t crystals 148,970 9.36 9.37 15,916 15,898 F i r s t mother liquor 77,507 1.06 0.75 73,119 103,343 Second crystals 99,320 7.75 7.70 12,815 32,899 Second mother liquor 47,555 1.69 1.39 28,339 34,232 Third crystals 73,520 6.91 6.87 10,640 10,702 Third mother l iquor 19,460 0.57 0.48 34,340 40,542 Fourth crystals 53,770 5.98 6.22 8,992 8,645 Fourth mother l iquor 18,485 0.80 0.74 23,106 24,980 F i f th crystals 36,290 5.21 5.15 6,965 7,047 F i f t h mother l iquor 12,475 1.01 0.83 32,351 15,030 Sixth crystals 28,045 4.64 4.64 6,044 6,044 Sixth mother l iquor 6,140 0.46 0.32 33,348 19,188 Seventh crystals 22,010 4.08 4.22 5,395 5,216 Seventh mother l iquor 4,528 0.32 0.59 34,150 7,675 Eighth crystals 8,875 3.29 3.28 2,698 2,706 Eighth mother l iquor 11,160 0.60 0.67 18,600 16,656 Ninth crystals 5,400 2.43 2.45 2,222 2,204 Ninth mother liquor 2,870 0.58 0.63 '4,948 4,556 Tenth crystals 3,368 1.63 1.62 2,066 2,079 Tenth mother l iquor 1,860 0.72 0.67 2,583 2,776 Eleventh crystals 2,178 1.27 1.17 1,715 1,862 Eleventh mother l iquor 610 0.33 0.24 1,848 2,542 fSee Table XII for explanation of abbreviations bSee Table XXXIV. TABLE XLII Substrate 3H-pregnenolone incubation: crystal l izat ions of estradiol-17/? Fraction Radioactivity (cpm) Weight 3 (mg) Specif ic ac t i v i t y (cpm/mg) Pool 436,925 10.42 41,931 F i r s t crystals 119,450 6.60 18,098 F i r s t mother liquor 278,620 3.44 80,994 Second crystals 49,665 6.08 8,169 Second mother liquor 48,125 0.39 123,397 Third crystals -32,460 5.14 6,315 Third mother l iquor 19,125 0.84 22,768 Fourth crystals 20,978 4.66 4,502 Fourth mother l iquor 9,275 0.60 15,200 F i f t h crystals 13,375 3.53 3,879 F i f t h mother l iquor 5,623 0.82 6,857 Sixth crystals 8,410 3.01 2,794 Sixth mother l iquor 2,808 0.47 5,974 Seventh crystals 2,068 2.20 940 Seventh mother l iquor 5,408 0.46 11,756 aWeight determined by weighing on an analyt ical balance. TABLE XL1II Subs t ra te %-pregneno lone i n c u b a t i o n : c r y s t a l l i z a t i o n s of es t rone F r a c t i o n R a d i o a c t i v i t y (cpm) We igh t 3 (mg) S p e c i f i c a c t i v i t y (cpm/mg) P o o l 1,030,150 9.08 113,453 F i r s t c r y s t a l s 32,925 6.46 5,097 F i r s t mother l i q u o r 839,600 2.65 316,830 Second c r y s t a l s 6,015 5.40 1,114 Second mother l i q u o r 26,345 0.97 27,160 T h i r d c r y s t a l s 1,850 3.76 492 T h i r d mother l i q u o r 4,615 1.62 2,849 F o u r t h c r y s t a l s 613 2.49 246 Fou r th mother l i q u o r 1,007 1.18 853 F i f t h c r y s t a l s 316 1.76 180 F i f t h mother l i q u o r 367 0.45 816 h e i g h t s determined by weigh ing on an a n a l y t i c a l b a l a n c e . TABLE XLIV Results of incubations ofE-testes from a patient with v i r i l i z i n g male pseudo-hermaphroditism Products Substrates •^-C-Androstenedione 14c -Progesterone ^H-Pregnenolone % Substrate Kg Sub- % Substrate Kg Sub- % Substrate Kg Sub-radioactivity strate con- radioactivity strate con- radioactivity strate con-verted verted verted Testosterone 16.4 17.7 12.9 13.7 21.9 3.94 Androstene-dione 11.1 11.6 10.2 1.84 16°C -Hydroxy-3.6 progesterone 3.4 0.1 0.02 17-Hydroxy-progesterone 2.8 3.0 0.07 0.01 Progesterone ~ 0 ~ 0 17-Hydroxy-pregnenolone 0.01 0.002 Dehydroepi-androsterone 2.0 0.36 Estrone ~ 0 ~ o - 0 ~ 0 ~ 0 Estradiol-17/3 ~ o ~0 ~o ~ 0 ~ o 100 Fig. 13. Substrate "^C-androstenedione incubation: paper chromato-graphy of the neutral fraction 0^6ocm (296,250 cpm) 0-7 cm (35,450 cpm) 0-9 cm (433,250 cpm) Neutral fraction (11,427,250 cpm) L/PG, 70 hrs Androstenedione3 Testosterone (4,255,000 cpm, 31.3 Kg, S.A. 135,942 cpm/Kg) 1 h Overflow (4,869,000 cpm) Bush A, 2 4 hrs ~7T~J— Testosterone (3,172,000 cpm) 21-40 cm (159,751 cpm) Bush A, 24 hrs Testosterone (2,136,875 cpm, 19.4 »* g, S.A. 110,148 cpm/Kg) c 1 23-4-0 cm-»-Overflow (367,900 cpm) ^Location of standard androstenedione chromatographed simultaneously. bSee Fig. 14. cSee Table XXVI. 101 Fig. 14. Substrate ^C-androstenedione incubation: paper chromato-graphy of androstenedione 0-18 cm (223,150 cpm) Overflow3 (4,869,000 cpm) L/PG, 20 hrs I Androstenedione (2,682,500 cpm, 22.4 K g , S.A. 119,754 cpm/K g ) i .t L/PG, 16 hrs Androstenedione (2,410,625 cpm, 23.9 * g , S.A. 100,863 cpm/K g ) Bush A, 7 hrs Androstenedione (1,810,500 cpm, 18.4 ug, S.A. 98,397 cpm/K.g) 1 25-40 cm -t-Overflow (1,018,000 cpm) aSee Fig. 13, footnote b. Fig. 15. Substrate -progesterone incubation: paper chromatography of the neutral fraction Origin (159,175 cpm) Neutral fraction (3,829,000 cpm) L/PG, 18 hrs Testosterone3 t Origin (929,600 cpm) (1,691,250 cpm)b L/PG, 72 hrs Androstenedione3, Progeste rone 3 I r (726,050 cpm, 165 Kg, S.A. 4,400 cpm/Kg) j R 1-4 cm Overflow (642,000 cpm) (21,710 cpm) Overflow (210,000 cpm) I Ac20,py L/PG, 16 hrs L/PG, 4 hrs Progesterone3 (144,650 cpm) T/PG, 8 hrs I 0-6 cm (12$, 000 cpm) I60C - HydroocyprQge'sterone3 6-13 cm (376,000 cpm)c 1 0-16 cm Androstenedione (64,600 cpm) (552,800 cpm, 158 Kg, S.A. 3,499 cpm/Kg) —F 25-40 cm + Overflow (66,125 cpm) 13-40 cm Overflow , (74,000 cpm) ,( 0-18 cm L/PG, 16 hrs T 1 Androstenedione 24-40 cm S.A/3,288 cpVKg) " ^ W c p m ) (25,190 cpm) (466,850 cpm, 142 K g , ^Locations of carrier steroids chromatographed simultaneously. bSee Fig. l£. cSee Table XXLX. dSee Table XXX. 103 Fig. 16. Substrate ^ (^progesterone incubation: paper chromatography of testosterone acetate and 17-hydroxyprogesterone Eluate from paper chroma-tography of neutral extract 3 * f Ac 2 0, py L/PG, 4 hrs 17-Hydroxypro-gesterone (740,450 cpm) T/PG, 4 hrs 200 Kg carrier 17-hydroxyprogesterone added Testosterone acetate (709,450 cpm, 159 S.A. 4462 cpm/ Kg) L/PG, 4 hrs \ : 0-13 cm 17-Hydroxypro-(172,000 cpm) gesterone (263,325 cpm, 188 Kg, S.A. 1401 c p n y V g ) 23-40 cm (210,625 cpm) Testosterone acetate (636,700 cpm, 1 4 9 ^ S.A. 4273 cpm/ug)B 0-12 cm (22,800 cpm) T/PG, 4 hrs 17-Hydroxyprogeste rone (219,125 cpm, 152 K g , S.A. 1442 cpm/ K g ) 2oLvO cm (12,530 cpm) See Fig. 15, footnote b. "See Table XXXI, cSee Table XXXIL F i g . 17. Substrate H-pregnenolone incubation: paper chromatography of the neutral f r a c t i o n O r i g i n 0 (2,596,000 cpm) il 0-4 cm (842,650 cpm) Neutral f r a c t i o n (23,075,000 cpm) Testosterone 3 17-Hydroxy-progesterone 3 L/PG, 24 hrs DHEA 1-4 cm (9,401,000 cpm) L/PG, 48 hrs T 4-8 cmc (1,887,000 cpm) Androstenedione 3 ?nolone a Progesterone a Pregner I1 13-21 cma Overflow (4,446,250 cpm) (3,624,000 cpm) L/PG, 4 hrs ^ Testosterone e 17-Hydroxypro-gesterone (7,623,?XX) cpm) 15-40 cm Overflow (446,330 cpm) I1 I' ~ II f 1-4 cm Progesterone-*- 21-40 cm (24,840 cpm) (688,600 cpm, (2,114,600 cpm) 221 K g ? S.A. 3yl l6 cpm/ >^g) ^Locations of car r i e r steroids chromatographed simultaneously. bSee F i g . 18. °See Table XXXIV". aSee F i g . 20. 6See F i g . 19. ISee Table XXXIV. F i g . 18. Substrate -^-pregnenolone incubation: paper chromatography of the eluate of the o r i g i n area of the neutral f r a c t i o n paper chromatogram Origin eluate 3 , (3,430,900 cpm) I B/F, 6 hrs f 0-7 cm (1,095,600 cpm) 16* -Hydroxypro-gesterone (860,400 cpm) 1 11-44 cm (953,480 cpm) T/PG, 18 hrs l/PG, 18 hrs ^-Hydroxy-pregnenolone 0 0-22 cm (285,500 cpm) j j 16*. -Hydroxyprogesterone 33-44 cm ( 3 , 3 l ' 3 ? ° o ^ ^ %*' Overflow S.A. 2,959 cpm/Kg) ( 6 3 ^ 5 0 c p m ) T/PG, 12 hrs 16* -Hydroxyprogesterone 4 (304,900 cpm, 101 Kg, S.A. 3,019 cpm/Kg) • 1 c 0-16 cm 16-21 cmc (362,100 cpm) (108,040 cpm) I 21-44 cm (300,46© cpm) See F i g . 17, footnote b. -Location of c a r r i e r 17-hydroxypregnenolone chromatographed simultaneously. cSee Table XXXV. dSee Table XXXVI. 106 Fig. 19. Substrate -'H-pregnenolone incubation: paper chromatography of testosterone - 17-hydroxyprogesterone eluate Testosterone-17-hydroxyprogesterone eluate 3 (7.623,700 cpm) Ac20,py L/PG, 4 hrs • 1 17-Hydroxyprogesterone (288,925 cpm, 145 K g , S.A. 1,993 cpm/Kg) — n : T — — r 3-16ccm Testosterone acetate" 26^44-,cm (212,180 cpm) (6,612,500 cpm, 142 ^  g , (348,810 cpm) S.A. 46,567 cpm/ K g ) T/PG, 6 hrs 0-16 cm (48,410 cpm) 17-HydroxyprogesteroneD (86,750 cpm, 146 Kg, S.A. 594 cpnyVg) 1 25-40 cm (123,430 cpm) fSee Fig. 17, footnote e. bSee Table XXXIV. 107 Fig. 20. Substrate %-pregnenolone incubation: of pregnenolone-androstenedione eluate paper chromatography n 0-16 cm (421,920 cpm) Pregnenolone-androstenedione eluate 3 , (4,446,250 cpm) Ac 20,py L/PG, 18 hrs 1 — i Androstenedione1-(2,658,850 cpm, 148 Kg, S.A. 17,965 cpm/ Kg) 1 23-44 cm (298,740 cpm) I 0-2 cm (64,650 cpm) p 2-30 cm (163,710 cpm) 1 Overflow (801,160 cpm) L/PG, 4 hrs Pregnenolone acetate 0 30-38 cm (405,200 cpm) fsee F i g . 17, footnote d. DSee Table XXXIV. °Location of ca r r i e r pregnenolone acetate chromatographed simultaneously. 108 Fig. 21. Substrate ^C-mevalonate incubations: paper chromatography of neutral fractions 0 I Origin (79,510 cpm) Neutral fraction, control incubation' (82,750 cpm) •L/PG, 48 hrs Testosterone 17^%droxyprdge sterone 1 1-5 cm (1,190 cpm) f Overflow (3,930 cpm) : H Origin (66,810 cpm) Neutral fraction, Pergonal incubation (115,000 cpm) L/PG, 48 hrs Testosterone" ^ 17-Hydroxyprogesterone —\ 1-5 cm (1,790 cpm) — | Overflow (6,070 cpm) aSee Table XXV. Locations of carrier steroids chromatographed simultaneously, 109 PART III STUDIES ON STEROID BIOSYNTHESIS IN VITRO BY THE RAT TESTIS PRELIMINARY EXPERIMENTS AND THE STUDIES (a) Introduction It has been established that preparations of rat testis i n vitro transform progesterone to androstenedione and testosterone (149, 189-194)j the formation and subsequent side-chain cleavage of 17-hydroxy-progesterone are essential reaction steps (195-198). Relatively minor transformation of progesterone to deoxycorticosterone, cortexolone, 17, 20 p -dihydroxypregn-4-en-3-one, 17,20<*. -dihydroxypregn-4-en-3-one and 16«.-hydroxyprogesterone has been reported (149,190,191,199) and substantial (8-4-0%) conversion to 5^-androstane-3^ ,17/S-diol has been observed (149). The results of incubations with ^C-progesterone and %-pregnenolone i n combination as substrates (192) have suggested that the predominant path-way of androgen biosynthesis i n the rat testis proceeds via progesterone and 17-hydroxyprogesterone rather than via 17-hydroxypregnenolone and dehydroepiandrosterone (Fig. 4). This section of the thesis reports preliminary observations on the metabolism i n vitro of radioactive progesterone by preparations of rat testes under several different experimental conditions. The results of these experiments were used to design time studies on the 17-hydroxy-lation of progesterone, the side-chain cleavage of 17-hydroxyprogesterone and the reduction of the 17-oxo group of androstenedione. 110 (b) Materials and methods (i) Rats: Long-Evans Hooded rats, approximately 3 months old and weigh-ing 200-250 g, were obtained from the Cancer Research Centre, University of B r i t i s h Columbia, Vancouver, Bri t i s h Columbia. The rats were stunned by a blow to the head and the testes were rapidly removed and placed in Petri dishes surrounded by crushed ice. ( i i ) Incubation with quartered testes: 4-^C-Progesterone (The Radiochemical Centre, Amersham, England) was purified prior to use by thin layer chromatography1 on East-man Kodak chroraatogram sheets (type K301R2) i n the solvent system benzene-ethyl acetate (1:1, by vol). The -^C-progesterone (556,056 cpm, 11.4 H-g) was transferred to a reaction flask i n a methanol solution, the solvent was evaporated and the radioactive substrate was redissolved i n 0.2 ml of propylene glycol. Four rat testes were quartered and placed i n the incu-bation flask and 10 ml of Krebs-Ringer phosphate buffer, pH 7.4, contain-ing glucose (0.01 M) and nicotinamide (0.04 M) were added. Incubation was performed for 1 hour i n a i r at 37° using a Dubnoff metabolic shaking incubator. At the end of the incubation period, the medium was removed and the tissue and the incubation flask were washed several times with d i s t i l l e d water. The aqueous washings were combined with the medium and the t o t a l volume was adjusted to 40 ml by the addition of d i s t i l l e d water. The tissue was stored at -19°. The aqueous fraction and the tissue were analyzed separately. 'See General Methods Section. I l l The aqueous fraction was extracted with ethyl acetate (5 x 40 ml) and with chloroform (4 x 40 ml). The organic extracts were evaporat-ed at 40° under reduced pressure and the combined extract was partitioned between hexane and 90$ aqueous methanol1. The aqueous methanol fraction was subjected to paper chromatography1 i n the solvent system l i g r o i n -propylene glycol. The zone of the experimental chroraatogram corresponding to the location of standard testosterone (chromatographed simultaneously) was eluted and the dry sample was treated with pyridine and acetic anhyd-r i d e 1 . Unlabeled testosterone acetate (200 Kg) and 17-hydroxyprogester-one (200 f-*-g) were added as c a r r i e r s 1 and the two steroids were resolved by paper chromatography i n the solvent system ligroin-propylene glycol.;; ' The eluate containing carrier 17-hydroxyprogesterone was evaporated and treated again with acetic anhydride and pyridine and then paper chroma-tography i n the solvent system toluene-propylene glycol was performed. Constant specific a c t i v i t y 1 was not attained. The eluate containing car-r i e r testosterone acetate was subjected to paper chromatography i n the solvent system ligroin-propylene glycol u n t i l constant specific a c t i v i t y was achieved. Five mg of unlabeled testosterone acetate were added to the eluate from the f i n a l paper chromatogram and crystallizations were performed u n t i l radiochemical homogeneity1 was observed. Unlabeled progesterone (100 p.g) a n c* androstenedione (200 \xg) were added to the overflow fraction of the i n i t i a l paper chromatogram of the 90$ aqueous methanol fraction. The two steroids were resolved by paper chromatography i n the solvent system ligroin-propylene glycol. The eluate containing androstenedione and the eluate containing progesterone were evaporated and each was treated with acetic anhydride and pyridine. 112 Further paper chromatography in the same solvent system was carried out u n t i l constant specific a c t i v i t y for both compounds was observed. The tissue was allowed to thaw, ground with sand i n a mortar with a pestle and extracted for one hour with hot absolute ethanol using a Soxlet apparatus. The ethanol was evaporated under reduced pressure at 40° and the extract was partitioned between hexane and 90$ aqueous methanol. The aqueous methanol extract was then partitioned between chloroform (100 ml) and d i s t i l l e d water (10 ml). Following equilibration, the aqueous phase was removed and the organic phase was extracted with d i s t i l l e d water (2 x 10 ml). The organic phase was evaporated under re-duced pressure at 40°, absolute ethanol was added and water was removed by azeotropic d i s t i l l a t i o n . Two hundred H-g of the following unlabeled steroids were then added: progesterone, 17-hydroxyprogesterone, and-rostenedione and testosterone. The extract was subjected to Column pa r t i -tion chromatography using s i l i c a g e l 1 . The fractions of the column ef-fluent that contained the carrier steroids were combined and the combined effluent was evaporated and subjected to paper chromatography i n the s o l -vent system ligroin-propylene glycol. Further resolution and purification of androstenedione, testosterone, progesterone and 17-hydroxyprogesterone were achieved using a combination of paper chromatography and treatment with acetic anhydride and pyridine i n the same manner that was employed to resolve and purify the steroids derived from the medium. Paper chroma-tography of each compound was repeated u n t i l constant specific a c t i v i t y was observed; no crystallizations were performed. Radioactivity was measured with a gas flow detector"'". 113 ( i i i ) Incubations with cell-free homogenates: The ^C-progesterone used as substrate i n the experiment with quartered testes was also used as substrate i n the incubations with cell-free homogenates. The 14 C -progesterone (452,254 cpm, 9.3 Kg) was transferred to each of two reaction flasks i n a methanol solution, the solvent was evaporated and the radioactive substrate was redissolved i n 0.2 ml of propylene glycol. Four rat testes were cut into small pieces and homogenized i n Krebs-Ringer phosphate buffer (pH 7.4), i n which Na"*" and K were interchanged, containing glucose (0.01 M) and nicotinamide (0.04 M)j a Potter-Elvehjem-type homogenizer was employed. The homogen-ate was centrifuged (755 x _g, 10 minutes, 4°) and the supernatant (20 ml) was divided equally between two incubation flasks. NADPH (7.89 mmoles;) was dissolved i n d i s t i l l e d water ( l ml) and added to one flask. Incuba-tions were performed for 1 hour i n a i r at 37° using a Dubnoff metabolic shaking incubator. At the end of the incubation period 200 ug of each of the following unlabeled steroids were added to each flask: progester-one, 17-hydroxyprogesterone, androstenedione and testosterone. The pro-cedures used for the extraction, resolution and purification of steroids i n both incubation media was the same. The contents of the incubation flask were diluted to 40 ml with d i s t i l l e d water and extracted with ethyl acetate (5 x 40 ml). The organic extracts were evaporated under reduced pressure at 40° and partitioned between hexane and JOfo aqueous methanol. The combined aqueous methanol extract was then subjected to column parti-tion chromatography on s i l i c a gel. The fractions of the column effluent that contained the carrier steroids were combined and evaporated under reduced pressure at 40°. Further resolution and purification of 114 progesterone, 17-hydroxyprogesterone, androstenedione and testosterone were achieved using a combination of paper chromatography and treatment with acetic anhydride and pyridine in the same manner that was employed to resolve and purify these compounds in the experiment with quartered testes. Paper chromatography of each compound was repeated until con-stant specific activity was observed; no crystallizations were performed. Radioactivity was measured with a gas flow detector, (iv) Time studies: Two sets of experiments were performed; each set consisted of 4 incubations. In one set, the incubations were stopped after 2,5,7 and 10 minutes and in the other set, the incubations were stopped after 10, 3 20,30 and 40 minutes. For the 2-10 minute group of experiments 7"* - H-progesterone (4,796,880 dpm, 12.60 nnK>les)vjands4-l^'C-17-hydroxyproges-terone (900,000 dpm, 12.23 nmoles) were both added to each of four incu-bation flasks and for the 10-40 minute group of experiments %-proges-terone (5,124,960 dpm, 13.46 nmoles) and "^C-17-hydroxyprogesterone (1,071, 420 dpm, 14.59 nmoles) were added to each of four incubation flasks. Both radioactive steroids were obtained from the Radiochemical Centre, Amersham, England and both were purified prior to use by paper chromato-graphy. The solvent system ligroin-propylene glycol was used to chroma-tograph the ^ H-progesterone, the solvent system toluene-propylene glycol was used to chromatograph the 14 C -17-hydroxyprogesterone. The radio-active steroids used as substrates were transferred to the reaction flasks in each instance in a methanol solution, the solvent was evaporated and the compounds were dissolved in 0.2 ml of propylene glycol. A cell-free homogenate was prepared in the same way from six 115 testes f o r each set of experiments. The testes were cut i n t o pieces and homogenized i n Krebs-Ringer phosphate b u f f e r (pH 7.4), i n which Na" and K were interchanged, containing glucose (0.01 M) and nicotinamide (0.C4 M); a Potter-Elvehjem-tvpe homogenizer was employed. The homogenate was f centrifuged (12,100 x g, 20 minutes, 4°) and the supernatant was d i l u t e d t o 40 ml with d i s t i l l e d water and divided e q u a l l y among f o u r reaction f l a s k s . NADPH (7.89 Kmoles) dissolved i n d i s t i l l e d water (0.4 ml) was added t o each f l a s k immediately p r i o r t o incubation. Incubations were performed i n a i r at 37° using a Dubnoff metabolic shaking incubator. Re-actions were stopped by the a d d i t i o n of e t h y l acetate (10 ml) previously c h i l l e d a t -19°. The f o l l o w i n g unlabeled compounds (100 Kg of each) were added to each f l a s k : progesterone, 17-hydroxyprogesterone, andros-tenedione and testosterone. The e x t r a c t i o n , r e s o l u t i o n and p u r i f i c a t i o n procedures were the same i n a l l experiments. The contents of each i n -cubation f l a s k were d i l u t e d to 40 ml with d i s t i l l e d water and extracted with e t h y l acetate (4 x 40 ml). The organic extracts were evaporated o under reduced pressure at 40 . The combined organic extract of each i n -cubation was then p a r t i t i o n e d between hexane and 70% aqueous methanol. Resolution and p u r i f i c a t i o n of progesterone, 17-hydroxyprogesterone, and-rostenedione and testosterone were achieved using a combination of paper chromatography and treatment with a c e t i c anhydride and pyridine i n the same manner that was employed to resolve and p u r i f y these compounds i n the experiment with quartered t e s t e s . Paper chromatography of each com-pound was repeated u n t i l constant s p e c i f i c a c t i v i t y was observed. R a d i o a c t i v i t y was measured with a l i q u i d s c i n t i l l a t i o n spec-tromatar 1. 116 The nmoles of substrate %-progesterone and substrate ^ +0-17-hydroxyprogesterone that were not metabolized and that were converted to products during each incubation were calculated from the per cent of sub-strate radioactivity present in each of the steroids identified at the end of the incubation period. Corrections were made for losses. The average concentrations of radioactive androstenedione and 17-hydroxypro-gesterone for the time intervals 0-2,2-5,5-7,7-10 and 10-20 minutes were calculated from the concentrations present at the beginning and at the 3 end of each time interval. The nmoles of ^ H-progesterone hydroxylated in the C17 position in the incubations of 2,5,7,10,10 and 20 minutes dura-3 tion were calculated by adding the nmoles of H-17-hydroxyprogesterone, 3 3 H-androstenedione and H-testosterone present at the end of each incuba-tion. The nmoles of radioactive 17-hydroxyprogesterone that underwent side-chain cleavage in each incubation were calculated by adding the nmoles of ^C- and 3H-androstenedione and ^ 0 - and ^ H-testosterone that were present at the end of each incubation. The ; nmoles of radioactive 17-hydroxyprogesterone that under-went side-chain cleavage during the time intervals 0-2,2-5,5-7,7-10 and 10-20 minutes were calculated from the nmoles that were cleaved follow-ing 0,2,5,7,10,10 and 20 minutes of incubation and the average rate of cleavage during each time interval was obtained by dividing the nmoles cleaved during each time interval by the number of minutes in the inter-3 val. The nmoles of H-progesterone that were hydroxylated following 0,2, 5,7,10,10 and 20 minutes of incubation were used to calculate the nmoles hydroxylated in the time intervals 0-2,2-5,5-7,7-10 and 10-20 minutes and 117 the result f o r each time interval was divided by the number of minutes i n the interval to obtain the average rate of 17-hydroxylation during each time interval. The average, rate of reduction of the 17-oxo group of androstenedione was calculated i n a similar manner using the nmoles of testosterone that were formed during each time interval. (?) Results Resolution and purification of progesterone, 17-hydroxyproges-terone, androstenedione and testosterone were achieved by a combination of paper chromatography and treatment with acetic anhydride and pyridine. The radiochemical homogeneity of the compounds i n the f i n a l eluates f o l -lowing paper chromatography to constant specific a c t i v i t y was evaluated i n some cases by the addition of milligram quantities of the appropriate unlabeled compound followed by crystallizations to constant specific a c t i -v i t y . The specific a c t i v i t y of the f i n a l crystals was observed to be with-i n 8% of the specific a c t i v i t y of the pool (radioactivity of the eluate from the f i n a l chromatography divided by the mg of carrier steroid added) for progesterone, 17-hydroxyprogesterone, androstenedione and testosterone acetate i n a l l cases i n which crystallizations were performed. The results of incubations of quartered rat testes and c e l l -free homogenates of rat testes with -progesterone as substrate are given i n Table XLV. Radioactive progesterone, 17-hydroxyprogesterone, androstenedione and testosterone were present i n small amounts i n both the tissue and the medium. Following incubations with cell-free homo-genates, the per cent of original substrate radioactivity present as ^C-progesterone was 1.3 i n the control incubation and less than 1 i n the i n -cubation with NADPH. Testosterone (42.5% of the i n i t i a l substrate 118 radioactivity) was the predominant radioactive metabolite identified i n the NADPH incubationj 5.6% of the i n i t i a l substrate radioactivitywas present as "^C-androstenedione and less than 1% was present as "^C-17-hydroxyprogesterone. In the control incubation the predominant metabolite 14 ' identified was C-androstenedione (36.5% of the i n i t i a l substrate radio-a c t i v i t y ) ; 15.3% of the i n i t i a l substrate radioactivity was present as 14c -testosterone and 11.5% as 17-hydroxyprogesterone. The sum of the per cent of the i n i t i a l substrate radioactivity present as progesterone, 17-hydroxyprogesterone, androstenedione and testosterone was 6 4 . 4 for the control experiment and 4 9 . 4 for the NADPH experiment. The results of the time studies are given i n Tables XLVI and XLVII and i n Figs. 22 to 2 4 . The t o t a l recovery of the i n i t i a l substrate radioactivity for 3 H and ^ KJ (when corrections are made for losses) for each incubation of the 0-10 minute set of experiments was greater than 90%. In the 0-40 minute set of experiments the t o t a l corrected recovery of the i n i t i a l substrate radioactivity for % and 14c was greater than 76% and 80%, respectively, i n each experiment. The to t a l corrected re-covery of the i n i t i a l substrate radioactivity i n a l l incubations of both sets of experiments was sli g h t l y greater than the tot a l corrected recovery of the i n i t i a l substrate % radioactivity; the same relation-ship between the values for the corrected recoveries of 3 H and "^ C radio-act i v i t y was observed for the ^ "C- and %-androstenedione and the ^ 0 -and %-testosterone i n a l l incubations of the 0-40 minute set of experi-ments. The data obtained i n the 2 , 5 , 7 and 10 minute set of experiments 3 show-a rapid decline i n the nmoles of substrate ^H-progesterone present 119 and a less rapid decline i n the nmoles of substrate ^C-17-hydroxy-3 progesterone present. The nmoles of ^H-17-hydroxyprogesterone present increased more rapidly than did the nmoles of radioactive androstenedione and testosterone. The results obtained i n the 10 minute experiment of the 0-40 minute set of experiments confirm the general trends observed i n the 0-10 minute set of experiments although the values for the nmoles of each com-pound present d i f f e r i n the two 10 minute experiments. In the 10 minute experiment of the 0-10 minute set of experiments less radioactive andros-tenedione and testosterone were identified and more %-progesterone was present than i n the 10 minute experiment of the 0-40 minute set of experi-ments. The quantity of radioactive 17-hydroxyprogesterone and %-proges-terone present at the end of 1 0 , 2 0 , 3 0 and 40 minutes of incubation pro-gressively declined with time while the quantity of radioactive testos-terone increased. The quantity of radioactive androstenedione present i n the 10 and 20 minute specimens of the 0-40 minute set of experiments were very similar; the quantity of radioactive androstenedione i n the 2 0 , 3 0 and 40 minute specimens progressively decreased with time. The average rates of 17-hydroxylation of %-progesterone and of side-chain cleavage of "^0- and 3H-17_hydroxyprogesterone were maximal during the f i r s t 2 minutes of incubation (Fig. 2 4 ) . The average rates of 3 17-hydroxylation of H-progesterone for the time intervals 0 - 2 , 2 - 5 , 5 - 7 and 7-10 minutes progressively decreased i n value but the average rates for the 5-7 and 7-10 minute intervals differed only slightly. In the time intervals 0 - 2 , 2 -5 and 5-7 minutes the average rates of 17-hydroxyla-tion of 3H-progesterone were greater than the average rates of side-120 chain cleavage of radioactive progesterone and i n the 7-10 minute inter-v a l the average rate of side-chain cleavage was greater than the average rate of 17-hydroxylation. The average rates of reduction of the 17-oxo group of andros-tenedione i n the 0-10 minute set of experiments reached a maximum value i n the 2-5 minute interval and the values for the 5-7 and 7-10 minute intervals differed s l i g h t l y from each other and from the value for the 2-5 minute interval (Fig. 24). The reverse reaction was not taken into account so that the average rates represent minimum values. The value for the average rate of side-chain cleavage for the 10-20 minute interval of the 0^40 minute set of experiments (Fig. 24) was the same as the value for the 7-10 minute interval of the 0-10 minute set of experiments. The value for the average concentration of radioactive 17-hydroxyprogesterone for the time interval 10-20 minutes was less than the value for the 7-10 minute interval and the values for the average concentration of radioactive androstenedione was greater. The average 3 rate of 17-hydroxylation of H-progesterone for the time interval 10-20 minutes was close to zero, (d) Discussion The results of the incubations of cell-free homogenates of rat testes with "^C-progesterone as substrate showed that this tissue prepara-tion i n the presence of NADFH was suitable for more elaborate studies of the reactions by which progesterone i s transformed to testosterone. The o 14 time studies with <H-progesterone and C-17-hydroxyprogesterone m com-bination as substrates were undertaken to evaluate the relative rates of 321 17-hydroxylation of progesterone and side-chain cleavage of 17-hydroxy-progesterone. In the time studies, the quantity of %-progesterone that was hydroxylated i n the C17 position during each incubation was estimated by summing the percentages of the i n i t i a l substrate radioactivity that were present i n 17-hydroxyprogesterone, androstenedione and testosterone at the end of the incubation period. The quantity of radioactive 17-hydroxyprogesterone that underwent side-chain cleavage during each incu-bation was estimated by summing the percentages of the i n i t i a l radioacti-v i t i e s of %-progesterone and "^C-17-hydroxyprogesterone that were present i n androstenedione and testosterone at the end of the incubation period. The methods that were employed to calculate the rates of 17-hydroxylation of progesterone and side-chain cleavage of 17-hydroxyprogesterone w i l l y i e l d valid results i f the following assumptions apply to the two enzy-matic reactions under investigation: the reactions are irreversible, endogenous unlabeled progesterone and 17-hydroxyprogesterone do not under-go reaction, the biosynthetic pathway proceeds exclusively from proges-terone via 17-hydroxyprogesterone to androstenedione and testosterone. Furthermore, a l l products of the two enzymatic reactions under study must be used to calculate the rates of the reactions. There i s no evidence that either enzymatic hydroxylations of steroid molecules or enzymatic scission of carbon-carbon bonds of the side-chains of steroid molecules are reversible reactions. The high levels of a c t i v i t y of the progesterone 17-hydroxylase and the 17-hydroxyprogesterone side-chain cleavage enzyme that were observed i n vitro suggest that negligible quantities of the corresponding substrates were present i n the cell-free homogenates prior to incubation; i t is also unlikely that substantial quantities of pro-gesterone and 17-hydroxyprogesterone were produced from endogenous 122 precursors during the incubation period. It has been suggested that re-duction of the 20-oxo group of 17-hydroxyprogesterone i s an obligatory step prior to side-chain cleavage i n the human testis (200) and i n the human and bovine ovary (201,202). In the hog adrenal (196), 17,20<*-dihydroxypregn-4-en-3-one may serve as the substrate for the side-chain cleavage enzymej however, i n the rat (196), mouse (198) and human testis (119) the evidence suggests that 17-hydroxyprogesterone i s the preferred substrate. The use of average concentrations and average rates i n attempts to describe and explain the results of the time studies i s subject to the following qualifications: the extent of the discrepancies between the actual events that occur during a period of observation and their assess-ment as average values depends on the length of the period of observa-tion and the rates of change of the characteristics under study. The shorter the period of observation and the less the rates of change of the phenomena under study, the more closely the average values correspond to the actual events that occur during the period of observation. In the 6 to 10 minute set of experiments, the sum of the per cent of i n i t i a l substrate radioactivity present as unreacted substrate plus the radioactive metabolites that were identified was greater than 90 for each isotope i n each incubation when the observed radioactivity was corrected 1 for losses incurred during the extraction, resolution and purification procedures. Because transformations of radioactive proges-terone, 17-hydroxyprogesterone, androstenedione and testosterone to uni-dentified metabolites occurred to a relatively minor extent, i t was possi-ble to calculate accurately the rates of 17-hydroxylation of progesterone 123 and side-chain cleavage of 17-hydroxyprogesterone from the specific a c t i -v i t i e s of the four compounds that were identified. In the incubations of more than 10 minutes duration, the sums of the per cent of i n i t i a l substrate radioactivity for each isotope present i n the compounds identi-f i e d were considerably less than the sums obtained i n the experiments of shorter duration. I t i s assumed that substantial quantities of unidenti-fied radioactive metabolites were produced i n the incubations of 20,30 and 40 minutes duration. This assumption is. supported by the observation that i n the experiments of 20 to 40 minutes duration substantial quanti-ties of radioactivity were present i n eluates from areas of the chroma-tograms corresponding i n location to the mobility of compounds more polar than 17-hydroxyprogesterone and testosterone. The unidentified polar metabolites may have included 16**- -hydroxyprogesterone, 17,20<* -dihydroxy-pregn-4-en-3-one and 5°< -andros tane -3 •=< ,17-diol (149). The metabolism of radioactive progesterone and 17-hydroxyprogesterone during the incuba-tion period via pathways that do not involve 17-hydroxylation of %-pro-gesterone or side-chain cleavage of radioactive 17-hydroxyprogesterone w i l l not affect the accuracy of the estimates of the 17* -hydroxylase and side-chain cleavage enzyme a c t i v i t i e s ; however, these activities w i l l be underestimated i f metabolites of radioactive androstenedione (other than testosterone) and testosterone (other than androstenedione) are formed. The formation of C21 metabolites of %-17-hydroxyprogesterone during the incubation period w i l l result i n an underestimation of the rate of 17-hydroxylation of %-pro gesterone but w i l l not affect the estimation of the rate of side-chain cleavage of radioactive 17-hydroxyprogesterone. The average rates of 17-hydroxylation of ^H-progesterone and 124 side-chain cleavagecof radioactive 17-hydroxyprogesterone for the time intervals 0-2,2-5,5-7,7-10 and 10-20 minutes are depicted in Fig. 24. The average rate of 17-hydroxylation of %-progesterone was more rapid than the average rate of side-chain cleavage of radioactive 17-hydroxy-progesterone during the 0-2,2-5, and 5-7 minute time intervals. During the 7-10 minute interval the average rate of side-chain cleavage exceed-ed the average rate of 17-hydroxylation. The decline in the average rate of 17-hydroxylation during the 2-5 and 5-7 minute time intervals may be attributed to the combined effect of decreasing concentration of substrate and increasing concentration of product. The low levels of substrate con-centration were probably responsible for the very low average rates of 17-hydroxylation that were observed during the 7-10 and 10-20 intervals. The observation that the average rates of 17-hydroxylation of 3 H-^progesterone were more rapid than the average rates of side-chain cleavage of 17-hydroxyprogesterone (during the period when substrate con-centration was not limiting) may not apply to the relative rates of the two enzymatic reactions in vivo or under different in vitro conditions. Data to be presented in another section of this thesis show that the pH optima for the 170C -hydroxylase and the side-chain cleavage enzyme lie in the region of 6.8. Others have reported that the pH optimum for the side-chain cleavage enzyme is about 7.0 (190,203). Loutfi and Hagerman (204) have presented data that purported to show that the side-chain cleavage reapt ioQ,: wascthe rate-limiting step in the latter part of andro-gen biosynthesis in the rat testis. These authors performed separate incubations at pH 7.4 with radioactive progesterone as substrate and with radioactive 17-hydroxyprogesterone as substrate and the rates of 125 17-hydroxylation of progesterone and side-chain cleavage of 17-hydroxy-progesterone were calculated from the differences i n the amount of each radioactive substrate present at the beginning and at the end of 30 min-utes of incubation. The assumption that the radioactive substrates were exclusively metabolized via the pathway under investigation was neither tested by Loutfi and Hagerman nor just i f i e d by the results of others (192) to which the authors referred. Data presented i n this thesis as well as i n other reports (149,191,193,197) show that testicular tissue, i n incubations of 20 minutes or more duration, transforms radioactive progesterone and 17-hydroxyprogesterone to a number of different meta-bolites. Some of these metabolites arise via 1704 -hydroxylation or side-chain cleavagej however, other compounds are formed that do not require these reactions. In the 0-10 minute set of experiments the average rate of side-chain cleavage of radioactive 17-hydroxyprogesterone was maximal i n the 0-2 minute interval and then declined to a constant value i n the 5-7 and 7-10 minute time intervals (Fig. 24). The average concentration of radio-active androstenedione increased rapidly in the 0-2 and 2-5 minute time intervals and then increased more slowly i n the 5-7 and 7-10 minute inter-vals. The data may be explained as follows: during the early minutes of incubation, product inhibition of the side-chain cleavage enzyme was not present; as the concentration of androstenedione increased, the rate of cleavage of 17-hydroxyprogesterone was retarded. It i s also postulated that the rate of reduction of the 17-oxo group of androstenedione increas-ed as the concentration of androstenedione increased and thus the rate of formation of androstenedione became very similar to the rate of transfor-mation of androstenedione to testosterone. 126 The existence of a steady state i n the formation and transform-ation of androstenedione is also suggested by the results of the 10 and 20 minute incubations of the 0-40 minute setuoftexperiments (Fig. 23). During the 10-20 minute time interval, the decline i n the per cent of % and "^ C substrate radioactivity present as radioactive 17-hydroxyproges-terone was accompanied by an increase i n the per cent of % and "^C sub-strate radioactivity present as radioactive testosterone; the per cent present as radioactive androstenedione changed very l i t t l e . The average rate of side-chain cleavage during the 10-20 minute time interval was the same as the average rate during the 7-10 minute interval of the 0-10 minute set of experiments although the average concentration of radio-active androstenedione was greater during the 10-20 minute time interval (Fig. 24). I t i s unlikely that the identical average rates of side-chain cleavage i n the two time intervals occurred by chance. A comparison of the results of the 10 minute incubation of the 0-10 minute set of experi-ments with the results of the 10 minute incubation of the 0-40 minute set of experiments (Tables XLVI and XLvII) shows that greater progesterone 17-hydroxylase and 17-hydroxyprogesterone side-chain cleavage a c t i v i t i e s • were present during the 10 minute incubation of the 0-40 minute set of experiments. If the quantity of side-chain cleavage enzyme present i n the 0-40 set of experiments was greater than the quantity present i n the 0-10 minute set of experiments, then the observation that similar average rates of side-chain cleavage occurred i n the 7-10 and 10-20 minute time intervals despite differences i n the average concentrations of androstene-dione i s consistent with the postulated relationship between the rate of side-chain cleavage and the concentration of androstenedione. An alternate 127 explanation for the average rates of side-chain cleavage of radioactive 17-hydroxyprogesterone that were observed i n the 0-2,2-5,5-7,7-10 and 10-20 minute time intervals i s that the conditions were optimal during the f i r s t two minutes of incubation and later a suboptimal steady state was present. Pyridine nucleotide coenzymes and molecular oxygen are required for side-chain cleavage of 17-hydroxyprogesterone (190,193,205). The concentration of molecular oxygen i n the incubation media may have declined to a level that limited the rate of side-chain cleavage. It i s unlikely that the concentration of NADPH was rate-limiting; a large excess was present. In a l l incubations of the 0-10 minute set of experiments, the quantities of "^''C-17-hydroxyprogesterone present at the end of each i n -3 cubation period were greater than the quantities of H-17-hydroxyproges-terone (Fig. 22, Table XLVI). Despite the disparity i n the concentrations of % - and ^ C-17-hydroxyprogesterone, the quantities of % - and ^"C-androstenedione present at the end of each incubation period were very similar as were the quantities of 3 H- and "^C-testosterone. Even in the 2 minute incubation i n which 12.23 nmoles of "^C-17-hydroxyprogesterone and no 3H_i7_hydroxyprogesterone were present i n i t i a l l y , the quantities of % - and ''"^C-androstenedione and 3 H- and -testosterone formed dur-ing the incubation period were very similar. These results suggest that the ^(^17-hydroxyprogesterone and the "^H-17-hydroxyprogesterone formed from %-progesterone did not mix i n a common pool prior to side-chain cleavage. Some of the characteristics of progesterone 17-hydroxylation 128 and side-chain cleavage of 17-hydroxyprogesterone that were observed can be explained by the following model: (i) At one site (site #1) progesterone i s hydroxylated i n the C17 position; the 17-hydroxyprogesterone that i s formed i s released. ( i i ) At a second site (site #2) progesterone can also undergo hydroxylation i n the C17 position; the 17-hydroxyprogesterone that i s formed i s a bound intermediate that serves as a substrate for side-chain cleavage at the same site. This site can also bind with 17-hydroxyproges-terone from the incubation medium and catalyze side-chain cleavage. The a f f i n i t y of this site i s the same for progesterone and 17-hydroxyproges-terone. Hypothetical mechanisms for the 17-hydroxylation of progesterone and for the side-chain cleavage of 17-hydroxyprogesterone must be consis-tent with the experimental observations that the ratio of the concentra-tions of the "^C products of cleavage to the concentrations of the ^H products was approximately unity i n the 0-10 minute set of experiments and that this ratio did not reflect the relative concentrations of "^C-3 and H-17-hydroxyprogesterone. These experimental observations are ex-plained by the proposed model i n which the compounds i n the incubation medium competing for binding at site #2 are 3n-progesterone, %-17-hydroxy-progesterone and "^C-17-hydroxyprogesterone. Site #2 must react with very similar numbers of ^H and "^C molecules per time interval. The data i n Table XLVI show that the sums of the nmoles of %-progesterone plus 17-hydroxyprogesterone were very similar to the nmoles of ^C-17-hydroxy-progesterone that were present at the end of 2,5,7 and 10 minutes of i n -cubation. The nmoles of %-progesterone and "^C-17-hydroxyprogesterone 129 present at the start of the incubations were also very similar. The very similar concentrations of and % products of side-chain cleavage that were observed following each incubation period may be explained by the propositions that the a f f i n i t y of site #2 is the same for ^H-progesterone, H-17-hydroxyprogesterone and 14C-17-hydroxyprogesterone and that a l l mole-cules that are bound to site #2 undergo cleavage. The experimental observation that the average rate of 17-hydroxyl-ation was greater than the average rate of side-chain cleavage (during the 0-2, 2-5 and 5-7 minute time intervals) is explained by the presence i n the model of site #1 that catalyzes the 17-hydroxylation of progesterone only. If, i n the model, only site #2 were present and the rate of hy-droxylation were more rapid than the rate of cleavage, an increase i n the quantity of bound %_17_nydroxyprogesterone would occur that would lead to the biosynthesis of more C19 products than 14C C19 products. If only site #2 were present and a l l or part of the 3H-17-hydroxyproges-terone formed were released into the medium, then the rate of side-chain cleavage of "^C-17-hydroxyprogesterone would he greater than the rate of cleavage of ^H-17-hydroxyprogesterone. These considerations are based on the assumption that the a f f i n i t y of site #2 for progesterone and 17-hydroxyprogesterone is the same. If the a f f i n i t y of site #2 for these two compounds differed, the concentrations of and products of side-chain cleavage would not be similar despite wide variations i n the re l a -tive concentrations of H-progesterone, JH-17-hydroxyprogesterone and C-17-hydroxyprogesterone. The model, i n which the same a f f i n i t y of site #2. for progesterone and 17-hydroxyprogesterone i s assumed, explains the simi-l a r i t y i n the concentrations of and 14c products of side-chain cleavage as a consequence of the.: similarity of the sum of the concentrations of 1 3 0 H-progesterone and -^H-^-hydroxyprogesterone to the concentration of 14C-17_hydroxyprogesterone. The model that i s proposed as an explanation for the results of the time studies could be tested by incubations i n which the i n i t i a l concentrations of substrate ^H-progesterone exceeded the i n i t i a l concen-trations of substrate ^C-17-hydroxyprogesterone. If the concentrations of % products of side-chain cleavage were observed to exceed the con-centrations of "^C products and, at the same time,.the concentrations did not reflect the relative concentrations of 3 H- and ^-C-17-hydroxy-progesterone, the liklihood would, be enhanced that the mechanism proposed herein corresponded to the events that occurred i n vitro. 131 TABLE XLV Results of incubations of preparations of rat testes with ^C-progesterone as substrate Per cent of substrate radioactivity Steroids isolated Quartered testes Cell-free homogenates Tissue Medium Control NADPH Progesterone 8.0 1.2 1.3 ^ 1 17-Hydroxypro-gesterone 1.6 1.5s 11.5 ^ 1 Androstenedione 1.6 1.3 36.5 6.9 Testosterone 3.0 6.1 15.3 42.5 Total 14.2 10.1 64.6 49.4 aConstant specific a c t i v i t y was not attained. TABLE XLVI Incubations with %-progesterone and ^ C-17-hydroxyprogesterone i n combination as substrates: results of time studies Compound and isotope Duration of incubation (minutes) 10 Proges-terone 3 H % Substrate nmolesa radio-activity 71.1 8.96 f0 Substrate nmoles radio-activity 34.0 4.28 % Substrate nmoles radio-a c t i v i t y 22.5 2.81 fo Substrate nmoles radio-a c t i v i t y 13.0 1.64 17-Hydroxy-progesterone 23.0 90.7 2.90 11.09 40.7 80.4 5.13 9.83 47.2 73.6 5.95 9.00 50.6 66.4 6.38 8.12 Andros-tenedione 3 H 5.6 5.6 0.71 0.68 10.3 11.2 1.30 1.37 12.3 12.1 1.55 1.48 13.5 14.2 1.70 1.74 Testosterone 3H 1.7 0.21 14C 1.7 0.23 6.7 7.1 0.84 0.87 9.6 9.6 1.21 1.17 14.2 14.5 1.79 1.77 Total 3H 14 C 101.4 98.2 12.78 12.00 91.7 98.7 11.55 12.01 91.4 95.3 11.52 11.65 91.3 95.1 11.51 11.63 The nmoles of radioactive steroid present at the end of each incubation period (See Text for the method of calculation). TABLE XLVII Incubations with ^H-progesterone and 14 G -17-hydroxyprogesterone i n combination as substrates: results of time studies Compound and isotope Duration of incubation (minutes) 10 20 30 40 Proges-terone % Substrate nmoles3 radio-ac t i v i t y 9 . 0 Andros-tenedione 3 H 14c 22.5 24.2 1.21 17-Hydroxy-progesterone 3H 3 6 . 0 4 . 8 5 14c 4 5 . 3 6 . 6 1 3 .03 3 . 5 3 Testosterone 3H 18 .6 2.50 ^ C 20.3 2 .96 % Substrate nmoles fo Substrate nmoles radio-ac t i v i t y 4 . 7 15.4 18 .2 22.9 25.4 3 6 . 3 40.2 0.63 2.07 2.66 3 . 0 8 3 . 7 1 4 . 8 9 5 .87 radio-activity 3 . 2 5 .9 6 . 8 17 .4 1 8 . 2 4 9 . 6 54.6 0 . 4 3 0 . 7 9 0 . 9 9 2 . 3 4 2 .66 6 . 6 8 7 . 9 7 fo Substrate nmoles radio-a c t i v i t y 2 . 7 2 .9 3 .6 14 .9 1 6 . 1 55 .9 6 1 . 2 0 .36 0 . 3 9 0 . 5 3 2 . 0 1 2 . 3 5 7.52 8.93 Total 3 H 14 C 8 6 . 8 8 9 . 8 11 .59 1 3 . 1 0 79 .3 8 3 . 8 1 0 . 6 7 12.24 7 6 . 1 79 .6 10.24 11.62 76 .4 80 .9 1 0 . 2 8 1 1 . 8 1 aThe nmoles of radioactive steroid present at the end of each incubation period (See Text for the method of calculation). 134 F i g . 22. incubations of ce l l - f ree homogenates of rat testes with ^ - p r o -gesterone and l 4 C - l 7 - h y d r o x y p r o g e s t e r o n e in combinat ion as subs t ra tes : resul ts of time studies 141 1 1 1 Minutes of incubation 3 H - P r o g e s t e r o n e , ( o — o ) ; 3 H - 17- hydroxyprogesterone , ( O — — 3 ) ; i 4 C - 1 7 - h y d r o x y p r o g e s t e r o n e , ( • — • ) ; 3 H - a n d i 4 C -androstenedione , (A A ) ; 3 H - a n d l 4 C - t e s t o s t e r o n e , (• • ) . 135 F ig . 23 . Incubation of cel l - f ree homogenates of rat testes with 3H-progesterone and i4c- l7 -hydroxyprogesterone in combinat ion as substrates: results of t ime studies Minutes of incubat ion 3H - Progesterone, (o——o) ; 3 H-17-hydroxyprogesterone , ( 0 — 3 ) ; w C - 1 7 - h y d r o x y p r o g e s t e r o n e , ( • — • ) ; 3H-andros tened ione , (A A ) ; l 4 C - a n d r o s t e n e d i o n e , (A • ) • 3H- tes tos te rone , (• • ); I4C- testosterone , (B • ). 136 F i g . 24 . Incubations of cel l - f ree homogenates of rat testes with 3H-progesterone and l 4 C - l 7 - h y d r o x y p r o g e s t e r o n e in com b inat ion as subst ra tes : resu l ts of time s tud ies 5-7 7-10 T i m e intervals (minutes) 10 -20 T h e methods employed to calculate the average concentrat ions of rad ioact ive androstenedione ( A — A ) and radioactive 17-hydroxyprogesterone (•——•) and the methods employed to calculate the average rate of 17 -hydroxy la t ion of 3H-pro-gesterone ( A — A ) , the average rate of reduction of the 17-oxo group of radioactive androstenedione ( O — O ) and the average rate of s ide -cha in c leavage of radioactive 17-hydroxyprogesterone ( • — Q ) are descr ibed in the text. 137 PREPARATION AND PROPERTIES OF A SOLUBLE SYSTEM OBTAINED FROM RAT TESTICU-LAR MICROSOMES THAT CATALYZES THE TRANSFORMATION OF PROGESTERONE TO 17-HYDROXYPROGESTERONE AND ANDROGENS (a) Introduction Progesterone 17-hydroxylase and 17-hydroxyprogesterone side-chain cleavage enzyme are known to reside i n the microsomal fraction of the rat testis (190,193). Young and coworkers (206) observed progesterone 17-hydroxylase and 21-hydroxylase act i v i t i e s and 20*-hydroxysteroid dehydro-genase a c t i v i t y i n supernatant fractions obtained by high speed centri-fugation of suspensions of acetone powders prepared from homogenates of bovine adrenal glands and L i t t l e et a l . (207) reported 1 to 2 per cent conversion of 14c -progesterone to androstenedione following incubations of supernatants obtained by ultracentrifugation of homogenates of human placentas. Attempts to obtain the testicular 17©fc-hydroxylase and the 17-hydroxyprogesterone side-chain cleavage enzyme i n an active, soluble form (defined as the supernatant following 2 hours of centrifugation at 105,000 x jg) have been hitherto unsuccessful (190,193,203). In this part of the thesis, the solubilization of the two enzymatic a c t i v i t i e s i s des-cribed and studies on the soluble system are reported. (b) Materials and methods (i) Rats: Long-Evans Hooded rats, weighing approximately 250 g and ap-proximately 3 months old, were used for a l l experiments. Animals obtain-ed from The National Laboratory. Animal Breeding Co., Edmonton, Alberta and Blue Spruce Farms,, Inc., Altamont, N.Y. were used for a l l experiments except the incubation with microsomes. In the latter experiment, the 1 3 8 microsomes were obtained from testes of Long-Evans Hooded rats bred at the Cancer Research Centre, University of B r i t i s h Columbia, Vancouver, Briti s h Columbia. ( i i ) Tissue preparation: The rats were stunned by a blow to the head and the testes were removed and immediately placed i n Petri dishes surrounded by crush-ed ice. Further procedures were carried out at 4°. Each testis was quartered and the tissue was homogenized i n 0.15 M KC1 using a Potter-Elvehjem-type homogenizer. The homogenate was centrifuged (10,500 x j * , 25 minutes) and the supernatant was then centrifuged (105,000 x £, 90-120 minutes) to yield the microsomal pellet which was immediately incubated, lyophilized or treated with acetone to yield an acetone powder. To pre-pare an acetone powder of microsomes or lyophilized microsomes, the mater-i a l was suspended i n a minimal volume of potassium phosphate buffer (0204 M, pH 7.4) and poured, with s t i r r i n g , into ten volumes of acetone (-19°). After 5 minutes the mixture was centrifuged (2,500 x _g, 10 minutes, 4°) and the precipitate was suspended i n 20 ml of acetone, centrifuged, and the precipitate was again suspended i n 20 ml of acetone and centrifuged. The precipitate from the f i n a l centrifugation was dried i n vacuo. The soluble fraction was obtained by suspending lyophilized microsomes i n 6 ml of a solution of Triton N-101 (Rohm and Haas, Philadelphia, Pa.) i n potassium phosphate buffer (0.04 M, pH 7.4). The mixture was swirled several times during the following 15 minutes and, after centrifugation (105,000 x jg, 2 hours), the supernatant (soluble fraction) was collected. Unless otherwise stated, the amount of lyophilized microsomes used to ob-tain the soluble fraction was 0.106 g (representing approximately the 139 material derived from two testes) and the buffer solution (6 ml) contain-ed 0.2% (v/v) Triton N-101. In one experiment, an attempt was made to obtain mitochondrial and microsomal fractions, each relatively free from contamination by other fractions. Twenty rat testes were cut up and homogenized i n 0.15 M KC1. The homogenate was centrifuged (700 x _ g , 15 minutes) and the precipitate was discarded. The supernatant was centrifuged (10,000 x jg, 45 minutes); the precipitate constituted the unwashed mitochondrial fraction and the supernatant was centrifuged (105,000 x £, 2 hours) to obtain the unwashed microsomal pellet. The mitochondrial fraction was suspended i n 15 ml of 0.15 M KC1 and centrifuged (700 x £, 15 minutes); the precipitate was dis-carded and the supernatant was centrifuged (10,000 x 25 minutes) to ob-tain the mitochondrial pellet. The mitochondrial pellet was washed twice by suspension i n 15 ml of 0.15 M KC1 followed by centrifugation (10,000 x j j , 15 minutes). The unwashed microsomal pellet was suspended i n 7 ml of 0.15 M KC1 and centrifuged (10,000 x £, 45 minutes) and the precipitate discarded. The supernatant was centrifuged (105,000 x £, 90 minutes); the precipitate constituted the washed microsomal fraction. Ammonium sulfate fractionation was performed at 4 ° . The soluble fraction was brought to 40% saturation (208) by the addition of solid ammonium sulfate. The material was allowed to stand for 10 minutes and was then centrifuged (10,500 x £, 15 minutes), ( i i i ) Radioactive substrates: The source and methods of purification of 7-^H-progesterone, 4-14c-progesterone and 4-^C-17-hydroxyprogesterone used as substrates are given i n the Preliminary Experiments and Time Studies section of this part 140 of the thesis. l,2-^H-Deoxycoi*ticosteronel was purified by paper chro-2 matography i n the solvent system toluene-propylene glycol prior to use as substrate; 7-%-pregnenolone (The Radiochemical Centre, Amersham, England) was not purified prior to use as substrate. The radioactive compounds used as substrates were transferred to the reaction flasks i n methanol solutions, the solvent was evaporated and the radioactive sub-strates were redissolved i n 0.2 ml of propylene glycol. (iv) Incubation conditions: Incubation mixtures were prepared using Krebs-Ringer phos-phate buffer (pH 7.4) i n which the Na"4" and K*" were interchanged or potas-sium phosphate buffer (0.04 M, pH 7.4). For incubations of the soluble fraction, 5 ml of the soluble fraction and 9 ml of phosphate buffer were added to each reaction flask. In a l l experiments, NADPH (7.89 Kmoles) was dissolved i n d i s t i l l e d water ( l ml) and added to the incubation mix-ture immediately prior to incubation. Unless otherwise stated, the tot a l volume of a l l incubation mixtures was 15 ml. Incubations were performed i n ai r for 40 minutes using a Dubnoff metabolic shaking incubator and were carried out at 37° unless otherwise stated. (v) Extraction, resolution and purification procedures: Incubations were stopped by the addition of ethyl acetate (15 ml) previously chilled at - 1 9 ° . Known quantities of carrier steroids^ (60-100 p-g of each) were added and the contents of each incubation flask were diluted to 40 ml with d i s t i l l e d water and extracted with ethyl acetate •'"A g i f t from the United States Public Health Service. 0 See General Methods section. 141 (4 x 40 ml). The organic extracts were evaporated under reduced pressure at 40°, absolute ethanol was added and the extracts were dried by azeo-tropic d i s t i l l a t i o n . Progesterone, 17-hydroxyprogesterone, androstenedione and testos-terone were separated and purified by paper chromatography i n the solvent systems ligroin-propylene glycol and toluene-propylene glycol. Following i n i t i a l chromatography, the eluted steroids were treated with pyridine and acetic anhydride^ and rechromatographed. 17-Hydroxyprogesterone and tes-tosterone do not separate i n the chromatographic systems used; hence, the 17-hydroxyprogesterone eluates were treated a second time with pyridine and acetic anhydride to assure that a l l of the testosterone was acetylated. Pregnenolone, deoxycorticosterone and 20oC-hydroxypregn-4-en-3-one were chromatographed both as the free compound and as their acetylated deriva-tives. Corticosterone, cortexolone and deoxycorticosterone were chromato-graphed on paper i n the solvent systems toluene-propylene glycol and Bush BI. Other steroids were resolved and purified by paper chromatography i n the solvent systems ligroin-propylene glycol and toluene-propylene glycol. Chromatography of the steroids containing the £\4-3-oxo grouping was re-peated u n t i l a difference of less than % occurred i n the specific a c t i -v ity^ for each isotope that the compound contained when compared to the value of the previous determination or u n t i l less than 1% of the i n i t i a l radioactivity of the substrate(s) was associated with the carrier steroid. Corrections were made for losses incurred during the isolation procedures. The specific a c t i v i t i e s of progesterone, 17-hydroxyprogesterone, andros-tenedione and testosterone acetate i n the eluates from the f i n a l chroma-tograms did not vary by more than 8$ when the compounds were subjected Ih2 to a series of crystallizations following the further addition of the corresponding unlabeled compounds. Pregnenolone, 17-hydroxypregnenolone and dehydroepiandrosterone were detected and assayed for radioactivity as follows: authentic re-ference compounds were chromatographed simultaneously with the experi-mental chromatograms and the standards were located by treatment with an ethanol solution of phosphomolybdic acid. The pattern of radioactivity of the experimental chromatograms was determined by scanning (Nuclear-Chicago Model 1032 4 p i Actigraph II chromatogram scanner) and the re-sults were compared with the locations of the steroids on the standard chromatograms. When i t was observed that the zones on the experimental chromatograms corresponding to the locations of standard dehydroepiandros-terone and 17-hydroxypregnenolone contained minimal radioactivity, the zones were excised and bisected longitudinally. One portion was treated with the phosphomolybdic acid reagent to verify the location of the ex-perimental steroid and the other portion was eluted with methanol and the radioactivity of the eluate was assayed by liquid s c i n t i l l a t i o n spectro-metry^. 20<-Hydroxypregn-4-en-3-one^ was purified by paper chromato-graphy i n the solvent system toluene-propylene glycol prior to addition as carrier; the 20/9 -epimer was purified by paper chromatography i n the solvent system ligroin-propylene glycol prior to addition as carrier, (vi) Column chromatography: Column chromatography was performed using Sephadex G-200, Sephadex G-25 (coarse) and DEAE-Sephadex A-25, a l l obtained from Phar-macia, Ltd., Montreal, Quebec. The Sephadex G-200 was allowed to swell 3 A g i f t from Dr. R. Neher, Basle,Switzerland. 143 i n d i s t i l l e d water for 4 days at 4° and was then added to a glass tube (2.5 x 45 cm) to a height of 39 cm. The void volume was 65 ml as deter-mined by chromatography of blue dextran. The column was run at a pres-sure of 15 cm of water. The eluent (potassium phosphate buffer, 0.04 M, pH 7.4) was allowed to flow through the column for 6 hours prior to ap-plication of the sample. The Sephadex G-25 (coarse) was allowed to swell i n 0.04 M ammonium bicarbonate for 4 hours at room temperature prior to use. The DEAE-Sephadex A-25 was allowed to swell i n d i s t i l l e d water and then washed with 0.5 N sodium hydroxide, d i s t i l l e d water, 0.5 N hydro-chloric acid and f i n a l l y with d i s t i l l e d water u n t i l constant pH of the washings was observed. The DEAE-Sephadex was equilibrated with a solution of potassium phosphate buffer (0.04 M, pH 7.4) and sodium chloride (0.1 M) and then packed i n a glass tube (1.5 x 30 cm) to a height of 25 cm. A l l columns were run at 4° i n a cold room, (vii) Other procedures: Protein was estimated by the method of Lowry and coworkers (209) as modified by Eggstein and Kreutz (210), with crystalline bovine serum albumin as standard. Non-heme iron was determined by the method of Massey (211) as modified by Simpson and Boyd (212), but using batho-phenanthroline. The molar extinction coefficient for the ferrous iron-bathophenanthroline complex (20,100) was determined by measuring the ab-sorbance at 235 nm of solutions of recrystallized ferrous ammonium sul -fate. Heme iron was determined according to the method of Diehl and Smith (213) i n the protein that was precipitated during the non-heme iron determination. NADPH dehydrogenase a c t i v i t y was measured by the method of Omura and coworkers (214) i n which changes i n the oxidation-reduction 3 4 4 states of dichlorophenolindophenol are used to measure dehydrogenase acti v i t y . A Sonifier c e l l disruptor (Heat Systems Co., Melville, N.Y.;).was employed for ultrasonic treatment and the specimens were chilled i n an ice-methanol bath during the procedure. Ijoc -Hydroxylase a c t i v i t y was estimated from the sum of the per cent of substrate radioactive progesterone dpm recovered as radioactive 17-hydroxyprogesterone, androstenedione and testosterone. 17-hydroxy-progesterone side-chain cleavage a c t i v i t y was estimated from the sum of the per cent of substrate radioactive progesterone dpm and substrate 34c-17-hydroxyprogesterone dpm (in incubations with two radioactive substrates) recovered as radioactive androstenedione and testosterone. Radioactivity was measured by liquid s c i n t i l l a t i o n spectrometry, (c) Results (i) Preparation of a soluble fraction containing 17«C-hydroxylase and side-chain cleavage enzyme activity: The progesterone 17-hydroxylase and 17-hydroxyprogesterone side-chain cleavage enzyme a c t i v i t i e s that were observed i n microsomes ' were only s l i g h t l y affected by lyophilization of the microsomes or by preparation of an acetone powder of lyophilized microsomes (Table XLVIII). large percentages of radioactive progesterone and 17-hydroxyprogesterone were converted to C19 compounds in a l l experiments. The t o t a l values shown i n Table XLVIII are greater than 90$ of the i n i t i a l radioactivity of the substrates when the radioactivity associated with uncharacterized material more polar than 17-hydroxyprogesterone and testosterone i s taken into account. The progesterone 17-hydroxylase and 17-hydroxyprogesterone 145 side-chain cleavage enzyme ac t i v i t i e s were not observed i n acetone powders of microsomes nor were these a c t i v i t i e s demonstrable i n the supernatants following centrifugation (105,000 x £, 90-120 minutes) and dialysis of 0.5 M NaCl or 2.0 M L i C l extracts of lyophilized microsomes. The possibility that progesterone 17-hydroxylase and 17-hydroxy-progesterone side-chain cleavage enzyme ac t i v i t i e s could be extracted from lyophilized microsomes with the detergent Triton N-101 was next investi-gated. The results of preliminary experiments (Table XLIX) suggested that maximal enzymatic a c t i v i t i e s could be obtained i n a soluble fraction (defined as the supernatant following centrifugation at 105,000 x for 2 hours) prepared by treatment of lyophilized microsomes with a solution of Triton N-101 of about 0.25% concentration (by vol). It was later found that a 0.20% solution was as effective as a 0.25% solution. The d i s t r i -bution of radioactivity following incubations of fractions obtained after centrifugation (105,000 x gf 2 hours) of lyophilized microsomes treated with 0.2% Triton N-101 i n potassium phosphate buffer (0.04 M, pH 7.4) i s presented i n Table L. Incubation of the precipitate obtained follow-ing high speed centrifugation of detergent-treated lyophilized microsomes 3 led to very active 17-hydroxylation of ^H-progesterone, a slight decrease i n side-chain cleavage of radioactive 17-hydroxyprogesterone and a marked dimunition i n reduction of 17-oxo group of androstenedione i n comparison to the results using lyophilized microsomes (Table XLVIII). Following incubation of the soluble fraction (obtained without ultrasonic treatment) 3 47 per cent of the radioactivity of H-progesterone was present as radio-active 17-hydroxyprogesterone, androstenedione and testosterone; only a small per cent of the original dpm of either radioactive substrate was 146 present as androstenedione or testosterone (Table L). Attempts were made to increase the enzymatic act i v i t i e s present i n the soluble fraction by ultrasonic treatment of lyophilized microsomes suspended i n 0.2$ Triton N-101 (Tables L and L l ) . The results suggested that ultrasonic treatment of 30 seconds to 4 minutes duration diminished the progesterone 17-hydroxylase and 17-hydroxyprogesterone side-chain cleavage enzyme ac t i v i t i e s of the soluble fraction; ultrasonic treatment for 2 minutes appeared to be least damaging. The enzymatic a c t i v i t i e s were not demonstrable i n the soluble fraction obtained following u l t r a -sonic treatment (2 minutes) of lyophilized microsomes suspended i n phos-phate buffer without Triton N-101. The soluble fraction obtained by treatment of lyophilized micro-somes with 0.2$ solution of Triton N-101 displayed considerably less side-chain cleavage a c t i v i t y and androstenedione 17-oxo reduction activ-i t y than did lyophilized microsomes. The possibility that these two en-zymatic act i v i t i e s i n the soluble fraction were inhibited by the presence of Triton N-101 was tested by incubating lyophilized microsomes i n the presence of the detergent. In addition, an experiment was performed to establish that tissue fractions were required for transformations of -^ H-progesterone and "^C-17-hydroxyprogesterone under the incubation conditions that were employed. The results of these investigations are given i n Table LII. No transformation of either radioactive substrate was observed following the incubation of a solution of Triton N-101 and potassium phos-phate buffer. The 17**--hydroxylase and side-chain cleavage enzyme activ-i t i e s of lyophilized microsomes were very similar i n incubations i n the presence (Table LII) and i n the absence (Table XLVIII) of Triton N-101. 1 4 7 The results of the two incubations differed i n that more radioactive androstenedione and less radioactive testosterone were present following the incubation i n the presence of Triton N-101. ( i i ) Other enzymatic a c t i v i t i e s i n the soluble fraction: The possible presence i n the soluble fraction of NADPH-stimulated enzymatic a c t i v i t i e s other than 17-hydroxylation of progesterone, side-chain cleavage of 17-hydroxyprogesterone and reduction of the 17-oxo group of androstenedione was investigated 4(Table LIII). A 17< -hydroxy-lase, a 3? -hydroxysteroid dehydrogenase and a steroid A-isomerase that u t i l i z e pregnenolone as substrate were not detected. A 20-hydroxysteroid dehydrogenase was present that reduced progesterone to the 20*. -hydroxy derivative; the corresponding 20^-epimer was not formed. Hydroxylation of progesterone at the C16 position was not detected, nor was 17-hydroxy-lation of deoxycorticosterone. In the three experiments i n which the con-version of ^ H-progesterone to 20<* -hydroxypregn-4-en-3-one, 20p -hydroxy-pregn-4-en-3-one, and 16<*. -hydroxyprogesterone was investigated, trans-formation of the radioactive substrates to 17-hydroxyprogesterone, andros-tenedione and testosterone also occurred to an extent similar to the values given i n Table L for the supernatant obtained following centrifugation of Triton N-101-treated lyophilized microsomes. Thus, the soluble fraction contains a 17«C -hydroxylase f o r which progesterone i s the preferred sub-strate rather than pregnenolone or deoxycorticosterone, a 17-hydroxypro-gesterone side-chain cleavage enzyme that does not cleave pregnenolone, a 17-hydroxysteroid dehydrogenase and a 20ot-hydroxysteroid dehydrogenase. ^The presence of NADPH dehydrogenase activity i n the soluble fraction i s reported i n another subsection of the Results section. 248 ( i i i ) Effects of changes of temperature and pH on the 17«*- -hydroxy-lase and side-chain cleavage enzyme acti v i t i e s of the soluble fraction: The results of incubations of the soluble fraction at d i f -ferent pH values and temperatures are shown i n Figs. 25 and 26, respec-tively. Both the 17*. -hydroxylase and the side-chain cleavage enzyme displayed maximal a c t i v i t y at 37° and at pH 6.8. (iv) Resolution and purification of components of the soluble fraction: The precipitate obtained by 40$ saturation of the soluble fraction with ammonium sulfate was dissolved i n 14 ml of potassium phos-phate buffer (0.04 M, pH 7.4) and incubated. Approximately one-quarter of the 17<* -hydroxylase a c t i v i t y exhibited by the untreated soluble frac-tion was observed; that i s , 12-15 per cent of the substrate %-proges-terone was converted to 17-hydroxyprogesterone (Table LI7). At pH 7.4, the activity of the side-chain cleavage enzyme was negligible i n the pre-cipitate obtained by 40$ saturation of the soluble fraction with ammonium sulfate. However, following an incubation performed at pH 6.7, the con-version of %-progesterone to androstenedione plus testosterone was found to be 11.4$ (Table LIV). In the latter experiment, the soluble fraction was prepared from 0.3 g of lyophilized microsomes rather than the usual 0.106 g. The supernatant following 40$ saturation of the soluble fraction with ammonium sulfate was inactive when incubated at pH 7.4 with %-pro-14 gesterone and C-17-hydroxyprogesterone i n combination as substrates and was also inactive when incubated after 4 hours of dialysis against potas-sium phosphate buffer (0.04 M, pH 7.4). When the dialyzed supernatant was combined with the precipitate, the formation of 17-hydroxyprogesterone was not increased over that which occurred when the precipitate was 149 incubated alone. The specific a c t i v i t y of the 17 <=<.-hydroxylase (as pmoles of pro-gesterone hydroxylated per minute per mg of protein) was found to be 10.4 for lyophilized microsomes and 1.38 for the soluble fraction (Table LV). The specific a c t i v i t y of the 17«< -hydroxylase present i n the precipitate obtained following 40% saturation of the soluble fraction with ammonium sulfate was determined twice and found to be 1.73 and 3.89. Both the soluble fraction and the precipitate obtained by 40% saturation of the soluble fraction with ammonium sulfate contained heme iron protein, non-heme iron protein and NADPH dehydrogenase activity. The hemo-proteins were shown to include cytochromes P-450 and P-420. A carbon monoxide difference spectrum under reducing conditions exhibited; a prominent absorption n&ximum at 450 nm with a small absorption maximum at 420 nm (Fig. 27). Attempts were made to resolve the non-heme iron protein, the cyto-chrome P-450 and the enzyme responsible for the NADPH dehydrogenase activ-i t y of the soluble fraction i n order to explore the relationship of these compounds to the 17<* -hydroxylase and side-chain cleavage enzyme activ-i t i e s that were present. The precipitate obtained by 40% saturation of the soluble fraction with ammonium sulfate was subjected to column chroma-tography on Sephadex G-200 (Fig. 28). One major and one minor peak, i n terms of the absorbance at 280 nm, were observed. In separate experiments i t was found that a portion of the Triton N-101 present i n the soluble fraction was precipitated by 40% saturation with ammonium sulfate. When Triton N-101 was subjected to column chromatography on Sephadex G-200, the detergent was present i n the fractions that immediately followed the 150 void volume. The ultraviolet spectrum of Triton N-101 (Fig. 29) served as a means of detection of the detergent. The absorption i n the region of 280 nm that i s characteristic of Triton N-101 interfered with the estimation of protein by measurement of the absorbance at 280 nm. Fractions 9 to 41 of the Sephadex G-200 column (Fig. 28) were pooled and the protein was precipitated by 40$ saturation of the pooled fractions with ammonium sulfate. A portion of the precipitate was dis-persed i n 15 ml of potassium phosphate buffer (0.04 M, pH 7.4) and i n -cubated with ^H-progesterone and "^C-17-hydroxyprogesterone i n combination as substrates. No transformations of the radioactive substrates were ob-served. The remainder of the precipitate was dissolved i n the same buffer and rechromatographed on the same column (Fig. 3 0 ) . Two peaks i n terms of the absorbance at 280 nm were observed. The position of peak #2 cor-responded to the positions of peaks i n the profiles of the non-heme iron content and NADPH dehydrogenase ac t i v i t y of the fractions. Heme iron was not demonstrated i n the effluent from the column and NADPH dehydrogenase activity was reduced i n comparison to the a c t i v i t y present i n the precip-itate obtained by 40$ saturation of the soluble fraction with ammonium sulfate. Non-heme iron protein could be obtained free of NADPH dehydrogenase ac t i v i t y by ion exchange chromatography. The precipitate obtained by 40$ saturation of the soluble fraction with ammonium sulfate was f i r s t sub-jected to gel f i l t r a t i o n chromatography using Sephadex G-200 as previously described. The elution profile i n terms of the absorbance at 280 nm was very similar to that depicted i n Fig. 28. The fractions containing peak #2 were pooled and passed through a Sephadex G-25 (coarse) column using 151 0.04 M ammonium bicarbonate as eluent. The protein-containing fractions were pooled and lyophilized. The lyophilized material was dissolved i n a minimal volume of a solution of potassium phosphate buffer (0.04 M, pH 7.4) and sodium chloride (0.1 M) and subjected to column chromatography on DEAE-Sephadex A-25 (Fig. 31). Peak #4 i n terms of the absorbance at 280 nm was observed to correspond i n location to the position of a peak i n the profile of the non-heme iron content of the fractions. NADPH dehydrogenase a c t i v i t y was low i n a l l fractions i n which i t was measured and was minimal i n the fraction containing the greatest amount of non-heme iron. A number of methods for the preparation of'microsomal non-heme iron protein were evaluated; the following procedure was found to yield the most highly purified product: an acetone powder of lyophilized micro-somes was dispersed in a solution of potassium phosphate buffer (0.04 M, pH 7.4) and sodium chloride (0.1 M) and the suspension was treated with ultrasound for 5 minutes. The supernatant following centrifugation (105,000 x _g, 2 hours) was applied to a DEAE-Sephadex A-25 column and gradient elution was performed using potassium phosphate buffer (0.04 M, pH 7.4) containing 0.1 to 0.7 M NaCl (Fig. 32). A prominant peak i n terms of the absorbance at 280 nm was observed that coincided i n location to the position of a peak in the profile of the non-heme iron content of the fractions. NADPH dehydrogenase act i v i t y was minimal i n a l l fractions i n which i t was measured. Fractions 18 to 31 from the column were pooled and passed through a Sephadex G-25 (coarse) column using 0.04 M ammonium bicarbonate as eluent. The protein-containing fractions were pooled and lyophilized. The lyophilized material was dissolved i n a minimal volumeoof 152 potassium phosphate buffer (0.04 M, pH 7.4) and subjected to gel f i l t r a -tion column chromatography on Sephadex G-200 (Fig. 33). Two peaks (in terms of the absorbance at 280 nm) were observed. The column fraction (#36) that displayed the greatest absorbance at 280 nm contained 6.0 p.g of non-heme iron. The procedure used to prepare microsomes included a minimal num-ber of centrifugation steps and no unusual precautions were taken to ex-clude contamination of the microsomal fraction by mitochondria. The non-heme iron content of more carefully prepared microsomal and mitochondrial fractions was investigated i n order to establish definitely the subcellular locations of the compound (Table LVI). The quantity of non-heme iron per gram of protein i n washed mitochondria and microsomes was greater than the quantity per gram of protein i n the corresponding unwashed preparations and the amount of non-heme iron per gram of protein i n washed microsomes was greater than the amount per gram of protein i n washed mitochondria, (d) Discussion The word "solution" and modifications of the term (such as "solu-ble fraction") are employed i n this part of the thesis to describe ma-t e r i a l that is not precipitated following a 2 hour period of centrifuga-tion at 105,000 x g. A solution may be defined formally as any phase con-taining more than one component; a phase is defined as a system that i s uniform throughout, not only i n chemical composition but also i n physi-c a l state (215). The observation that a substance i s not precipitated by high speed centrifugation provides evidence that the material is i n solution. A system designated as a solution according to the results of high speed centrifugation may include material that is not i n "true" solu-tion according to a formal definition. On the other hand, substances 153 t h a t are i n t r u e s o l u t i o n may be p r e c i p i t a t e d by prolonged c e n t r i f u g a t i o n . Other workers have at tempted t o s o l u b i l i z e the t e s t i c u l a r p roges-te rone 17-hydroxyla.se and 17-hydroxyprogesterone s i d e - c h a i n c leavage e n -zyme by t reatment of microsomes w i t h detergent (193,203). The success of the method repo r ted h e r e i n may be a t t r i b u t e d t o the use of l y o p h i l i -zed microsomesj however, no at tempt was made t o s o l u b i l i z e the 1 7 « . -hydroxy lase and s i d e - c h a i n c leavage enzyme by treatment of microsomes w i t h T r i t o n N-101. I t i s p o s s i b l e tha t the concen t ra t i ons of the detergents used by o ther workers p rec luded the presence o r the e x p r e s s i o n of the e n -zymat ic a c t i v i t i e s i n the s o l u b l e f r a c t i o n (Table X L I X ) . The f o rma t i on o f 17-hydroxypregnenolone has been repor ted i n i n -cubat ions of homogenates o f r a t t e s t e s w i t h 3n_pregnenolone as s u b s t r a t e (216). S h i k i t a and coworkers , however, were unable t o recover r a d i o a c t i v e 17-hydroxypregnenolone o r dehydroepiandrosterone f o l l o w i n g i ncuba t i ons of 3 r a t t e s t i c u l a r microsomes w i t h H-pregnenolone as s u b s t r a t e (192). S h i k i t a and coworkers a l s o performed i ncuba t i ons w i t h •^•'C-progesterone and 3 H -pregnenolone i n combinat ion as subs t ra tes and w i t h Hc-17-hydroxyproges-terone and 3H_l7-hydroxypregnenolone i n combinat ion as s u b s t r a t e s . From the r e s u l t s o f these exper iments the au thors conc luded t h a t t e s t o s t e r o n e i s formed f rom pregnenolone by r a t t e s t i c u l a r microsomes ma in l y v i a p r o -gesterone and andros tened ione . The i n c u b a t i o n o f the s o l u b l e f r a c t i o n w i t h "^C-progesterone and -^H-pregnenolone i n combinat ion as s u b s t r a t e s repo r ted h e r e i n (Tab le L I I I ) was performed t o a s c e r t a i n the s u b s t r a t e s p e c i f i c i t y of the 17t*- - hyd roxy -l a s e ; ^H-pregnenolone served as the t e s t subs t ra te and -p roges te rone se rved as the c o n t r o l . NAD^~ , a coenzyme r e q u i r e d f o r the conve rs i on of 154 pregnenolone to progesterone (217,218) was omitted from the incubation medium. Negligible transformation of -^-pregnenolone to 17-hydroxypreg-nenolone, dehydroepiandrosterone, progesterone, 17-hydroxyprogesterone, testosterone and androstenedione was observed. The results are i n accord with the conclusion of Shikita et a l . and also show that progesterone i s the preferred substrate for the 17*-hydroxylase. A pH optimum of 6.8 was observed for the ac t i v i t i e s of the 17ot-hydroxylase and the side-chain cleavage enzyme i n the soluble fraction (Fig. 25). A pH optimum of 7.0 has been observed for the side-chain cleavage enzyme ac t i v i t y i n incubations of rat (203) and guinea pig (190) testicular microsomes. Lynn and Brown (190) reported that the progesterone 17-hydroxylase of guinea pig microsomes was quite active at pH 8.5. When the act i v i t y of the 17* -hydroxylase i s estimated from the sum of the per cent of i n i t i a l 21-^C-progesterone present as radioactive acetate plus 17-hydroxyprogesterone, the data of Lynn and Brown show a pH optimum of 7.0. Young et a l . (206) observed progesterone 17-hydroxylase a c t i v i t y in a supernatant obtained by centrifugation (105,000 x j_, 60 minutes) of a suspension of an acetone powder prepared from a homogenate of bovine adrenal glands. The a c t i v i t y of the 17*-hydroxylase increased as the pH of the incubation media was increased to 9.1. It i s of interest that the act i v i t i e s of the 17* -hydroxylase and the side-chain cleavage enzyme i n the soluble fraction were similar i n incubations at 23°,32° and 37° (Fig. 26). The effect of temperature on these enzymatic a c t i v i t i e s has not been reported previously. 17°^-Hydroxylase, side-chain cleavage enzyme and 17-hydroxysteroid dehydrogenase act i v i t i e s were present i n the precipitate obtained by 40 155 per cent saturation of the soluble fraction with ammonium sulfate (Table LIV). The presence of 17-hydroxysteroid dehydrogenase and side-chain cleavage enzyme i n the precipitate was demonstrated i n an incubation per-formed at pH 6.7 but not i n an incubation performed at pH 7.4. The spe-c i f i c a c t i v i t y of the 17*-hydroxylase was greater i n the precipitate ob-tained by 40 per cent saturation of the soluble fraction than i n the un-treated soluble fraction; however, considerably less enzymatic ac t i v i t y was observed i n the precipitate (Table LV). The highest specific activ-i t y of the 17«- -hydroxylase was observed i n lyophilized microsomes. The results suggest that the enzyme was p a r t i a l l y inactivated during the i s o l a -tion procedures. The precipitate obtained by 40 per cent saturation of the soluble fraction with ammonium sulfate contained NADPH dehydrogenase activity, cytochrome P-450 and non-heme iron protein. The NADPH dehydrogenase and the cytochrome P-450 were unstable and i t was not possible to isolate these compounds for further investigations. Kimura and Ohno extracted non-heme iron protein from an acetone powder of homogenized pig testes (219). The authors postulated that the non-heme iron protein was present i n testicular mitochondria although their method of preparation did not exclude a microsomal source. The results reported herein establish the presence of non-heme iron protein i n both microsomes and mitochondria of rat testes. In bovine adrenal mitochondria the 11^ -hydroxylase (220-223) and the cholesterol side-chain cleavage enzyme (212,224) systems are considered to consist of NADPH dehydrogenase, non-heme iron protein and cytochrome P-450. The progesterone 17* -hydroxylase and the 17-hydroxy-progesterone side-chain cleavage enzyme of rat testicular microsomes may 156 function by a similar mechanism. This hypothesis cannot be tested con-clusively u n t i l NADPH dehydrogenase, cytochrome P-450 and non-heme iron protein are isolated i n active forms. A soluble preparation that catalyzes the transformation of pro-gesterone to 17-hydroxyprogesterone and to androgens should provide a useful experimental system for further investigation of the components and properties of the 17* -hydroxylase and the 17-hydroxyprogesterone side-chain cleavage enzyme of rat testicular microsomes. TABLE XLVIII Distribution of radioactivity following incubations of preparations of rat testes Per cent of substrate radioactivity Steroids isolated Microsomes Lyophilized microsomes Acetone powder of lyophilized microsomes 3 H \ h Progesterone ^1 1 1 2 <1 1 17-Hydroxyproge sterone < 1 C 1 <• 1 < 1 £ 1 *1 Androstenedione 22 21 24 21 Testosterone 3,2 31 54 49 50 49 Total 54 53 7a 72 In the incubations of microsomes (obtained from two testes) and lyophilized micro-somes (0.106 g), ^ H-progesterone (4,638,960 dpm, 3.71 K g ) and ^ C-17-hydroxyprogesterone (892,200 dpm, 3.72 Kg) were used i n combination as substrates. In the incubation of the ace-tone powder (0.053 g) of lyophilized microsomes, ^H-progesterone (4,400,880 dpm, 3.64 Kg) and -^C-17-hydroxyprogesterone (723,700 dpm, 3.64 Kg) were used i n combination as substrates. The microsomes were transferred to the reaction flask i n 5 ml of 0.15 M KC1 and 5 ml of Krebs-Ringer phosphate buffer (pH 7.4), in which the Na+ and K*" were interchanged, were added. The lyophilized microsomes and the acetone powder were each transferred to reaction flasks and incubated in 10 ml of the Krebs-Ringer buffer. In the experiment with the acetone powder testosterone acetate was not chromatographed to constant specific activity and androstenedione was not purified extensively. 158 TABLE XLIX Results of incubations of supernatants obtained following high speed centrifugation of Triton N-101-treated lyophilized microsomes Per cent solution of Triton N-101 used to treat lyophilized microsomes (v/v) Per cent of i n i t i a l radioactivity of substrate 3H-progesterone pres-ent as ^ H-progesterone following incubation 0.125 56 0.250 45 .0.500 71 1.00 86 2.00 93 Equal quantities (0.053 g) of lyophilized microsomes were treated with solutions of Triton N-101 i n 0.15 M KC1. The supernatants (5 ml i n each experiment) following centrifugation (105,000 x 90 minutes) were transferred to reaction flasks con-taining ^-progesterone (4,400,880 dpm, 3.64 t^g) and ^C-^-hy-droxyprogesterone (723,700 dpm, 3.64 Kg) i n combination as substrates; 5 ml of Krebs-Ringer phosphate buffer (pH 7.4),iin which the Na* and K + were interchanged, were added and incubations were performed. 159 TABLE L Distribution of radioactivity following incubations of fractions obtained after centrifugation (105,000 x _g, 2 hours) of lyophilized microsomes treated with 0.2% Triton N-101 Per cent of substrate radioactivity Precipitate Supernatant (soluble fraction) Steroids From micro- From micro-isolated somes •+• T r i - somes 4- Triton ton N-101 N-101 + u l t r a -sonic treatment 3 H 14 c 3 H 14 c 3H Progesterone C 1 6 c l 33 47 17-Hydroxyprogesterone 21 19 82 38 89 31 Androstenedione 62 53 10 7 3 4 Testosterone 1 1 3 2 1 1 Total 84 79 95 80 93 83 In a l l experiments 0.106 g of lyophilized microsomes were treat-ed with 6 ml of a 0.2% (v/v) solution of Triton N-101 i n potassium phos-phate buffer (0.04 M, pH 7.4). 3H_.progesterone (2,732,800 dpm, 2.35 Kg) and "^G-17-hydroxyprogesterone (372,360 dpm, 2.12 Kg) were used i n com-bination as substrates i n a l l experiments. Incubations of the soluble fractions were performed following the addition of 9 ml of Krebs-Ringer phosphate buffer (pH 7.4) i n which Na* and K + were interchanged. The precipitate was incubated i n 14 ml of the same buffer. Ultrasonic treat-ment (20 kilocycles per second, 70 watts, 2 minutes) was performed on the detergent-lyophilized microsome mixture prior to centrifugation. 160 TABLE LI Distr ibution of radioact iv i ty following incubations of the soluble f ract ion obtained af ter treatment of lyophil ized microsomes with 0.2$ Tr i ton N-101;and ultrasound Duration of ultrasonic treatment (minutes) Per cent of i n i t i a l radioact iv i ty of substrate 3H-progesterone pre-sent as ^H-progesterone following incubations 0 33 0.5 65 1 61 2 47 4 78 In a l l experiments 0.106 g of lyophil ized microsomes were suspended in 6 ml of a 0.2$ (v/v) solution of Triton uN-101- isi potassium phosphate buffer (0.04 M, pH 7.4). Ultrasonic treatment of the suspension was performed as described i n the Text. The con-dit ions of incubation of the soluble' fract ions and the radioactive substrates used are given in Table L. TABLE LII Studies on the effects of the composition of the incubation medium on progesterone 17-hydroxylation, 17-hydroxyprogesterone side-chain cleav-age and androstenedione 17-oxo reduction Per cent of substrate radioactivity Steroids isolated Lyophilized microsomes + Triton N-101 Blank 3 H 34c 3 H Progesterone < 1 6 * 1 92 17-Hydroxyprogesterone 2 1 94 < 1 Androstenedione 52 48 < 1 < 1 Testosterone 21 20 < 1 < 1 Total 75 75 94 92 Lyophilized microsomes (0.106 g) were dispersed i n 8 ml of potassium phosphate buffer (0.04 M, pH 7*4) and 6 ml of 0.2% (v/v) Triton N-101 dissolved i n the buffer were added immediately prior to incubation. The blank contained the same quantities of buffer and Triton N-101 and no lyophilized microsomes.,. In both incubations 3 H -progesterone (2,732,800 dpm, 2.35 Kg) and "^C-17-hydroxyprogesterone (372,360 dpm, 2.12 Kg) were used i n combination as substrates. 162 TABLE LIII The per cent of substrate radioactivity i n isolated compounds formed dur-ing incubations of the soluble fraction with radioactive steroids Per cent of substrate radioactivity Steroid isolated Substrate ^H-Proges-terone ^-Deoxy-cortic-osterone 14C -Progesterone -^-Pregnenolone Progesterone 64 < 1 17-Hydroxy-<1 progesterone 11 17-Hydroxy-pregnenolone < 1 < 1 Dehydroe piandrosterone <1 < 1 Andros tenedione 1.8 < 1 Testosterone < 1 < 1 20«- -Hydroxypregn-4-ene-3-one 2.4 20,8 -Hydroxypregn-4-ene-3-one < 1 16-Hydroxy-progesterone < 1 Cortexolone < 1 Corticosterone < 1 In a l l incubations, potassium phosphate buffer (0.04 M, pH 7.4) was used. The substrates used singly were 7-^H-progesterone (1,676,208 dpm- 1.2 Kg) and l,2-3H-deoxycorticosterone (6,809,270 dpm, 3.88 Kg); 4-14o-progesterone (390,000 dpm, 4.32 Kg) and 7-^H-pregnenolone (13, 080,000 dpm, 3.99 g) were used i n combination as substrates. 163 TABLE LI7 Distribution of radioactivity following incubations of the precipitate obtained by 40$ saturation of the soluble fraction with ammonium sul -fate Per cent of substrate radioactivity Steroids Incubations at pH 7.4 Incubation identified Experiment 1 Experiment 2 at pH 6.7 3 H ^0 3 H Progesterone < 1 80 < 1 86 62 17-Hydroxyproge sterone 95 12 100 15 19 Androstenedione 1 C 1 < 1 < 1 5 Testosterone < 1 < 1 < 1 < 1 7 Total 96 92 100 101 93 In the incubations at pH 7.4, the soluble fractions were pre-pared from 0.106 g of lyophilized microsomes and 3H-progesterone (2, 286.400cdpm> 1.96 K g ) and 14C-17-hydroxyprogesterone (284,660u4pm, 1.62 /u.g; were used as combined substrates i n each incubation. In the i n -cubation at pH 6.7, the soluble fraction was prepared from 0.3 g of l y -ophilized microsomes and 3H-progesterone (l,676,208u,dpm, 1.45 K g ) was used as the sole substrate. A l l precipitates were dispersed in 14 ml of potassium phosphate buffer (0.04 M, pH 7.4). For the incubation at pH 6.7, the pH of the f i n a l incubation mixture was adjusted by the ad-dition of one drop of dilute HC1. TABLE LV Specific activity of the progesterone 17-hydroxylase5 Fraction Treatment Subfraction Protein (mg) % Substrate ^H-progesterone hydroxylated Specific a c t i v i t y 1 3 Lyophilized microsomes Triton N-101 10.5 70 10.4 Lyophilized microsomes Triton N-101, Sentrifugation (105,000 x 2 hrs) Supernatant Precipitate 9.0 42.0 36 57 1.38 9.80 Soluble fraction 4056 (NH 4) 2S0 4 Precipitate l c Precipitate 2° 10.8 6.0 12 15 1.73 3.89 aEach determination Of the specific activity represents a separate experiment i n which different weights of lyophilized microsomes were used as starting material. kPicomoles of -^-progesterone hydroxylated per minute per mg protein i n the incubation medium. °The specific activity of the precipitate was determined i n two separate experiments. 165 TABLE LVI Non-heme iron and protein content of washed and unwashed mitochondria and microsomes Tissue Non-heme Fe Protein K g Non-heme Fe preparation (Kg) (g) per g protein Microsomes Unwashed 95.9 8.25 11.6 Washed 2.6 0.05 51.0 Mitochondria Unwashed 31.1 1.42 21.9 Washed 1.8 0.05 36.0 166 Fig.25. The effect of pH on the activities of the 17a -hydroxylase and the side-chain cleavage enzyme in the soluble fraction Each incubation flask contained 3 H-progesterone ( l , 6 7 6 , 2 0 8 d p m , 1.2^.g) as substrate. The pH of the incubation mixtures was adjusted by the addition of small quantities of dilute HCl or K O H . The conditions of incubation and the methods used to calculate the enzymatic activities are described in the text. 17a - Hydroxylase activity, ( o — - o ) ; s i d e -chain cleavage a c t i v i t y , ( • — — • ) . 167 Fig. 26. T h e ef fect of temperature on the act ivi t ies of the 17a-hydroxylase and the . s i d e - c h a i n cleavage enzyme in the soluble f rac t ion 2 8 r ' 1 _ 24 -T e m p e r a t u r e ("Cent igrade) E a c h incubation f lask contained 3H -progesterone ( 1 , 6 7 6 , 2 0 8 d p m , 1.2 fig) as substrate. The conditions of incubation and the methods used to ca lcu la te the enzymat ic activit ies are d e s c r i b e d in the text. 17a -Hydroxy lase ac t i v i t y , (o o) ; s i d e - c h a i n a c t i v i t y , (•——•). 163 Fig. 27 Absorbance spectrum of the precipitate obtained by 4 0 % saturation of the soluble fraction with ammonium sulfate > a o < i i i i 1 1 380 4 0 0 420 4 4 0 4 6 0 4 8 0 5 0 0 Wavelength (nm) The precipitate obtained by 4 0 % saturation of the soluble fraction with ammonium sulfate was redissolved in potassium phosphate buffer ( 0 .04 M, pH 7.4) and treated with sodium dithionite and carbon monoxide. An untreated redissolved precipitate was employed as the blank and absorbance was measured with a Unicam S P 8 0 0 spectro-photometer. 169 Fig. 2 8 . Sephadex G - 2 0 0 column chromatography 170 Fig. 29. Ultraviolet spectrum of Triton N-IOI i . 8 h i i i i _ i 2 0 0 2 2 5 2 5 0 2 7 5 3 0 0 Wavelength (nm) The absorbance of a 0.006 % (v/v) solution of Triton N-IOI in distilled water was measured with a Unicam SP800 spectrophotometer. 171 Fig. 30 Sephadex G -200 column chromatography Volume NADPH dehydrogenase activity measured as the ^g of dichloro-phenolindophenol reduced per minute Absorbance, 280 nm, ( ), NHI, non-heme iron, (o o ) ; NADPH dehydrogenase activity, (• • ) . Absorbance , 280 nm F i g . 3 3 . S e p h a d e x G - 2 0 0 c o l u m n c h r o m a t o g r a p h y H F r a c t i o n ( 8 m l ) V o i d V o l u m e A b s o r b a n c e , 2 8 0 n m , ( ); N H I , non-heme iron , ( o ) 175 STUDIES ON THE PYRIDINE NUCLEOTIDE COENZYME SPECIFICITIES OF THE PROGES-TERONE 17-HYDROXYLASE, 17-HYDROXYPROGESTERONE SIDE-CHAIN CLEAVAGE ENZYME AND 17£ -HYDROXYSTEROID DEHYDROGENASE OF THE RAT TESTIS (a) Introduction Pyridine nucleotide coenzymes are utilized as reductants i n en-zymatic side-chain cleavage of steroid hormones as well as i n enzymatic hydroxylations of these compounds (225,226). The view i s widely held that NADPH i s sp e c i f i c a l l y required for these reactions i n animal t i s -sues; however, a distinction may be drawn between a requirement and a preference for NADPH. Many reports purporting to show a NADPH require-ment have not included comparative studies with NADH (for example, 227, 228,229). In other investigations i n which the results of incubations i n the presence of NADPH and NADH were compared, the observation that, larger quantities of a hydroxylated derivative were formed i n incubations with NADPH led the authors to conclude that the reaction required NADPH, despite substantial formation of the hydroxylated product in incubations performed i n the presence of NADH (for example, 230). It has been observ-ed that certain reactions i n vitro, such as the 11)5 -hydroxylation of deoxycorticosterone (231), are stimulated by the addition of NADPH but not by the addition of NADH. This section of the thesis reports comparative studies on the act i v i t i e s of progesterone 17-hydroxylase, 17-hydroxyprogesterone side-chain cleavage enzyme and YJfi -hydroxysteroid dehydrogenase during i n -cubations of tissue preparations derived from rat testes performed in the presence of NADH and NADPH. The results demonstrate that NADPH i s required for the enzymatic reduction of the 17-oxo group of androstene-dione and that either NADH or NADPH may be ut i l i z e d as a reductant by 176 the 17*. -hydroxylase and the side chain-cleavage enzyme, (b) Materials and Methods (i) Rats: Long-Evans Hooded rats, weighing approximately 250 g and approximately 3 months old, were used for a l l experiments. The animals were purchased from Blue Spruce Farms, Inc., Altamont, N.Y. ( i i ) Tissue preparation: The rats were stunned by a blow to the head and the testes were removed and immediately placed i n Petri dishes surrounded by crush-ed ice. Further procedures were carried out at 4° . Each testis was quartered and the tissue was homogenized i n 0.15 M KC1 using a Potter-Elvehjem-type homogenizer. The homogenate was centrifuged (10,500 x j , 25 minutes) and the supernatant was then centrifuged (105,000 x j,- 2 hours) to yield the microsomal pellet which was immediately incubated or lyophi-lized. The soluble fraction was obtained by suspending 0.106 g of lyo-philized microsomes (representing approximately the material derived from two testes) i n 6 ml of a 0.2% (by vol) solution of Triton N-101 i n potassium phosphate buffer (0.04 M, pH 7.4). The mixture was swirled several times during the following 15 minutes and, after centrifugation (105,000 x _g, 2 hours), the supernatant (soluble fraction) was collected. ( i i i ) Radioactive substrates: Unless otherwise stated, incubations were performed with 7-^H-progesterone (2,351,440 dpm, 1.7 M-g) as substrate. Four incubations were performed with 4-"^fC-androstenedione (757,800 dpm, 1.9 V*-g) a s S U D ~ strate. The source and method of purification of the 7-^H-progesterone 177 used as substrate are given i n the Preliminary Experiments and Time Studies sectionnof this part of the thesis. 4-14C-Androstenedione (The Radiochemical Centre, Amersham, England) was treated with pyridine and acetic anhydride 1 and subjected to paper chromatography1 i n the solvent system ligroin-propylene glycol prior to use as substrate. The radio-active compounds used as substrates were transferred to the reaction flasks i n each instance i n methanol solutions, the solvent was evaporat-ed and the radioactive substrates were redissolved i n 0.2 ml of propy-lene glycol. (iv) Incubation conditions: The microsomes or lyophilized microsomes were dispersed i n potassium phosphate buffer (0.04 M, pH 7.4) and 14 ml were added to each reaction flask. For incubations of the soluble fraction, 6 ml of the soluble fraction and 8 ml of the buffer were added to each reaction flask. In a l l experiments the amount of tissue present i n each reaction flask approximated that derived from two testes. The following pyridine nu-cleotide coenzymes were used: NADPH (Calbiochem, l o t #802207), NADH (Calbiochem, lot #64057 and lot #900114) and NADP+ (Calbiochem, lot #800099). Unless otherwise stated, 7.89 u moles of NADPH or 9.17 (mmoles of NADH (lot #64057) were dissolved i n 1 ml of d i s t i l l e d water and added to the incubation mixture immediately prior to incubation. In control incubations, 1 ml of d i s t i l l e d water was added. Incubations were performed i n a i r at 37° for 40 minutes using a Dubnoff metabolic shaking incubator. "^See General Methods section. 178 (v) Extraction, resolution and purification procedures: Incubations were stopped by the addition of 15 ml of ethyl acetate previously chilled at -19° and known quantities (60-100 |m.g) of 1 3 carrier steroids were added. In the experiments with H-progesterone as substrate, unlabeled progesterone, 17-hydroxyprogesterone, andros-tenedione and testosterone were added; i n the experiments with "^C-an-drostenedione as substrate, unlabeled androstenedione and testosterone were added. The contents of each incubation flask were diluted to 40 ml with d i s t i l l e d water and extracted with ethyl acetate (4 x 40 ml). The organic extracts were evaporated under reduced pressure at 40°, ab-solute ethanol was added and the extracts were dried by azeotropic dis-t i l l a t i o n . Progesterone, 17-hydroxyprogesterone, androstenedione and tes-tosterone, i n the experiments with %-progesterone as substrate, were resolved and purified by paper chromatography i n the solvent systems ligroin-propylene glycol and toluene-propylene glycol. Following i n i t i a l chromatography, the eluted steroids were treated with pyridine and acetic anhydride and rechromatographed. 17-Hydroxyprogesterone and testosterone do not separate i n the chromatographic systems used; hence, the 17-hy-droxyprogesterone eluates were treated a second time with pyridine and acetic anhydride to assure that a l l of the testosterone was acetylated. Chromatography of each steroid was continued u n t i l constant specific activity*" was observed or u n t i l less than 1$ of the i n i t i a l radioactivity 3 of the H-progesterone was associated with the carrier steroid. Andros-tenedione and testosterone i n the experiments with ^"C-androstenedione as substrate were resolved and purified by paper chromatography i n the 179 solvent system ligroin-propylene glycol. Following i n i t i a l chromato-: graphy, the eluted steroids were treated with pyridine and acetic anhy-dride and chromatography was continued u n t i l constant specific a c t i v i t y was observed for each steroid. (vi) Chromatography of pyridine nucleotide coenzymes; A 50 mg sample of NADH (lot #64057) and a 30 mg sample of NADPH were each subjected to column ion exchange chromatography on DEAE cellulose, carbonate form (Selectacel, no.70, standard, Carl Schleicher and Schuell Co., Keene, N.H.). A glass tube (1.5 x 30 cm) was packed with DEAE cellulose i n d i s t i l l e d water to a height of 29 cm and, follow-ing application of the sample, gradient elution with 0 to 0.25 M ammo-nium bicarbonate (total volume 3000 ml) was performed (232,233). Paper chromatography of NADH (lot #64057 and lot #900114), NADPH and NADP+ was performed i n the system 0.1 M sodium phosphate buffer (pH 6.8)-solid am-monium sulfate-l-propanol (100:60:2, vol/wt/vol) (234). Compounds were located by viewing the chromatograms under short and long wave ultr a -violet light''". ( v i i ) Other procedures: 17*.-Hydroxylase a c t i v i t y was estimated from the sum of the per cent of the i n i t i a l ^H-progesterone dpm recovered as radioactive 17-hydroxyprogesterone, androstenedione and testosterone and 17-hydroxy-progesterone side-chain cleavage enzyme ac t i v i t y was estimated from the 3 sum of the per cent of the i n i t i a l H-progesterone dpm present as radio-active androstenedione and testosterone. Corrections were made for losses incurred during isolation procedures. Radioactivity was measured''" by liqu i d s c i n t i l l a t i o n spectrometry. 180 (c) Results The chemical purity of the pyridine nucleotide coenzymes that were employed in the incubations was assessed by column ion exchange chromatography and paper chromatography. The elution profile obtained by chromatography of a 50 mg sample of NADH (lot #64057) on a' DEAE cel-lulose column is shown in Fig. 34. One major and one minor peak in terms of the absorbance at 340 nm were observed. No increase in absor-bance above the baseline value occurred in the region where NADPH was eluted. The elution profile of a 30 mg. sample of NADPH obtained by the same chromatographic procedure is also depicted in Fig. 34. One major and one minor peak were observed and no increase in absorbance above the baseline value occurred in the region where NADH was eluted. The positions of the minor peaks in the two chromatograms were similar; the positions of the two major peaks were widely separated. NADH (lot #900114) and NADP+ migrated as single compounds when subjected to paper chromatography. Small quantities of uncharacterized fluorescent material were separated from NADPH and NADH (lot #64057) by paper chromatography; however, there was no evidence of contamination of either coenzyme by the other. It was observed (Table L 7 I I ) that the enzymatic reduction of the 17-oxo group of "^C-androstenedione was stimulated by the addition of NADPH to the incubation medium but not by the addition of NADH (lot #64057). In addition to providing a means of assessing the chemical purity of the NADH, incubations with "^C-androstenedione as substrate were performed to test the possibility that NADPH was formed during the incubation period. The per cent of the i n i t i a l "^C-androstenedione dpm 181 recovered as radioactive testosterone following an incubation with NADH and NADP*" was comparable to the per cent of i n i t i a l substrate dpm present as testosterone following the incubation with NADH alone and following the control incubation (Table LVII). The sum of the per cent of i n i t i a l substrate radioactivity recovered as radioactive androstenedione and testosterone was greatest i n the control incubation and declined progres-sively i n the incubations with NADH, NADH and NADP+ , and NADPH. The results of comparative studies on the effects of NADH and NADPH on the distribution of radioactivity following incubations of microsomes, lyophilized microsomes and the soluble fraction derived from rat testes with %-progesterone as substrate are summarized i n Tables LVIII and LIX. In incubations of microsomes with NADH, the major metab-olite of %-progesterone was 17-hydroxyprogesterone (Table LVIII); andros-tenedione was formed to a lesser extent and the formation of radioactive testosterone was not observed. Following incubations of microsomes with NADPH, the per cent of i n i t i a l substrate radioactivity present as proges-terone and 17-hydroxyprogesterone was low; androstenedione and testosterone contained large percentages of the i n i t i a l substrate radioactivity. In-cubation of microsomes without the addition of either coenzyme resulted i n a 5% conversion of the i n i t i a l substrate radioactivity to 17-hydroxy-progesterone; the formation of radioactive androstenedione and testos-terone was not observed. Following incubations of lyophilized microsomes with NADH or 3 NADPH, most of the i n i t i a l H-progesterone radioactivity was recovered as androstenedione and testosterone (Table LIX). The formation of radio-active androstenedione and testosterone was not observed i n an incubation 182 of lyophilized microsomes without the addition of either coenzyme; 12% 3 of the i n i t i a l substrate radioactivity was recovered as "^H-17-hydroxy-progesterone. The fractions from the ion exchange chromatography of NADH (lot #64057) that contained the major peak i n terms of the absorbance at 340 nm (Fig. 34) were pooled and the combined fraction was taken to dryness using a rotary evaporator at 40°. The residuum was dissolved i n a small volume of d i s t i l l e d water, transferred to another vessel and lyophiliza-tion was performed. A 7 mg sample (9.17 Kmoles) of the purified NADH was then used i n an incubation of lyophilized microsomes with ^H-proges-terone (1,676,208 dpm, 1.2 Kg) as substrate. The results were very similar to those shown in Table LIX for the incubation of lyophilized microsomes with NADH of the same lot number that had not been subjected to ion exchange chromatography. Results that were also very similar to those shown i n Table LIX for the incubation of lyophilized microsomes with NADH were observed following an incubation of lyophilized microsomes with %-progesterone (1,676,208 dpm, 1.2 Kg) as substrate and 8.47 ttmoles of NADH of lot number 900114. 3 Following incubations of the soluble fraction with ^H-progesterone as substrate (Table LIX), the per cent of the i n i t i a l substrate radio-3 a c t i v i t y recovered as -^-17-hydroxyprogesterone was 68 i n the presence of NADH and 34 i n the presence of NADPH. The sums of the per cent of the i n i t i a l substrate radioactivity recovered as radioactive androstenedione and testosterone were very similar i n incubations of the soluble fraction with either coenzyme. In the control incubation, minimal metabolism of 3 H-progesterone was observed. 183 In the incubations of lyophilized microsomes with ^H-progesterone as substrate (Table LIX) the concentrations of NADH and NADPH i n the i n -cubation media at the start of the incubations were 0.60 and 0.53 Kmoles per ml, respectively. Incubations were performed subsequently '(Table LX) in which the concentrations of the coenzymes were one-fifth and one-f i f t i e t h the concentrations of the preceding experiments. When the con-centration of NADH i n the incubation medium was decreased to 0.12 mmoles per ml, the 17-hydroxylase ac t i v i t y during incubation was not affected; however, the side-chain cleavage enzyme a c t i v i t y decreased markedly. A further decrease i n NADH concentration to 0.012 tumbles, per;ml-was"asso-ciated with a decline i n the activities of both the 17«-hydroxylase and the side-chain cleavage enzyme during the incubation period. When the NADPH concentration was decreased to 0.11 mmoles per ml, the activities of the 17* -hydroxylase and the side-chain cleavage enzyme were not decreased i n comparison to the ac t i v i t i e s that were ob-served when the incubation medium contained 0.53 M- moles of NADPH per ml. The ac t i v i t y of the 17/* - o l dehydrogenase (estimated as the ratio of the per cent of i n i t i a l substrate dpm present as testosterone to the per cent of i n i t i a l substrate dpm present as androstenedione) was less at the low-er NADPH concentration. At an NADPH concentration of 0.011 Kmbles per ml of incubation medium, the major radioactive metabolite formed during incubation was 17-hydroxyprogesterone (57% conversion of i n i t i a l Substrate dpm). The activities of the side-chain cleavage enzyme and the 17 £ - o l dehydrogenase were decreased i n comparison to the ac t i v i t i e s that were observed when the incubation medium contained 0.11 mmoles of NADPH per ml. 184 (d) Discussion Lynn and Brown (190) reported that the 17* -hydroxylase and the 17-hydroxyprogesterone side-chain cleavage enzyme were inactive i n i n -cubations of microsomes obtained from guinea pig testes performed i n the presence of NADH (0.5 /mmoles per ml). In incubations performed;.in the presence of NADPH (0.5 M-moles per ml), 15$ of;,5substrate 21-^'C-proges-terone was recovered as 17-hydroxyprogesterone and 30$ of substrate 17-hydroxyprogesterone was converted to testosterone. The results of incu-bations of microsomes derived from rat testes with 4-"^C-17-hydroxypro-gesterone as substrate i n the presence of NADH and NADPH have been re-ported by Shikita and Tamaoki (193). Following an incubation with NADH (0.1 mmoles per ml) as reductant, 86.6$ of the i n i t i a l substrate radio-activity was recovered as ^ C-17-hydroxyprogesterone and less than 2$ was transformed to testosterone and androstenedione. Following an incu-bation i n the presence of NADPH (0.1 f^moles per ml), 6$ of the radio-act i v i t y of substrate "^C-17-hydroxyprogesterone was present as testos-terone and 38.1$ was present as androstenedione. The results of two incubations of microsomes obtained from rat 3 testes with H-progesterone as substrate performed i n the presence of NADH (0.6 mmoles per ml) are reported i n this section of the thesis (Table LVIII). In one experiment the 17*-hydroxylase activity was very similar to that observed i n incubations of microsomes performed in the presence of NADPH (0.53 Kmoles per ml). In the other experiment the act i v i t y of the 17*-hydroxylase was considerable, but less than that ob-served i n the experiment with NADPH (Table LVIII). In both experiments with NADH, less side-chain cleavage enzyme ac t i v i t y was observed than i n 18$ comparable experiments with NADPH. The activities of the 17* -hydroxy-lase and the side-chain cleavage enzyme were considerably different i n the two incubations of microsomes with NADH despite attempts to maintain uniform methods of tissue preparation and identical conditions of incu-bation. The microsomes used i n experiment #1 with NADH and NADPH (Table LVIII) were prepared as a single sample (a portion of which was also used for the control incubation) as were the microsomes employed for experiment #2. In a l l incubations shown in Table LVIII approximately the same quant-i t y of microsomes was added to each incubation flask. The v a r i a b i l i t y of the results of the two incubations of microsomes with NADH contrasts with the similar findings observed i n the two incubations with NADPH (Table LVIII). The range of experimental conditions under which NADH can be uti l i z e d as a reductant by the microsomal 17<* -hydroxylase and side-chain cleavage enzyme may be restricted i n comparison to the range of conditions under which NADPH can serve as the reductant. This suggestion may explain the failure of previous investigators (190,193) to observe substantial 17* -hydroxylase and side-chain cleavage enzyme acti v i t i e s i n incubations of microsomes with NADH. The differences between the results reported herein and those of Lynn and Brown (190) may also be attributed to species differences. The observation that the testicular 17£ -hydroxysteroid dehydrogenase requires NADPH i s i n agreement with the findings of other investigators (190,193). It i s established that the 20*-hydroxysteroid dehydrogenase of rat testes requires NADPH (197). In the incubation of lyophilized micro-3 somes with ^H-progesterone as substrate performed i n the presence of NADH (Table LIX), 74$ of the i n i t i a l substrate radioactivity was recovered as 1 8 6 androstenedione. This finding supports the view ( 1 9 6 ) that the 2 0 < * -hydroxy derivative of 17-hydroxyprogesterone is not an obligatory sub-strate for the side-chain cleavage enzyme. In the incubations of the soluble fraction (Table LIX), the major radioactive metabolite recovered i n the experiments with NADH and NADPH was 17-hydroxyprogesterone and the side-chain cleavage ac t i v i t y was very similar i n both experiments. The t o t a l per cent of the i n i t i a l substrate radioactivity recovered i n the incubation with NADH was higher than i n the incubation with NADPH and a lesser per cent of the substrate radioactivity was recovered as 17_hydroxyprogesterone i n the experiment with NADPH. I t i s suggested that in the incubation with NADPH a substantial portion of radioactive 17-hydroxyprogesterone was transformed to 17,20<<-dihydroxypregn-4-en-3-one. The hypothesis that the 2 0 * -reduction of 17-hydroxyprogesterone serves as a regulatory mechanism in androgen biosynthesis i n the rat testis has been proposed by Inano and coworkers ( 2 3 5 ) . The effects of changes i n the concentrations of NADH and NADPH on the activities of the 17°<- -hydroxylase and the side-chain cleavage enzyme (Table LX) show that higher concentrations of NADH than NADPH are required for maximum ac t i v i t i e s . The results of the incubation per-formed at an i n i t i a l NADPH concentration of 0 . 0 1 1 K- moles per ml may be explained as follows: minimal quantities of testosterone and (presumably) 1 7 , 2 0 * -dihydroxypregn-4-en-3-one were present at the end of the incu-bation period because NADPH was ut i l i z e d for 17-hydroxylation of proges-terone and side-chain cleavage of 17-hydroxyprogesterone and the equil-i b r i a of the two dehydrogenase reactions"favored ; the oxidized .products. Several reservations must be considered with regard to the 187 experiments reported herein. None of these reservations invalidates the experimental observations or the major conclusions that are drawn from the studies. The methods used to purify the androstenedione and testos-terone following incubations with ^C-androstenedione as substrate were not assessed by crystallizations of the products. Testosterone was sub-jected to paper chromatography both as the free compound and as the acety-lated derivative and androstenedione was treated with pyridine and acetic anhydride as a part of the purification procedure. It i s unlikely that substantial quantities of impurities were present i n the testosterone acetate and androstenedione samples that displayed constant specific a c t i v i t y following paper chromatography. The procedures used to isolate progesterone, 17-hydroxyprogesterone, androstenedione and testosterone 3 following incubations with H-progesterone as substrate were assessed by crystallizations of the compounds eluted from the f i n a l paper, chromato-grams (see Preliminary Experiments and Time Studies section). In the discussion of some of the results reported herein i t i s assumed that the direction i n which the 17/8 -hydroxysteroid dehydrogenase reaction proceeds i s very responsive to the concentrations of the products and reactants. Although i t is established that the reaction i s reversible, the inter-conversion of androstenedione and testosterone does not display the characteristics of a f a c i l e equilibrium under certain i n vitro conditions (216). The experiments with %-progesterone as substrate were conduct-ed at pH 7.4; the i n vi t r o pH optima for the 17*- -hydroxylase and the side-chain cleavage enzyme are i n the region of 6.8 (see the previous section of this thesis). The pH of the incubation medium may be of par-ti c u l a r significance i n reactions involving oxidation and reduction of 188 pyridine nucleotide coenzymes because H + i s a stoichiometric reactant or product i n these reactions. The results observed following incu-bations at pH 7.4 may be quite unlike the results observed at another pH. The observations reported herein may be reconciled with the widely held view that NADPH i s required for hydroxylations and side-chain cleav-age of steroid molecules by invoking any of the following hypotheses: the NADH added to the incubation media was contaminated with NADPH, phos-phorylation of NADH occurred during the incubation period, transhydro--t-genation of endogenous NADP occurred during the incubation period, a barrier that impedes spe c i f i c a l l y the transport of NADH to the enzymes was destroyed, the s p e c i f i c i t y of the enzymes for NADPH was altered. The i n a b i l i t y of the NADH employed i n the experiments with % _ progesterone as substrate to support 17-oxo reduction of substrate -^ C— androstenedione (Table LVII) i s strong evidence that NADPH was absent from the NADH that was added and that a freely accessible pool of NADPH was not formed during the incubations. No evidence of contamination of the NADH by NADPH was found i n column and paper chromatograms of the NADH. Moreover, a sample of NADH purified by column ion exchange chroma-tography stimulated 17<* -hydroxylase and side-chain cleavage enzyme activ-i t i e s to an extent similar to that observed for the unpurified coenzyme. It i s highly unlikely that sufficient ATP or the requisite- enzymes for phosphorylation of NADH were present i n the microsomal preparations or i n the soluble fraction. The experimental results with "^"C-androstenedione as substrate -I-do not eliminate the pos s i b i l i t y that NADH -NADP transhydrogenase activ-i t y was present as a component of the ^"'--hydroxylase and side-chain 189 cleavage enzyme systems and not as a component of the Yip -hydroxysteroid dehydrogenase. The hypothesis that substantial amounts of NADPH were produced during the incubations with NADH by enzymatic transfer of re-ducing equivalents from NADH to a freely accessible pool of endogenous NADP"*" i s incompatible with the very low yield of radioactive testosterone that was observed i n the incubation of lyophilized microsomes with NADH and -^C-androstenedione as substrate (Table LVII) or ^ H-progesterone as substrate (Table LIX). It i s possible that slight transhydrogenase a c t i v i t y was present that required NADP added exogenously for expression. The t o t a l per cent of i n i t i a l substrate radioactivity recovered i n each of the four experiments with "^'C-androstenedione was : control incubation, 101; incubation with NADH, 9 3 ; incubation with NADH and NADP+ , 8 6 ; incu-bation with NADPH, 7 0 . The results suggest that the metabolism of andros-tenedione and testosterone to other compounds such as androstane metabo-l i t e s (149) proceeds at a greater rate i n the presence of NADPH than i n the presence of NADH. The observation that the t o t a l per cent i n i t i a l substrate radioactivity present as androstenedione plus testosterone was less i n the incubation performed with NADH and NADP"** than i n the incu-bation with NADH alone may be explained as follows: slight NADH - NADP + transhydrogenase act i v i t y was present; the small amount of NADPH that was formed did not influence significantly the NADPH + androstenedione testosterone NADP+ equilibrium because of the relatively large quant-i t y of NADP+ that was added. However, the NADPH that was formed per-mitted the metabolism of small quantities of •^'C-androstenedione to pro-ducts other than testosterone and, therefore, the per cent of the i n i t i a l substrate radioactivity present as testosterone plus androstenedione was 190 diminished. The stimulation of 17* -hydroxylase and side-chain cleavage enzyme ac t i v i t i e s that was observed i n incubations with NADH may have been due to loss of a selective NADPH transport system or to alterations i n the inherent specificities of the enzymes. A comparison of the re-sults of the incubations of microsomes with NADH and NADPH (Table LVIII) and a comparison of the results of the incubations of lyophilized micro-somes with NADH (0.12 p-moles per ml) and NADPH (0.11 (^ .moles per ml) (Table LX) demonstrate that the preference of the side-chain cleavage enzyme for NADPH was greater than that of the 17-hydroxylase. These observations may be used as evidence against the loss of a selective transport system for NADPH. The results of the experiments reported herein demonstrate that NADH can serve as a reductant for active 17-hydroxylation of progesterone and for side-chain cleavage of 17-hydroxyprogesterone. Under certain ex-perimental conditions, the quantity of products formed i n incubations per-formed in the presence of NADH were very similar to the quantities formed i n the presence of NADPH. The spec i f i c i t i e s of the 17<* -hydroxylase and the side-chain cleavage enzyme for pyridine nucleotide coenzymes that were observed are different from the spec i f i c i t i e s reported by others for the same enzymes as well as for other steroid hydroxylases and side-chain cleavage enzymes. The results reported herein may be artifactual; i f so, alteration of the intrinsic s p e c i f i c i t i e s of the enzymes appears to be the most l i k e l y explanation. On the other hand, the observations reported herein may reflect the spe c i f i c i t i e s of the enzymes i n vivo. Hayaishi,Iin a recent review of monooxygenase-catalyzed reactions (226), 191 questions the generally held belief that i n animal systems NADPH, rather than NADH, i s u t i l i z e d more frequently as the reductant i n enzymatic hydroxy la tions. SUMMARY AND CONCLUSIONS (i) The side-chain cleavage of 17-hydroxyprogesterone i s the rate-limiting reaction i n the biosynthesis of testosterone from progesterone i n the rat testis. ( i i ) 17-Hydroxyprogesterone is present as a bound intermediate (at least i n part). ( i i i ) The progesterone 17-hydroxylase and the 17-hydroxyprogesterone side-chain cleavage enzyme can be solubilized by treatment of lyophilized microsomes with Triton N-101. (iv) '> Both the 17* -hydroxylase and the side-chain cleavage enzyme i n the soluble fraction displayed maximal act i v i t y at pH 6.8 and at 37°. (v) Progesterone rather than pregnenolone i s the preferred substrate for the 17* -hydroxylase. The rat testis contains enzymes necessary to con-vert pregnenolone to progesterone. The speci f i c i t y of the 17«- -hydroxy-lase for progesterone rather than for pregnenolone helps to explain the observation that the major route of testosterone biosynthesis from preg-nenolone proceeds via progesterone, 17-hydroxyprogesterone and andros-tenedione rather than via 17-hydroxypregnenolone and dehydroepiandrosterone. (vi) The soluble fraction and the precipitate obtained by 40% saturation of the soluble fraction with ammonium sulfate contain NADPH dehydrogenase, non-heme iron and cytochrome P-450. The presence of these compounds i n association with 17* -hydroxylase and side-chain cleavage enzyme a c t i v i t i e s 192 suggests that these reactions are catalyzed by elaborate enzymatic sys-tems analogous to those required by the 11,8 -hydroxylase and the choles-t e r o l side-chain cleavage enzyme of adrenal mitochondria, (vii ) NADH can serve as a reductant for active 17-hydroxylation of pro-gesterone and for side-chain cleavage of 17-hydroxyprogesterone. The spec i f i c i t i e s of these enzymes for NADH and NADPH that were observed are different than the spe c i f i c i t i e s reported by others for the same enzymes as well as for other steroid hydroxylases and side-chain cleavage enzymes. The results reported herein may be attributed to alterations of the i n -herent specificities of the enzymes. On the other hand, the observations reported herein may reflect the spe c i f i c i t i e s of the enzymes i n vivo. 193 TABLE L V I I . Effects of pyridine nucleotide coenzymes on the distribution of radioactivity following incubations of lyophilized microsomes with -^C-androstenedione as substrate Steroid isolated Per cent of substrate radioactivity NADH NADPH NADPf NADH3 Control Androstenedione 88 18 82 99 Testosterone 5 52 4 2 Total 93 70 86 101 aThe incubation medium (15 ml) contained NADH (4.8 nmoles) and NADP (4.5 v-moles). The contents of the other incubation flasks are described i n the Text. TABLE LVIII Effects of NADH and NADPH on the distribution of radioactivity following incubation of microsomes with ^H-progesterone as substrate Per cent of substrate radioactivity Steroid isolated NADHa . NADPH3, Control 1 2 1 2 Progesterone 11 24 8 4 89 17-Hydroxyprogesterone 62 55 2 < 1 5 Androstenedione 26 7 29 39 < 1 Testosterone <i 1 <1 40 28 < 1 Total 99 86 79 71 94 'Two separate experiments were performed. TABLE LIX Effects of NADH and NADPH on the distribution of radioactivity following incubations of lyophilized microsomes and soluble fractions with 3H-progesterone as substrate i Per cent of substrate radioactivity Steroids isolated Lyophilized microsomes 1 Soluble fraction NADH NADPH Control NADH NADPH Control Progesterone 5 2 81 23 32 102 17-Hydroxyprogesterone 2 < 1 12 68 34 2 Androstenedione 74 21 < 1 12 4 < 1 Testosterone 3: 49 < 1 < 1 6 < 1 Total 84 72 93 103 76 104 In the incubation with lyophilized microsomes and NADPH H-progesterone (4,038,960 dpm> 3.71 H-g) and Mc-17-hydroxyprogesterone (892,200 dpm> 3.72 M-g) were used as combined substratesj~ results are given i n terms of the per cent of i n i t i a l 3H-progesterone radioacti-v i t y . In the other incubations with lyophilized microsomes and i n the experiments with the soluble fraction ^H-progesterone (1,676,208, dpm, 1.2 n-g) was used as substrate. TABLE LX Effects of NADH and NADPH concentrations on the distribution of radioactivity following incubations of lyophilized microsomes with ^H-progesterone as substrate Per cent of substrate radioactivity Steroids NADH concentration NADPH concentration isolated ( Kmoles/ml) ( JJL moles/ml) 0.60 0.12 0.012 0.53 0.11 0.011 Progesterone 5 8 30 2 5 6 17-Hydroxyprogesterone 2 46 48 < 1 < 1 57 Andros te nedione 67 23 6 21 56 22 Testosterone < 1 < 1 49 13 1 Total 77 77 84 72 74 86 The nucleotide coenzymes were dissolved in d i s t i l l e d water (1 ml) and added to the incubation flasks. The concentrations of the coenzymes i n the incubation media at the start of the incubations are given. 197 Fig.34. Column ion exchange chromatography of NADH and NADPH Fraction (10 ml) 198 BIBLIOGRAPHY 1. Zaffaroni, A., Burton, R.B., and Keutmann, E.H., Science, 111, 6 (1950). 2. Savard, K., J. B i o l . Chem., 202, 457 (1953). 3 . Bush, I.E., Biochem. J., 50, 370 (1952). 4. 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