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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., U n i v e r s i t y 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 t h i s t h e s i s as conforming t o the required standard  THE UNIVERSITY OF BRITISH COLUMBIA December, 1969  In p r e s e n t i n g  t h i s thesis 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 o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y I f u r t h e r agree t h a  a v a i l a b l e f o r r e f e r e n c e and study.  permission f o r extensive  copying o f t h i s  thesis  f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s .  I t i s understood t h a t c o p y i n g o r p u b l i c a t i o n  of t h i s thesis f o r f i n a n c i a l written  gain  permission.  Department o f  B vo cA/te-wus^vi  The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada  Date  f^rewvVer Z H  ;  s h a l l not be a l l o w e d w i t h o u t my  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 carcinoma 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 a c t i v i t y was present.  The excretion rates i n urine of  t o t a l 17-ketosteroids, 17-hydroxycorticoids and 17-ketogenic steroids were elevated; the excretion rates of testosterone, dehydroepiandrosterone, pregn a n d i o l , pregnanetriol and free C o r t i s o l 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 pseudohermaphroditism.  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 ^Cprogesterone. The results are similar to those of others who have  ii  i n v e s t i g a t e d the steroidogenic c a p a c i t y of gonadal t i s s u e i n p a t i e n t s w i t h male pseudohermaphroditism and f e m i n i z a t i o n a t puberty. A defect i n the formation of progesterone from pregnenolone has been suggested to e x p l a i n the r e s u l t s of a previous study i n which the gonadal t i s s u e obtained from a p a t i e n t w i t h v i r i l i z i n g male pseudohermaphroditism was 3  incubated with  H-pregnenolone as substrate.  I n the i n v e s t i g a t i o n s r e -  ported herein, transformationsof %-pregnenolone t o testosterone and androstenedione occurred both v i a 17-hydroxypregnenolone and dehydroepiandrosterone and v i a progesterone and 17-hydroxyprogesterone.  The f a i l u r e  of p a t i e n t s with v i r i l i z i n g male pseudohermaphroditism t o 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 t r a n s i e n t e f f e c t during embryonic development.  A l t e r n a t i v e l y , the s e n s i t i v i t y t o andro-  genic hormones may be subnormal i n c e r t a i n t i s s u e s and normal i n other t i s s u e s of patients with v i r i l i z i n g male pseudohermaphroditism. The b i o s y n t h e s i s of testosterone from progesterone and pregnenolone was i n v e s t i g a t e d i n the r a t t e s t i s .  Time s t u d i e s were performed using  c e l l - f r e e homogenates and % - p r o g e s t e r o n e and i n combination as substrates.  -17-hydroxyprogesterone  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 r e a c t i o n i n the biosynthesis of testosterone from progesterone and the evidence suggested that 17-hydroxyprogesterone was present as a bound intermediate (at l e a s t i n part). The progesterone 17-hydroxylase and the 17-hydroxyprogesterone side, chain cleavage enzyme of the r a t 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 T r i t o n N-101. Both enzymes displayed maximal a c t i v i t y a t pH 6.8 and a t 37°.  Progesterone rather than pregnenolone  i s the preferred substrate f o r the 17 * -hydroxylase. 6  E i t h e r NADH o r  iii  NADPH can serve as the reductant f o r active 17-hydroxylation of progesterone and f o r side-chain cleavage of 17-hydroxyprogesterone.  The solu-  ble f r a c t i o n contains NADPH dehydrogenase, non-heme i r o n protein and cytochrome P-450.  The presence of these compounds i n association with the  17«. -hydroxylase and the side-chain cleavage enzyme a c t i v i t i e s suggests that these reactions are catalyzed by elaborate enzymatic systems analogous t o those required f o r 11£ -hydroxylation and cholesterol side-chain cleavage i n adrenal mitochondria.  iv  TABLE OF CONTENTS Page ABSTRACT  i  TABLE OF CONTENTS  iv  LIST OF TABLES  ix  LIST OF FIGURES  xiv  GLOSSARY  xvii  ACKNOWLEDGEMENTS  xx  GENERAL INTRODUCTION  1  GENERAL METHODS  7  (a)  7  Chromatography (i)  S i l i c a g e l column chromatography  7  (ii)  Paper chromatography  7  (iii)  Thin layer chromatography  8  (b)  Detection and quantitation of steroids  8  (c)  C r i t e r i a employed to assess the purity of compounds and methods used to calculate the minimal per cent conversion of substrate t o product  10  (d)  (e)  (i)  Crystallizations  10  (ii)  Paper chromatography  10  Assay of r a d i o a c t i v i t y  11  (i)  Liquid s c i n t i l l a t i o n spectrometry  11  (ii)  Gas flow detection  11  Separation by solvent p a r t i t i o n  12  (i)  Hexane-aqueous methanol p a r t i t i o n  12  (ii)  P a r t i t i o n into neutral, "estrone-estradiol" and " e s t r i o l " fractions  12  V  Page (f) Reactions  1 3  (i)  Acetylation  1 3  (ii)  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 STEROID METABOLISM BY THE TUMOR IN VITRO !  INTRODUCTION  16  (a) Metabolism of adrenal steroids i n normal subjects and i n patients with adrenocortical neoplasms (b)  Hypoglycemia associated with extra-pancreatic neoplasms that are not of adrenal o r i g i n (i)  (c)  17  17  (ii)  Excessive glucose consumption by the tumor  IB  (iii)  Deficient gluconeogenesis due to a specific effect of tryptophan metabolites  IS  (iv)  Other hypotheses  19  Hypoglycemia associated with adrenocortical tumors  Patient  '. (b) Tumor (c)  16  Enhanced glucose disappearance due to the presence of excessive i n s u l i n or i n s u l i n l i k e material i n the blood  MATERIALS AND METHODS (a)  16  Incubation conditions  19 21 21 22 22  (d) Extraction, resolution and purification procedures  23  (e) Assay of steroids i n urine  24  RESULTS.  25  DISCUSSION  26  SUMMARY AND CONCLUSIONS  30  vi  Page PART I I  STEROID BIOSYNTHESIS IN VITRO BY THE GONADS OF A PATIENT WITH VIRILIZING MALE PSEUDOHERMAPHRODITISM  58  INTRODUCTION  58  (a)  General considerations and c l a s s i f i c a t i o n  58  (b)  Steroid biosynthesis by the normal t e s t i s  59  (c)  Studies i n v i t r o of gonads obtained from patients with male pseudohermaphroditism  MATERIALS AND  59  METHODS  6  0  (a)  Patient  (b)  Gonads  61  (c)  Incubation conditions  61  (d)  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  62  6 0  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 PART I I I  CONCLUSIONS  73  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  (ii)  Incubations with quartered testes  110  (iii)  Incubations with c e l l - f r e e homogenates  113  (iv)  Time studies  U4  vii Page (c)  Results  117  (d)  Discussion  120  PREPARATION AND PROPERTIES OF A SOLUBLE SYSTEM OBTAINED FROM RAT TESTICULAR MICROSOMES THAT CATALYZES THE TRANSFORMATION OF PROGESTERONE TO 17-HYDROXYPROGESTERONE AND ANDROGENS '  137  (a)  Introduction  137  (b)  Materials and methods  137  (c)  (i)  Rats  137  (ii)  Tissue preparation  138  (iii)  Radioactive substrates  139  (iv)  Incubation conditions  140  (v)  Extraction, resolution and p u r i f i c a t i o n procedures '  140  (vi)  Column chromatography  142  (vii)  Other procedures  143  Results (i)  (ii) (iii)  144 Preparation of a soluble f r a c t i o n cont a i n i n g 17** -hydroxylase and side-chain cleavage enzyme a c t i v i t y  144  Other enzymatic a c t i v i t i e s i n the soluble fraction  147  E f f e c t s 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  Resolution and p u r i f i c a t i o n of components of the soluble f r a c t i o n  148  (iv) (d)  Discussion  STUDIES ON THE PYRIDINE NUCLEOTIDE COENZYME SPECIFICITIES OF THE PROGESTERONE 17-HYDROXYLASE, 17-HYDROXYPROGESTERONE SIDECHAIN CLEAVAGE ENZYME AND 17/8 -HYDROXYSTEROID DEHYDROGENASE OF THE RAT TESTIS  152  175  viii  Page (a)  Introduction  175  (b)  Materials and methods  176  (i)  Rats  176  (ii)  Tissue preparation  176  (iii)  Radioactive substrates  176  (iv)  Incubation conditions  177  (v)  Extraction, r e s o l u t i o n and p u r i f i c a t i o n procedures  178  (vi)  (vii)  Chromatography of pyridine nucleotide coenzymes  179  Other procedures  179  (c)  Results  180  (d)  Discussion  184  SUMMARY AND CONCLUSIONS  191  BIBLIOGRAPHY  198  ix  LIST OF TABLES Table I II  III  IV V VI  VII  VIII  IX  X XI  Page Data concerning previously reported cases of adrenocortical tumor and hypoglycemia  32  Excretion rates of 17-hydroxysteroids i n the urine of patients with adrenocortical tumors and hypoglycemia  33  Excretion rates of 17-ketosteroids i n the urine of patients with adrenocortical tumors and hypoglycemia  34  Excretion rates of steroids i n the urine of patients with adrenocortical tumors and hypoglycemia  35  Studies on the etiology of the hypoglycemia i n patients with adrenocortical tumors  36  Substrate "^C-progesterone incubation: column absorption chromatography of the 90% aqueous methanol fraction  37  Substrate -^C-progesterone incubation: further paper chromatography of eluates containing carrier androstenedione, 17-hydroxyprogesterone and testosterone acetate  38  Substrate %-pregnenolone incubation: column absorption chromatography of the aqueous methanol fraction  39  Substrate -'H-pregnenolone incubation: further paper chromatography of eluates containing testosterone acetate, 17-hydroxyprogesterone, C o r t i s o l and progesterone  40  Substrate %-pregnenolone incubation: chromatography  41  t h i n layer  Substrate %-pregnenolone incubation: paper chromatography of compounds previously separated by thin layer chromatography  42  XII  Substrate %-pregnenolone incubation: zations of androstenedione  crystalli-  43  XIII  Substrate %-pregnenolone incubation: zations of testosterone acetate  crystalli-  44  X  Page XIV  Substrate H-pregnenolone incubation: zations of dehydroepiandrosterone 3  crystalli-  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 C o r t i s o l , aldosterone and cortisone  47  Results of incubations of tumor t i s s u e with subs t r a t e •^•C-progesterone and substrate 3H-pregnenolone  48  S t e r o i d s excreted i n the urine  49  Products i d e n t i f i e d f o l l o w i n g incubations of gonads from patients with male pseudohermaphrod i t i s m and f e m i n i z a t i o n with r a d i o a c t i v e progesterone as substrate  74  Products i d e n t i f i e d f o l l o w i n g incubations of gonads from patients with male pseudohermaphroditism and f e m i n i z a t i o n with substrate pregnenolone  75  Products i d e n t i f i e d f o l l o w i n g incubations of gonads from patients with male pseudohermaphroditism and f e m i n i z a t i o n with r a d i o a c t i v e testosterone arid r a d i o a c t i v e androstenedione as substrates  76  Products i d e n t i f i e d f o l l o w i n g incubations of gonads from patients with male pseudohermaphroditism and f e m i n i z a t i o n with r a d i o a c t i v e dehydroepiandrosterone and r a d i o a c t i v e dehydroepiandrosterone s u l f a t e as substrates  77  Products i d e n t i f i e d f o l l o w i n g incubations of gonads from patients w i t h male pseudohermaphroditism and f e m i n i z a t i o n with r a d i o a c t i v e acetate, r a d i o a c t i v e 17-hydroxyprogesterone and r a d i o a c t i v e 17-hydroxypregnenolone as substrates  78  Results of incubations of gonads from patients w i t h v i r i l i z i n g male pseudohermaphroditism w i t h r a d i o a c t i v e pregnenolone and r a d i o a c t i v e 17hydroxypregnenolone as substrates  79  Per cent of substrate r a d i o a c t i v i t y present i n f r a c t i o n s f o l l o w i n g e x t r a c t i o n , p a r t i t i o n and separation prodedures  80  Substrate •^C-androstenedione incubation: zations of testosterone  81  XVII  XVIII XLX  XX  XXI  XXII  XXIII  XXIV  XXV  XXVI.  crystalli-  xi  Page XXVII XXVIII XXIX XXX XXXI XXXII XXXIII XXXIV  XXXV XXXVI XXXVII XXXVIII  Substrate "^C-androstenedione incubation: t a l l i z a t i o n s of estradiol-17/9  crys-  Substrate •^C-androstenedione incubation: t a l l i z a t i o n s of estrone  crys-  82  Substrate -^C-progesterone incubation: zations of 16<* -hydroxyprogesterone  crystalli-  Substrate "^C-progesterone incubation: zations of androstenedione  crystalli-  Substrate "^C-progesterone incubation: zations of testosterone acetate  crystalli-  Substrate "^C-progesterone incubation: zations of 17-hydroxyprogesterone  crystalli-  Substrate -^C-progesterone incubation: zations of estrone and estradiol-17/3  crystalli-  84 85 86 87  89  Substrate %-pregnenolone incubation: zations of 17-hydroxypregnenolone  crystalli90  Substrate %-pregnenolone incubation: zations of 16**- -hydroxyprogesterone  crystalli-  Substrate %-pregnenolone incubation: zations of dehydroepiandrosterone  crystalli-  Substrate ^H-pregnenolone incubation: zations of androstenedione  crystalli-  3  91 92 93  Substrate ^H-pregnenolone incubation: zations of testosterone acetate  crystalli-  XL  Substrate ^H-pregnenolone incubation: zations of 17-hydroxyprogesterone  crystalli-  Substrate ^H-pregnenolone incubation: zations of progesterone  crystalli-  Substrate %-pregnenolone incubation: zations of estradiol-17/9  crystalli-  XLII  88  Substrate %-pregnenolone incubation: further paper chromatography of eluates containing carrier steroids  XXXIX  XLI  83  94 95 96 97  xii  Substrate -^-pregnenolone incubation: l i z a t i o n s of estrone  crystal-  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-17hydroxyprogesterone i n combination as substrates: results of time studies Incubations with %-progesterone and "^G-17hydroxyprogesterone i n combination as substrates: results of time studies f  Distribution of radioactivity following incubations of preparations of rat testes Results of incubations of supernatants obtained following high speed centrifugation of Triton N-101-treated lyophilized microsomes 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 f  Distribution of radioactivity following incubations 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 17hydroxylation, 17-hydroxyprogesterone sidechain 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 steroids Distribution of radioactivity following incubations of the precipitate obtained by t%Ofo saturation of the soluble fraction with ammonium sulfate  xiii  Specific a c t i v i t y of the progesterone 17-hydroxylas e Non-heme ironeand protein content of washed 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 1.  Page General outline of steroid biosynthetic pathways i n the adrenal  3  2.  Metabolism of C o r t i s o l  4  3.  Metabolism of androgens  5  4. 5.  Biosynthesis of androgens Substrate ^"C-progesterone incubation: flow sheet for extraction, 90% aqueous methanol-hexane p a 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  6  6. 7.  8.  9.  10.  Substrate •^ C-progesterone incubation: and purification of progesterone f  50  resolution 51  Substrate "^C-progesterone incubation: paper chromatography of selected eluates prior to and following the addition of unlabeled carrier androstenedione, testosterone and 17-hydroxyprogesterone Substrate ^C-progesterone incubation: paper chromatography of fractions 8 and 9 from the column chromatogram 3 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 3  52  53  54  Substrate ^H-pregnenolone incubation: paper chromatography of eluates containing pregnenolone and androstenedione  55  Substrate %-pregnenolone incubation: paper chromatography of fractions 8 and 9 of the column chromatogram  56  Substrate ^H-pregnenolone incubation: paper chromatography of eluates containing carrier testosterone and dehydroepiandrosterone  57  13.  Substrate "^C-androstenedione incubation: chromatography of the neutral fraction  paper  1D0  14.  Substrate -^-C-androstenedione incubation: chromatography of androstenedione  paper  11.  12.  101  XV  Page 15. 16.  17.  18.  19.  20. 21. 22.  23.  24.  25.  26.  27.  Substrate -progesterone incubation: chromatography of the neutral f r a c t i o n  paper  102  Substrate -^C-progesterone incubation: paper chromatography of testosterone acetate and 17-hydroxyprogesterone  103  Substrate ^H-pregnenolone incubation: tography of the neutral f r a c t i o n  104  paper chroma-  Substrate ^H-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  105  Substrate ^H-pregnenolone incubation: paper chromatography of testosterone-17-hydroxyprogesterone eluate  106  Substrate %-pregnenolone incubation: paper chromatography of pregnenolone-androstenedione eluates  107  Substrate •^C-mevalonate incubations: tography of n e u t r a l f r a c t i o n s  108  paper chroma-  Incubations of c e l l - f r e e homogenates of r a t testes with 3H_progesterone and l^C-17-hydroxyprogesterone i n combination as substrates: results of time studies  134  Incubations of c e l l - f r e e homogenates of r a t testes with ^H-progesterone and ^C-17-hydroxyprogesterone i n combination as substrates: results of time studies  135  Incubations of c e l l - f r e e homogenates of r a t testes with 3H-progesterone and 14C-17-hydroxyprogesterone i n combination as substrates: results of time studies  136  The e f f e c t of pH on the a c t i v i t i e s of the 17* hydroxylase and the side-chain cleavage enzyme i n the soluble f r a c t i o n  166  The e f f e c t of temperature on the a c t i v i t i e s of the 17<sc -hydroxylase and the side-chain cleavage enzyme i n the soluble f r a c t i o n  167  Absorbance spectrum of the precipitate obtained by saturation of the soluble f r a c t i o n with ammonium sulfate  168  1+Ofo  xvi  Page 28. 29.  Sephadex G-200 column chromatography ' U l t r a v i o l e t spectrum of T r i t o n N-101  I69 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 i o n exchange chromatography of NADH and NADPH  197  xvii  GLOSSARY (a) Steroids Aldosterone, 11/8, 21-dihydroxy-18-oxo-pregn-4-ene-3,  20-dione.  Androstenediol, androst-5-ene-3,r3 , 17/ - d i o l . 3  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 -yl-sulfate. 8  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. C o r t i s o l , 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. E q u i l i n i n , 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 - h y d r o x y - 5 p  -androstan-17-one.  xviii  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 17-Hydroxypregnenolone, 3 p,  17  16*6 -Hydroxyprogesterone, 16«< 17-Hydroxyprogesterone, 17  -dihydrcex:ypregn-5-en-20-one. -dihydroxypregn-5-en-20-6ne.  -hydroxypregn-4-ene-3,20-dione.  -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 s u l f a t e , 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. L i g r o i n , l i g h t petroleum ether (b.p. 80-100°). NAD , oxidized nicotinamide-adenine dinucleotide. NADH, reduced NAD . NADP , oxidized nicotinamide-adenine dinucleotide 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 g l y c o l .  T/ > toluene--propylene g l y c o l . PG  (d)  Miscellaneous  Ac, a c e t y l group, b.p., b o i l i n g point, cpm, counts per minute. dpm, disintegrations per minute. S.A., s p e c i f i c a c t i v i t y , v;, v o l ; volume, w, wt; weight.  phosphate.  XX  ACKNOWLEDGEMENTS I am deeply grateful to Dr. V.J. 0 Donnell f o r his guidance ,  during the course of the investigations reported herein and for his assistance i n the preparation of t h i s 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 f o r f i n a n c i a l support i n the form of fellowships awarded to me during these studies. The research 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 c o r t i c a l 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 pseudohermaphroditism (Part II) and i n the t e s t i s 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 steroid metabolism i n the rare disorders that were present and to search f o r biochemical explanations f o r 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 catabolism of steroid hormones were studied i n the patient with adrenal c o r t i c a l carcinoma and hypoglycemia; the investigations encompassed the steroid biosynthetic 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) constitutes the segment of the pathway of androgen biosynthesis that i s the principal subject of the studies reported i n Part I I I of the thesis. The results of preliminary experiments on the metabolism of radioactive progesterone and 17-hydroxyprogesterone by the rat t e s t i s i n v i 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-hydroxyprogesterone and the reduction of the 17-oxo group of androstenedione.  CORTISOL  ESTRONE  Fig. 2. Metabolism of Cortisol  Itfl-HYDROXYANDROSTERONE  Hj8-HYDROXY-5/9ANDROSTERONE  CORTOL  DIHYDROCORTISOL I  0-CORTOL  DIHYDROCORTISONE I  CORTOLONE  M-KETOANDROSTERONE  /3-CORTOLONE  ll-KETO-5/3ANDROSTERONE  F i g . 3.  A N D R O S T A N E - 3 a , 17/3-DIOL  Metabolism of androgens  5 / 3 - A N D R O S T A N E - 3 a , l 7 / J - DIOL  6  Fig. 4. Biosynthesis of  PROGESTERONE  17-HYDROXYPROGESTERONE  TESTOSTERONE  androgens  PREGNENOLONE  I7-HYDR0XYPREGNEN0L0NE  ANDROSTENEDIOL  7  GENERAL METHODS (a)  Chromatography (i)  S i l i c a g e l column chromatography: S i l i c a g e l (Davidson, grade 923) was dried i n an oven at  90° f o r 2 hours and added to the column as a s l u r r y i n hexane. Hexane was then run through the column f o r 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 added to the column. Stepwise elution was then performed using hexane-benzene (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 e l u -  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. (ii)  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 glycol (2); ligroin-methanol-water (5:4:1, by v o l ) , Bush A (3); benzene-methanol-water (2:1:1, by v o l ) , Bush B5 (3); and toluene-ligroin-methanol-water. (5*5:7:3, by v o l ) , Bush BI (3).  Paper s t r i p s were impregnated with a 40-  50$ methanol solution of formamide or propylene glycol prior t o the a p p l i cation of the sample. Following chromatography tfietchromatograms were  8  dried for 12-24 hours at room temperature. Chromatography using the Bush systems was i n i t i a t e d after 6-18 hours equilibration.  A l l paper chroma-  tography was performed at room temperature i n a descending direction. Following chromatography, the compounds were eluted i n the following manner:  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. (iii)  Thin layer chromatography: Glass plates coated with s i l i c a g e l G (according to Stahl)  and Eastman Kodak chromatogram sheets (type  K301R2)  were employed f o r t h i n  layer chromatography. Radelin GS-115 phosphor was extracted with methanol 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 d i s 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 methanol ( 3 x 5  ml).  The chromatogram sheets were cut and eluted i n the same  way that was used f o r paper chromatograms. (b)  Detection and quantitation of steroids Steroids containing the £>^-3-ketone grouping were detected i n  paper and t h i n layer chromatograms by viewing the chromatogram under short wave ultraviolet l i g h t (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 phosphomolybdic acid (4). In some cases, a narrow s t r i p cut from the experimental chromatogram was used; otherwise, chromatograms containing standard A  compounds were run simultaneously and the positions of the ex-  perimental steroids were estimated from the locations of the corresponding standards as ascertained by treatment with an ethanol solution of phosphomolybdic acid. Absorbance was measured with a Beckman DU spectrophotometer matched quartz c e l l s of 1 cm light path.  using  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 s t r i p of paper and the  experimental chromatogram were prepared and run together. The values used as molar extinction coefficients (at 240 nm wavelength) were as follows: 20«.-acetoxypregn-4-en-3-one, 16,000; androstenedione, 16,000; aldosterone, 15,800; cortexolone, 16,800; cortexolone acetate, 16,000; C o r t i s o l , 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)  C r i t e r i a 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 c r y s t a l l i n e 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 procedures and both results were used to assess the purity of the fractions. When i t appeared that a compound was radiochemically homogeneous, the results attained from the procedure f o r 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 conversion 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 f o r the crystals and the mother liquors i n two successive c r y s t a l l i z a t i o n s did not d i f f e r by more than f i v e per cent. I n calculating the per cent minimal conversion of substrate to product, the average values of the s p e c i f i c a c t i v i t i e s of the crystals from the f i n a l two c r y s t a l l i z a t i o n s 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. (ii)  Paper chromatography: In most cases, chromatography of the steroids containing  the &^-3-ketone grouping was repeated u n t i l constant specific a c t i v i t i e s  11  were attained. The s p e c i f i c a c t i v i t y of a compound was considered to be constant i f the values f o r the s p e c i f i c a c t i v i t i e s of the compound i n the eluates of two successive chromatograms d i d not d i f f e r by more than f i v e per cent. (d)  Assay of r a d i o a c t i v i t y (i)  L i q u i d s c i n t i l l a t i o n spectrometry: A Nuclear-Chicago l i q u i d s c i n t i l l a t i o n  spectrometer  (model 725) was employed f o r assay of r a d i o a c t i v i t y by l i q u i d s c i n t i l l a t i o n spectrometry.  Steroid samples i n methanol solution were pipet-  ted into counting v i a l s and the solvent evaporated.  Each residuum  then dissolved i n 5 ml of toluene containing 2,5-diphenyloxazole  was  (0.4%),  1,4-bis ^2-(5~phenoxyloxazolyl)j -••benzene (0.01%) and absolute ethanol (2%).  The counting e f f i c i e n c y was 25% f o r \  containing a single isotope.  and 85% f o r -^C i n samples  In samples containing both isotopes, the  e f f i c i e n c y was 20% f o r t r i t i u m and 40$ f o r •^C.  In samples containing  both H and "^C, the dpm f o r each isotope was calculated according to the 3  simultaneous equation method of Okita and coworkers (5). (ii)  Gas flow detection: A Nuclear-Chicago gas flow detector (model D47) operating  i n the Geiger region was used without a window.  Copper planchets were  cleaned with concentrated s u l f u r i c acid saturated with sodium dichromate. A f t e r r i n s i n g with d i s t i l l e d water, the planchets were wiped with acetone and d r i e d .  A 0.1 or 0.2 ml portion of a methanol solution of the  s t e r o i d was pipetted on to the planchet and dried i n a i r at room temperature.  The gas flow detector was operated at an e f f i c i e n c y of approxi-  mately 45% f o r  and approximately 20% f o r % .  12  (e)  Separation by solvent p a r t i t i o n (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 f r a c t i o n was removed and the hydrocarbon 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 (ii)  distillation. P a r t i t i o n into neutral, "estrone-estradiol" and " e s t r i o l "  fractions (6): The dried steroid mixture was transferred to a separatory funnel i n absolute ethanol (2.5 ml).  Benzene (50 ml) and l i g 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 y i e l d the " e s t r i o l " f r a c t i o n . The benzene-ligroin f r a c t i o n 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 y i e l d the "estrone-estradiol" fraction. The remaining benzene-ligroin mixture constituted the neutral f r a c t i o n . The solvents were removed from the neutral, "estrone-estradiol" and " e s t r i o l " fractions under reduced pressure at 40°, absolute ethanol was added and the fractions were dried by azeotropic  distillation.  13  (f)  Reactions (i)  Acetylation: The dry substrate was treated with anhydrous pyridine  (0.3 nil)  and a c e t i c anhydride (0.15 ml) overnight at room temperature  i n a stoppered t e s t tube.  Following the reaction period, several drops  of methanol were added to the reaction mixture and the solvents were evaporated under a gentle stream of nitrogen a t 40°.  Several a d d i t i o n a l  drops of methanol were then added and evaporated i n a s i m i l a r manner. (ii)  Girard (7): Girard's T reagent (8) was c r y s t a l l i z e d from absolute  ethanol p r i o r to use. ide  The s t e r o i d mixture was dried over calcium c h l o r -  i n an evacuated dessicator; 0.5 ml of g l a c i a l a c e t i c a c i d and 100  of Girard's T reagent were added.  mg  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 a t 95° f o r 20 minutes. The tube was removed from the o i l bath and 15 ml of i c e water were added and the reaction mixture was immediately transferred to a small separat o r y funnel.  S u f f i c i e n t 10$ sodium hydroxide was added to neutralize nine-  tenths of the a c e t i c a c i d . were made.  Three extractions with 20 ml portions of ether  The ether extracts were combined and washed once with 10 ml  of i c e water.  This aqueous wash was combined with the rest of the aque-  ous ketonic f r a c t i o n .  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. tained the non-ketonic f r a c t i o n .  The ether con-  The aqueous ketonic f r a c t i o n was  acidi-  f i e d with 3 ml of concentrated hydrochloric acid, allowed to stand a t room temperature f o r 2 hours and then extracted with ether (3 x 10 ml).  1U  This ether extract was washed with 2.5$ d i s t i l l e d water (3 x 10 ml). ketonic f r a c t i o n .  sodium carbonate (1 x 10 ml) and  This second ether extract contained the  The ketonic and non-ketonic f r a c t i o n s were evaporated  under reduced pressure a t 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)  P u r i f i c a t i o n of unlabeled steroids added as c a r r i e r s Unless otherwise stated, unlabeled steroids added as c a r r i e r s  were p u r i f i e d by a series of c r y s t a l l i z a t i o n s and t h e i r p u r i t y was assessed by melting point determinations using a Kofler hot stage apparatus and polarized l i g h t . (h)  Solvent p u r i f i c a t i o n Solvents (AR grade) were p u r i f i e d as follows:  acetone was  reflux-  ed f o r 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  distillation  from calcium hydride under anhydrous conditions. Chloroform was shaken with concentrated s u l f u r i c a c i d , washed with d i s t i l l e d water, d r i e d 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  f o r 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 d i l u t e , weakly a c i d i c s o l u t i o n of ferrous sulfate (3 x one-tenth v o l ) , washed to n e u t r a l i t y with d i s t i l l e d water and d i s t i l l e d .  Anhydrous  e t h y l acetate was prepared by drying i t over calcium chloride f o r 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 f o r 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 anhydride, 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 HYPOGLYCEMIA: 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 F i g . 1 (9,10,11,12). At present, l i t t l e i s known regarding which routes i n t h i s complex network of steroid transformations 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 C o r t i s o l formation proceeded from pregnenolone v i a 17whydroxypregnenolone rather than v i a progesterone (13). Most studies i n v i t r o of adrenal steroid biosynthesis have been performed on tissue from animals other than man or on glands obtained from diseased human subjects. Studies i n v i t r o 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%), C o r t i s o l (10.3%), androstenedione (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 f o r C o r t i s o l 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 r e f l e c t , 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 c a l manifestations, marked qualitative and quantitative differences i n steroids excreted v i a the kidney and i n steroid transformations i n v i t r o 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 hypoglycemia that occurs i n some patients with extra-pancreatic neoplasms (53-59). (i)  Enhanced glucose disappearance due to the presence of excessive  i n s u l i n or i n s u l i n - l i k e material i n the blood: Increased i n s u l i n or i n s u l i n - l i k e a c t i v i t y i n the circulation could be the result of secretion of i n s u l i n or i n s u l i n - l i k e material by the tumor, pancreatic stimulation by the tumor, supression of physiologic i n s u l i n antagonists, or delayed degradation of i n s u l i n .  Insulin or i n -  s u l i n - l i k e 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 i n s u l i n and  18  i n s u l i n - l i k e 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 i n s u l i n secretion. (ii)  Excessive glucose consumption by the tumor: Evidence f o r 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 f a t t y 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 uptake by the tumor tissue i n v i t r o (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 v i t r o (61,74). (iii)  Deficient gluconeogenesis due to a specific effect of trypto-  phan metabolites: Certain metabolites of tryptophan cause inhibition of phosphoenolpyruvate  carboxykinase i n v i t r o (75). Increases i n the concentra-  tions of several indole compounds i n the blood and urine were observed during episodes of severe hypoglycemia i n a group of patients with neoplasms of extra-pancreatic origin, but not i n patients with l o c a l 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 hypoglycemic episodes have been found i n other patients with tumors of extrapancreatic origin (53,77).  19  (iv)  Other hypotheses: Other hypotheses advanced to explain the hypoglycemia i n cer-  t a i n patients with extra-pancreatic neoplasms include hepatic insufficiency 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 concerning 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 v i a the kidney i n patients with hypoglycemia and adrenocortical tumor are l i s t e d i n Tables I I to IV. With one exception, the excretion rates of 17-ketosteroids and 17-hydroxysteroids 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 obtained by alkaline p a r t i t i o n of a carbon tetrachloride extract of acidhydrolyzed urine.  The aldosterone excretion rate was normal i n one sub-  ject and elevated i n another. The excretion rate of 3<* ,17,20-trihydroxy5P -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 C o r t i s o l 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 elevated:  16°C-hydroxypregnenolone, pregn-5-ene-3 £ ,16* , 20<* - t r i o l , preg-  n a n d i o l , free corticosterone and pregnanetriol plus 17-hydroxypregnenolone 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 hypoglycemia 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 i n s u l i n - l i k e a c t i v i t y i n extracts of the tumor have given normal or negative 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" i n s u l i n - l i k e a c t i v i t y i n acidalcohol extracts of tumor tissue when tested by rat hemidiaphragm and epididymal f a t pad technics.  Serum i n s u l i n - l i k e a c t i v i t y has been normal  or diminished i n a l l f i v e 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-phosphatase 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 tryptophan 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 v i t r o 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 hypoglycemia has not been previously assessed i n v i 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 widespread metastases were present.  22  (b)  Tumor  1  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 c e l l s with prominent cytoplasmic walls, f i n e l y granular eosinophilic cytoplasm and central irregular nuclei. The nuclei varied i n size and small nucleoli.  contained  Occasional mitotic a c t i v i t y was observed. The c e l l s  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-^ C-Progesterone (The Radiof  chemical Centre, Amersham, England) was purified by thin layer chroma2 , tography using the solvent system ethyl acetate-benzene (1:1, by vol;* N  2.528 x 10^ cpm and 50.2 flasks.  \~g were added to each of three incubation  The radioactive 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 g l y c o l immediately prior to the addition of the c e l l - f r e e homogenate. The tumor was stored at -19° following excision.  Seven days  later, i t was p a r t i a l l y thawed and a section was taken f o r 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 f o r the macroscopic and microscopic description of the tumor. 2see General Methods section.  23  homogenized at pH 7.4 i n Krebs-Ringer phosphate buffer (90), Na"^ and K  +  i n which  were interchanged, containing glucose (0.01 M) and n i c o t i n a -  mide (0.04 M); a Potter-Elvehjem-type  homogenizer was used (91).  The  homogenate was centrifuged (1500 x _g, 10 minutes, 4 ° ) and the supernatant (150 ml) was divided equally among f i v e incubation f l a s k s .  In-  cubations were performed i n a gas phase of 95% oxygen-5% carbon dioxide f o r 3 hours at 3 7 ° using a Dubnoff metabolic shaking incubator, (d)  E x t r a c t i o n , resolution and p u r i f i c a t i o n procedures Data concerning the extraction, resolution and p u r i f i c a t i o n pro-  cedures employed are summarized i n F i g s . 5 to 8 and Tables VI and  VII  14 f o r the experiment using  C-progesterone as substrate and i n F i g s . 9 to  12 and Tables VIII to XVI f o r the experiment using ^H-pregnenolone substrate.  a  s  At the end of the incubation period the contents of the i n -  cubation f l a s k s containing "^C-progesterone were combined and d i l u t e d to 200 ml with d i s t i l l e d water.  The contents of the f l a s k s containing % -  pregnenolone were treated i n a s i m i l a r manner.  E x t r a c t i o n with e t h y l  acetate (4 x 200 ml) was then performed; the combined incubation medium from the experiment using "^C-progesterone was a l s o extracted with methylene chloride (4 x 200 ml) and the e t h y l acetate and methylene chloride extracts were combined.  The extracts were evaporated under reduced p r e s -  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 H 3  prejgnenolone were each partitioned between hexane and 90% aqueous metha2 nol.  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 f o r 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 chromatography^ i n the solvent systems ligroin-propylene glycol, toluenepropylene glycol and Bush B5.  Thin layer chromatography on coated glass 2  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 substrate.  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 negligible radioactivity remained with the carrier steroid.  or u n t i l In instances  where constant specific a c t i v i t y was observed f o r A^-3-ketosteroids and after several paper chromatograms of the  steroids, more purified car2  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 negl i g i b l e radioactivity. 2 Radioactivity was measured  with a l i 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 i n the Bush A s o l 2 vent system using Whatman no. 1 paper and t h i n layer chromatography i n the solvent system ethyl acetate-benzene (1:3, by vol) were employed  25  instead of the s i n g l e chromatography on Whatman no. 42 paper as described i n the o r i g i n a l procedure.  Quantitative estimation of testosterone was  was performed by measuring the absorbance a t 595 nm following treatment of the f i n a l eluate with a 0.5$ s o l u t i o n of v a n i l l i n i n ethanol-sulfuric a c i d (93).  An A l l e n correction (94) using the absorbances a t 510 and  680 nm was applied to the absorbance a t 595 nm.  Absorbance was measured  i n a Unicam SP 800 spectrophotometer with quartz cuvettes of 1 cm o p t i c a l path. The rates of excretion i n urine of 17-ketosteroids hydroepiandrosterone  (95-97), de-  (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 f r e e C o r t i s o l (106) were measured by Biomedical Assay Laboratories, Worcester, Massachusetts.  The excretion rate  i n urine of 3 * ,17,21-trihydroxy-5/B -pregnan-20-one was measured by both C  commercial laboratories using the same method (107). RESULTS The results of incubations of the tumor t i s s u e with radioactive progesterone and pregnenolone are given i n Table XVII.  No radioactive  steroids were i d e n t i f i e d 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 i d e n t i fied.  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 steroids were elevated; the excretion rates of dehydroepiandrosterone, pregnanetriol, pregnandiol and free C o r t i s o l were within the normal range. The excretion rate of 3 , lY^l-trihydroxy-S/s -pregnan-20-one i n the same o C  urine specimen was found to be elevated i n two determinations by both commercial laboratories. An elevated excretion rate of t o t 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 substrates to identifiable metabolites.  A large percentage of both radio-  active substrates was recovered unchanged and three radioactive metabolites 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 f a i l u r e to demonstrate active steroid biosynthesis in v i t r o may be attributed to some aspect of the experimental procedure rather than t o 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 a c 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 f o r incubation or the quantity of tissue that was incubated may have been too small to transform measureable amounts of the radioactive substrates.  The tumor was large  (1,285  g) and contained areas of necrosis and the rate of steroid biosynthesis per gram of tissue was undoubtedly low.  The tumor was frozen f o r one  week between excision and incubation and during that time i n t e r v a l damage to enzymes may have occurred.  Deficiencies of essential coenzymes  during the incubation period may have caused low enzymatic a c t i v i t y ; i n cubations were performed i n a Krebs-Ringer phosphate buffer containing only nicotinamide and glucose. The minimal metabolism of  3 lit H-pregnenolone and C-progesterone  by the tumor i n v i t r o may not have reflected quantitatively the i n vivo a c t i v i t y of the tumor. In contrast to the results of the experiments i n v i 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 biosynthetic capabilities of the tumor. The predominant pathway of C o r t i s o l biosynthesis from progesterone i n the normal adrenal cortex procedes v i a successive hydroxylations of progesterone at positions C 1 7 , C 2 1 , and C l l (9-12''and F i g . l ) . Studies i n patients with deficiencies i n hydroxylase a c t i v i t y and observations following the administration of steroids to normal subjects have shown that the major metabolites i n urine of progesterone, 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/toC-  and  N  21-hydroxylase a c 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. cretion i n urine of 3 * ,17,21-trihydroxy-5/B 1  The elevated rate of ex-  -pregnan-20-one i n the patient  reported herein suggests that a defect i n llj& -hydroxylase a c t i v i t y was present. 3*,17,21-Trihydroxy-5/ -pregnan-20-one, the major ketol metab o l i t e of cortexolone (109), i s normally found i n very small quantities i n the urine (38) and i n adrenal vein blood (110).  The rate of excret-  ion i n urine of 3* ,17,21-trihydroxy-5 3 -pregnan-20-one has been elevated r  i n a l l patients with an adrenocortical tumor and hypoglycemia i n whom i t has been measured (Table I I ) . I t 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 f r a c t i o n of steroids consists of compounds with the dihydroxyacetone side-chain at C17; the 17-ketogenic f r a c t i o n 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 i n many patients with adrenoc o r t i c a l 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 measured (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 f o r gluconeogenesis.  It  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 f o r the hypoglycemia that was present i n the patient reported herein; however, i t i s possible that the tumor produced other steroids 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 d i f f e r among patients (113).  Patients with adrenocortical tumors and hypogly-  cemia are a r a r i t y 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 adr e n o c o r t i c a l neoplasms and hypoglycemia have yielded results that have a l s o been observed i n patients with adrenocortical tumors without hypoglycemia and no d i s t i n c t i v e 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 unrecognized steroid produced by some adrenocortical tumors.  Steroid metabolism  has not been studied extensively i n patients with hypoglycemia and extrapancreatic tumors that a r i s e from organs and tissues other than the adrenal.  I t 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 ^"Cprogesterone as substrates.  Transformation of %-pregnenolone t o pro-  gesterone, dehydroepiandrosterone and androstenedione was observed; no metabolism of "^"C-progesterone was detected. (ii)  The excretion rate i n urine of 3 ,17,21-trihydroxy-5/S oL  -pregnan-20-  one was elevated which suggests that a defect i n 11/J -hydroxylase a c t i v i t y was present.  The excretion rates i n urine of t o t a l  17-hydroxycorticosteroids  17-ketosteroids,  and 17-ketogenic steroids were elevated; the  excretion rates of testosterone, dehydroepiandrosterone, pregnanetriol, pregnanediol and free C o r t i s o l were not elevated.  31  (iii)  The e t i o l o g y of the hypoglycemia that may accompany adrenocortical  neoplasms i n some patients remains unknown.  I t was not possible to relate  the r e s u l t s of the investigations of s t e r o i d metabolism reported herein to the hypoglycemia that was present.  TABLE I  >  Data concerning previously reported cases of adrenocortical tumor and hypoglycemia  Authors Anderson(78) Lawrence (79) Broster & Patterson(80) S t a f f i e r i et a l { 8 l ) Thannhauser (8"2) Case # 1 Case # 2 Case # 3 Dohan et a l . (15)  Age  Sex  33 24 14 25  M F F M  48  F M F F  Schamaun et al(83) Askanazy et aL(85) Kuhnlein & Meythaler(84) Boss (86) Williams et al.(l6) Case f l  37 26 45 64  M F M M  24  F  Case # 2 Eymontt et a l . (87) Wikman & McCracken (88) Scholz et al.(89) Cara (40 F~ Silverstein et al.(77)  48 19 59  M F M F F F  16  12 25  h e i g h t of tumor plus kidney.  Signs of steroid hormone excess None Hypertension, acne Virilism Gynecomastia  Tumor Size 0.4 kg "Grapefruit" 2.98 kg* 20x11x9 cm  Side  Histology  L L L R  Carcinoma Carcinoma Carcinoma Sarcoma •-  Virilism,hypokalemic alkalosis,' 2.2 kg hypertension, gynecomastia None 1.7 kg None 2.2 kg None 2.6 kg None "Grapefruit" V i r i l i s m , acne, hypertension, hypokalemic alkalosis Hypertension V i r i l i s m , acne, hypertension None None Hypertension, v i r i l i s m  2.0 kg 1.1 kg 1.4 kg 2.4 kg 1.8 k g 0.7 kg  a  ........  -  L  Carcinoma Carcinoma Carcinoma Carcinoma  R L R L  Adenoma Carcinoma Carcinoma Carcinoma  L L L L L . R  Carcinoma Carcinoma Carcinoma Carcinoma Carcinoma Carcinoma  TABLE I I Excretion rates of 17-hydroxysteroids mors and hypoglycemia > a  Authors Gara (40) Willaims et a l . (16) Case Wl Case # 2  B  i n the urine of patients with adrenocortical t u -  mg/24 hrs Total  THS  31°  7.2  162 (7-16) C  >4CO  C  Eymontt et a l . (87)  35.7-48.9°  Scholz et a l . (89)  12.2  Increased (trace)  193  THE  p-g/24 hrs THF  P» t r i o l  Free F  2.4  0.9  16.8 (1.6)  6.3  (oa-1.9)  367 (9-82)  (2.8)  2,199  d  P ' t r i o l , pregnanetriol; F, C o r t i s o l . See Glossary f o r other abbreviations and f o r 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 E x c r e t i o n rates of 17-ketosteroids i n the urine of p a t i e n t s with a d r e n o c o r t i c a l tumors and hypoglycemia * 31  mg/24 hrs  Authors B r o s t e r & Patterson (80) Dohan e t a l . (15)  Total  D  1,980  Increased  80  A  E  ll-0xy  25-27  Thannhauser (82) Case # 1 Case # 2 Cara (40) W i l l i a m s e t a l . (16) Case F " l Case # 2 Eymontt e t a l . (87) Shamaun e t a l . (83)  43 56.8 (4-14) 130.1 85.8-23.6  17.0 (4-6) 11.3  19.9 (1-1.4) 80.8  9.1 (2^) 15.2  43% (16-20)  16% (38-50)  c  c  4.8 (OiA-0.9)  13.9  41$ (30-47) c  33-41  Kuhnlein & Meythaler (84)  166  Scholz e t a l . (89)  7-31  S t a f f i e r i et a l . (81)  1.3 7.6 (0.1-0.3)  23.2  D, dehydroepiandrosterone; A, androstenedione; E, etiocholanolone; 11-Oxy, 11-oxygenated 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. a  35  TABLE 17 Excretion rates of steroids i n the urine of patients with adrenocortical tumors and hypoglycemia  Authors Dohan et a l (15) Cara (40) Williams et a l . (16) Case F l  Case # 2 Eymontt et al.(87)  Steroids  0  Estrogens increased 16-Hydroxypregnenolone, 8.7 mg/24 hrs Pregn-5-ene-3/5 ,3i><>c , 2 0 * - - t r i o l , 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  f o r m a l 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 a c t i v i t y Fasting plasma  Tumor Askanazy et al.(85) Negative Boss (86) Negative Williams et al.(l6) Case Wl Negative Case # 2 Kuhnlein and Meythaler(84) P o s i t i v e Eymontt et al.(87) 350^g  Normal Normal  No glucose-6-phosphatase i n tumor  0  3  Wikman & McCracken (88) Scholz et al.(89)  "Significant"  180K /ml u  b  Normal 2^U/ml  S i l v e r s t e i n et al.(77)  fValue i s within the normal range for muscle, l i v e r and kidney. Normal range 60-250 y*. U/ml. See Text f o r details. b  c  Other studies  No arteriovenous difference i n gluccose concentration across tumor Normal concentrations metabolites i n blood Normal concentrations metabolites i n blood  of tryptophan and urine of tryptophan and urine  ON  TABLE VI  Substrate ^C-progesterone incubation: methanol f r a c t i o n  Fraction number 1 2 3 4 5 6 7 8 9 10 11 12 13 Total  column a d s o r p t i o n chromatography o f t h e 90$ aqueous  Eluent composition • (v/v)  Eluate  :  Hexane-benzene (1:1) Benzene E t h y l a c e t a t e - b e n z e n e (1:99) M .i i« (2:98) » " (4:96) " " " (10:90) " » " (20:80) » " " (40:60) E t h y l acetate M e t h a n o l : e t h y l acetate(2:98) " (5:95) " " " (10:90) Methanol 11  Dry weight (mg) 89.92 0.38 4.90 17.96 6.68 5.45 5.12 8.04 6.64 66.04 98.70 13.23 49.65 371.71  —  —-  • — r  R a d i o a c t i v i t jr (cpm) 707,500 3,450 4,886,500 608,900 57,100 31,850 42,850 426,300 6,764,450  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 a s s a y o f r a d i o a c t i v i t y .  a  38  TABLE V I I Substrate -progesterone incubation: further paper chromatography of eluates containing carrier androstenedione, 17-hydroxyprogesterone and testosterone acetate a  „ , Compound  ™ , , . Chromatographic solvent system  Androstenedione  17-Hydroxyprogesterone  Testosterone acetate  Contents of eluate Radioactivity (cpm)  Weight (|^g)  Specific activity (cpm/Kg)  L/PG L/PG L/PG L/PG L/PG L/PG  23,840 16,610 11,790 8,100 6,860 4,870 2,660  93.8 79.1 78.9 60.3 57.2 49.9 42.0  254 -210: 150 133 120 98 63  T/PG T/PG  28,960 20,025 8,950  66.2 58.2 50.1  437 353 175  L/PG L/PG L/PG L/PG L/PG  62,240 9,930 7,540 5,020 3,850 3,235  58.0 47.6 38.8 35.9 26.4  171 158 129 107 122  b  See F i g . 7 f o r prior paper chromatography. "The f i n a l testosterone acetate eluate was subjected to c r y s t a l l i z a t i o n following the addition of unlabeled testosterone acetate; the crystals contained negligible radioactivity.  a  TABIE VIII Substrate -'H-pregnenolone incubation: methanol f r a c t i o n a  Fraction number 1 2 3 4 5 6 7 8 9 10 11 12 13 Total  column adsorption chromatography of the aqueous  Eluate  Eluent• composition (v/v) 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 .. ( .95) " " " (10:90) Methanol 5  Dry weight (mg)  Radioa ct ivity* (cpm)  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  242.47  . 163,350 Hi5S?;500 2,987,400 370,050 194,800 169,920 687,500 16,685,770  kSee Text f o r details. f r a c t i o n s 2 and 3; 5,6 and 7; 8 and 9 were combined prior to assay of radioactivity.  5  40  TABLE IX Substrate -^-pregnenolone incubation: further paper chromatography of eluates containing testosterone acetate, 17-hydroxyprogesterone, C o r t i s o l and progesterone  Contents of eluate Chromatographic solvent system  Compound  Radioactivity  Weight (Vg)  cpm Testosterone acetate  150,500 68,075 43,650 29,006 17,250 12,110  230 209 189 183 143 104  654 326 231 159 121 ll£>  T/PG T/PG  32,200 13,425 8,280  69.0 41.2  201°  B5  49,050 32,720  99.0 63.0  a  Cortisol** f Progesterone  See See °See See ®See See  a  b  f  cpnyVg  L/PG L/PG L/PG L/PG L/PG  a  17-Hydroxyprogesterone  Specific activity  L/PG L/PG L/PG L/PG  F i g . 12, footnote f . Table X I I I . Table XV. F i g . 11, footnote g. Table XVI. F i g . 9, footnote f .  dpm 1,099,950 737,360 655,263 418,263  261 193 180 113  e 519  4 9 5  dpm/M.g 4,214 3,821 3,650 3,705 c  TABLE X Substrate %-pregnenolone incubation:  Distance from origin (cm) 0-1 1-2.9 2.9-3.9 3.9-5.6 5.6-6.5 6.5-7.3 7.3-8.1 8.1-9.1 9.1-10 10-12 12-15 15-17 Total a  b  Thin layer chromatography  i Contents of combined methanol extract  Compound or zone  Radioactivity (cpm) Origin Aldosterone*  47,920 51,270  34,640 120,440  5  Corticosterone Cortisone  213,790 190,850 80,990  b  0  Cortexolone^ 17-Hydroxypregnenolone  D  Solvent front  86,060 38,970 39,900 3,490  Weight (Kg)  Specific a c t i v i t y (cpm/Kg)  94.7  366  84.1 64.8  2,945  83.1  1,036  2,542  450  908,770  The source of the sample subjected to thin layer chromatography i s depicted i n P i g . 11, footnote e. See Table XI.  42  TABLE XI  Substrate ^H-pregnenolone incubation: paper chromatography of compounds previously separated by t h i n layer chromatography 3.  Compound  Chromatographic solvent system  Radioactivity (cpm)  Weight (H-g)  • B5 B5 B5  34,640 8,000 6,960 6,100  94.7 65.0 48.4 38.3  366 123 144 159  T/PG T/PG T/PG  213,790 25,800 12,890 7,285  84.1 45.0 33.4 25.7  2,542 573 386 283  B5 B5  190,850 6,000 4,221  64.8 20.0 13.6  2,945 300, 310  T/PG T/PG T/PG T/PG  86,060 3,620 7,830 4,025 1,760  83.1 32.0 27.0 17.5 9.7  1,036 113 290 230 181  T/PG  39,900 8,510  Aldosterone  Corticosterone  Cortisone  Cortexolone  Contents of eluate '  C  17-IHydroxypregnenolone  Specific activity (cpm/Kg)  v b  b  See Table X. See Table XVI. Overflow and eluate containing carrier cortexolone from the f i r s t chromatogram were combined prior to the second chromatogram.  a b  c  TABLE X I I Substrate %-pregnenolone incubation:  Fraction  Radioactivity (cpm)  c r y s t a l l i z a t i o n s of androstenedione Weight (mg) Balance  Pool  19,475  15  UV  3  Specific a c t i v i t y (cpm/mg) C  10.13  Balance  13  UV  C  1,923  F i r s t crystals F i r s t mother l i q u o r  5,100 9,030  8.03 2.12  7.75 2.08  635 4,259  658 4,341  Second c r y s t a l s Second mother l i q u o r  2,470  6.76 1.33  6.74 0.95  365 1,416  396 1,684  Third crystals T h i r d mother l i q u o r  1,810  5.60 1.01  5.79 0.73  323 867  1,200  Fourth c r y s t a l s Fourth mother l i q u o r  1,170 705  3.67 1.91  3.42 1.50  319 369  342 470  F i f t h crystals F i f t h mother l i q u o r  745 373  2.32  2.24 0.90  321 316  333 414  Sixth crystals S i x t h mother l i q u o r  385 255  1.14  1.01 0.65  337 319  381 392  1,600  876  1.18  0.80  313  ^See F i g . 10, footnote b. ^Weight of s t e r o i d measured by weighing on a balance. h e i g h t of s t e r o i d 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:  Fraction Pool  Radioactivity (cpm) 12,110  F i r s t crystals F i r s t mother liquor  crystallizations of testosterone acetate > ,c a  Weight (rag) Balance UV  b  Specific a c t i v i t y (cpm/mg) Balance UV 1,183  10.15  970 9,560  6.49 2.74  6.51  Second crystals Second mother liquor  338 65$  3.39  3.40  100  2.72  3.53  242  259  Third crystals Third mother liquor  105 220  0.97 2.14  0.94 2.20  108 103  112 100  Pool  758  4.35  F i r s t crystals F i r s t mother liquor  305 488  3.03 1.09  2.96 0.83  101 448  103 588  Second crystals Second mother liquor  130 133  1.90  0.86  1.93 0.71  68 155  67 187  Third crystals Third mother liquor  55 34  0.93 0.56  60 49  59 61  d  0.92  0.69  2.67  149 3,489  149 3,581 99  174  fSee Table XII f o r explanation of abbreviations. h e i g h t and specific a c t i v i t i e s calculated for free testosterone. °See Table IX. ^Second and third mother liquors and third crystals combined.  TAB IE XIV Substrate %-pregnenolone incubation:  c r y s t a l l i z a t i o n s of dehydroepiandrosterone  Specific activity (cpm/mg)  Radioactivity (cpm)  Weight (mg)  Pool  213,600  15.19  14,062  F i r s t crystals F i r s t mother liquor  58,475 102,425  13.91 2.15  4,204 47,640  Second crystals Second mother liquor  22,600 35,450  10.48 3.23  2,156 10,975  Third crystals Third mother liquor  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  720 2,490  4.10 1.41  1,756 1,766  Fraction  F i f t h crystals F i f t h mother liquor a b c  See Table XII f o r explanation of abbreviations. S e e F i g . 12, footnote e. Weight determined by weighing on a n a l y t i c a l balance.  0  a  46  TABLE XV Substrate H-pregnenolone incubation: c r y s t a l l i z a t i o n s of 17-hydroxyprogesterone and progesterone 3.  Compound and fraction  Radioactivity  Specific a c t i v i t y  Weight (mg) Balance  UV  Balance  UV  cpm/mg 17-Hydroxyprogesterone  cpm  Pool  8,280  10.15  F i r s t crystals F i r s t mother l i q u o r  2,310 4,380  6.75 3.59  342 7.13 3.51 1,220 '  Second c r y s t a l s Second mother l i q u o r  710 938  3.84 2.78  3.98 2.70  185 337  178 347  Third c r y s t a l s Third mother l i q u o r  700 246  3.21 0.46  3.25 0.35  218 535  215 703  Fourth c r y s t a l s Fourth mother l i q u o r  443 203  1.93 0.93  2.02 , 0.55  230 218  219 369  F i f t h crystals F i f t h mother l i q u o r  269 234  1.24 0.84  1.33 0.44  217 279  202 532  Progesterone  0  816  dpm/mg  dpm  Pool  418,263  15.23  27,463  F i r s t crystals F i r s t mother l i q u o r  208,030 206,409  8.97 6.50  23,192 31,775  Second c r y s t a l s Second mother l i q u o r  185,470 21,850  7.81 0.59  23,747 37,034  Third c r y s t a l s Third mother l i q u o r  146,757 28,535  6.12 1.16  23,980 24,599  3. b  See Table XII f o r explanation of abbreviations. See Table IX.  324 1,248  TABLE XVI Substrate %-pregnenolone incubation: crystallizations  Compound  Fraction  Radioactivity (cpm)  of C o r t i s o l , aldosterone and cortisone Weight (mg) Balance  Cortisol  b  Aldosterone  0  a b c  0  Specific a c t i v i t y (cpm/mg) Balance  UV  2,181  Pool  32,720  15.00  F i r s t crystals F i r s t 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  Pool  6,100  12.00  10  10.49 1.58  Pool  4,221  10.00  F i r s t crystals F i r s t mother liquor  75 2,223  4.64 4.30  F i r s t crystals F i r s t mother liquor Cortisone  UV  Lost  See Table XII for explanation of abbreviations. See Table IX. See Table XI.  508 10.80 1.41  < 1  < 1  422 5.25 5.84  3  16 517  14 381  48  TABLE XVII  R e s u l t s o f i n c u b a t i o n s o f tumor t i s s u e w i t h subs t r a t e -^C-progesterone and s u b s t r a t e 3H-pregnenolone  Steroids isolated  P e r cent o f s u b s t r a t e r a d i o a c t i v i t y -Progesterone  17-Hydroxypregnenolone  -^-Pregnenolone ~  0 1.0  Progesterone 17-Hydroxyprogesterone  ~  0  Androstenedione  ~  0  Testosterone  ~  0  ~  0 0.05  .~  0 0.2  Dehydroepiandrosterone Cortisol  0  ~  0  Cortisone  ~  0  ^  0  Cortexolone  ~ 0  *~  0  Corticosterone  -~ 0  ~  0  Aldosterone  ~ 0  ~  0  TABLE X V I I I  Steroids excreted i n the u r i n e * a  Urine specimen  D  M-g/24 h r s  mg/24 h r s 17-KS 20° (6-15)  " DHEA < l  c  17-0HS  17-KGS  P'triol  25.6° (3-9)  62 (3-15)  3.1  c  Q  (<4)  P'diol  4.4 «  THS  Free F  25.4  d  7)  59.0 (20-90) U  15.5? 14.0° (<D 14.5  C  2  16°  62°  3  IT  58  c  17-KS, t o t a l 1 7 - k e t o s t e r o i d s ; 17-0HS, t o t a l 1 7 - h y d r o x y s t e r o i d s ; 17-KGS, t o t a l 17-ketogenic o i d s ; P.Jtriol, p r e g n a n e t r i o l ; P ' d i o l , pregnanediol: F, C o r t i s o l ; T, t e s t o s t e r o n e . ^Normal v a l u e s shown i n parentheses. °Assay performed b y B i o s c i e n c e L a b o r a t o r i e s (See T e x t ) . Assay performed b y B i o m e d i c a l Assay L a b o r a t o r i e s (See T e x t ) . a  < 1 < 1  ster-  50  F i g . 5 . Substrate -^C-progesterone incubation: flow sheet f o r ext r a c t i o n , 9 0 $ aqueous methanol-hexane p a r t i t i o n , column adsorption chromatography and i n i t i a l paper chromatography of f r a c t i o n s 5 - 7 of the column eluate  Incubation mixture. ( 7 , 5 8 4 , 0 0 0 cpm, 1 5 1 K g progesterone)  E t h y l acetate 4methylene chloride extraction iCombined extract (6,687,500  cpm)  Hexane-90% aqueous methanol p a r t i t i o n  f  Hexane (17,900  Aqueous methanol  cpm)  (6,988,500  cpm)  Column adsorption chromatography 3.  I  1  Fractions 5 - 7 Combined (4,888,500  cpm)  Fractions 8,9 Combined (608,000 cpm)  Other f r a c t i o n s (1,269,050 cpm)  L/PG, 5 7 hrs Testosterone  ^ Androstenedione Progesterone  (  c  0  i 0 - 4 cm ( 2 4 5 , 2 0 0 cpm)  4—14 cm (54,075 cpm)  1 4 - 4 4 cnr ( 1 4 9 , 9 0 0 cpm)  S e e Table VI. See F i g . 8 . Locations of standards chromatographed simultaneously. ^Eluates combined; see F i g . 7 , footnote a. See F i g . 6 .  a  b  c  e  Overflow (4,139,750  6  cpm)  51  F i g . 6. Substrate -^C- progesterone incubation: r e s o l u t i o n and p u r i f i c a t i o n o f progesterone Overflow (4,139,750 cpm) 3  L/PG, 4 hrs  Androstenedione  f  Progesterone (4,221,000 cpm)  0-6 cm (107,300 cpm) c  17-42 cm (70,200 cpm)  L/PG, 4 hrs  / 0-13 cm (555,725 cpm)  c  1 Progesterone (3,313,250 cpm, 64.3 K g , S.A. 51,528 cpm/w-g)  20-40 cm (181,550 cpm)  L/PG, 4 h r s  P  I  0-12 cm (178,000 cpm)  a  c  c  _  Progesterone (2,751,500 cpm, 54,7 ug, S.A. 50,302 cpm/ug)  —r  20-4.0 cm (49,625 cpm)  See F i g . 5, footnote e. L o c a t i o n of standard androstenedione chromatographed simultaneously. E l u a t e s 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 (1,045,000 cpm)  a  L/PG, 15 hrs Testosterone  I  r  Origin (451,590 cpm)  1-6 cm (258,610 cpm)  Androstenedione T 6-25 cm  (192,060 cpm)  Progesterone  25-40 cm -T-  & Overflow  (136,050 cpm)  Combined (710,200 cpm) Carrier testosterone (100 M-g) and carrier 17-hydroxyprogesterone (100 v*- g) added  L/PG, 15 hrs Androstenedione (23,840 cpm, 93.8 K g , S.A. 254 cpm/ M.g)  T/PG, 4 hrs  H  Origin (269,150 cpm) 4  Testosterone, 17-hydroxyprogesterone (118,100 cpm)  18-40 cm (187,000 cpm)  Ac 0,py 2  T/PG, 4 hrs  1  17-Hydroxypr ogesterone (28,960 cpm, 66.2 K g , S.A. 437 cpm/»^g)  Testosterone acetate (62,240 cpm)  fsee Fig. 5, footnote d,.and Fig. 6, footnote c. "Locations of standards chromatographed simultaneously. °See Table VII., See Fig. 8, footnote b. 5  0  F i g . 8. Substrate ^ - p r o g e s t e r o n e incubation: column chromatogram  paper chromatography of f r a c t i o n s 8 and 9 from the  3.  Fractions 8 and 9 from column chromatogram combined and o r i g i n eluate from paper chromatogram added (877,150 cpm) D  T/PG, 33 hrs Cortisol OCortisone  0  1  Origin (153,040 cpm)  1-6 cm (106,075 cpm)  r 0-9 cm  (15,960 cpm)  "1  6-19 cm (17,210 cpm)  B5, 5 hrs Cortisol Cortisone 0  Cortexolone  P  0  19-25 cm (11,735 cpm)  Corticosterone i 25^-33 cm (7,680 cpm) 0  f  33-40 cm (5,930 cpm)  F  Overflow (477,000 cpm) T/PG, 12 hrs  0  1 9-16 cm  (10,023 cpm)  16-32 cm  (13,686 cpm)  22-42 cm  (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) a b  See Table VI. See F i g . 7, footnote d. Locations of standards chromatographed simultaneously.  54  F i g . 9. Substrate %-pregnenolone incubation: flow sheet f o r extraction, 90% aqueous methanol-hexane p a r t i t i o n , 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 p a r t i t i o n  r  Hexane (419,620 cpm)  Aqueous methanol (14,490,000  cpm)  Carrier Cortisol 200 (utg added Column adsorption chromatography »  Fractions 5-7 ^Combined", cfrV  (11,587,500  cpm)  L/PG, 24 hrs  r  Origin  DHEA  (172,300 cpm)  C  15-14 cmd (295,800 cpm)  f  Fractions 8 & 9 Combined 15  (2,987,400  Androstenedione Pregnenolone Jgne  cpm)  0  Progesterone* 24I4O cm (672,000 cpm)  See Table VIII. See Fig. 11. °Locations of standards chromatographed simultaneously. See F i g . 12, footnote d. |See F i g . 10. See Table IX.  a b  d  I  Other fractions (2,110,870 cpm)  0  14-24 cme (8,822,000 cpm)  a  0verflow (1,091,000 cpm) f  55  Fig. 10. Substrate -'H-pregnenolone incubation: paper chromatography of eluates containing pregnenolone and androstenedione  Pregnenolone •+• androstenedione (8,822,000 cpm)  3  200 M.g carrier androstenedione added Ac 0„ py 2  L/PG, 17 hrs  1  0-15 cm (748,600 cpm)  r-—:  Androstenedione (202,250 cpm, 176 K g , S.A. 1,150 cpm/(Ag)  1  r  25-40 cm Overflow (135,250 cpm) Pregnenolone acetate (6,978,000 cpm)  Ac20,py L/PG, 16 hrs :  Jj  0-14 cm (22,830 cpm)  Androstenedione (23,450 cpm, 118 K g , SI A. 199 cpm/Kg)  1  L/PG, 16 hrs 1 b Androstened ione (19,475 cpm, 101 K g ,  S.A. 193 cpm/Kg)  See Fig. 9. 'See Table X I I .  1  22i40 cm Overflow (8,925 cpm) (73,325 cpm)  Fig. 11. Substrate %-pregnenolone incubation: chromatogram  paper chromatography of fractions 8 and 9 of column  3  Fractions 8 and 9 of column chromatogram combined (2,987,400 cpm)  Cortisol 17-Hydroxypregnen. olone  T/PG, 5 hrs DHEA  b  Origin  (715,000 cpm)  1  2-10 cm  Testosterone  (729,300 cpm)  |  (104,500 cpm)  0  (700,875 cpm, 307 Kg,  S.A. 2,283 cpm/Kg)  T/PG, 16 hrs  Origin  j  I  Cdrtisone  17-Hydroxypregnenolone"  c  L/PG, 48 hrs  J  Cortisol  (161,300 cpm, 182, M-g, S.A.  3-9 cme  (52,400 cpm)  9-40 cme  -tOverflow  (571,500 cpm)  886 cpm/Kg)  Testosterone*  B5, 3 hrs  0-4 cm 4-7 cm (74,230 cpm) (75,980 cpm) d  fl 0-6 cm (43,950 cpm)  22^42 cm (643,000 cpm)  Cortisol^  (49,050 cpm, 99 wg, S.A.  1  f -  7-12 cm (36,510 cpm)  DHEA }  12-16 cm" (57,300 cpm) 1  10^40 cm (52,750 cpm) e  495 cpm/Kg)  fsee Table 8 and Fig. 9, footnote b. Locations of standards chromatographed simultaneously. °See F i g . 12, footnote a. See F i g . 12, footnote b. d  Eluates combined, see Table X. % e e F i g . 12, footnote d. gSee Table IX.  •  16-40 cm -+• Overflow (243,475 cpm)  Fig. 12. Substrate -'H-pregnenolone incubation: paper chromatography of eluates containing carrier testosterone and dehydroepiandrosterone  Testosterone (700,875 cpm, 307 K g , 9,  S.A. 2,283 cpm/Kg)  L/PG, 48 hrs 0-3 cm (187,920 cpm)  Testosterone (229,700 cpm, 250 K g , S.A. 918 cpm/Kg)  7-12 cm (22,150 cpm)  Eluate of testosterone zone added 119 K g Carrier 17-hydroxyprogesterone added b  Ac 0,py L/PG, 4 hrs 2  1 Origin (16,730 cpm)  17-Hydroxyprogesterone* (32,200 cpm)  ~1 3-22 cm (24,480 cpm)  DHEA  16-42 cm -tOverflow (6,250 cpm)  Eluates of DHEA zones added L/PG, 18 hrs d  DHEA (213,600 cpm)  7  1  Testosterone acetate- (150,500 cpm, 230 K g , S.A. 654 cpm/Kg)  fsee F i g . 11, footnote c. £See F i g . 11, footnote d. Location of standard dehydroepiandrosterone chromatographed simultaneously. See F i g . 9, footnote d and F i g . U , footnote f . |See Table XIV. See Table IX. d  1  T  12-16 cm (10,500 cpm)  1  r 30-40 cm (59,125 c]  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 i s defined as that condition i n  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-scrot a l fusion and penile development. Wilkens (115) has divided male pseudohermaphroditism into two major groups:  (i) those with external genitalia  that simulate the male or are ambiguous and ( i i ) those with external genitalia that resemble the female.  In the Latter group, feminization usually  occurs at puberty and the syndrome has been termed "male pseudohermaphroditism with testicular feminization" (116). Subjects i n whom the external genitalia resemble the male or are ambiguous have been designated as presenting an "incomplete" form of testicular feminization (117); this term i s misleading because some patients with ambiguous genitalia w i l l v i r i l ize at puberty.  In this part of the thesis, patients with external geni-  t a l i a resembling the female who undergo feminization or i n whom only minimal changes occur at puberty w i l l be designated as exhibiting male pseudohermaphroditism with feminization; patients who v i r i l i z e at puberty w i l l be designated as exhibiting v i r i l i z i n g male pseudohermaphroditism.  59  (b) Steroid biosynthesis by the normal t e s t i s 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 biosynthesis i n v i t r o by the normal human t e s t i s ; 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 v i c t i m 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 ;  3,17^-  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 v i t r o of gonads obtained from patients with male pseudo-  hermaphroditism Results of studies i n v i t r o 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 f o r 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 v i t r o 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 r a d i o a c t i v i t y was found i n the "phenolic" f r a c t i o n s ( a l kaline washes of e t h y l 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 androstenedione, testosterone and dehydroepiandrosterone were formed, but radioactive progesterone and 17-hydroxyprogesterone were not detected. The authors suggested that androgen synthesis proceeded from pregnenolone v i a 17-hydroxypregnenolone and dehydroepiandrosterone rather than v i a progesterone and 17-hydroxyprogesterone. In t h i s 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.  i n t e r n a l g e n i t a l i a were found.  At a previous laparotomy no  The vagina ended i n a b l i n d pouch, the  l a b i a had a s c r o t a l appearance, the c l i t o r i s was markedly" enlarged and i n g u i n a l gonads were present. There was no inguinal, a x i l l a r y or f a c i a l h a i r , nor was there breast development. sent.  Leukocytic sex chromatin was ab-  The immediate cause f o r excision of the gonads was deepening of the  voice and masculinization of body b u i l d .  61  (b)  Gonads  1  p  The two gonads were collected i n ice.  Microscopic examination  of a small wedge from each t e s t i s 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  (35.43  x 10° cpm, 18 K g ) , 4- C-androstene14  dione (10,80 x 10^ cpm, 108 K g ) and 4-^C-progesterone ( 5 . 5 7 x 10° cpm, 106 ^g) were each added to a different reaction flask. lonic acid, lactone form, (0.05 mCi,5.85 s) m  action flasks.  2-^C-D, L-Meva-  was added to two other r e -  The "^-progesterone (The Radiochemical Centre, Amersham,  England) was purified prior to use by Celite column p a r t i t i o n chromatography using an iso-octane-90% aqueous methanol solvent system. The androstenedione (New England Nuclear Corporation, Boston, Mass.) was subjected to paper p a r t i t i o n 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 radioactive 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 homogenizer was used.  The homogenate was centriguged (650 x _y 20 minutes,  4°) and the supernatant (110 ml) was divided equally among f i v e 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 f o l l i c l e - s t i m u l a t i n g 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 f o r 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 f i v e 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 3H-pregnenolone and 1A "^'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 f o r 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 f r a c t i o n of each incubation was separated into "estrone-estradiol", " e s t r i o l " and neutral f r a c t i o n s . The " e s t r i o l " 4  fractions were not processed further.  The "estrone-estradiol" fractions  were separated into ketonic and non-ketonic portions and estrone and es4  tradiol-17/8 were purified by a series of c r y s t a l l i z a t i o n s . The neutral fractions were further resolved by paper chromatography i n the solvent systems ligroin-propylene g l y c o l , toluene-propy4  lene g l y c o l , benzene-formamide and Bush A.  Chromatography of steroids  containing the A. -3-ketone grouping was usually repeated i n the l a s t 4  system used u n t i l constant specific a c t i v i t y was attained . 4  Milligram  quantities of unlabeled c a r r i e r were then added to the eluates and cryst a l l i z a t i o n s were performed u n t i l radiochemical homogeneity was accomplished  4  or u n t i l the crystals contained negligible r a d i o a c t i v i t y . In a l l experiments unlabeled estrone and estradiol-17,* (9-11 mg  of each) were added immediately prior to hexane-aqueous methanol p a r t i tion.  The amounts of neutral steroid carriers added for crystallizations  are given as the weights of the pools i n 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-  tallization.  In the experiment with ^ C-progesterone as substrate, un4  labeled androstenedione (200 ^g) and testosterone (200 v*g) were added prior to hexane-aqueous methanol p a r t i t i o n and 16* -hydroxyprogesterone and 17-hydroxyprogesterone were added immediately prior to c r y s t a l l i z a t i o n . In the other experiments 200 K g of each of the following unlabeled steroids 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 l i q u 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, 14  C  -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 substrate converted. No demonstrable biosynthesis of estrone or estradiol17/9 from ^(^androstenedione occurred (Tables XXVII and XXVIII). The specific a c t i v i t y 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 transformed 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-hydroxyprogesterone. Synthesis of estrone and estradiol-17fl could not be demonstrated (Table XXXIII).  65  Only 2 . 6 % of the o r i g i n a l recovered unchanged.  C-progesterone r a d i o a c t i v i t y was  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 f r a c t i o n s ( F i g . 1 5 ) . (c)  Substrate ^H-pregnenolone incubation Testosterone  ( 3 . 9 4 y-g) was the major r a d i o a c t i v e metabolite r e 3  covered f o l l o w i n g i n c u b a t i o n of the t i s s u e with substrate The formation of r a d i o a c t i v e androstenedione ( 1 . 8 4 gesterone ( 0 . 0 2 Kg),  K g ) , 16«* -hydroxypro-  17-hydroxyprogesterone ( 0 . 0 1 Kg),  nenolone ( 0 . 0 0 2 v*-g) and dehydroepiandrosterone  H-pregnenolone.  (0.36  17-hydroxypreg-  yxg) was a l s o demon-  3  strated.  The transformation of  H-pregnenolone t o r a d i o a c t i v e progesterone  (Table XLI), estrone (Table XLIII) and e s t r a d i o l - 1 7 / 3 (Table XLIl) was  not  observed. (d)  Substrate -^•C-mevalonate incubations Transformation  of -^C-mevalonate t o s t e r o i d s was not observed  f o l l o w i n g incubations performed i n the presence or absence of Perganol (Table XXV and F i g . 2 1 ) . DISCUSSION  I t i s e s t a b l i s h e d that the gonads of p a t i e n t s with male pseudohermaphroditism and f e m i n i z a t i o n ( 1 1 7 , 1 2 0 - 1 2 5 , 1 2 7 - 1 3 1 , 1 3 3 - 1 3 6 ) or v i r i l i zation (114)  are capable of transforming a v a r i e t y of r a d i o a c t i v e sub-  s t r a t e s t o testosterone i n v i t r o (Tables XIX-XXIV).  The r e s u l t s reported  h e r e i n u t i l i z i n g the gonads of a p a t i e n t with v i r i l i z i n g male pseudohermaphroditism are i n accord with these f i n d i n g s ; r e l a t i v e l y l a r g e amounts of substrate progesterone,  pregnenolone and androstenedione were con-  v e r t e d t o testosterone (Table XLIV).  Green et a l . (114), who have r e -  ported studies i n v i t r o on the gonads of two p a t i e n t s w i t h 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 substrate ^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 v i a 17-hydroxypregnenolone and dehydroepiandrosterone (Fig. 4).  An alternate explanation of the findings of  Green et a l . (114) i s that radioactive progesterone and 17-hydroxyprogesterone were formed and then rapidly metabolized.  The p l a u s i b i l i t y of  t h i s 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$) and 17hydroxyprogesterone (2.8$) 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-hydroxyprogesterone incurred during the extraction, resolution and p u r i f i c a t i o n procedures (Table XXV, Figs. 15 and 16). Fallowing the incubation with ^Epregnenolone as substrate (Table XLIV), 10.2 per cent of the substrate radioactivity was recovered as androstenedione and 21.9 per cent as testosterone.  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 radioa c t i v i t y 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 e f f i c i e n t l y metabolized by the tissue and  67  the data are i n accord with the concept t h a t the metabolic a c t i v i t y of a compound cannot n e c e s s a r i l y be i n f e r r e d from i t s r e l a t i v e abundance, part i c u l a r l y when the compound i s an intermediate i n a complex metabolic pathway.  The r e s u l t s suggest that the t i s s u e was capable of transforming  pregnenolone to testosterone both v i a 17-hydroxypregnenolone and dehydroepiandrosterone and v i a progesterone, 17-hydroxyprogesterone and androstenedione ( F i g . 4 ) .  The data do not permit assessment of the r e l a t i v e  importance of the two major b i o s y n t h e t i c pathways because the s i z e s 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 t o 16<*  -hydroxypro-  gesterone has been demonstrated i n v i t r o using human ovarian (137,138), p l a c e n t a l (139), a d r e n a l (140,141,142,143,144) and t e s t i c u l a r (123-125, 128,131,145-147) t i s s u e 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 w i t h the t e s t e s of some p a t i e n t s with male pseudohermaphroditism and f e m i n i z a t i o n (Table XLX); t h i s compound has not been reported as a product of incubations of gonadal t i s s u e from these p a t i e n t s with substrate pregnenolone (Table XX) nor has i t been observed p r e v i o u s l y 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  Adadevoil and Engel have suggested (144) that  i n the t e s t i s i s unknown. l£><  -hydroxylation of pro-  gesterone i n the a d r e n a l represents a regulatory process which serves to withdraw substrate t h a t would otherwise undergo 17-hydroxylation and hydroxylation.  C a l v i n and Lieberman (150) ad^iinistered r a d i o a c t i v e  21-  68  16<*• -hydroxyprogesterone to a normal male subject and found 3°^hydroxy17-iso-pregnan-20-one as a metabolite i n the urine; the evidence a  suggested  A"^ intermediate. The conversion of substrate mevalonate t o steroids i n v i t r o hf,  preparations of endocrine tissues that form s t e r o i d hormones has been observed 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 f a i l u r e to obtain s i g n i f i c a n t conversions of substrate mevalonate i n rat t e s t i s preparations i n v o l v i n g unbroken c e l l s was due t o the i n a b i l i t y of mevalonate to penetrate i n t a c t c e l l s .  These workers a l s o pos-  tulated that the rapid destruction of adenosine triphosphate by a microsomal adenosine triphosphatase was responsible f o r 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 c e l l - f r e e homogenates of r a t testes and a l s o reported that the microsomal f r a c t i o n contained a pyrophosphate pyrophosphohydrolase that catalyzed the hydrolysis of pyrophosphorylated  intermediates of s t e r o l biosynthesis.  The conversion of substrate mevalonate t o squalene was enhanced by the presence of an adenosine triphosphate-generating system (pyruvate kinase, phosphoenolpyruvate and adenosine diphosphate).  In the experiments r e -  ported i n t h i s section of the t h e s i s , an adenosine triphosphate-generating system was not included i n the incubation media and the f a i l u r e t o demonstrate transformation of substrate •^C-mevalonate to steroids may be due to the presence of adenosine triphosphatase and pyrophosphate pyrophosphohydrolase a c t i v i t y i n the c e l l - f r e e homogenate. The e t i o l o g y of the syndrome of male pseudohermaphroditism i s  69  not understood.  The following hypotheses have been suggested:  deficient  androgen synthesis by the t e s t i s (159), excessive peripheral conversion of androgens to estrogens, rapid metabolism of c i r c u l a t i n g androgens to b i o l o g i c a l l y inactive compounds, i n a c t i v a t i o n of androgens by antagonists (160)  and diminished target-organ s e n s i t i v i t y 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 d i f f e r e n t i a t i o n  of  the external g e n i t a l i a i s complete by the twelfth week of gestation (163). Experiments on animals have shown that the development of the male ext e r n a l g e n i t a l i a i s dependent on the presence of the f e t a l t e s t i s ; c a s t r a t i o n of the male fetus i n utero leads to female urogenital sinus development (164).  Androgenic steroids do not appear to i n h i b 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 p r e sence of the M u l l e r i a n i n h i b i t o r y f a c t o r (165)  that prevents the formation  of the uterus i n the male. The results of a number of studies provide substantial evidence that male pseudohermaphroditism i s usually not associated with d e f i c i e n t androgen production or excessive estrogen production.  Testosterone con-  centrations i n peripheral venous plasma p r i o r to c a s t r a t i o n have f r e quently been found to be i n the range of normal males (120,121,133, 166-169);  lower values were observed postoperatively when plasma t e s -  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 f a t e have a l s o been reported (117,133); these values declined following  70  castration.  The value f o r the plasma testosterone concentration was r e -  ported to be i n the range of normal females i n one subject (117) and between the upper l i m i t f o r normal females and the lower l i m i t f o r normal males i n another subject (130).  Two groups of investigators have report-  ed that the values f o r 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 f o r normal males (114,120, 133,170) and normal females (114,129,171). In one study (117), dehydroepiandrosterone and androstenedione were isolated from the gonadal tissue; testosterone and estrogens were not found. The values f o r the excretion rate of estrogens v i a the kidney prior t o castration have usually been i n the overlapping region of low normal for nonpregnant females and high normal f o r males and postoperative values were frequently lower than preoperative values (121,123,128,129,132,134,167,172-176). Estrone and estradiol-17/5 concentrations i n peripheral venous plasma were observed to be i n 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 pseudohermaphroditism. The metabolic clearance rate and the h a l f - l i f e of testosterone i n plasma were reported to be normal (166-169). Following administration of "^C-testosterone to one patient (167), the pattern of radioactive metabolites excreted i n 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 i n urine during the administration of large doses of testosterone. At present, the evidence favors the hypothesis that a defect i n target-organ s e n s i t i v i t y 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 i n 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 i n search of an explanation for a target-organ defect i n patients with the syndrome of male pseudohermaphroditism and feminization have studied the metabolism of testosterone i n peripheral tissues of these patients. I t 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 a f t e r incubation of r a t p r o s t a t i c tissue with testosterone (183). Furthermore, 17 £ -hydroxy-5<* -androstane steroids have been observed to be more potent androgens i n c e r t a i n tests than testosterone i t s e l f  185).  (184,  These findings have l e d 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 of these patients when compared to normal subjects.  i n the urine  Northcutt and cowork-  ers (188) incubated portions of abdominal skin, vas deferens and epididymus  and pubic h a i r f o l l i c l e s obtained from normal subjects and from patients  with male pseudohermaphroditism and feminization with radioactive testosterone as substrate and observed that the tissues from the patients transformed less testosterone to 17fi -hydroxy-5«< -androstan-3-one than did corresponding tissue from the normal subjects.  Both groups of investigators  suggested that the f a i l u r e of patients with the syndrome of male pseudohermaphroditism and feminization to respond to testosterone may a defect i n testosterone 5  0t  -reductase  be due to  a c t i v i t y 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 t h i s 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 androgens i n v i t r o from substrate H-C-progesterone and substrate % - p r e g 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 i t s 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 i n vitro was investigated i n testes obtained  during puberty from a patient with v i r i l i z i n g male pseudohermaphroditism. 3  Cell-free homogenates of gonadal tissue efficiently metabolized -'H-pregnenolone, "^C-progesterone and "^C-androstenedione to testosterone; formation 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 i n patients with male pseudohermaphroditism and feminization at puberty.  Green and coworkers (114), who  have reported the only other detailed study of subjects with v i r i l i z i n g 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 (iii)  17-hydroxyprogesterone. The failure 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 tran-  sient effect during embryonic development. Alternatively, the sensitivity to androgenic hormones may be subnormal i n certain tissues and normal i n other tissues of patients with v i r i l i z i n g male pseudohermaphroditism.  TABLE XIX Products identified following incubations of gonads from patients with male pseudohermaphroditism and feminization with radioactive progesterone as substrate Product 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  D  X X X  X X X  X X X  6  A  B  X X X X  X X X X X  a  X  xd xd xd  X  X X X  X X  X  X  X  X  H  I  J  X X X X X  X X X X  X X  c  K  X  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 qualit i v e l y the same i n both experiments. Gonads from two patients were incubated i n two separate experiments. Testosterone was formed i n both experiments; the i d e n t i f i c a t i o n of androstenedione was not established i n one experiment. Identification tentative. A , ( l 2 l ) ; B,(122); C,(l23); D,(l24); E,(l25); F, (127); G,(128); H,(117); 1,(129); J,(l30); K,(131). c  e  G  Reference E F (p  75  TABLE XX Products identified following incubations of gonads from patients with male pseudohermaphroditism and feniinization with substrate pregnenolone Reference  Product A Progesterone Andros tenedione De hydroepiandrosterone Testosterone 17-Hydroxyprogesterone Estrone Estradiol-17 £ 17-Hydroxypregnenolone Androstenediol Pregn-5-ene-3 P ,17,20 * - t r i o l 20* -Hydroxypregn-4-ene-3-one 19-Hydroxyandrostenedione Pregnenolone sulfate Dehydroepiandrosterone sulfate  X X X X X X X  a  6  B  c  D>  E  pC  G  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  d  x xd d  X X X  X  X  A  x x x  Gonads from three patients were incubated i n three separate experiments. 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. Gonads from two patients were incubated i n two separate experiments. The results were qualitatively the same i n botheexperiments. Non-radioactive pregnenolone used as substrate, dldentification tentative. % ( U 2 2 ) B,(125); C,(l27); D,(l28). E,(117); F,(l32) G,(129). a  b  c  ;  ;  76  TABLE 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 and radioactive androstenedione as substrates  Substrate  Reference  Product  A B O D E Testosterone  Androstenedione  Estradiol-17/S Estrone Androstenedione 19-Hydroxytestosterone 6/6 -Hydroxytestosterone  Testosterone 19-Hydroxytestosterone 19-Hydroxyandrostenedione 6l* -Hydroxytestosterone 6p -Hydroxyandrostenedione Estrone Estradiol-17/? Equilinin  c  F G H 3,  x X X  X  X x x x  x x x  x xb b  x x b  13  5>  X X  X X  X" X" 1  Gonads from two patients were incubated i n two separate experiments. The results were q u a l i t a t i v e l y the same i n both experiments, i d e n t i f i c a t i o n tentative. C  A,(121); B,(l23)j C,(U24); D,(l25); E,(U27); F,(133); G,(128); H,(117).  77  TABLE 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 s u l fate as substrates  Substrate  Product  , A  Dehydroepiandrosterone  Dehydroepiandrosterone sulfate  Androstenedione Testosterone Estrone Estradiol-17£ Androstenediol 19-Hydroxyandrostenedione DHEA-sulfate Androstenediol sulfate  x x x x  a  Reference  b  B  C  D  E  F  x x  x x  x x  x x x x  x x  x  Androstenedione DHEA Testosterone Androstenediol Androstenediol sulfate  x x x x x  x  x x x x x  Gonads from three patients were incubated i n three separate experiments. Estrone was formed i n one experiment, the other products -^listed were formed i n a l l three experiments. a  'A, (122); B,(125); C,(l34); D,(l27); E , ( l l 7 ) , F,(l35).  78  \  TABLE 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 17hydroxypregnenolone as substrates j N  Substrate Acetate  Reference  0  Product A x x x x x x x  Pregnenolone Progesterone 17-Hydroxyprogesterone Dehydroepiandrosterone Androstenedione Testosterone Estradiol-17p Estrone Cholesterol Cholesterol sulfate  B  C x  IT  E  x x x x  17-Hydroxy- None progesteroneiTestosterone 17,20 - -Dihydroxypregn-4-en3-one 17,20^ -Dihydroxypregn-4-en3-one Androstenedione  x  x  ot  17-Hydroxy- Dehydroepiandrosterone pregnenolone Pregn-5-ene-3P ,17,20* - t r i o l Androstenedione Testosterone 17-Hydroxyprogesterone  x x x  D  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. "Identification tentative. A,(120); B,(123); C,(l34); D,(228) E,(l36).  C  5  TABLE 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 ^-hydroxypregnenolone as substrates  Substrate  Product  H-Pregnenolone D  H-17-Hydroxypregnenolone  3  a  0  Androstenedione Dehydroepiandrosterone Testosterone Progesterone 17-Hydroxyprogesterone Androstenedione Dehydroepiandrosterone Testosterone 17-Hydroxyprogesterone  Reference ' 114. 1.0 j*Gi per incubation flask. 100 K-Ci per incubation flask.  b  c  cpm/g of tissue incubated Case l 464,348 40,935 67,478 0 0 202,428 153,325 61,725 0  a  Case 2  d  129,868 107,763 29,860 0  TABLE XXV Per cent of substrate radioactivity present i n fractions following extraction, partition and separation procedures 3  Per cent of substrate radioactivity^ Procedure  Fraction Androstenedione  Extraction of incubation medium  Organic extract  Hexane-aqueous methanol p a r t i tion  Hexane  P a r t i t i o n of aqueous methanol fraction  Neutral "Estrone-Estradiol" "Estriol"  Aq. methanol  Girard separation Ketonic of the "estrone- Non-ketonic estradiol" fraction a  See the Text f o r details of the procedures  Radioactive substrate ProgestMevalonate Pregnenerone (control) olone  Mevalonate (Pergonal)  101  86  22  1  2  £ 1  97  99  86  20  15  96 * 1 < 1  70 6 12  65 6 11  £ 1 < 1 1  < 1 < 1 1  <1 < 1  3  3 1  96 < 1  C  22 t  1  TABLE XXVI Substrate -^C-androstenedione incubation: crystallizations of testosterone -' 3  Fraction  Radioactivity (cpm)  Weight (mg) Balance  uv  Specific a c t i v i t y (cpm/mg) Balance  UV  Pool  2,136,875  14.78  144,579  F i r s t crystals F i r s t mother liquor  1,286,500 767,500  9.69 5.14  132,767 149,319  Second crystals Second mother liquor  1,091,000 127,800  8.91 1.43  122,446 89,371  Third crystals Third mother liquor  1,027,250 122,825  7.90 1.11  130,031 110,653  Fourth crystals Fourth mother liquor  932,250 67,600  7.46 0.59  7.19  124,966 114,576  129,659  F i f t h crystals F i f t h mother liquor  592,700 243,750  5.33 2.30  5.17 2.00  111,201 105,978  114,642 121,875  Sixth crystals Sixth mother liquor  217,525 381,500  2.01 3.59  1.75 3.19  108,221 106,267  124,300 119,592  See Table XII for explanation of abbreviations 'See F i g . 13, footnote c.  TABLE XXVII Substrate ^C-^drostenedione incubation: T" 7"! ft""*""  Radioactivity (cpm)  crystallizations of estradiol-17,5 Weight (mg)  3  Specific a c t i v i t y (cpm/mg)  Pool  16,850  4.75  3,547  F i r s t crystals F i r s t mother liquor  5,350 12,350  2.60 1.95  2,058 6,333  1.94 0.44  711 8,155  Second crystals Second mother liquor  1,380 3,588  Third crystals Third mother liquor  489  1.56  688  0.30  Fourth crystals Fourth mother liquor  311 184  1.36 0.24  229 767  F i f t h crystals F i f t h mother liquor  194 68  1.19 0.17  163 400  iia  Weight determined by weighing on an analytical balance  313  2,293  TABLE XXVIII Substrate ^G-androstenedione incubation:  Fraction  Radioactivity (cpm)  c r y s t a l l i z a t i o n s of estrone  Weight (mg)  3  Specific activity (cpm/mg)  Pool  98,350  4.10  23,988  F i r s t crystals F i r s t mother l i q u o r  4,700 87,150  3.16  0.72  1,487 121,041  Second c r y s t a l s Second mother liquor  655 4,065  2.26 0.87  290 4,672  Third crystals Third mother l i q u o r  314 359  1.67 0.41  188  Fourth crystals Fourth mother liquor  190 89  1.32 0.30  144 297  F i f t h crystals F i f t h mother liquor  122 75  0.89 0.44  137 170  h e i g h t determined by weighing on an a n a l y t i c a l balance  876  TABLE XXIX-  1  Substrate -^C-progesterone incubation:  Fraction  c r y s t a l l i z a t i o n s of 16*- -hydroxyprogesterone'  Radioactivity (cpm)  Weight (mg) Balance  UV  Specific activity (cpm/mg) Balance  UV  £ool  376,000  10.38  F i r s t crystals F i r s t mother liquor  165,575 182,230  8.11 2.21  8.07 2.09  20,416 82,457  20,517 87,191  Second c r y s t a l s Second mother liquor  111,425 54,510  5.87 2.22  5.98 2.08  19,982 24,554  18,633 26,207  Third crystals Third mother liquor  69,800 36,670  3.82 2.10  3.78 2.10  18,272 17,462  18,466 17,462  Fourth crystals Fourth mother liquor  45,390 20,195  2.35 1.15  2.53 1,18  19,315 17,561  17,941 17,114  a b  See Table XII f o r explanation of abbreviations. S e e F i g . 15, footnote c .  36,224  TABLE XXX Substrate ^C-progesterone incubation: crystallizations ofandrostenedione » a  c  Fraction  Radioactivity (cpm)  Weight (mg) Balance UV  Specific a c t i v i t y ?J (cpm/mg) Balance UV  Pool  467,000  15.00  F i r s t crystals F i r s t mother liquor  297,325 173,050  9.74  10.10  30,526  29,438  5.47  5.36  31,636  32,285  Second crystals Second mother liquor  163,525 119,625  5.65  55U96  4.13  3.95  28,942 28,965  27,437 30,285  Third crystals Third mother liquor  49,050 109,400  28,517 28,416  27,250  See Table X I I for explanation of abbreviations See F i g . 1 5 , footnote d.  a b  31,133  1.72  1.80  3.85  3.90  28,051  TABLE XXXI Substrate -^C-progesterone incubation:  Fraction  Radioactivity (cpm)  crystallizations of testosterone acetate^, ' 0  Weight (mg) Balance  UV  Specific a c t i v i t y (cpm/mg) Balance  UV  Pool  636,700  17.45  F i r s t crystals F i r s t mother liquor  349,200 259,200  11.10 6.70  11.85 6.41  31,459 38,687  29,468 40,437  Second crystals Second mother liquor  283,275 52,430  8.94 1.62  9.18 1.71  31,686 32,364  30,858 30,661  Third crystals Third mother liquor  221,575 43,890  7.56 1.43  7.65 1.31  29,309 30,692  28,964 33,504  36,487  fSee Table X I I f o r explanation of abbreviations. "Weights and specific a c t i v i t i e s calculated for free testosterone See F i g . 16, footnote b. c  TABLE XXXII Substrate 14  Fraction  c  -progesterone incubation: crystallizations of 17-hydroxyprogesterone Radioactivity Weight Specific a c t i v i t y (cpm) (mg) (cpm/mg) Balance Balance UV UV 14,608  Pool  219,125  15/.00  F i r s t crystals F i r s t mother liquor  104,125  11.04 4.30  11.80  127,460  4.19  9,432 29,642  8,824 30,420  Second crystals Second mother liquor  77,110 26,240  8.72 2.42  9.01 2.12  8,843 10,843  8,558 12,377  Third crystals Third mother liquor  50,200 20,720  5.97 2.32  6.26 2.42  8,409  8,019 8,562  Fourth crystals Fourth mother liquor  31,100 13,655  3.97 1.76  4.14  7,834  7,758  7,512 7,462  F i f t h crystals F i f t h mother liquor  18,770 10,675  2.36  2.35 1.24  7,953 8,087  7,987 8,609  See Table X I I for explanation of abbreviations See F i g . 16, footnote c.  a b  1.32  1.83  8,931  TABLE XXXIII Substrate  14  C  -progesterone incubation: c r y s t a l l i z a t i o n s of estrone and estradiol 17/S Radioactivity Weight Specific a c t i v i t y (cpm) (mg) (cpm/mg) -  a  Fraction Estrone Pool  172,825  7.97  21,684  First crystals F i r s t mother l i q u o r  3,243 169,975  6.37 1.89  509 89,934  135  2.40  1,934  1.64  56 1,156  9,625  9.21  1,045  665 7,465  4.30 4.78  156 1,562  243  2.87  85  465  1.04  4447  Second c r y s t a l s Second mother l i q u o r Estradiol-17^ Pool First crystals F i r s t mother l i q u o r Second c r y s t a l s Second mother l i q u o r  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: eluates containing carrier steroids  Compound  Chromatographic solvent system  DHEA  a  L/PG L/EG  Androstenedione  Progesterone  See See °See See See  a  b  d e  Fig. Fig. Fig. Fig. Fig.  Weight (Kg)  Specific activity  (cpm/K-g)  1,887,000 1,276,425 921,875 17,965 21,246  134  19,917  L/PG L/PG L/PG  6,612,500 5,930,875 5,292,000 5,210,000  342 348  46,567 40,073 37,532 38,592  T/PG T/PG  86,750 57,100 54,625  346  15  17, footnote 20, footnote 19, footnote 19, footnote 17, footnote  Radioactivity (cpm)  148 121 127  L/PG L/PG L/PG  c  17-Hydroxyprogesterone  Contents of eluate  2,658,850 2,570,750 2,423,850  13  Testosterone acetate  further paper chromatography of  L/PG L/PG L/PG L/PG c b b b f  2,270,550  688,600  428,175 332,550 274,950 249,625  and Table XXXVII. and Table XXXVIII. and Table XXXIX. and Table XL. and Table XLI.  341  135  336  136  221 145 123 116 106  19,085  594 420 402 3,316  2,953 2,704 2,370 2,355  TABLE XXXV Substrate ^H-pregnenolone incubation:  c r y s t a l l i z a t i o n s of 17-hydroxypregnenolone  Radioactivity (cpm)  Weight (mg)  108,040  15.00  7,203  10,800 84,560  12.69 2.43  851 34,798  Second c r y s t a l s Second mother l i q u o r  3,790  8,425  10.38 2.08  365 4,050  Third c r y s t a l s Third mother liquor  2,030 920  7.58 1.61  268 571  Fourth crystals Fourth mother liquor  1,260 498  6.16 1.31  205 380  F i f t h crystals F i f t h mother liquor  1,020 272  4.90  1.27  208 214  Sixth crystals Sixth mother liquor  801 154  3.99 0.74  201 208  Fraction Pool F i r s t crystals F i r s t mother liquor  See F i g . 18, footnote c. "Weight determined by weighing on an a n a l y t i c a l balance.  0  Specific a c t i v i t y (cpm/mg)  a  TABLE XXXVI Substrate ^H-pregnenolone incubation: crystallizations of lo**--hydroxyprogesterone > a  Radioactivity (cpm)  Fraction  Weight (mg) Balance  i  UV  Specific a c t i v i t y (cpm/mg) Balance  UV  Pool  304,900  8.87  F i r s t crystals F i r s t mother liquor  61,030 194,010  6.01 3.36  10,155  2.63  57,741  11,302 73,768  22,025  34,374  5.40  Second crystals Second mother liquor  37,785  3.13 2.75  3.03 2.65  7,037 13.740  7,269 14,258  Third crystals Third mother liquor  8,063  1.25 1.88  1.29  12,295  Fourth crystals Fourth mother liquor  6,180 1,205  6.24 1.15  F i f t h crystals F i f t h mother liquor  3,730 2,475  3.70 2.41  a b c  c  6,450  1.59  6,540  6,250 7,733  6.27  990 1,048  986 1,148  1,008  1,051 1,053  1.05 3.55 2.35  1,027  See Table X I I for explanation of abbreviations. See F i g . 18, footnote d. 6.13 *wvg of additional unlabeled 16*> -hydroxyprogesterone added prior to fourth crystallization.  TABLE XXXVII Substrate %-pregnenolone incubation:  crystallizations of dehydroepiandrosterone  Radioactivity (cpm)  Weight (mg)  Pool  921.875  20.30  45,413  F i r s t crystals F i r s t mother liquor  650,000 258,000  15.72 4.89  41,348 52,761  Second crystals Second mother liquor  532,000 68,500  14.30 1.02  37,203 67,157  Third crystals Third mother liquor  456,000 46,400  13.04 1.12  34,969 41,429  Fourth crystals Fourth mother liquor  437,000 14,600  12.38 0.41  35,299 35,561  F i f t h crystals F i f t h mother liquor  392,000 34,000  11.26 1.01  35,813 33,663  Fraction  See Table XXXIV. ^Weight determined by weighing on an analytical balance. a  b  Specific a c t i v i t y (cpm/mg)  3  TABLE XXXVIII o  a  Substrate •'H-pregnenolone incubation:  Fraction  c r y s t a l l i z a t i o n s of androstenedione '  Radioactivity (cpm)  Weight (mg) Balance  UV  Specific a c t i v i t y (cpm/mg) Balance  UV  Pool  2,270,550  20.20  F i r s t crystals F i r s t mother l i q u o r  1,344,000 863,775  12.67 7.62  13.15 7.96  106,077 113,356  102,205 108,514  Second c r y s t a l s Second mother l i q u o r  1,028,850 291,850  9.55 2.90  10.13 2.85  107,733 100,638  101,565 102,404  748,175 224,730  7.20 2.23  7.43 2.20  103,913 100,776  100,697 102,150  Third crystals T h i r d mother l i q u o r a b  S e e Table X I I f o r explanation of abbreviations See Table XXXIV.  112,403  TABLE XXXIX Substrate %-pregnenolone incubation:  Fraction  Radioactivity (cpm)  crystallizations of testosterone acetate > , a  Weight (mg) Balance  UV  b  Specific a c t i v i t y (cpm/mg) Balance  UV  Pool  5,210,000  12.63  F i r s t crystals F i r s t mother liquor  3,782,700 1,341,650  10.15 2.77  8.63 2.51  372,680 484,350  438,320 534,522  Second crystals Second mother liquor  2,885,000 666,250  8.12 1.79  8.60 1.81  355,296 372,207  335,465 368,094  Third crystals Third mother liquor  2,350,950 634,520  6.33 1.76  6.26 1.75  371,398 360,522  375,551 362,583  412,509  See Table X I I f o r explanation of abbreviations. See Table XXXIV. cWeights and specific a c t i v i t i e s calculated f o r free testosterone.  a  b  c  TABLE XL Substrate 3H-pregnenolone incubation:  Fraction  c r y s t a l l i z a t i o n s of 17-hydroxyprogesterone'  Radioactivity (cpm)  Weight (mg) Balance  UV  Specific activity (cpm/mg) Balance  UV  5,408  Pool  54,625  10.10  F i r s t crystals F i r s t mother l i q u o r  14,400 29,890  6.87 3.09  7.60 3.04  2,096 9,673  1,895 9,832  Second c r y s t a l s Second mother l i q u o r  8,080 5,290  4.38 2.28  4.85 2.48  1,845 2,320  1,666 2,133  Third crystals Third mother l i q u o r  5,165 2,023  3.09 1.18  3.06 1.12  1,672 1,714  1,688 1,806  Fourth c r y s t a l s Fourth mother l i q u o r  2,855 2,168  1.73 1.33  1.74 1.32  1,650 1,630  1,6U 1,642  F i f t h crystals F i f t h mother l i q u o r  1,508 1,083  0.93 0.66  0.97 0.71  1,622 1,641  1,555 1,525  a b  See Table X I I f o r explanation of abbreviations. S e e Table XXXIV.  TABLE XLI Substrate %-pregnenolone Incubation:  Fraction  Pool F i r s t crystals F i r s t mother liquor Second crystals Second mother liquor Third crystals Third mother liquor Fourth crystals Fourth mother l i q u o r F i f t h crystals F i f t h mother l i q u o r Sixth crystals Sixth mother liquor Seventh crystals Seventh mother l i q u o r Eighth crystals Eighth mother liquor Ninth crystals Ninth mother liquor Tenth crystals Tenth mother liquor Eleventh crystals Eleventh mother liquor  c r y s t a l l i z a t i o n s of progesterone ' a  Radioactivity (cpm)  -  Balance  UV  10.06 9.36 1.06 7.75 1.69 6.91 0.57 5.98 0.80 5.21 1.01 4.64 0.46 4.08 0.32 3.29 0.60 2.43 0.58 1.63 0.72 1.27 0.33  9.37 0.75 7.70 1.39 6.87 0.48 6.22 0.74 5.15 0.83 4.64 0.32 4.22 0.59 3.28 0.67 2.45 0.63 1.62 0.67 1.17 0.24  249,625 148,970 77,507 99,320 47,555 73,520 19,460 53,770 18,485 36,290 12,475 28,045 6,140 22,010 4,528 8,875 11,160 5,400 2,870 3,368 1,860 2,178 610  fSee Table XII f o r explanation of abbreviations See Table XXXIV. b  Weight (mg)  Specific activity (cpm/mg) Balance 24,814 15,916 73,119 12,815 28,339 10,640 34,340 8,992 23,106 6,965 32,351 6,044 33,348 5,395 34,150 2,698 18,600 2,222 '4,948 2,066 2,583 1,715 1,848  UV  15,898 103,343 32,899 34,232 10,702 40,542 8,645 24,980 7,047 15,030 6,044 19,188 5,216 7,675 2,706 16,656 2,204 4,556 2,079 2,776 1,862 2,542  TABLE XLII Substrate 3H-pregnenolone incubation:  Fraction  Radioactivity (cpm)  c r y s t a l l i z a t i o n s of estradiol-17/?  Weight (mg)  3  Specific activity (cpm/mg)  Pool  436,925  10.42  41,931  F i r s t crystals F i r s t mother liquor  119,450 278,620  6.60 3.44  18,098  Second crystals Second mother liquor  49,665 48,125  6.08  0.39  123,397  Third crystals Third mother liquor  -32,460 19,125  5.14 0.84  6,315 22,768  Fourth crystals Fourth mother l i q u o r  20,978 9,275  4.66 0.60  4,502 15,200  F i f t h crystals F i f t h mother liquor  13,375 5,623  3.53 0.82  3,879 6,857  Sixth crystals Sixth mother liquor  2,808  3.01 0.47  2,794 5,974  2.20 0.46  940 11,756  Seventh crystals Seventh mother l i q u o r  a  8,410 2,068 5,408  Weight determined by weighing on an a n a l y t i c a l balance.  80,994 8,169  TABLE XL1II Substrate %-pregnenolone incubation:  Fraction  Radioactivity (cpm)  c r y s t a l l i z a t i o n s of estrone  Weight (mg)  3  Specific activity (cpm/mg)  1,030,150  9.08  113,453  32,925 839,600  6.46 2.65  5,097 316,830  6,015 26,345  5.40 0.97  1,114 27,160  Third crystals T h i r d mother l i q u o r  1,850 4,615  3.76 1.62  492 2,849  Fourth crystals F o u r t h mother l i q u o r  613 1,007  2.49 1.18  246 853  316 367  1.76 0.45  180 816  Pool First crystals F i r s t mother l i q u o r Second c r y s t a l s Second mother l i q u o r  F i f t h crystals F i f t h mother l i q u o r  h e i g h t s determined by weighing 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 pseudohermaphroditism Substrates  Products •^-C-Androstenedione % Substrate radioactivity Testosterone Androstenedione 16°C -Hydroxyprogesterone 17-Hydroxyprogesterone Progesterone 17-Hydroxypregnenolone Dehydroepiandrosterone Estrone Estradiol-17/3  16.4  ~ o  Kg Substrate converted 17.7  ~ 0  14c -Progesterone % Substrate radioactivity  Kg Substrate converted  ^H-Pregnenolone % Substrate radioactivity  Kg Substrate converted  12.9  13.7  21.9  3.94  11.1  11.6  10.2  1.84  3.4  3.6  0.1  0.02  2.8  3.0  0.07 ~ 0  0.01 ~ 0  ~ o ~0  - 0 ~o  0.01  0.002  2.0 ~ 0 ~ 0  0.36 ~ 0 ~ o  100  Fig. 13. Substrate "^C-androstenedione incubation: paper chromatography of the neutral fraction  Neutral f r a c t i o n (11,427,250  cpm)  L/PG, 70 hrs Androstenedione 0^6ocm  (296,250 cpm)  1  Testosterone  (4,255,000 cpm, 31.3 K g , S.A. 135,942 cpm/Kg)  3  h  Overflow (4,869,000 cpm)  Bush A, 2 4 hrs  0-7 cm (35,450 cpm)  ~7T~J—  21-40 cm (159,751 cpm)  Testosterone  (3,172,000 cpm)  Bush A, 24 hrs  0-9 cm (433,250 cpm)  1  Testosterone  (2,136,875 cpm, 19.4 »* g, S.A. 110,148 cpm/Kg) c  23-4-0 cm-»-  Overflow  (367,900 cpm)  ^Location of standard androstenedione chromatographed simultaneously. bSee F i g . 14. See Table XXVI. c  101  Fig. 14. Substrate ^C-androstenedione incubation: graphy of androstenedione  paper chromato-  Overflow (4,869,000 cpm) 3  L/PG, 20 hrs 0-18 cm (223,150 cpm)  I Androstenedione (2,682,500 cpm, 22.4 K g , S.A. 119,754 c p m / K g ) i  .t  L/PG, 16 hrs Androstenedione (2,410,625 cpm, 23.9 * g , S.A. 100,863 c p m / K g ) Bush A, 7 hrs Androstenedione (1,810,500 cpm, 18.4 u g , S.A.  a  See Fig. 13, footnote b.  98,397 cpm/K.g)  1  25-40 cm -tOverflow (1,018,000 cpm)  Fig. 15. Substrate  -progesterone incubation:  paper chromatography of the neutral fraction  Neutral fraction (3,829,000 cpm) L/PG, 18 hrs Testosterone  t Origin (929,600 cpm)  Androstenedione  3  (1,691,250 cpm)  b  3,  Progeste rone  I  3  r  (726,050 cpm, Overflow 165 K g , S.A. (210,000 cpm) 4,400 cpm/Kg) L/PG, 4 hrs Ac20,py Progesterone (144,650 cpm) L/PG, 16 hrs  I  L/PG, 72 hrs  3  R  j  Origin (159,175 cpm)  1-4 cm (642,000 cpm)  Overflow (21,710 cpm)  0-16 cm (64,600 cpm)  T/PG, 8 hrs I60C - HydroocyprQge'sterone  3  I  0-6 cm (12$, 000 cpm)  6-13 cm (376,000 cpm)  Androstenedione (552,800 cpm, 158 Kg, S.A. 3,499 cpm/Kg)  25-40 cm + Overflow (66,125 cpm) L/PG, 16 hrs  1  c  13-40 cm Overflow (74,000 cpm)  , ,(  0-18 cm (25,190 cpm)  —F  T  1 Androstenedione 24-40 cm (466,850 cpm, 142 K g , S.A/3,288 c p V K g ) " ^ W  c  ^Locations of carrier steroids chromatographed simultaneously. See F i g . l£. See Table XXLX. See Table XXX. b  c  d  p  m  )  103  Fig. 16. Substrate ^(^progesterone incubation: paper chromatography of testosterone acetate and 17-hydroxyprogesterone Eluate from paper chromatography of neutral e x t r a c t *  f  3  A c 0 , py 2  L/PG, 4 hrs Testosterone acetate  17-Hydroxyprogesterone  (709,450 cpm, 159 S.A. 4462 cpm/ Kg)  (740,450 cpm)  \  0-13 cm  (172,000 cpm)  L/PG, 4 hrs  T/PG, 4 hrs 200 K g carrier 17hydroxyprogesterone added  :  17-Hydroxyprogesterone  (263,325 cpm, 188 K g , S.A. 1401 c p n y V g )  Testosterone 23-40 cm acetate (210,625 cpm) (636,700 cpm, 1 4 9 ^ S.A. 4273 cpm/ug) B  T/PG, 4 hrs 0-12 cm (22,800 cpm)  17-Hydroxyprogeste rone (219,125 cpm, 152 K g , S.A. 1442 cpm/ K g )  See F i g . 15, footnote b. "See Table XXXI, See Table XXXIL  c  2oLvO cm  (12,530 cpm)  F i g . 17. Substrate  H-pregnenolone incubation: paper chromatography of the n e u t r a l f r a c t i o n  Neutral fraction  (23,075,000 cpm) L/PG, 24 hrs  Testosterone 17-Hydroxyprogesterone  3  3  Origin (2,596,000 cpm) 0  il  0-4 cm (842,650 cpm)  DHEA c  15-40 cm  13-21 cm (4,446,250 cpm) a  I  1-4 cm (24,840 cpm) (446,330 cpm)  Overflow  ^Locations of c a r r i e r s t e r o i d s chromatographed simultaneously. See F i g . 18. °See Table XXXIV". See F i g . 20. See F i g . 19. See Table XXXIV. a  6 I  Overflow (3,624,000 cpm)  L/PG, 4 h r s I'  ~  f  Progesterone *(688,600 cpm, 221 K g S.A. -  ?  3 y l l 6 cpm/ >^g)  b  a  I  4-8 cm (1,887,000 cpm)  1  e  Progesterone 1  L/PG, 48 hrs  Testosterone 17-Hydroxyprogesterone (7,623,?XX) cpm)  3  a  T  1-4 cm (9,401,000 cpm)  ^  Androstenedione ?nolone Pregner  II 21-40 cm (2,114,600 cpm)  F i g . 18. Substrate -^-pregnenolone incubation: paper chromatography o f the eluate of the o r i g i n area of the n e u t r a l f r a c t i o n paper chromatogram  O r i g i n eluate (3,430,900 cpm) 3,  I B/F, 6 h r s  1  f  16* -Hydroxyprogesterone (860,400 cpm)  0-7 cm (1,095,600 cpm)  11-44 cm (953,480 cpm)  T/PG, 18 hrs l/PG, 18 h r s 0-22 cm (285,500 cpm)  j 16*. -Hydroxyprogesterone , l '2,959 ? ° o ^cpm/Kg) ^ %*' S.A.  ( 3  3  j 33-44 cm  3  (  6  3  ^  Overflow 5  0  c  p  m  • 0-16 cm (362,100 cpm)  ^-Hydroxypregnenolone  c 16-21 cm (108,040 cpm)  )  T/PG, 12 hrs 16* -Hydroxyprogesterone (304,900 cpm, 101 Kg, S.A. 3,019 cpm/Kg) 4  See F i g . 17, footnote b. -Location of c a r r i e r 17-hydroxypregnenolone chromatographed simultaneously. See Table XXXV. See Table XXXVI. c  d  0  1  c  I  21-44 cm (300,46© cpm)  106  F i g . 19. Substrate -'H-pregnenolone incubation: of testosterone - 17-hydroxyprogesterone eluate  paper chromatography  Testosterone-17-hydroxyprogesterone eluate (7.623,700 cpm)  3  Ac 0,py 2  • 1  17-Hydroxyprogesterone (288,925 cpm, 145 K g , S.A. 1,993 cpm/Kg) T/PG,  : 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 )  6 hrs  0-16 cm (48,410 cpm)  fSee F i g . 17, footnote e. See Table XXXIV. b  — n  L/PG, 4 hrs  17-Hydroxyprogesterone  D  (86,750 cpm, 146 Kg, S.A. 594 cpnyVg)  1  25-40 cm (123,430 cpm)  107  paper chromatography  F i g . 20. Substrate %-pregnenolone incubation: of pregnenolone-androstenedione eluate  Pregnenolone-androstenedione e l u a t e (4,446,250 cpm)  3,  Ac 0,py 2  L/PG, 18 h r s  n  0-16 cm (421,920 cpm)  — i Androstenedione (2,658,850 cpm, 148 Kg, S.A. 17,965 cpm/ Kg) 1  1  1 23-44 cm ( 2 9 8 , 7 4 0 cpm)  1 Overflow (801,160 cpm)  L/PG, 4 h r s Pregnenolone acetate 0  I 0-2 cm (64,650 cpm)  p  2-30 cm (163,710 cpm)  30-38 cm (405,200 cpm)  fsee F i g . 17, footnote d. See Table XXXIV. °Location of c a r r i e r pregnenolone acetate chromatographed simultaneously. D  108  Fig. 21. Substrate ^C-mevalonate incubations: of neutral fractions  paper chromatography  0  Neutral fraction, control incubation' (82,750 cpm) •L/PG, 48 hrs Testosterone 17^%droxyprdge sterone  I  Origin (79,510 cpm)  1  1-5 cm (1,190 cpm)  f Overflow (3,930 cpm)  Neutral fraction, Pergonal incubation (115,000 cpm) L/PG, 48 hrs Testosterone" ^ 17-Hydroxyprogesterone : H Origin (66,810 cpm)  a  —\  1-5 cm (1,790 cpm)  —  |  Overflow (6,070 cpm)  See Table XXV. Locations of carrier steroids chromatographed simultaneously,  109  PART I I I  STUDIES ON STEROID BIOSYNTHESIS IN VITRO BY THE RAT TESTIS  PRELIMINARY EXPERIMENTS AND T H E STUDIES (a) Introduction It has been established that preparations of r a t t e s t i s i n v i t r o transform progesterone to androstenedione and testosterone (149, 189-194)j the formation and subsequent side-chain cleavage of 17-hydroxyprogesterone are e s s e n t i a l reaction steps (195-198).  R e l a t i v e l y minor  transformation of progesterone t o 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 t o 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 r a t t e s t i s proceeds v i a progesterone and 17-hydroxyprogesterone  rather than v i a 17-hydroxypregnenolone  and  dehydroepiandrosterone ( F i g . 4 ) . This section of the thesis reports preliminary observations on the metabolism i n v i t r o of radioactive progesterone by preparations of r a t testes under several d i f f e r e n t experimental conditions.  The r e s u l t s  of these experiments were used t o design time studies on the 17-hydroxyl a t i o n 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 r a t s , approximately 3 months o l d and  weigh-  ing 200-250 g, were obtained from the Cancer Research Centre, University of B r i t i s h Columbia, Vancouver, B r i t i s h Columbia.  The rats were stunned  by a blow to the head and the testes were r a p i d l y removed and placed i n P e t r i dishes surrounded by crushed i c e . (ii)  Incubation with quartered testes: 4-^C-Progesterone  (The Radiochemical Centre, Amersham,  England) was p u r i f i e d p r i o r to use by t h i n layer chromatography on E a s t 1  man Kodak chroraatogram sheets (type K301R2) i n the solvent system benzenee t h y l acetate (1:1, by v o l ) .  The -^C-progesterone  (556,056 cpm,  11.4  H-g)  was transferred to a reaction f l a s k i n a methanol s o l u t i o n , the solvent was evaporated and the radioactive substrate was redissolved i n 0.2 ml of propylene g l y c o l .  Four r a t testes were quartered and placed i n the incu-  bation f l a s k and 10 ml of Krebs-Ringer phosphate buffer, pH 7.4, containing glucose (0.01 M) and nicotinamide (0.04 M) were added.  Incubation  was performed f o r 1 hour i n a i r a t 3 7 ° using a Dubnoff metabolic shaking incubator.  At the end of the incubation period, the medium was removed  and the tissue and the incubation f l a s k 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 t i s s u e was stored a t - 1 9 ° .  The aqueous f r a c t i o n and the tissue were  analyzed separately.  'See General Methods Section.  Ill  The aqueous f r a c t i o n was extracted with e t h y l 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 methanol .  The aqueous methanol f r a c t i o n  1  was subjected to paper chromatography propylene g l y c o l .  1  i n the solvent system l i g r o i n -  The zone of the experimental chroraatogram corresponding  to the l o c a t i o n of standard testosterone (chromatographed simultaneously) was eluted and the dry sample was treated with pyridine and acetic anhydride . 1  Unlabeled testosterone acetate (200 Kg) and 17-hydroxyprogester-  one (200 f-*-g) were added as c a r r i e r s by paper chromatography  1  and the two steroids were resolved  i n the solvent system ligroin-propylene glycol.;; '  The eluate containing c a r r i e r 17-hydroxyprogesterone was evaporated and treated again with a c e t i c anhydride and pyridine and then paper chromatography i n the solvent system toluene-propylene g l y c o l was Constant s p e c i f i c a c t i v i t y  1  was not attained.  performed.  The eluate containing car-  r i e r testosterone acetate was subjected to paper chromatography i n the solvent system ligroin-propylene g l y c o l u n t i l constant s p e c i f i c 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 c r y s t a l l i z a t i o n s were performed u n t i l radiochemical homogeneity was observed. 1  Unlabeled progesterone (100 p.g)  a n c  * androstenedione (200  \xg)  were added to the overflow f r a c t i o n of the i n i t i a l paper chromatogram of the 90$ aqueous methanol f r a c t i o n . paper chromatography  The two steroids were resolved by  i n the solvent system ligroin-propylene g l y c o l .  The  eluate containing androstenedione and the eluate containing progesterone were evaporated and each was treated with acetic anhydride and pyridine.  112  Further paper chromatography i n the same solvent system was carried out u n t i l constant s p e c i f i c a c t i v i t y f o r both compounds was  observed.  The t i s s u e was allowed to thaw, ground with sand i n a mortar with a pestle and extracted f o r 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 e q u i l i b r a t i o n ,  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 r e -  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 . steroids were then added:  Two hundred H-g of the following unlabeled progesterone, 17-hydroxyprogesterone,  rostenedione and testosterone.  and-  The extract was subjected to Column p a r t i -  t i o n chromatography using s i l i c a g e l . The f r a c t i o n s of the column e f 1  fluent that contained the c a r r i e r steroids were combined and the combined e f f l u e n t was evaporated and subjected to paper chromatography i n the s o l vent system ligroin-propylene g l y c o l .  Further resolution and p u r i f i c a t i o n  of androstenedione, testosterone, progesterone and  17-hydroxyprogesterone  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 the steroids derived from the medium.  Paper chroma-  tography of each compound 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; no c r y s t a l l i z a t i o n s were performed. measured with a gas flow detector"'".  Radioactivity was  113  (iii)  Incubations with c e l l - f r e e homogenates: The ^C-progesterone used as substrate i n the experiment  with quartered testes was a l s o used as substrate i n the incubations with c e l l - f r e e homogenates.  The 14 C -progesterone  (452,254 cpm, 9.3 Kg)  was  transferred to each of two reaction f l a s k s i n a methanol solution, the solvent was evaporated and the radioactive substrate was redissolved i n 0.2 ml of propylene g l y c o l .  Four rat testes were cut into small pieces  and homogenized i n Krebs-Ringer and K  were interchanged, containing glucose (0.01 M) and  (0.04 M)j a Potter-Elvehjem-type ate was  phosphate b u f f e r (pH 7.4), i n which Na"*" nicotinamide  homogenizer was employed.  The homogen-  centrifuged (755 x _g, 10 minutes, 4°) and the supernatant  was divided equally between two incubation f l a s k s .  (20 ml)  NADPH (7.89 mmoles;)  was dissolved i n d i s t i l l e d water ( l ml) and added to one f l a s k .  Incuba-  tions were performed f o r 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 the following unlabeled steroids were added to each f l a s k : one, 17-hydroxyprogesterone, androstenedione  and testosterone.  of each progesterThe  pro-  cedures used f o r the extraction, r e s o l u t i o n and p u r i f i c a t i o n of steroids i n both incubation media was the same.  The contents of the incubation  f l a s k were diluted to 40 ml with d i s t i l l e d water and extracted with e t h y l acetate (5 x 40 ml).  The organic extracts were evaporated under reduced  pressure at 40° and p a r t i t i o n e d between hexane and  JOfo  aqueous methanol.  The combined aqueous methanol extract was then subjected to column p a r t i t i o n chromatography on s i l i c a g e l .  The f r a c t i o n s of the column effluent  that contained the c a r r i e r steroids were combined and evaporated under reduced pressure at 40°.  Further r e s o l u t i o n and p u r i f i c a t i o n of  114  progesterone, 17-hydroxyprogesterone, androstenedione and testosterone 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 these compounds i n 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 i n the other set, the incubations were stopped after 10, 3  20,30 and 40 minutes. For the 2-10 minute group of experiments 7"* - Hprogesterone (4,796,880 dpm, 12.60 nnK>les)vjands4-l^'C-17-hydroxyprogesterone (900,000 dpm, 12.23 nmoles) were both added to each of four incubation flasks and for the 10-40 minute group of experiments %-progesterone (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 chromatography.  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 -17-hydroxyprogesterone. The radioC  active steroids used as substrates were transferred to the reaction flasks i n each instance i n a methanol solution, the solvent was evaporated and the compounds were dissolved i n 0.2 ml of propylene glycol. A cell-free homogenate was prepared i n the same way from six  115  t e s t e s f o r each s e t o f experiments. homogenized i n Krebs-Ringer  The t e s t e s were c u t i n t o p i e c e s and  phosphate b u f f e r (pH 7.4),  were i n t e r c h a n g e d , c o n t a i n i n g g l u c o s e (0.01 a P o t t e r - E l v e h j e m - t v p e homogenizer was centrifuged  (12,100 x g, 20  minutes,  M) and n i c o t i n a m i d e (0.C4  employed.  4°)  i n which Na" and  M);  The homogenate was  and t h e s u p e r n a t a n t was  K  f  diluted  t o 40 ml w i t h d i s t i l l e d water and d i v i d e d e q u a l l y among f o u r r e a c t i o n flasks.  NADPH (7.89  Kmoles) d i s s o l v e d i n d i s t i l l e d water (0.4  added t o each f l a s k immediately performed  i n a i r a t 37°  prior to incubation.  ml)  was  I n c u b a t i o n s were  u s i n g a Dubnoff m e t a b o l i c shaking i n c u b a t o r .  Re-  a c t i o n s were stopped by the a d d i t i o n of e t h y l a c e t a t e (10 ml) p r e v i o u s l y c h i l l e d a t -19°.  The f o l l o w i n g u n l a b e l e d compounds (100  were added t o each f l a s k :  K g of each)  p r o g e s t e r o n e , 17-hydroxyprogesterone, a n d r o s -  tenedione and t e s t o s t e r o n e .  The e x t r a c t i o n , r e s o l u t i o n and  procedures were the same i n a l l experiments.  purification  The c o n t e n t s of each i n -  c u b a t i o n f l a s k were d i l u t e d t o 40 m l w i t h d i s t i l l e d water and e x t r a c t e d w i t h e t h y l a c e t a t e (4 x 40 m l ) .  The o r g a n i c e x t r a c t s were  evaporated  o under reduced p r e s s u r e a t 40 c u b a t i o n was  .  The combined o r g a n i c e x t r a c t of each i n -  then p a r t i t i o n e d between hexane and 70% aqueous methanol.  R e s o l u t i o n and p u r i f i c a t i o n o f progesterone,  17-hydroxyprogesterone, and-  r o s t e n e d i o n e and t e s t o s t e r o n e were a c h i e v e d u s i n g a combination  of paper  chromatography and treatment w i t h a c e t i c anhydride and p y r i d i n e i n the same manner t h a t was the experiment pound was  employed t o r e s o l v e and p u r i f y t h e s e compounds i n  with quartered t e s t e s .  r e p e a t e d u n t i l c o n s t a n t s p e c i f i c a c t i v i t y was R a d i o a c t i v i t y was  tromatar . 1  Paper chromatography o f each com-  measured w i t h a l i q u i d  observed.  scintillation  spec-  116  The nmoles of substrate %-progesterone and substrate ^+0-17hydroxyprogesterone that were not metabolized and that were converted to products during each incubation were calculated from the per cent of substrate radioactivity present i n 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-hydroxyprogesterone f o r 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 end of each time interval.  3  The nmoles of ^H-progesterone hydroxylated  i n the C17 position i n the incubations of 2,5,7,10,10 and 20 minutes duration were calculated by adding the nmoles of  3  3  H-17-hydroxyprogesterone,  3  H-androstenedione and H-testosterone present at the end of each incubation.  The nmoles of radioactive 17-hydroxyprogesterone that underwent  side-chain cleavage i n 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 underwent 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 following 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 i n the inter3  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 i n the time intervals 0-2,2-5,5-7,7-10 and 10-20 minutes and  117  the result f o r each time i n t e r v a l was divided by the number of minutes i n the i n t e r v a l t o obtain the average rate of 17-hydroxylation during each time i n t e r v a l . of androstenedione  The average, rate of reduction of the 17-oxo group was calculated i n a s i m i l a r manner using the nmoles  of testosterone that were formed during each time i n t e r v a l . (?)  Results Resolution and p u r i f i c a t i o n of progesterone,  terone, androstenedione  17-hydroxyproges-  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 t o constant s p e c i f i c a c t i v i t y was evaluated i n some cases by the a d d i t i o n of milligram quantities of the appropriate unlabeled compound followed by c r y s t a l l i z a t i o n s to constant s p e c i f i c a c t i vity.  The s p e c i f i c a c t i v i t y of the f i n a l crystals was observed t o be with-  i n 8% of the s p e c i f i c a c t i v i t y of the pool ( r a d i o a c t i v i t y of the eluate from the f i n a l chromatography divided by the mg of c a r r i e r steroid added) f o r progesterone,  17-hydroxyprogesterone, androstenedione  and testosterone  acetate i n a l l cases i n which c r y s t a l l i z a t i o n s were performed. The r e s u l t s of incubations of quartered r a t testes and c e l l f r e e homogenates of r a t testes with given i n Table XLV. androstenedione  -progesterone  Radioactive progesterone,  as substrate are  17-hydroxyprogesterone,  and testosterone were present i n small amounts i n both  the t i s s u e and the medium. Following incubations with c e l l - f r e e homogenates, the per cent of o r i g i n a l substrate r a d i o a c t i v i t y present as ^Cprogesterone was 1.3 i n the c o n t r o l 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-17hydroxyprogesterone.  I n the control incubation the predominant metabolite '  14  i d e n t i f i e d was C-androstenedione (36.5% of the i n i t i a l substrate radioa 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, 1 7 hydroxyprogesterone, androstenedione and testosterone was 6 4 . 4 f o r the control experiment and 4 9 . 4 f o r 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 f o r 3 and ^KJ (when corrections are made f o r losses) f o r H  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 f o r % and 14c  was greater than 76% and 80%, respectively, i n each experiment. The t o t a l corrected recovery of the i n i t i a l substrate  radioactivity i n a l l incubations of  both sets of experiments was s l i g h t l y greater than the t o t a l corrected recovery of the i n i t i a l substrate % radioactivity; the same r e l a t i o n ship between the values f o r the corrected recoveries of 3 and "^C radioH  a c t i v i t y was observed f o r the ^"C- and %-androstenedione and the ^ 0 and %-testosterone i n a l l incubations of the 0-40 minute set of experiments. 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 l e s s rapid decline i n the nmoles of substrate ^C-17-hydroxy3 progesterone present. The nmoles of ^H-17-hydroxyprogesterone present increased more r a p i d l y than did the nmoles of radioactive androstenedione and testosterone. The r e s u l t s 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 f o r the nmoles of each compound 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 androstenedione and testosterone were i d e n t i f i e d and more %-progesterone was present than i n the 10 minute experiment of the 0-40 minute set of experiments.  The quantity of radioactive 17-hydroxyprogesterone and % - p r o g e s -  terone present a t the end of 1 0 , 2 0 , 3 0 and 40 minutes of incubation prog r e s s i v e l y declined with time while the quantity of radioactive t e s t o s 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 during the f i r s t 2 minutes of incubation (Fig. 2 4 ) .  were maximal  The average rates of  3 17-hydroxylation of  H-progesterone f o r the time i n t e r v a l s 0 - 2 , 2 - 5 , 5 - 7  and 7-10 minutes progressively decreased i n value but the average rates f o r the 5-7 and 7-10 minute i n t e r v a l s d i f f e r e d only s l i g h t l y .  In the  time intervals 0 - 2 , 2 - 5 and 5-7 minutes the average rates of 17-hydroxylat i o n of 3H-progesterone were greater than the average rates of side-  120  chain cleavage of radioactive progesterone and i n the 7-10 minute i n t e r 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 androstenedione i n the 0-10 minute set of experiments reached a maximum value i n the 2-5 minute i n t e r v a l and the values f o r the 5-7 and 7-10 minute intervals differed s l i g h t l y from each other and from the value f o r 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 f o r the average rate of side-chain cleavage f o r the 10-20 minute interval of the 0^40 minute set of experiments (Fig. 24) was the same as the value f o r the 7-10 minute interval of the 0-10 minute set of experiments. The value for the average concentration of radioactive 17-hydroxyprogesterone f o r the time interval 10-20 minutes was less than the value f o r the 7-10 minute interval and the values f o r the average concentration of radioactive androstenedione was greater.  The average  3  rate of 17-hydroxylation of H-progesterone f o r 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 preparat i o n i n the presence of NADFH was suitable f o r 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 combination as substrates were undertaken to evaluate the relative rates of  321  17-hydroxylation of progesterone and side-chain cleavage of 17-hydroxyprogesterone.  In the time studies, the quantity of %-progesterone that  was hydroxylated i n the C17 p o s i t i o n during each incubation was estimated by summing the percentages of the i n i t i a l substrate r a d i o a c t i v i t y 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 incubation was estimated by summing the percentages of the i n i t i a l r a d i o a c t i v i t i e s of %-progesterone and "^C-17-hydroxyprogesterone  that were present  i n androstenedione and testosterone a t 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  will  y i e l d v a l i d results i f the following assumptions apply to the two enzymatic reactions under investigation:  the reactions are i r r e v e r s i b l e ,  endogenous unlabeled progesterone and 17-hydroxyprogesterone do not undergo reaction, the biosynthetic pathway proceeds e x c l u s i v e l y from progesterone v i a 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 s c i s s i o n of carbon-carbon bonds of the side-chains of steroid molecules are  reversible reactions.  The high l e v e l s 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 v i t r o suggest that n e g l i g i b l e quantities of the corresponding substrates were present i n the c e l l - f r e e homogenates p r i o r to incubation; i t i s also u n l i k e l y that substantial quantities of progesterone and 17-hydroxyprogesterone were produced from endogenous  122  precursors during the incubation period.  I t has been suggested that r e -  duction of the 20-oxo group of 17-hydroxyprogesterone i s an obligatory step prior to side-chain cleavage i n the human t e s t i s (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 f o r the side-chain cleavage enzymej however, i n the rat (196), mouse (198) and human t e s t i s (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 t h e i r assessment as average values depends on the length of the period of observat i o n 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 f o r each isotope i n each incubation when the observed radioactivity was corrected f o r losses incurred during the extraction, resolution and 1  purification procedures. Because transformations of radioactive progesterone, 17-hydroxyprogesterone, androstenedione and testosterone to unidentified metabolites occurred to a r e l a t i v e l y minor extent, i t was possible 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 f o r each isotope present i n the compounds i d e n t i 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-  f i e d 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 quantit i e s of radioactivity were present i n eluates from areas of the chromatograms 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<* -dihydroxypregn-4-en-3-one and 5°< -andros tane -3 •=< ,17-diol  (149).  The metabolism  of radioactive progesterone and 17-hydroxyprogesterone during the incubat i o n period v i a pathways that do not involve 17-hydroxylation of % - p r o 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 a c t i v i t i e s 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 17hydroxylation of % - p r o 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 i n Fig. 24. The average rate of 17-hydroxylation of %-progesterone was more rapid than the average rate of side-chain cleavage of radioactive 17-hydroxyprogesterone 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 exceeded the average rate of 17-hydroxylation.  The decline i n 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 17hydroxylation 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 concentration was not limiting) may not apply to the relative rates of the two enzymatic reactions i n vivo or under different in vitro conditions. Data to be presented i n another section of this thesis show that the pH optima for the 170C -hydroxylase and the side-chain cleavage enzyme l i e i n the region of 6.8.  Others have reported that the pH optimum for the  side-chain cleavage enzyme i s 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 i n the latter part of androgen biosynthesis i n 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-hydroxyprogesterone were calculated from the differences i n the amount of each radioactive substrate present a t the beginning and at the end of 30 minutes of incubation.  The assumption that the radioactive substrates were  e x c l u s i v e l y metabolized v i a the pathway under i n v e s t i g a t i o n was neither tested by L o u t f i and Hagerman nor j u s t i f i e d by the results of others (192) to which the authors referred.  Data presented i n t h i s thesis as  w e l l as i n other reports (149,191,193,197) show that t e s t i c u l a r t i s s u e , i n incubations of 20 minutes or more duration, transforms radioactive progesterone and 17-hydroxyprogesterone to a number of d i f f e r e n t metabolites.  Some of these metabolites a r i s e v i a 1704 -hydroxylation or s i d e -  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 s i d e -  chain cleavage of radioactive 17-hydroxyprogesterone was maximal i n the 0-2 minute i n t e r v a l and then declined to a constant value i n the 5-7 7-10  minute time i n t e r v a l s ( F i g . 24).  and  The average concentration of r a d i o -  active androstenedione increased r a p i d l y i n the 0-2 and 2-5 minute time i n t e r v a l s and then increased more slowly i n the 5-7 and 7-10 vals.  The data may be explained as follows:  minute i n t e r -  during the e a r l y minutes  of incubation, product i n h i b i t i o n of the side-chain cleavage enzyme was not present; as the concentration of androstenedione increased, the rate of cleavage of 17-hydroxyprogesterone was retarded.  I t i s also postulated  that the rate of reduction of the 17-oxo group of androstenedione increased as the concentration of androstenedione increased and thus the rate of formation of androstenedione became very s i m i l a r to the rate of t r a n s f o r mation of androstenedione to testosterone.  126  The existence of a steady state i n the formation and transformation of androstenedione i s 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 i n t e r v a l , the decline i n the per cent of  %  and "^C substrate radioactivity present as radioactive 17-hydroxyprogesterone was accompanied by an increase i n the per cent of % and ^"C substrate 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 radioactive androstenedione was greater during the 10-20 minute time i n t e r v a l (Fig. 24). I t i s unlikely that the i d e n t i c a l 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 experiments 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.  I f 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 010 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 androstenedione i s consistent with the postulated relationship between the rate of side-chain cleavage and the concentration of androstenedione. An alternate  127  explanation f o r 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 i n t e r v a l s i s that the conditions were optimal during  the f i r s t two minutes of incubation and l a t e r a suboptimal steady state was present.  Pyridine nucleotide coenzymes and molecular oxygen are  required f o r side-chain cleavage of 17-hydroxyprogesterone (190,193,205). The concentration o f molecular oxygen i n the incubation media may have declined to a l e v e l that l i m i t e d the rate of side-chain cleavage.  It  i s u n l i k e l y that the concentration of NADPH was r a t e - l i m i t i n g ; 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 a t the end of each i n -  3 cubation period were greater than the quantities of H-17-hydroxyprogesterone ( F i g . 22, Table XLVI).  Despite the d i s p a r i t y i n the  concentrations  of % - and ^C-17-hydroxyprogesterone, the quantities of % - and ^"Candrostenedione present a t the end of each incubation period were very s i m i l a r as were the quantities of 3 - and "^C-testosterone. H  Even i n 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 - and H  ing  -testosterone formed dur-  the incubation period were very s i m i l a r . These results suggest that  the ^(^17-hydroxyprogesterone and the "^H-17-hydroxyprogesterone formed from %-progesterone did not mix i n a common pool p r i o r to side-chain cleavage. Some of the c h a r a c t e r i s t i c s 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 s i t e ( s i t e #1) progesterone i s hydroxylated i n the C17  position; the 17-hydroxyprogesterone (ii)  that i s formed i s released.  At a second s i t e ( s i t e #2) progesterone can a l s o undergo  hydroxylation i n the C17 position; the 17-hydroxyprogesterone  that i s  formed i s a bound intermediate that serves as a substrate f o r side-chain cleavage a t the same s i t e .  This s i t e can also bind with  17-hydroxyproges-  terone from the incubation medium and catalyze side-chain cleavage. a f f i n i t y of t h i s s i t e i s the same f o r progesterone and  The  17-hydroxyproges-  terone. Hypothetical mechanisms f o r the 17-hydroxylation of progesterone and f o r the side-chain cleavage of 17-hydroxyprogesterone  must be consis-  tent with the experimental observations that the r a t i o of the concentrations 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 t h i s r a t i o did not r e f l e c t the r e l a t i v e concentrations of "^C3 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 f o r binding at s i t e #2 are 3n-progesterone, %-17-hydroxyprogesterone and "^C-17-hydroxyprogesterone.  Site #2 must react with very  s i m i l a r numbers of ^H and "^C molecules per time i n t e r v a l .  The data i n  Table XLVI show that the sums of the nmoles of %-progesterone plus 17-hydroxyprogesterone  were very s i m i l a r 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 s t a r t of the incubations were also very s i m i l a r . s i m i l a r concentrations  of  and %  The  very  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 s i t e #2 i s the same f o r ^H-progesterone, H-17-hydroxyprogesterone and 14C-17-hydroxyprogesterone and that a l l molecules that are bound to s i t e #2 undergo cleavage. The experimental observation that the average rate of 17-hydroxyla t i o n was 0-2,  greater than the average rate of side-chain cleavage (during the  2-5 and 5-7 minute time i n t e r v a l s ) i s explained by the presence i n  the model of s i t e #1 that catalyzes the 17-hydroxylation only.  of progesterone  I f , i n the model, only s i t e #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  14  C  C19  products.  If only s i t e #2 were present and a l l or part of the 3H-17-hydroxyprogesterone 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 s i t e #2 f o r progesterone and hydroxyprogesterone i s the same.  17-  I f the a f f i n i t y of s i t e #2 f o r these  two compounds d i f f e r e d , the concentrations of  and  products of s i d e -  chain cleavage would not be s i m i l a r despite wide variations i n the r e l a t i v e concentrations of 17-hydroxyprogesterone.  H-progesterone, H-17-hydroxyprogesterone and J  C-  The model, i n which the same a f f i n i t y of s i t e #2.  f o r progesterone and 17-hydroxyprogesterone i s assumed, explains the s i m i l a r i t y i n the concentrations of  and 14c products of side-chain cleavage  as a consequence of the.: s i m i l a r i t y of the sum of the concentrations of  130  H-progesterone and -^H-^-hydroxyprogesterone to the concentration of 14C-17_hydroxyprogesterone. The model that i s proposed as an explanation f o r 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 concentrations of substrate ^C-17-hydroxyprogesterone. of %  I f the concentrations  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 - and ^-C-17-hydroxyH  progesterone, the l i k l i h o o d would, be enhanced that the mechanism proposed herein corresponded to the events that occurred i n v i t r o .  131  TABLE XLV Results of incubations of preparations of r a t testes with ^C-progesterone as substrate  Per cent of substrate r a d i o a c t i v i t y Steroids isolated  C e l l - f r e e homogenates  Quartered testes Tissue  Medium  Control  NADPH  Progesterone  8.0  1.2  1.3  ^ 1  17-Hydroxyprogesterone  1.6  1.5  11.5  ^ 1  Androstenedione  1.6  1.3  36.5  6.9  Testosterone  3.0  6.1  15.3  42.5  14.2  10.1  64.6  49.4  Total a  Constant  s  s p e c i f i c 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: of time studies Compound and isotope  Progesterone 3 H  17-Hydroxyprogesterone  Androstenedione 3 H  Testosterone 3H  14C Total 3H  14  C  results  Duration of incubation (minutes) % Substrate nmoles radioactivity  a  f Substrate nmoles radioactivity 0  % Substrate nmoles radioactivity  10 fo Substrate nmoles radioactivity  71.1  8.96  34.0  4.28  22.5  2.81  13.0  1.64  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  5.6 5.6  0.71 0.68  10.3 11.2  1.30 1.37  12.1  12.3  1.55 1.48  13.5 14.2  1.70 1.74  1.7 1.7  0.21 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  91.7 98.7  11.55 12.01  91.4 95.3  91.3 95.1  11.51 11.63  101.4 98.2  12.78 12.00  11.52 11.65  The nmoles of radioactive steroid present at the end of each incubation period (See Text f o r the method of calculation).  TABLE XLVII Incubations with ^H-progesterone of time studies  Compound and isotope  9.0  Androstenedione 3 H  14c Testosterone 3H ^C Total 3  14  a  H  C  i n combination as substrates:  20  10  Progesterone  14c  G  results  Duration of incubation (minutes)  % Substrate radioactivity  17-Hydroxyprogesterone 3H  and 14 -17-hydroxyprogesterone  36.0 45.3  nmoles  1.21  4.85 6.61  22.5 24.2  3.03 3.53  18.6  2.50  20.3 86.8 89.8  2.96 11.59 13.10  3  30  40  % Substrate radioactivity  nmoles  fo Substrate radioactivity  nmoles  fo Substrate radioactivity  nmoles  4.7  0.63  3.2  0.43  2.7  0.36  15.4 18.2  2.07 2.66  5.9 6.8  0.79 0.99  2.9 3.6  0.39 0.53  22.9 25.4  3.08 3.71  17.4 18.2  2.34 2.66  14.9 16.1  2.01 2.35  36.3  4.89 5.87  49.6 54.6  6.68 7.97  55.9 61.2  8.93  10.67  76.1 79.6  10.24 11.62  76.4 80.9  10.28 11.81  40.2 79.3 83.8  12.24  7.52  The nmoles of radioactive steroid present at the end of each incubation period (See Text f o r the method of calculation).  134  F i g . 22.  incubations  gesterone a n d results  l 4  of c e l l - f r e e h o m o g e n a t e s of rat t e s t e s with  C-l7-hydroxyprogesterone  in c o m b i n a t i o n as  ^ - p r o -  substrates:  of time s t u d i e s  141  1  1  1  Minutes of i n c u b a t i o n 3  H-Progesterone,(o—o)  ;  3 H - 17- hydroxyprogesterone, ( O — — 3 ) ;  i C-17-hydroxyprogesterone , ( • — • ) ; 4  3  H-and  l 4  C - t e s t o s t e r o n e , (•  •).  3 H - a n d i 4 C - a n d r o s t e n e d i o n e , (A  A)  ;  135  F i g . 2 3 . Incubation of c e l l - f r e e homogenates of rat testes with 3H-progesterone and i 4 c - l 7 - h y d r o x y p r o g e s t e r o n e in c o m b i n a t i o n as substrates: results of time studies  Minutes of  incubation  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 , ( • — • ) ; 3 H - a n d r o s t e n e d i o n e , (A A) ; C - a n d r o s t e n e d i o n e , (A • ) • 3 H - t e s t o s t e r o n e , (• • ); I4C- testosterone , (B • ). ;  l 4  ;  136  Fig. 24. l 4  Incubations of c e l l - f r e e h o m o g e n a t e s of rat testes with 3 H - p r o g e s t e r o n e a n d  C - l 7 - h y d r o x y p r o g e s t e r o n e in c o m b i n a t i o n a s s u b s t r a t e s : r e s u l t s of time s t u d i e s  5-7  7-10  10-20  T i m e intervals ( m i n u t e s ) T h e methods e m p l o y e d to c a l c u l a t e the a v e r a g e c o n c e n t r a t i o n s of r a d i o a c t i v e androstenedione ( A — A ) methods  a n d radioactive  1 7 - h y d r o x y p r o g e s t e r o n e (•——•) and the  employed to c a l c u l a t e the a v e r a g e rate of  1 7 - h y d r o x y l a t i o n of  3H-pro-  g e s t e r o n e ( A — A ) , the a v e r a g e rate of r e d u c t i o n of the 17-oxo group of radioactive androstenedione ( O — O )  and the a v e r a g e rate of s i d e - c h a i n c l e a v a g e of radioactive  17-hydroxyprogesterone ( • — Q )  are  d e s c r i b e d in the  text.  137  PREPARATION AND PROPERTIES OF A SOLUBLE SYSTEM OBTAINED FROM RAT TESTICULAR MICROSOMES THAT CATALYZES THE TRANSFORMATION OF PROGESTERONE TO 17HYDROXYPROGESTERONE AND ANDROGENS (a) Introduction Progesterone 17-hydroxylase and 17-hydroxyprogesterone side-chain cleavage enzyme are known to reside i n the microsomal f r a c t i o n of the rat testis  (190,193).  Young and coworkers  (206)  observed progesterone  hydroxylase and 21-hydroxylase a c t i v i t i e s and 20*-hydroxysteroid  17dehydro-  genase a c t i v i t y i n supernatant fractions obtained by high speed c e n t r i 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 u l t r a c e n t r i f u g a t i o n of homogenates of human placentas.  Attempts to obtain the t e s t i c u l a r 17©fc-hydroxylase and the  17-hydroxyprogesterone side-chain cleavage enzyme i n an a c t i v e , soluble form (defined as the supernatant following 2 hours of centrifugation a t  105,000 x  jg)  have been hitherto unsuccessful  (190,193,203).  In t h i s part  of the t h e s i s , the s o l u b i l i z a t i o n of the two enzymatic a c t i v i t i e s i s described and studies on the soluble system are reported. (b)  Materials and methods (i)  Rats: Long-Evans Hooded rats, weighing approximately 250  proximately 3 months o l d , were used f o r a l l experiments.  g and ap-  Animals obtain-  ed from The National Laboratory. Animal Breeding Co., Edmonton, Alberta and Blue Spruce Farms,, Inc., Altamont, N.Y. except the incubation with microsomes.  were used f o r a l l experiments  In the l a t t e r experiment, the  138  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, B r i t i s h Columbia. (ii)  Tissue preparation: The rats were stunned by a blow to the head and the testes  were removed and immediately placed i n P e t r i dishes surrounded by crushed ice.  Further procedures were carried out at 4°. Each t e s t i s was  quartered and the tissue was homogenized i n 0.15 M KC1 using a PotterElvehjem-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 y i e l d the microsomal pellet which was immediately incubated, lyophilized or treated with acetone to y i e l d an acetone powder. To prepare an acetone powder of microsomes or lyophilized microsomes, the materi 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 obt a i n the soluble fraction was 0.106 g (representing approximately the  139  material derived from two testes) and the buffer solution (6 ml) contained 0.2% (v/v) Triton N-101. In one experiment, an attempt was made to obtain mitochondrial and microsomal fractions, each r e l a t i v e l y 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 p e l l e t .  The mitochondrial fraction was suspended i n 15 ml of  0.15 M KC1 and centrifuged (700 x £, 15 minutes); the precipitate was d i s carded and the supernatant was centrifuged (10,000 x t a i n the mitochondrial pellet.  25 minutes) t o ob-  The mitochondrial pellet was washed twice  by suspension i n 15 ml of 0.15 M KC1 followed by centrifugation (10,000 x The unwashed microsomal pellet was suspended i n 7 ml of  j j , 15 minutes).  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 s o l i d ammonium sulfate.  The material was allowed to stand for 10 minutes and  was then centrifuged (10,500 x £, 15 minutes), (iii)  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 t h e s i s .  l,2-^H-Deoxycoi*ticosteronel  was p u r i f i e d by paper chro-  2 matography  i n the solvent system toluene-propylene g l y c o l p r i o r t o use  as substrate; 7-%-pregnenolone (The Radiochemical Centre, Amersham, England) was not p u r i f i e d p r i o r to use as substrate.  The radioactive  compounds used as substrates were transferred to the reaction f l a s k s i n methanol solutions, the solvent was evaporated and the radioactive substrates were redissolved i n 0.2 ml of propylene g l y c o l . (iv)  Incubation conditions: Incubation mixtures were prepared using Krebs-Ringer phos-  phate b u f f e r (pH 7.4) i n which the Na"" and K*" were interchanged or potas4  sium phosphate b u f f e r (0.04 M, pH 7.4).  For incubations of the soluble  f r a c t i o n , 5 ml of the soluble f r a c t i o n and 9 ml of phosphate buffer were added to each reaction f l a s k .  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 mixture immediately p r i o r to incubation.  Unless otherwise stated, the t o t a l  volume of a l l incubation mixtures was 15 ml. Incubations were performed i n a i r f o r 40 minutes using a Dubnoff metabolic shaking incubator and were c a r r i e d out a t 3 7 ° unless otherwise stated. (v)  Extraction, resolution and p u r i f i c a t i o n procedures: Incubations were stopped by the addition of e t h y l acetate  (15 ml) previously c h i l l e d a t - 1 9 ° . Known quantities of c a r r i e r s t e r o i d s ^ (60-100 p-g of each) were added and the contents of each incubation 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 •'"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 azeotropic d i s t i l l a t i o n . Progesterone, 17-hydroxyprogesterone,  androstenedione and t e s t o s -  terone were separated and p u r i f i e d by paper chromatography i n the solvent systems ligroin-propylene g l y c o l and toluene-propylene g l y c o l .  Following  i n i t i a l chromatography, the eluted steroids were treated with pyridine and a c e t i c anhydride^ and rechromatographed.  17-Hydroxyprogesterone and t e s -  tosterone do not separate i n the chromatographic 17-hydroxyprogesterone  systems used; hence, the  eluates were treated a second time with pyridine  and a c e t i c 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 f r e e compound and as t h e i r acetylated derivatives.  Corticosterone, cortexolone and deoxycorticosterone were chromato-  graphed on paper i n the solvent systems toluene-propylene g l y c o l and Bush BI.  Other steroids were resolved and p u r i f i e d by paper chromatography i n  the solvent systems ligroin-propylene g l y c o l and toluene-propylene g l y c o l . Chromatography of the steroids containing the £\ -3-oxo grouping was r e 4  peated u n t i l a difference of less than %  occurred i n the s p e c i f i c a c t i -  v i t y ^ f o r each isotope that the compound contained when compared t o the value of the previous determination or u n t i l less than 1% of the i n i t i a l r a d i o a c t i v i t y of the substrate(s) was associated with the c a r r i e r s t e r o i d . Corrections were made f o r losses incurred during the i s o l a t i o n procedures. The s p e c i f i c 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 chromatograms d i d not vary by more than 8$ when the compounds were subjected  Ih2  to a series of c r y s t a l l i z a t i o n s  following the further addition of the  corresponding unlabeled compounds. Pregnenolone,  17-hydroxypregnenolone and dehydroepiandrosterone  were detected and assayed f o r r a d i o a c t i v i t y as follows: authentic r e ference compounds were chromatographed simultaneously with the experimental chromatograms and the standards were located by treatment with an ethanol s o l u t i o n of phosphomolybdic acid.  The pattern of r a d i o a c t i v i t y  of the experimental chromatograms was determined by scanning (NuclearChicago Model 1032 4 p i Actigraph I I chromatogram scanner) and the r e 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 r a d i o a c t i v i t y , the zones were excised and bisected l o n g i t u d i n a l l y .  One portion was treated  with the phosphomolybdic a c i d reagent to v e r i f y the l o c a t i o n of the experimental s t e r o i d and the other portion was eluted with methanol and the r a d i o a c t i v i t y of the eluate was assayed by l i q u i d s c i n t i l l a t i o n spectrometry^.  20<-Hydroxypregn-4-en-3-one^ was p u r i f i e d by paper chromato-  graphy i n the solvent system toluene-propylene g l y c o l p r i o r to a d d i t i o n as c a r r i e r ; the 20/9 -epimer was p u r i f i e d by paper chromatography i n the solvent system ligroin-propylene g l y c o l p r i o r to addition as c a r r i e r , (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 Pharmacia, Ltd., Montreal, Quebec.  3  The Sephadex G-200 was allowed t o swell  A g i f t from Dr. R. Neher, Basle,Switzerland.  143  i n d i s t i l l e d water f o r 4 days a t 4° and was then added t o 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. sure of 15 cm of water.  The column was run a t a pres-  The eluent (potassium phosphate buffer, 0.04 M,  pH 7.4) was allowed to flow through the column f o r 6 hours p r i o r t o app l i c a t i o n of the sample.  The Sephadex G-25 (coarse) was allowed to swell  i n 0.04 M ammonium bicarbonate f o r 4 hours a t room temperature p r i o r t o use.  The DEAE-Sephadex A-25 was allowed t o 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 hydroc h l o r i c a c i d 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 c r y s t a l l i n e bovine serum albumin as standard.  Non-heme i r o n was determined by the method  of Massey (211) as modified by Simpson and Boyd (212), but using bathophenanthroline.  The molar e x t i n c t i o n c o e f f i c i e n t f o r the ferrous i r o n -  bathophenanthroline complex  (20,100) was determined by measuring the ab-  sorbance a t 235 nm of solutions of r e c r y s t a l l i z e d ferrous ammonium s u l fate.  Heme i r o n was determined according to the method of Diehl and  Smith (213) i n the p r o t e i n that was precipitated during the non-heme i r o n 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  344  states of dichlorophenolindophenol are used to measure dehydrogenase activity. A S o n i f i e r c e l l disruptor (Heat Systems Co., M e l v i l l e , N.Y.;).was employed f o r u l t r a s o n i c treatment and the specimens were c h i l l e d 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 34c17-hydroxyprogesterone dpm ( i n incubations with two radioactive substrates) recovered as radioactive androstenedione and testosterone. Radioactivity was measured  by l i q u i d s c i n t i l l a t i o n spectrometry,  (c) Results (i)  Preparation of a soluble f r a c t i o n containing 17«C-hydroxylase  and side-chain cleavage enzyme a c t i v i t y : 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 l y o p h i l i z a t i o n of the microsomes or by preparation of an acetone powder of l y o p h i l i z e d microsomes (Table XLVIII). large percentages of radioactive progesterone and  17-hydroxyprogesterone  were converted to C19 compounds i n 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 r a d i o a c t i v i t y of the substrates when the r a d i o a c t i v i t y associated with uncharacterized material more polar than 17-hydroxyprogesterone and testosterone i s taken i n t o account.  The progesterone 17-hydroxylase and  17-hydroxyprogesterone  145  side-chain cleavage enzyme a c 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 d i a l y s i s of  0.5 M NaCl or 2.0 M L i C l extracts of l y o p h i l i z e d microsomes. The p o s s i b i l i t y that progesterone 17-hydroxylase and 17-hydroxyprogesterone side-chain cleavage enzyme a c t i v i t i e s could be extracted from l y o p h i l i z e d microsomes with the detergent T r i t o n N-101 was next i n v e s t i 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 f r a c t i o n (defined as the supernatant following centrifugation at 105,000 x  for  2 hours) prepared by treatment of l y o p h i l i z e d microsomes with a solution of T r i t o n N-101 that a 0.20%  of about 0.25%  concentration (by v o l ) .  solution was as e f f e c t i v e as a 0.25%  I t was l a t e r found  solution.  The d i s t r i -  bution of r a d i o a c t i v i t y following incubations of f r a c t i o n s obtained a f t e r centrifugation (105,000 x g  f  with 0.2% T r i t o n N-101  2 hours) of l y o p h i l i z e d microsomes treated  i n potassium phosphate buffer (0.04 M, pH  i s presented i n Table L.  7.4)  Incubation of the precipitate obtained follow-  ing high speed centrifugation of detergent-treated l y o p h i l i z e d microsomes 3 l e d to very active 17-hydroxylation of ^H-progesterone, a s l i g h t 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 l y o p h i l i z e d microsomes (Table XLVIII). Following incubation of the soluble f r a c t i o n (obtained without ultrasonic treatment) 3 47 per cent of the r a d i o a c t i v i t y of active 17-hydroxyprogesterone,  H-progesterone was present as radio-  androstenedione and testosterone; only a  small per cent of the o r i g i n a l dpm of e i t h e r radioactive substrate was  146  present as androstenedione or testosterone (Table L ) . Attempts were made to increase the enzymatic a c t i v i t i e s present i n the soluble f r a c t i o n by ultrasonic treatment of l y o p h i l i z e d microsomes suspended i n 0.2$ T r i t o n 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 a c t i v i t i e s of the soluble f r a c t i o n ; ultrasonic treatment f o r 2 minutes appeared to be l e a s t damaging.  The enzymatic a c t i v i t i e s  were not demonstrable i n the soluble f r a c t i o n obtained following u l t r a sonic treatment (2 minutes) of l y o p h i l i z e d microsomes suspended i n phosphate buffer without T r i t o n N-101. The soluble f r a c t i o n obtained by treatment of l y o p h i l i z e d microsomes with 0.2$  solution of T r i t o n N-101  displayed considerably less  side-chain cleavage a c t i v i t y and androstenedione 17-oxo reduction a c t i v i t y than d i d l y o p h i l i z e d microsomes.  The p o s s i b i l i t y that these two en-  zymatic a c t i v i t i e s i n the soluble f r a c t i o n were i n h i b i t e d by the presence of T r i t o n N-101 was tested by incubating l y o p h i l i z e d microsomes i n the presence of the detergent.  In addition, an experiment was performed t o  e s t a b l i s h that tissue fractions were required f o r transformations of -^Hprogesterone and "^C-17-hydroxyprogesterone that were employed. Table L I I .  under the incubation conditions  The results of these investigations are given i n  No transformation of either radioactive substrate was observed  following the incubation of a solution of T r i t o n N-101 and potassium phosphate b u f f e r .  The 17**--hydroxylase and side-chain cleavage enzyme a c t i v -  i t i e s of l y o p h i l i z e d microsomes were very similar i n incubations i n the presence (Table LII) and i n the absence (Table XLVIII) of T r i t o n N-101.  147  The results of the two incubations d i f f e r e d i n that more radioactive androstenedione and less radioactive testosterone were present following the incubation i n the presence of T r i t o n N-101. (ii)  Other enzymatic a c t i v i t i e s i n the soluble f r a c t i o n : The possible presence i n the soluble f r a c t i o n 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 (Table L I I I ) . A 17< -hydroxy4  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 a t the C16 p o s i t i o n was not detected, nor was 17-hydroxyl a t i o n 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, transformation of the radioactive substrates to 17-hydroxyprogesterone, androstenedione and testosterone also occurred to an extent s i m i l a r to the values given i n Table L f o r the supernatant obtained following centrifugation of T r i t o n N-101-treated l y o p h i l i z e d microsomes.  Thus, the soluble f r a c t i o n  contains a 17«C -hydroxylase f o r which progesterone i s the preferred substrate rather than pregnenolone or deoxycorticosterone, a 17-hydroxyprogesterone side-chain cleavage enzyme that does not cleave pregnenolone, a 17-hydroxysteroid dehydrogenase and a 20ot-hydroxysteroid dehydrogenase.  ^The presence of NADPH dehydrogenase a c t i v i t y i n the soluble f r a c t i o n i s reported i n another subsection of the Results section.  248  (iii)  Effects of changes of temperature and pH on the 17«*- -hydroxy-  lase and side-chain cleavage enzyme a c t i 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, respectively.  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 phosphate 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 f r a c t i o n was observed; that i s , 12-15 per cent of the substrate %-progesterone was converted to 17-hydroxyprogesterone (Table LI7).  At pH 7.4,  the a c t i v i t y of the side-chain cleavage enzyme was negligible i n the precipitate 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 l a t t e r 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 % - p r o 14 gesterone and  C-17-hydroxyprogesterone i n combination as substrates and  was also inactive when incubated after 4 hours of d i a l y s i s against potassium 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 s p e c i f i c a c t i v i t y of the 17 <=<.-hydroxylase (as pmoles of progesterone hydroxylated per minute per mg of protein) was found to be  10.4  f o r l y o p h i l i z e d microsomes and 1.38 f o r the soluble f r a c t i o n (Table LV). The s p e c i f i c a c t i v i t y of the 17«< -hydroxylase present i n the precipitate obtained following 40% saturation of the soluble f r a c t i o n with ammonium s u l f a t e was determined twice and found to be 1.73  and  3.89.  Both the soluble f r a c t i o n and the p r e c i p i t a t e obtained by 40% saturation of the soluble f r a c t i o n with ammonium sulfate contained heme i r o n protein, non-heme i r o n p r o t e i n and NADPH dehydrogenase a c t i v i t y . 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 a t 450 nm with a small absorption maximum at 420 nm ( F i g . 27). Attempts were made to resolve the non-heme i r o n protein, the cytochrome P-450 and the enzyme responsible f o r the NADPH dehydrogenase a c t i v i t y of the soluble f r a c t i o n i n order to explore the r e l a t i o n s h i p of these compounds to the 17<* -hydroxylase and side-chain cleavage enzyme a c t i v i t i e s that were present.  The p r e c i p i t a t e obtained by 40% saturation of  the soluble f r a c t i o n with ammonium s u l f a t e was subjected to column chromatography on Sephadex G-200 ( F i g . 28).  One major and one minor peak, i n  terms of the absorbance a t 280 nm, were observed. i t was found that a portion of the T r i t o n N-101  In separate  experiments  present i n the soluble  f r a c t i o n was precipitated by 40% saturation with ammonium s u l f a t e . T r i t o n N-101  When  was subjected to column chromatography on Sephadex G-200,  the detergent was present i n the f r a c t i o n s that immediately followed the  150  The u l t r a v i o l e t spectrum of Triton N-101 (Fig. 29) served  void volume.  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 d i s -  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. served.  No transformations of the radioactive substrates were ob-  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 corresponded to the positions of peaks i n the profiles of the non-heme iron content and NADPH dehydrogenase a c t i v i t y of the fractions. Heme i r o n was not demonstrated i n the effluent from the column and NADPH dehydrogenase a c t i v i t y was reduced i n comparison to the a c t i v i t y present i n the precipi t a t e obtained by 40$ saturation of the soluble f r a c t i o n with ammonium sulfate. Non-heme iron protein could be obtained free of NADPH dehydrogenase a c t i v i t y by ion exchange chromatography.  The precipitate obtained by 40$  saturation of the soluble f r a c t i o n with ammonium sulfate was f i r s t subjected to gel f i l t r a t i o n chromatography using Sephadex G-200 as previously described.  The elution p r o f i l e i n terms of the absorbance at 280 nm was  very similar to that depicted i n F i g . 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 f r a c t i o n s  were pooled and l y o p h i l i z e d . The l y o p h i l i z e d material was  dissolved i n  a minimal volume of a s o l u t i o n of potassium phosphate buffer (0.04 M, 7.4)  pH  and sodium chloride (0.1 M) and subjected to column chromatography  on DEAE-Sephadex A-25 280 nm was  ( F i g . 31).  Peak #4 i n terms of the absorbance at  observed to correspond i n l o c a t i o n to the p o s i t i o n of a peak  i n the p r o f i l e of the non-heme i r o n content of the f r a c t i o n s . 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 f r a c t i o n containing the greatest amount of  non-  heme i r o n . A number of methods f o r the preparation of'microsomal non-heme i r o n protein were evaluated;  the following procedure was found to y i e l d  the most highly p u r i f i e d product: somes was pH 7.4)  an acetone powder of l y o p h i l i z e d micro-  dispersed i n a s o l u t i o n of potassium phosphate buffer (0.04  and sodium chloride (0.1 M) and the suspension was  ultrasound f o r 5 minutes.  M,  treated with  The supernatant following centrifugation  (105,000 x _g, 2 hours) was applied to a DEAE-Sephadex A-25 column and gradient e l u t i o n was pH 7.4)  performed using potassium phosphate buffer (0.04  containing 0.1 to 0.7 M NaCl ( F i g . 32).  of the absorbance at 280 nm was  M,  A prominant peak i n terms  observed that coincided i n l o c a t i o n to  the p o s i t i o n of a peak i n the p r o f i l e of the non-heme i r o n content of the fractions. which i t was  NADPH dehydrogenase a c t i v i t y was measured.  Fractions 18 to 31 from the column were pooled  and passed through a Sephadex G-25 bicarbonate lyophilized.  as eluent.  minimal i n a l l f r a c t i o n s i n  (coarse) column using 0.04  M ammonium  The protein-containing f r a c t i o n s were pooled and  The l y o p h i l i z e d material was  dissolved i n a minimal volumeoof  152  potassium phosphate buffer (0.04 M, pH 7.4)  and subjected to g e l f i l t r a -  t i o n column chromatography on Sephadex G-200 ( F i g . 33). terms of the absorbance a t 280 nm) were observed. (#36)  Two  peaks ( i n  The column f r a c t i o n  that displayed the greatest absorbance at 280 nm contained 6.0  p.g  of non-heme i r o n . The procedure used to prepare microsomes included a minimal number of centrifugation steps and no unusual precautions were taken to exclude contamination of the microsomal f r a c t i o n by mitochondria. heme i r o n content of more c a r e f u l l y prepared microsomal and  The  non-  mitochondrial  f r a c t i o n s was investigated i n order to e s t a b l i s h d e f i n i t e l y the s u b c e l l u l a r locations of the compound (Table LVI). gram of protein i n washed mitochondria  The quantity of non-heme i r o n per and microsomes was  quantity per gram of protein i n the corresponding  greater than the  unwashed preparations  and the amount of non-heme i r o n per gram of protein i n washed microsomes was (d)  greater than the amount per gram of protein i n washed  mitochondria,  Discussion The word "solution" and modifications of the term (such as "solu-  ble f r a c t i o n " ) are employed i n t h i s part of the thesis to describe  ma-  t e r i a l that i s not precipitated following a 2 hour period of centrifugat i o n at 105,000 x g.  A s o l u t i o n may be defined formally as any phase con-  t a i n i n g more than one component; a phase i s defined as a system that i s uniform throughout, not only i n chemical composition but a l s o i n physic a l state (215).  The observation that a substance i s not p r e c i p i t a t e d  by high speed centrifugation provides evidence that the material i s i n solution.  A system designated as a s o l u t i o n according to the results of  high speed c e n t r i f u g a t i o n may  include material that i s not i n "true" s o l u -  t i o n according to a formal d e f i n i t i o n .  On the other hand, substances  153  t h a t a r e 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 b y p r o l o n g e d  centrifugation.  O t h e r workers have a t t e m p t e d t o s o l u b i l i z e t h e t e s t i c u l a r p r o g e s t e r o n e 17-hydroxyla.se and 17-hydroxyprogesterone zyme b y t r e a t m e n t o f microsomes w i t h d e t e r g e n t  s i d e - c h a i n cleavage e n (193,203).  The s u c c e s s  of t h e method r e p o r t e d h e r e i n may be a t t r i b u t e d t o t h e use o f l y o p h i l i zed microsomesj however, no a t t e m p t was made t o s o l u b i l i z e t h e 1 7 « . h y d r o x y l a s e and s i d e - c h a i n c l e a v a g e enzyme b y treatment o f 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 that the concentrations of t h e detergents  used b y o t h e r workers p r e c l u d e d t h e presence o r t h e e x p r e s s i o n o f t h e e n z y m a t i c 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 ( T a b l e X L I X ) . The f o r m a t i o n o f 17-hydroxypregnenolone  has been r e p o r t e d i n i n -  c u b a t i o n s o f homogenates o f r a t t e s t e s w i t h 3n_pregnenolone a s s u b s t r a t e (216).  S h i k i t a and c o w o r k e r s , however, were unable t o r e c o v e r 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 incubations 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).  Shikita  and coworkers a l s o performed i n c u b a t i o n s w i t h •^•'C-progesterone and 3 H pregnenolone i n c o m b i n a t i o n as s u b s t r a t e s and w i t h H c - 1 7 - h y d r o x y p r o g e s t e r o n e and 3H_l7-hydroxypregnenolone  i n c o m b i n a t i o n as s u b s t r a t e s .  From  t h e r e s u l t s o f t h e s e experiments the a u t h o r s c o n c l u d e d t h a t t e s t o s t e r o n e i s formed f r o m pregnenolone b y r a t t e s t i c u l a r microsomes m a i n l y v i a p r o g e s t e r o n e and a n d r o s t e n e d i o n e . The i n c u b a t i o n o f t h e 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 c o m b i n a t i o n a s s u b s t r a t e s r e p o r t e d h e r e i n ( T a b l e L I I I ) was performed t o a s c e r t a i n t h e s u b s t r a t e s p e c i f i c i t y o f t h e 17t*- - h y d r o x y l a s e ; ^ H - p r e g n e n o l o n e s e r v e d a s t h e t e s t s u b s t r a t e and s e r v e d as t h e c o n t r o l .  -progesterone  NAD^~ , a coenzyme r e q u i r e d f o r t h e c o n v e r s i o n o f  154  pregnenolone t o 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 r e s u l t s are i n accord  with the conclusion of S h i k i t a et a l . and also show that progesterone i s the preferred substrate f o r the 17*-hydroxylase. A pH optimum of 6.8 was observed f o r the a c t i v i t i e s of the 17othydroxylase and the side-chain cleavage enzyme i n the soluble f r a c t i o n ( F i g . 25).  A pH optimum of 7.0 has been observed f o r the side-chain  cleavage enzyme a c t i v i t y i n incubations of r a t (203) and guinea p i g (190) t e s t i c u l a r microsomes. 17-hydroxylase  Lynn and Brown (190) reported that the progesterone  of guinea p i g microsomes was quite active a t pH 8.5.  When  the a c t 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 17-hydroxyprogesterone, 7.0.  present as radioactive acetate plus  the data of Lynn and Brown show a pH optimum of  Young et a l . (206) observed progesterone 17-hydroxylase  activity  i n 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  of the incubation media was increased to 9.1.  increased as the pH  I t i s of i n t e r e s t that the  a c t i v i t i e s of the 17* -hydroxylase and the side-chain cleavage enzyme i n the soluble f r a c t i o n were s i m i l a r i n incubations a t 23°,32° and 37° ( F i g . 26).  The e f f e c t 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 a c t i v i t i e s were present i n the p r e c i p i t a t e obtained by 40  155  per cent saturation of the soluble f r a c t i o n with ammonium s u l f a t e (Table LIV).  The presence of 17-hydroxysteroid dehydrogenase and side-chain  cleavage enzyme i n the p r e c i p i t a t e was demonstrated  i n an incubation per-  formed at pH 6.7 but not i n an incubation performed a t pH 7.4. c i f i c a c t i v i t y of the 17*-hydroxylase  The spe-  was greater i n the p r e c i p i t a t e ob-  tained by 40 per cent saturation of the soluble f r a c t i o n than i n the untreated soluble f r a c t i o n ; however, considerably less enzymatic a c t i v i t y was observed i n the p r e c i p i t a t e (Table LV).  The highest s p e c i f i c a c t i v -  i t y of the 17«- -hydroxylase was observed i n l y o p h i l i z e d microsomes.  The  r e s u l t s suggest that the enzyme was p a r t i a l l y inactivated during the i s o l a t i o n procedures. The p r e c i p i t a t e obtained by 40 per cent saturation of the soluble f r a c t i o n with ammonium sulfate contained NADPH dehydrogenase a c t i v i t y , cytochrome P-450 and non-heme i r o n protein.  The NADPH dehydrogenase and  the cytochrome P-450 were unstable and i t was not possible to i s o l a t e these compounds f o r further investigations.  Kimura and Ohno extracted  non-heme i r o n protein from an acetone powder of homogenized pig testes (219).  The authors postulated that the non-heme i r o n protein was present  i n t e s t i c u l a r mitochondria although t h e i r method of preparation did not exclude a microsomal source.  The r e s u l t s reported herein establish the  presence of non-heme i r o n protein i n both microsomes and mitochondria of r a t testes.  In bovine adrenal mitochondria the 11^ -hydroxylase (220-  223) and the c h o l e s t e r o l side-chain cleavage enzyme (212,224) systems are considered to consist of NADPH dehydrogenase, non-heme i r o n protein and cytochrome P-450.  The progesterone 17* -hydroxylase and the 17-hydroxy-  progesterone side-chain cleavage enzyme of r a t t e s t i c u l a r microsomes may  156  function by a s i m i l a r mechanism.  This hypothesis cannot be tested con-  c l u s i v e l y u n t i l NADPH dehydrogenase, cytochrome P-450 and non-heme i r o n protein are i s o l a t e d i n active forms. A soluble preparation that catalyzes the transformation of progesterone to 17-hydroxyprogesterone and to androgens should provide a u s e f u l experimental system f o r further i n v e s t i g a t i o n of the components and properties of the 17* -hydroxylase and the 17-hydroxyprogesterone side-chain cleavage enzyme of r a t t e s t i c u l a r microsomes.  TABLE XLVIII Distribution of radioactivity following incubations of preparations of rat testes  Per cent of substrate radioactivity Steroids isolated  Microsomes  3 Progesterone 17-Hydroxyproge sterone  ^1  < 1  C  Lyophilized microsomes  \  H  h  1  1  2  <1  1  1  <• 1  <1  £1  *1  50  49  Androstenedione  22  21  24  21  Testosterone  3,2 54  31 53  54 7a  49 72  Total  Acetone powder of l y o p h i l i z e d microsomes  In the incubations of microsomes (obtained from two testes) and lyophilized microsomes (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 acetone powder (0.053 g) of lyophilized microsomes, ^H-progesterone (4,400,880 dpm, 3.64 K g ) 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 KrebsRinger phosphate buffer (pH 7.4), i n which the Na and K*" were interchanged, were added. The lyophilized microsomes and the acetone powder were each transferred to reaction flasks and incubated i n 10 ml of the Krebs-Ringer buffer. In the experiment with the acetone powder testosterone acetate was not chromatographed to constant specific a c t i v i t y and androstenedione was not purified extensively. +  158  TABLE XLIX  Results of incubations of supernatants obtained following high speed centrifugation of T r i t o n N-101-treated l y o p h i l i z e d microsomes  Per cent solution of T r i t o n N-101 used t o treat l y o p h i l i z e d microsomes (v/v)  Per cent of i n i t i a l r a d i o a c t i v i t y of substrate 3H-progesterone present 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 l y o p h i l i z e d microsomes were treated with solutions of T r i t o n 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 t o reaction f l a s k s cont a i n i n g ^-progesterone (4,400,880 dpm, 3.64 t^g) and ^ C - ^ - h y droxyprogesterone (723,700 dpm, 3.64 Kg) i n combination as substrates; 5 ml of Krebs-Ringer phosphate b u f f e r (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 a f t e r 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 isolated  3  H  From microsomes •+• T r i ton N-101  From microsomes 4- Triton N-101 + u l t r a sonic treatment  14  c  3  14  c  l  33  H  c  3H  C 1  6  17-Hydroxyprogesterone  21  19  82  38  89  31  Androstenedione  62  53  10  7  3  4  1  1  3  2  1  1  84  79  95  80  93  83  Progesterone  Testosterone Total  47  In a l l experiments 0.106 g of lyophilized microsomes were treated with 6 ml of a 0.2% (v/v) solution of Triton N-101 i n potassium phosphate buffer (0.04 M, pH 7.4). 3H_. gesterone (2,732,800 dpm, 2.35 Kg) and "^G-17-hydroxyprogesterone (372,360 dpm, 2.12 Kg) were used i n combination 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 treatment (20 kilocycles per second, 70 watts, 2 minutes) was performed on the detergent-lyophilized microsome mixture prior to centrifugation. pro  +  160  TABLE LI D i s t r i b u t i o n of r a d i o a c t i v i t y following incubations of the soluble f r a c t i o n obtained a f t e r treatment of l y o p h i l i z e d microsomes with 0.2$ T r i t o n N-101;and ultrasound  Duration of u l t r a s o n i c treatment (minutes)  Per cent of i n i t i a l r a d i o a c t i v i t y of substrate 3H-progesterone p r e 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 l y o p h i l i z e d microsomes were suspended i n 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 cond i t i o n s of incubation of the s o l u b l e ' f r a c t i o n s and the radioactive substrates used are given i n Table L.  TABLE LII 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  Per cent of substrate radioactivity Steroids isolated  Blank  Lyophilized microsomes + Triton N-101 3  H  34c  3  *  1  92  H  < 1  6  2  1  94  < 1  Androstenedione  52  48  < 1  < 1  Testosterone  21  20  < 1  < 1  Total  75  75  94  92  Progesterone 17-Hydroxyprogesterone  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 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. H  162  TABLE LIII The per cent of substrate r a d i o a c t i v i t y i n i s o l a t e d compounds formed during incubations of the soluble f r a c t i o n with radioactive steroids  Per cent of substrate r a d i o a c t i v i t y Steroid isolated  Substrate ^H-Progesterone  ^-Deoxycorticosterone  14  C  -Progesterone -^-Pregnenolone  Progesterone 17-Hydroxyprogesterone 17-Hydroxypregnenolone Dehydroe piandrosterone Andros tenedione Testosterone 20«- -Hydroxypregn4-ene-3-one 20,8 -Hydroxypregn4-ene-3-one 16-Hydroxyprogesterone Cortexolone Corticosterone  64  <1  11  <1  <1 <1 1.8 < 1  < < < <  1 1 1 1  2.4 < 1 < 1 <1 <1  In a l l incubations, potassium phosphate b u f f e r (0.04 M, pH 7.4) was used. The substrates used s i n g l y were 7-^H-progesterone (1,676,208 dpm- 1.2 K g ) and l,2-3H-deoxycorticosterone (6,809,270 dpm, 3.88 K g ) ; 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 D i s t r i b u t i o n of r a d i o a c t i v i t y following incubations of the precipitate obtained by 40$ saturation of the soluble f r a c t i o n with ammonium s u l fate  Per cent of substrate r a d i o a c t i v i t y Steroids identified  Incubations at pH 7.4 Experiment 1  3 Progesterone 17-Hydroxyproge sterone Androstenedione Testosterone Total  H  Experiment 2  Incubation at pH 6.7  3  ^0  H  <1  80  <1  86  62  95  12  100  15  19  1  <1  < 1  5  < 1  <1  <1  <1  7  96  92  100  101  93  1  C  In the incubations at pH 7.4, the soluble f r a c t i o n s were prepared from 0.106 g of l y o p h i l i z e d microsomes and 3H-progesterone (2, 286.400cdp 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 a t pH 6.7, the soluble f r a c t i o n was prepared from 0.3 g of l y o p h i l i z e d microsomes and 3H-progesterone (l,676,208u,dpm, 1.45 K g ) was used as the sole substrate. A l l precipitates were dispersed i n 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 add i t i o n of one drop of d i l u t e HC1. m>  TABLE LV Specific a c t i v i t y of the progesterone 17-hydroxylase  Fraction  Treatment  Subfraction  5  Protein (mg)  % Substrate ^H-progesterone hydroxylated  Specific activity  10.4  Lyophilized microsomes  Triton N-101  10.5  70  Lyophilized microsomes  Triton N-101, Sentrifugation Supernatant (105,000 x 2 hrs) Precipitate  9.0 42.0  36  10.8 6.0  12  Soluble fraction  4056 (NH ) S0 4  2  Precipitate l Precipitate 2° c  4  57  15  1.38 9.80 1.73  3.89  Each determination Of the specific a c t i v i t y 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 a c t i v i t y of the precipitate was determined i n two separate experiments.  a  13  165  TABLE LVI Non-heme i r o n and protein content of washed and unwashed mitochondria and microsomes  Tissue preparation  Non-heme Fe (Kg)  Protein (g)  K g Non-heme Fe per g protein  Microsomes Unwashed Washed  95.9  8.25  11.6  2.6  0.05  51.0  31.1  1.42  21.9  0.05  36.0  Mitochondria Unwashed Washed  1.8  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 H - p r o g e s t e r o n e ( 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 H C l 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 c h a i n cleavage a c t i v i t y , ( • — — • ) . 3  ;  167  F i g . 2 6 . T h e e f f e c t of t e m p e r a t u r e on the a c t i v i t i e s of the 17a-hydroxylase a n d the . s i d e - c h a i n c l e a v a g e e n z y m e in the s o l u b l e f r a c t i o n  28 r  _  '  1  24 -  Temperature  Each  i n c u b a t i o n f l a s k c o n t a i n e d 3 H - p r o g e s t e r o n e ( 1 , 6 7 6 , 2 0 8 d p m , 1.2 fig)  as substrate. the (o  ("Centigrade)  enzymatic  The  conditions of i n c u b a t i o n a n d t h e m e t h o d s u s e d to c a l c u l a t e  activities  are d e s c r i b e d in the text.  o ) ; s i d e - c h a i n a c t i v i t y , (•——•).  17a - H y d r o x y l a s e  activity,  163  Fig. 27 Absorbance spectrum of the precipitate obtained by 4 0 % saturation of the soluble fraction with ammonium sulfate  > a  o  <  380  i  400  i  420  i  440  i  460  1  1  480  500  Wavelength (nm) The precipitate obtained by 4 0 % saturation of the soluble fraction with ammonium sulfate was redissolved in potassium phosphate buffer ( 0 . 0 4 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 spectrophotometer.  169  Fig. 2 8 .  Sephadex G - 2 0 0 column chromatography  170  Fig. 29. Ultraviolet spectrum of Triton N-IOI  i.8 h  i  200  i 225  i 250  i 275  _i  300  Wavelength (nm) The absorbance of a 0.006 % (v/v) solution of Triton N-IOI in distilled water was measured with a Unicam S P 8 0 0 spectrophotometer.  171  Fig. 30  Sephadex G - 2 0 0 column chromatography  Volume NADPH dehydrogenase activity measured as the ^ g of dichlorophenolindophenol reduced per minute Absorbance, 2 8 0 nm, ( ), NHI, non-heme iron, (o o ) ; NADPH dehydrogenase activity,  (•  •).  Absorbance , 2 8 0 nm  Fig. 33.  S e p h a d e x G - 2 0 0 column  Void Volume  H  Absorbance, 2 8 0 n m , (  chromatography  Fraction  (8ml)  ); N H I , n o n - h e m e iron , ( o )  175  STUDIES ON THE PYRIDINE NUCLEOTIDE COENZYME SPECIFICITIES OF THE PROGESTERONE 17-HYDROXYLASE, 17-HYDROXYPROGESTERONE SIDE-CHAIN CLEAVAGE ENZYME AND 17£ -HYDROXYSTEROID DEHYDROGENASE OF THE RAT TESTIS (a)  Introduction Pyridine nucleotide coenzymes are u t i l i z e d as reductants i n en-  zymatic side-chain cleavage of steroid hormones as w e l l as i n enzymatic hydroxylations of these compounds (225,226).  The view i s widely held  that NADPH i s s p e c i f i c a l l y required f o r these reactions i n animal t i s sues; however, a d i s t i n c t i o n may be drawn between a requirement and a preference f o r NADPH. Many reports purporting to show a NADPH requirement have not included comparative studies with NADH ( f o r 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 l e d the authors to conclude that the reaction required NADPH, despite substantial formation of the hydroxylated  product i n incubations  performed i n the presence of NADH ( f o r example, 230).  I t has been observ-  ed that c e r t a i n reactions i n v i t r o , 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 a c t i v i t i e s of progesterone 17-hydroxylase, 17-hydroxyprogesterone sidechain cleavage enzyme and YJfi -hydroxysteroid dehydrogenase during i n cubations of t i s s u e preparations derived from rat testes performed i n the presence of NADH and NADPH.  The results demonstrate that NADPH i s  required f o r the enzymatic reduction of the 17-oxo group of androstenedione and that either NADH or NADPH may be u t 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 o l d , were used f o r a l l experiments.  The animals  were purchased from Blue Spruce Farms, Inc., Altamont, N.Y. (ii)  Tissue preparation: The rats were stunned by a blow to the head and the testes  were removed and immediately placed i n P e t r i dishes surrounded by crushed ice.  Further procedures were carried out at 4°.  Each t e s t i s was  quartered and the tissue was homogenized i n 0.15 M KC1 using a PotterElvehjem-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 lyophilized. The soluble f r a c t i o n was obtained by suspending 0.106 g of lyophilized 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. (iii)  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-"^ C-androstenedione (757,800 dpm, 1.9 V*-g) f  strate.  a s  S U D  ~  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 and subjected to paper chromatography i n the solvent 1  1  system ligroin-propylene g l y c o l 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 evaporated and the radioactive substrates were redissolved i n 0.2 ml of propylene 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 f r a c t i o n 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 f l a s k approximated that derived from two testes.  The following pyridine nu-  cleotide coenzymes were used: NADPH (Calbiochem, l o t #802207), NADH (Calbiochem, l o t #64057 and l o t #900114) and NADP (Calbiochem, l o t +  #800099). Unless otherwise stated, 7.89 u moles of NADPH or 9.17 (mmoles of NADH  ( l o t #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° f o r 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 c h i l l e d at -19° and known quantities (60-100 |m.g) of carrier steroids 1 were added. In the experiments with 3 H-progesterone as substrate, unlabeled progesterone, 17-hydroxyprogesterone, androstenedione and testosterone were added; i n the experiments with "^C-androstenedione 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°, absolute ethanol was added and the extracts were dried by azeotropic d i s tillation. Progesterone, 17-hydroxyprogesterone, androstenedione and testosterone, 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-hydroxyprogesterone 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 anhydride and chromatography was continued u n t i l constant specific a c t i v i t y was observed f o r 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, following application of the sample, gradient elution with 0 to 0.25 M ammonium bicarbonate ( t o t a l volume 3000 ml) was performed (232,233).  Paper  chromatography of NADH (lot #64057 and l o t #900114), NADPH and NADP was +  performed i n the system 0.1 M sodium phosphate buffer (pH 6.8)-solid ammonium sulfate-l-propanol (100:60:2, vol/wt/vol) (234). Compounds were located by viewing the chromatograms under short and long wave u l t r a v i o l e t light''". (vii)  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-hydroxyprogesterone side-chain cleavage enzyme a c 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 f o r losses  incurred during i s o l a t i o n procedures. Radioactivity was measured''" by l i q u 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 i n 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 c e l lulose column i s shown i n Fig. 34. One major and one minor peak i n terms of the absorbance at 340 nm were observed.  No increase i n absor-  bance above the baseline value occurred i n 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 i n Fig. 3 4 . One major and one minor peak were observed and no increase i n absorbance above the baseline value occurred i n the region where NADH was eluted. The positions of the minor peaks i n 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  L7II)  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 progressively 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-  o l i t e 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 progesterone and 17-hydroxyprogesterone was low; androstenedione and testosterone contained large percentages of the i n i t i a l substrate radioactivity. I n 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-hydroxyprogesterone; the formation of radioactive androstenedione and testosterone 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 l y o p h i l i z e d microsomes without the addition of either coenzyme; 12% 3 of the i n i t i a l substrate r a d i o a c t i v i t y was recovered as "^H-17-hydroxyprogesterone. The f r a c t i o n s from the i o n exchange chromatography of NADH ( l o t  #64057)  that contained the major peak i n terms of the absorbance a t 340  nm ( F i g . 34) were pooled and the combined f r a c t i o n was taken to dryness using a rotary evaporator a t 40°.  The residuum was dissolved i n a small  volume of d i s t i l l e d water, transferred to another v e s s e l and l y o p h i l i z a t i o n was performed.  A 7 mg sample (9.17  Kmoles) of the p u r i f i e d NADH  was then used i n an incubation of l y o p h i l i z e d microsomes with ^H-progesterone  (1,676,208  dpm,  1.2  Kg) as substrate. The results were very  s i m i l a r to those shown i n Table LIX f o r the incubation of l y o p h i l i z e d microsomes with NADH of the same l o t number that had not been subjected to ion exchange chromatography.  Results that were also very s i m i l a r to  those shown i n Table LIX f o r the incubation of l y o p h i l i z e d microsomes with NADH were observed following an incubation of l y o p h i l i z e d microsomes with %-progesterone of NADH of l o t number  (1,676,208  dpm,  1.2  Kg) as substrate and  8.47  ttmoles  900114.  3 Following incubations of the soluble f r a c t i o n with ^H-progesterone as substrate (Table LIX), the per cent of the i n i t i a l substrate r a d i o 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 r a d i o a c t i v i t y recovered as radioactive  androstenedione  and testosterone were very s i m i l a r i n incubations of the soluble f r a c t i o n with e i t h e r coenzyme.  3  In the control incubation, minimal metabolism of  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)  i n which the concentrations of the coenzymes were one-fifth and onef 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  a c 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"associated with a decline i n the a c t i v i t i e s 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 a c t i v i t i e s of the 1 7 * -hydroxylase and the side-chain cleavage enzyme were not decreased i n comparison to the a c t i v i t i e s that were observed when the incubation medium contained 0.53 M- moles of NADPH per ml. The a c t i v i t y of the 17/* - o l dehydrogenase (estimated as the r a t i o 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 lower 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 a c t i v i t i e s of the side-chain cleavage enzyme and the 17 £ - o l  dehydrogenase were decreased i n comparison to the a c 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). presence of NADPH (0.5  In incubations performed;.in the  M-moles per ml), 15$ of , substrate 21-^'C-proges;  5  terone was recovered as 17-hydroxyprogesterone and 30$ of substrate 17hydroxyprogesterone was converted to testosterone.  The r e s u l t s of i n c u -  bations of microsomes derived from rat testes with 4-"^C-17-hydroxyprogesterone as substrate i n the presence of NADH and NADPH have been r e ported by S h i k i t a and Tamaoki  (0.1  (193).  mmoles per ml) as reductant,  Following an incubation with NADH  86.6$  of the i n i t i a l substrate radio-  a c t i v i t y 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 radioa c t i v i t y of substrate "^C-17-hydroxyprogesterone was present as t e s t o s terone and 38.1$ was present as androstenedione. The results of two incubations of microsomes obtained from r a t  3 testes with  H-progesterone as substrate performed i n the presence of  NADH (0.6 mmoles per ml) are reported i n t h i s section of the thesis (Table LVIII).  In one experiment the 17*-hydroxylase a c t i v i t y was very  s i m i l a r to that observed i n incubations of microsomes performed i n the presence of NADPH (0.53  Kmoles per ml).  In the other experiment the  a c t i v i t y of the 17*-hydroxylase was considerable, but less than that observed i n the experiment with NADPH (Table LVIII).  In both experiments  with NADH, less side-chain cleavage enzyme a c t i v i t y was observed than i n  18$  comparable experiments with NADPH.  The a c t i v i t i e s of the 17* -hydroxy-  lase and the side-chain cleavage enzyme were considerably d i f f e r e n t i n the two incubations of microsomes with NADH despite attempts to maintain uniform methods of t i s s u e preparation and i d e n t i c a l conditions of i n c u 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 a l s o used f o r the control incubation) as were the microsomes employed f o r experiment #2.  In a l l incubations shown i n Table LVIII approximately the same quant-  i t y of microsomes was added t o each incubation f l a s k .  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 s i m i l a r findings observed i n the two incubations with NADPH (Table LVIII).  The range of experimental conditions under which NADH can be  u t i l i z e d as a reductant by the microsomal 17<* -hydroxylase and side-chain cleavage enzyme may be r e s t r i c t e d i n comparison to the range of conditions under which NADPH can serve as the reductant.  This suggestion may explain  the f a i l u r e of previous investigators (190,193) to observe s u b s t a n t i a l 17* -hydroxylase and side-chain cleavage enzyme a c t i 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 a t t r i b u t e d to species differences.  The observation that the t e s t i c u l a r 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 r a t testes requires NADPH (197).  dehydrogenase of  In the incubation of l y o p h i l i z e d 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 r a d i o a c t i v i t y was recovered as  186  androstenedione.  This finding supports the view  (196)  that the  20<*-  hydroxy derivative of 17-hydroxyprogesterone i s not an obligatory substrate f o r 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 a c 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 i n the incubation with NADPH a substantial portion of radioactive 17-hydroxyprogesterone was transformed to 17,20<<-dihydroxypregn-4-en3-one.  The hypothesis that the 2 0 * -reduction of 17-hydroxyprogesterone  serves as a regulatory mechanism i n androgen biosynthesis i n the rat t e s t i s has been proposed by Inano and coworkers  (235).  The effects of changes i n the concentrations of NADH and NADPH on the a c t i v i t i e s of the 17°<- -hydroxylase and the side-chain cleavage enzyme (Table LX) show that higher concentrations of NADH than NADPH are  required f o r maximum a c 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 explained as follows: 17,20*  K- moles per ml may be  0.011  minimal quantities of testosterone and (presumably)  -dihydroxypregn-4-en-3-one were present at the end of the incu-  bation period because NADPH was u t i l i z e d for 17-hydroxylation of progesterone and side-chain cleavage of 17-hydroxyprogesterone and the e q u i l 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 t o p u r i f y the androstenedione and testos-  terone following incubations with ^C-androstenedione as substrate were not assessed by c r y s t a l l i z a t i o n s of the products.  Testosterone was sub-  jected t o paper chromatography both as the f r e e compound and as the acetylated derivative and androstenedione was treated with pyridine and a c e t i c anhydride as a part of the p u r i f i c a t i o n procedure.  I t i s u n l i k e l y that  substantial quantities of impurities were present i n the testosterone acetate and androstenedione samples that displayed constant a c t i v i t y following paper chromatography. progesterone,  specific  The procedures used t o i s o l a t e  17-hydroxyprogesterone, androstenedione and testosterone 3  following incubations with  H-progesterone as substrate were assessed  by c r y s t a l l i z a t i o n s of the compounds eluted from the f i n a l paper, chromatograms (see Preliminary Experiments and Time Studies section).  In the  discussion of some of the r e s u l t s reported herein i t i s assumed that the d i r e c t i o n 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 i s established that the reaction i s r e v e r s i b l e , the i n t e r conversion of androstenedione and testosterone does not d i s p l a y the c h a r a c t e r i s t i c s of a f a c i l e equilibrium under c e r t a i n i n v i t r o conditions (216).  The experiments with %-progesterone as substrate were conduct-  ed a t pH 7.4; the i n v i t r o pH optima f o r the 17*- -hydroxylase  and the  side-chain cleavage enzyme are i n the region of 6.8 (see the previous section of t h i s t h e s i s ) .  The pH of the incubation medium may be of par-  t i c u l a r s i g n i f i c a n c e i n reactions involving oxidation and reduction of  188  pyridine nucleotide coenzymes because H or product i n these reactions.  +  i s a stoichiometric reactant  The r e s u l t s observed following incu-  bations a t pH 7.4 may be quite unlike the r e s u l t s observed at another pH. The observations held view that NADPH  reported herein may be reconciled with the widely  i s required f o r 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, phosphorylation of NADH occurred during the incubation period,  transhydro-  -tgenation of endogenous NADP  occurred during the incubation period, a  b a r r i e r that impedes s p e 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 f o r NADPH was a l t e r e d . 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 f r e e l y 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 p u r i f i e d by column i o n exchange chromatography stimulated 17<* -hydroxylase and side-chain cleavage enzyme a c t i v i t i e s t o an extent s i m i l a r t o that observed f o r the unpurified coenzyme. I t i s h i g h l y u n l i k e l y that s u f f i c i e n t ATP o r the requisite- enzymes f o r phosphorylation of NADH were present i n the microsomal preparations or i n the soluble f r a c t i o n . The experimental results with "^"C-androstenedione as substrate -Ido not eliminate the p o s s i b i l i t y that NADH -NADP  transhydrogenase a c t i v -  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 s u b s t a n t i a l amounts of NADPH were  produced during the incubations with NADH by enzymatic transfer of r e ducing equivalents from NADH t o a f r e e l y accessible pool of endogenous NADP"*" i s incompatible with the very low y i e l d of radioactive testosterone that was observed i n the incubation of l y o p h i l i z e d microsomes with NADH and -^C-androstenedione as substrate (Table LVII) or ^H-progesterone as substrate (Table LIX).  I t i s possible that s l i g h t transhydrogenase  a c t i v i t y was present that required NADP  added exogenously f o r expression.  The t o t a l per cent of i n i t i a l substrate r a d i o a c t i v i t y recovered i n each of the four experiments with "^'C-androstenedione was : c o n t r o l incubation, 101;  incubation with NADH, 9 3 ; incubation with NADH and NADP , 8 6 ; i n c u +  bation with NADPH, 7 0 .  The results suggest that the metabolism of andros-  tenedione and testosterone t o other compounds such as androstane metabol i t e s (149) proceeds a t 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 r a d i o a c t i v i t y present as androstenedione plus testosterone was l e s s i n the incubation performed with NADH and NADP"** than i n the i n c u bation with NADH alone may be explained as follows:  s l i g h t NADH - NADP  +  transhydrogenase a c t i v i t y was present; the small amount of NADPH that was formed did not influence s i g n i f i c a n t l y the NADPH + androstenedione testosterone  NADP equilibrium because of the r e l a t i v e l y 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 products other than testosterone and, therefore, the per cent of the i n i t i a l substrate r a d i o a c t i v i t y present as testosterone plus androstenedione was  190  diminished. The stimulation of 17* -hydroxylase  and side-chain cleavage  enzyme a c t i v i t i e s that was observed i n incubations with NADH may have been due to l o s s of a s e l e c t i v e NADPH transport system or to a l t e r a t i o n s i n the inherent s p e c i f i c i t i e s of the enzymes.  A comparison of the r e -  sults of the incubations of microsomes with NADH and NADPH (Table LVIII) and a comparison of the r e s u l t s of the incubations of l y o p h i l i z e d microsomes 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 f o r NADPH was greater than that of the 1 7 - h y d r o x y l a s e .  These  observations may be used as evidence against the loss of a s e l e c t i v e transport system f o r NADPH. The results of the experiments reported herein demonstrate that NADH can serve as a reductant f o r a c t i v e 17-hydroxylation of progesterone and f o r side-chain cleavage of 17-hydroxyprogesterone.  Under c e r t a i n ex-  perimental conditions, the quantity of products formed i n incubations  per-  formed i n the presence of NADH were very s i m i l a r to the quantities formed i n the presence of NADPH.  The s p e c i f i c i t i e s of the 17<* -hydroxylase and  the side-chain cleavage enzyme f o r pyridine nucleotide coenzymes that were observed are d i f f e r e n t from the s p e c i f i c i t i e s reported by others f o r the same enzymes as w e l l as f o r other s t e r o i d hydroxylases chain cleavage enzymes.  and s i d e -  The r e s u l t s reported herein may be a r t i f a c t u a l ;  i f so, a l t e r a t i o n of the i n t r i n s i c 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 r e f l e c t the s p e c i f i c i t i e s of the enzymes i n v i v o . Hayaishi,Iin a recent review o f monooxygenase-catalyzed reactions (226),  191  questions the generally held b e l i e f 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 l a tions.  SUMMARY AND CONCLUSIONS (i)  The side-chain cleavage of 17-hydroxyprogesterone  i s the rate-  l i m i t i n g reaction i n the biosynthesis of testosterone from progesterone i n the r a t t e s t i s . (ii)  17-Hydroxyprogesterone  i s present as a bound intermediate (at l e a s t  i n part). (iii)  The progesterone 17-hydroxylase and the 17-hydroxyprogesterone  side-chain cleavage enzyme 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 T r i t o n N-101. (iv)  ' > Both the 17* -hydroxylase and the side-chain cleavage enzyme i n  the soluble f r a c t i o n displayed maximal a c t i v i t y a t pH 6.8 and a t 37°. (v)  Progesterone rather than pregnenolone i s the preferred substrate f o r  the 17* -hydroxylase.  The r a t t e s t i s contains enzymes necessary to con-  v e r t pregnenolone t o progesterone.  The s p e c i f i c i t y of the 17«- -hydroxy-  lase f o r progesterone rather than f o r pregnenolone helps to explain the observation that the major route of testosterone biosynthesis from pregnenolone proceeds v i a progesterone, 17-hydroxyprogesterone tenedione rather than v i a 17-hydroxypregnenolone (vi)  and andros-  and dehydroepiandrosterone.  The soluble f r a c t i o n and the p r e c i p i t a t e obtained by 40% saturation  of the soluble f r a c t i o n with ammonium sulfate contain NADPH dehydrogenase, non-heme i r o n 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 systems analogous t o those required by the 11,8 -hydroxylase and the cholest e r o l side-chain cleavage enzyme of adrenal mitochondria, (vii)  NADH can serve as a reductant f o r active 17-hydroxylation of pro-  gesterone and f o r side-chain cleavage of 17-hydroxyprogesterone. The s p e c i f i c i t i e s of these enzymes f o r NADH and NADPH that were observed are d i f f e r e n t than the s p e c i f i c i t i e s reported by others f o r the same enzymes as w e l l as f o r other s t e r o i d hydroxylases and side-chain cleavage enzymes. The results reported herein may be a t t r i b u t e d t o a l t e r a t i o n s of the i n herent s p e c i f i c i t i e s of the enzymes.  On the other hand, the observations  reported herein may r e f l e c t the s p e c i f i c i t i e s of the enzymes i n vivo.  193  TABLE L V I I .  E f f e c t s of pyridine nucleotide coenzymes on the d i s t r i b u t i o n of r a d i o a c t i v i t y following incubations of l y o p h i l i z e d microsomes with -^C-androstenedione as substrate  Steroid isolated  Per cent of substrate r a d i o a c t i v i t y NADH  NADPH  NADP  f  NADH  3  Androstenedione Testosterone Total a  Control  88  18  82  99  5  52  4  2  93  70  86  101  The incubation medium (15 ml) contained NADH (4.8 nmoles) and NADP (4.5 v-moles). The contents of the other incubation f l a s k s are described i n the Text.  TABLE LVIII E f f e c t s of NADH and NADPH on the d i s t r i b u t i o n of r a d i o a c t i v i t y following incubation of microsomes with ^H-progesterone as substrate  Per cent of substrate r a d i o a c t i v i t y  Steroid isolated  NADH  .  a  NADPH  Control  3,  1  2  1  2  Progesterone  11  24  8  4  89  17-Hydroxyprogesterone  62  55  2  <1  5  Androstenedione  26  7  29  39  <1  <i 1  <1  40  28  <1  99  86  79  71  94  Testosterone Total  '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 Steroids isolated  Per cent of substrate radioactivity  Lyophilized microsomes NADH NADPH Control  1  Soluble fraction NADH NADPH Control  Progesterone  5  2  81  23  32  102  17-Hydroxyprogesterone  2  <1  12  68  34  2  74  21  < 1  12  4  < 1  3:  49  < 1  < 1  6  < 1  84  72  93  103  76  104  Androstenedione Testosterone Total  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 radioactiv 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 E f f e c t s of NADH and NADPH concentrations on the d i s t r i b u t i o n of r a d i o a c t i v i t y following incubations of lyophilized microsomes with ^H-progesterone as substrate  Per cent of substrate r a d i o a c t i v i t y Steroids isolated  NADH concentration ( Kmoles/ml)  NADPH concentration ( JJL moles/ml)  0.60  0.12  0.012  0.53  0.11  Progesterone  5  8  30  2  5  6  17-Hydroxyprogesterone  2  46  48  <1  <1  57  67  23  6  21  56  22  <1  < 1  49  13  1  77  84  72  74  86  Andros te nedione Testosterone Total  77  0.011  The nucleotide coenzymes were dissolved i n d i s t i l l e d water (1 ml) and added to the incubation f l a s k s . The concentrations of the coenzymes i n the incubation media at the s t a r t of the incubations are given.  197  Fig.34. 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