18]

BIOSYNTHESIS OF 5 - A N D R O S T E N E D I O L BY

HUMAN T E S T I S IN V I T R O

J . M . R o s n e r and ~I. C. M a c o m e

I n s t i t u t o L a t i n o a m e r i c a n o de Fis io log~ 'a de la R e p r o d u c c i 6 n ( I L A F I R ) C a s i l l a de C o r r e o 10, San Migue l , P c i a . de Bs . As . A r g e n t i n a

Received March Ii, 1969

A B S T R A C T

The b i o s y n t h e s i s o f a n d r o s t e n e d i o l by s e v e n n o r m a l h u m a n t e s t e s i n c u b a t e d in v i t r o w i t h l a b e l e d p r e g n e n o l o n e and d e h y d r o - e p i a n d r o s t e r o n e is d e m o n s t r a t e d in a l l the c a s e s .

W h e n e q u a l a m o u n t s of 7a( - 3 H - d e h y d r o e p i a n d r o s t e r o n e and 1 7 - ~ h y d r o x y p r o g e s t e r o n e - 4 - 1 4 C w e r e i n c u b a t e d w i th n o r m a l t e s t e s the 3 , H / 1 4 C r a t i o w a s h i g h e r in t e s t o s t e r o n e than a n d r o s t e n e - d ione (p~' 0 . 0 1 ) . T h i s s u g g e s t s the e x i s t e n c e of a p a t h w a y to t e s t o s t e r o n e p r o d u c t i o n b y p a s s i n g a n d r o s t e n e d i o n e . By i n c u b a t i n g b i o s y n t h e s i z e d a n d r o s t e n e d i o l - 14C wi th n o r m a l h u m a n t e s t e s i t s c o n v e r s i o n to t e s t o s t e r o n e in v i t r o is d e m o n s t r a t e d . The b i o l o g i c a l s i g n i f i c a n c e of t h e s e r e s u l t s is d i s c u s s e d .

I N T R O D U C T I O N

The e x i s t e n c e of two m a i n p a t h w a y s f o r the b i o s y n t h e s i s

of s t e r o i d s b y the t e s t i s ha s b e e n e s t a b l i s h e d in m a n y s p e c i e s

including man (I) (g) (3) (4) (5).

The ~ 5-3/ /~ ol p a t h w a y t h r o u g h p r e g n e n o l o n e , 1 7 ~ O H -

h y d r o x y - p r e g n e n o l o n e and d e h y d r o e p i a n d r o s t e r o n e ha s l a t e l y r e c e i v e d

a g r e a t d e a l of a t t en t i on , and in the r a b b i t and m o n k e y it h a s b e e n

d e m o n s t r a t e d tha t d e h y d r o e p i a n d r o s t e r o n e is f u r t h e r c o n v e r t e d to

a n d r o s t e n e d i o l , a n o t h e r s t e r o i d wi th the ~ 5 - 3 / ~ ol c o n f i g u r a t i o n (5)

(6) (7).

182 S T E R 0 I D S 15:1

The b i o s y n t h e s i s of 5 - a n d r o s t e n e d i o l b y n o r m a l and a b n o r m a l

h u m a n t e s t e s h a s b e e n s c a r c e l y e x p l o r e d and c o n s t i t u t e s a m a t t e r

of c o n t r o v e r s y (8) (9) (10).

F u r t h e r m o r e , i t has no t y e t b e e n d e m o n s t r a t e d tha t

a n d r o s t e n e d i o l ' c a n be c o n v e r t e d to t e s t o s t e r o n e by n o r m a l o r a b n o r m a l

h u m a n t e s t e s b e c a u s e no t i m e e x p e r i m e n t s o r i n c u b a t i o n s w i th

a n d r o s t e n e d i o l as s u b s t r a t e h a v e b e e n p e r f o r m e d .

In th i s p a p e r we h a v e i n c u b a t e d s e v e n n o r m a l t e s t e s w i th l a b e l e d

p r e g n e n o l o n e and d e h y d r o e p i a n d r o s t e r o n e and the p r o d u c t i o n of

a n d r o s t e n e d i o l w a s d e m o n s t r a t e d in e v e r y one of the p a t i e n t s and,

w h e n h u m a n t e s t e s w e r e i n c u b a t e d wi th b i o s y n t h e s i z e d a n d r o s t e n e d i o l

the p r o d u c t i o n of t e s t o s t e r o n e f r o m a n d r o s t e n e d i o l w a s shown.

MATERIAL AND METHODS

S e v e n h i s t o l o g i c a l l y n o r m a l h u m a n t e s t e s o b t a i n e d f r o m 57 to 72 y e a r s old o r c h i e c t o m i z e d p a t i e n t s due to p r o s t a t i c c a r c i n o m a w e r e p r e p a r e d fo r be ing h o m o g e n i z e d by r e m o v i n g fa t , t u n i c a a l b u g i n e a , e p i d i d y m u s and e p i d i d y m a l fa t pad . The t e s t e s w e r e k e p t on i ce and l e s s than an h o u r l a t e r t h e y w e r e i n c u b a t e d . The t e s t e s w e r e w e i g h e d , cu t in s m a l l p i e c e s , h o m o g e n i z e d in a L o u r d e s M M - I A m e d h a n i c a l h o m o g e n i z e r in the cold . S u f f i c i e n t 0. 1 M p h o s p h a t e b u f f e r , pH 7 . 4 w a s a d d e d to m a k e a 10 % c o n c e n t r a t i o n of the h o m o g e n a t e .

P r e g n e n o l o n e - 4 - 14C s p e c i f i c a c t i v i t y 50 m C i / m M , d e h y d r o - e p i a n d r o s t e r o n e - 4 14C s p e c i f i c a c t i v i t y 50 m C i / m M , d e h y d r o e p i - a n d r o s t e r o n e 7 ~ 3 H s p e c i f i c a c t i v i t y 10 C i / m M , 1 7 ~ h y d r o x y - p r o g e s t e r o n e - 4 - 14C s p e c i f i c a c t i v i t y 50 m C i / m M ; and a n d r o s t e n e d i o l 14C s p e c i f i c a c t i v i t y 35 m C i / m M w e r e e m p l o y e d as s u b s t r a t e s , in the fo l lowing e x p e r i m e n t s .

The s u b s t r a t e s w e r e c h r o m a t o g r a p h e d in W h a t m a n p a p e r ~ 1 in s y s t e m E and r e c r y s t a l l i z e d to c o n s t a n t s p e c i f i c a c t i v i t y . Whole t e s t e s h o m o g e n a t e s w e r e u s e d in e a c h i n c u b a t i o n f l a s k and A T P (6 .56 , ,u m o l e s ) . N A D P (3 .60 .,~ m o l e s ) NAD (3 .77 ~ m o l e s ) g l u c o s e - 6 - p h o s p h a t e (9 .63 ,lu m o l e s and 50 ,~ag g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e w e r e a d d e d to

Jan. 1970 s T E R O I D S 183

e a c h f l a s k . The f i na l v o l u m e of 3 m l w a s i n c u b a t e d fo r two h o u r s a t 37 ° C u n d e r a i r in a Dubnoff m e t a b o l i c s h a k e r . The i n c u b a t i o n w a s s t o p p e d by the a d d i t i o n of 5 ~ of d i e t h y l e t h e r .

The h o m o g e n i z e d t i s s u e was e x t r a c t e d fou r t i m e s w i t h 30 m l of d i e t h y l e t h e r . The c o m b i n e d e t h e r e x t r a c t s w e r e w a s h e d t h r e e t i m e s wi th Na OH and wi th w a t e r un t i l n e u t r a l i t y . The w a s h e d e t h e r e x t r a c t was f i l t r a t e d o v e r s o d i u m s u l p h a t e and b r o u g h t to d r y n e s s u n d e r r e d u c e d p r e s s u r e .

The r a d i a c t i v i t y of the s t e r o i d s a m p l e was coun t ed in a l i qu id s c i n t i l l a t i o n c o u n t e r , P a c k a r d I n s t r u m e n t s Co. Model 314 X, u s i n g 10 m l s c i n t i l l a t i o n f lu id con t a in ing 4 g Z. 5 d e p h e n y l o x a z o l e and 4o mg p-bis Z-15-Z S phenyloxazolyl)) ben-one / 1 to luene The e f f i c i e n c y f o r 4 C w a s 67% and fo r 3H was 38 %. When a double i s o t o p e t e c h n i q u e was u s e d the e f f i c i e n c y f o r 14C was 45 % and fo r 3H was 16 %.

The S t u d e n t ' s T'tv' t e s t was u s e d fo r the s t a t i s t i c a l a n a l y s i s of the r e s u l t s .

EXPERIMENTS

1) Incuba t i on wi th p r e g n e n o l o n e = 4 - 1 4 C : to the d r y e x t r a c t w e r e a d d e d 100 jug e a c h of p r o g e s t e r o n e , a n d r o s t e n e d i o n e , t e s t o s t e r o n e and

a n d r o s t e n e d i o l ; the e x t r a c t was app l i ed to W h a t m a n p a p e r ~ 1 and c h r o m a t o g r a p h e d in s y s t e m B fo r t h r e e h o u r s . The s t r i p s w e r e e x a m i n e d fo r r a d i a c t i v i t y in a N u c l e a r Ch i cago A c t i g r a p h III and the c a r r i e r s t a n d a r d s w e r e l o c a t e d by an u l t r a v i o l e t s c a n n e r . Z) I n c u b a t i o n w i t h d e h y d r o e p i a n d r o s t e r o n e - 4 - 14C: the p r o c e d u r e w a s p e r f o r m e d e x a c t l y as in E x p e r i m e n t 1, e x c e p t t h a t no p r o g e s t e r o n e s t a n d a r d was added . 3) I n c u b a t i o n wi th 7 ~ - 3 H d e h y d r o e p i a n d r o s t e r o n m and 17 ~ - h y d r o x y -

p r o g e s t e r o n e - 14C ; h u m a n t e s t e s h o m o g e n a t e s w e r e i n c u b a t e d w i t h 1 #ug e a c h of 7 ~ - 3 H - d e h y d r o e p i a n d r o s t e r o n e and 1 7 ~ - h y d r o x y - p r o g e s t e r o n e - 4 - 14(3.

E n o u g h u n l a b e l e d d e h y d r o e p i a n d r o s t e r o n e w a s a d d e d to the t r i t i a t e d one to a c h i e v e a s p e c i f i c a c t i v i t y of 1 ~t~g= 3 .8 X 106 d . p . m .

The i n i t i a l r a t i o of the s u b s t r a t e a d d e d to the i n c u b a t i o n w a s 11.5 3H/14(3. 4) I ncuba t i on w i t h 5 = a n d r o s t e n e d i o l 14(3 : by i ncuba t i ng r a b b i t t e s t e s

w i t h d e h y d r o e p i a n d r o s t e r o n e - 4 - 1 4 C i t was o b t a i n e d b i o s y n t h e s i z e d 5- a n d r o s t e n e d i o l 14C t h a t was p u r i f i e d by p a p e r c h r o m a t o g r a p h y in s y s t e m s D; A ; F and E.

The s p e c i f i c a c t i v i t y of the b i o s y n t h e s i z e d a n d r o s t e n e d i o l 14 C w a s 35 m C i / m M and was d e t e r m i n e d by gas C h r o m a t o g r a p h y u s i n g a

184 S T E R O I D S 15:1

P a c k a r d gas c h r o m a t o g r a p h wi th a f r a m e i o n i z a t i o n d e t e c t o r .and coun t ing the r a d i a c t i v i t y in a P a c k a r d l iqu id s c i n t i l l a t i o n C o u n t e r .

A f t e r e s t a b l i s h i n g i ts r a d i o c h e m i c a l p u r i t y by r e c r y s t a l l i z a t i o n to c o n s t a n t s p e c i f i c a c t i v i t y the p u r i f i e d 1 4 C - a n d r o s t e n e d i o l was i n c u b a t e d w i t h h u m a n t e s t e s , to p r o v e i f a n d r o s t e n e d i o l was c o n v e r t e d to t e s t o s t e r o n e .

T A B L E I

Composition of C h r o m a t o g r a p h i c S y s t e m s Used

S y s t e m Compositionby v o l u m e R e f e r e n c e

'A B C D E F

H e x a n e / F o r m a m i d e 13 H e x a n e - B e n z e n e ( 1 : 1 ) / F o r m a m i d e 13 H e p t a n e / F o r m a m i d e 13 T o l u e n e / P r o p y l e n e g l y c o l 13 C y c l o h e x a n e - m e t h a n o l - w a t e r ( 100:100:10) 14 Bush "A" ( Petroleum-ether-methanol- w a t e r 100: 8 0 : 2 0 ) 15

IDENTIFICATION OF FORMED COMPOUNDS

A n d r o s t e n e d i o l

The m a t e r i a l behav ing l ike t e s t o s t e r o n e and a n d r o s t e n e d i o l in the f i r s t c h r o m a t o g r a p h y was c h r o m a t o g r a p h e d in s y s t e m D f o r f o u r hours .

A redloactive area, that had the same Rf as the authentic a n d r o s t e n e d i o l , w a s found. The a n d r o s t e n e d i o l s t a n d a r d was l o c a l i z e d by expos ing the c h r o m a t o g r a p h i c s t r i p s to iod ine v a p o r s .

The m a t e r i a l behav ing l ike a n d r o s t e n e d i o l was c h r o m . a t o g r a p h e d in s y s t e m s E and F f o r f ive and t e n h o u r s r e s p e c t i v e l y .

An a l i q u o t of th i s m a t e r i a l w a s a c e t y l a t e d wi th a c e t i c a n h y d r i d e in p y r i d i n e ( 1:5, V:V) and c h r o m a t o g r a p h e d in s y s t e m A fo r 3½ h o u r s , A u t h e n t i c a n d r o s t e n e d i o l d i a c e t a t e was c h r o m a t o g r a p h e d in a s e p a r a t e s t r i p , and had i d e n t i c a l Rf as the unknown s a m p l e .

The m a t e r i a l behav ing l ike a n d r o s t e n e d i o l d i a c e t a t e was e lu t ed , e v a p o r a t e d to d r y n e s s and d i s s o l v e d in 1 m l of 0 .1 N e t h a n o l i c s o d i u m h y d r o x y d e and i n c u b a t e d a t 37 ° C f o r 30 m i n u t e s .

F i v e m l of w a t e r w e r e a d d e d and e x t r a c t e d wi th f ive v o l u m e s of e t h y l a c e t a t e . The e t h y l a c e t a t e e x t r a c t was c h r o m a t o g r a p h e d in s y s t e m C f o r t w e l v e h o u r s , and b e h a v e d l ike a n d r o s t e n e d i o l s t a n d a r d . The m a t e r i a l behav ing l ike a n c l r o s t e n e d i o l was r e c r y s t a l l i z e d to c o n s t a n t s p e c i f i c a c t i v i t y f r o m t h r e e d i f f e r e n t s o l v e n t s .

Jan. 1970 s T E R O I D S 185

T e s t o s t e r o n e

A f t e r the c h r o m a t o g r a p h y in s y s t e m B w h e r e t e s t o s t e r o n e has the s a m e Rf as a n d r o s t e n e d i o l , the a r e a was c h r o m a t o g r a p h e d in s y s t e m D f o r fou r h o u r s . The r a d i a c t i v e a r e a c o r r e s p o n d i n g to the a d d e d t e s t o s t e r o n e s t a n d a r d w a s r e c h r o m a t o g r a p h e d in s y s t e m F f o r e igh t h o u r s . An a l i quo t was a c e t y l a t e d and c h r o m a t o g r a p h e d in s y s t e m A f o r t h r e e h o u r s . A n o t h e r a l i quo t was o x i d i z e d w i t h 0. Z rnl 0 .5 % c h r o m i u m t r i o x i d e in 95 % g l a c i a l a c e t i c a c i d fo r two h o u r s in the d a r k a t r o o m t e m p e r a t u r e . The o x i d i z e d m a t e r i a l was c h r o m a t o g r a p h e d in s y s t e m A f o r t w e n t y f o u r h o u r s and i t b e h a v e d l ike a u t h e n t i c a n d r o s t e n e d i . o n e s t a n d a r d c h r o m a t o g r a p h e d in a s e p a r a t e s t r i p .

A n o t h e r a l i quo t of the m a t e r i a l behav ing l ike t e s t o s t e r o n e w a s r e c r y s t a l l i z e d to c o n s t a n t s p e c i f i c a c t i v i t y f r o m at l e a s t t h r e e d i f f e r e n t s o l v e n t s un t i l the s p e c i f i c a c t i v i t y did not v a r y m o r e t h a n 5 90 b e t w e e n two r e c r y s t a l l i z a t i o n s .

Androstenedione

M a t e r i a l b e h a v i n g l ike d e h y d r o e p i a n d r o s t e r o n e and a n d r o s t e n e d i o n e in s y s t e m B was r e c h r o m a t o g r a p h e d in s y s t e m F f o r e igh t h o u r s and the a n d r o s t e n e d i o n e a r e a was l o c a t e d by u l t r a v i o l e t a b s o r p t i o n and the d e h y d r o e p i a n d r o s t e r o n e zone by iod ine v a p o r s . The m a t e r i a l hav ing the s a m e Rf as a u t h e n t i c a n d r o s t e n e d i o n e was a c e t y l a t e d and r e c h r o m a t o g r a p h e d in s y s t e m A f o r t h r e e h o u r s w h e r e it b e h a v e d as a n d r o s t e n e d i o n e s t a n d a r d . Th i s m a t e r i a l was o x i d i z e d and c h r o m a t o g r a p h e d in s y s t e m D f o r 3½ h o u r s and had the s a m e Rf as a u t h e n t i c a n d r o s t e n e d i o n e .

Th i s m a t e r i a l w a s r e c r y s t a l l i z e d to c o n s t a n t s p e c i f i c a c t i v i t y f r o m t h r e e d i f f e r e n t s o l v e n t s un t i l the s p e c i f i c a c t i v i t y d id not change m o r e t han 5 90 b e t w e e n two r e c r y s t a l l i z a t i o n s .

RESULTS AND DISCUSSION

The " in vitro" and " in vivo" data on androstenediol

biosynthesis by human testes are very few and controversial,

because the isolation of androstenediol from normal and pathological

testes was obtained by some researchers and not by others (8) (9) (I0)

(11).

186 S T E R 0 1 D S 15:1

R E C R Y S T A L L I Z A T I O N TO CONSTANT S P E C I F I C ACTIVITY

TABLE II ANDR OS TENE DIOL

C r y s t a l l i z a t i o n No. Solven t Spec i f i c activity (dpm/m~) Mother liquor crystals

1 E t h a n o l 1, 123 1, 115 Z A c e t o n e 897 903 3 M e t h a n o l 9 Z5 915

F i f t e e n m i l l i g r a m s of a u t h e n t i c a n d r o s t e n e d i o l w e r e a d d e d to the unknown p r e v i o u s to c r y s t a l l i z a t i o n .

T A B L E III TES T OS TER ON E

Crystallization No. Solven t Spec i f i c a c t i v i t y M o t h e r l i quo r

( d p m / m $ ) c r y s t a l s

1 M e t h a n o l 1 ,543 1,429 2 A c e t o n e I, 482 1 ,470 3 E t h a n o l 1 ,489 1,477

F o u r t e e n m i l l i g r a m s of a u t h e n t i c t e s t o s t e r o n e w e r e a d d e d to the unknown p r e v i o u s to c r y s t a l l i z a t i o n .

T A B L E IV ANDROS TENEDIONE

C r y s t a l l i z a t i o n No. So lven t Spec i f i c a c t i v i t y ( d p m / m ~ ) M o t h e r l i q u o r c r y s t a l s

1 M e t h a n o l 1 ,770 1,253 Z Hexane - c h l o r o -

f o r m 3:1 1 ,514 1, 102 3 E t h a n o l 1, 109 1, 115 4 M e t h a n o l 1, 10Z 994

Ten m i l l i g r a m s of a u t h e n t i c a n d r o s t e n e d i o n e w e r e added to the unknown p r e v i o u s to c r y s t a l l i z a t i o n .

Jan. 1970 s T E R O I D S 187

In th i s r)aper it is d e m o n s t r a t e d t ha t a n d r o s t e n e d i o l was

p r o d u c e d " in v i t r o " in s e v e n out of s e v e n n o r m a l t e s t e s i n c u b a t e d

e i t h e r wi th l a b e l e d p r e g n e n o l o n e or d e h y d r o e p i a n d r o s t e r o n e . (Table

V and VI).

When equa l a m o u n t s of 7o<3 H - d e h y d r o e p i a n d r o s t e r o n e and

17- ~( - h y d r o x y p r o g e s t e r o n e - 4 - 14C w e r e i n c u b a t e d wi th n o r m a l

t e s t e s and the 3 H / 1 4 C r a t i o d e t e r m i n e d in a n d 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 , h i g h e r r a t i o s of 3 H t o 14C w e r e found in the l a t t e r

s t e r o i d (p~0.01).

This e x p e r i m e n t a c t u a l l y i nvo lved t h r e e p a t h w a y s to t e s t o s t e -

rone .

1- 1 7 ~ - h y d r o x y p r o g e s t e r o n e - ~ a n d r o s t e n e d i o n e - ) t e s t o s t e r o n e .

Z- d e h y d r o e p i a n d r o s t e r o n e - ~ a n d r o s t e n e d i o n e - ) t e s t o s t e r o n e .

3- d e h y d r o e p i a n d r o s t e r o n e - 9 a n d r o s t e n e d i o l - ) t e s t o s t e r o n e .

If a n d r o s t e n e d i o n e would be an o b l i g a t o r y i n t e r m e d i a t e f o r

the b i o s y n t h e s i s of t e s t o s t e r o n e f r o m d e h y d r o e p i a n d r o s t e r o n e as i t

is f r o m 1 7 - ~ ( - h y d r o x y p r o g e s t e r o n e the 3 H / 14C r a t i o shou ld be the

s a m e in a n d r o s t e n e d i o n e than in t e s t o s t e r o n e .

The ob t a ined h i g h e r r a t i o s of 3 H / 14C in t e s t o s t e r o n e than in

a n d r o s t e n e d i o n e only s u g g e s t the e x i s t e n c e of a n o t h e r p a t h w a y to

t e s t o s t e r o n e b y p a s s i n g a n d r o s t e n e d i o n e , but b r i n g s no e v i d e n c e to

the r e l a t i v e i m p o r t a n c e of t h e s e p a t h w a y s .

188 S T E R O I D S 15:1

A s t h e p r e s e n c e of a n d r o s t e n e d i o l ]~y i t s e l f i s no t s u f f i c i e n t .

e v i d e n c e t o p r o v e t h a t t h i s s t e r o i d i s a c t u a l l y c o n v e r t e d t o t e s t o s t e r o n e

b i o s y n t h e s i z e d a n d r o s t e n e d i o l w a s i n c u b a t e d w i t h h u m a n t e s t e s " in

v i t r o " a n d t h e p r o d u c t i o n of t e s t o s t e r o n e w a s d e m o n s t r a t e d ( T a b l e V I I I )

Yamayi et al. (12) have recently identified androstenediol in canine

spermatic vein blood and demonstrated its increase after LH

( l u t e i n i z i n g h o r m o n e ) a d m i n i s t r a t i o n .

The present "in vitro" results could not be extrapolated to

" in vivo" situations, they only indicate the presence of the necessary

enzymes for the biosynthesis of androstenediol and its conversion to

testosterone. Further investigations are being done in human males

" in vivo" to see if these findings could be reproduced under these

circumstances and time experiments are performed " in vitro" to

establish the importance of the conversion of androstenediol to

testosterone.

Jan. 1970 s T E R O I D S 189

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Jan. 1970 s T E R O I D S 191

TAB L E VII

3- INCDBATION OF HUMAN TESTIS HOMOGENATES* WITH 7 ¢~ 3 H DEH3 DROEPIANDROSTERONE AND 17 o~ - H~/DROX ~] - PROGESTERONE-4- 14 C

P a t i e n t No. T e s t o s t e r o n e *** A n d r o s t e n e d i o n e

d p m 3 H 3H/14C ra t i o** d p m 3H 3 H / 1 4 C r a t i o** d p m 14C d p m 14C

1 760,000_ 15 247,900__ 4 51 ,300 58 ,300

843 000 168,800 ' = 23 : 5

37,000 35,800

3 719 ,000 557 ,000 15 25?600 = 28 37 ,300 =

4 745,000 = 23 102,700 32,400 22,300

5 790,.000__ 16 89,00______~0__ 3 50,900 33,700

6 830,000= 30 596,600= 27,800 43,200

14

7 676 ,000 22 506 ,600 31,000: 84,700" 6

* F i v e h u n d r e d m i l l i g r a m s of t e s t i c u l a r t i s s u e , 1 ug d e h y d r o - e p i a n d r o s t e r o n e - 7 ¢ ~ - 3 H , and 1 ug 1 7 - ~ 4 - h y d r o x y p r o g e s t e r o n e - 4 - 1 4 C w e r e u s e d in e a c h i n c u b a t i o n f l a s k . ** The i n i t i a l 3H / 1 4 C r a t i o was 11.5 *** The d i f f e r e n c e in the 3 H / 1 4 C r a t i o s b e t w e e n t e s t o s t e r o n e and a n d r o s t e n e d i o n e was s i g n i f i c a n t for p < 0 . 0 1 .

192 S T E R O I D S 15:1

T A B L E VII I

4 - I N C U B A T I O N O F H U M A N T E S T I S H O M O G E N A T E S * W I T H B I O - S Y N T H E S I Z E D - 1 4 C A N D R O S T E N E D I O L ( S . A . 35 m C i / m M )

P R O D U C T I O N O F T E S T O S T E R O N E **

P a t i e n t No . T e s t o s t e r o n e 1 15 2 8 B 11 4 19 5 27 6 17 7 6

* T h r e e h u n d r e d m i l l i g r a m s of t e s t i c u l a r t i s s u e a n d 5X 10 4 d p m of 14 C a n d r o s t e n e d i o l w e r e u s e d in e a c h f l a s k . ** R e s u l t s a r e e x p r e s s e d in d p m / m g t i s s u e .

* T h e f o l l o w i n g t r i v i a l n a m e s a n d a b b r e v i a t i o n s w e r e u s e d in t h i s p a p e r : P r o g e s t e r o n e : 4 - p r e g n e n e , 3 - 2 0 , d i o n e .

P r e g n e n o l o n e : 3 ~ - h y d r o x y - 5 - p r e g n e n e - 2 0 one .

17 ¢ ~ - h y d r o x y - p r o g e s t e r o n e : 17 ~ h y d r o x y , 4 - D r e g n e n e , 3 - 2 0 , d i o n e

D e h y d r o e p i a n d r o s t e r o n e : 3 /3 - h y d r o x y - 5 - a n d r o s t e n - 17 o n e .

A n d r o s t e n e d i o l : 5 - a n d r o s t e n e - 3 / ~ , 17 ] ~ - d i o l .

T e s t o s t e r o n e : 17 //3 - h y d r o x y - 4 - a n d r o s t e n - 3 one .

A n d r o s t e n e d i o n e : 4 - a n d r o s t e n b B , 17 d i o n e .

N A D : N i c o t i n a m i d e A d e n i n e D i n u c l e o t i d e .

N A D P : N i c o t i n a m i d e A d e n i n e D i n u c l e o t i d e P h o s p h a t e

A T P : A d e n o s i n e 5 T r i p h o s p h a t e .

A C K N O W L E D G E M E N T S

T h i s w o r k w a s s u p p o r t e d by a g r a n t of t h e F o r d F o u n d a t i o n .

Jan. 1970 s T E R O I D S 193

REFERENCES

(1) KAHNT, F . W . , N E H E R , K . , SCHMID and W E T T S T E I N E x ~ e r i e n t i a , 17: 19, (1961).

(2) E I K - N E S , K . B . and K E K R E , M. , B i o c h i m . B i o o h y s . A c t a 78: 449, ( 1 9 6 3 )

¢3) ELLIS, L.C. and BERLINGER, D.L., Endocrinology 76:591

(1965)

(4) DAVID, R . R . , W IENER, M . , ROSS, L . , and LANDAU, R . L . , 5. Cl in . E n d o c r i n o l o g y & Metab . 2 5 : 1 3 9 3 (1965)

(5) TAMAOKI, B . I . and SHIKITA, M. , S t e r o i d D y n a m i c s , e d i t e d by G. PINCUS, T. NAKAO and J . F . TAIT , A c a d e m i c P r e s s , New Y o r k . p. 493 (1966)

(6) HOSCHOIAN, J.C. and BROWNIE, A.C. , Steroids I_O0:49 (1967)

(7) ROSNER, J. M., HORITA, S. and FORSHAM, .P.H., Endocrinology

75: Z99, (1964)

(8) NEHER, K. , KAHNT, F. W. , Ghent symposium, June 1965, p. 130

Excerpta Medica Foundation.

(9) AXELROD: Bioch. Biophys, Acta 97:551 (1965)

(I0) GUAL, C. , SANCHEZ, J. , DORFMAN, R. and ROSENTHAL, J. Clin. Endocrin. ZZ: 1040, (1962)

I.

(I 1 ) KNAPSTEIN, P. , WENDLBERGER, F. , G. and TOUCHSTONE, J.C., Steroids

MENZEL, P., OERTEL, Iz:Igl (1968)

(12) YAMAJI, T. , MOTOHASHI, K. , TANIOKA, T. and IBAYASHI, H.

Endocrinology 83:99Z (1968)

(13) ZAFFARONI, A. Recent Progr. Hormone Res. 8:51 (1953)

(14) PETERSON, R.E. Personal Communication . (1961)

(15) BUSH, I.E. Biochem. ff. 50:370 (195Z)