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The conversion of 1,3-N15-,2-c14-adenine to polynucleotide adenine and guanine in the adult male rat Tomlinson, Raymond Valentine 1956

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TO POLYNUCLEOTIDE ADENINE AND GUANINE IN THE ADULT MALE RAT  by Raymond Tomlinson B.A., U n i v e r s i t y o f B r i t i s h Columbia, 1954.  A Thesis submitted i n P a r t i a l F u l f i l m e n t of the r e q u i r e m e n t s f o r t h e degree o f Master o f S c i e n c e i n t h e Department of Biochemistry.  We a c c e p t t h i s t h e s i s as conforming t o t h e required standard  The u n i v e r s i t y o f B r i t i s h October 1956.  Columbia  In presenting  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  the r e q u i r e m e n t s f o r an advanced degree a t t h e  University  of 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 freely  a v a i l a b l e f o r r e f e r e n c e and  agree t h a t p e r m i s s i o n f o r e x t e n s i v e t h e s i s f o r s c h o l a r l y purposes may of my  study.  I further  copying of  this  be g r a n t e d by the Head  Department o r by h i s r e p r e s e n t a t i v e .  I t i s under-  stood 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 t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my permission.  Department The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada.  written  ABSTRACT. 15 1,3-N administered  14 -,2-C  -adenine was s y n t h e s i z e d and  t o r a t s by i n t r a p e r i t o n e a l i n j e c t i o n .  The  compound was i n c o r p o r a t e d as b o t h adenine and guanine i n t o the v i s c e r a l n u c l e i c a c i d s .  Comparison o f t h e C  1 4  /N^ ratios  found i n t h e adenine and guanine i s o l a t e d from t h e v i s c e r a l n u c l e i c a c i d s w i t h t h a t i n t h e i n j e c t e d adenine i n d i c a t e d t h a t 14 f o r b o t h compounds t h e r e had been a l o s s o f C i n the C^/N^  5  .  The decrease^  r a t i o o f t h e p o l y n u c l e o t i d e guanine was much  greater than that i n t h e p o l y n u c l e o t i d e adenine.  The i n f e r -  ence drawn from t h i s marked d i f f e r e n c e was t h a t on a t l e a s t one pathway f o r t h e c o n v e r s i o n o f adenine t o guanine t h e 14 p u r i n e n u c l e u s does n o t remain i n t a c t . The l a b i l e 2-C was 14 found a s C Og i n t h e a n i m a l s ' e x p i r e d a i r . E v i d e n c e t h a t 14 p a r t o f t h e C Og r e s u l t e d from p u r i n e i n t e r c o n v e r s i o n was a f f o r d e d b y examining t h e a l l a n t o i n e x c r e t e d i n t h e a n i m a l s 14 15 u r i n e . T h i s compound had a C /N r a t i o consistent with the source from w h i c h i t was d e r i v e d , t h e n u c l e i c a c i d p u r i n e s . 14. 14 1^ The d e f i c i t o f C , apparent from t h e C /N ratio, i n this e x c r e t o r y p r o d u c t accounted f o r 47% o f t h e C 0 e x p i r e d b y the a n i m a l s . 1  g  ACKNOWLEDGEMENTS  The author w i s h e s t o e x p r e s s h i s a p p r e c i a t i o n o f the encouragement, a d v i c e , and p e r s o n a l a s s i s t a n c e i n t h e l a b o r a t o r y g i v e n by Dr. S. H. Zbarsky d u r i n g t h e course o f t h i s research. T h i s r e s e a r c h was supported b y a g r a n t from t h e N a t i o n a l Research C o u n c i l .  TABLE OF CONTENTS. Page 1  INTRODUCTION EXPERIMENTAL A.  Chemical (1)  Synthesis foreword i) 2-C adenine ii) 1,3-N:~-adenine iii) l , 3 - N - , 2 - C - a d e n i n e .......  10 10 11 11  Proof il ii) iii)  11 12  1 4  T  ± 0  (2)  (3)  X 4  of I d e n t i t y . C r i t e r i a of Purity chromatography spectrophotometry d e r i v a t i v e and i t s m e l t i n g point  Measurement o f I s o t o p e C o n c e n t r a t i o n il C~r d e t e r m i n a t i o n ............ ii; N determination 1 5  B.  (1)  Technique o f Metabolism  (2)  Treatment o f Samples general i ) t i s s u e ....................... ii) acid-soluble fraction iii) c a r b o n d i o x i d e ............... iv) urine ................. v) f a e c e s ....................... blood  Experiment I Results  D.  15 16  Metabolic  vi) C.  12  Experiment I I Results  Experiments.  16 17 17 19 20 21 22 22 22 23 24 25  DISCUSSION ..  31  SUMMARY  45  BIBLIOGRAPHY  48  TABLES I.  Number and n a t u r e o f t h e samples saved i n 'Experiments I and I I .......................... II. C and N contents of the v i s c e r a l n u c l e i c a c i d p u r i n e s i n Experiment I ..•,.« III. C and N contents of the v i s c e r a l n u c l e i c a c i d p u r i n e s i n Experiment I I IV. C " c o n t e n t o f t h e e x p i r e d carbon d i o x i d e i n Experiment I I V. C** c o n t e n t o f t h e u r i n e i n Experiment I I ..... VI. C and N c o n t e n t o f t h e u r e a i n Experiment I I ,,.,..,..>.......,«..........«.....£........ V I I . fo i n c o r p o r a t i o n o f t h e i n j e c t e d 1,3-N - , 2 - C adenine i n t o t h e v i s c e r a l n u c l e i c a c i d s , E x p e r i m e n t s I and I I , e x p r e s s e d i n terms o f C , N and C / N ratios 1 4  1 4  -  1  Page  17 23 25 26 27  l 0  A4  1 5  1 4  1 5  28  32  FIGURES 1. 2. 3. 4.  C o n t a c t p r i n t o f chromatogram used t o v e r i f y t h e i d e n t i t y o f t h e s y n t h e t i c adenine A comparison o f t h e u l t r a v i o l e t a b s o r p t i o n c u r v e s o f a u t h e n t i c and s y n t h e t i c a d e n i n e . . . . . . A comparison between t h e e x c r e t i o n s o f C 0 g and C - U r e a i n Experiment I I ................. Account o f t h e d a i l y i n j e c t e d C ^ as t h e amount e x p i r e d o r e x c r e t e d and t h e amount r e t a i n e d by the a n i m a l s i n Experiment I I .................. 14:  1 4  1  13 14 39 44  INTRODUCTION The q u e s t i o n o f how p u r i n e b a s e s a r e i n t e r c o n v e r t e d i s a problem t h a t has o c c u p i e d years. (1).  i n v e s t i g a t o r s f o r the past t e n  I t s o r i g i n i s t o be found i n t h e work o f Schoenheimer When i s o t p p e s became r e a d i l y a v a i l a b l e i n t h e e a r l y war  y e a r s Schoenheimer, Buchanan, Hevesy and o t h e r s began e x p e r i m e n t a t i o n on t h e b i o s y n t h e s i s o f n u c l e i c a c i d s and t h e i r component p u r i n e and p y r i m i d i n e b a s e s .  These i n v e s t i g a t i o n s have  c l a r i f i e d many o f t h e q u e s t i o n s c o n c e r n i n g  possible precursor  m o l e c u l e s f o r t h e v a r i o u s atoms i n t h e p u r i n e n u c l e u s  during  t h e course o f »de novo' s y n t h e s i s w i t h i n t h e body. I n 1944, Schoenheimer and P l e n t l (3) p u b l i s h e d t h e • r e s u l t s o f i n v e s t i g a t i o n s w h i c h had extended t h e work beyond t h e s e a r c h f o r p r e c u r s o r compounds t o t h e u t i l i z a t i o n o f t h e f r e e bases t h e m s e l v e s . aware o f M i e s c h e r ' s  A l t h o u g h t h e s e i n v e s t i g a t o r s were  e a r l y o b s e r v a t i o n t h a t preformed p u r i n e s  o r p y r i m i d i n e s were n o t e s s e n t i a l f o r t h e s y n t h e s i s o f n u c l e i c a c i d s ( 2 ) , i t seemed w o r t h w h i l e  t o them t o d e t e r m i n e  whether t h e f r e e bases would be u t i l i z e d f o r t h e s y n t h e s i s o f n u c l e i c a c i d s when f e d t o an a n i m a l . T h e i r e x p e r i m e n t s i n c l u 15 15 ded t h e f e e d i n g o f 1,3-N -, 2 amino-N -guanine as w e l l as t h e l a b e l l e d p y r i m i d i n e s , thymine and u r a c i l , t o r a t s . t h e b a s i s o f t h e r e s u l t s o b t a i n e d from t h e s e Schoenheimer and P l e n t l s t a t e d :  On  experiments,  "Neither purines nor p y r i -  m i d i n e s s u p p l i e d i n t h e d i e t a r e u t i l i z e d by t h e body f o r t h e synthesis of nucleoprotein" (3).  - 2  -  Because t h i s appeared t o be an e x c e p t i o n t o t h e dynamic concept o f metabolism, Brown and h i s co-workers ( 4 ) , i n 1947, r e i n v e s t i g a t e d t h e p o s s i b l e u t i l i z a t i o n o f f r e e bases f e d t o an a n i m a l .  While t h e y were c h e c k i n g t h e r e s u l t s  o b t a i n e d when r a t s were f e d l a b e l l e d guanine t h e y d e c i d e d t o i n v e s t i g a t e the r e s u l t s of feeding l a b e l l e d adenine.  I t was  f e l t t h a t a d e n i n e , s i n c e i t o c c u p i e d a more c e n t r a l p o s i t i o n i n an a n i m a l ' s m e t a b o l i s m , might show r e s u l t s d i f f e r e n t from 15 t h o s e o f guanine.  T h e r e f o r e , 1,3-N  -adenine was s y n t h e s i z e d  and i n c l u d e d i n one o f t h e d i e t s f e d t o t h e r a t s .  The r e s u l t s  o f t h i s experiment were a s t a r t l i n g c o n t r a s t t o t h o s e obt a i n e d when guanine was f e d . I t was found t h a t t h e f r e e base adenine was i n c o r p o r a t e d i n t o t h e mixed n u c l e i c a c i d s o f t h e v i s c e r a and a l s o t h a t i t had been c o n v e r t e d t o g u a n i n e , w h i c h had l i k e w i s e been i n c o r p o r a t e d i n t o t h e mixed v i s c e r a l n u c l e i c acids.  A p p r o x i m a t e l y t h i r t e e n p e r c e n t o f t h e adenine and  e i g h t p e r c e n t o f t h e guanine e x t r a c t e d from t h e mixed  visceral  n u c l e i c a c i d s had been d e r i v e d from t h e 1,3-N-^-adenine w h i c h had been f e d .  F i g u r e s o f t h i s magnitude  l e f t no p o s s i b i l i t y  f o r the p o s t u l a t i o n of experimental e r r o r .  The d a t a of Brown  and h i s co-workers l e d t o two c o n c l u s i o n s :  f i r s t ; that the  f r e e p u r i n e bases c o u l d be used by an a n i m a l f o r t h e s y n t h e s i s o f t i s s u e n u c l e i c a c i d s , second; t h a t t h e y were i n t e r c o n v e r tible (4). The e x p e r i m e n t s mentioned had a l l been c a r r i e d out u s i n g compounds s y n t h e s i z e d from t h e s t a b l e i s o t o p e o f n i t r o g e n , 15 N . When s t a b l e i s o t o p e s a r e used t o l a b e l compounds t h e  - 3 d i f f i c u l t y of excessive  d i l u t i o n must be a v o i d e d .  The  degree  o f d i l u t i o n encountered i n t h e s e b i o l o g i c a l e x p e r i m e n t s r e s u l t e d i n t r a c e i n c o r p o r a t i o n o f the l a b e l l e d b a s e s i n t o 14  the n u c l e i c a c i d s b e i n g o v e r l o o k e d . i s o t o p e , was  When C  , a radioactive  employed t o l a b e l the t e s t compound as was  done .  by B a l i s e t a l . ( 5 ) , u s i n g 8 l a b e l l e d g u a n i n e , or by Abrams ( 6 ) , u s i n g 2 l a b e l l e d g u a n i n e , i t was  e a s i l y demonstrated  t h a t f r e e guanine when f e d t o an a n i m a l was manner s i m i l a r t o f r e e a d e n i n e .  The  of guanine i n t o the n u c l e i c a c i d s was  metabolised i n a  degree o f  incorporation  so . s l i g h t t h a t i t had  been masked i n the e a r l i e r e x p e r i m e n t s .  T h i s masking  had  o c c u r r e d as a r e s u l t o f t h e l i b e r a t i o n o f the l a b e l l e d 8-amino group i n t o the g e n e r a l amino group p o o l on the c a t a b o l i s m  of  l , 3 - N - , g amino-N -guanine t o a l l a n t o i n . 15  i.e.  1 5  the N  15  The  free N , 1 5  Hg of the f r e e amino p o o l , l a b e l l e d a l l the p u r i n e s  an e x t e n t s u f f i c i e n t t o c o n c e a l the s l i g h t i n c o r p o r a t i o n guanine i n t o t h e n u c l e i c a c i d s w i t h t h e a i d o f C^  4  (g).  I t ¥/as  also  of  discovered,  l a b e l l e d g u a n i n e / t h a t when guanine  f e d as t h e f r e e b a s e , i n a d d i t i o n t o b e i n g i n c o r p o r a t e d the n u c l e i c a c i d s , a s m a l l p o r t i o n o f i t was  to  was into  converted to  adenine (6). The  e x p e r i m e n t a l e v i d e n c e from the  previously  mentioned i n v e s t i g a t i o n s r e i n f o r c e d t h e concept t h a t a t some p o i n t i n t h e i r a n a b o l i c pathways a l l p u r i n e s common i n t e r m e d i a t e .  a r o s e from a...  T h i s t h e o r y had been t e s t e d by  feeding  v a r i o u s common p u r i n e bases i n t h e i r f r e e s t a t e t o see i f one c o u l d be found w h i c h would be c o n v e r t e d t o e i t h e r adenine or  - 4 guanine i n t h e course o f i t s metabolism.  I s o g u a n i n e was  i n v e s t i g a t e d by B e n d i c h and h i s c o l l e a g u e s ( 7 ) , h y p o x a n t h i n e and x a n t h i n e by G e t l e r e t a l . ( 8 ) , and u r i c a c i d by Brown and h i s group ( 9 ) .  None o f t h e s e compounds were c o n v e r t e d t o  t i s s u e n u c l e i c a c i d adenine o r g u a n i n e . t e s t e d by B e n d i c h e t a l .  2,6-diamino p u r i n e ,  ( 1 0 ) , was found t o c o n t r i b u t e  a p p r e c i a b l y t o p o l y n u c l e o t i d e guanine.  As ffl$ch as f o u r p e r -  c e n t o f t h e guanine e x t r a c t e d from t h e n u c l e i c a c i d s had come from t h i s compound when i t was i n c l u d e d i n an a n i m a l ' s d i e t . There was, however, no c o n v e r s i o n o f t h e compound i n t o adenine by t h e r a t . I n 1950 Brown and h i s co-workers  ( I I ) published the  f o l l o w i n g m e t a b o l i c map showing t h e v a r i o u s s u b s t a n t i a t e d m e t a b o l i c c o n v e r s i o n s w h i c h might c o n c e i v a b l y be r e l a t e d t o the i n t e r c o n v e r s i o n o f adenine and g u a n i n e :  guanine  hypoxanthine  xanthine  -  5  -  On t h i s map t h r e e t y p e s o f arrows a r e used t o d e s i g n a t e one o f t h e t h r e e d i f f e r e n t r e l a t i o n s h i p s an i n d i v i d u a l r e a c t i o n might have t o t h e pathway w h i c h f r e e adenine f o l l o w s d u r i n g i t s conversion  t o n u c l e i c a c i d guanine.  The c o n v e n t i o n a l  arrows-  denote r e a c t i o n s w h i c h had been demonstrated and w h i c h p r e sumably c o u l d l e a d t o p o l y n u c l e o t i d e arrows w h i c h have been c r o s s e d  guanine.  The  conventional  out denote r e a c t i o n s w h i c h  would l e a d t o n u c l e i c a c i d guanine b u t w h i c h had been demonstra* t e d as n o t o c c u r r i n g w i t h i n t h e body.  The d o t t e d arrows show  p o s s i b l e r e l a t i o n s h i p s , proven and unproven, amongst t h e v a r i o u s p u r i n e bases w h i c h had been t e s t e d as p r e c u r s o r s  of  n u c l e i c a c i d guanine. Brown and h i s c o - w o r k e r s , when a n a l y s i n g  t h i s meta-  b o l i c map, argue t h a t s i n c e n e i t h e r f r e e h y p o x a n t h i n e n o r f r e e x a n t h i n e f u n c t i o n as a p r e c u r s o r  of n u c l e i c a c i d purines i n  the r a t ( 8 ) , a p r e l i m i n a r y h y d r o l y s i s a t t h e 6-amino group o f adenine i s e x c l u d e d as t h e i n i t i a l s t e p i n i t s c o n v e r s i o n t o polynucleotide  guanine.  Whether t h e r e c e n t l y demonstrated  i n t e r c o n v e r t i b i l i t y of adenylic a c i d , i n o s i n i c a c i d , xanthosine monophosphate and g u a n y l i c a c i d w i l l f o r c e a r e c o n s i d e r a t i o n of t h i s route f o r the production  o f n u c l e i c a c i d guanine from  f r e e adenine remains t o be seen (12) ( 1 3 ) . A l t h o u g h i s o g u a n i n e occurs i n nature, i t i s not u t i l i z e d f o r n u c l e i c a c i d  purine  s y n t h e s i s when i t i s f e d i n t h e f r e e s t a t e ( 7 ) .  fact,  This  Brown and h i s co-workers f e e l , r e n d e r s an i n i t i a l  oxidation  a t t h e 2 - p o s i t i o n o f adenine a l s o i m p r o b a b l e as t h e s t a r t i n g p o i n t on i t s c o n v e r s i o n  to polynucleotide  guanine.  A=direct  r e a c t i o n between adenine and ammonia i s u n l i k e l y on a c h e m i c a l  - 6  basis  (10)  and  -  even more so i n v i e w of R e i c h a r d ' s (14)  evidence  t h a t the 2-amino group of guanine i s d e r i v e d f o r the most p a r t from g l y c i n e .  Brown and  h i s colleagues  c o n c l u d e t h a t the most  p r o b a b l e i n i t i a l s t e p i s a r e a c t i o n between adenine and Brown e t a l . (11)  make no mention of the  r o l e t h a t an i n c o m p l e t e p u r i n e might p l a y as an .in  t h i s i n t e r c o n v e r s i o n o f adenine and g u a n i n e .  glycine.  possible  intermediate This f a c t i s  i n agreement w i t h the v i e w t h e y h e l d on the e s s e n t i a l i n t e g r i t y of the p u r i n e Buchanan and group (16)  on  s k e l e t o n d u r i n g the i n t e r c o n v e r s i o n . h i s group (15)  or t h a t o f Greenberg and  his  'de novo' s y n t h e s i s of r i b o t i d e s from i n c o m p l e t e  p u r i n e r i n g compounds would make one between some carboxamide r i b o t i d e and intermediate  Siork of  reaction.  suspect t h a t a r e a c t i o n glycine i s a plausible  T h i s s u s p i c i o n would r e q u i r e t h a t  i n i t i a l step i n the i n t e r c o n v e r s i o n be a r u p t u r e  o f the  the  purine  skeleton. I n 1954 metabolic  plan.  R o l l and Brown (2) p u b l i s h e d T h i s map  t o o k c o g n i z a n c e of new  another experimental  e v i d e n c e w h i c h had  accumulated on p o s s i b l e pathways f o r  i n t e r c o n v e r s i o n of  purines.  Exogenous adenine « 'De novo'  A c t i v e aaenme  v  Nucleici acid Adenine  s y n t h e s i s —*• Incompletes: •Common // Purine Intermediate i n o s m i c Acid  I I I  w  Exogenous guanine  the  Nucleic acid Guanine  I  > A c t i v e guanine  - 7 I t has been i n f e r r e d from e x p e r i m e n t a l  e v i d e n c e t h a t even i n  the courses of'de novo' s y n t h e s i s a t l e a s t p a r t o f one p u r i n e may a r i s e v i a t h e o t h e r  (2). T h i s f a c t i s d i f f i c u l t t o  r a t i o n a l i z e i f the only path f o r i n t e r c o n v e r s i o n presented i s t h a t t h r o u g h a common i n t e r m e d i a t e ,  c o n s e q u e n t l y , an a l t e r n a t i v e  direct interconversion i s also offered.  As y e t t h e r e i s no  i r r e f u t i b l e evidence i n d i c a t i n g a t w h i c h s t a g e , i . e . f r e e base, r i b o s i d e o r r i b o t i d e , t h e i n t e r c o n v e r s i o n o f adenine and guanine takes place.  C u r r e n t work on t h e b i o s y n t h e s i s o f p u r i n e s  favor  the p r o b a b i l i t y o f t h e i n t e r c o n v e r s i o n t a k i n g p l a c e as t h e ribotides.  There i s , however, o t h e r e v i d e n c e i n d i c a t i n g t h a t  the i n t e r c o n v e r s i o n may t a k e p l a c e as t h e f r e e bases o r as the r i b o s i d e s ( 1 7 ) .  Considering  r e c e n t work o f Ochoa e t a l .  ( 1 8 ) , i t would appear t h a t t h e n a t u r e o f a c t i v e adenine o r guanine i n t h e case o f Pentose N u c l e i c A c i d s y n t h e s i s i s t h a t of a d i p h o s p h a t e r i b o s i d e . The problem under i n v e s t i g a t i o n i n t h i s t h e s i s i n v o l v e s t h e p o s s i b l e l a b i l i t y o f t h e 2-C- atom o f adenine on i t s conversion non  t o p o l y n u c l e o t i d e guanine.  i s i n a l l p r o b a b i l i t y due t o a complex s e r i e s o f r e a c t i o n s ,  t h e r e a r e two p o s s i b l e o v e r a l l p r o c e s s e s :  (-)  (2)  W h i l e t h i s phenome-  HN*C HN'  V  (1) .  T h i s i n v o l v e s no d i s r u p t i o n o f t h e b a s i c p u r i n e s t r u c t u r e  and a d i r e c t a m i n a t i o n a t t h e 2 p o s i t i o n o f a d e n i n e .  This,  as s t a t e d e a r l i e r , Brown and h i s c o l l e a g u e s (10) c o n s i d e r t o be  unlikely.  (2) .  This i n v o l v e s a rupture of the purine nucleus a t the 2  p o s i t i o n w i t h subsequent a m i n a t i o n and r e c l o s u r e o f t h e r i n g * T h i s second r o u t e p e r m i t s t h e p o s s i b i l i t y o f exchange o r o x i d a t i o n r e a c t i o n s t a k i n g place a t the 2 p o s i t i o n . In 1953 P a t e r s o n and Zbarsky C^ -adenine. 4  (19) s y n t h e s i z e d 2-  T h i s compound a l l o w e d a d i r e c t . i n v e s t i g a t i o n o f  the f a t e o f t h e 2-C-atom o f adenine d u r i n g t h e c o u r s e o f i t s metabolism  and c a t a b o l i s m .  There was no p r e v i o u s knowledge '  o f t h i s p a r t i c u l a r atom a l t h o u g h i t had been t h e s u b j e c t o f conjecture regarding i t s possible l a b i l i t y  (41) ( 4 2 ) .  The  r e s u l t s o b t a i n e d by P a t e r s o n (20) on f e e d i n g 2 - C - a d e n i n e t o 14  r a t s would i n d i c a t e t h a t t h i s p a r t i c u l a r atom does posess aa g r e a t e r l a b i l i t y t h a n t h e o t h e r C atoms o f adenine.  However,  no d i r e c t c o r r e l a t i o n was p o s s i b l e between t h i s evidence f o r l a b i l i t y and t h e i n t e r c o n v e r s i o n o f p u r i n e b a s e s .  I t was f e l t ,  by t h e a u t h o r , t h a t a m e t a b o l i c s t u d y i n v o l v i n g 1,3-N -, 2 - C 15  14  adenine might y i e l d t h e d e s i r e d i n f o r m a t i o n .  I t would be  p o s s i b l e t o c o r r e l a t e t h e r e l a t i v e percentage  incorporation of  f r e e a d e n i n e . i n t o p o l y n u c l e o t i d e guanine i n terms o f b o t h and NA " on f e e d i n g such a compound. 0  C^  4  I f t h e two p e r c e n t a g e s  are i d e n t i c a l t h i s would f u r n i s h d i r e c t p r o o f o f t h e i n t e g r i t y of the purine skeleton during the i n t e r c o n v e r t i o n process. I f t h e two p e r c e n t a g e s  vary t o the extent that there i s  v i r t u a l l y no i n c o r p o r a t i o n i n terms o f C-^, a r u p t u r e o f t h e  p u r i n e s k e l e t o n and subsequent exchange r e a c t i o n would  be  indicated.  F i n a l l y , i f the two p e r c e n t a g e s v a r y such t h a t t h e 14 i n c o r p o r a t i o n e x p r e s s e d as C i s s i g n i f i c a n t l y lower than the 15 i n c o r p o r a t i o n e x p r e s s e d as N  the i n t e r p r e t a t i o n could  be  t h a t s e v e r a l r o u t e s e x i s t , a t l e a s t one o f them r e q u i r i n g a r u p t u r e o f the p u r i n e n u c l e u s . The experiments p r e s e n t e d i n t h i s t h e s i s were u n d e r t a k e n t o r e s o l v e t h e problem o f whether t h e p u r i n e n u c l e u s remained i n t a c t on t h e c o n v e r s i o n o f exogenous adenine t o polynucleotide  guanine.  T h i s was a c c o m p l i s h e d by comparing  the N"^ and C"*" i n c o r p o r a t i o n v a l u e s t o d i s c o v e r w h i c h o f t h e 4  t h r e e above mentioned p o s s i b i l i t i e s was t o be found on i n j e c t i n g l,5-K ^-,S-C' " -adenine 1  l  4  into rats.  The r e s u l t s i n d i -  c a t e d t h a t b o t h i n t h e case o f t h e i n c o r p o r a t i o n o f exogenous adenine as adenine and i n t h e case o f i t s c o n v e r s i o n t o guanine and i n c o r p o r a t i o n as t h a t compound t h e p e r c e n t a g e i n c o r p o r a t i o n i n t o t h e n u c l e i c a c i d s was l e s s when measured as a f u n c t i o n o f t h e C  1 4  content of the i n j e c t e d l , 3 - N - 2 1 5  C ^ - a d e n i n e t h a n when measured as a f u n c t i o n o f i t s N"" 4  content.  1 5  Otherwise s t a t e d , t h e i n j e c t e d l,3-N^-,2-C" " i  4  14  adenine appears t o l o s e C  b o t h on i t s i n c o r p o r a t i o n i n t o  the v i s c e r a l n u c l e i c a c i d s as adenine and a l s o on i t s conv e r s i o n and i n c o r p o r a t i o n as guanine.  The degree o f t h i s  l o s s i s such as t o " i n d i c a t e t h a t t h e t h i r d p o s s i b i l i t y i s t h e p r o b a b l e one, i . e . more t h a n one r o u t e e x i s t s f o r t h e conv e r s i o n o f adenine t o g u a n i n e .  The t a c i t assumption a r i s i n g  from t h i s c h o i c e out o f t h e t h r e e p o s s i b i l i t i e s i s t h a t on a t l e a s t one r o u t e t h e p u r i n e n u c l e u s i s not m a i n t a i n e d i n t a c t during the  interconversion.  - 10 EXPERIMENTAL A.  Chemical Synthesis The proposed s y n t h e s i s o f d o u b l y l a b e l l e d  adenine  i n v o l v e d i n p r a c t i c e t h e s y n t h e s i s o f two s e p a r a t e compounds. 14 F i r s t ; 2-C  -adenine was s y n t h e s i z e d by t h e method o f P a t e r s o n  and Z b a r s k y (19) adapted from Shaw ( 2 1 ) , second; was produced by t h e method o f B e n d i c h e t a l .  l,3-N -adenine 15  (22) m o d i f i e d  from t h a t o f Todd and h i s co-workers ( 2 3 ) . 14  I t was e s s e n t i a l t h a t t h e 2-C  -adenine be o f h i g h  s p e c i f i c a c t i v i t y o t h e r w i s e on m i x i n g t h e two compounds, 2-C"*"4  adenine and 1,3-N  - a d e n i n e , t h e atom % e x c e s s N  would have 14 been e x c e s s i v e l y d i l u t e d . F u r t h e r m o r e , t h e h i g h e r t h e 2-C adenine was i n s p e c i f i c a c t i v i t y , t h e e a s i e r i t would be t o 14 t r a c e t h e b y p r o d u c t s by means o f t h e i r C  c o n t e n t upon t h e  a d m i n i s t r a t i o n o f a minimum amount o f t h e t e s t i)  compound.  2-C -adenine 14  The p r e l i m i n a r y s t e p i n t h e s y n t h e s i s o f 2-C" - 14 1  adenine was t h e s y n t h e s i s o f C  -formate.  4  T h i s was a c h i e v e d  by a c o m b i n a t i o n o f t h e method o f Jeanes (32) and t h a t o f Abrams and h i s co-workers ( 2 4 ) . N a C 0 1 4  g  BaC 0 .  The B a C  1 4  3  l 4  0  g  was c o n v e r t e d t o  was reduced t o B a ( C ? N ) 1 4  3  g  by h e a t i n g i n  a f u r n a c e i n t h e presence o f Na-Zn amalgam w h i l e a t t h e same time d r y ammonia gas was passed over t h e hot m e l t . Ba(C iN) 1 4  The  was c o n v e r t e d t o C - f o r m i c a c i d by means o f a c i d 1 4  g  hydrolysis.  The C - f o r m a t e was condensed w i t h 4-amino, 14  5 - i m i d a z o l e c a r b o x i m i d i n e h y d r o c h l o r i d e by t h e method o f  - 11 P a t e r s o n and Zbarsky (19) t o g i v e 2 - C - a d e n i n e . 14  (1)  Nc^C'V,  "Ba.(C £N) W  D-XHCP  (*)  KC-100K f  ii)  1,3-N -adenine l^  a  —>-  .  HC"bOH  |  O  15  1,3-N  -adenine was  Bendich et a l . (22).  s y n t h e s i z e d by the method of  N - F o r m a m i d i n e h y d r o c h l o r i d e was  pared and condensed w i t h p h e n y l - a z o m a l o n o n i t r i l e , the 4,6-diaraino-5-phenyl-azopyrimidine, triamino-pyrimidine.  was  product,  reduced t o g i v e 4,5,6-  F o r m y l a t i o n , f o l l o w e d by r i n g c l o s u r e  of t h i s compound y i e l d e d 1,3-N  iii)  pre-  15  -adenine.  1,3-N -,2-C -adenine 15  The  14  two compounds, 2-C  14  -adenine and 1,3-N  l^S  adenine were d i s s o l v e d w i t h a c a l c u l a t e d e q u i v a l e n t of  -  HC1.  T h i s s o l u t i o n c o u l d t h e n be l o o k e d upon as c o n s i s t i n g of 1,3-N -,2-C -adenine hydrochloride, a doubly l a b e l l e d 15  14  molecule. P r o o f of I d e n t i t y . C r i t e r i a of P u r i t y i)  Chromatography A comparison was  the s y n t h e t i c p r o d u c t  made between the R j  values  of  and a sample of a u t h e n t i c a d e n i n e w h i c h  had b o t h been run s i m u l t a n e o u s l y on a paper chromatogram employing  -  12  -  a s o l v e n t o f i s o p r o p a n o l , H C 1 , H 0 as d e s c r i b e d by Wyatt ( 3 1 ) . g  The R j v a l u e quoted f o r adenine i n t h i s s o l v e n t i s 0 . 3 2  (31).  F i g u r e 1 shows a c o n t a c t p r i n t o f t h e chromatogram as d e v e l o p e d under u l t r a v i o l e t l i g h t  (34).  The s y n t h e t i c p r o d u c t  the same Rj v a l u e as t h e a u t h e n t i c a d e n i n e .  shows  The appearance  o f b u t a s i n g l e spot i s e v i d e n c e o f t h e e s s e n t i a l l y uncontaminated ii)  nature of the s y n t h e t i c product.  Spectrophotometry T h e e p u r i n e s , p y r i m i d i n e s and t h e i r d e r i v a t i v e s  have v e r y w e l l d e f i n e d a b s o r p t i o n maxima and minima i n t h e u l t r a v i o l e t region.  The method o f e s t a b l i s h i n g t h e wave-  l e n g t h a t w h i c h t h e maximum a b s o r p t i o n o c c u r s , t h e wavel e n g t h a t w h i c h t h e minimum a b s o r p t i o n o c c u r s and t h e r a t i o of t h e o p t i c a l d e n s i t i e s o b t a i n e d a t any two a r b i t r a r y wavel e n g t h s i s a v a l u a b l e a i d i n c h a r a c t e r i z i n g t h e s e compounds. When t h i s method o f a n a l y s i s was a p p l i e d t o t h e s y n t h e s i z e d product was  i t was observed t h a t t h e p o i n t o f maximum a b s o r p t i o n  recorded  a t 2 6 2 . 5 m/*and t h e p o i n t o f minimum a b s o r p t i o n  at 229.0 m y .  These d a t a agree w i t h t h e v a l u e s r e c o r d e d  adenine ( 4 3 ) .  Further, the r a t i o of the o p t i c a l d e n s i t i e s  recorded  a t 260 m/^and 280 m/*, '  i . e . A280  was 0 . 3 7 5 .  for  This  A260  v a l u e i s a l s o i n agreement w i t h t h e v a l u e quoted f o r a d e n i n e . F i g u r e 2 shows t h e a b s o r p t i o n curves from w h i c h these  values  were c a l c u l a t e d . iii)  D e r i v a t i v e and i t s m e l t i n g p o i n t The p i c r a t e s a l t o f adenine was prepared  derivative.  as a  I t was r e c r y s t a l l i z e d t h r e e times from 25$ a c e t i c  a c i d , washed w i t h d i s t i l l e d w a t e r , and i t s m e l t i n g p o i n t  - IS -  FIGURE 1  Contact p r i n t of a Chromatogram  snowing  a comparison  of the  Ri v a l u e s o i t n e synthesized  adenine  ( r i g n t ) and a n authentic  sample  (left).  The homogeneous nature of tne spot v e r i f i e s tne absence of any s i g n i f i c a n t amount o f c o n t a m i n a t i o n o f the synthetic  proauct.  FIGURE 2  1.500 1.400 1.300  OPTICAL DENSITY 230  240  250  WAVE LENGTH  260  270  280  290  300  Aj-  A comparison o f t h e U.V. a b s o r p t i o n curves o f a u t h e n t i c adenine and the s y n t h e s i z e d p r o d u c t .  These r e a d i n g s were  taken w i t h a Beckman q u a r t z s p e c t r o p h o t o m e t e r Model DK 2.  - 15 determined.  T h i s v a l u e i s r e c o r d e d as 285-28.6°C ( w i t h d e -  composition)  (S3).  The m e l t i n g p o i n t range o b t a i n e d was  283-287°C ( w i t h d e c o m p o s i t i o n ) . Measurement o f I s o t o p e  Concentration  14 i)  C  determination Measurement o f t h e s p e c i f i c a c t i v i t y o f t h e s y n t h e -  s i z e d adenine was a c c o m p l i s h e d  by t h e f o l l o w i n g  procedure.  An a l i q u o t was t r e a t e d by t h e wet o x i d a t i o n t e c h n i q u e o f Van S l y k e and F o l c h ( 2 8 ) .  The COg e v o l v e d was t r a p p e d i n c a r b o n a t e -  f r e e a l k a l i and p r e c i p i t a t e d w i t h B a ( C l ) g .  The r a d i o a c t i v i t y  o f t h e t h i c k sample o f BaC03 was determined  by c o u n t i n g i n a  gas f l o w windowless c o u n t e r .  B o t h the t e c h n i q u e and t h e  c o u n t e r employed i n t h i s l a b o r a t o r y have been d e s c r i b e d by P a t e r s o n (20) and Wright  (26).  The s p e c i f i c a c t i v i t y o f  the s y n t h e t i c adenine was found t o be 1.66 x 1 0 The 2.24  s p e c i f i c a c t i v i t y expressed  4  c.p.m./mg.  as c.p.m./mM. o f adenine was  x 10 . 6  The s y n t h e s i z e d adenine was shown t o be f r e e o f any s i g n i f i c a n t amount o f c o n t a m i n a t i n g r a d i o a c t i v e m a t e r i a l by means o f an a u t o r a d i o g r a m . o f Zbarsky and Wright  (44).  T h i s was done u s i n g t h e method A developed  paper chromatogram  was p l a c e d i n c o n t a c t w i t h X-ray f i l m i n a d a r k room f o r one week.  When t h e f i l m was developed  r a d i o a c t i v i t y appeared as  a s i n g l e s p o t , i n d i c a t i n g o n l y one r a d i o a c t i v e component was present.  The  v a l u e o f 0.32 corresponded  v a l u e o f adenine ( 3 1 ) .  t o t h e quoted R j  - 16 ii)  N  1 5  determination The atom % e x c e s s N  1 5  of t h e s y n t h e t i c p r o d u c t was  e s t i m a t e d on samples o f Ng p r e p a r e d by the method o f R i t t e n b e r g (29).  An a l i q u o t of t h e compound was c o n v e r t e d t o  by the K j e l d a h l procedure.-  (NH ) S0 4  g  4  T h i s (NH" )gSB was t h e n t r e a t e d 4  4  w i t h NaOBr i n vacuo y i e l d i n g Ng w h i c h was t r a p p e d and s t o r e d i n breakseal containers.  The Ng was a n a l y s e d on a mass  s p e c t r o m e t e r f o r the atom % e x c e s s N 15 t r a t i o n of N  1 5  present.  The  concen-  i n the s y n t h e t i c adenine as d e t e r m i n e d by  this  method was 2.897 atom % e x c e s s . B.  Metabolic Technique o f M e t a b o l i s m E x p e r i m e n t s 15 14 The 1,3-N  v  -,2-C  phosphate b u f f e r (pH 3.8).  -adenine was d i s s o l v e d i n a T h i s s o l u t i o n was a d m i n i s t e r e d a t  t w e l v e hour i n t e r v a l s by means o f i n t r a p e r i t o n e a l i n j e c t i o n s . W h i l e an experiment was i n p r o g r e s s the r a t s were k e p t i n a metabolism cage s i m i l a r t o t h a t d e s c r i b e d by Wright (26) and Paterson (20).  The arrangement  o f t h i s metabolism cage i s  s u c h as t o p e r m i t s e p a r a t e c o l l e c t i o n o f e i t h e r o r b o t h , expired C0  g  and, u r i n e and f a e c e s , f o r any g i v e n t i m e p e r i o d ,  w i t h a minimum o f c o n t a m i n a t i o n . I n a p r e l i m i n a r y experiment t h r e e r a t s were u s e d . They were i n j e c t e d w i t h adenine a t a l e v e l o f 100 m g . / k i l o . body weight/day.  T h i s experiment was t e r m i n a t e d a t the end  of t h e f i r s t t w e n t y - f o u r hours by t e c h n i c a l d i f f i c u l t i e s . I n a second experiment two r a t s were used.  They  were i n j e c t e d w i t h adenine a t a l e v e l of 50 m g . / k i l o . body  - 17 weight/day f o r a p e r i o d o f n i n e t y - s i x h o u r s .  The samples  c o l l e c t e d f o r a n a l y s i s from t h e s e two e x p e r i m e n t s a r e t a b u l a t e d below. TABLE I Number and Nature o f t h e Samples Saved i n E x p e r i m e n t s I and I I Number o f Samples  Nature o f Sample  Experiment I Blood Viscera 12 hour u r i n e specimen 24 hour f a e c e s specimen 12 hour e x p i r e d a i r specimen  Experiment I I  —  —  1 0 1  8 4 8  Treatment o f Samples At t h e t e r m i n a t i o n o f an experiment each r a t was k i l l e d by a b l o w on i t s head. bled.  I t was t h e n d e c a p i t a t e d and  The v i s c e r a were i m m e d i a t e l y removed and f r o z e n i n a  dry i c e - a l c o h o l mixture.  They were t h e n homogenized  c c . o f c o l d water i n a Waring B l e n d o r .  w i t h 10  T h i s was f o l l o w e d by  t h r e e s u c c e s s i v e e x t r a c t i o n s w i t h 100 c c . p o r t i o n s o f c o l d 5$ t r i c h l o r o a c e t i c a c i d .  A f t e r each e x t r a c t i o n t h e t i s s u e  was c e n t r i f u g e d a t 0°C. and t h e s u p e r n a t a n t was d e c a n t e d . I t was presumed t h a t t h i s 300 c c . o f c o l d y e l l o w i s h  tri-  chloroacetic acid extract contained a l l the f r e e nucleotides as w e l l as o t h e r a c i d - s o l u b l e f a c t o r s , i)  Tissue The e x t r a c t e d t i s s u e was t r e a t e d w i t h 95$ e t h a n o l ,  - 18 100$  e t h a n o l , 100$  alone.  e t h a n o l and e t h e r and f i n a l l y w i t h e t h e r  T h i s t r e a t m e n t removed a l l o f the r e s i d u a l water and  fatty material.  A f t e r t h i s treatment the t i s s u e was  a i r dried.  The d r y t i s s u e was next e x t r a c t e d f o r e i g h t hours a t 80°C. w i t h 150 c c . o f 10$ N a C l , t h i s t r e a t m e n t was  repeated t w i c e ,  t h e n the f i l t r a t e s were combined and the t i s s u e d i s c a r d e d .  Two  and o n e - h a l f volumes o f 95$ e t h a n o l were added t o the combined f i l t r a t e and the s o l u t i o n was  allowed to stand o v e r n i g h t .  f i n e w h i t e p r e c i p i t a t e o f crude sodium n u c l e a t e s e t t l e d during t h i s period.  T h i s p r e c i p i t a t e was r e c o v e r e d  f u g a t i o n and, a f t e r w a s h i n g , was s m a l l volume o f :htir,tl0$ N a C l .  immediately  I t was  by  A  out centri-  resuspended i n a  once a g a i n r e p r e c i p i t a t e d  by the a d d i t i o n o f a l c o h o l , washed, and t h i s time d r i e d . The pure sodium n u c l e a t e was one hour i n 5 c c . o f 1 N. HC1 a t 100°C. b r o w n i s h h y d r o l y s a t e was r e s i n and e l u t i o n was  adsorbed  then hydrolysed f o r The r e s u l t a n t s l i g h t l y  on Dowex-50 i o n exchange  c a r r i e d out w i t h 1 N. HC1 u s i n g an  matic f r a c t i o n c o l l e c t o r .  T h i s e l u a t e was d i v i d e d i n t o t h r e e  main f r a c t i o n s on the b a s i s o f o p t i c a l d e n s i t y r e a d i n g s a t 260 my w i t h a model DU Beckman Spectrophotometer. f r a c t i o n s were reduced  auto-  t o 10 c c . volume each.  taken  These  At t h i s  stage  paper chromatograms and a b s o r p t i o n s p e c t r a were done t o v e r i f y the suspected i d e n t i t y o f the components o f each fraction.  The t h r e e f r a c t i o n s were shown t o c o n s i s t o f ade-  n i n e , guanine and a m i x t u r e o f p y r i m i d i n e s .  The  ultraviolet  a b s o r p t i o n measurements p e r m i t t e d c a l c u l a t i o n o f the concent r a t i o n o f each component from i t s molar e x t i n c t i o n  - 19 coefficient.  -  Carbon o x i d a t i o n s were c a r r i e d out on a l i q u o t s  of t h e s e f r a c t i o n s t o o b t a i n the t o t a l and 14 with respect to C  .  ii)  activities  F u r t h e r a l i q u o t s were t r e a t e d by  method o f R i t t e n b e r g (29) t o determine N of atom % e x c e s s  specific  1 5  the  v a l u e s i n terms  N . 1 5  Acid-soluble fraction The 300  c c . of a c i d - s o l u b l e e x t r a c t was  succes-  s i v e l y e x t r a c t e d w i t h e t h e r u n t i l pH d e t e r m i n a t i o n s  showed  the major p o r t i o n of the t r i c h l o r o a c e t i c a c i d t o have been removed. was  now  The s o l u t i o n was  s t i l l yellow at t h i s stage.  It  reduced t o a volume of 10 c c . and 1 c c . of t h i s  removed f o r paper chromatography and a u t o r a d i o g r a p h y . chromatography proved t o be u n s u c c e s s f u l . s o l u t i o n was  was The  If sufficient  a p p l i e d t o the chromatogram t o d e t e c t  the  r a d i o a c t i v i t y , s e p a r a t i o n of the components became i m p o s s i b l e ; on the o t h e r hand w i t h l e s s s o l u t i o n per chromatogram s e p a r a t i o n was  p o s s i b l e , but the r a d i o a c t i v i t y c o u l d not  be  detected. The r e m a i n i n g one hour i n 1 N. HC1  9 c c . of e x t r a c t was  d i v i d e d i n t o t h r e e main f r a c t i o n s .  This eluate  One" tube was  moved from each of these f r a c t i o n s c o n c e n t r a t e d of a p p r o x i m a t e l y  for  a t 100°C. t h e n adsorbed on Dowex-50  i o n exchange r e s i n and e l u t e d w i t h 1 N. HC1. was  hydrolysed  re-  t o a volume  J c c . and a p p l i e d t o a paper chromatogram.  Rj v a l u e s r e v e a l e d t h a t these t h r e e f r a c t i o n s c o n t a i n e d r e s p e c t i v e l y , a d e n i n e , .guanine and a m i x t u r e p y r i m i d i n e s and n y p o x a n t h i n e .  c o n s i s t i n g of  The r a d i o a c t i v e compounds  - 20 were l o c a t e d by s c a n n i n g the chromatograms w i t h an a u t o m a t i c windowless gas f l o w chromatogram c o u n t e r . 14 showing C  Those compounds  a c t i v i t y by t h i s method were t h e n punched out o f  the chromatogram and counts were r e c o r d e d f o r the paper d i s c s w i t h the c o n v e n t i o n a l counter.  Following determination  of t h e i r a c t i v i t y , t h e s e paper d i s c s were, e x t r a c t e d f o r e i g h t hours w i t h 0.1 N. HC1.  A b s o r p t i o n s p e c t r a were t h e n  r e c o r d e d f o r t h e s e e x t r a c t s t o v e r i f y t h e i d e n t i t y and t o e s t a b l i s h t h e c o n c e n t r a t i o n of t h e component p r e s e n t . These f i n d i n g s checked w i t h t h e i d e n t i t i e s e s t a b l i s h e d by means o f R j  v a l u e s on t h e chromatograms.  With these data  i t was p o s s i b l e t o c a l c u l a t e s p e c i f i c and t o t a l a c t i v i t i e s 14 for C  b u t no attempt was made t o c o r r e l a t e t h e  results  w i t h c o u n t s o b t a i n e d by t h e c a r b o n o x i d a t i o n t e c h n i q u e . The remainder o f each f r a c t i o n , a f t e r t h e removal of the s i n g l e t u b e , was c o n c e n t r a t e d t o 10 c c . volume. A b s o r p t i o n s p e c t r a were t a k e n from w h i c h the c o n c e n t r a t i o n of t h e component i n each f r a c t i o n was e s t a b l i s h e d .  Aliquots  were t h e n o x i d i z e d t o determine t h e r a d i o a c t i v i t y p r e s e n t . I n the f i r s t experiment o n l y t h e t o t a l a c t i v i t y p r e s e n t estimated.  was  This value then provided a guide f o r the t r e a t -  ment t o be accorded the a c i d - s o l u b l e f r a c t i o n o f t h e experiment.  second  I n the ease of t h e second experiment b o t h t h e  t o t a l r a d i o a c t i v i t y and t h e s p e c i f i c a c t i v i t y o f each  fraction  were d e t e r m i n e d , iii)  Carbon d i o x i d e The t w e l v e hour samples o f e x p i r e d COg had been  c o l l e c t e d i n 10$ NaOH.  A f t e r making t h e s o l u t i o n up t o a  s p e c i f i c volume, 1 l i t e r , 2 c c . a l i q u o t s were removed and t h e NagCOg was p r e c i p i t a t e d from t h e s e a l i q u o t s as BaCOg. The r a d i o a c t i v i t y o f t h e s e t h i c k samples o f BaCOg was d e t e r m i n e d i n t h e manner d e s c r i b e d b y P a t e r s o n ( 2 0 ) . iv)  Urine Each t w e l v e hour sample o f u r i n e was made up t o  a volume o f 50 c c . w i t h d i s t i l l e d water and 3 c c . a l i q u o t s  14 were withdrawn f o r d e t e r m i n a t i o n o f t h e t o t a l C c o n v e n t i o n a l carbon o x i d a t i o n t e c h n i q u e ( 2 8 ) .  by the  Further 5 cc.  a l i q u o t s o f u r i n e were t r e a t e d w i t h u r e a s e i n t h e manner d e s c r i b e d by Wright  (26) f o r t h e d e t e r m i n a t i o n o f t h e r a d i o -  a c t i v i t y present i n the urea,  ©hen t h i s p r o c e d u r e was com-  p l e t e t h e d i g e s t e d samples were made a l k a l i n e a f t e r t h e method o f R i t t e n b e r g (29) and t h e e v o l v e d NH 15 and c o n v e r t e d t o Ng f o r N  determination.  g  was t r a p p e d  A l l a n t o i n was  measured on'each t w e l v e hour sample o f u r i n e by t h e method of Larson (27).  C r y s t a l l i n e a l l a n t o i n was i s o l a t e d - from  an a l i q u o t o f p o o l e d u r i n e a c c o r d i n g t o t h e method o f Brown and h i s c o l l e a g u e s ( 4 ) .  A l i q u o t samples o f p o o l e d  u r i n e were used f o r t h i s i s o l a t i o n because i t was d e s i r e d t h a t t h e JE*" and N !  4  1 5  v a l u e s from t h e a n a l y s i s o f t h e com-  pound be r e p r e s e n t a t i v e o f t h e e x c r e t i o n over t h e n i n e t y s i x hour p e r i o d .  R e a l i z i n g that the p r o b a b i l i t y existed  t h a t a c o n s i d e r a b l e p o r t i o n o f t h e i n j e c t e d adenine might be e x c r e t e d u n a l t e r e d i n t h e u r i n e , a n a l y s i s t o determine i f t h i s was so was c a r r i e d out by means o f c a r r i e r t e c h n i q u e  - 22 cn p o o l e d samples o f u r i n e .  The c a r r i e r adenine was  i s o l a t e d as the p i c r a t e s a l t .  re-  T h i s p i c r a t e s a l t was  d i z e d and i t s r a d i o a c t i v i t y was d e t e r m i n e d .  The  oxi-  paper  chromatograms w h i c h had been developed w i t h the u r i n e were t r e a t e d i n a v a r i e t y o f ways t o l o c a t e and i d e n t i f y  the  14 sources of C  a c t i v i t y present.  Amongst the  treatments  accorded t h e s e chromatograms were photography under u l t r a v i o l e t i r r a d i a t i o n (34), autoradiography w i t h para-dimethylaminobenzaldehyde  ( 4 4 ) , and  (30), d i a z o t i z e d  n i l i c a c i d (30) and ammoniacal s i l v e r n i t r a t e v)  spraying sulfa-  (30).  Faeces The f a e c e s , a f t e r d e h y d r a t i o n , were t r e a t e d by  the c o n v e n t i o n a l o x i d a t i o n t e c h n i q u e and the r a d i o a c t i v i t y p r e s e n t was  measured as t h i c k sample counts on the BaCOg  produced ( 2 8 ) . vi)  Blood The b l o o d samples were t r e a t e d by homogenizing  c l o t t e d specimens and w i t h d r a w i n g a l i q u o t s f o r o x i d a t i o n 14 / and subsequent C determination (28). C.  Experiment I I n experiment  I , t h r e e male r a t s o f the l i s t a r  s t r a i n , h a v i n g a combined weight  o f 515 gms.,  were i n j e c t e d  i n t r a p e r i t o n e a l l y w i t h the b u f f e r e d a d e n i n e s o l u t i o n . i n d i v i d u a l dose per r a t was mgs.  o f adenine.  0.91  T h i s dosage was  cc. which contained  The 8.62  administered twice d a i l y ,  - 23 thus t h e r a t s were b e i n g i n j e c t e d a t a l e v e l o f 100 m g . / K i l o . / day.  Since the s p e c i f i c a c t i v i t y of the administered  adenine  was 1.66 x 10 c.p.m./mg., t h e r a t s r e c e i v e d a t o t a l dosage 4  o f 8.77 x 10 c.p.m.  D u r i n g t h e course o f t h e experiment t h e  r a t s were a l l o w e d f r e e a c c e s s t o f o o d and water.  This ex-  periment was t e r m i n a t e d a t t h e end o f t w e n t y - f o u r  hours.  Results The r e s u l t s o f t h e a n a l y s i s o f t h e t i s s u e a c i d p u r i n e s and p y r i m i d i n e m i x t u r e from t h i s  nucleic  experiment  a r e p r e s e n t e d i n Table I I . TABLE I I C and N V a l u e s o f t h e Adenine, Guanine and Mixed Pyrimidines of the V i s c e r a l Nucleic Acids a f t e r I n t r a p e r i t oneal I n j e c t i o n of l,3-Ni.-,2-C -Adenine (49.32 mgm., 8.77 x 10 c.p.m.). 1 4  1 5  14  5  Compound  f4  R a d i o a c t i v i t y Found  15  T o t a l c.p.m. S p e c i f i c a c t i v i t y Atom % c.p.m./mg. Cpd. excess Adenine  17,200  745  0.163  Guanine  8,030  175  0.106  Pyrimidine m i x t u r e (from i o n exchange)  1,750  -  -  The  s i g n i f i c a n c e o f these values i s e l a b o r a t e d  upon i n t h e d i s c u s s i o n .  There, f o r t h e purpose o f c l a r i t y ,  they are converted t o values r e p r e s e n t i n g percentage i n c o r p o r a t i o n i n t o t h e v i s c e r a l n u c l e i c a c i d s and C  1 4  /N  1 5  - 24 r a t i o s i n the i n d i v i d u a l purine  bases.  The s p e c i f i c a c t i v i t i e s o f t h e t h r e e p u r i n e s s e p a r a t e d from t h e a c i d - s o l u b l e f r a c t i o n were hypoxanthine-0.97, and guanine-0.29.  adenine=l,  These v a l u e s a r e e x -  p r e s s e d as r e l a t i v e s p e c i f i c a c t i v i t i e s because t h e y were d e r i v e d from counts o b t a i n e d by a t e c h n i q u e o f c o u n t i n g w h i c h was n o t c o r r e l a t e d w i t h t h e s t a n d a r d BaCOg method o f c o u n t i n g . Examination  of the expired a i r f o r the f i r s t  hours o f t h i s experiment were p r e s e n t .  twelve  showed t h a t a t o t a l o f 60,000 c.p.m.  T h i s r e p r e s e n t s 14$ o f t h e a c t i v i t y  injected  during t h i s period. The r e s u l t s o f examining  t h e u r i n e showed t h a t  49.5$ o f t h e t o t a l i n j e c t e d a c t i v i t y was e l i m i n a t e d from t h e animals by t h i s r o u t e . 14 of the t o t a l C  The o n l y f u r t h e r s u b d i v i s i o n made  a c t i v i t y p r e s e n t i n t h e u r i n e was t h a t  p r e s e n t as u r e a .  Urea was shown t o c o n t r i b u t e o n l y 1$ o f  the t o t a l o f 49.5$.  The N  1 5  c o n t e n t o f t h e u r e a was a l s o  shown t o be n e g l i g a b l e , o n l y 0.009 N"" atom $ e x c e s s . 1  5  E x a m i n a t i o n o f t h e b l o o d showed a s p e c i f i c  acti-  v i t y o f 320 c.p.m./cc. D.  Experiment I I I n experiment  I I , two male r a t s o f t h e W i s t a r  s t r a i n h a v i n g a combined weight o f 462 gms. were i n j e c t e d intraperitoneally with the test solution.  The i n d i v i d u a l  dose p e r r a t was 0.62 c c . w h i c h c o n t a i n e d 5.80 mgms. o f adenine.  T h i s dosage was a d m i n i s t e r e d t w i c e d a i l y ,  the r a t s were b e i n g i n j e c t e d a t a l e v e l o f 50  thus  mg./kilo./day.  - 25 The  s p e c i f i c a c t i v i t y o f t h e a d m i n i s t e r e d adenine b e i n g  1.66 x 1 0 c.p.m./mg. adenine, t h e two r a t s r e c e i v e d a t o t a l 4  o f 1.57 x 1 0 c.p.m. 6  D u r i n g t h e course o f t h e experiment  the r a t s r e c e i v e d t h e i r n o r m a l r a t i o n s and had f r e e a c c e s s to water. ninty-six  T h i s experiment was c a r r i e d on f o r a t o t a l o f hours.  Results The v a l u e s o b t a i n e d from t h e C  1 4  and N ^ a n a l y s i s 1  o f t h e p u r i n e and p y r i m i d i n e bases r e c o v e r e d from t h e n u c l e i c a c i d s a r e p r e s e n t e d i n Table I I I TABLE I I I C"'" and N^°" Values o f t h e Adenine, Guanine and Mixed P y r i m i dines o f the V i s c e r a l N u c l e i c Acids a f t e r I n t r a p e r i t o n e a l I n j e c t i o n o f 1,3-N -, 2 - C - a d e n i n e (92.8 mgm., 1.57 x 1 0 c.p.m.) 4  15  14  6  R a d i o a c t i v i t y Found Compound  N  1 5  T o t a l c.p.m. S p e c i f i c a c t i v i t y Atom % c.p.m./mg. Cpd. Excess  Adenine  12,800  1,575  0.336  Guanine  10,300  398  0.168  Pyrimidine mixture (from i o n exchange)  2,000  These v a l u e s a r e combined w i t h those o b t a i n e d i n experiment  I and a r e r e c o r d e d a g a i n i n a more s i g n i f i c a n t  form i n Table V I I . I n t h i s experiment  i t was p o s s i b l e t o c a l c u l a t e  b o t h t h e r e l a t i v e and t h e a b s o l u t e s p e c i f i c a c t i v i t i e s f o r the t h r e e p u r i n e s r e c o v e r e d from t h e a c i d - s o l u b l e f r a c t i o n .  - 26 The d i f f e r e n c e between t h i s experiment and t h e f i r s t b e i n g t h a t t h e counts on t h e s e bases were o b t a i n e d from b o t h paper d i s c and t h i c k sample BaCOg c o u n t s .  I t was e x p l a i n e d  e a r l i e r t h a t t h e paper d i s c counts b e a r no d i r e c t r e l a t i o n s h i p t o any o f t h e o t h e r counts r e c o r d e d i n t h e d a t a , a l l o f t h o s e counts h a y i n g been done on BaCOg p r e c i p i t a t e s .  The r e l a t i v e  s p e c i f i c . a c t i v i t i e s o b t a i n e d were a d e n i n e = l , n y p o x a n t h i n e ^ 0.92 and guanine=0.24.  The a b s o l u t e s p e c i f i c a c t i v i t i e s were  f o r adenine 4040 c.p.m./mg. cpd. and f o r guanine 931 c.p.m./ mg. c p d . There i s no a b s o l u t e s p e c i f i c a c t i v i t y r e c o r d e d f o r hypoxanthine s i n c e i t was p r e s e n t i n a m i x t u r e o f p y r i m i d i n e s and was o n l y s e p a r a t e d by paper  chromatography.  The r e s u l t s o f examining t h e e x p i r e d a i r a r e p r e sented i n Table I V . TABLE I V e x p i r e d by Rats a f t e r R e c e i v i n g , by I n t r a p e r i t o n e a l I n j e c t i o n , 92.8 mgm. o f l , 3 - N j - . j 2 - C - a d e n i n e (1.57 x 1 0 c.p.m.) 5  14  b  Period of Collection hr.  0-12 12 - 24 24 - 36 36 - 48 48 - 60 60 - 72 72 - 84 84 - 96 Total  R a d i o a c t i v i t y Found T o t a l c.p.m.  Specific Activity c.p.m./mgm. 0  44,000 33,666 43,666 70,300 32,666 44,666 34,000 41,666  11.39 19.25 11.25 17.80 8.90 12.65 8.80 10.41  344,630  -  - 27 The p o s s i b l e s o u r c e o f t h e s e 344,630 c.p.m. found i n the expired a i r i s discussed l a t e r .  A relationship  will  be drawn i n t h e d i s c u s s i o n between t h e r e s u l t s i n T a b l e I V and those i n T a b l e s I I and I I I . The r e s u l t o f examining t h e u r i n e f o r i t s t o t a l C  1 4  c o n t e n t i s p r e s e n t e d i n Table V. TABLE V  Excretion of C i n the Urine o f t h e Rats a f t e r I n t r a p e r i t o n e a l I n j e c t i o n o f 92.8 mgm. o f l , 3 - N - , 2 - C - a d e n i n e (1.57 x 1 0 c.p.m.). 1 5  1 4  6  Period of Collection hr.  Volume cc.  Radioactivity Excreted T o t a l c.p.m.  Specific activity c.p.m./mgm. C  0-12 12 - 24 24 - 36 36 - 48 48 - 60 60 - 72 72 - 84 84-96  20 16 18 28 18 25 18 20  69,400 67,100 109,900 115,700 129,100 131,600 116,700 93,700  231 266 384 303 461 376 459 205  Total  163  833,200  -  The t o t a l o f 833,200 c.p.m. r e c o r d e d r e p r e s e n t s 53$ o f t h e t o t a l i n j e c t e d a c t i v i t y .  Both the t o t a l  excretion  and t h e s p e c i f i c a c t i v i t i e s r i s e s l o w l y over t h e f i r s t t h r e e days t h e n d e c l i n e somewhat on t h e f o u r t h day. The r e s u l t s o f u r e a a n a l y s i s on t h e u r i n e a r e p r e s e n t e d i n Table V I .  - 28 TABLE VI Excretion of C and N i n t h e Urea o f t h e R a t s ' U r i n e a f t e r I n t r a p e r i t o n e a l I n j e c t i o n o f 92.8 mem. o f 1 . 3 - I \ f - , 2 - C adenine (1.57 x 10° c.p.m.) 1 4  1 5  15  Period of Collection hr.  14  R a d i o a c t i v i t y Excreted T o t a l c.p.m.  N  Atom % Excess  Specific Activity c.p.m./mgm. C  0-24 24 - 48 48 - 72 72 - 96  5,440 1,020 1,720 1,200  35.05 17.20 10.20 8.80  Total  9,480  - -  15  0.014 6'." 014 0.012 0.014  -  W i t h t h e e x c e p t i o n o f t h e f i r s t day t h e t o t a l counts e x c r e t e d p e r day were o f a s i m i l a r o r d e r o f magnitude. T h i s t a b l e shows a s t e a d y d e c l i n e i n t h e s p e c i f i c of the urea. to  activity  T h i s d e c l i n e i n s p e c i f i c a c t i v i t y i s comparable  t h a t found f o r t h e e x p i r e d  C 0 . l 4  g  E s t i m a t i o n of the a l l a n t o i n content of t h e u r i n e r e v e a l e d t h a t t h e r e were 340.4 mgm. o f t h i s compound i n t h e t o t a l u r i n e volume. t o t a l o f 258,200 c.p.m.  present  These 340.4 mgm. c o n t a i n e d a  The s p e c i f i c a c t i v i t y was c a l c u -  l a t e d t o be 758 c.p.m./mg. Cpd. The s p e c i f i c a c t i v i t y i n 15 terms o f atom % excess N  was 0.223.  I t w i l l be d i s c u s s e d  l a t e r t h a t t h e s e f i g u r e s r e p r e s e n t a d e f i c i t o f 159,800 c.p.m. i f t h e a l l a n t o i n i s c o n s i d e r e d as a r i s i n g  solely  from p u r i n e s o f t h e same i s o t o p e c o n t e n t as t h a t o f t h e i n jected l,3-N -,2-C -adenine. 1 5  1 4  - 29 The a d d i t i o n o f c a r r i e r adenine t o an a l i q u o t o f t h e u r i n e and i t s subsequent r e - i s o l a t i o n l e d t o t h e r e c o v e r y o f adenine w i t h a s p e c i f i c a c t i v i t y o f 55 c.p.m./mg. Cpd. I f a l l t h e c a r r i e r adenine had been r e c o v e r e d from t h e a l i quot, t h e t o t a l counts r e c o r d e d f o r a l l t h e u r i n e e x c r e t e d would have been 100,625 c.p.m.  T h i s r e p r e s e n t s 6.02 mg. o f  t h e adenine i n j e c t e d , i . e . adenine o f s p e c i f i c  activity  1.66 x 1 0 c.p.m./mg. Cpd. 4  The paper chromatograms w h i c h had been developed on t h e u r i n e , showed u l t r a v i o l e t a b s o r p t i o n over t h e i r  entire  l e n g t h , c o n s e q u e n t l y no c o n c l u s i o n s c o u l d be drawn from t h i s approach c o n c e r n i n g p u r i n e s w h i c h might o r might n o t be present.  Scanning f o r r a d i o a c t i v i t y by means o f t h e a u t o -  m a t i c chromatogram c o u n t e r i n d i c a t e d t h a t t h e major p o r t i o n 14 of C  a c t i v i t y l a y between t h e R j  v a l u e s 0.2 - 0.5. T h i s  r e s u l t was v e r i f i e d b y means o f an a u t o r a d i o g r a m .  Spraying  t h e chromatograms w i t h para-dimethylaminobenzaldehyde, a r e a g e n t w h i c h r e a c t s w i t h u r e a and a l l a n t o i n , i n d i c a t e d t h a t t h e s e substances were b o t h p r e s e n t on t h e chromatograms. The Rj 0.57.  v a l u e determined f o r u r e a was 0.71, f o r a l l a n t o i n The a l l a n t o i n t a i l e d i n t o t h e r a d i o a c t i v e r e g i o n o f  the chromatograms, Rj  0.2 - 0.5. S p r a y i n g w i t h ds'iazotized  s l u l p h a n i l i c a^cid, a r e a g e n t r e a c t i n g w i t h t h e p u r i n e b a s e s , was u n s u c c e s s f u l because t h e r e a g e n t was c o u p l i n g w i t h o t h e r compounds t o g i v e a complete s t r e a k i n g o f t h e chromatograms, t h u s no c l u e was a f f o r d e d as t o t h e presence o r q u a n t i t y o f p u r i n e s on t h e chromatograms, w h i c h might be c o n t r i b u t i n g t o  - 30 their radioactivity.  Use o f ammoniacal s i l v e r n i t r a t e  as a  spray r e a g e n t gave n e g a t i v e o r i n a p p r e c i a b l e r e a c t i o n f o r t h e presence o f u r i c  acid.  The f a e c e s were a n a l y s e d o n l y f o r t h e i r content.  C  1 4  T h i s v a l u e was r e a s o n a b l y c o n s t a n t over each 24 hour 14  p e r i o d b e i n g a p p r o x i m a t e l y 9000 c.p.m.  The t o t a l C  activity  e x c r e t e d i n t h e f a e c e s over t h e f o u r day p e r i o d was 32,930 c.p.m.  T h i s v a l u e r e p r e s e n t s 2% o f t h e t o t a l a c t i v i t y  injected  i n t o the animals. 14 The b l o o d when examined f o r i t s C an a c t i v i t y o f 1,280 c.p.m./cc.  c o n t e n t had  - 31 DISCUSSION When adenine i s a d m i n i s t e r e d t o a r a t i t becomes incorporated i n t o the t i s s u e n u c l e i c a c i d s , appearing both as adenine and as g u a n i n e . 15 f o r 1,3-N  T h i s f a c t has been demonstrated  - l a b e l l e d adenine by Brown and h i s c o l l e a g u e s  ( 4 ) , and f o r 2 - C - l a b e l I e d adenine by P a t e r s o n ( 2 0 ) . Two 1 4  p o s s i b i l i t i e s suggest themselves f o r t h i s c o n v e r s i o n o f adenine t o g u a n i n e , f i r s t ; i t i s a c c o m p l i s h e d by a d i r e c t a m i n a t i o n p r o c e s s w i t h no d i s r u p t i o n o f t h e p u r i n e n u c l e u s , second; t h e p u r i n e n u c l e u s opens a t t h e 2 p o s i t i o n t o g i v e an i n c o m p l e t e r i n g compound w h i c h i s c o n v e r t e d t o guanine by a m i n a t i o n and subsequent r i n g  closure.  The p r o j e c t u n d e r t a k e n i n t h i s t h e s i s was t o 15 14 i n j e c t 1,3-N  -,2-C  -adenine i n t o r a t s and t o examine t h e  p u r i n e s r e c o v e r e d from t h e v i s c e r a l n u c l e i c a c i d s t o d e t e r mine i f any change had o c c u r r e d i n t h e C t h a t o f t h e i n j e c t e d compound.  1 4  /N  r a t i o from  1 5  The o n l y r a t i o n a l by w h i c h  such a change i n t h e i s o t o p e r a t i o c o u l d be e x p l a i n e d i s t h a t d u r i n g t h e i n c o r p o r a t i o n o f adenine as adenine o r d u r i n g i t s c o n v e r s i o n t o guanine and i t s subsequent  incorporation  i n t o t h e v i s c e r a l n u c l e i c a c i d s as t h a t compound t h e p u r i n e n u c l e u s must be broken a t t h e 2 p o s i t i o n . 14 occurred then C  I f t h i s event  would be l i b e r a t e d and s h o u l d appear a t  some o t h e r p o i n t i n t h e a n i m a l ' s m e t a b o l i s m .  This l i n e of  r e a s o n i n g would be s u b s t a n t i a t e d by f i n d i n g C^ 0g i n t h e 4  expired a i r of the animal.  To f u r t h e r r e l a t e a change i n  t h e i s o t o p e r a t i o o f t h e n u c l e i c a c i d p u r i n e s and t h e  - 32 14  appearance o f C  Og i n t h e a n i m a l ' s e x p i r e d a i r t o one a n o t h e r ,  14  a d e f i c i t of C The C  1 4  bolism  /N  1 5  s h o u l d be observed i n t h e e x c r e t e d a l l a n t o i n .  r a t i o i n t h i s u r i n a r y by-product o f p u r i n e c a t a -  s h o u l d r e f l e c t t h e f a c t t h a t i t . i s d e r i v e d from b o t h  the adenine and t h e guanine o f t h e n u c l e i c a c i d s . r e l a t i n g t o the C  l 4  /N  1 5  The d a t a  r a t i o s a r e p r e s e n t e d i n Table V I I * TABLE V I I  The Percentage o f t h e I n j e c t e d l , 3 - N ^ - , 2 - C - a d e n i n e I n corporated i n t o the Purines of the V i s c e r a l Nucleic Acids of the Rats e x p r e s s e d i n terms o f i t s C-- and N content. 1  14  1  Compound  Synthetic  % Incorporation C a 1 4  4  % Incorporation N ^ b 1  Adenine  C  1 4  /N  1 5  a/b 1.000  Experiment I Nucleic Acid  Adenine  4.48  5.49  0.826  Nucleic Acid  Guanine  1.05  3.54  0.298  Nucleic I c i d  Adenine  9.50  Nucleic Acid  Guanine  2.39  Experiment 2 11.22 5.81  a  -  14 % incorporation C  b  =  % i n c o r p o r a t i o n N'15 . atom % e x c e s s N ^ atom % excess N -  C  1 4  /N  1 5  0.427  c.p.m./mg.Cpd. xlOO c.p.m./mg,Cpd. ( i n j e c t e d a d e n i n e ) 1 5  a/b  0.852  xlOO ( i n j e c t e d adenine)  c.p.m./mg.Cpd. / ( i n j e c t e d adenine) atom % e x c e s s N /c.p.nu/mg.Cpd. atom %. e x c e s s N ( i n j e c t e d adenine) 1£>  1 5  The C  1 4  /N  r a t i o i n the i n j e c t e d  1 5  l,3-N -,2-C 1 5  adenine was e s t a b l i s h e d as u n i t y and a l l o t h e r r a t i o mations were r e f e r r e d t o t h i s v a l u e .  1 4  esti-  I n b o t h experiments a l l  t h e p u r i n e s i s o l a t e d from t h e n u c l e i c a c i d s showed a C r a t i o l e s s t h a n 1.  l 4  /N  1 5  The adenine r e c o v e r e d from t h e v i s c e r a l  n u c l e i c acids, had, i n b o t h e x p e r i m e n t s , a v a l u e o f a p p r o x i l A 15 m a t e l y 0.83 f o r i t s C / N 4  ratio.  I n o t h e r words a t l e a s t  17$ o f t h e i n j e c t e d a d e n i n e w h i c h was i n c o r p o r a t e d i n t o t h e n u c l e i c a c i d s as adenine had passed t h r o u g h an i n t e r m e d i a r y s t a g e w h i c h p e r m i t t e d t h e 2-C- -atom t o be exchanged f o r a 1-4  n o n - i s o t o p i c atom.  The guanine r e c o v e r e d from t h e n u c l e i c 14 / 15  a c i d s showed an average v a l u e o f 0*36 f o r i t s C  /N  ratio.  T h i s would i n d i c a t e some 64$ o f t h e i n j e c t e d adenine w h i c h had been c o n v e r t e d t o guanine and t h e n been i n c o r p o r a t e d . i n t o t h e v i s c e r a l n u c l e i c a c i d s had f a i l e d t o m a i n t a i n i t s purine nucleus i n t a c t during the process.  Although a  c r i t i c a l comparison o f d a t a cannot be made between independent b i o l o g i c a l e x p e r i m e n t s , t h e i n c o r p o r a t i o n v a l u e s compare w i t h those o b t a i n e d b y t h e two p r e v i o u s l y mentioned e x p e r i m e n t o r s (4) ( 2 0 ) .  There i s a s i m i l a r i t y i n o r d e r o f magnitude f o r  the i n c o r p o r a t i o n as adenine b u t t h e c o n v e r s i o n t o and i n c o r p o r a t i o n as guanine i s l e s s marked. M e n t i o n was made e a r l i e r o f t h e t h r e e p o s s i b l e values the C  1 4  /N  1 5  r a t i o f o r guanine might t a k e i n an e x -  periment such as t h i s one. 1)  No C appears i n t h e p o l y n u c l e o t i d e g u a n i n e , i . e . t h e 14 15 C /N r a t i o becomes 0. 1 4  g)  The C  c o n t e n t o f t h e p o l y n u c l e o t i d e guanine i s t h e  1  same as t h a t o f t h e a d e n i n e , i . e . t h e C  l 4  /N  1 5  ratio  remains a t 1. 3)  The Cp- c o n t e n t o f t h e guanine i s lower t h a n t h a t o f 4  the adenine b u t s t i l l r e p r e s e n t s a s i g n i f i c a n t  frac-  t i o n o f t h e p o l y n u c l e o t i d e guanine i s o l a t e d , i . e . t h e C  l 4  /N  1 5  The  r a t i o becomes some f r a c t i o n between 0 and 1. assumption i s made i n a l l t h e s e  postulated  r a t i o s t h a t t h e r e i s no l o s s o f N ^ on t h e c o n v e r s i o n o f 1  adenine t o g u a n i n e .  T h i s assumption i s made on the.,basis  of t h e work o f Brown and h i s c o l l e a g u e s  ( 4 ) , and would be  v a l i d whether o r n o t t h e t r a n s f o r m a t i o n i n v o l v e d a r u p t u r e of t h e p u r i n e  nucleus.  A p l a u s i b l e explanation f o r the f i r s t p o s s i b i l i t y , C  l 4  /K  1 5  » 0, i s t h a t an exchange r e a c t i o n t a k e s p l a c e i n -  v o l v i n g an open c h a i n a l i p h a t i c compound and some p r e c u r s o r m o l e c u l e w h i c h s e r v e s b o t h as a source o f t h e 2*amino group and t h e 2-C- atom o f guanine. molecule.  I n 1949 R e i c h a r d  G l y c i n e i s such a p r e c u r s o r  (14) showed t h a t t h e 2-amino •'  group o f guanine was d e r i v e d l a r g e l y from t h i s s o u r c e .  More  r e c e n t experiments o f Van P o t t e r e t a l . (35) would i n d i c a t e t h a t t h e 2-C-atom o f guanine i s a l s o d e r i v e d from g l y c i n e . The  r e s u l t s o f b o t h t h e s e experiments r e p r e s e n t  anabolism,  consequently  'de novo'  t h i s p a t h may be p o s t u l a t e d f o r t h e  s y n t h e s i s o f guanine, but i t i s not n e c e s s a r i l y f o l l o w e d f o r t h e i n t e r c o n v e r s i o n o f t h e two bases and c e r t a i n l y i s not t h e s o l e p a t h f o r t h e i r  transformation.  The second p o s s i b l e r a t i o  mentioned, l  C  4 / N  15 .  l f  14 i s that i n which the C c o n t e n t o f t h e adenine and guanine s e p a r a t e d from t h e mixed n u c l e i c a c i d s i s o f t h e same o r d e r o f magnitude i n b o t h compounds. T h i s r e s u l t would i n d i c a t e 14 no l o s s o f C  during the conversion, a v i r t u a l  impossibility  u n l e s s t h e p u r i n e n u c l e u s remains i n t a c t and t h e c o n v e r s i o n t a k e s p l a c e by a d i r e c t a m i n a t i o n a t t h e 2 p o s i t i o n , preceded by a p r i o r o x i d a t i o n a t t h i s p o i n t . The t h i r d p o s s i b i l i t y , t h a t t h e C r * c o n t e n t o f t h e guanine i s l o w e r t h a n t h a t o f t h e adenine but s t i l l  repre-  s e n t s a s i g n i f i c a n t f r a c t i o n o f t h e p o l y n u c l e o t i d e guanine i s o l a t e d , i . e . , C ^ / N ^ ^ l , c o u l d be e x p l a i n e d i n s e v e r a l ways.  A rupture o f the purine nucleus a t the 2 p o s i t i o n t o  y i e l d a 4-formamido-, compound wouldnleave t h e 2-C-atom e x posed, a s t a t e i n w h i c h o x i d a t i o n and a m i n a t i o n become more p l a u s i b l e , but i n w h i c h exchange  a l s o becomes more l i k e l y .  E i t h e r exchange o r o x i d a t i o n would r e s u l t i n a l i b e r a t i o n of C  1 4  w i t h a r e s u l t a n t drop i n t h e C  1 4  /N  1 5  ratio.  Two compounds a r e t h e p o s s i b l e r e s u l t o f s u c h a s p l i t t i n g o f t h e p u r i n e n u c l e u s o f adenine i n i t s c o n v e r s i o n t o g u a n i n e , 4-formamido-, 5 - i m i d a z o l e carboxamide o r 4formamido-, 5 - i m i d a z o l e c a r b o x i m i d i n e , depending on whether deamination a t the 6 p o s i t i o n precedes o r f o l l o w s of t h e r i n g .  splitting  Compounds o f t h i s n a t u r e a r e n o t unknown b o t h  as b i o l o g i c a l and c h e m i c a l e n t i t i e s .  4 amino-5-imidazole  c a r b o x i m i d i n e i s t h e p r o d u c t r e s u l t i n g from a c i d  hydrolysis  o f adenine ( 3 6 ) , t h u s showing a l a b i l i t y o f t h e 2 p o s i t i o n  -36.i n a chemical been the  the  4 amino-5-imidazole  subject of experimentation  Greenberg ted  sense.  (38).  The  formation  products  Any  by Buchanan  e x p e r i m e n t s o f b o t h men  of i n o s i n i c  when t h i s  acid  and  i n v o l v i n g an  must o f n e c e s s i t y e x p l a i n t h e  has  (37)  and-  have d e m o n s t r a -  h y p o x a n t h i n e as  compound i s i n c u b a t e d  theory  carboxamide  with  formate.  open c h a i n  evidence  the  intermediate  o f Brown e t a l .  (4)  15 for  the r e t e n t i o n o f N  the  purine  nucleus.  s i d e r e d and  no  restrictions the  to one-half  ments m i g h t be  put  are put  isotope  forward  pound, b u t  free,  h a v i o r by  an  same n a t u r e  decarboxylation Acid  as and  This  lies  a carboximidine This  t h a t d e s c r i b e d by  First; pro-  com-  situation resonance  s t r u c t u r e may  not  be  p l a c e d upon i t s b e -  over-all  conversion  suggested l i m i t a t i o n Ogaton  of  w o u l d be  (39)  for  h y d r a t i o n r e a c t i o n s i n the Krebs  of  the Citric  Cycle. Another e x p l a n a t i o n  the  i s not  enzyme i n v o l v e d i n t h e  argu-  suggestion.  opening, i . e . the  limitation  1  Two  ring  carboximidine  have a s t e r i c  adenine to guanine. the  this  o f the n e c e s s i t y f o r c o n s i d e r i n g  Second; the  i t may  of the  concentration.  r a t h e r a c a r b o x a m i d e compound.  would d i s p o s e effect.  s t r u c t u r e i s con-  upon i t , r e s o n a n c e  to defeat  intermediate  3 p o s i t i o n s of  concentration  i t s original  t h a t d e a m i n a t i o n precedes the posed a l i p h a t i c  1 and  I f a carboximidine  immediately d i l u t e s position  i n b o t h the  C"^N^ i n the  not by  r a t i o upon the  for a significant  conversion  p o s s i b i l i t y that this  a single  route but  by  fall  of adenine to  transformation  a variety  of r o u t e s .  in  guanine,  takes In  place some  - 37 of these paths a l i b e r a t i o n of the 2-C-atom, might w h i l e i n others the t r a n s f o r m a t i o n might be w i t h the r i n g i n t a c t .  accomplished  T h i s theory was suggested  i n the  i n t r o d u c t i o n by r e f e r e n c e t o the second metabolic p u b l i s h e d by R o l l and Brown ( 2 ) .  occur  map  In t h i s map the authors  show two routes f o r the c o n v e r s i o n o f a c t i v e adenine t o a c t i v e guanine.  One p a t h denotes a d i r e c t c o n v e r s i o n o f  the two bases, while the other i n v o l v e s a b a c k t r a c k i n g towards more g e n e r a l p r e c u r s o r molecules.  This region of  p r e c u r s o r i n t e r m e d i a t e s i s one i n which incomplete  purine  n u c l e i a r e known t o p l a y a prominent r o l e . E i t h e r o f those suggested  p o s s i b i l i t i e s , a single  r e a c t i o n or a v a r i e t y o f r e a c t i o n s f o r accomplishing the c o n v e r s i o n o f adenine to guanine, c o u l d be used t o e x p l a i n the observed  f a c t t h a t the i n c o r p o r a t i o n o f l , 3 - N - , 2 - C 1 5  1 4  adenine i n t o the t i s s u e n u c l e i c a c i d s as guanine was l e s s 14 15 when measured i n terms o f C than when measured as N . Other r e s u l t s observed 3-N  15 1)  -,2-C  14  from the i n j e c t i o n o f I ,  -adenine i n t o a r a t a r e :  A s i g n i f i c a n t amount o f C  1 4  appears i n the e x p i r e d  a i r o f the animal. 2)  Large amounts o f C  a c t i v i t y are found i n the  animal's u r i n e . 3)  An a c i d - s o l u b l e e x t r a c t o f the t i s s u e s c o n t a i n s purine n u c l e o t i d e s t h a t a r e of h i g h e r  specific  a c t i v i t y than the p o l y n u c l e o t i d e p u r i n e s . 4)  No a p p r e c i a b l e q u a n t i t y o f r a d i o a c t i v i t y i s found i n the animal's  blood.  - 38 5)  R a d i o a c t i v i t y excreted i n the faeces i s n e g l i g i b l e 14  compared t o t h e t o t a l i n j e c t e d amount o f C A t e c h n i c a l f l a ? / must be t h e f i r s t p o s s i b i l i t y 14  c o n s i d e r e d w i t h r e g a r d t o t h e l a r g e amounts o f C i n the expired a i r of the animals.  Og found  Because a g r o s s amount  o f r a d i o a c t i v i t y i s e x c r e t e d i n t h e form o f b y - p r o d u c t s i n t h e a n i m a l ' s u r i n e , t h e p o s s i b i l i t y e x i s t s t h a t t h e s e byp r o d u c t s a r e undergoing b a c t e r i a l d e c o m p o s i t i o n r e s u l t i n g 14  i n the l i b e r a t i o n of C  Og.  I f t h i s were t h e c a s e , t h e n  14  the C  Og i n t h e e x p i r e d a i r samples would bear no r e l a t i o n 14  s h i p t o t h e r a t ' s metabolism.  Three f e a t u r e s o f t h i s C  p r o d u c t i o n might be c i t e d t o r e l a t e i t t o t h e r a t ' s r a t h e r than t o b a c t e r i a l decomposition.  0  g  metabolism  The C 0 g shows a 1 4  marked d i u r n a l v a r i a t i o n , w h i c h i s i n agreement w i t h t h e r a t ' s n o c t u r n a l h a b i t s ( F i g u r e 3 ) . The s p e c i f i c a c t i v i t y of t h e u r e a e x c r e t e d i n t h e u r i n e shows a p a r a l l e l i s m t o that of the expired C 0 g (25). 1 4  T h i s would f o l l o w from t h e  Krebs-Henseleit theory f o r the formation of urea.  Finally  t h e q u a n t i t y o f r a d i o a c t i v i t y found i n t h e e x p i r e d a i r , 25$ of t h e t o t a l i n j e c t e d a c t i v i t y , would argue a g a i n s t t h i s ' t e c h n i c a l f l a w ' e x p l a n a t i o n f o r t h e presence o f C^ 0g i n 4  the expired a i r .  I f the C  1 4  0  g  i s a product of t h e r a t ' s  metabolism t h e n i t becomes d i r e c t e v i d e n c e f o r a of t h e 2-C-atom o f a d e n i n e .  lability  As such t h e inferenceii;imi±sfcd3e  drawn t h a t adenine undergoes some major r e a c t i o n i n v o l v i n g t h i s 2-C-atom.  The r e a c t i o n might be a t o t a l d e g r a d a t i o n  of t h e adenine m o l e c u l e .  T h i s p o s s i b i l i t y would n o t be i n  - 39 -  FIGURE 3  Histogram showing t h e R e l a t i v e E x c r e t i o n of C 0 and C - U r e a . 1 4  X4t  g  9 19.3 17.8 100,000  \  S P E C I F I C ACTIVITY c.p.m./mg.C.  >  '  i \  80,000  \  *  60,000  8.9  ^  3  -  1°8  40,000 RADIOACTIVITY c.p.m. 20,000  0  24  48  72  96  TIME (hours) C -Urea 1 4  C  1 4  0,  4  C  1 4  0p  - 40 agreement w i t h t h e t h e o r y f o r t h e main pathway o f p u r i n e c a t a b o l i s m , w h i c h i s thought t o l e a d t o t h e end p r o d u c t , a l l a n t o i n , by means o f t h e f o l l o w i n g  steps:  Adenine  *» n y p o x a n t h i n e  Guanine  * xanthine uric  acid  allantoin At no p o i n t on t h i s p a t h would t h e r e be a l i b e r a t i o n o f C  1 4  0  from 1 , 3 - N - , 2 - C - a d e n i n e . 15  g  14  Since the r e a c t i o n  sought i n v o l v e s some 25$ o f t h e adenine i n j e c t e d i t does n o t seem l i k e l y t h a t a d e g r a d a t i o n p a t h o f t h i s would have escaped n o t i c e .  magnitude  A substantiated reaction that  adenine i s known t o undergo, i n v o l v i n g t h i s s i t e , i s i t s t r a n s f o r m a t i o n t o guanine.  Moreover t h i s r e a c t i o n i s o f  s u f f i c i e n t b i o l o g i c a l importance t o be c o n s i d e r e d as t h e source f o r t h i s l a r g e amount o f r a d i o a c t i v i t y .  Opening o f  the p u r i n e n u c l e u s a t t h e 2- p o s i t i o n t o y i e l d a 4-formamidocompound exposes t h e 2-C-atom o f adenine t o e i t h e r  chance  o x i d a t i o n o r exchange  o r t o planned s u b s t i t u t i o n , e i t h e r 14 possibility : p o s s i b i l i t y r e s u l t i n g i n t h e appearance o f C 0,. i n t h e expired a i r . The source o f a l a r g e f r a c t i o n o f t h e C  1 4  activity  i n t h e u r i n e i s u n i d e n t i f i e d a f t e r s u b s t r a c t i n g t h e compounds i n which one would e x p e c t t o f i n d r a d i o a c t i v i t y .  That i s ,  the most l o g i c a l compounds w h i c h one would e x p e c t t o c o n t a i n r a d i o a c t i v i t y a f t e r administering 1,3-N -,2-C -adenine 15  account f o r o n l y 42$ o f t h e a c t i v i t y p r e s e n t .  14  The t o t a l  - 41 14  urine C  c o n t e n t c a n be a p p o r t i o n e d  1)  allantoin  28.0$  2)  adenine  11.6$  3)  urea.  4)  unknown  thus:  1.4$ 58.0$  The unknown 58$ o f t h e a c t i v i t y c o u l d n o t be accounted  for  by any major component t h a t c o u l d be d e t e c t e d by chromato14 graphic, autoradiographic or C techniques.  chromatographic  The a c t i v i t y w h i c h was accounted  scanning  f o r appears  as t h e compounds one would expect t o a r i s e from t h e n o r m a l metabolism  o f t h e f r e e base adenine.  A l l a n t o i n i s t h e major  end-product o f p u r i n e c a t a b o l i s m i n t h e r a t and a s such i t 14 appears as t h e major C  c o n t a i n i n g component w h i c h c o u l d  be i d e n t i f i e d i n t h e u r i n e .  S i n c e adenine i s n n o t ; ; a r r e q u i r e d  m e t a b o l i t e i n t h e d i e t o f t h e r a t , t h e r e f o r e , one would expect i t might be e x c r e t e d e i t h e r unchanged o r i n some c o n j u g a t e d form i f i t i s f e d i n e x c e s s .  This s u s p i c i o n i s 14  v e r i f i e d by t h e r e c o v e r y o f adenine c o n t a i n i n g C the t e c h n i q u e o f c a r r i e r a d d i t i o n .  by u s i n g  U r e a , t h e t h i r d component  investigated, although i t contributed very l i t t l e t o the o v e r - a l l a c c o u n t i n g f o r r a d i o a c t i v i t y does h e l p t o i d e n t i f y 14 the source o f t h e C itself.  0  g  i n t h e e x p i r e d a i r as b e i n g t h e r a t  A f u r t h e r examination o f the a l l a n t o i n excreted  shows t h a t i t s C  1 4  /N  1 5  r a t i o i s 0.618.  I f i t had been d e -  r i v e d from t h e p u r i n e s formed from t h e i n j e c t e d and t h e i r C  l 4  /N  1 5  adenine  r a t i o s had remained a t 1 t h e n t h e a l l a n -  t o i n s h o u l d a l s o have had a C" /N" '^ r a t i o o f 1. L4  1  In t h i s  event f o r a l l a n t o i n w i t h a s p e c i f i c a c t i v i t y o f 0.223 atom  - 42 ^  15  % excess N  , 340.4 mgm. s h o u l d have c o n t a i n e d 418,000 c.p.m.  Only 258,200 c.p.m. a r e p r e s e n t .  T h i s v a l u e r e p r e s e n t s a.  d e f i c i t o f 159,800 c.p.m., a d e f i c i t t h a t c a n be used t o 14 account f o r a p p r o x i m a t e l y 50% o f t h e C animals' expired a i r .  0  g  found i n t h e  This, furthermore, d i r e c t l y  relates  the C 0 g found i n t h e e x p i r e d a i r t o t h e drop i n C 1 4  l 4  /N  1 5  r a t i o found i n t h e n u c l e i c a c i d p u r i n e s . I n v i e w o f t h e r e c e n t work o f Ochoa e t a l . (18) and t h a t o f Kornberg and h i s group (40) t h e h i g h s p e c i f i c a c t i v i t y shown b y t h e f r e e n u c l e o t i d e s i s n o t s u r p r i s i n g . The e x p e r i m e n t s o f b o t h men have shown t h a t t h e phosphoryl a t e d r i b o t i d e s and d e s o x y r i b o t i d e s would appear t o be t h e u n i t s i n c o r p o r a t e d i n t o R i b o s e N u c l e i c a c i d and D e s o x y r i b o s e 14 Nucleic acid.  The importance o f 2-C  -hypoxanthine r i b o -  t i d e as an i n t e r m e d i a r y p r o d u c t concerned w i t h t h e c o n v e r s i o n of adenine t o guanine cannot be e s t i m a t e d .  I f i t does  f i g u r e i n t h i s r o l e , i t i s a l s o w e l l known as a breakdown p r o d u c t o f adenine i n t h e c o u r s e of i t s d e g r a d a t i o n t o a l l a n t o i n , and t h e r e s p e c t i v e c o n t r i b u t i o n o f e i t h e r 14 to t h e o v e r a l l t o t a l o f C  reaction  -hypoxanthine p r e s e n t cannot be  measured i n t h i s e x p e r i m e n t .  However, Abrams and B e n t l e y  (13) have shown t h a t t h e r o u t e l e a d i n g from a d e n y l i c a c i d t h r o u g h i n o s i n i c a c i d t o x a n t h o s i n e r i b o t i d e phosphate and from t h e r e t o g u a n y l i c a c i d can be c o n s i d e r e d as a v a l i d s e r i e s o f r e a c t i o n s w h i c h c o u l d l e a d from adenine t o guanine. A b l o c k e d r e a c t i o n o r s a t u r a t i o n t e c h n i q u e might be used t o s o l v e t h e q u e s t i o n o f t h e importance o f t h i s r o u t e .  If  - 43  -  l a b e l l e d adenine and u n l a b e l l e d i n o s i n i c a c i d were a d m i n i s t e r e d s i m u l t a n e o u s l y , the degree of i n c o r p o r a t i o n o f adenine as guanine i n t o the t i s s u e n u c l e i c a c i d s s h o u l d d e c r e a s e i f i n o s i n i c a c i d i s a d i r e c t i n t e r m e d i a t e upon the pathway o f t h i s conversion. Very l i t t l e can be d i s c u s s e d about any r o l e  the  b l o o d might p l a y i n t h i s t r a n s f o r m a t i o n phenomenon s i n c e the o n l y a n a l y s i s c a r r i e d out was 14 total C  activity.  an e s t i m a t i o n o f the  T h i s , i n the case of b o t h  t u r n e d out t o be n e g l i g i b l e w i t h r e s p e c t t o the  experiments, total  a c t i v i t y i n j e c t e d and c o n s i d e r i n g the r e s u l t s o b t a i n e d i n the e x p i r e d a i r was  p r o b a b l y m a i n l y i n the form o f b i -  carbonate. The f a e c e s , l i k e w i s e , showed v e r y l i t t l e of the t o t a l a c t i v i t y i n j e c t e d , i n d i c a t i n g , as might be  expected,  t h a t f a e c a l e x c r e t i o n p l a y s a v e r y minor r o l e i n the  dis-  p o s a l o f the i n j e c t e d 1 , 3 - N - , 2 - C - a d e n i n e . 15  14  F i g u r e IV p r e s e n t s an o v e r a l l a c c o u n t i n g o f the t o t a l r a d i o a c t i v i t y i n j e c t e d each day i n terms o f  percentages  c o n t r i b u t e d by each o f the t h r e e e x c r e t o r y r o u t e s p l u s a f o u r t i i d i v i s i o n l a b e l l e d unknown.  T h i s unknown f r a c t i o n i s  t h a t p o r t i o n of the i n j e c t e d dose w h i c h presumably remains w i t h i n the a n i m a l and e n t e r s the v a r i o u s pathways o f t h e animal's metabolic  processes.  - 44 FIGURE 4 D a i l y a c c o u n t a b l e Radioactivity:.!oiand :Llnthe Three E x c r e t o r y P r o d u c t s e x p r e s s e d a s _ a Percentage oi' t h e R a t s ' 24 h o u r l y dose o f l , 3 - N - , 2 - C - - a d e n i n e (392,500 c.p.m.) ±B  L4  100  100  80  80  60  C0  60  2  Percentage  Percentage 40  40  20  20  100 80  FAECES  72 24 48 Time (hours)  =1=  96 100 80  URINE  60  Percentage  Percentage  40  20  20  96  96  UNKNOWN  60  40  24 48 72 Time (hours)  24 48 72 Time (hours)  24 48 72 Time (hours)  96  - 45 SUMMARY A.  Doubly l a b e l l e d a d e n i n e , 1 , 3 - N - , 2 - C - a d e n i n e , 15  14  was s y n t h e s i z e d and a d m i n i s t e r e d t o r a t s by means o f i n t r a peritoneal injections.  A t t h e end o f an experiment t h e  mixed v i s c e r a l n u c l e i c a c i d s were i s o l a t e d and h y d r o l y s e d . The adenine and guanine o b t a i n e d from t h i s s o u r c e were t h e n examined f o r t h e i r i s o t o p e c o n t e n t i n terms o f t h e i r r a t i o r e l a t i v e to the C B.  1 4  /N  The r a t i o o f C  1 4  C  1 4  /N  1 5  r a t i o i n t h e adenine i n j e c t e d .  1 5  /N  1 5  i n t h e adenine r e c o v e r e d from  t h e v i s c e r a l n u c l e i c a c i d s was 0,326 i n t h e f i r s t and 0.S52 i n t h e second e x p e r i m e n t .  experiment  Therefore, approximately  17$ o f t h e i n j e c t e d , 1 , 3 - N - , 2 - C - a d e n i n e w h i c h was i n 15  14  c o r p o r a t e d i n t o t h e n u c l e i c a c i d s as adenine had exchanged i t s 2 - C - a t o m f o r a n o n - i s o t o p i c atom. 14  C.  The r a t i o o f C  1 4  /N  1 5  i n t h e guanine r e c o v e r e d from  t h e v i s c e r a l n u c l e i c a c i d s was 0.298 i n experiment I and 0.427 i n experiment I I . T h i s i n d i c a t e s t h a t a p p r o x i m a t e l y 64$ o f t h e i n j e c t e d 1 , 3 - N - , 2 - C - a d e n i n e w h i c h was i n 15  14  c o r p o r a t e d i n t o t h e v i s c e r a l n u c l e i c a c i d s i n t h e form o f 14 guanine had exchanged i t s 2-C -atom f o r a n o n - i s o t o p i c atom. D.  I n the purines of the v i s c e r a l nucleic acids  t h e r e was 47$ l e s s C  1 4  found i n t h e guanine t h a n i n . t h e  adenine d e s p i t e t h e f a c t t h a t t h e y were b o t h d e r i v e d from t h e same s o u r c e , 1 , 3 - N - , 2 - C - a d e n i n e w h i c h had a C 15  14  c o n t e n t c o n s i d e r e d t o be 100$.  1 4  - 46 E.  Large amounts o f C  14  a c t i v i t y were found i n t h e  e x p i r e d a i r upon i n j e c t i o n o f l,3-N^-,g-C^" -adenine i n t o 14 4  a rat.  The amount o f r a d i o a c t i v i t y found as C  s e n t e d 25$ o f t h e t o t a l i n j e c t e d a c t i v i t y .  0  g  repre-  The t o t a l  14  a c t i v i t y . o f the C F.  0  g  was 344,630 c.p.m.  The r a d i o a c t i v i t y found i n t h e u r i n e a c c o u n t e d  f o r 58$ o f t h e i n j e c t e d a c t i v i t y . degradation  A l l a n t o i n , the f i n a l  product of purine catabolism,  represented  the  14  largest i d e n t i f i a b l e pool of C  a c t i v i t y contributing to  the t o t a l r a d i o a c t i v i t y p r e s e n t i n t h e u r i n e . G.  The C  1 4  /N  1 5  r a t i o i n t h e a l l a n t o i n was 0.618.  This r a t i o i n d i c a t e d a t o t a l 0 H.  1 4  d e f i c i t o f 159,800 c.p.m.  T h i s d e f i c i t o f counts i n t h e a l l a n t o i n was used  t o account f o r 47$ o f t h e C"*" 0 found i n t h e a n i m a l s ' 4  g  expired a i r . I. purines  The r e l a t i v e s p e c i f i c a c t i v i t i e s found f o r t h e o f t h e a c i d - s o l u b l e f r a c t i o n were a d e n i n e = l ,  hypoxanthine=-0.92 and guanine-0.24 i n d i c a t i n g t h a t hypoxant h i n e i n some form r e p r e s e n t s  a possible intermediate  on  the r o u t e o f i n t e r c o n v e r s i o n o f a d e n i n e t o p o l y n u c l e o t i d e guanine.  The a b s o l u t e  s p e c i f i c a c t i v i t i e s found i n t h e  a c i d - s o l u b l e f r a c t i o n were much h i g h e r t h a n t h o s e found f o r t h e p u r i n e s o f the n u c l e i c a c i d s .  Therefore,  i t could  be i n f e r r e d t h a t i n t e r c o n v e r s i o n t o o k p l a c e a t t h i s s t a g e r a t h e r t h a n a f t e r i n c o r p o r a t i o n i n t o t h e n u c l e i c a c i d s had taken place.  - 47 J.  -  The d i s c r e p a n c y between the s p e c i f i c a c t i v i t i e s  of the C 0 g and the C - u r e a , (the s p e c i f i c a c t i v i t y o f 1 4  1 4  the u r e a i s h i g h e r t h a n t h a t o f t h e COg),  indicated that  t h e r e i s p o s s i b l y another s o u r c e f o r t h i s u r e a i n a d d i t i o n to the K r e b s - H e n s e l e i t c y c l e .  - 48 BIBLIOGRAPHY B a r n e s , F.W., J r . , and Schoenheimer, R., J . B i o l . Chem.. 151. 123 (1943). Brown, G.B., R o l l , P.M., i n C h a r g a f f , E., and Davidson,J.N., The N u c l e i c A c i d s , V o l . I I , Academic P r e s s , I n c . (1955). P l e n t l , A.A., and Schoenheimer, R., J . B i o l . Chem., 155. 203 (1944). 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