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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 of 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 requirements f o r the degree of Master of Science i n the Department of Biochemistry. We accept t h i s t h e s i s as conforming to the required standard The u n i v e r s i t y of B r i t i s h Columbia October 1956. 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 requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree th a t permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e . I t i s under-stood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada. ABSTRACT. 15 14 1,3-N -,2-C -adenine was synthesized and administered 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 incorporated as both 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 of the C 1 4 / N ^ r a t i o s found i n the adenine and guanine i s o l a t e d from the v i s c e r a l n u c l e i c a c i d s w i t h that i n the i n j e c t e d adenine i n d i c a t e d that 14 f o r both compounds there had been a l o s s of C . The decrease^ i n the C^/N^ 5 r a t i o of the p o l y n u c l e o t i d e guanine was much greater than that i n the 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 tha t on at l e a s t one pathway f o r the conversion of adenine t o guanine the 14 purine nucleus does not remain i n t a c t . The l a b i l e 2-C was 14 found as C Og i n the animals' expired a i r . Evidence t h a t 14 part of the C Og r e s u l t e d from purine i n t e r c o n v e r s i o n was afforded by examining the a l l a n t o i n excreted i n the anim a l s 1 14 15 u r i n e . This compound had a C /N r a t i o c o n s i s t e n t w i t h the source from which i t was d e r i v e d , the 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 of C , apparent from the C /N r a t i o , i n t h i s e x c r e t o r y product accounted f o r 47% of the C 0 g expired by the animals. ACKNOWLEDGEMENTS The author wishes to express h i s a p p r e c i a t i o n of the encouragement, advice, and personal a s s i s t a n c e i n the la b o r a t o r y given by Dr. S. H. Zbarsky during the course of t h i s research. This research was supported by a grant from the N a t i o n a l Research C o u n c i l . INTRODUCTION TABLE OF CONTENTS. Page 1 EXPERIMENTAL A. Chemical (1) Synthesis foreword 10 i ) 2-C 1 4 Tadenine 10 i i ) 1,3-N:~-adenine 11 i i i ) l,3-N ± 0-,2-C X 4-adenine ....... 11 (2) Proof 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 l chromatography 11 i i ) spectrophotometry 12 i i i ) d e r i v a t i v e and i t s melting p o i n t 12 (3) Measurement of Isotope Concentration i l C~r determination ............ 15 i i ; N 1 5 determination 16 B. Metabolic (1) Technique of Metabolism Experiments. 16 (2) Treatment of Samples general 17 i ) t i s s u e ....................... 17 i i ) a c i d - s o l u b l e f r a c t i o n 19 i i i ) carbon d i o x i d e ............... 20 i v ) u r i n e ................. 21 v) faeces ....................... 22 v i ) blood 22 C. Experiment I 22 Resu l t s 23 D. Experiment I I 24 Results 25 DISCUSSION .. 31 SUMMARY 45 BIBLIOGRAPHY 48 TABLES Page I . Number and nature of the samples saved i n 'Experiments I and I I .......................... 17 I I . C 1 4 and N contents of the v i s c e r a l n u c l e i c a c i d purines i n Experiment I ..•,.« 23 I I I . C 1 4 and N contents of the v i s c e r a l n u c l e i c a c i d purines i n Experiment I I 25 IV. C -" content of the expired carbon d i o x i d e i n Experiment I I 26 V. C** content of the u r i n e i n Experiment I I ..... 27 VI. C 1 and N l 0 content of the urea i n Experiment I I ,,.,..,..>.......,«..........«.....£........ 28 V I I . fo i n c o r p o r a t i o n of the i n j e c t e d 1,3-N -,2-C A 4-adenine 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 , Experiments I and I I , expressed i n terms of C , N 1 5 and C 1 4 / N 1 5 r a t i o s 32 FIGURES 1. Contact p r i n t of chromatogram used to v e r i f y the i d e n t i t y of the s y n t h e t i c adenine 13 2. A comparison of the u l t r a v i o l e t a b s o r p t i o n curves of authentic and s y n t h e t i c adenine...... 14 3. A comparison between the exc r e t i o n s of C 1 4 :0g and C 1 4-Urea i n Experiment I I ................. 39 4. Account of the d a i l y i n j e c t e d C 1^ as the amount expired or excreted and the amount r e t a i n e d by the animals i n Experiment I I .................. 44 INTRODUCTION The q u e s t i o n of how purine bases are i n t e r c o n v e r t e d i s a problem t h a t has occupied i n v e s t i g a t o r s f o r the past t e n years. I t s o r i g i n i s t o be found i n the work of Schoenheimer ( 1 ) . When isotppes became r e a d i l y a v a i l a b l e i n the e a r l y war years Schoenheimer, Buchanan, Hevesy and others began e x p e r i -mentation on the b i o s y n t h e s i s of n u c l e i c a c i d s and t h e i r com-ponent purine and pyri m i d i n e bases. 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 of the questions concerning p o s s i b l e precursor molecules f o r the var i o u s atoms i n the purine nucleus during the course of »de novo' synthesis w i t h i n the body. In 1944, Schoenheimer and P l e n t l (3) published the • r e s u l t s of i n v e s t i g a t i o n s which had extended the work beyond the search f o r precursor compounds t o the u t i l i z a t i o n of the f r e e bases themselves. Although these i n v e s t i g a t o r s were aware of Miescher's e a r l y observation t h a t preformed purines or p y r i m i d i n e s were not e s s e n t i a l f o r the s y n t h e s i s of n u c l e i c a c i d s ( 2 ) , i t seemed worthwhile t o them t o determine whether the f r e e bases would be u t i l i z e d f o r the sy n t h e s i s of n u c l e i c a c i d s when fed t o an animal. Their experiments i n c l u -15 15 ded the feeding of 1,3-N -, 2 amino-N -guanine as w e l l as the 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 . On the b a s i s of the r e s u l t s obtained from these experiments, Schoenheimer and P l e n t l s t a t e d : "Neither purines nor p y r i -midines supplied i n the d i e t are u t i l i z e d by the body f o r the synth e s i s of n u c l e o p r o t e i n " ( 3 ). - 2 -Because t h i s appeared to be an exception to the dynamic concept of 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 the p o s s i b l e u t i l i z a t i o n of f r e e bases fed to an animal. While they were checking the r e s u l t s obtained when r a t s were fed l a b e l l e d guanine they decided to 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 hat adenine, since i t occupied a more c e n t r a l p o s i t i o n i n an animal's metabolism, might show r e s u l t s d i f f e r e n t from 15 those of guanine. Therefore, 1,3-N -adenine was synthesized and included i n one of the d i e t s f e d to the r a t s . The r e s u l t s of t h i s experiment were a s t a r t l i n g c o n t r a s t to those ob-t a i n e d when guanine was f e d . I t was found that the f r e e base adenine was incorporated i n t o the mixed n u c l e i c a c i d s of the v i s c e r a and a l s o t h a t i t had been converted to guanine, which had l i k e w i s e been incorporated i n t o the mixed v i s c e r a l n u c l e i c a c i d s . Approximately t h i r t e e n percent of the adenine and ei g h t percent of the guanine e x t r a c t e d from the mixed v i s c e r a l n u c l e i c a c i d s had been derived from the 1 ,3-N-^-adenine which had been f e d . Figures of 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 data of Brown and h i s co-workers l e d to two con c l u s i o n s : f i r s t ; t h a t the f r e e purine bases could be used by an animal f o r the syn t h e s i s of t i s s u e n u c l e i c a c i d s , second; t h a t they were i n t e r c o n v e r -t i b l e ( 4 ). The experiments mentioned had a l l been c a r r i e d out using compounds synthesized from the s t a b l e i s o t o p e of n i t r o g e n , 15 N . When st a b l e isotopes are used to l a b e l compounds the - 3 -d i f f i c u l t y of excessive d i l u t i o n must be avoided. The degree of d i l u t i o n encountered i n these b i o l o g i c a l experiments 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 of the l a b e l l e d bases i n t o 14 the n u c l e i c a c i d s being overlooked. When C , a r a d i o a c t i v e i s o t o p e , was employed to l a b e l the t e s t compound as was done . by B a l i s et a l . ( 5 ) , using 8 l a b e l l e d guanine, or by Abrams (6 ) , using 2 l a b e l l e d guanine, i t was e a s i l y demonstrated tha t f r e e guanine when fed t o an animal was metabolised i n a manner s i m i l a r t o f r e e adenine. The degree of i n c o r p o r a t i o n of guanine i n t o the n u c l e i c a c i d s was so .slight that i t had been masked i n the e a r l i e r experiments. This masking had occurred as a r e s u l t of the l i b e r a t i o n of the l a b e l l e d 8-amino group i n t o the general amino group pool on the catabolism of l ,3-N 1 5-,g amino-N 1 5-guanine t o a l l a n t o i n . The f r e e N 1 5 , i . e . 15 the N Hg of the f r e e amino p o o l , l a b e l l e d a l l the purines t o an extent s u f f i c i e n t t o conceal the s l i g h t i n c o r p o r a t i o n of guanine i n t o the n u c l e i c a c i d s ( g ) . I t ¥/as a l s o d i s c o v e r e d , w i t h the a i d of C^ 4 l a b e l l e d guanine/ th a t when guanine was fed as the f r e e base, i n a d d i t i o n t o being in c o r p o r a t e d i n t o 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 of i t was converted t o adenine ( 6 ) . The experimental evidence from the p r e v i o u s l y 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 the concept th a t at some point i n t h e i r a n a b o l i c pathways a l l purines arose from a... common intermediate. This theory had been t e s t e d by feeding v a r i o u s common purine bases i n t h e i r f r e e s t a t e t o see i f one could be found which would be converted t o e i t h e r adenine or - 4 -guanine i n the course of i t s metabolism. Isoguanine was i n v e s t i g a t e d by Bendich and h i s colleagues ( 7 ) , hypoxanthine and xanthine by G e t l e r et a l . (8), and u r i c a c i d by Brown and h i s group ( 9 ) . None of these compounds were converted t o t i s s u e n u c l e i c a c i d adenine or guanine. 2,6-diamino pur i n e , t e s t e d by Bendich et a l . (10), 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 to p o l y n u c l e o t i d e guanine. As ffl$ch as four per-cent of the guanine ex t r a c t e d from the n u c l e i c a c i d s had come from t h i s compound when i t was included i n an animal's d i e t . There was, however, no conversion of the compound i n t o adenine by the r a t . f o l l o w i n g metabolic map showing the v a r i o u s s u b s t a n t i a t e d metabolic conversions which might conceivably 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 of adenine and guanine: In 1950 Brown and h i s co-workers ( I I ) published the guanine hypoxanthine xanthine - 5 -On t h i s map three types of arrows are used t o designate one of the three 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 the pathway which f r e e adenine f o l l o w s during i t s conversion t o n u c l e i c a c i d guanine. The conventional arrows-denote r e a c t i o n s which had been demonstrated and which pre -sumably could l e a d t o p o l y n u c l e o t i d e guanine. The conventional arrows which have been crossed out denote r e a c t i o n s which would lead t o n u c l e i c a c i d guanine but which had been demonstra* ted as not oc c u r r i n g w i t h i n the body. The dotted 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 the var i o u s purine bases which had been t e s t e d as precursors of n u c l e i c a c i d guanine. Brown and h i s co-workers, when an a l y s i n g t h i s meta-b o l i c map, argue th a t s i n c e n e i t h e r f r e e hypoxanthine nor f r e e xanthine f u n c t i o n as a precursor 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 at the 6-amino group of adenine i s excluded as the i n i t i a l step i n i t s conversion to po l y n u c l e o t i d e guanine. Whether the 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 ad e n y l i c a c i d , i n o s i n i c a c i d , xanthosine monophosphate and guanylic 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 of n u c l e i c a c i d guanine from f r e e adenine remains t o be seen (12) (13). Although isoguanine 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 synthesis when i t i s f e d i n the f r e e s t a t e ( 7). This f a c t , Brown and h i s co-workers f e e l , renders an i n i t i a l o x i d a t i o n at the 2 - p o s i t i o n of adenine a l s o improbable as the s t a r t i n g p o i n t on i t s conversion t o p o l y n u c l e o t i d e 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 chemical - 6 -b a s i s (10) and even more so i n view of Reichard's (14) evidence that the 2-amino group of guanine i s d e r i v e d f o r the most part from g l y c i n e . Brown and h i s colleagues conclude t h a t the most probable i n i t i a l step i s a r e a c t i o n between adenine and g l y c i n e . Brown et a l . (11) make no mention of the p o s s i b l e r o l e that an incomplete purine might p l a y as an intermediate . i n t h i s i n t e r c o n v e r s i o n of adenine and guanine. This f a c t i s i n agreement w i t h the view they held on the e s s e n t i a l i n t e g r i t y of the purine s k e l e t o n during the i n t e r c o n v e r s i o n . Siork of Buchanan and h i s group (15) or t h a t of Greenberg and h i s group (16) on 'de novo' s y n t h e s i s of r i b o t i d e s from incomplete purine r i n g compounds would make one suspect t h a t a r e a c t i o n between some carboxamide r i b o t i d e and g l y c i n e i s a p l a u s i b l e intermediate r e a c t i o n . This s u s p i c i o n would r e q u i r e t h a t the 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 rupture of the purine s k e l e t o n . In 1954 R o l l and Brown (2) published another metabolic p l a n . This map took cognizance of new experimental evidence which had accumulated on p o s s i b l e pathways f o r the i n t e r c o n v e r s i o n of p u r i n e s . Exogenous adenine « 'De novo' synthesis Exogenous guanine —*• Incompletes: Purine •Common // Intermediate A c t i v e aaenme v inosmic Acid w I I I I N u c l e i c i a c i d Adenine N u c l e i c a c i d Guanine > A c t i v e guanine - 7 -I t has been i n f e r r e d from experimental evidence that even i n the courses of'de novo' syn t h e s i s at l e a s t p a r t of one purine may a r i s e v i a the other (2). This f a c t i s d i f f i c u l t to 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 that through a common intermediate, consequently, an a l t e r n a t i v e i r r e f u t i b l e evidence i n d i c a t i n g at which stage, i . e . f r e e base, r i b o s i d e or r i b o t i d e , the i n t e r c o n v e r s i o n of adenine and guanine takes p l a c e . Current work on the b i o s y n t h e s i s of purines favor the p r o b a b i l i t y of the i n t e r c o n v e r s i o n t a k i n g place as the r i b o t i d e s . There i s , however, other evidence i n d i c a t i n g that the i n t e r c o n v e r s i o n may take place as the f r e e bases or as the r i b o s i d e s (17). Considering recent work of Ochoa et a l . (18), i t would appear that the nature of a c t i v e adenine or guanine i n the case of Pentose N u c l e i c A c i d s y n t h e s i s i s that of a diphosphate r i b o s i d e . volves the p o s s i b l e l a b i l i t y of the 2-C- atom of adenine on i t s conversion to p o l y n u c l e o t i d e guanine. While t h i s phenome-non i s i n a l l p r o b a b i l i t y due to a complex s e r i e s of r e a c t i o n s , d i r e c t i n t e r c o n v e r s i o n i s a l s o o f f e r e d . As yet there i s no 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 -there are two p o s s i b l e o v e r a l l processes: ( - ) (2) HN*CV HN' (1) . This i n v o l v e s no d i s r u p t i o n of the b a s i c purine s t r u c t u r e and a d i r e c t amination at the 2 p o s i t i o n of adenine. T h i s , as stated e a r l i e r , Brown and h i s colleagues (10) consider to be u n l i k e l y . (2) . This i n v o l v e s a rupture of the purine nucleus at the 2 p o s i t i o n w i t h subsequent amination and r e c l o s u r e of the r i n g * This second route permits the p o s s i b i l i t y of exchange or 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 at the 2 p o s i t i o n . In 1953 Paterson and Zbarsky (19) synthesized 2-C^ 4-adenine. This compound allowed a d i r e c t . i n v e s t i g a t i o n of the f a t e of the 2-C-atom of adenine during the course of i t s metabolism and catabolism. There was no previous knowledge ' of t h i s p a r t i c u l a r atom although i t had been the subject of conjecture regarding i t s p o s s i b l e l a b i l i t y (41) (42). The r e s u l t s obtained by Paterson (20) on feeding 2-C 1 4-adenine t o 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 greater l a b i l i t y than the other C atoms of 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 the i n t e r c o n v e r s i o n of purine bases. I t was f e l t , by the author, that a metabolic study i n v o l v i n g 1,3-N 1 5-, 2-C 1 4-adenine might y i e l d the 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 the r e l a t i v e percentage i n c o r p o r a t i o n of f r e e adenine.into p o l y n u c l e o t i d e guanine i n terms of both C^ 4 and NA0" on feeding such a compound. I f the two percentages 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 proof of the i n t e g r i t y of the purine s k e l e t o n during the i n t e r c o n v e r t i o n process. I f the two percentages vary t o the extent t h a t 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 of C-^, a rupture of the purine s k e l e t o n and subsequent exchange r e a c t i o n would be i n d i c a t e d . F i n a l l y , i f the two percentages vary such that the 14 i n c o r p o r a t i o n expressed 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 expressed 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 routes e x i s t , at l e a s t one of them r e q u i r i n g a rupture of the purine nucleus. The experiments presented i n t h i s t h e s i s were undertaken t o r e s o l v e the problem of whether the purine nucleus remained i n t a c t on the conversion of exogenous adenine t o p o l y n u c l e o t i d e guanine. This was accomplished by comparing the N"^ and C"*"4 i n c o r p o r a t i o n values t o d i s c o v e r which of the three 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 1^-,S-C' l" 4-adenine i n t o r a t s . The r e s u l t s i n d i -cated t h a t both i n the case of the i n c o r p o r a t i o n of exogenous adenine as adenine and i n the case of i t s conversion t o guanine and i n c o r p o r a t i o n as that compound the percentage i n c o r p o r a t i o n i n t o the 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 of the C 1 4 content of the i n j e c t e d l , 3 - N 1 5 - 2 -C^ 4-adenine than when measured as a f u n c t i o n of i t s N"1"5 content. Otherwise s t a t e d , the 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 both 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 con-v e r s i o n and i n c o r p o r a t i o n as guanine. The degree of t h i s l o s s i s such as t o "indicate that the t h i r d p o s s i b i l i t y i s the probable one, i . e . more than one route e x i s t s f o r the con-v e r s i o n of adenine t o guanine. The t a c i t assumption a r i s i n g from t h i s choice out of the three p o s s i b i l i t i e s i s t h a t on at l e a s t one route the purine nucleus i s not maintained i n t a c t during the i n t e r c o n v e r s i o n . - 10 -EXPERIMENTAL A. Chemical Synthesis The proposed s y n t h e s i s of doubly 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 the syn t h e s i s of two separate compounds. 14 F i r s t ; 2-C -adenine was synthesized by the method of Paterson and Zbarsky (19) adapted from Shaw (21), second; l,3-N 1 5-adenine was produced by the method of Bendich et a l . (22) modified from t h a t of Todd and h i s co-workers (23). 14 I t was e s s e n t i a l t h a t the 2-C -adenine be of high s p e c i f i c a c t i v i t y otherwise on mixing the two compounds, 2-C"*"4-adenine and 1,3-N -adenine, the atom % excess N would have 14 been e x c e s s i v e l y d i l u t e d . Furthermore, the higher the 2-C -adenine was i n s p e c i f i c a c t i v i t y , the e a s i e r i t would be t o 14 t r a c e the byproducts by means of t h e i r C content upon the a d m i n i s t r a t i o n of a minimum amount of the t e s t compound. i ) 2-C 1 4-adenine The p r e l i m i n a r y step i n the synthesis of 2-C"1-4-14 adenine was the s y n t h e s i s of C -formate. This was achieved by a combination of the method of Jeanes (32) and th a t of Abrams and h i s co-workers (24). N a g C 1 4 0 g was converted t o B a C 1 4 0 3 . The B a C l 4 0 3 was reduced t o B a ( C 1 4 ? N ) g by heating i n a furnace i n the presence of Na-Zn amalgam w h i l e at the same time dry ammonia gas was passed over the hot melt. The B a ( C 1 4 i N ) g was converted t o C 1 4 - f o r m i c a c i d by means of a c i d h y d r o l y s i s . The C 1 4-formate was condensed w i t h 4-amino, 5-imidazole carboximidine hydrochloride by the method of - 11 -Paterson and Zbarsky (19) t o give 2-C 1 4-adenine. (1) N c ^ C ' V , "Ba.(CW£N)a — > - HC"bOH (*) KC-100K f D-XHCP . | O i i ) 1,3-N 1 5-adenine l ^ 1,3-N -adenine was synthesized by the method of Bendich et a l . (22). N 1 5-Formamidine hydrochloride was p r e -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 product, 4,6-diaraino-5-phenyl-azopyrimidine, was reduced to give 4,5,6-tr i a m i n o - p y r i m i d i n e . Formylation, 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 -adenine. i i i ) 1,3-N 1 5-,2-C 1 4-adenine 14 l^S The two compounds, 2-C -adenine and 1,3-N -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 equivalent of HC1. This s o l u t i o n could then be looked upon as c o n s i s t i n g of 1,3-N 1 5-,2-C 1 4-adenine h y d r o c h l o r i d e , a doubly l a b e l l e d molecule. Proof 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 made between the R j values of the s y n t h e t i c product and a sample of authentic adenine which had both been run simultaneously on a paper chromatogram employing - 12 -a solvent of is o p r o p a n o l , HC1, H g 0 as described by Wyatt ( 3 1 ) . The R j value quoted f o r adenine i n t h i s s olvent i s 0 . 3 2 ( 3 1 ) . Figure 1 shows a contact p r i n t of the chromatogram as developed 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 product shows the same Rj value as the authentic adenine. The appearance of but a s i n g l e spot i s evidence of the e s s e n t i a l l y uncon-taminated nature of the s y n t h e t i c product. i i ) Spectrophotometry Theepurines, pyrimidines and t h e i r d e r i v a t i v e s have very w e l l defined absorption maxima and minima i n the u l t r a v i o l e t r e g i o n . The method of e s t a b l i s h i n g the wave-l e n g t h at which the maximum absorption occurs, the wave-l e n g t h at which the minimum absorption occurs and the r a t i o of the o p t i c a l d e n s i t i e s obtained at any two a r b i t r a r y wave-lengths i s a valuable a i d i n c h a r a c t e r i z i n g these compounds. When t h i s method of a n a l y s i s was a p p l i e d to the synthesized product i t was observed that the po i n t of maximum absorption was recorded at 2 6 2 . 5 m/*and the p o i n t of minimum absorption at 2 2 9 . 0 m y . These data agree w i t h the values recorded f o r 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 at 260 m/^and 280 m/*, i . e . A280 was 0 . 3 7 5 . This ' A260 value i s a l s o i n agreement w i t h the value quoted f o r adenine. Figure 2 shows the abso r p t i o n curves from which these values were c a l c u l a t e d . i i i ) D e r i v a t i v e and i t s melting p o i n t The p i c r a t e s a l t of adenine was prepared as a d e r i v a t i v e . I t was r e c r y s t a l l i z e d three 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 water, and i t s melting p o i n t - IS -FIGURE 1 C o n t a c t p r i n t o f a Chromatogram snowing a comparison o f the Ri v a l u e s o i t n e s y n t h e s i z e d adenine ( r i g n t ) and an a u t h e n t i c sample ( l e f t ) . The homogeneous n a t u r e o f tne sp o t v e r i f i e s tne absence of any s i g n i f i c a n t amount of c o n t a m i -n a t i o n of the s y n t h e t i c p r o a u c t . FIGURE 2 1.500 1.400 1.300 OPTICAL DENSITY 230 240 250 260 270 280 290 300 WAVE LENGTH Aj-A comparison of the U.V. absorption curves of au t h e n t i c adenine and the synthesized product. These readings were taken w i t h a Beckman quartz spectrophotometer Model DK 2. - 15 -determined. This value i s recorded as 285-28.6°C (wi t h de-composition) (S3). The melting p o i n t range obtained was 283-287°C (wi t h decomposition). Measurement of Isotope Concentration 14 i ) C determination Measurement of the s p e c i f i c a c t i v i t y of the synthe-s i z e d adenine was accomplished by the f o l l o w i n g procedure. An a l i q u o t was tr e a t e d by the wet o x i d a t i o n technique of Van Slyke and F o l c h (28). The COg evolved was trapped i n carbonate-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 of the t h i c k sample of BaC03 was determined by counting i n a gas f l o w windowless counter. Both the technique and the counter employed i n t h i s l a b o r a t o r y have been described by Paterson (20) and Wright (26). The s p e c i f i c a c t i v i t y of the s y n t h e t i c adenine was found to be 1.66 x 1 0 4 c.p.m./mg. The s p e c i f i c a c t i v i t y expressed as c.p.m./mM. of adenine was 2.24 x 10 6. The synthesized adenine was shown to be f r e e of any s i g n i f i c a n t amount of contaminating r a d i o a c t i v e m a t e r i a l by means of an autoradiogram. This was done using the method of Zbarsky and Wright (44). A developed paper chromatogram was placed i n contact w i t h X-ray f i l m i n a dark room f o r one week. When the 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 spot, i n d i c a t i n g only one r a d i o a c t i v e component was present. The value of 0.32 corresponded to the quoted Rj value of adenine (31). - 16 -i i ) N 1 5 determination The atom % excess N 1 5 of the s y n t h e t i c product was estimated on samples of Ng prepared by the method of R i t t e n b e r g (29). An a l i q u o t of the compound was converted to ( N H 4 ) g S 0 4 by the K j e l d a h l procedure.- This (NH"4)gSB4 was then t r e a t e d w i t h NaOBr i n vacuo y i e l d i n g Ng which was trapped and stored i n b r e a k s e a l c o n t a i n e r s . The Ng was analysed on a mass spectrometer f o r the atom % excess N 1 5 present. The concen-15 t r a t i o n of N i n the s y n t h e t i c adenine as determined by t h i s method was 2.897 atom % excess. B. Metabolic Technique of Metabolism Experiments 15 14 The 1,3-N v-,2-C -adenine was d i s s o l v e d i n a phosphate b u f f e r (pH 3.8). This s o l u t i o n was administered at twelve hour i n t e r v a l s by means of 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 . While an experiment was i n progress the r a t s were kept i n a metabolism cage s i m i l a r to that described by Wright (26) and Paterson (20). The arrangement of t h i s metabolism cage i s such as to permit separate c o l l e c t i o n of e i t h e r or both, expired C0 g and, u r i n e and faeces, f o r any given time p e r i o d , w i t h a minimum of contamination. In a p r e l i m i n a r y experiment three r a t s were used. They were i n j e c t e d w i t h adenine at a l e v e l of 100 mg./kilo. body weight/day. This experiment was terminated at the end of the f i r s t twenty-four hours by t e c h n i c a l d i f f i c u l t i e s . In 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 at a l e v e l of 50 mg./kilo. body - 17 -weight/day f o r a p e r i o d of n i n e t y - s i x hours. The samples c o l l e c t e d f o r a n a l y s i s from these two experiments are tabu-l a t e d below. TABLE I Number and Nature of the Samples Saved i n Experiments I and I I Nature of Sample Number of Samples Experiment I Experiment I I Blood V i s c e r a — — 12 hour u r i n e specimen 1 8 24 hour faeces specimen 0 4 12 hour expired a i r 1 8 specimen Treatment of Samples At the t e r m i n a t i o n of an experiment each r a t was k i l l e d by a blow on i t s head. I t was then decapitated and b l e d . The v i s c e r a were immediately 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 then homogenized w i t h 10 cc. of c o l d water i n a Waring Blendor. This was fo l l o w e d by three successive e x t r a c t i o n s w i t h 100 cc. p o r t i o n s of 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 the t i s s u e was c e n t r i f u g e d at 0°C. and the supernatant was decanted. I t was presumed that t h i s 300 cc. of c o l d y e l l o w i s h t r i -c h l o r o a c e t i c a c i d e x t r a c t contained a l l the f r e e n u c l e o t i d e s as w e l l as other 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$ etha n o l , - 18 -100$ ethanol, 100$ ethanol and ether and f i n a l l y w i t h ether alone. This treatment removed a l l of the r e s i d u a l water and f a t t y m a t e r i a l . A f t e r t h i s treatment the t i s s u e was a i r d r i e d . The dry t i s s u e was next extracted f o r e i g h t hours at 80°C. w i t h 150 cc. of 10$ NaCl, t h i s treatment was repeated t w i c e , then 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 one-half volumes of 95$ ethanol were added to the combined f i l t r a t e and the s o l u t i o n was allowed to stand overnight. A f i n e white p r e c i p i t a t e of crude sodium nucleate s e t t l e d out during t h i s p e r i o d . This p r e c i p i t a t e was recovered by c e n t r i -f u g a t i o n and, a f t e r washing, was immediately resuspended i n a s m a l l volume of :htir,tl0$ NaCl. I t was once again r e p r e c i p i t a t e d by the a d d i t i o n of a l c o h o l , washed, and t h i s time d r i e d . The pure sodium nucleate was then hydrolysed f o r one hour i n 5 cc. of 1 N. HC1 at 100°C. The r e s u l t a n t s l i g h t l y brownish hydrolysate was adsorbed on Dowex-50 i o n exchange r e s i n and e l u t i o n was c a r r i e d out w i t h 1 N. HC1 using an auto-matic f r a c t i o n c o l l e c t o r . This e l u a t e was d i v i d e d i n t o three main f r a c t i o n s on the b a s i s of o p t i c a l d e n s i t y readings taken at 260 my w i t h a model DU Beckman Spectrophotometer. These f r a c t i o n s were reduced t o 10 cc. volume each. At t h i s stage paper chromatograms and absorption spectra were done to v e r i f y the suspected i d e n t i t y of the components of each f r a c t i o n . The three f r a c t i o n s were shown to c o n s i s t of ade-ni n e , guanine and a mixture of p y r i m i d i n e s . The u l t r a v i o l e t a b s o r p t i o n measurements permitted c a l c u l a t i o n of the concen-t r a t i o n of each component from i t s molar e x t i n c t i o n - 19 -c o e f f i c i e n t . 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 these f r a c t i o n s to o b t a i n the t o t a l and s p e c i f i c a c t i v i t i e s 14 w i t h respect to C . Further a l i q u o t s were t r e a t e d by the method of R i t t e n b e r g (29) t o determine N 1 5 values i n terms of atom % excess N 1 5 . i i ) A c i d - s o l u b l e f r a c t i o n The 300 cc. 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 ether u n t i l pH determinations 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 to have been removed. The s o l u t i o n was s t i l l y e l l o w at t h i s stage. I t was now reduced to a volume of 10 cc. and 1 cc. of t h i s was removed f o r paper chromatography and autoradiography. The chromatography proved to be u n s u c c e s s f u l . I f s u f f i c i e n t s o l u t i o n was a p p l i e d to the chromatogram to detect the r a d i o a c t i v i t y , separation of the components became im p o s s i b l e ; on the other hand w i t h l e s s s o l u t i o n per chromatogram sepa-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 could not be detected. The remaining 9 cc. of e x t r a c t was hydrolysed f o r one hour i n 1 N. HC1 at 100°C. then 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. This e l u a t e was d i v i d e d i n t o three main f r a c t i o n s . One" tube was r e -moved from each of these f r a c t i o n s concentrated to a volume of approximately J c c . and a p p l i e d to a paper chromatogram. Rj values revealed that these three f r a c t i o n s contained r e s p e c t i v e l y , adenine, .guanine and a mixture c o n s i s t i n g of pyrimidines and nypoxanthine. The r a d i o a c t i v e compounds - 20 -were l o c a t e d by scanning the chromatograms w i t h an automatic windowless gas f l o w chromatogram counter. Those compounds 14 showing C a c t i v i t y by t h i s method were then punched out of the chromatogram and counts were recorded f o r the paper d i s c s w i t h the conventional counter. F o l l o w i n g determination of t h e i r a c t i v i t y , these paper d i s c s were, e x t r a c t e d f o r eig h t hours w i t h 0.1 N. HC1. Absorption s p e c t r a were then recorded f o r these e x t r a c t s to v e r i f y the i d e n t i t y and to e s t a b l i s h the c o n c e n t r a t i o n of the component present. These f i n d i n g s checked w i t h the i d e n t i t i e s e s t a b l i s h e d by means of R j values on the chromatograms. With these data i t was p o s s i b l e to 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 f o r C but no attempt was made to c o r r e l a t e the r e s u l t s w i t h counts obtained by the carbon o x i d a t i o n technique. The remainder of each f r a c t i o n , a f t e r the removal of the s i n g l e tube, was concentrated to 10 cc. volume. Absorption spectra were taken from which the c o n c e n t r a t i o n of the component i n each f r a c t i o n was e s t a b l i s h e d . A l i q u o t s were then o x i d i z e d to determine the r a d i o a c t i v i t y present. In the f i r s t experiment only the t o t a l a c t i v i t y present was estimated. This value then provided a guide f o r the t r e a t -ment to be accorded the a c i d - s o l u b l e f r a c t i o n of the second experiment. In the ease of the second experiment both the t o t a l r a d i o a c t i v i t y and the s p e c i f i c a c t i v i t y of each f r a c t i o n were determined, i i i ) Carbon d i o x i d e The twelve hour samples of 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 the s o l u t i o n up to a s p e c i f i c volume, 1 l i t e r , 2 cc. a l i q u o t s were removed and the NagCOg was p r e c i p i t a t e d from these a l i q u o t s as BaCOg. The r a d i o a c t i v i t y of these t h i c k samples of BaCOg was de-termined i n the manner described by Paterson (20). i v ) Urine Each twelve hour sample of u r i n e was made up to a volume of 50 cc. w i t h d i s t i l l e d water and 3 cc. a l i q u o t s 14 were withdrawn f o r determination of the t o t a l C by the conventional carbon o x i d a t i o n technique (28). Further 5 cc. a l i q u o t s of u r i n e were tr e a t e d w i t h urease i n the manner described by Wright (26) f o r the determination of the r a d i o -a c t i v i t y present i n the urea, ©hen t h i s procedure was com-p l e t e the digested samples were made a l k a l i n e a f t e r the method of Ritt e n b e r g (29) and the evolved NH g was trapped 15 and converted to Ng f o r N determination. A l l a n t o i n was measured on'each twelve hour sample of u r i n e by the 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 is o l a t e d - from an a l i q u o t of pooled u r i n e according to the method of Brown and h i s colleagues (4). A l i q u o t samples of pooled 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 that the !JE*"4 and N 1 5 values from the a n a l y s i s of the com-pound be r e p r e s e n t a t i v e of the e x c r e t i o n over the 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 e x i s t e d that a considerable p o r t i o n of the i n j e c t e d adenine might be excreted u n a l t e r e d i n the u r i n e , a n a l y s i s to determine i f t h i s was so was c a r r i e d out by means of c a r r i e r technique - 22 -cn pooled samples of u r i n e . The c a r r i e r adenine was r e -i s o l a t e d as the p i c r a t e s a l t . This p i c r a t e s a l t was o x i -d i z e d and i t s r a d i o a c t i v i t y was determined. The paper chromatograms which 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 of ways to 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 these 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 (44), and spraying w i t h para-dimethylaminobenzaldehyde (30), d i a z o t i z e d s u l f a -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 (30). v) Faeces The faeces, a f t e r dehydration, were t r e a t e d by the conventional o x i d a t i o n technique and the r a d i o a c t i v i t y present was measured as t h i c k sample counts on the BaCOg produced (28). v i ) Blood The blood samples were t r e a t e d by homogenizing c l o t t e d specimens and withdrawing 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 In experiment I , three male r a t s of the l i s t a r s t r a i n , having a combined weight of 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 adenine s o l u t i o n . The i n d i v i d u a l dose per r a t was 0.91 cc. which contained 8.62 mgs. of adenine. This dosage was administered twice d a i l y , - 23 -thus the r a t s were being i n j e c t e d at a l e v e l of 100 mg./Kilo./ 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 104c.p.m./mg., the r a t s r e c e i v e d a t o t a l dosage of 8.77 x 10 c.p.m. During the course of the experiment the r a t s were allowed f r e e access to food and water. This ex-periment was terminated at the end of twenty-four hours. R e s u l t s The r e s u l t s of the a n a l y s i s of the t i s s u e n u c l e i c a c i d purines and pyri m i d i n e mixture from t h i s experiment are presented i n Table I I . TABLE I I C 1 4 and N 1 5 Values of the Adenine, Guanine and Mixed Pyrimidines of 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 -oneal I n j e c t i o n of l,3-Ni.-,2-C 1 4-Adenine (49.32 mgm., 8.77 x 10 5c.p.m.). Compound R a d i o a c t i v i t y Found f4 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 c.p.m./mg. Cpd. Atom % excess Adenine 17,200 745 0.163 Guanine 8,030 175 0.106 Pyrimidine mixture (from i o n exchange) 1,750 - -The s i g n i f i c a n c e of these values i s elaborated upon i n the d i s c u s s i o n . There, f o r the purpose of c l a r i t y , they are converted to values representing percentage 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 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 of the three purines separated from the a c i d - s o l u b l e f r a c t i o n were adenine=l, hypoxanthine-0.97, and guanine-0.29. These values are ex-pressed 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 they were derived from counts obtained by a technique of counting which was not c o r r e l a t e d w i t h the standard BaCOg method of counting. Examination of the expired a i r f o r the f i r s t twelve hours of t h i s experiment showed th a t a t o t a l of 60,000 c.p.m. were present. This represents 14$ of the a c t i v i t y i n j e c t e d during t h i s p e r i o d . The r e s u l t s of examining the u r i n e showed th a t 49.5$ of the t o t a l i n j e c t e d a c t i v i t y was el i m i n a t e d from the animals by t h i s r o u t e . The only f u r t h e r s u b d i v i s i o n made 14 of the t o t a l C a c t i v i t y present i n the u r i n e was t h a t present as urea. Urea was shown t o c o n t r i b u t e only 1$ of the t o t a l of 49.5$. The N 1 5 content of the urea was a l s o shown t o be n e g l i g a b l e , only 0.009 N"1"5 atom $ excess. Examination of the blood showed a s p e c i f i c a c t i -v i t y of 320 c.p.m./cc. D. Experiment I I In experiment I I , two male r a t s of the Wistar s t r a i n having a combined weight of 462 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 t e s t s o l u t i o n . The i n d i v i d u a l dose per r a t was 0.62 cc. which contained 5.80 mgms. of adenine. This dosage was administered twice d a i l y , thus the r a t s were being i n j e c t e d at a l e v e l of 50 mg./kilo./day. - 25 -The s p e c i f i c a c t i v i t y of the administered adenine being 1.66 x 1 0 4 c.p.m./mg. adenine, the two r a t s r e c e i v e d a t o t a l of 1.57 x 1 0 6 c.p.m. During the course of the experiment the r a t s r e c e i v e d t h e i r normal r a t i o n s and had f r e e access to water. This experiment was c a r r i e d on f o r a t o t a l of n i n t y - s i x hours. Results The values obtained from the C 1 4 and N 1^ a n a l y s i s of the purine and pyri m i d i n e bases recovered from the n u c l e i c a c i d s are presented i n Table I I I TABLE I I I C"'"4 and N^°" Values of the Adenine, Guanine and Mixed P y r i m i -dines of 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 of 1,3-N 1 5-, 2-C 1 4-adenine (92.8 mgm., 1.57 x 10 6 c.p.m.) Compound R a d i o a c t i v i t y Found 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 c.p.m./mg. Cpd. Atom % Excess Adenine Guanine Pyrimidine mixture (from i o n exchange) 12,800 10,300 2,000 1,575 398 0.336 0.168 These values are combined w i t h those obtained i n experiment I and are recorded again i n a more s i g n i f i c a n t form i n Table V I I . In t h i s experiment i t was p o s s i b l e to c a l c u l a t e both the r e l a t i v e and the absolute s p e c i f i c a c t i v i t i e s f o r the three purines recovered from the 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 the f i r s t being that the counts on these bases were obtained from both paper d i s c and t h i c k sample BaCOg counts. I t was explained e a r l i e r t h a t the paper d i s c counts bear no d i r e c t r e l a t i o n s h i p t o any of the other counts recorded i n the data, a l l of those counts haying 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 obtained were adenine=l, nypoxanthine^ 0.92 and guanine=0.24. The absolute 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. cpd. There i s no absolute s p e c i f i c a c t i v i t y recorded f o r hypoxanthine since i t was present i n a mixture of p y r i m i -dines and was only separated by paper chromatography. The r e s u l t s of examining the expi r e d a i r are pre-sented i n Table IV. TABLE IV expired 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. of l , 3 - N j 5 - . j 2-C 1 4-adenine (1.57 x 10 b c.p.m.) R a d i o a c t i v i t y Found Period of C o l l e c t i o n h r. T o t a l c.p.m. S p e c i f i c A c t i v i t y c.p.m./mgm. 0 0 - 1 2 44,000 11.39 12 - 24 33,666 19.25 24 - 36 43,666 11.25 36 - 48 70,300 17.80 48 - 60 32,666 8.90 60 - 72 44,666 12.65 72 - 84 34,000 8.80 84 - 96 41,666 10.41 T o t a l 344,630 -- 27 -The p o s s i b l e source of these 344,630 c.p.m. found i n the expired a i r i s discussed l a t e r . A r e l a t i o n s h i p w i l l be drawn i n the d i s c u s s i o n between the r e s u l t s i n Table IV and those i n Tables I I and I I I . The r e s u l t of examining the u r i n e f o r i t s t o t a l C 1 4 content i s presented i n Table V. TABLE V E x c r e t i o n of C i n the Urine of the 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 of 92.8 mgm. of l,3-N 1 5-,2-C 1 4-adenine (1.57 x 1 0 6 c.p.m.). Period of Volume R a d i o a c t i v i t y Excreted C o l l e c t i o n cc. h r . T o t a l c.p.m. S p e c i f i c a c t i v i t y c.p.m./mgm. C 0 - 1 2 20 69,400 231 12 - 24 16 67,100 266 24 - 36 18 109,900 384 36 - 48 28 115,700 303 48 - 60 18 129,100 461 60 - 72 25 131,600 376 72 - 84 18 116,700 459 8 4 - 9 6 20 93,700 205 T o t a l 163 833,200 -The t o t a l of 833,200 c.p.m. recorded represents 53$ of the 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 e x c r e t i o n and the 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 the f i r s t three days then d e c l i n e somewhat on the f o u r t h day. The r e s u l t s of urea a n a l y s i s on the u r i n e are presented i n Table V I. - 28 -TABLE VI E x c r e t i o n of C 1 4 and N 1 5 i n the Urea of the Rats' Urine 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 of 92.8 mem. of 1.3-I\f 1 5-,2-C 1 4-adenine (1.57 x 10° c.p.m.) Period of C o l l e c t i o n hr. R a d i o a c t i v i t y Excreted N15 T o t a l c.p.m. S p e c i f i c A c t i v i t y c.p.m./mgm. C Atom % Excess 0 - 2 4 24 - 48 48 - 72 72 - 96 T o t a l 5,440 1,020 1,720 1,200 35.05 17.20 10.20 8.80 0.014 6'." 014 0.012 0.014 9,480 - - -With the exception of the f i r s t day the t o t a l counts excreted per day were of a s i m i l a r order of magnitude. This t a b l e shows a steady d e c l i n e i n the s p e c i f i c a c t i v i t y of the urea. This 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 to that found f o r the expired C l 4 0 g . E s t i m a t i o n of the a l l a n t o i n content of the u r i n e revealed that there were 340.4 mgm. of t h i s compound present i n the t o t a l u r i n e volume. These 340.4 mgm. contained a t o t a l of 258,200 c.p.m. 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 of atom % excess N was 0.223. I t w i l l be discussed l a t e r that these f i g u r e s represent a d e f i c i t of 159,800 c.p.m. i f the a l l a n t o i n i s considered as a r i s i n g s o l e l y from purines of the same isotope content as tha t of the i n -jected l,3-N 1 5-,2-C 1 4-adenine. - 29 -The a d d i t i o n of c a r r i e r adenine t o an a l i q u o t of the u r i n e and i t s subsequent r e - i s o l a t i o n l e d to the recovery of adenine w i t h a s p e c i f i c a c t i v i t y of 55 c.p.m./mg. Cpd. I f a l l the c a r r i e r adenine had been recovered from the a l i -quot, the t o t a l counts recorded f o r a l l the u r i n e excreted would have been 100,625 c.p.m. This represents 6.02 mg. of the adenine i n j e c t e d , i . e . adenine of s p e c i f i c a c t i v i t y 1.66 x 1 0 4 c.p.m./mg. Cpd. The paper chromatograms which had been developed on the u r i n e , showed u l t r a v i o l e t a b sorption over t h e i r e n t i r e l e n g t h , consequently no conclusions could be drawn from t h i s approach concerning purines which might or might not be present. Scanning f o r r a d i o a c t i v i t y by means of the auto-matic chromatogram counter i n d i c a t e d t h a t the major p o r t i o n 14 of C a c t i v i t y l a y between the Rj values 0.2 - 0.5. This r e s u l t was v e r i f i e d by means of an autoradiogram. Spraying the chromatograms w i t h para-dimethylaminobenzaldehyde, a reagent which r e a c t s w i t h urea and a l l a n t o i n , i n d i c a t e d that these substances were both present on the chromatograms. The Rj value determined f o r urea was 0.71, f o r a l l a n t o i n 0.57. The a l l a n t o i n t a i l e d i n t o the r a d i o a c t i v e r e g i o n of the chromatograms, Rj 0.2 - 0.5. Spraying w i t h ds'iazotized s l u l p h a n i l i c a^cid, a reagent r e a c t i n g w i t h the purine bases, was uns u c c e s s f u l because the reagent was coupling w i t h other compounds to give a complete s t r e a k i n g of the chromatograms, thus no clue was afforded as to the presence or qu a n t i t y of purines on the chromatograms, which might be c o n t r i b u t i n g to - 30 -t h e i r r a d i o a c t i v i t y . Use of ammoniacal s i l v e r n i t r a t e as a spray reagent gave negative or i n a p p r e c i a b l e r e a c t i o n f o r the presence of u r i c a c i d . The faeces were analysed only f o r t h e i r C 1 4 content. This value was reasonably constant over each 24 hour 14 p e r i o d being approximately 9000 c.p.m. The t o t a l C a c t i v i t y excreted i n the faeces over the four day pe r i o d was 32,930 c.p.m. This value represents 2% 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 t o the animals. 14 The blood when examined f o r i t s C content had an a c t i v i t y of 1,280 c.p.m./cc. - 31 -DISCUSSION When adenine i s administered to 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 guanine. This f a c t has been demonstrated 15 f o r 1,3-N - l a b e l l e d adenine by Brown and h i s colleagues (4 ) , and f o r 2 - C 1 4 - l a b e l I e d adenine by Paterson (20). Two p o s s i b i l i t i e s suggest themselves f o r t h i s conversion of adenine to guanine, f i r s t ; i t i s accomplished by a d i r e c t amination process w i t h no d i s r u p t i o n of the purine nucleus, second; the purine nucleus opens at the 2 p o s i t i o n t o give an incomplete r i n g compound which i s converted to guanine by amination and subsequent r i n g c l o s u r e . The p r o j e c t undertaken i n t h i s t h e s i s was to 15 14 i n j e c t 1,3-N -,2-C -adenine i n t o r a t s and to examine the purines recovered from the 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 occurred i n the C 1 4 / N 1 5 r a t i o from th a t of the i n j e c t e d compound. The only r a t i o n a l by which such a change i n the isot o p e r a t i o could be explained i s that during the i n c o r p o r a t i o n of adenine as adenine or during i t s conversion to guanine and i t s subsequent 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 tha t compound the purine nucleus must be broken at the 2 p o s i t i o n . I f t h i s event 14 occurred then C would be l i b e r a t e d and should appear at some other p o i n t i n the animal's metabolism. This l i n e of reasoning would be su b s t a n t i a t e d by f i n d i n g C^ 40g i n the 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 the isotope r a t i o of the n u c l e i c a c i d purines and the - 32 -14 appearance of C Og i n the animal's expired a i r to one another, 14 a d e f i c i t of C should be observed i n the excreted a l l a n t o i n . The C 1 4 / N 1 5 r a t i o i n t h i s u r i n a r y by-product of purine c a t a -bolism should r e f l e c t the f a c t that i t . i s d e r i v e d from both the adenine and the guanine of the n u c l e i c a c i d s . The data r e l a t i n g to the C l 4 / N 1 5 r a t i o s are presented i n Table V I I * TABLE V I I The Percentage of the I n j e c t e d l,3-N 1^-,2-C 1 4-adenine I n -corporated i n t o the Purines of the V i s c e r a l N u c l e i c Acids of the Rats expressed i n terms of i t s C-1-4 and N content. Compound % Incorpo- % Incorpo-r a t i o n C 1 4 r a t i o n N 1^ C 1 4 / N 1 5 a b a/b Synthetic Adenine 1.000 Experiment I Nu c l e i c A c i d Adenine N u c l e i c A c i d Guanine 4.48 1.05 5.49 3.54 0.826 0.298 Experiment 2 Nuc l e i c I c i d Adenine N u c l e i c Acid Guanine 9.50 2.39 11.22 5.81 0.852 0.427 a - % i n c o r p o r a t i o n C 14 b = % i n c o r p o r a t i o n N' a/b - C 1 4 / N 1 5 15 . c.p.m./mg.Cpd. xlOO  c.p.m./mg,Cpd. ( i n j e c t e d adenine) atom % excess N ^ xlOO atom % excess N 1 5 ( i n j e c t e d adenine) c.p.m./mg.Cpd. / ( i n j e c t e d adenine) atom % excess N1£>/c.p.nu/mg.Cpd. atom %. excess N 1 5 ( i n j e c t e d adenine) The C 1 4 / N 1 5 r a t i o i n the i n j e c t e d l , 3 - N 1 5 - , 2 - C 1 4 -adenine was e s t a b l i s h e d as u n i t y and a l l other r a t i o e s t i -mations were r e f e r r e d to t h i s v alue. In both experiments a l l the purines i s o l a t e d from the n u c l e i c a c i d s showed a C l 4 / N 1 5 r a t i o l e s s than 1. The adenine recovered from the v i s c e r a l n u c l e i c acids, had, i n both experiments, a value of a p p r o x i -l A 15 mately 0.83 f o r i t s C 4/N r a t i o . In other words at l e a s t 17$ of the i n j e c t e d adenine which was incorporated i n t o the n u c l e i c a c i d s as adenine had passed through an intermediary stage which permitted the 2-C-1-4-atom to be exchanged f o r a non - i s o t o p i c atom. The guanine recovered from the n u c l e i c 14 / 15 a c i d s showed an average value of 0*36 f o r i t s C /N r a t i o . This would i n d i c a t e some 64$ of the i n j e c t e d adenine which had been converted t o guanine and then been incorporated . 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 had f a i l e d to maintain 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 of data cannot be made between independent b i o l o g i c a l experiments, the i n c o r p o r a t i o n values compare w i t h those obtained by the two p r e v i o u s l y mentioned experimentors (4) (20). There i s a s i m i l a r i t y i n order of magnitude f o r the i n c o r p o r a t i o n as adenine but the conversion to and i n -c o r p o r a t i o n as guanine i s l e s s marked. Mention was made e a r l i e r of the three 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 take i n an ex-periment such as t h i s one. 1) No C 1 4 appears i n the p o l y n u c l e o t i d e guanine, i . e . the 14 15 C /N r a t i o becomes 0. g) The C 1 content of the p o l y n u c l e o t i d e guanine i s the same as that of the adenine, i . e . the C l 4 / N 1 5 r a t i o remains at 1. 3) The Cp-4 content of the guanine i s lower than t h a t of the adenine but s t i l l represents a s i g n i f i c a n t f r a c -t i o n of the 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 . the C l 4 / N 1 5 r a t i o becomes some f r a c t i o n between 0 and 1. The assumption i s made i n a l l these p o s t u l a t e d r a t i o s that there i s no l o s s of N 1^ on the conversion of adenine to guanine. This assumption i s made on the.,basis of the work of Brown and h i s colleagues ( 4 ) , and would be v a l i d whether or not the transformation i n v o l v e d a rupture of the purine nucleus. A p l a u s i b l e e x p l a n a t i o n 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 that an exchange r e a c t i o n takes place i n -v o l v i n g an open chain a l i p h a t i c compound and some precursor molecule which serves both as a source of the 2*amino group and the 2-C- atom of guanine. Glycine i s such a precursor molecule. In 1949 Reichard (14) showed th a t the 2-amino •' group of guanine was de r i v e d l a r g e l y from t h i s source. More recent experiments of Van Po t t e r et a l . (35) would i n d i c a t e t h a t the 2-C-atom of guanine i s a l s o derived from g l y c i n e . The r e s u l t s of both these experiments represent 'de novo' anabolism, consequently t h i s path may be po s t u l a t e d f o r the synthesis of guanine, but i t i s not n e c e s s a r i l y followed f o r the i n t e r c o n v e r s i o n of the two bases and c e r t a i n l y i s not the sole path f o r t h e i r t r a n s f o r m a t i o n . The second p o s s i b l e r a t i o mentioned, C l 4 / N 1 5 . l f 14 i s t h a t i n which the C content of the adenine and guanine separated from the mixed n u c l e i c a c i d s i s of the same order of magnitude i n both compounds. This r e s u l t would i n d i c a t e 14 no l o s s of C during the conversion, a v i r t u a l i m p o s s i b i l i t y unless the purine nucleus remains i n t a c t and the conversion takes place by a d i r e c t amination at the 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 at 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 , that the Cr* content of the guanine i s lower than t h a t of the adenine but s t i l l r e p r e -sents a s i g n i f i c a n t f r a c t i o n of the 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 , could be explained i n s e v e r a l ways. A rupture of the purine nucleus at the 2 p o s i t i o n t o y i e l d a 4-formamido-, compound wouldnleave the 2-C-atom ex-posed, a s t a t e i n which o x i d a t i o n and amination become more p l a u s i b l e , but i n which exchange a l s o becomes more l i k e l y . E i t h e r exchange or 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 the C 1 4 / N 1 5 r a t i o . Two compounds are the p o s s i b l e r e s u l t of such a s p l i t t i n g of the purine nucleus of adenine i n i t s conversion to guanine, 4-formamido-, 5-imidazole carboxamide or 4-formamido-, 5-imidazole carboximidine, depending on whether deamination at the 6 p o s i t i o n precedes or f o l l o w s s p l i t t i n g of the r i n g . Compounds of t h i s nature are not unknown both as b i o l o g i c a l and chemical e n t i t i e s . 4 amino-5-imidazole carboximidine i s the product r e s u l t i n g from a c i d h y d r o l y s i s of adenine (36), thus showing a l a b i l i t y of the 2 p o s i t i o n - 3 6 . -i n a chemical sense. 4 amino-5-imidazole carboxamide has been the s u b j e c t of e x p e r i m e n t a t i o n by Buchanan (37) and-Greenberg (38). The experiments of b o t h men have demonstra-ted the f o r m a t i o n of i n o s i n i c a c i d and hypoxanthine as the products when t h i s compound i s incubated w i t h formate. Any theory i n v o l v i n g an open c h a i n i n t e r m e d i a t e must of n e c e s s i t y e x p l a i n the evidence of Brown et a l . (4) 15 f o r the r e t e n t i o n of N i n b o t h the 1 and 3 p o s i t i o n s of the p u r i n e n u c l e u s . I f a carboximidine s t r u c t u r e i s con-s i d e r e d and no r e s t r i c t i o n s are put upon i t , resonance immediately d i l u t e s the i s o t o p e c o n c e n t r a t i o n of the 1 p o s i t i o n t o one-half i t s o r i g i n a l c o n c e n t r a t i o n . Two argu-ments might be put forward to d e f e a t t h i s s u g g e s t i o n . F i r s t ; t h a t deamination precedes the r i n g opening, i . e . the p r o -posed a l i p h a t i c i n t e r m e d i a t e i s not a carboximidine com-pound, but r a t h e r a carboxamide compound. T h i s s i t u a t i o n would di s p o s e of the n e c e s s i t y f o r c o n s i d e r i n g resonance e f f e c t . Second; the carboximidine s t r u c t u r e may not be f r e e , i t may have a s t e r i c l i m i t a t i o n p l a c e d upon i t s be-h a v i o r by an enzyme i n v o l v e d i n the o v e r - a l l c o n v e r s i o n of adenine to guanine. T h i s suggested l i m i t a t i o n would be of the same nature as t h a t d e s c r i b e d by Ogaton (39) f o r the d e c a r b o x y l a t i o n and h y d r a t i o n r e a c t i o n s i n the Krebs C i t r i c A c i d C y c l e . Another e x p l a n a t i o n f o r a s i g n i f i c a n t f a l l i n the C"^N^ r a t i o upon the c o n v e r s i o n of adenine to guanine, l i e s i n the p o s s i b i l i t y t h a t t h i s t r a n s f o r m a t i o n takes p l a c e not by a s i n g l e route but by a v a r i e t y of r o u t e s . In some - 37 -of these paths a l i b e r a t i o n of the 2-C-atom, might occur while i n others the transformation might be accomplished with the ring i n t a c t . This theory was suggested i n the introduction by reference to the second metabolic map published by R o l l and Brown (2). In t h i s map the authors show two routes f o r the conversion of active adenine to active guanine. One path denotes a d i r e c t conversion of the two bases, while the other involves a backtracking towards more general precursor molecules. This region of precursor intermediates i s one i n which incomplete purine n u c l e i are known to play a prominent r o l e . Either of those suggested p o s s i b i l i t i e s , a single reaction or a variety of reactions f o r accomplishing the conversion of adenine to guanine, could be used to explain the observed fact that the incorporation of l,3-N 1 5-,2-C 1 4-adenine into the tissue nucleic acids as guanine was les s 14 15 when measured i n terms of C than when measured as N . Other r e s u l t s observed from the i n j e c t i o n of I, 15 14 3-N -,2-C -adenine into a rat are: 1) A s i g n i f i c a n t amount of C 1 4 appears i n the expired a i r of the animal. 2) Large amounts of C a c t i v i t y are found i n the animal's urine. 3) An acid-soluble extract of the tissues contains purine nucleotides that are of higher s p e c i f i c a c t i v i t y than the polynucleotide purines. 4) No appreciable quantity of 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 to the t o t a l i n j e c t e d amount of C A t e c h n i c a l fla?/ must be the f i r s t p o s s i b i l i t y 14 considered w i t h regard t o the l a r g e amounts of C Og found i n the expired a i r of the animals. Because a gross amount of r a d i o a c t i v i t y i s excreted i n the form of by-products i n the animal's u r i n e , the p o s s i b i l i t y e x i s t s that these by-products are undergoing b a c t e r i a l decomposition 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 the case, then 1 4 the C Og i n the expired a i r samples would bear no r e l a t i o n -14 s h i p t o the r a t ' s metabolism. Three f e a t u r e s of t h i s C 0 g production might be c i t e d t o r e l a t e i t t o the r a t ' s metabolism r a t h e r than t o b a c t e r i a l decomposition. The C 1 40g shows a marked d i u r n a l v a r i a t i o n , which i s i n agreement w i t h the r a t ' s n o c t u r n a l h a b i t s (Figure 3 ) . The s p e c i f i c a c t i v i t y of the urea excreted i n the u r i n e shows a p a r a l l e l i s m t o tha t of the expired C 1 40g (25). This would f o l l o w from the Krebs-Henseleit theory f o r the formation of urea. F i n a l l y the q u a n t i t y of r a d i o a c t i v i t y found i n the expired a i r , 25$ of the t o t a l i n j e c t e d a c t i v i t y , would argue against t h i s ' t e c h n i c a l f l a w ' e xplanation f o r the presence of C^ 40g i n the expired a i r . I f the C 1 4 0 g i s a product of the r a t ' s metabolism then i t becomes d i r e c t evidence f o r a l a b i l i t y of the 2-C-atom of adenine. As such the inferenceii;imi±sfcd3e drawn that 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 degradation of the adenine molecule. This p o s s i b i l i t y would not be i n - 39 -FIGURE 3 Histogram showing the R e l a t i v e E x c r e t i o n of C 1 4 0 g and C X 4 t-Urea. 100,000 80,000 60,000 40,000 RADIOACTIVITY c.p.m. 20,000 9 19.3 17.8 \ > ' i \ \ SPECIFIC ACTIVITY C 1 4 0 p c.p.m./mg.C. 0 24 TIME (hours) * 8.9 ^ 3 - 8 1 ° - 4 48 72 96 C 1 4-Urea C 1 40, - 40 -agreement w i t h the theory f o r the main pathway of purine catabolism, which i s thought to lead to the end product, a l l a n t o i n , by means of the f o l l o w i n g steps: Adenine *» nypoxanthine Guanine * xanthine u r i c a c i d a l l a n t o i n At no poin t on t h i s path would there be a l i b e r a t i o n of C 1 4 0 g from 1,3-N 1 5-,2-C 1 4-adenine. Since the r e a c t i o n sought i n v o l v e s some 25$ of the adenine i n j e c t e d i t does not seem l i k e l y t hat a degradation path of t h i s magnitude would have escaped n o t i c e . A s u b s t a n t i a t e d r e a c t i o n t h a t adenine i s known to undergo, i n v o l v i n g t h i s s i t e , i s i t s transformation to guanine. Moreover t h i s r e a c t i o n i s of s u f f i c i e n t b i o l o g i c a l importance to be considered as the source f o r t h i s l a r g e amount of r a d i o a c t i v i t y . Opening of the purine nucleus at the 2- p o s i t i o n to y i e l d a 4-formamido-compound exposes the 2-C-atom of adenine to e i t h e r chance o x i d a t i o n or exchange or to planned s u b s t i t u t i o n , e i t h e r p o s s i b i l i t y : expired a i r . 14 p o s s i b i l i t y r e s u l t i n g i n the appearance of C 0,. i n the The source of a lar g e f r a c t i o n of the C 1 4 a c t i v i t y i n the 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 the compounds i n which one would expect to 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 which one would expect to co n t a i n r a d i o a c t i v i t y a f t e r a d m i n i s t e r i n g 1,3-N 1 5-,2-C 1 4-adenine account f o r only 42$ of the a c t i v i t y present. The t o t a l - 41 -14 u r i n e C content can be apportioned thus: 1) a l l a n t o i n 28.0$ 2) adenine 11.6$ 3) urea. 1.4$ 4) unknown 58.0$ The unknown 58$ of the a c t i v i t y could not be accounted f o r by any major component th a t could be detected by chromato-14 gra p h i c , autoradiographic or C chromatographic scanning techniques. The a c t i v i t y which was accounted f o r appears as the compounds one would expect to a r i s e from the normal metabolism of the f r e e base adenine. A l l a n t o i n i s the major end-product of purine catabolism i n the r a t and as such i t 14 appears as the major C co n t a i n i n g component which could be i d e n t i f i e d i n the u r i n e . Since adenine isnnot;;arrequired metabolite i n the d i e t of the r a t , t h e r e f o r e , one would expect i t might be excreted e i t h e r unchanged or i n some conjugated form i f i t i s fed i n excess. This s u s p i c i o n i s 14 v e r i f i e d by the recovery of adenine c o n t a i n i n g C by using the technique of c a r r i e r a d d i t i o n . Urea, the t h i r d component i n v e s t i g a t e d , although i t c o n t r i b u t e d very l i t t l e to the o v e r - a l l accounting f o r r a d i o a c t i v i t y does help to i d e n t i f y 14 the source of the C 0 g i n the expired a i r as being the r a t i t s e l f . A f u r t h e r examination of the a l l a n t o i n excreted shows that i t s C 1 4 / N 1 5 r a t i o i s 0.618. I f i t had been de-r i v e d from the purines formed from the i n j e c t e d adenine and t h e i r C l 4 / N 1 5 r a t i o s had remained at 1 then the a l l a n -t o i n should a l s o have had a C"L4/N"1'^ r a t i o of 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 of 0.223 atom - 42 -^ 1 5 % excess N , 340.4 mgm. should have contained 418,000 c.p.m. Only 258,200 c.p.m. are present. This value represents a. d e f i c i t of 159,800 c.p.m., a d e f i c i t t h a t can be used to 14 account f o r approximately 50% of the C 0 g found i n the animals' expired a i r . T h i s , furthermore, d i r e c t l y r e l a t e s the C 1 40g found i n the expired a i r to the drop i n C l 4 / N 1 5 r a t i o found i n the n u c l e i c a c i d p u r i n e s . In view of the recent work of Ochoa et a l . (18) and that of Kornberg and h i s group (40) the high s p e c i f i c a c t i v i t y shown by the f r e e n u c l e o t i d e s i s not s u r p r i s i n g . The experiments of both men have shown that the phosphory-l a t e d r i b o t i d e s and deso x y r i b o t i d e s would appear to be the u n i t s incorporated i n t o Ribose N u c l e i c a c i d and Desoxyribose 14 N u c l e i c a c i d . The importance of 2-C -hypoxanthine r i b o -t i d e as an intermediary product concerned w i t h the conversion of adenine to guanine cannot be estimated. 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 product of adenine i n the course of i t s degradation t o a l -l a n t o i n , and the r e s p e c t i v e c o n t r i b u t i o n of e i t h e r r e a c t i o n 14 to the o v e r a l l t o t a l of C -hypoxanthine present cannot be measured i n t h i s experiment. However, Abrams and Be n t l e y (13) have shown that the route l e a d i n g from a d e n y l i c a c i d through i n o s i n i c a c i d to xanthosine r i b o t i d e phosphate and from there to guanylic a c i d can be considered as a v a l i d s e r i e s of r e a c t i o n s which could lead from adenine to guanine. A blocked r e a c t i o n or s a t u r a t i o n technique might be used to solve the question of the importance of t h i s route. I f - 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 administered simultaneously, the degree of i n c o r p o r a t i o n of 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 should decrease i f i n o -s i n i c a c i d i s a d i r e c t intermediate upon the pathway of t h i s conversion. Very l i t t l e can be discussed about any r o l e the blood might play i n t h i s t r a nsformation phenomenon since the only a n a l y s i s c a r r i e d out was an e s t i m a t i o n of the 14 t o t a l C a c t i v i t y . T h i s , i n the case of both experiments, turned out to be n e g l i g i b l e w i t h respect to the t o t a l 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 obtained i n the e x p i r e d a i r was probably mainly i n the form of b i -carbonate. The faeces, l i k e w i s e , showed very 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, that f a e c a l e x c r e t i o n plays a very minor r o l e i n the d i s -p o s a l of the i n j e c t e d 1,3-N 1 5-,2-C 1 4-adenine. Figure IV presents an o v e r a l l accounting of 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 of percentages c o n t r i b u t e d by each of the three e x c r e t o r y routes plus 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. This 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 which presumably remains w i t h i n the animal and enters the v a r i o u s pathways of the animal's metabolic processes. - 44 -FIGURE 4 D a i l y accountable Radioactivity:.!oiand :Llnthe Three E x c r e t o r y Products expressed as_a Percentage oi' the Rats' 24 hou r l y dose of l,3-N ± B-,2-C- L 4-adenine (392,500 c.p.m.) 100 80 60 Percentage 40 20 C0 2 Percentage 24 48 100 80 60 Percentage 40 20 Time (hours) URINE 100 80 60 40 20 72 96 Percentage 100 80 60 40 20 24 48 72 Time (hours) 96 FAECES =1= 24 48 72 96 Time (hours) UNKNOWN 24 48 72 Time (hours) 96 - 45 -SUMMARY A. Doubly l a b e l l e d adenine, 1,3-N 1 5-,2-C 1 4-adenine, was synthesized and administered t o r a t s by means of 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 . At the end of an experiment the 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 hydrolysed. The adenine and guanine obtained from t h i s source were then examined f o r t h e i r isotope content i n terms of t h e i r C 1 4 / N 1 5 r a t i o r e l a t i v e to the C 1 4 / N 1 5 r a t i o i n the adenine i n j e c t e d . B. The r a t i o of C 1 4 / N 1 5 i n the adenine recovered from the 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 the f i r s t experiment and 0.S52 i n the second experiment. Therefore, approximately 17$ of the i n j e c t e d , 1,3-N 1 5-,2-C 1 4-adenine which was i n -corporated i n t o the n u c l e i c a c i d s as adenine had exchanged i t s 2-C 1 4-atom f o r a n o n - i s o t o p i c atom. C. The r a t i o of C 1 4 / N 1 5 i n the guanine recovered from the 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 . This i n d i c a t e s that approximately 64$ of the i n j e c t e d 1,3-N 1 5-,2-C 1 4-adenine which was i n -corporated 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 i n the form of 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. In the purines of the v i s c e r a l n u c l e i c a c i d s there was 47$ l e s s C 1 4 found i n the guanine than i n . t h e adenine d e s p i t e the f a c t that they were both d e r i v e d from the same source, 1,3-N 1 5-,2-C 1 4-adenine which had a C 1 4 content considered t o be 100$. - 46 -14 E. Large amounts of C a c t i v i t y were found i n the expired a i r upon i n j e c t i o n of l,3-N^-,g-C^" 4-adenine i n t o 14 a r a t . The amount of r a d i o a c t i v i t y found as C 0 g r e p r e -sented 25$ of the t o t a l i n j e c t e d a c t i v i t y . The t o t a l 14 a c t i v i t y . o f the C 0 g was 344,630 c.p.m. F. The r a d i o a c t i v i t y found i n the u r i n e accounted f o r 58$ of the i n j e c t e d a c t i v i t y . A l l a n t o i n , the f i n a l degradation product of purine catabolism, represented the 14 l a r g e s t i d e n t i f i a b l e p o o l of C a c t i v i t y c o n t r i b u t i n g to the t o t a l r a d i o a c t i v i t y present i n the u r i n e . G. The C 1 4 / N 1 5 r a t i o i n the 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 1 4 d e f i c i t of 159,800 c.p.m. H. This d e f i c i t of counts i n the a l l a n t o i n was used to account f o r 47$ of the C"*"40g found i n the animals' expired a i r . I . 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 the purines of the a c i d - s o l u b l e f r a c t i o n were adenine=l, hypoxanthine=-0.92 and guanine-0.24 i n d i c a t i n g t h a t hypoxan-t h i n e i n some form represents a p o s s i b l e intermediate on the route of i n t e r c o n v e r s i o n of adenine to p o l y n u c l e o t i d e guanine. The absolute s p e c i f i c a c t i v i t i e s found i n the a c i d - s o l u b l e f r a c t i o n were much higher than those found f o r the purines of 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 took place at t h i s stage ra t h e r than a f t e r i n c o r p o r a t i o n i n t o the n u c l e i c a c i d s had taken p l a c e . - 47 -J . 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