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The metabolism of 2-C¹⁴-adenine in the adult male rat Paterson, Alan Robb Phillips 1952

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/ THE METABOLISM OF £-C -ADENINE  IN THE ADULT MALE RAT by Alan Robb P h i l l i p s Patarson i A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n the Department of Biochemistry. We accept t h i s t h e s i s as conforming to the standard required from candidates f o r the degree of MASTER OF ARTS. Members of the Department of Biochemistry. The U n i v e r s i t y of B r i t i s h Columbia A p r i l , 1952. ABSTRACT Iaotopic adenine, labeled wi th C 1 4 i n p o s i t i o n 2, has been prepared i n three steps; (a) formylat ion of 4-amino,- 5-imidazolecarboxamidine i n aqueous G 1 4 - f ormic a c i d , (b) r i n g closure of the r e s u l t i n g formamido compound to form 2-C 1 4 -adenine , (c) p u r i f i c a t i o n by sublimation i n vacuo. The o v e r a l l y i e l d f o r the three operations was 60 percent. Proof of i d e n t i t y of the adenine prepared i n t h i s manner was obtained from the preparation of a d e r i v a -t i v e , combustion a n a l y s i s , paper chromatography and u l t r a -v i o l e t spectrophotometry. The metabolism of 2-C 1 4 -adenine was studied i n the adult male r a t . The labeled compound was administered to the experimental animals 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 isotope of the administered adenine was found d i s t r i b u t e d i n the purines of the v i s c e r a l n u c l e i c ac ids , the expired carbon dioxide and ur ine , where part of the a c t i v i t y was found i n both urea and a l l a n t o i n . Nucleic a c i d adenine and guanine were synthesized to the extent of 7.7 percent and 5.5 percent respect ive ly from administered 2-G -adenine. The adenine renewal i s lower than s i m i l a r values derived from l , 3 - N 1 5 - a d e n i n e as reported i n the l i t e r a t u r e . Expired carbon dioxide was found to contain over 8 percent of the administered isotope. Combustion analyses of whole urine indicated that 28 percent of the administered isotope was contained t h e r e i n . Urea and a l l a n t o i n together accounted f o r 16-29 percent of the t o t a l r a d i o a c t i v i t y i n u r i n e . The presence of radioact ive carbon dioxide i n the expired a i r of the experimental animals, when considered i n the l i g h t of other evidence, i s regarded as being i n d i c a t i v e of a b i o l o g i c a l l a b i l i t y i n p o s i t i o n 2 of the purines<, ACKNOWLEDGMENTS The author g r a t e f u l l y acknowledges personal assistance from the N a t i o n a l Research Counci l i n the form of a Bursary, a Summer Supplementary Allowance and a Studentship. The author a lso wishes to express h i s appreciat ion of the encouragement, advice, and personal assistance i n the laboratory given by Dr. S. H. Zbarsky during the course of t h i s research. This research was supported by a grant from the Nat ional Research C o u n c i l . TABLE OF CONTENTS Page INTRODUCTION 1 EXPERIMENTAL A. METHODS. I . Synthesis of 2-C 1 4 -adenine 19 ( i ) Preparation of 4-amino-5-imidazole-carboxamidine dihydrochloride . . . 21 ( i i ) Preparation of Cl*- formic ac id . . 22 (a) Procedure f o r the p u r i f i c a t i o n and determination of formic ac id 24 ( i i i ) Synthesis of 2-C 1 4 -adenine . . . . 27 (a) Procedure f o r the synthesis of 2-C 1 4 -adenine 28 ( iv) Proof of i d e n t i t y of synthetic adenine 31 (a) Combustion analys is 31 (b) Der ivat ive 32 (c) Chromatography 32 (d) Spectrophotometry 35 I I . Measurement of R a d i o a c t i v i t y 36 I I I . Metabolism Experiments 39 ( i ) Respiratory carbon dioxide; c o l l e c t i o n and analysis . . . . . 43 ( i i ) Urine; c o l l e c t i o n and analys is . . 45 (a) Whole urine 45 (b) Urea determination 46 (c) A l l a n t o i n 46 ( i i i ) I s o l a t i o n of v i s c e r a l n u c l e i c acids and the preparation of the purines . 47 B. RESULTS. I . Experiment 1. ( i ) Administrat ion of 2-C 1 4 -adenine . . 49 ( i i ) Expired carbon dioxide 49 ( i i i ) Urine 51 ( iv) Nucleic acids and purines 51 I I . Experiment 2. ( i ) Administrat ion of 2-C 1 4 -adenine . . 52 ( i i ) Expired carbon dioxide . . . . . . 54 Page ( i i i ) Urine 54 ( iv) Nucleic acids and purines 54 DISCUSSION 59 SUMMARY 69 BIBLIOGRAPHY 71 TABLES I . G 1 4 contents of ur ine , ur inary urea and a l l a n t o i n , Experiment 1 50 I I . C 1 4 contents of n u c l e i c acids and purines , Experiment 1 53 I I I . C 1 4 contents of expired carbon d i o x i d e , Experiment 2 . 55 IV. C 1 4 contents of ur ine, ur inary urea and a l l a n t o i n , Experiment 2 56 V. contents of nuc le ic acids and purines, Experiment 2 58 VT.. Renewals of polynucleotide purines . 61 V I I . D i s t r i b u t i o n of C X 4 b from in jec ted 2-C-L*-adenine . . 63 FIGURES 1. S t r u c t u r a l formulae of some purines and pyrimidines 5 2. The synthesis of 2-G^ 4-adenine from m a l o n o n i t r i l e . 20 3. Apparatus f o r the wet combustion of organic compounds 25 4. Apparatus f o r the p u r i f i c a t i o n and recovery of formic ac id by steam d i s t i l l a t i o n . . . . . . . . . 26 5. Apparatus f o r the p u r i f i c a t i o n of adenine by sublimation * . 30 6. Contact p r i n t of a chromatogram of a hydrolysate of yeast, n u c l e i c ac id . . 33 7. Contact p r i n t of a chromatogram used to v e r i f y the i d e n t i t y of synthet ic adenine 34 8. A comparison of absorption curves of authentic and synthetic adenine 37 9. Metabolism cage f o r the separate c o l l e c t i o n of expired carbon d iox ide , urine and feces . . . . . 44 i INTRODUCTION Interest i n nucleic acids dates back to 1868 when Fried e r i c h Miescher began the fundamental investigations which led to t h e i r discovery. The subsequent research, which f i r s t revealed the chemical nature of the nucleic acids and which i s now bringing to l i g h t something of the role these compounds play i n l i f e processes, constitutes a long and complex chapter of science. Although the o r i g i n a l enquiries into t h i s f i e l d were of a biochemical nature, the f i r s t important contributions to our knowledge of the nucleic acids were made by organic chemists. In the course of i n v e s t i g a t i o n into the properties and structure of uric acid by the early German organic chemists, the chemistry of the purines and pyrimidines became well-established. Representatives of both classes of these nitrogenous bases are present i n nucleic acids. The early biochemists active i n nucleic acid research were concerned p r i n c i p a l l y with the nature of the nucleic acid components. The German school, notably Kossel and h i s group, were l a r g e l y responsible f o r the i s o l a t i o n and characterization of the nitrogenous bases of the nucleic acids. The pentoses of the nucleic acids were f i r s t investigated by the German chemists, but t h e i r i d e n t i t y was established p r i n c i p a l l y through the e f f o r t s of the American investigators, Levene and Jacob, That the nucleic acids contained phosphorous had been known since Miescher's early investigations. L o g i c a l l y following t h i s stage i n the h i s t o r y of nucleic acid research came investigations into the manner i n which the constituent parts of the nucleic acid molecule are linked to form i t s small s t r u c t u r a l units, the nucleosides and the nucleotides. During the l a s t t h i r t y years there has been -considerable work done on the structure of the nucleic acid polymer employing a wide v a r i e t y of techniques. The development of a n a l y t i c a l methods fo r the estimation of nucleic acids constituted the next major step i n t h i s f i e l d . A n a l y t i c a l techniques based on phosphorous determination and the colorimetric estimation of pentoses were the f i r s t developed and are s t i l l i n wide use today. Purine estimations, at f i r s t done chemically, are now made almost exclusively by u l t r a v i o l e t absorption techniques. Two important achievements, the establishment of suitable a n a l y t i c a l methods and the development of h i s t o -chemical tests f o r nucleic acids, then set the stage f o r fundamental developments i n the biology and biochemistry of the nucleic acids. The necessary chemical background having been supplied, biochemists and c y t o l o g i s t s began examining the nucleic acids i n the l i g h t of t h e i r - 3 p a r t i c i p a t i o n i n processes of c e l l growth, c e l l reproduction and c e l l f u n c t i o n . In recent years, the biology of the nucle ic acids has advanced to the point where the fundamental importance of these compounds has been t r u l y appreciated. For example, there i s now a w e l l establ ished body of evidence which i n d i c a t e s that the chromosomes are const i tuted mainly of nucleoprotein (1) and that desoxyribose n u c l e i c a c i d might w e l l be the actual genie mater ia l (£). The changes i n the nuc le ic a c i d content of r a p i d l y growing t i s s u e s and the fact that the v iruses and bacteriophages have i n v a r i a b l y proven to be nucleoprotein i n nature (1) i n d i c a t e that the nuc le ic acids p a r t i c i p a t e i n growth processes. I t i s widely appreciated today that the n u c l e i c acids play important, i f not fundamental r o l e s i n the economy of the c e l l . Recognition of t h i s fact by workers i n the b i o l o g i c a l sciences i s l a r g e l y responsible f o r the great r e v i v a l of i n t e r e s t i n n u c l e i c aoids w i t h i n the l a s t decade. r The nuc le ic acids occur i n the i n t a c t c e l l as p r o t e i n conjugates termed nucleoprotein. The p r o t e i n , usua l ly a simple basic p r o t e i n such as a histone or p r o t a -mine, i s removed during the process of i s o l a t i o n of the free nuc le ic a c i d s . There are two p r i n c i p a l types of nuc le ic a c i d , the chemical d i s t i n c t i o n between them being made on the basis of the sugar moiety of each type. - 4 -Ribonucleic a c i d (often abbreviated RNA) has as i t s c h a r a c t e r i s t i c sugar, /S -D-ribofuranose. The other n u c l e i c ac id type, desoxyribonucleic ac id (DNA), incorporates i n i t s s tructure (3 -D-2-desoxyribofuranose. A f u r t h e r dif ference between RNA and DNA may be found i n t h e i r con-s t i t u e n t nitrogenous bases (See Figure 1). The purines , adenine and guanine, are found i n both types of n u c l e i c a c i d . With respect to the pyr imidines , eytosine i s present i n both types of n u c l e i c a c i d , but u r a c i l i s found only i n RNA and i t s methylated d e r i v a t i v e , thymine, occurs only i n DNA. The b i o l o g i c a l d i s t i n c t i o n between DNA and RNA l i e s not i n t h e i r sources, as e a r l y workers thought, but i n t h e i r l o c a t i o n s w i t h i n the c e l l . Desoxyribonucleic a c i d i s confined e x c l u s i v e l y to the nucleus while r i b o n u c l e i c aoid occurs p r i n c i p a l l y i n the cytoplasm and to a small extent i n the nucleus where i t i s located i n the nucleolus and i t s v i c i n i t y . The n u c l e i c acids are h i g h l y polymerized sub-stances w i t h molecular weights ranging from 300,000, as reported f o r tobacco v i r u s RNA (3), to 3,000,000 f o r thymus gland DNA (4) . The monomer units of these s tructures , the nucleot ides , are joined by phosphoric ester l inkages . Cold, weakly a l k a l i n e conditions are H , C V 5 II ^ I N 2 h V > / h 8 3 9 P U R I N E A D E N I N E N H N H N H S \ G U A N I N E 2 , 6 - O I A M I N O P U R I N E N H 0 .H O.H I S O G U A N I N E H Y P O X A N T H I N E X A N T H I N E P U R I N E S U R I C A C I D 1 N C 5 2 C I 4 H N N H 3 P Y R I M I D I N E C Y T O S I N E P Y R I M I D I N E S N H ^ . ^ N V C H O H c / ° \ l 2 Y E A S T A D E N Y L I C A C I D 'w^ - H \ H H / C H ( A D E N O S I N E - 3 - P H O S P H A T E ) N 9 - 9 o,H H O 0 — P - O H 11 0 A R I B O N U C L E O T I D E F I G U R E I S T R U C T U R A L F O R M U L A E O F S O M E P U R I N E S A N D P Y R I M I D I N E S . - 6 -s u f f i c i e n t to hydrolyse RNA i n t o i t s constituent nucleo-t i d e s , but the more stable DNA i s best depolymerized to i t s nucleotides by enzymatic h y d r o l y s i s . With reference to s t ructure , the r ibonucleot ides (Figure 1) are purine and pyrimidine r i b o s i d e s phosphorylated i n p o s i t i o n 3 of the r ibose molecule. Recently, two isomeric forms f o r each of the r ibonucleot ides have been i s o l a t e d ; i t i s not known whether the new isomer, designated " a " , d i f f e r s from the f i r s t discovered form " b " , i n having the phosphate located at the 2 - p o s i t i o n i n r ibose or whether the dif ference i s due to * , isomerism about the g l y c o s i d i c l inkage ( 5 , 6 , 7 ) . The desoxyribo^nucleotides are phosphorylated i n the 3 - p o s i t i o n of the desoxyribose molecule. The sugar moiety i n the purine nucleotides i s attached to the ni trogen i n p o s i t i o n 9 (Figure 1) i n both adenine and guanine; i n the pyrimidine nucleosides, the sugar l inkage i s d i rec ted to nitrogen 3 i n a l l cases. Nucle ic acids are common d i e t a r y constituents and i n v e s t i g a t i o n s i n t o the manner i n which they are handled by the i n t a c t animal have provided us w i t h much valuable information concerning the metabolism of these compounds. Dietary n u c l e i c acids are hydrolysed to nucleotides by enzymes (nucleases) of the duodenal secret ions . The nucleotides are, i n t u r n , degraded to nucleosides and phosphoric a c i d by i n t e s t i n a l nucleosidases. The i nucleosides apparently are absorbed as such from the upper part of the i n t e s t i n e (8) . The purine nucleosides, once absorbed, may be s p l i t into purines and sugar i n various organs, notably the l i v e r , by purine nucleosidase. Studies of the metabolism of the free purine and pyrimidine bases, have been slanted heavily i n favour of the purines. P r a c t i c a l l y a l l that i s known of the metabolic fate of the pyrimidines i s that pyrimidine nitrogen i s con-verted to urea and ammonia (9) . The course of purine metabolism varies widely amongst the d i f f e r e n t animal species. The primates, birds and some r e p t i l e s convert purines to uric acid, the form i n which they are excreted. In the lower mammals, purine degradation i s carried a step further with uric acid being oxidized to the excretory product a l l a n t o i n . In some fishes a l l a n t o i n i s oxidized, i n an additional step, to a l l a n t o i c acid; however, most of the fishes and the amphibia degrade purines beyond t h i s point to urea and g l y o x y l i c acid. The conversion of adenine and guanine into u r i c acid and a l l a n t o i n may be summarized by the following diagram (10). adenase adenine *- hypoxanthine xanthine oxidase guanase J guanine *- xanthine | xanthine oxidase uric acid — a l l a n t o i n urico oxidase Purines from both exogenous and endogenous sources are metabolised i n t h i s manner. Birds not only convert purines to uric acid, but also synthesize ur i c acid f o r the purpose of nitrogen excretion. Uric acid i s the p r i n c i p a l form i n which nitrogen i s eliminated i n birds; that portion of i t a r i s i n g from purine breakdown amounts to approximately 5 percent of a l l the nitrogen excreted (10). The purine hypoxanthine has been shown by Krebs and his co-workers (11) to be an intermediate i n the biosynthesis of ur i c acid i n birds. Thus i t may be seen that purine formation i s a major synthetic pathway i n the b i r d . These facts make i t apparent that the b i r d i s a convenient animal i n which to study the metabolism and biosynthesis of the purine bases. Pigeon l i v e r homogenates and s l i c e s have been used extensively as purine-synthesizing systems, since i t has been found that hypoxanthine tends to accumulate i n these preparations. As one aspect of enquiries into the manner i n which the animal i s able to elaborate tissue polynucleotides extensive studies have been made of the biosynthesis of the purines. Early approaches to t h i s problem, made through the c l a s s i c a l feeding techniques, have not provided much information of a s p e c i f i c nature concerning the pre-cursors of the purine r i n g . However, i t was found by such methods that the animal does not depend upon dietary - 9 -sources f o r purines but i s able to synthesize them de.novo (8). Only information of a general character has arisen from the use of these methods and i t has remained f o r those using the recently-developed tracer techniques to provide s p e c i f i c evidence as to the precursors which are involved i n the synthesis of the purine r i n g . Since the animal has been shown to be independent of dietary sources f o r purines, i t i s evident that i t must synthesize these compounds from smaller molecules. Barnes and Schoenheimer (12) demonstrated that N 1 5 - l a b e l e d ammonium s a l t s were incorporated into the tissue nucleic acids and the purine excretory products. Buchanan notes that " t h i s paper was l a r g e l y responsible f o r c a l l i n g attention to the fact that the purines are b u i l t up from small carbon and nitrogen units rather than l a r g e r , pre-formed metabolic units from the d i e t " (13). In the course of t h e i r investigations of purine biosynthesis, Buchanan and his associates have determined the sources of the various atoms of the purine r i n g by feeding pigeons i s o t o p i c a l l y labeled compounds which were incorporated into u r i c acid. The excreted u r i c acid was degraded by pro-cedures which permitted the separate examination of each of the d i f f e r e n t atoms of i t s molecule. B r i e f l y , t h e i r work showed that of the nitrogen atoms, N]_, N3 and % were derived from the metabolic nitrogen pool, while Ny was - 10 -provided by g l y c i n e . Of the carbon atoms, C 2 and C Q were derived from formate, C 4 and C 5 from g l y c i n e , and C g was derived from carbon dioxide (13). Recently these f indings have been extended, demonstrating that ammonium s a l t s , g l y c i n e , formate and carbon dioxide contribute i n the same manner to the biosynthesis of polynucleotide adenine and guanine i n the rat and yeast (14, 15). In order to determine what steps are intermediate i n the incorporat ion of the e a r l y precursors i n t o the purine molecule, various c y c l i c compounds have been examined to determine whether they p a r t i c i p a t e i n purine b iosynthes is . The pyrimidines would appear to be l i k e l y precursors of the purines since the 6-membered r i n g i s common to both. But experiments w i t h the N 1 5 - l a b e l e d pyrimidine r i n g have shown that these compounds do not p a r t i c i p a t e i n purine synthesis (9). S i m i l a r l y , N 1 5 - l a b e l e d h i s t i d i n e Has shown not to be a precursor (16), i n sp i te of ear ly i n d i c a t i o n s of i n -volvement i n purine metabolism (17). An i n t e r e s t i n g c y c l i c intermediate has been found i n the compound 4-amino-5-imidazolecarboxamide: - C-N H The imidazolecarboxamide, when injected i n t o r a t s , has been - 11 -shown to be incorporated i n large measure i n t o the t i s s u e nucle ic a c i d purines (18). There are strong i n d i c a t i o n s that t h i s compound, i n a conjugated form, may be an i n t e r -mediate i n purine synthesis (13). In a recent paper, Greenberg has elaborated on the r o l e of the imidazolecarboxamide i n purine synthesis (19). In demonstrating the de novo synthesis of hypo-xanthine w i t h C 1 4 - formate i n pigeon l i v e r homogenates, he has shown that hypoxanthine i s preceded by i n o s i n e - 5 -phosphate and i n o s i n e , i n that order. Inosine-5-phosphate appeared to be the f i r s t complete purine synthesized i n the system used and Greenberg postulated that the immediate precursor of t h i s compound was a r i b o t i d e of incomplete s t r u c t u r e , l i k e l y 4-amino-5-imidazolecarboxamide r i b o t i d e . Although the animal i s able to synthesize i t s own purines, i t i s l o g i c a l to expect that administered purines would be u t i l i z e d by the organism i n anabolic processes. However, on the basis of feeding experiments w i t h labeled guanine, u r a c i l and thymine, F l e n t l and Schoenheimer con-cluded that "neither purines nor pyrimidines supplied i n the d i e t are u t i l i z e d by the body f o r the synthesis of nucleoproteins" (9) . Because adenine i s much more important i n a biochemical sense than i s guanine, Brown and h i s associates (20) considered that adenine merited a separate i n v e s t i g a t i o n before the general statement of - 12 -P l e n t l and Sehoenheimer could be accepted. Accordingly , these workers synthesized adenine labeled w i t h N 1 5 i n p o s i t i o n s 1 and 3. They found i n feeding experiments w i t h t h i s compound that the isotope was incorporated i n t o polynucleotide adenine and guanine. Adenine, therefore , appears to be the only one of the free n u c l e i c a c i d bases that p a r t i c i p a t e s i n polynucleotide synthesis . Because the isotope was found i n p o s i t i o n s 1 and 3 of i s o l a t e d adenine and guanine, Brown and h i s c o l l a b -orators concluded that the purine r i n g remained i n t a c t during the conversion of adenine to guanine. More recent evidence on the conversion of 8- -adenine i n t o n u c l e i c acid guanine by yeast (21) and the rat (22) has been c i t e d by Brown (23) as fur ther i n d i c a t i o n that the purine r i n g remains i n t a c t . In experiments w i t h adenine labeled i n p o s i t i o n s 1 and 3 w i t h N 1 5 and i n p o s i t i o n 8 w i t h C 1 4 , Marrian et a l . (22) have shown that polynucleotide renewal i n the ra t i s the same whether measured by the uptake of N 1 5 or G 1 4 . They concluded from t h i s that the purine r i n g remains i n t a c t during the transformation of adenine i n t o polynucleotide guanine. Other purines, namely hypoxanthine, xanthine and isoguanine, were synthesized to contain and were found not to p a r t i c i p a t e i n the biosynthesis of polynucleotide purines (24, 25). The only purine, other than adenine, - 13 -which haa been shown to p a r t i c i p a t e to any extent i s 2,6-diaminopurine. This compound was considered by Bendich, Furst and Brown (26) to be a l i k e l y intermediate i n the con-vers ion of adenine to guanine i n v i v o . These workers syn-thesized the diaminopurine labeled w i t h N 1 5 i n the 2-amino group and r i n g nitrogens 1 and 3. A f t e r o r a l adminis trat ion of the tagged diaminopurine to r a t s , the polynucleotide purines were found to have taken up the isotope only i n guanine. The isotope was incorporated to approximately the same extent as that from 1 , 3 - N ^ - l a b e l e d adenine administered i n s i m i l a r experiments. Hence, i t was considered that the diaminopurine may p a r t i c i p a t e as an intermediate i n the conversion of adenine to guanine. In a p a r a l l e l experiment, c a r r i e d out using 2,6-diaminopurine-2-C 1 3 , ' the isotope i n the polynucleotide purines again was found to be confined to the guanine f r a c t i o n but w i t h a notably smaller incorpora-15 t i o n (1.5 percent as opposed to 4 .0 percent f o r the N experiment). Bendich and h i s co-workers d i d not o f fer an explanation f o r t h i s discrepancy but Gordon (27) has suggested that i t may be due to a b i o l o g i c a l l a b i l i t y at the 2-pos i t ion i n the purine r i n g during i t s i n c o r p o r a t i o n . He also pointed out that the evidence offered by Brown et a l . i n support of the re tent ion of the intac t r i n g system does not r u l e out the p o s s i b i l i t y that the purine r i n g may open between nitrogens laand 3 or i n the imidazole - 14 -r i n g . Marsh has interpreted the f indings reported i n several other papers as i n d i c a t i n g that carbon 2 of the n u c l e i c ac id purines might be r e l a t i v e l y more l a b i l e than some of the other r i n g carbons (28). Barnes and Sehoenheimer observed that the 2-amino nitrogen of po ly-nucleotide guanine from pigeon v i s c e r a had a higher turnover than d i d the r i n g nitrogens (12). Confirmation of t h i s observation comes from Reichard (29) i n h i s experiments on the incorporat ion of N 1 5 - l a b e l e d g lyc ine i n t o RNA purines . A l a b i l e 2-pos i t ion i n the purines could quite conceivably increase the rate of turnover of the 2 - p o s i t i o n subst i tuent . The previously mentioned p a r t i c i p a t i o n of 4-amino-5-imidazolecarboxamide or one of i t s der ivat ives i n purine synthesis (18, 13) supports the idea of l a b i l e 2-posi t ions i n the purines . In a purely chemical sense, adenine has been shown to possess an act ive 2 - p o s i t i o n . In degradation studies on adenine, C a v a l i e r i et a l . (30) showed t h a t , by hydrolys is w i t h HC1, formate can be s p l i t out of the adenine molecule leav ing 4-amino-5-imidazolecarboxamidine. Since the concept of a l a b i l e 2 - p o s i t i o n suggests an i n t e r -change between the purines and some 1-carbon compound or a l a b i l e substituent of a l a r g e r molecule i n the t i s s u e s , the observation that formate i s an excel lent precursor of - 15 -carbon 2 and 8 of the purines (13) would appear t o support t h i s postu late . There i s , therefore, considerable i n d i r e c t evidence favouring t h i s concept, but evidence which does not depend on inference has been l a c k i n g . Since administered adenine p a r t i c i p a t e s i n polynucleotide formation and i s excreted i n a manner i d e n t i c a l to that of the endogenous purines, i t seemed that a study of the metabolism of 2-labeled adenine might provide s p e c i f i c evidence for or against the postulated l a b i l i t y of carbon 2 i n the pur ines . A method for the synthesis of 2-C 1 4 -adenine was developed using, as i t s b a s i s , the formylat ion of ^amino-s-imidazole carboxamidine and the r i n g closure of the r e s u l t i n g formamido d e r i v a t i v e to form adenine (31). Radioactive carbon was incorporated i n t o the molecule by 14 carrying out the formylation w i t h C -formic a c i d . C y c l i c z a t i o n of the formamido-imidazole compound resul ted i n the formation of 2-C 1 4 -adenine as may be seen i n Figure 2. By t h i s method, 2-C 1 4 -adenine may be prepared i n y i e l d s of 40-65 percent, depending on the formylat ing 14 conditions used, and the excess formylating agent, C -formic a c i d , may be recovered e a s i l y f o r further use. The i d e n t i t y of adenine prepared i n t h i s manner was v e r i f i e d by paper chromatography, u l t r a v i o l e t spectrophotometry and the preparation of a d e r i v a t i v e . r 16 -Metabolic studies of 2-labeled adenine were then undertaken using the ra t as the experimental animal. The postulated b i o l o g i c a l l a b i l i t y of the 2-carbon of the purines impl ies an interchange between t h i s carbon and some 1-carbon compound (or a l a b i l e group of some l a r g e r mole-cule) i n the t i s s u e s . I f t h i s were so, administered 2-labeled adenine and the purine intermediates i n i t s meta-bolism would be expected to lose isotope from the 2 - p o s i t i o n to some t i s s u e constituent and reincorporate non-isotopic carbon. The compound involved i n the interchange react ion w i t h carbon 2 would l i k e l y undergo ox idat ion i n the course of i t s p a r t i c i p a t i o n i n other metabolic processes. For these reasons i t was considered desirable to examine the expired a i r of the experimental animals f o r the presence of radioact ive carbon d iox ide . Therefore, 2-C 1 4 -adenine was administered to ra ts which were placed i n a metabolism cage that permitted the separate c o l l e c t i o n of expiratory carbon d iox ide , urine and feces. The labe led compound was in jected i n t r a p e r i t o n e a l l y i n d a i l y doses f o r four days, during which time regular c o l l e c t i o n s of expiratory C 0 2 and excreta were made. At the completion of the experiment the animals were s a c r i f i c e d and the n u c l e i c acids extracted from the pooled v i s c e r a . The purines, adenine and guanine, and the combined pyrimidines were prepared from the nuc le ic acids and assayed f o r r a d i o a c t i v i t y . Analys is of the expired carbon dioxide revealed that i t contained a s i g n i f i c a n t amount of the r a d i o a c t i v e carbon. Approximately 8 percent of the administered isotope appeared i n the r e s p i r a t o r y gases. This f i n d i n g i s con-sidered to be d i r e c t evidence that the E - p o s i t i o n i n the purines i s b i o l o g i c a l l y l a b i l e . A l l a n t o i n , . prepared from the urine of the e x p e r i -mental animals, had incorporated the isotope, as would be expected. I t was observed that ur inary urea had also incorporated some of the radioact ive carbon. The s p e c i f i c a c t i v i t i e s of the expired carbon dioxide and urinary urea made i t apparent that the isotope could not have been incorporated s o l e l y from the carbonate of the t i s s u e f l u i d s (32). The combined a c t i v i t y of the a l l a n t o i n and urea accounted for 16-29 percent of the t o t a l a c t i v i t y i n the ur ine . I f carbon 2 of the purine r i n g i s l a b i l e , the renewals of polynucleotide adenine and guanine, ca lculated 14 from,the incorporat ion of isotope from administered 2-C -adenine, would be expected to be lower than the corres-ponding renewals measured by the uptake of isotope from l , 3 - N 1 5 - a d e n i n e . Accordingly , the proportions of the nuc le ic a c i d purines synthesized from 2-C 1 4 -adenine i n the experiments reported herein were compared w i t h the r e s u l t s of s i m i l a r experiments w i t h 1 ,3-N 1 5 -adenine reported i n the - 18 -l i t e r a t u r e (20, 21, 25). This comparison i s of l i m i t e d value only since these l a t t e r experiments d i f f e r i n the l e v e l at which the adenine was administered and i n the manner of adminis t ra t ion . I t was noted that n u c l e i c ac id adenine renewal from 2-C 1 4 -adenine was s i g n i f i c a n t l y lower 15 than that measured by the uptake of isotope from 1,3-N -adenine (25), although guanine renewals were e s s e n t i a l l y the same f o r these two experiments. The reason f o r t h i s apparent anomaly i s not evident. The question of whether carbon 2 has a higher turnover than the other atoms of the purine r i n g would undoubtedly be c l a r i f i e d by experiments i n 15 14 which N and C labeled adenine were administered under i d e n t i c a l condi t ions . EXPERIMENTAL A. METHODS I . Synthesis of 2-C 1 4 -adenine A p p r a i s a l of the evidence c i t e d i n favour of a b i o l o g i c a l l y l a b i l e 2 - p o s i t i o n i n the purines showed that there had been no experiments reported i n which the turnover rate of carbon 2 had been measured d i r e c t l y , and no reports had been made of attempts to f o l l o w carbon 2 i n the course of purine metabolism. The knowledge that administered adenine p a r t i c i p a t e s i n nuc le ic a c i d formation and i s excreted apparently i n the same manner as endogenous purines suggested that studies of the metabolism of adenine, labeled i n the 2 - p o s i t i o n , might provide evidence relevant to the question of 2-pos i t ion l a b i l i t y i n the t i s s u e purines. The synthesis of various purines from imidazole precursors described by Shaw (31) suggested a method by which adenine could be prepared w i t h i s o t o p i c carbon i n -corporated i n t o the 2 - p o s i t i o n . A method f o r the synthesis of 2-C 1 4 -adenine was developed having as i t s basis the formylat ion of 4-amino-5-imidazolecarboxamidine and c y c l i z a t i o n of the r e s u l t i n g compound (31). The synthesis of the imidazolecarboxamidine from m a l o n o n i t r i l e i s i l l u s t r a t e d i n Figure 2. 1 - 20 -"V H N\ HN\ H N^ ; C H z \ \-NH2 H N > N H 2 H 2 N C - N H 2 . . . HN _ u r H N N HN NH (I) . 2 H C L || . (III) 6 6 H (IV) .2 HCL I _ STEP. H 0 H \ N C - N H , H N C - N H , H , N C - N H , \ / \ STEP 6 \ / 2 .STEPS 2 \ / 2 c = c -+ c = c — c = c / \ / \ Hc'*OOH / \ "V" "N/" " " V " H H H (VII) ' (VI) ( V ) F I G U R E 2 . T H E S Y N T H E S I S OF 2 - C 1 4 — A D E N i N E FROM MALONON ITRILE. Radioactive carbon i s incorporated i n t o the 2-p o s i t i o n of adenine by formylating 4-amino-5-imidazole-carboxamidine (V) w i t h C 1 4 - f o r m i c ac id (Step 5) . The formylation r e a c t i o n , when performed according to the method of Shaw, requires a large excess of 98 percent formic a c i d and acet ic anhydride and, consequently, i s quite i m p r a c t i c a l for the i n t r o d u c t i o n of the radioact ive formyl group. The cost of the i s o t o p i c formic ac id required to produce adenine having a s p e c i f i c a c t i v i t y high enough to be usefu l , would be p r o h i b i t i v e i f t h i s method were fol lowed. I t was found, however, that the 4-amino group could be formylated under much milder condi t ions , namely, i n d i l u t e aqueous formic a c i d . The reduction i n the amount of formic ac id employed i n the formylation react ion made the use of radioact ive - 21 -formic a c i d p r a c t i c a l , and the e l i m i n a t i o n of acet ic anhydride from the react ion mixture made i t possible to recover the excess C 1 4 - f o r m i c ac id f o r further use. Ring 14 closure of the 4-formamido-C -5-imidazolecarboxamidine to form 2-C 1 4 -adenine was effected by r e f l u x i n g i n a l k a l i n e s o l u t i o n as described by Shaw. ( i ) Preparat ion of 4-amino-5-imidazole- carboxamidine dihydroohloride The preparation of t h i s compound, according to the method of Shaw (31), i s out l ined i n Figure 2. M a l o n o n i t r i l e (I) was converted to malonamidine (II) which coupled r e a d i l y wi th benzene diazonium chlor ide to form phenylazomalonamidine ( I I I ) . Reduction of the azo compound i n formic ac id produoed formamidomalonamidine (TV), which underwent c y c l i z a t i o n on heating to become 4-amino-5-imidazolecarboxamidine (V). In spi te of s t r i c t adherence to the experimental procedures described by Shaw, the best y i e l d of f i v e attempts was 2 percent (based on malononitr i le) , whereas the reported y i e l d was 25 percent. The p r i n c i p a l d i f f i c u l t y appeared to be i n the coupling of benzene diazonium chlor ide wi th malonamidine (Step 2 ) . Personal communication w i t h Dr. Shaw indicated that the coupling react ion would proceed under conditions l e s s a c i d i c than the value of pH 4 reported, namely, between pH 4 and 7. By carrying out the coupling react ion at a pH of 5 - 5.2 - 22 -the o v e r a l l y i e l d from m a l o n o n i t r i l e was increased to 9 percent. ( i i ) Preparation of C 1 4 - f o r m i c a c i d In pre l iminary experiments w i t h non-isotopic formic ac id i t was demonstrated that the formylat ion of the amino-imidazole could be effected i n d i l u t e formic a c i d . The use of radioact ive formic ac id f o r the i n t r o d u c t i o n of a labeled carbon atom i n the 2 - p o s i t i o n of adenine therefore became f e a s i b l e . In consequence, the synthesis of C 1 4 - f o r m i e a c i d was undertaken employing the method of M e l v i l l e , Rachelle and K e l l e r (33). In t h i s procedure radioact ive potassium bicarbonate i s reduced to radioact ive potassium formate by hydrogen i n the presence of palladium black. The r e a c t i o n sequence, w i t h barium carbonate as the s t a r t i n g m a t e r i a l , i s as f o l l o w s : H +• KOH ,„ HpPd ->A B a C 1 4 G 5 - d 4 0 2 ^ E H C 1 4 0 3 — - H C 1 4 0 0 K ° 70°C,100 atm. Radioactive potassium bicarbonate was prepared from r a d i o -act ive barium carbonate i n a gas t ransfer apparatus described by the same authors. The pal ladium black cata lys t used i n pre l iminary hydrogenation experiments was prepared by the reduction of palladous chlor ide w i t h formaldehyde i n the presence of a l k a l i (34), but use of t h i s ca ta lys t r e s u l t e d i n h igh ly v a r i a b l e y i e l d s of formate. More s a t i s f a c t o r y - 23 y i e l d s were obtained wi th a pal ladium cata lyst prepared by the method of Shriner and Adams (35). In t h i s procedure, palladous oxide i s formed from palladous chlor ide by oxida-t i o n w i t h sodium n i t r a t e . The oxide was added to the s o l u t i o n of radioact ive bicarbonate and became reduced to the act ive c a t a l y s t , pal ladium black, when subjected to the conditions of the hydrogenation r e a c t i o n . In e f fec t , t h i s step involved the preparation of the palladium black cata lys t by reduction i n the presence of the substance to be hydro-genated. The hydrogenation was c a r r i e d out i n an Aminco high pressure hydrogenator.^ Since the hydrogenation reactions d i d not go to completion, i t was found necessary to p u r i f y the formate and recover the unreduced radioact ive bicarbonate. This was accomplished by a c i d i f y i n g the hydrogenation mixture i n the combustion apparatus shown i n Figure 3. The procedure used was s i m i l a r to that followed f o r combustion a n a l y s i s , d i f f e r i n g only i n the respect that phosphorie ac id was added i n place of combustion f l u i d and no heat was a p p l i e d . The formic ac id was recovered from the a c i d i f i e d hydrogenation mixture by steam d i s t i l l a t i o n i n the apparatus shown i n Figure 4. The steam d i s t i l l a t e was then t i t r a t e d w i t h ^ Permission to use t h i s equipment was generously granted by the Department of Chemistry. 24 -a l k a l i and evaporated to dryness under reduced pressure. By t h i s procedure, the i s o t o p i c formic ac id was both p u r i f i e d and determined. (a) Procedure f o r the p u r i f i c a t i o n and  determination of formic a c i d The react ion mixture from the hydrogenation was placed i n Tube C of the combustion apparatus shown i n Figure 3, and carbon dioxide - free 1 N sodium hydroxide (25 ml . ) (36) was added to Tube D which was then q u i c k l y attached to the apparatus. The apparatus was then evacuated w i t h a water pump and stopcock F c losed. Syrupy phosphoric a c i d (2.5 ml . ) was placed i n Tube A and added dropwise to the mixture i n Tube C. T h i r t y minutes were allowed f o r the absorption of the l i b e r a t e d carbon dioxide and then a i r was allowed to enter the apparatus s lowly through a soda-lime tube placed on Tube A. The rece iver D was lowered and, a f t e r the gas i n l e t tube E had been r i n s e d r a p i d l y i n t o D with f r e s h l y - b o i l e d water, was closed t i g h t l y w i t h a rubber stopper. The radioact ive carbonate was recovered from t h i s s o l u t i o n by p r e c i p i t a t i o n as barium carbonate (37). The a c i d i f i e d mixture i n Tube G and the washings were transferred q u a n t i t a t i v e l y to the apparatus i l l u s t r a t e d i n Figure 4 and C 1 4 - f o r m i c a c i d recovered by steam d i s t i l l a t i o n . During the steam d i s t i l l a t i o n the volume i n the f l a s k was kept to approximately 15 m l . by Figure 3. Apparatus f o r the wet combustion of organic compounds (37). This apparatus was used f o r the recovery of unreduced Bl^-bicarbonate i n the synthesis of C 1 4 -potass ium formate according to the method of M e l v i l l e et a l . (33). - 26 -STEAM " Figure 4. Apparatus for the p u r i f i c a t i o n and recovery of formic acid by steam d i s t i l l a t i o n * - 27 -heating wi th a small flame. When the volume of the d i s -t i l l a t e was 50 - 60 m l . the recovery of the formic a c i d was e s s e n t i a l l y complete. The d i s t i l l a t e was then t i t r a t e d w i t h standard a l k a l i to the phenolphthalein end-point and con-centrated to a small volume by evaporation i n vacuo. I n the development of t h i s method, t r i a l recoveries of added formate showed that the method was near ly q u a n t i t a t i v e . In f i v e experiments, i n which 200 mgm. of potassium formate were added, an average of 97 percent recovery was obtained. ( i i i ) Synthesis of 2-C 1 4 -adenine Since the incorporat ion of radioact ive carbon i n t o the adenine molecule i s achieved p r i m a r i l y by the formylat ion of the amino group of 4-amino-5-imidazole-carboxamidine, the s p e c i f i c a c t i v i t y of the product w i l l be the same as that of the formylating agent, C^ 4 -formic a c i d . As adenine of a high s p e c i f i c a c t i v i t y was e s s e n t i a l f o r the metabolism experiments planned, i t followed that a p r a c t i c a l method f o r i t s synthesis must employ the smallest poss ib le amount of formylating agent. I t was found, as the r e s u l t of experiments w i t h various concentrations of formic a c i d , that adenine could be prepared i n y i e l d s of 36 - 50 percent using 14 percent formic ac id i n 6 molar excess; y i e l d s were increased to 61 - 65 percent by increas ing the concentration of formie ac id to 24 percent and the molar - 28 -excess to 11 times. As the imidazolecarboxamidine had been prepared as the dihydrochloride s a l t , the free base was liber a t e d i n the formylation mixture by the addition of 2 equivalents of potassium formate: imidazolecarboxamidine dihydrochloride •+- *- imidazolecarboxamidine 2 HC^OOK + 2 H ( , 1 4 0 0 H ^ 2 m The potassium formate used f o r t h i s purpose contained a l l of the radioactive carbon used. The isotopic formic acid re-leased by the above reaction became equilibrated with and increased the concentration of the formic acid solution i n i t i a l l y added to the reaction mixture. (a) Procedure f o r the synthesis of 2-C 1 4- adenine The experiment described below was that i n which the 2-C 1 4-adenine f o r Experiment 2 was prepared. A solu-t i o n of C 1 4-potassium formate (0.17 gm., with an a c t i v i t y of 5.41 x 10 6 c.p.m.) was placed i n the reaction vessel, which was a 60 mm. test-tube made from a 24/40 9 outer member j o i n t , and evaporated to dryness under a stream of hot, dry a i r . To the residue was added 4-amino-5-imid-azolecarboxamidine dihydrochloride (0.20 gm.) and 20 percent formic acid (2.0 ml.) and the solution refluxed f o r 4 hours. This procedure constitutes the formylation reaction. - 29 -Ring closure was effected by r e f l u x i n g an a l k a l i n e i s o l u t i o n of the formamido d e r i v a t i v e . The formylation s o l u -t i o n was made a l k a l i n e by the slow a d d i t i o n of potassium bicarbonate (1.69 gm.) and subsequently d i l u t e d to 8.0 m l . , which made the s o l u t i o n 0.5 M with respect to bicarbonate. A b o i l i n g tube was added and the s o l u t i o n ref luxed f o r 1 hour. The s o l u t i o n was then almost n e u t r a l i z e d by the a d d i t i o n of s l i g h t l y l e s s than the equivalent amount of hydrochloric a c i d . The s o l u t i o n was t ransferred to a 100 m l . R.B. f l a s k and concentrated to 3 m l . by evaporation under reduced pressure. A f t e r cool ing i n an i c e - b a t h , the p r e c i p i t a t e of crude adenine was c o l l e c t e d by c e n t r i f u g a t i o n , washed three times wi th cold water (0.5 - 1.0 ml .) and d r i e d i n vacuo. The supernatant l i q u i d and washings contained the excess C 1 4 - f o r m i c a c i d which was recovered and determined by the prev ious ly described methods. The crude adenine was p u r i f i e d by sublimation (38) i n the apparatus shown i n Figure 5. In pre l iminary e x p e r i -ments w i t h t h i s apparatus, adenine recovered i n the sublimed form averaged 93 percent of the o r i g i n a l m a t e r i a l . The d r i e d , crude adenine was placed i n the outer tube and ground to powder w i t h a glass rod. When the pressure was reduced to approximately 1 mm. and the temperature maintained at 220°G by heating i n a wax bath, the p u r i f i e d adenine - 30 -1 1 u "COLD-FINGER' 1 - CON DENSER 160 X 25 MM. VACUUM " PUMP OUTER TUBE 130 X 32 MM. 3 0 MM. Figure 5. Apparatus f o r p u r i f i c a t i o n of adenine by sublimation. - 31 -appeared as a white deposit on the " c o l d f i n g e r " condenser, leaving a l i g h t , brownish residue i n the outer tube. In t h i s manner 83.0 mg. of adenine (containing 4,358 c.p.m. per mg.) were obtained, representing an o v e r a l l y i e l d of 61 percent f o r the three steps, formylat ion, c y c l i z a t i o n and p u r i f i c a t i o n , 14 / The excess C -formic ac id (11.24 m i l l i e q u i v a l e n t s , a recovery of 93.5 percent) recovered as the potassium s a l t , was used i n the preparation of a second l o t of i s o t o p i c adenine. As i n the f i r s t preparat ion, a s o l u t i o n of r a d i o a c t i v e formate was evaporated to dryness i n the react ion v e s s e l . To t h i s was added 4-amino-5-imidazole-carboxamidine dihydrochloride (0.200 gm.) and 5.18 N hydrochloric ac id (1.77 ml . ) and the s o l u t i o n ref luxed f o r 4 hours. The remaining steps of t h i s preparation were s i m i l a r to those described above. The sublimed adenine from t h i s procedure weighed 88.1 mg., an o v e r a l l y i e l d of 64.5 percent. ( iv) Proof of i d e n t i t y of synthetic adenine The i d e n t i t y of adenine, prepared by the above method was establ ished by combustion a n a l y s i s , preparat ion of a d e r i v a t i v e , paper chromatography and u l t r a v i o l e t absorption spectrophotometry. .(.a) Combustion analys is Adenine: C 5 H 5 % . Calculated, C 44.44; found, C 44.27. - 32 -(b) Der ivat ive The product formed a p i c r a t e , mp. 286 - 287°C (with decomposition), and admixture of t h i s p i c r a t e w i t h that of an authentic sample d i d not depress the melt ing p o i n t . I t must be noted that t h i s melt ing point i s not a p a r t i c u l a r l y good c r i t e r i o n , since the concomitant decom-p o s i t i o n makes the melt ing point d i f f i c u l t to observe w i t h p r e c i s i o n . (c) Chromatography P a r t i t i o n chromatography on f i l t e r paper, employing the techniques of Markham and Smith (39, 40) was used to v e r i f y the i d e n t i t y of synthetic adenine as w e l l as adenine and guanine i s o l a t e d from experimental animals and to tes t the p u r i t y of these compounds. The solvent system employed was the t e r t i a r y butanol-hydrochloric acid-water system described by these authors (39) and a modi f i ca t ion of t h e i r photographic method (40) was used to locate purine components on the chromatogram. Since Whatman No. 1 f i l t e r paper i s reasonably transparent to u l t r a v i o l e t l i g h t , and since the purines absorb s trongly i n the 2600 A region of the spectrum, i t i s poss ible to locate these compounds on a chromatogram by making a contact p r i n t of i t w i t h u l t r a -v i o l e t l i g h t on s e n s i t i z e d paper. A low pressure germicidal lamp (General E l e c t r i c 15 watt Germicidal lamp) which emits 90 percent of i t s r a d i a t i o n at 2537 A was found to be a - 33 -Figure 6. Contact print of a chromatogram made with l i g h t from a General E l e c t r i c 15 watt Germicidal lamp. The chromatogram i s of a hydrolysate of yeast ribonucleic acid and shows, i n descending order, spots of guanine, adenine, c y t i d y l i c acid and u r i d y l i c acid. The solvent system used was the t e r t i a r y butanol-HCl-water system described by Smith and Markham (39). The hydrolysate was made by placing 14.8 mg. yeast ribonucleic acid i n a small sealed tube with 1 ml. N HC1 and heating the mixture f o r 1 hour at 100°. 15.4 mi c r o l i t r e s of the hydrolysate were used f o r t h i s chromatogram. - 34 -Figure 7. Contact print of a chromatogram com-paring the Rf values of adenine synthesized as described above with authentic adenine. The synthetic product (right spot) i s homo-geneous and has the same Rf value as the authentic adenine ( l e f t spot). - 35 -simple, cheap, and e n t i r e l y s a t i s f a c t o r y subst i tute f o r the high pressure lamp with gas and l i q u i d f i l t e r s used by Markham and Smith. Examples of chromatograms p r i n t e d w i t h a 15 watt germicidal lamp are shown i n Figures 6 and 7. The photographic paper used was Ansco "Scona" Reflex paper or Kodak A-4 Kodabromide paper. On chromatograms run i n the above manner synthetic adenine, prepared and p u r i f i e d as described above, had the same Rf value as authentic adenine, that i s , 0.40 (Figure 7) . Further , these chromatograms showed only one spot f o r the synthetic product and r a d i o a c t i v i t y could be detected only at t h i s l o c a t i o n . As the Rf value of 4-amino-5-imidazolecarboxamidine i n - t h i s solvent system was deter-mined to be 0 . 3 , the presence of t h i s substance i n the synthetic adenine would have been detected as another spot. This technique proved valuable i n confirming the i d e n t i t y of adenine and guanine i s o l a t e d from b i o l o g i c a l sources and i n determining whether such compounds were pure. A sample of adenine, f o r example, i f i t were con-taminated wi th guanine would show two spots on i t s chrom-atogram w i t h Rf values 0.S7 (guanine) and 0.40 (adenine). (d) Spectrophotometry The purines, pyrimidines and t h e i r d e r i v a t i v e s have very wel l -def ined absorption maxima i n the u l t r a v i o l e t which have proven to be valuable c h a r a c t e r i s t i c s i n - 36 -a n a l y s i s . Adenine and guanine have maxima i n the v i c i n i t y of 2600 A; the absorption s p e G t r a of authentic adenine and adenine prepared as described above are compared i n Figure 8 and are seen to be i d e n t i c a l . The presence of e i t h e r 4-amino-5-imidazolecarboxamidine or i t s formamido d e r i v a t i v e as impuri t ies i n the synthetic adenine would have d i s t o r t e d the shape of the absorption curve since these compounds have maxima at 2850 A\ and 2720 1 respec-t i v e l y (31). Observations were made w i t h the Beckman Model DU Quartz spectrophotometer. I I . Measurement of R a d i o a c t i v i t y A l l determinations of radioact ive carbon were made w i t h the carbon i n the form of barium carbonate. Samples to be analysed f o r r a d i o a c t i v i t y were ox id ized by the wet combustion method; the carbon dioxide so produced was trapped i n a l k a l i and p r e c i p i t a t e d as barium carbonate (37). I n the case of r e s p i r a t o r y carbon d i o x i d e , the carbonate was p r e c i p i t a t e d d i r e c t l y from the a l k a l i i n which i t was trapped. The barium carbonate samples were mounted on paper- l ined, brass f i l t e r i n g dishes f o r counting. The preparation of barium carbonate samples for counting and the determination of r a d i o a c t i v i t y have been described i n d e t a i l by Wright (37). R a d i o a c t i v i t y was determined by means of a s e l f -- 37 -j -I L I . I I I L _ 2 4 0 2 5 0 2 6 0 2 7 0 2 8 0 2 9 0 3 0 0 WAVE LENGTH (m>J) Figure 8. A comparison of the absorption curves of authentic adenine, concentration 8.9 a per m 1. w i t h ' adenine prepared as described above, concentration 11.1 5 per JJL 1. The solvent used i n both cases was phosphate buffer , pH 6.5, and the observations were made i n a Beckman quartz spectrophotometer Model DU. - 38 -quenching Geiger-Muller counter, having a mica end-window with a thickness of 1.6 mg. per sq. cm. The G-.M. counter was connected to a commercial scaling unit. 3^ The barium carbonate samples f o r radioactive assay were prepared i n such a manner that t h e i r weights were kept above the minimum weight required to produce an " i n f i n i t e l y t h i c k " sample, which was found to be 132.7 mg. fo r the p a r t i c u l a r assembly used (37). Since the a c t i v i t y observed from an " i n f i n i t e l y t h i c k " sample i s proportional to the s p e c i f i c a c t i v i t y of the sample, no self-absorption corrections were necessary. A l l observed a c t i v i t i e s were corrected f o r coincidence error, background and counter performance (36). The t o t a l a c t i v i t y of the sample was then determined by multiplying the observed a c t i v i t y , corrected i n t h i s manner, by the factor Sample (BaC05) weight i n mg. 132.7 A s u f f i c i e n t number of counts was recorded to insure that the error involved i n counting was not greater than 2 percent (36). The wet combustion technique employed i n the pre-paration of the barium carbonate samples was that described by Wright (37) and permitted the determination of both ^ Nuclear Instruments and Chemicals Corp. Sealing Unit Model 163. 39 -t o t a l and radioact ive carbon of organic substances. In t h i s procedure, the sample was oxidized under reduced pressure w i t h the Van Slyke-Folch combustion mixture and the evolved carbon dioxide was trapped i n carbonate-free a l k a l i (36) contained i n a centrifuge tube. The carbonate was then p r e c i p i t a t e d as the barium s a l t from hot s o l u t i o n , washed and c o l l e c t e d i n a paper- l ined, brass f i l t e r i n g d i s h . The apparatus used i s shown i n Figure 3. I I I . Metabolism Experiments Dietary or p a r e n t e r a l l y administered adenine (20, 25) i s incorporated, i n p a r t , i n t o the t i s s u e n u c l e i c acids and i s degraded, i n p a r t , to the purine excretory products. In studying the metabolism of 2-C^ 4-adenine administered to r a t s , i t was therefore necessary to i s o l a t e and examine the polynucleotide purines and the purine end-product, a l l a n t o i n . The expired carbon dioxide and ur inary urea from the experimental animals were also examined f o r r a d i o a c t i v i t y since the presence of the isotope i n these compounds would i n d i c a t e that the purine r i n g was broken i n v ivo at p o s i t i o n 2. The r a t s used i n these experiments were adult males of the Wistar s t r a i n . I s o t o p i c a l l y labeled adenine hydrochloride dissolved i n rat serum was administered to each ra t i n d a i l y doses 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 . - 40 -During the experiments the animals were kept i n a metabolism cage (Figure 9) which permitted the separate c o l l e c t i o n of expired carbon dioxide, urine and feces. The t o t a l carbon dioxide was collected f o r each 12-hour period of the experi-ment and the urine was collected f o r each 24-hour period. The animals were fed t h e i r usual diet (U.B.C. ra t i o n 18) ad l i b i t u m throughout the experiment. Two experiments, each of the type described, were performed. In Experiment 1, adenine containing 75,550 c.p.m. was administered; analysis of the expired carbon dioxide indicated that a small amount of radioactive carbon was present therein. However, the observed a c t i v i t i e s were so close to background that l i t t l e reliance could be placed on these findings. Methods are available f o r determining such a c t i v i t i e s with accuracy, but the counting times i n -volved are so long as to make the method impractical i n t h i s case (36). Consequently, Experiment 2 was undertaken i n which the amount of isotope administered was 6.6 times that given i n the f i r s t experiment. The observed a c t i v i t y i n the respiratory carbon dioxide collected i n Experiment 2 was s i g n i f i c a n t l y higher than background. In Experiment 1, the animals received labeled adenine at the l e v e l of 17 mg. per k i l o of body weight per day, administered i n single d a i l y injections f o r 4 days. They showed only a s l i g h t drop i n weight (1*4$) and - 41 displayed no signs of t o x i c i t y due to the administered adenine (41, 42, 25). In the second experiment, i n order to increase as much as possible the amount of radioactive carbon administered, adenine of a higher s p e c i f i c a c t i v i t y was prepared and administered at a higher l e v e l per k i l o of body weight than i n Experiment 1. The d a i l y dose of adenine was increased to 42 mg. per k i l o and was given at t h i s l e v e l f o r 4 days. This dose i s approaching the l e v e l at which toxic effects may appear. Raska (41) observed that adenine administered o r a l l y at the l e v e l of 1G0 mg. per k i l o per day over a period of 1-2 weeks induced i n dogs a syndrome which resembled avitaminosis. The oral or parenteral administra-t i o n of smaller amounts of adenine (30-50 mg. per k i l o per day) to both dogs and rats produced changes i n the blood picture, namely, marked increases i n non-protein nitrogen, urea, uric acid, and creatinine (42). Parenchymatous de-generation of the kidneys had also occurred i n these animals. Extensive renal damage due to the deposition of 2,8-dioxyadenine crystals i n the d i s t a l tubules has been shown to occur i n rats within 5 days when adenine i s injected i n single doses at a l e v e l of 87 mg. per k i l o per day (25). I t was considered that the short term nature of t h i s experi-ment and the administration of 21 mg. of adenine per k i l o at 12-hour i n t e r v a l s rather than twice t h i s amount at 24-r 42 -hour i n t e r v a l s would reduce the p o s s i b i l i t y that t o x i c mani-festations might appear. The animals l o s t 6 percent of t h e i r weight i n the 4 days of the second experiment. The gross appearance of •the-the abdominal cavity was noted at Atime the animals were s a c r i f i c e d . The walls of the abdominal cavity, the mesenteries and the surface of the intestines were notably reddish i n appearance due to the engorgement of the blood vessels. The kidneys appeared to be s l i g h t l y enlarged and the brown colour was more pronounced than usual. In the absence of a detailed examination of the blood picture and h i s t o l o g i c a l examinations of the kidneys, i t cannot be stated with certainty whether or not these animals were showing some signs of t o x i c i t y from the administered adenine. However, i t was f e l t that although the amount of adenine administered probably would have produced tox i c effects over a longer period, the d a i l y dose was s u f f i c i e n t l y small that any of the above-described manifestations of adenine i n t o x i c a t i o n that might have appeared would not be s u f f i c i e n t l y advanced to affect the v a l i d i t y of the experimental result s obtained. I t was considered desirable to administer the adenine as a solution which was nearly neutral. Since adenine i s a very weak base, solutions of adenine hydro-chloride w i l l be strongly a c i d i c . I t was found that the pH - 43 -of solutions of adenine hydrochloride could be raised to approximately 6, without the p r e c i p i t a t i o n of the free base, by the addition of serum. Solutions of labeled adenine administered i n these experiments were prepared by dissolving the free base i n s l i g h t l y more than the equivalent amount of d i l u t e acid, followed by the addition of s u f f i c i e n t rat serum to raise the pH to approximately 5 or 6. In Experiment 1, the animals received four i n -jections of labeled adenine spaced 24 hours apart and were s a c r i f i c e d 24 hours af t e r the l a s t i n j e c t i o n . The adenine was administered at 12-hour i n t e r v a l s i n Experiment 2; each animal received 8 i n j e c t i o n s and was k i l l e d 12 hours a f t e r the l a s t i n j e c t i o n . Each animal was anesthetized with ether and as much blood as possible obtained from the dorsal aorta by means of a syringe. The heart, lungs, thymus, l i v e r , kidneys, spleen and small i n t e s t i n e were removed immediately, and frozen i n a dry ice-ethanol mixture. Before freezing, the small i n t e s t i n e was s l i t lengthwise, washed and cut into small pieces (20). The nucleic acids were is o l a t e d sub-sequently from the pooled organs. ( i ) Respiratory carbon dioxide; c o l l e c t i o n  and analysis Respiratory carbon dioxide was collected f o r each 12-hour period of the metabolism experiments i n the a l k a l i scrubbing towers of the metabolism cage (Figure 9). One - 44 -i _ • • Figure 9. Metabolism cage. The experimental animals were placed i n the wire basket A, resting inside vessel B which was made from a 2G l i t r e b o t t l e . The l u c i t e top C was seated on a greased rubber gasket and the j o i n t made a i r t i g h t by securing the top down f i r m l y with clamps. Tube D was attached to a water pump and a i r was drawn through the apparatus, passing successively through towers E and F which contained 10 percent sodium hydroxide and saturated barium hydroxide, respectively. Tube G contained saturated sodium chloride and served to control the humidity of the a i r . Carbon dioxide expired by the animals was swept out of the cage by the current of carbon dioxide-free a i r and trapped as the a i r passed up through the a l k a l i scrubbing towers H or J. By manipulation of stopcock K and clamp L the a i r flow could be switched from one tower to the other without i n -terrupting the experiment. Tube M contained a saturated barium hydroxide solution which indicated any incomplete absorption of C0 2 i n towers H or J. Urine was collected i n Tube N and feces accumulated i n 0 with only s l i g h t contami-nation from urine. - 45 -tower only was used f o r each c o l l e c t i o n ; 600 ml. of 12 percent carbonate-free sodium hydroxide was found adequate to absorb the carbon dioxide produced by the animals and yet o f f e r a margin of safety. At the end of each c o l l e c t i o n period the a i r flow through the cage was switched to the alternate tower which had been f i l l e d previously with fresh a l k a l i . The tower containing the absorbed carbon dioxide was then drained and washed; the a l k a l i , together with the washings, was d i l u t e d to 1 l i t r e . The carbonate from aliquots (2.0 ml.) of t h i s solution was precipitated as barium carbonate and collected f o r weighing and counting as previously described. ( i i ) Urine; c o l l e c t i o n and analysis The metabolism cage (Figure 9) used i n these experiments permitted the separate c o l l e c t i o n of urine and feces. As p a r t i c l e s of food dropped by the animals frequently found t h e i r way into the urine, i t was necessary to centrifuge the urine before examination. The t o t a l urine excreted f o r each 24-hour period of both experiments was col l e c t e d , d i l u t e d to 100 ml. and aliquots taken f o r the preparation of a l l a n t o i n , the determination of urea and the isotope content of whole urine. (a) Whole urine. Aliquots (3.0 ml.) of each urine c o l l e c t i o n were evaporated to dryness i n combustion - 46 -tubes (Figure 3, Tube C), converted to barium carbonate by the wet combustion procedure and counted, as described previously. (b) Urea determination. Urinary urea was deter-mined by the urease method described by Wright (37). In t h i s procedure an aliquot of urine was incubated with glycerol-urease (43) i n a phosphate-buffered solution. Urinary urea was hydrplysed to ammonium carbonate by the enzyme urease i n t h i s step. The incubation vessel was then connected to 2 a l k a l i traps i n such a way that a i r could be drawn through the urine-urease solution, sweeping the i n -cubation vessel and then passing through the a l k a l i traps. The ammonium carbonate i n the urine-urease solution was decomposed by the addition of phosphoric acid and the carbon dioxide thus l i b e r a t e d was swept from the reaction vessel and trapped i n the a l k a l i . In t h i s manner, the carbon of urea was converted to carbonate which then was recovered from the a l k a l i as barium carbonate, weighed and analysed f o r r a d i o a c t i v i t y as previously described. The enzyme urease i s highly s p e c i f i o i n i t s action and deter-minations of t o t a l and radioactive carbon i n urea by t h i s method are i n excellent agreement with analyses made using the wet combustion technique (37). (c) A l l a n t o i n . A l l a n t o i n was iso l a t e d from urine by the method described by Brown et a l . (20). The urine 47 -was treated with phoaphotungstic acid to remove i n t e r f e r i n g substances and basic lead acetate was then added to remove the excess phosphotungstic acid. Excess lead was p r e c i p i -tated by the addition of sulphuric acid and the solution neutralized with a l k a l i . A l l a n t o i n was precipitated from t h i s solution as the mercury s a l t . Mercury allantoinate was decomposed with hydrogen sulphide and the free a l l a n t o i n recovered by c r y s t a l l i z a t i o n . The C 1 4 contents of the a l l a n t o i n samples so obtained were determined by conversion to barium carbonate. ( i i i ) I s o l a t i o n of v i s c e r a l nucleic acids and the  preparation of the purines The pooled i n t e r n a l organs were homogenized, dried and defatted according to the method of Brown et a l . (20). The remainder of the procedure followed i n the preparation of the nucleic acids and purines was that des-cribed by P l e n t l and Schoenheimer (9). The tissue powder prepared as above was extracted with hot, 10 percent sodium chloride solution and the sodium s a l t s of the mixed nucleic acids precipitated from t h i s solution by the addition of ethanol. The free nucleic acids were obtained by a c i d i f i c a -t i o n of a solution of t h e i r sodium s a l t s . The isotopic carbon content of the free nucleic acids was determined as above. The nucleic acids were hydrolysed by treatment - 48 -with hydrogen chloride i n methanolic solution. Adenine and guanine, l i b e r a t e d i n t h i s process, were precipitated from solution as the hydrochloride s a l t s . The mixture of the purine hydrochlorides was resolved by dissolving i t i n water and r a i s i n g the pH to 5, at which point free guanine pre-c i p i t a t e d out of solution. Adenine was recovered from the solution by p r e c i p i t a t i o n as the pic r a t e . The purines pre-pared i n t h i s manner were p u r i f i e d by several r e c r y s t a l l i -zations, adenine as the picrate and guanine as the sulphate. The p u r i t y and i d e n t i t y of these compounds were tested by paper chromatography. For chromatography, adenine hydro-chloride was prepared from the picrate by d i s s o l v i n g a small amount of the l a t t e r i n d i l u t e hydrochloric acid and ex-t r a c t i n g the solution with ether u n t i l colourless. Repeated applications of small volumes of t h i s solution were placed on a s t r i p of f i l t e r paper i n a small spot u n t i l approx-imately 10-20 S of adenine had been added. Authentic adenine was applied i n an adjacent spot and the chromatogram run and printed as previously described. In the case of guanine, solutions of the sulphate were applied to the chromatogram. The pyrimidines were not i s o l a t e d since only a n e g l i g i b l e amount of r a d i o a c t i v i t y could be detected i n the nucleic acid hydrolysate following the removal of the purine hydrochlorides. - 49 -B. RESULTS I . Experiment 1 (i ) Administration of 2 - C 1 4-adenine. Radioactive adenine (56 mg., containing 82,550 c.p.m.) was dissolved i n 0.28 N hydrochloric acid (1.5 ml.) and the solution d i l u t e d with rat serum (11.5 ml.). C 1 4 determinations carried out on aliquots of t h i s solution showed i t to have an a c t i v i t y of 6,350 c.p.m. per ml. Two rats were each given 4 i n t r a -peritoneal injections of 1.0 ml. of t h i s s olution spaced 24 hours apart. A t h i r d rat received i d e n t i c a l treatment except that the f i n a l i n j e c t i o n was 0.9 ml. The combined weight of the rats was 758 gm. and they received a t o t a l of 11.9 ml. of the adenine-serum so l u t i o n which contained 51.3 mg. of 2-G 1 4-adenine with an a c t i v i t y of 75,550 c.p.m. ( i i ) Expired carbon dioxide. "Analysis of the respiratory carbon dioxide indicated that some radioactive carbon was present, but, i n general, the counts obtained from samples of the precipitated carbonate were so close to background that the r e s u l t s were unreliable. The carbon-dioxide collected from the 48-60 hour period was the only sample which displayed an a c t i v i t y s i g n i f i c a n t l y greater than background. 48-60 hour carbon dioxide: t o t a l a c t i v i t y 2,495 c.p.m. spec, a c t i v i t y 0.42 c.p.m./mg.C TABLE I G analyses of urine, Experiment 1. 2-C -adenine, having an a c t i v i t y of 75,550 c.p.m., was administered by intraperitoneal i n j e c t i o n to three male adult rats whose aggregate weight was 758 gm., i n doses of 17 mg. per k i l o per day f o r 4 days. The t o t a l urine exoreted by these animals was collected at 24-hour i n t e r v a l s and the C 1 4 content of urea, a l l a n t o i n and whole urine i n each sample was determined. Col l e c t i o n Period Urea Total Specific a c t i v i t y a c t i v i t y c.p.m. c.p.m./mg.C Al l a n t o i n Total Specific a c t i v i t y a c t i v i t y c.p.m. c.p.m./mg. C Whole Urine Total Specific a c t i v i t y a c t i v i t y c.p.m. c.p.m./mg.C. 0-24 hour* 24-48 " 48-72 " 72-96 " Total i 95 0.27 77 0.21 2,390 8.13 178 0.56 2,740 553 44.6 668 73.7 827 80.5 1,445 84.7 3,493 5,410 6.32 5,550 7.07 5,430 8.75 5,000 6.61 21,390 - 51 -This experiment showed that i n any succeeding experiments which were intended to demonstrate conc lus ive ly whether the expired a i r contained any radioact ive carbon d iox ide , the amount of isotope administered must be i n -creased by several t imes. ( i i i ) Ur ine . Radioactive carbon determinations on urine and ur inary constituents are presented i n Table I . I t should be mentioned that the method employed f o r the i s o l a t i o n of a l l a n t o i n was not q u a n t i t a t i v e . I t w i l l be noted that the sum of the t o t a l a c t i v i t i e s found i n urea and a l l a n t o i n account f o r 16-29 percent of the t o t a l a c t i v i t y i n whole u r i n e . ( iv ) Nucleic acids and purines. The combined v i s c e r a of the three r a t s weighed 109 gm. and y ie lded 17.4 gm. of dr ied t i s s u e powder. From t h i s powder 1.6 gm. of crude sodium nuc le ic acids were extracted which when r e -p r e c i p i t a t e d as the free n u c l e i c acids weighed 0.602 gm. The combined purine hydrochlorides obtained from the nuc le ic acids by hydro lys i s weighed 129 mg. and r e -s o l u t i o n of t h i s mixture y ie lded 44.4 mg. of guanine sul fate and 47.1 mg. of adenine p i c r a t e . By paper chrom-atography i t was shown that the adenine was contaminated w i t h a small amount of a substance having the same Rf value as guanine. An attempt was made to p u r i f y the adenine, but i n the course of t h i s operation i t was l o s t . A - 52 chromatogram of the guanine sulfate showed only one com-ponent and that had an Rf value the same as that of authentic guanine. An examination of the nucleic acid hydrolysate a f t e r the removal of the purines showed that only n e g l i g i b l e a c t i v i t y was present. Consequently, i t may be concluded that the administered adenine did not contribute appreciably to pyrimidine biosyntheses. In Table I I the re s u l t s of iso t o p i c carbon deter-minations on the free nucleic acids and guanine are shown. I I . Experiment 2 ( i ) Administration of 2-C 1 4-adenine. Radioactive adenine (122.5 mg., with an a c t i v i t y of 4358 c.p.m. per mg.) was dissolved i n 0.343 N hydrochloric acid (2.80 ml.) and di l u t e d with rat serum (14.2 ml.). As a small amount of par t i c u l a t e matter appeared on standing, the sol u t i o n was heated to 45°C and c l a r i f i e d by centrifugation. Aliquots of t h i s solution analysed f o r C 1 4 were shown to have an a c t i v i t y of 32,630 c.p.m. per ml. The two rats used i n t h i s experiment had a combined weight of 692 gm.; each rat received 7 intraperitoneal i n j e c t i o n s of 1.0 ml. of t h i s solution at 12-hour i n t e r v a l s . An eighth i n j e c t i o n of 0.65 ml. was given to each rat 12 hours l a t e r and the animals s a c r i f i c e d at the end of the next 12-hour period. - 53 -TABLE I I content of v i s c e r a l nucleic acids, guanine and adenine# i s o l a t e d i n Experiment 1. 2_cl4-adenine having an a c t i v i t y of 75,550 c.p.m. was administered by intraperitoneal i n j e c t i o n to three adult male r a t s , whose aggregate weight was 758 gm., each receiving four d a i l y doses of 17 mg. per k i l o . The animals were s a c r i f i c e d 24 hours a f t e r the l a s t i n j e c t i o n , t h e i r viscera immediately removed and frozen. The mixed nucleic acids and nucleic acid purines were prepared from the pooled organs. Substance isolated C 1 4 Content Specific Total a c t i v i t y c.p.m. c.p.m./mg. C mixed nucleic acids guanine sulfate adenine p i c r a t e ^ 2,814 23.5 347 26.3 .805 49.5 "The adenine picrate sample was l o s t during p u r i f i c a t i o n . The values reported are calculated using the a n a l y t i c a l data of P l e n t l and Sehoenheimer (9) and the C l 4 contents observed f o r the nucleic acids and guanine. -• 54 -The rata received, therefore, a t o t a l of 15.3 ml. of the i n j e c t i o n solution which had an a c t i v i t y of 499,300 c.p.m. The adenine was administered at a l e v e l of 41.3 mg. per k i l o per day. ( i i ) Expired carbon dioxide. The amount of isotope administered i n t h i s experiment was 6.6 times greater than that given i n Experiment 1. The presence of C 1 4 i n the expired carbon dioxide, indicated i n the f i r s t experiment, was confirmed and measured; the r e s u l t s obtained are presented i n Table I I I . ( i i i ) Urine. Determinations of the r a d i o a c t i v i t y present i n urine and urinary constituents are shown i n Table IV. (iv) Nucleic acids and purines. The pooled viscera, when homogenized, dried and defatted, yielded 11.1 gm. of tissue powder. The crude sodium nucleic acids extracted from t h i s powder weighed l.£7 gm. and when re-precipitated as the free nucleic acids weighed 0.569 gm. The combined purine hydrochlorides, obtained from'the nucleic acids by hydrolysis, weighed 161 mg. and yielded 48.6 mg. of adenine picrate and 55.7 mg. of guanine sulfa t e . Paper chromatography was used to v e r i f y the i d e n t i t y of these compounds and showed them to be pure. An examination of the nucleic acid hydrolysate 55 -TABLE I I I C content of expired carbon dioxide, Experiment 2. 2-C 1 4-adenine, having an a c t i v i t y of 499,300 c.p.m. was administered by intraperitoneal i n j e c t i o n to two male adult rats with an aggregate weight of 692 gm.; each rat received 8 injections of adenine at the l e v e l of 21 mg. per k i l o per 12-hour period. The t o t a l expired carbon dioxide f o r each 12-hour period following i n j e c t i o n was trapped i n the a l k a l i towers of a metabolism cage (Figure 9), d i l u t e d to 1 l i t r e and aliquots taken f o r analysis. C o l l e c t i o n period Expired C02 Total a c t i v i t y c.p.m. Specific a c t i v i t y c.p.m./mg. C 0-12 hours 2,250 0.68 12-24 " 1,650 0.38 24-36 M 3,000 0.73 36-48 " 4,500 0.91 48-60 " 12,000 3.03 60-72 " 5,750 0.99 72-84 " 6,250 1.02 84-96 " 7,000 1.46 Total 42,400 TABLE IV analyses of urine, Experiment 2. 2-C -adenine, having an a c t i v i t y of 499,300 c.p.m, was administered by intraperitoneal, i n j e c t i o n to two male adult rats whose aggregate weight was: 692 gm. Each rat received 8 doses adenine given at the l e v e l of 21 mg. per k i l o per 12-hour period. The t o t a l urine excreted was collected at 24-hour i n t e r v a l s and the Cl4 content of a l l a n t o i n , urea and whole urine determined. Urea Al l a n t o i n .Whole Urine Collection Period Total a c t i v i t y c.p.m. Specific a c t i v i t y c.p.m./mg.C Total a c t i v i t y c.p.m. Specific a c t i v i t y c.p.m./mg.C Total a c t i v i t y c.p.m. Specific a c t i v i t y c.p.m./mg.C 0-24 hours 363 1.29 3,860 694 41,000 59.7 24-48 M 225 0.76 3,505 431 29,260 56.4 48-72 " 3,300 10.90 5,040 909 28,450 44.5 72-96 " 2,687 9.79 4,370 872 43,300 71.2 Total 6,575 16,775 142,010 57 a f t e r the removal of the purines showed that only a small amount of r a d i o a c t i v i t y was present. Since t h i s was probably due to r e s i d u a l amounts of purines, i t was con-sidered that the administered adenine had not contributed to pyrimidine synthesis . The " t o t a l a c t i v i t i e s " f o r the mixed n u c l e i c acids and the combined purine hydrochlorides reported i n Table V provide a measurement of the recovery of the purine hydro-chlor ides from the n u c l e i c a c i d hydrolysate. The C 1 4 content of the purine f r a c t i o n i s seen to be 8G percent of that of the nueleic aeids. I f the assumption i s made that there has been no incorporat ion of the isotope i n t o the pyrimidines of the n u c l e i c a c i d s , t h i s value may be sa id to represent the f r a c t i o n of the purines recovered from the n u c l e i c a c i d hydrolysate. - 58 -TABLE V C x content of v i s c e r a l nucleic acids and purines i s o l a t e d i n Experiment 2. 2-C 1 4-adenine, having an a c t i v i t y of 499,300 c.p.m., was administered hy intraperitoneal i n j e c t i o n to two adult male rats whose aggregate weight was 692 gm. Each rat received 8 i n -jections of adenine at the l e v e l of 21 mg. per k i l o per 12-hour period. The animals were s a c r i f i c e d 12 hours a f t e r the l a s t i n j e c t i o n , t h e i r viscera immediately removed and frozen. Nucleic acids and nucleic acid purines were prepared from the pooled organs. c14 Content Substance is o l a t e d Total a c t i v i t y c.p.m. Speci f i c a c t i v i t y c.p.m./mg. C free nucleic acids 21,070 88.5 combined purines 16,940 322 guanine sulfate 4,150 270 adenine picrate 4,759 344 adenine (calculated from picrate) 757 59 -DISCUSSION Brown and his co-workers observed that adenine, labeled i n nitrogens 1 and 3 with N 1 5, when fed to rats i s incorporated into adenine and guanine of the tissue nucleic acids. The nitrogen isotope of the nucleic acid guanine was found also i n positions 1 and 3; from t h i s fact Brown et a l . concluded that the purine r i n g remained intact during the conversion of adenine to guanine (20). Further evidence that the rin g remains intact has recently been furnished by Marrian et a l . who fed rats adenine labeled with H 1 5 i n positions 1 and 3, and with C 1 4 i n position 8 (1,3-N 1 5, 8-C 1 4-adenine) and showed that there was an e s s e n t i a l l y equal incorporation of the N^ 5 and C^4 isotopes into polynucleotide guanine (22). However, as has been discussed previously, t h i s concept was questioned by Gordon (27) and Marsh (28) who suggested that the purine r i n g may not remain i n t a c t during metabolic processes, but rather may be b i o l o g i c a l l y l a b i l e at po s i t i o n 2. I f the concept of a b i o l o g i c a l l y l a b i l e 2-position i n the purines i s v a l i d , a loss of isotope 14 from t h i s carbon would be expected when administered 2-C -adenine underwent any metabolic transformations, such as incorporation into the nucleic acid purines. A comparison i s indicated, therefore, between experiments with 1,3-labeled adenine and 2-labeled adenine whieh measure the extent to - 60 -which the isotope of the administered adenine i s incorporated i n t o the polynucleotide purines. Such a comparison i s made i n Table VI between the experiments of Brown and c o l l a b -orators who used l , 3 - N 1 5 - a d e n i n e and the experiments on the metabolism of 2-C 1 4 -adenine described h e r e i n . This compari-son i s of l i m i t e d value since the method of adminis trat ion of the labeled adenine and the amounts administered per k i l o of body weight vary i n the experiments described. I t w i l l be noted that the percentage of n u c l e i c ac id adenine synthesized from i n j e c t e d 2-G 1 4 -adenine i s lower than the 15 corresponding value reported f o r in jected 1,3-N -adenine (25). Guanine renewals are e s s e n t i a l l y the same i n these two experiments. The reason f o r the dif ference between the adenine and guanine renewals i s not apparent. Before s i g n i f i c a n c e i s attached to t h i s observation, confirmation by fur ther experiments of a s i m i l a r nature i s necessary. The question of the 2-pos i t ion l a b i l i t y could undoubtedly be 15 c l a r i f i e d by an experiment i n which a s o l u t i o n of 1,3-NJ-*'-adenine and 2-G**"4-adenine i s administered to the e x p e r i -mental animals. Adenine administered i n t h i s manner would be, i n e f fec t , doubly l a b e l e d . A comparison made of the n u c l e i c ac id purine renewals ca lculated from N 1 5 and C 1 4 uptake very l i k e l y would reveal any dif ferences due to the proposed l a b i l i t y of carbon 2. TABLE VI A comparison of the renewal of polynucleotide adenine and guanine as measured by the incorporation of the isotope from 1,3-Nl5-adenine and 2-C 1 4-adenine. The values shown are the percentages of the nucleic acid purines derived from the administered labeled adenine, calculated i n the ease of Cl4 l a b e l i n g , as the r a t i o of the speoific a c t i v i t y per mole of the polynucleotide purine to that of administered adenine, expressed as a percent. The renewals shown f o r N 1 5 l a b e l i n g are calculated as the r a t i o of "atom percent excess N^5" of the isolated purine to that of the administered adenine, expressed as a percent. A l l of the experiments were 4 days i n length and the rats used were adult males. 2-G 1 4-adenine, intraperitoneal t l,3-N 1 5-adenine, intraperitoneal 1 15 ,3-N -adenine, o r a l 17 mg. per k i l o per day Expt. 1. 42 mg. per k i l o per day Expt. 2. 87 mg. per k i l o per day (Ref.25) 27 mg. per k i l o per day (Ref.20) 38 mg. per k i l o per day (Ref.21) 200 mg. per k i l o per day TRef.20) Adenine t 7.69 11.7 5.4 2.1 13.7 Guanine 1.1 5.51 5.2 3.2 1.3 8.2 62 -Bendich et . a l . (25) observed a higher renewal of polynucleotide guanine from dietary 2,6-diaminopurine-l,3-N 1 5 than from dietary 2,6-diaminbpurine-2-C 1 3 i n a p a r a l l e l experiment. Approximately 4.0 percent of the nucleic acid guanine was synthesized" from N 1 5 - l a b e l e d diaminopurine, whereas only 1.5 percent was derived from the C 1 3 - l a b e l e d compound, as measured by the uptake of isotope i n each case. This difference i s c i t e d by Gordon (27) as evidence f o r a l a b i l e 2-position. In the preceding discussion i t was suggested that there may w e l l be a loss of isotope i n the tissues from administered 2-labeled adenine or the compounds intermediary i n i t s metabolism. This would imply that the purine r i n g was broken at pos i t i o n 2 and that the i s o t o p i c a l l y labeled carbon 2, as a l a b i l e substituent, was p a r t l y transferred to some compound i n the tissues and replaced by non-isotopic carbon. This i s i n accord with the concept of a dynamic equilibrium of the t i s s u e constituents (44). Some of the C-^-label of adenine presumably would be found i n the tissues as a 1-carbon compound, or as a l a b i l e substituent of some lar g e r molecule, and thereby would become d i s t r i b u t e d throughout the organism according to whatever metabolic processes i t underwent. For these reasons, the expired a i r of the experimental animals was examined f o r the presence of radioactive carbon dioxide. As may be seen i n Table VII, - 63 -TABLE VII Di s t r i b u t i o n of C x* following the adminis-t r a t i o n of 2-Cl4-adenine D y intraperitoneal i n j e c t i o n . Experiment 1 Experiment 2 Total c.p.m. Percent of administered adenine Total c.p.m. Percent of administered adenine Adenine (injected) 75,550 100 499,300 100 Ezpired carbon dioxide trace 42,400 8.4 Urea 2,740 3.62 6,450 1.29 A l l a n t o i n 3,493 4.62 16,775 3.36 Whole urine 21,390 28.3 142,010 28.40 Combined nucleic acids 2,814 3.82 21,070 4.22 - 64 -the expired carbon dioxide collected i n Experiment 2 con-tained 8.4 percent of the isotope administered. In considering what compound was the immediate precursor of the C 1 4-carbon dioxide i n the expired a i r , note must be made of the presence of the isotope i n urinary urea. Recently i t has been shown that urea i s active i n i n t e r -mediary metabolism and may contribute part of i t s carbon to the carbon dioxide of the t i s s u e s . Wright (37) has shown that C 1 4-urea, when injected into r a t s , may contribute as much as 30 percent of i t s isotope to expired carbon dioxide. Since, i n the experiments described herein, the expired carbon dioxide contained almost 7 times the amount of isotope found i n urinary urea (Table V I I ) , i t i s considered that the expired carbon dioxide was not derived p r i n c i p a l l y from urea. The metabolic fate of adenine, as f a r as i s known, appears to consist of transformations into the nucleic acid purines, a l l a n t o i n , adenosine triphosphate, and 2,8-dioxyadenine (23). The p o s s i b i l i t y also exists that the adenine molecule could be extensively degraded and the carbon atoms oxidized to carbon dioxide. Such a process could conceivably account f o r the presence of C 1 4 i n the expired carbon dioxide observed i n Experiment 2. However, i t appears u n l i k e l y that a sizeable breakdown of adenine occurred since, i n the experiments of Brown et a l . (45), only f r a c t i o n a l percentages of ammonia and urea were - 65 -derived from N 1 5 - l a b e l e d adenine, isoguanine, hypoxanthine, xanthine and uric acid. The presence of the radioisotope i n expired carbon dioxide can be explained by the postulated l a b i l i t y of carbon 2 i n the purines. As suggested previously, i t i s l i k e l y that the C 1 4-carbon dioxide arose from the oxidation of some tissue constituent which picked up by i n t e r -change with the l a b i l e 2-carbon of 2-G 1 4-adenine or with some compound intermediate i n i t s metabolism. The demon-st r a t i o n that formate i s an excellent precursor to pos i t i o n 2 and the implication of 4-amino-5-imidazolecarboxamide i n purine biosynthesis (13) suggest that formate or some metabolic derivate of formate, may be the compound i n equilibrium with the l a b i l e 2-carbon of the purines. Recent work has shown that formate i s an important intermediate i n metabolism (13) and that i t may be produced i n the body from the oc carbon of glycine (46) and the <S carbon of serine (47). The presence of free formaldehyde and formate have been demonstrated i n the tissues by Mackenzie (48). Formate has been shown to be oxidized by the rat but the mechanism of t h i s process has not yet been elucidated (46). An experiment which might shed some l i g h t on the postulated interchange reaction between the purine 2-carbon and some tissue constituent i s suggested by the work of du Vigneaud et a l . (49). These authors showed that C 1 4 - l a b e l e d methanol, 66 formaldehyde and formate were u t i l i z e d by the rat i n the biosynthesis of the l a b i l e methyl group i n choline; G 1 4 - b i -earbonate was not used f o r t h i s process. I f the appearance of C 1 4 i n the methyl groups of choline were found as a 14 result of the metabolism of 2-C -adenine, t h i s would suggest that formate, or a derivative of formate, was l i b e r a t e d from po s i t i o n 2 by the opening of the purine r i n g . The f i n d i n g of C^-urea i s somewhat more d i f f i c u l t to i n t e r p r e t . Since the t o t a l amount of C 1 4 i n urea was only 1-4 percent of that present i n the administered adenine (Table vTI), the labeled urea could have arisen from several minor reactions. Urinary urea consistently showed a small uptake of isotope i n metabolism experiments with various N 1 5-labeled purines (45) and with 1,3-N 1 5-adenine, admin-istered both o r a l l y and parenterally (20, 25). In these experiments the isotope could have been derived from a small degradation of the purine or of any of the intermediates i n the course of i t s oxidation to a l l a n t o i n . A portion of the C 1 4 found i n urea i n Experiments 1 and 2 described herein could be due to such factors. I t i s wel l established that carbon dioxide of the tissues i s incorporated into urea (50, 32); a large part of the C 1 4 found i n the urea could be accounted f o r by t h i s process. However, i t must be noted that the s p e c i f i c a c t i v i t y of t h i s urea was higher than that of the expired carbon dioxide of the corresponding - 67 -period. This does not agree with the r e s u l t s obtained by Mackenzie and du Vigneaud (32) who found that r a t s , which had received methionine labeled with C 1 4 i n the methyl group, excreted urea and expired carbon dioxide having the same s p e c i f i c a c t i v i t i e s during the same period. This anomaly may be due, i n part, to the fact that the C 1 4 i n the urea i s o l a t e d i n these experiments i s undoubtedly derived from several precursors. I t w i l l be noted i n Tables I and I I I that there i s a decided increase i n the s p e c i f i c a c t i v i t y of urea i n the 48-72 hour period of both Experiments 1 and 2; i t i s probably s i g n i f i c a n t that a s i m i l a r increase occurs i n the 48-60 hour carbon dioxide of Experiment 2. The only s i g n i f i c a n t l y radioactive carbon dioxide found i n the f i r s t experiment was that collected i n t h i s same period. This p a r a l l e l increase i n the r a d i o a c t i v i t y of carbon dioxide and urea suggests that at least part of the urea was derived from carbon dioxide. As may be seen i n Tables I and I I , the sum of the r a d i o a c t i v i t y present i n the urea and a l l a n t o i n f r a c t i o n s of urine f a l l s considerably short of the t o t a l r a d i o a c t i v i t y present i n whole urine. An investigation of the nature of the other radioactive compounds present i n urine i s therefore indicated. Although the finding of C 1 4 i n the expired carbon dioxide of animals to which 2-C 1 4-adenine had been t - 68 -administered would seem to be direct evidence of a b i o l o g i c a l l a b i l i t y i n the 2-position of the purines, a more d e f i n i t e answer to t h i s question would undoubtedly be provided by studies of the renewals of the polynucleotide purines measured by G 1 4 and N 1 5 uptake from doubly-labeled adenine (1,3-N 1 5, 2-C 1 4-adenine). - 69 -SUMMARY 1. A method has been developed f o r the synthesis of 2-C 1 4-adenine. This compound was prepared by the formylation of 4-amino-5-imidazolecarboxamidine i n aqueous C^4-formic acid, followed by c y c l i z a t i o n of the r e s u l t i n g formamido compound. This procedure i s a modification of the synthesis of adenine described by Shaw (31), which i n i t s o r i g i n a l form was unsuitable f o r the i n -corporation of i s o t o p i c carbon into position 2 of the adenine molecule. The formylation procedure described herein permits an almost complete recovery of the 14 excess formylating agent, C -formic acid. Isotopic adenine, having an a c t i v i t y of 4,358 c.p.m. per mg., was prepared i n 60 percent y i e l d by t h i s method, with a 93 percent recovery of the unused C 1 4-formic acid. 2. A method f o r the p u r i f i c a t i o n and determination of small amounts of formic acid has been developed f o r use i n the preparation of C 1 4-formic acid required i n the synthesis of 2-C 1 4-adenine. 3. A modification of the photographic technique of Markham and Smith (40) has been used f o r the l o c a t i o n of nucleic acid derivatives on f i l t e r paper chromatograms. The apparatus used by these authors has been greatly - 70 -s i m p l i f i e d by using as a source of u l t r a v i o l e t l i g h t a low pressure germicidal lamp which emits 90 percent of i t s r a diation at 2537 1. The use of t h i s lamp obviates the need f o r a system of gas and l i q u i d f i l t e r s . 4. The metabolism of 2-C 1 4-adenine was studied i n the adult male r a t . Isotopic adenine, administered by i n t r a p e r i -toneal i n j e c t i o n , was incorporated into adenine and guanine of the nucleic acids, confirming the findings of Bendich et a l . (25). Radioactive carbon dioxide was found i n s i g n i f i c a n t amounts i n the expired a i r of the experimental animals and accounted f o r 8 percent of the administered r a d i o a c t i v i t y . Whole urine was found to contain 28 percent of the administered isotope, while urinary urea and a l l a n t o i n together accounted f o r 16-29 percent of the t o t a l a c t i v i t y found i n urine. 5. The renewals of nucleic acid purines, measured by the uptake of C 1 4 from 2-C 1 4-adenine, were compared with renewals measured by the uptake of N 1 5 from 1,3-N15--adenine as reported i n the l i t e r a t u r e (20, 21, 25). These findings and the presence of C 1 4 carbon dioxide i n expired a i r are discussed i n connection with the postu-lated b i o l o g i c a l l a b i l i t y of the 2-position i n the purines. - 71 -BIBLIOGRAPHY 1. Davidson, J. N., The biochemistry of the nucleic acids. Methuen and Co., London (1950). 2. P o l l i s t e r , A. W., Swift, H., and A l f r e t , M., J. C e l l , and Comp. Physiol., 38, Supp. 1, 101 (1951). 3. Gulland, J. M., Jordan, D. 0., and T h r e l f a l l , C. J . , J. Chem. S o c , 1129 (1947). 4. Cohen, S. S., and Stanley, W. M., J. B i o l . Chem., 144, 589 (1942). 5. Conn,. W. E., J. Am. Chem. S o c , 72, 1471 (1950). 6. Conn, W. E., J. Am. Chem. S o c , 72, 2811 (1950). 7. Cohn, W. E., J. C e l l , and Comp. Physiol., 38, Supp. 1, 21 (1951). 8. West, E. S., and Todd, W. R., Textbook of biochemistry. The Macmillan Company, New York (1951). 9. P l e n t l , A. A., and Sehoenheimer, R., J. B i o l . Chem., 155. 203 (1944). 10. 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