Metabolism of caffeine to nucleic acid precursors in mammalian cells

105

Mutation Research, 36 ( 1 9 7 6 ) 1 0 5 - - 1 1 4 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in The N e t h e r l a n d s

METABOLISM OF CAFFEINE TO NUCLEIC ACID P R E C U R S O R S IN MAMMALIAN CELLS *

R E G I N E G O T H ** and J.E. C L E A V E R

Laboratory of Radiobiology, University of California, San Francisco, Calif. 94143 (U.S.A.) (Received N o v e m b e r 3rd, 1975) (Revision received J a n u a r y 2 9 t h , 1 9 7 6 ) (Accepted February 16th, 1976)

Summary Caffeine is rapidly metabolized in human and mouse cells in culture by demethylation: within 1--3 h of exposure to millimolar concentrations of labeled caffeine, more than 90% of the pool consists of labeled products of metabolism and less than 10% is still caffeine. The methyl groups seem to b e transferred and used in the de novo synthesis of thymine, guanine, and adenine in nucleic acids. Normal fibroblasts, Lesch-Nyhan fibroblasts, xeroderma pigmentosum fibroblasts, HeLa cells, wild type mouse cells, and adenine phosphoribosyltransferase-deficient mouse cells all seem to metabolize caffeine similarly.

Introduction

Caffeine and other methylated xanthines have pleotropic effects in many organisms and cell culture systems. These effects include mutagenesis [12], chromosome aberration induction [15,17], inhibition of post-replication repair in UV-irradiated mammalian cells [6,18,19], altered mutation frequencies induced by radiations [1,24] or chemicals [ 2 1 ] , reduced UV-induced skin carcinogenesis [ 3 0 ] , increased in vitro transformation by chemical carcinogens [ 1 0 ] , and altered cyclic AMP levels [23,26,27]. These effects all imply that caffeine may interact with the genetic material, but the molecular mechanism(s) are n o t well understood. Some effects may be mediated by caffeine itself, whereas others may be mediated by its metabolic products, and effects seen in the whole animal may involve different biochemical pathways from * Work p e r f o r m e d in part u n d e r the auspices o f the U.S. Energy R e s e a r c h and D e v e l o p m e n t Administration. ** The work r e p o r t e d in this p a p e r w a s u n d e r t a k e n during the t e n u r e of a Research Training Fellowship a w a r d e d b y the I n t e r n a t i o n a l Agency for Research on Cancer.

106

those in cell culture systems. The catabolic pathway of caffeine in vivo is known to be similar to that of normal purines, and a major breakdown product is 1-methyl uric acid [7,14], but little information is available in tissue culture systems. We therefore investigated the metabolism of labeled caffeine in cultured mammalian cells and found that it is rapidly and extensively metabolized and acts as a methyl donor in pathways probably involving tetrahydrofolic acid. Materials and methods Human and mouse cell lines were grown in Eagle's minimum essential medium with 10% fetal calf serum. The h u m a n lines used were normal fibroblasts (GM637) and Lesch-Nyhan syndrome fibroblasts transformed by SV40 virus, HeLa cells, and primary fibroblasts from a normal person (SF629) and from xeroderma pigmentosum (XP) patients. The XP cell lines were excision-repairdefective complementation group A cells (XPPK, XP12RO) [4,5,16] and a post-replication-repair-defective XP variant line (XP4BE) [19]. Mouse lines were 3T3 and an adenine-phosphoribosyl-transferase-deficient line (3T3/DF8) derived from 3T3. Cells were incubated with generally labeled [3H] caffeine (Amersham/Searle Corp. Ill., 10--100 pCi/ml, 8.4 Ci/mmol} or [1-14C-methyl]caffeine (Amersham/Searle Corp. Ill., 50 #Ci/ml, 52 mCi/mmol) for 1--24 h. The caffeine concentration was increased to 10 -3 M with nonradioactive carrier to reproduce the high concentration c o m m o n l y used in studies of radiation-damaged cells [6, 20]. The purity of the labeled caffeine was c o n f r m e d by column and paper chromatography before use; there were no detectable amounts of contaminating purines. In some experiments, [3H] caffeine was supplemented with fluorodeoxyuridine (2 × 10 -s M), amethopterin (10 -s M), azaserine (2 X 1 0 -6 M), thymidine (4 × 10 -6 M), and glycine (10 -4 M). At the end of chosen labeling periods the medium was removed, the cultures were rinsed in physiological saline, the cells were harvested and processed either for determination of the radioactivity in the acid-soluble, RNA, and DNA fractions or for isolation and purification of the DNA. For determination of radioactivity, cultures were fixed for 7 min at 0°C in 0.2 N HC1 to obtain the acidsoluble fraction, centrifuged, rinsed in SSC (0.15 M sodium chloride, 0.015 M sodium citrate), and digested with 1 N NaOH to dissolve RNA; DNA and protein were precipitated by adding 1/5 of the volume of 6 N HC1; and then DNA bases were dissolved by digestion for 7 min at 60°C with 10% perchloric acid as described previously [3]. The 0.2 N HC1 supernatant was completely evaporated by bubbling air through it at room temperature. It was then digested with 0.5 ml of trifluoroacetic acid at 180°C for 90 min in sealed tubes, and the digest was analyzed by Sephadex G10 column chromatography. For isolation and purification of DNA, cells were lysed in 0.1% sodium dodecylsulfate in SSC, and RNA and protein were degraded by adding RNase (final strength, 0.1 mg/ml) and pronase (final strength, 0.1 mg/ml) and incubating the mixture at 37°C for 1--3 h. The DNA was then deproteinized by being shaken with chloroform : isoamyl alcohol (24 : 1, v/v) and was dialyzed against two changes of 500 ml each of SSC. Finally, cells were centrifuged in isopycnic

107 cesium chloride gradients and then dialyzed to remove the cesium chloride. The specific activity of the DNA was determined from the 3H activity and the 260 nm absorbance. Purified DNA was precipitated by adding 2 volumes of ethanol, centrifuged, and air dried. The DNA was digested with 0.5 ml of trifluoroacetic acid at 180°C for 90 min in sealed tubes, and the digest was analyzed by Sephadex G10 column chromatography to determine which DNA bases were labeled with 3H (ref. [13] ). The products of trifluoroacetic acid digestion were dried, redissolved in 1 ml of 0.05 M ammonium formate (pH 6.8) containing 0.001--0.01 mg of non-radioactive markers (caffeine, thymine, xanthine, hypoxanthine, guanine, adenine), and loaded onto an 80 × 1.5 cm Sephadex G10 column equilibrated with 0.05 M ammonium formate (pH 6.8). The digest was eluted with ammonium formate at 25 ml/h, 5 ml fractions were collected, and the optical density at 260 and 280 nm was recorded. The radioactivity in 1 or 2 ml aliquots of each fraction was counted in Aquasol. The optical density peaks of the non-radioactive markers were identified by spectral analysis and by prior chromatography of each marker individually to determine elution volumes. The identity of 3H activity in the caffeine region of column fractions and in mixtures of 3H-labeled c o m p o u n d s was confirmed by determining the solubility of the 3H-labeled material in chloroform. Caffeine, unlike all the normal bases found in DNA and RNA, is soluble in chloroform [26], so this additional property served as a ready means of identifying [3H] caffeine in mixtures. Results

Distribution and identity o f 3H in various cell fractions A crude analysis of normal human cells labeled with [3HI- or [I4C] caffeine (Table I) showed that radioactivity was distributed generally throughout each of the major cell fractions assayed: the acid-soluble, the RNA, and the DNA. This indicates that caffeine either is strongly bound to each fraction or is extensively metabolized. In subsequent investigations we concentrated on the 3H in the acid-soluble fraction and in DNA. Column chromatography of trifluoroacetic acid digests of the acid-soluble fraction and of purified DNA indicated that very little of the 3H was in the form of caffeine (Fig. 1). Both the acid-soluble fraction and the DNA contained 3H-labeled thymine, guanine, and adenine. The acid-soluble fraction also TABLE I D I S T R I B U T I O N O F R A D I O A C T I V I T Y IN V A R I O U S C E L L F R A C T I O N S OF H U M A N C E L L S G R O W N I N L A B E L E D C A F F E I N E (50 p C i / m l , 10 -3 M) Fraction

Acid-soluble RNA DNA

R a d i o a c t i v i t y ( c p m / 6 0 m m 2 p e t r i dish X 1 0 3 ) X P 1 2 R O cells

L e s c h - N y h a n cells

Hela cells

[ 14 C] c a f f e i n e , 24 h

[3H] caffeine, 6 h

[ 3 H ] c a f f e i n e , 24 h

131 8.5 I.I

53.4 17.6 4.1

88.2 57.2 19

108 1400 -

ACID SOLUBLE FRACTION

1200

uJ Z I

1000

Z --

~ Z

Z --

~ Z

Z

$

$

$

A

J%

800

$$1

600 400

200 I-

800 DNA

6oo I

4OO ~ 200 j-

-/ 10

20

30

40

.,..,A

50

60

70

80

FRACTION NUMBER Fig. 1. 3 H r a d i o a c t i v i t y p r o f i l e s f r o m S e p h a d e x G 1 0 c o l u m n c h r o m a t o g r a p h y o f t r i f l u o r a c e t i c acid digests of t h e acid-soluble f r a c t i o n a n d p u r i f i e d D N A f r o m n o r m a l h u m a n ( G M 6 3 7 ) cells g r o w n f o r 12 h in [ 3 H I c a f f e i n e ( 2 0 p C i / m l , 10 -3 M). T h e a r r o w s d e n o t e t h e p o s i t i o n s of n o n - r a d i o a c t i v e m a r k e r s a d d e d t o identify the n a t u r e o f the 3 H - l a b e l e d peaks. C y t o s i n e ( n o t s h o w n in t h e figure) e l u t e s at f r a c t i o n 18.

contained a trace (less than 10% of the total activity} of [3H]caffeine and hypoxanthine and a major peak of 3H-labeled material that eluted early from the column. We have not been able to identify this peak, but it must represent some intermediate p r o d u c t of caffeine metabolism. More than 95% of the 3H radioactivity applied to columns was recovered between the front and the adenine position.

Time course of [3HI caffeine metabolism The acid-soluble fraction was analyzed at various times after the beginning of exposure to [3H] caffeine to determine the time course of caffeine metabolism (Table II). At all sampling times after labeling, most of the 3H in the acidsoluble pool was in c o m p o u n d s other than caffeine, and column profiles were qualitatively similar to those shown for 12 h of labeling (Fig. 1). After 1 h of exposure to [3HI caffeine, only 10% of the pool was still [3H] caffeine, and this proportion decreased during subsequent incubation (Table II).

109 T A B L E II P R O P O R T I O N O F 3H A C T I V I T Y T H A T IS [ 3 H ] C A F F E I N E IN T H E A C I D - S O L U B L E F R A C T I O N S O F N O R M A L H U M A N ( G M 6 3 7 ) C E L L S A N D H e L a C E L L S G R O W N F O R V A R I O U S P E R I O D S IN THE P R E S E N C E O F [ 3 H I C A F F E I N E ( 1 0 0 ttCi/ml, 10 -3 M) I n c u b a t i o n t i m e (h)

3H activity N o r m a l h u m a n cells (GM 6 3 7 )

H e L a ceUs c p m / 1 0 6 cells

P r o p o r t i o n as caffeine (%)

c p m / 1 0 6 cells

P r o p o r t i o n as caffeine (%)

1

--

--

35,786

i0.0

2

--

--

50,734

6.4

8.0

56,651

4.7

3 S 12 24

23,860 84,568 -108,544

0.7

--

--

65,981

0.6

--

-2.0 --

Caffeine metabolism in various cell types Because label from caffeine appeared to enter nucleic acid bases, we investigated caffeine metabolism in various cell types with known defects in nucleic acid metabolism or distinctive responses to caffeine in order to determine the biochemical pathways involved. Various human and mouse cell lines all metabolized [3H] caffeine to nucleic acid bases {Table III). The primary cell lines seemed to put relatively more 3H into thymine than did established or transformed cell types (the GM637 and Lesch-Nyhan cells) (Table III), and the primary human cell lines all seemed to be less efficient at incorporating label from caffeine into DNA than other lines were. The absence of hypoxanthine-guanine phosphoribosyl transferase in LeschNyhan cells and of adenine phosphoribosyl transferase in mouse 3 T 3 / D F 8 cells had no influence on the a m o u n t of distribution of label found in DNA. Also, although excision-repair-defective (XPPK, XP12RO) and post-replication-repair-defective {XP4BE) XP cell lines are more sensitive to inhibition of postreplication repair by caffeine than normal lines are [ 1 9 ] , their metabolism of caffeine seemed to be qualitatively similar. Variations that occurred in the proportions of 3H in thymine, guanine, and adenine in DNA of various cell types seemed to be minor and no more than what would be expected for a metabolic pathway that involves numerous steps all influenced by the sizes of intracellular pools and cell growth rates. Detailed investigations of these variations were not performed. Influence o f metabolic inhibitors on the incorporation o f 3H from [3H]caffeine The data thus far imply that 3H from [3H] caffeine enters pathways that lead to several of the DNA bases. Because two main pathways exist for the synthesis of nucleic acid precursors, the de novo and salvage pathways, we attempted to discriminate between them with metabolic inhibitors to determine whether the 3H in the DNA originated from the transfer of methyl groups or from label in the xanthine ring incorporated by the salvage pathways. Fluorodeoxyuridine, amethopterin, and azaserine all reduced the specific

110

T A B L E III D I S T R I B U T I O N O F 3 H A C T I V I T Y IN T H Y M I N E , G U A N I N E , A N D A D E N I N E IN S E P H A D E X G 1 0 C H R O M A T O G R A P H S OF DNA DIGESTS FROM P U R I F I E D DNA OF V A R I O U S H U M A N AND M O U S E C E L L T Y P E S G R O W N F O R 24 h IN [ 3 H ] C A F F E I N E ( 1 0 ~ C i / m l , 10 -3 M) a Cell t y p e

Normal human (GM637) (SV40 transformed) Normal h u m a n (GM637) + 2 X 10 -s M fluorodeoxyuridine (SV40 transformed) Lesch-Nyhan (SV40 transformed) L e s c h - N y h a n + 2 X 10 -5 M fluorodeoxyuridine (SV40 transformed) Human fibroblast (SF629) XPPK b XP12RO (SV40 transformed) b XP4BE b Mouse ( 3 T 3 ) M o u s e ( 3 T 3 ) + 5 X 10 - 4 M t h y m i d i n e M o u s e ( 3 T 3 ) + 10 -5 M f l u o r o d e o x y u r i d i n e + 5 × 10 -4 M t h y m i d i n e Mouse ( 3 T 3 / D F 8 ) Mouse ( 3 T 3 / D F 8 ) + 5 X 10 -4 M t h y m i d i n e Mouse ( 3 T 3 / D F 8 ) + 10 -5 M f l u o r o d e o x y u r i d i n e + 5 X 10 -4 M t h y m i d i n e

Specific a c t i v i t y (cpm#zg)

P e r c e n t a g e of r a d i o a c t i v i t y in bases ..................... thymine

guanine

adenine

292

64 + 11

10 ' 3

27 ± 8

40 284

_

r

_

31

21

49

0 100 100 92 87 77 78

25 0 0 3 0 4 4

75 0 0 5 13 19 18

84 82

0 1

16 17

89 4.5 2.3 6.3 346 414 59 230 216 4

a E x c e p t X P 1 2 R O , w h i c h w a s l a b e l e d w i t h [ 1 4 C . l . m e t h y l ] c a f f e i n e ( 2 . 5 p C i / m l , 10 -3 M). b X P P K a n d X P 1 2 R O are e x c i s i o n - r e p a i r - d e f e c t i v e , c o m p l e m e n t a t i o n g r o u p A cell lines; X P 4 B E is a postr e p l i c a t i o n - r e p a l r ~ d e f e c t i v e XP v a r i a n t cell line.

activity of DNA labeled by [3H]caff~ine, and each caused a characteristic change in the relative distribution of radioactivity in thymine, guanine, and adenine (Table IV): fluorodeoxyuridine reduced the proportion of 3H in DNA found in thymine, azaserine reduced the proportion of 3H found in guanine and adenine, and amethopterin reduced the incorporation of 3H into all three bases. These changes induced with inhibitors are consistent with the idea that most of the 3H in DNA arose from the de novo synthetic pathways. T A B L E IV D I S T R I B U T I O N O F 3 H A C T I V I T Y IN T H Y M I N E , G U A N I N E . A N D A D E N I N E I N S E P H A D E X G 1 0 C H R O M A T O G R A P H S OF DNA DIGESTS FROM P U R I F I E D DNA OF N O R M A L H U M A N (GM637) C E L L S G R O W N F O R 2 4 h I N [ 3 H ] C A F F E I N E ( 1 0 # C i / m l , 10 -3 M) W I T H V A R I O U S I N H I B I T O R S Additive

None F l u o r o d e o x y u r i d i n e (10 -5 M) + t h y m i d i n e ( 4 X 1 0 - 6 M) 1 A m e t h o p t e r i n ( 1 0 -5 M) + t h y m i d i n e (4 X 1 0 - 6 M) + g l y c i n e ( 1 0 -6 M) Az~erine ( 2 X 10 -6 M)

Specific a c t i v i t y (cpm/~g)

P e r c e n t a g e of r a d i o a c t i v i t y thymine

guanine

adenine

556

62

10

27

134

3

13

84

7 230

73 96

6 1

22 3

! A s i m i l a r s t u d y o f H e L a cells l a b e l e d w i t h [ 3 H ] c a f f e i n e in the p r e s e n c e of f l u o r o d e o x y u r i d i n e ( 1 0 -5 M ) a n d b r o m o d e o x y u r i d i n e ( 1 0 -5 M) gave a d i s t r i b u t i o n of r a d i o a c t i v i t y w h i c h was 5% t h y m i n e , 41% guanine, a n d 54% a d e n i n e .

111 Discussion An essential feature of many interpretations of caffeine's pleotropic effects in animal and plant cells is that caffeine is active as caffeine [9,15,19,25]. Our results, however, show that caffeine is rapidly and extensively metabolized in human and mouse cells in culture, which suggests that at least some of the effects attributed to caffeine might actually be due to its metabolic products. Similar results have been reported for mice that were given [ 3H] caffeine [ 2]. One difficulty that we had to contend with in our studies is our incomplete knowledge of the initial locations of aH in generally labeled caffeine molecules. [3H]caffeine has 10 potential 3H sites: the 1, 3 and 7 methyl groups and the 8-H. If we assume, in the absence of fuller information, that all sites are equally likely to contain 3H, then 90% of the 3H activity that we see should originate in the methyl groups. This assumption can account both for the appearance of 3H in thymine, adenine, and guanine of DNA (Fig. 1) and for the results obtained with various cell types and metabolic inhibitors. The methyl groups of caffeine could provide 3H-labeled formyl groups for the 2- and 8-carbons of the purine ring and the [3H] methyl group of thymine during de novo synthesis by means of tetrahydrofolic acid derivatives. This pathway from [3H]caffeine must involve a specific methyl transfer rather than a random oxidative breakdown of the methyl groups, because oxidation would produce tritiated water and formic acid, which enters the one-carbon pool, and would result in non-specific labeling of macro-molecules. The absence of any detectable 3H in cytosine in DNA argues against significant amounts of oxidation being involved in the patterns of 3H labeling that we observed. The results obtained with various cell types suggest that defects in the purine salvage pathways, namely, the absence of either hypoxanthine, guanine, or adenine phosphoribosyl transferases, had no influence on the pattern of 3H labeling in DNA. This is consistent with our view that most of the 3H enters DNA by methyl transfer rather than by salvage of the purine ring after demethylation. Less 3H was incorporated into the DNA of primary human fibroblasts than into the DNA of other cell types. This might be explained by the slower growth rate of these fibroblasts, b u t it does n o t appear to be related to the genetic defects in repair carried b y any of the XP cell lines used. Therefore, the high sensitivity of XP variant cells to caffeine [19] may depend on the sensitivity of their defective repair systems to caffeine or to its breakdown products rather than on a distinctive pathway for caffeine metabolism. The results obtained with metabolic inhibitors (Tables III, IV) show the effects of blocking one or another of the multiple pathways by which 3H in the form of methyl groups can enter DNA. Fluorodeoxyuridine blocks thymidine m o n o p h o s p h a t e synthetase, thus diverting most of the 3H to the de novo purine pathway; azaserine blocks the de novo purine pathway, thus diverting most of the 3H into thymine; amethopterin blocks the tetrahydrofolate pathway, thus reducing the incorporation of 3H from caffeine into all three DNA bases. These results for various cell types and metabolic inhibitors indicate that the first step of caffeine metabolism in manlmalian cells is rapid demethylation. This will probably result in the accumulation of xanthine in cells in culture, whereas, in vivo, xanthine can be oxidized to uric acid by the action of liver

112

and intestinal mucosal xanthine oxidase [7,29]. Conversion of xanthine to xanthosine in the cell is probably inefficient [29], so xanthine will be an end product of caffeine demethylation. The reason no [3H]xanthine was detected in the acid-soluble pool (Fig. 1) is presumably that the a m o u n t of 3H in position 8 of the caffeine ring was insignificant. The accumulation of xanthine in cells may be involved in some of the effects of caffeine in radiation- and chemical carcinogen-damaged cells. But such a mechanism would be difficult to test directly because xanthine's low solubility and high charge would prevent extracellular xanthine from entering cells at high concentrations. Up till now, the main explanations have been based on the ability of caffeine to bind to single-stranded DNA [8,9,19,22] or damaged sites in DNA [22] or to inhibit phosphodiesterase and to increase cyclic AMP levels [23,25,27,28]. Evidence that caffeine would sensitize mouse L cells to UV light when added after irradiation but not if removed immediately before irradiation [20] supports explanations based on binding between DNA and caffeine itself. But increased cyclic AMP levels do not now appear to be involved in the inhibitory effects of caffeine on DNA replication in damaged cells [ 11]. We show here that caffeine is extensively metabolized, so whether there is enough caffeine inside cells to bind to DNA or to damaged sites in the way that it has been shown to bind in vitro [8,22] remains an open question. In the absence of direct in vivo evidence for mechanisms of action involving caffeine itself, the possibility of more complex mechanisms involving high concentrations of metabolic products of caffeine should also be entertained.

Acknowledgements We are grateful to Dr. A. Greene, National Institute for Medical Research, Camden, N.J., for providing the SV40-transformed normal and Lesch-Nyhan cells; to Dr. G. Veldhuisen, TNO, Rijswijk, The Netherlands, for providing the XP12RO cell line, and to Dr. H. Green, Massachusetts Institute of Technology, Cambridge, for providing the mouse cell lines. We are also grateful for advice received from Dr. D. Martin, Department of Biochemistry and Biophysics, University of California, San Francisco. References 1 A r l e t t , C.F. a n d S.A. H a r c o u r t , T h e i n d u c t i o n o f 8 - a z a g u a n i n e - r e s i s t a n t m u t a n t s in c u l t u r e d Chinese h a m s t e r cells b y u l t r a v i o l e t light. T h e e f f e c t o f c h a n g e s in post-irradiation c o n d i t i o n s , M u t a t i o n Res., 14 (1972) 431--437. 2 B u r g , A.W. a n d E. W e r n e r , Tissue d i s t r i b u t i o n o f c a f f e i n e and its m e t a b o l i t e s in the m o u s e , B i o c h e m . P h a r m a c o l . , 21 ( 1 9 7 2 ) 9 2 3 - - 9 3 6 . 3 Cleaver, J . E . , R e p a i r r e p l i c a t i o n and d e g r a d a t i o n o f b r o m o u r a c i l - s u b s t i t u t e d D N A in m a m m a l i a n cells after irradiation w i t h u l t r a v i o l e t light, B i o p h y s . J., 8 ( 1 9 6 8 ) 7 7 5 - - 7 9 1 . 4 Cleaver, J . E . a n d D. B o o t s m a , X e r o d e r m a p i g m e n t o s u m : b i o c h e m i c a l and g e n e t i c characteristics, A n n . Rev. G e n e t . , 9 ( 1 9 7 5 ) 1 9 - - 3 8 . 5 Cleaver, J . E . , D. B o o t s m a a n d E. F r i e d b e r g , H u m a n diseases w i t h g e n e t i c a l l y altered D N A r e p a i r p r o cesses, G e n e t i c s , 7 9 ( 1 9 7 5 ) 2 1 5 - - 2 2 5 . 6 Cleaver, J . E . a n d G . H . T h o m a s , Single strand i n t e r r u p t i o n s in D N A and the e f f e c t s o f c a f f e i n e in C h i n e s e h a m s t e r cells irradiated w i t h u l t r a v i o l e t Hght, B i o c h e m . B i o p h y s . Res. C o m m u n . , 3 6 ( 1 9 6 9 ) 203--208. 7 C o r n i s h , H . H . a n d A . A . C h r i s t m a n , A s t u d y o f t h e m e t a b o l i s m o f t h e o b r o m i n e , t h e o p h y l l i n e , and caffeine in m a n , J. Biol. C h e m . , 2 2 8 ( 1 9 5 7 ) 3 1 5 - - 3 2 3 .

113

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