Metabolism of Caffeine and Related Purine Alkaloids in Leaves of Tea

Plant Cell Physiol. 38(4): 413-419 (1997)JSPP © 1997

Metabolism of Caffeine and Related Purine Alkaloids in Leaves of Tea{Camellia sinensis L.)

Hiroshi Ashihara1, Fiona M. Gillies2 and Alan Crozier2'3

1 Department of Biology, Faculty of Science, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo, 112 Japan2 Bower Building, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow,

Glasgow G12 8QQ, U.K.

Purine alkaloid catabolism pathways in young, ma-ture and aged leaves of tea (Camellia sinensis L.) wereinvestigated by incubating leaf sections with l4C-labelledtheobromine, caffeine, theophylline and xanthine. Incor-poration of label into CO2 was determined and methanol-soluble metabolites were analysed by high-performanceliquid chromatography-radiocounting and thin layer chro-matography. The data obtained demonstrate that theobro-mine is the immediate precursor of caffeine, which accumu-lates in tea leaves because its conversion to theophylline isthe rate limiting step in the purine alkaloid catabolism path-way. The main fate of [8-14C]theophylline incubated withmature and aged leaves, and to a lesser extent young leaves,is conversion to 3-methylxanthine and onto xanthine whichis degraded to 14CO2 via the purine catabolism pathway.However, with young leaves, sizable amounts of [8-14C]-theophylline were salvaged for the synthesis of caffeine viaa 3-methylxanthine —»• theobromine —* caffeine pathway.Trace amounts of [2-14C]xanthine were also salvaged forcaffeine biosynthesis in young leaves, by conversion to 3-methylxanthine, and this was enhanced in the presence of5 mM allopurinol which inhibits purine catabolism. Feedsof [2-14C]xanthine to young leaves also indicated that 3-methylxanthine, as well as being salvaged for theobromineand caffeine production, is also converted, via TV-l-methyla-tion, to theophylline.

Key words: Caffeine — Camellia sinensis — Purinealkaloids — Theobromine — Theophylline — Xanthine.

The mono-, di- and trimethylxanthines that are foundin a limited number of higher plant species, includingcoffee (Coffea arabica L.), tea (Camellia sinensis L.) andmate (Ilex paraguariensis), are referred to as purine alka-loids. The biosynthesis of the purine alkaloids caffeine(1,3,7-trimethylxanthine) and theobromine (3,7-dimethyl-xanthine) has been investigated in tracer studies that haveutilised radiolabelled precursors such as [8-14C]adenine and[8-14C]guanine (see Suzuki et al. 1992). In tea, the leaves ofwhich typically contain ca. 2-5% caffeine (g dry weight)"1

(Takeda 1994, Ashihara et al. 1995), caffeine biosynthesis

3 To whom correspondence should be addressed.

involves an AMP —*• IMP —• XMP —* (or GMP —*• guano-sine) -»• xanthosine -+ 7-methylxanthosine —• 7-methyl-xanthine -> theobromine ->• caffeine pathway (Fig. 1, seeSuzuki et al. 1992). In addition to this major route, there isevidence from in vitro studies that several minor path-ways may also operate (Kato et al. 1996). Furthermore,Schulthess et al. (1996) have recently proposed that caffeinebiosynthesis in C. arabica starts with the metabolicallychannelled conversion of XMP -*• 7-methyl-XMP —• 7-methylxanthosine. In tea, the highest levels of caffeine bio-synthesis occur in young leaves (Ashihara and Kubota1986, Fujimori et al. 1991), flowers (Fujimori and Ashihara1990) and fruits (Terrasaki et al. 1994) and these ratesdecline markedly as the tissues age.

In contrast to caffeine biosynthesis, relatively little isknown about caffeine degradation and the catabolism ofpurine alkaloids in tea. In Coffea arabica, it has beenshown that caffeine is metabolised very slowly, via theo-phylline and 3-methylxanthine, to xanthine which is degrad-ed by the purine catabolism pathway to CO2 and NH3

(Kalberer 1964, 1965, Ashihara et al. 1996b). This paperreports on a study of caffeine catabolism in which the invivo metabolism of 14C-labelled xanthine, theobromine,theophylline and caffeine were investigated in young, ma-ture and aged leaves of Darjeeling tea. Incorporation oflabel in I4CO2 was determined and methanol-soluble me-tabolites were analysed by reversed phase HPLC-radio-counting (RC) and TLC. In some experiments allopurinolwas used to inhibit xanthine dehydrogenase/oxidase activ-ity (Nguyen 1986), thereby preventing the degradation ofxanthine by the purine catabolism pathway (see Ashiharaet al. 1996b).

Materials and Methods

Plant material—Leaves of tea (Camellia sinensis L. cv. Dar-jeeling) were obtained, in July to October 1995, from two-year-old plants growing under a natural photoperiod in a greenhouse atthe University of Glasgow. The developmental stages of the leaveswas categorized as (i) young leaves, (ii) mature leaves and (iii) agedleaves. Young leaves were the most recently emerged, weighed ca.50 mg (FW) and were ca. 28 mm long and 13 mm in width. Matureleaves comprised the fully expanded second and third leaf belowthe apex (ca. 80 mm long and 30 mm in width, weight ca. 400 mg),while similarly sized aged leaves, from near the base of the shoot,were dark green and weighed ca. 500 mg.

Chemicals—The following radiochemicals were purchased

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414 Metabolism of caffeine in tea leaves

from the commercial sources indicated: [8-I4C]caffeine (specific ac-tivity 1.91 MBq/jmor1, Moravek Biochemicals Inc., Brea, CA,U.S.A.), [2-14C]theobromine (2.07 MBq^mol"1, Moravek), [2-14C]xanthine (1.94MBq//mol"', Moravek) and [8-l4C]theophyl-line (2.04MBq^mol~', American Radiolabelled Chemicals, Inc.St. Louis, MO, U.S.A.). Radiolabelled uric acid, allantoin andallantoic acid were prepared in vitro from [2-l4C]xanthine usingxanthine oxidase (buttermilk), uricase (Candida utilis) and allan-toinase (peanut). These enzymes and all other chemicals were pur-chased from Sigma (St. Louis, MO, U.S.A.).

Metabolism ofradiolabelled purine alkaloids and xanthine—Segments of tea leaves (ca. 4 mm x 4 mm, 100 mg fresh weight,unless noted otherwise in Table legends) were incubated in 2 mlmedium, comprising 30 mM potassium phosphate, pH5.6, 10mM sucrose and a radiolabelled substrate, in a 30 ml Erlenmeyerflask, in a shaking water bath at 27°C. The 30-ml Erlenmeyer flaskhad a centre well containing a small glass tube into which was in-serted a piece of filter paper wetted with 0.1 ml of 20% potassiumhydroxide (w/v). At the end of the incubation period, the glasstube and filter paper from the centre well were transferred to a 50ml-flask containing 10 ml of distilled water and, after thoroughshaking, radioactivity in a 0.5 ml-aliquot was determined by liq-uid scintillation counting in order to estimate the amount of I4CO2released. The tea leaf segments were separated from the incuba-tion medium by filtering through a tea strainer, washed with 50 mldistilled water then transferred to 1 ml of extraction medium, com-prising 20 mM sodium diethyldithiocarbamate in 80% metha-nol and stored at — 20° C. The leaf segments were subsequentlyground in a pestle and mortar with the same extraction medium(ca. 5 ml) after which the resultant tissue homogenate was centri-fuged at 12,000 xg for 5 min and the supernatant and pellet sepa-rated. The pellet was resuspended in extraction medium (ca. 5 ml)and recentrifuged. The supernatant fractions, containing the meth-anol-soluble metabolites, were combined, reduced to dryness invacuo and aliquots were analysed by HPLC-RC and TLC.

HPLC-RC analysis of radiolabelled metabolites—An Altex332 liquid chromatograph (Berkeley, CA, U.S.A.) was used todeliver a 25 min, 0-40% gradient of methanol in 50 mM sodiumacetate, pH5.0, at a flow rate of 1 ml min"1. Samples were in-troduced off-column via an Altex 210 valve with a 1 ml loop.Reversed phase HPLC utilised a 250- x 4.6-mm i.d. universal fer-ruleless column (Capital HPLC Specialists, Broxburn, Lothian,U.K.), packed in-house with a 5-j/m ODS Hypersil support (Shan-don, Runcorn, Cheshire, U.K.). Column eluate was directed firstto a Spectra Physics 8450 absorbance monitor operating at 270nm and then to a Reeve Analytical 9701 radioactivity monitor(Glasgow, U.K.) fitted with a 200-//1 heterogeneous glass flow cellcontaining a cerium-activated lithium glass scintillator. Signalsfrom both detectors were processed by a dual channel 2700 datahandling system (Reeve Analytical). The gradient elution, revers-ed phase HPLC system separates 11 different purine derivativesand is also able to resolve 14C-labelled uric acid, allantoin andallantoic acid produced enzymically from [2-l4C]xanthine (Ashi-hara et al. 1996b). Samples containing mono-methylxanthineswere also analysed isocratically using a mobile phase of 10% meth-anol in 50 mM sodium acetate, pH5.0, which provides an en-hanced separation of 1-, 3- and 7-methylxanthine (Ashihara et al.1996b).

TLC analysis of radiolabelled metabolites—The methanol-soluble metabolites were also analysed by TLC using 200x200mm sheets of micro crystalline cellulose (Spotfilm, Tokyo KaseiKogyo Co., Tokyo, Japan). The solvent system used was n-butanol/acetic acid/water ( 4 : 1 : 2 , v/v) which separates the radio-

active metabolites which had been identified with the HPLC-RC.The Rf values of allantoin, xanthine, 3-methylxanthine, theobro-mine, theophylline and caffeine were 0.23, 0.35, 0.47, 0.59, 0.68and 0.78, respectively. Radiolabelled spots, detected after 2-3weeks exposure using Kodak X-OMAT AR film (Eastman KodakCo., Rochester, NY, U.S.A.), were scraped off the TLC plates,eluted from the cellulose support with distilled water and the radio-activity was measured by liquid scintillation counting (Ashihara etal. 1996a).

Results

Metabolism of [8-"]caffeine and [2-'4C]theobromine—Incubation of tea leaf segments with [2-'4C]theobromineresulted in the conversion of the radiolabelled substrate to[l4C]caffeine, with no detectable incorporation of radio-activity into other metabolites, including CO2 (Table 1,Fig. 2A). Theobromine, thus, appears to act as the imme-diate precursor of caffeine. The highest rate of [14C]caffeineproduction from [2-14C]theobromine was observed in ma-ture leaves. Only a small amount of 14CO2 was releasedfrom [8-l4C]caffeine incubated with young, mature and ag-ed tea leaves over a 48 h period and no methanol-solubleradiolabelled metabolites were detected (Table 1, Fig.2B).These observations indicate that the rate of caffeine ca-tabolism is extremely low and as a consequence caffeine ac-cumulates as the major purine alkaloid in tea leaves.

Incubation of tea leaf segments with 5 mM allopurinolresulted in reduced conversions of [2-14C]theobromine to[14C]caffeine (Table 1). This suggests that allopurinol, an in-hibitor of xanthine dehydrogenase/oxidase (Nguyen 1986),also inhibits S-adenosylmethionine:theobromine-7V-l-meth-yltransferase activity (see Fig. 1).

Metabolism of [8-'4C]theophylline-Unlike [2-14C]-caffeine and [2-l4C]theobromine, [8-14C]theophylline wasmetabolized to a variety of compounds (Table 2, Fig. 3).Metabolism of [8-l4C]theophylline was especially rapidin mature and aged leaves where more than 75% of thetotal radioactivity taken up by the tea leaf segments wascatabolised and recovered as 14C-labelled CO2, 3-methyl-xanthine, xanthine and allantoin, with trace amounts of la-bel also being incorporated into theobromine and caffeine.The addition of 5 mM allopurinol to the incubation medi-um resulted in a marked decline in 14CO2 production and aconcomitant increase in [l4C]xanthine, with little effect onthe level of radioactivity associated with theobromine andcaffeine in the mature and aged leaf incubations. Thesedata indicate that theophylline is catabolised, via 3-methylxanthine, to xanthine which is degraded to CO2 bythe conventional purine catabolic pathway.

Young tea leaves catabolised [8-14C]theophylline moreslowly than mature and aged leaves, with a marked reduc-tion in 14CO2 output, indicating that this is a consequenceof the purine catabolism pathway being less active than inthe older leaves. Approximately 20% of total radioactivity


Metabolism of caffeine in tea leaves 415

Table 1 Summary of the metabolism of [2-14C]theobromine and [8-14C]caffeine by young, mature and aged leaves ofCamellia sinensis

Substrate Leaves TreatmentDistribution of recovered radioactivity

(% of total ±SE)Total radioactivity

recovered

[2-l4C]Theobromine

[8-14C]Caffeine

Young

Mature

Aged

Young

Aged

Mature

ControlAllopurinol

ControlAllopurinol

ControlAllopurinol

Control

Control

Control

Tb

76.8 + 2.686.2±1.8

61.6±0.277.4±2.1

95.9±0.999.9±0.1

—

—

Cf

23.2±1.813.8±2.6

38.4±0.322.6±2.1

4.5±1.50.1±0.1

99.9 ±0.1

99.9 ±0.1

99.6±0.1

CO2

n.d.n.d.

n.d.n.d.

n.d.n.d.

0.1±0.0

0.1±0.1

0.4±0.1

(kBq±S.E.)

0.64±0.030.76±0.03

0.90±0.020.91 ±0.08

O.58±O.O8O.58±O.O3

6.90±0.04

8.42±0.51

8.18±1.0O

Leaf segments incubated with [2-14C]theobromine (3.7 IcBq) in the presence and absence of 5 mM allopurinol for 24 h and with [8-HC]-caffeine (36.7 kBq) in the absence of allopurinol for 48 h. Total radioactivity recovered presented as kBq±S.D. (n=3). Levels oftheobromine (Tb) and caffeine (Cf) and CO2 are expressed as a percentage of the total radioactivity recovered at the end of the incuba-tion period, n.d. = not detected.

taken up by the young leaf segments was associated with[l4C]caffeine and, to a lesser extent, [l4C]theobromine. Theconversion of [8-l4C]theophylline to caffeine, but not theo-bromine, was inhibited, and the size of the [14C]3-methyl-

xanthine pool enhanced, by the addition of 5 mM allo-purinol to the incubation medium (Table 2). This is inkeeping with the observation made in the previous section,that allopurinol lowers the rate of conversion of theobro-

/ XanthosineNRibose

SAM --.

O

7-Methylxanthosine

Ribose

Ribose-II

N rSAM

rv

SAHrSAM

SAH

7-Methylxanthine 3,7-Dimethylxanthine(Theobromine)

CH3

1,3,7-Trimethy Ixan thine(Caffeine)

Fig. 1 Pathway for the biosynthesis of caffeine from xanthosine in leaves of C. sinensis L. The numbers relate to the enzymes involvedin the individual steps. (I) S-adenosylmethionine: xanthosine-N-7-methyltransferase; (II) Af-methyl nucleosidase; (III) S-adenosylmethio-nine:7-methylxanthine-/V-3-methyltransferase; (IV) S-adenosylmethionine:theobromine-7V-l-methyltransferase. EC numbers have notyet been assigned to these enzymes.



5 10 15 20 25

Retention time (mins)30

Fig. 2 Reversed-phase HPLC-RC analysis of aliquots of metha-nol extracts from young leaves of C. sinensis following incubationwith 3.67 kBq [2-14C]theobromine for 24 h (A) and 36.7 kBq [8-l4C]caffeine for 48 h (B). Tb, theobromine; Cf, caffeine. Samplesize: trace A-230 Bq, trace B-300 Bq.

mine to caffeine. The increased incorporation of label into3-methylxanthine is probably a consequence of the reducedrate of turnover of theobromine.

Metabolism of [2-'4C]xanthine—The incubation ofyoung, mature and aged tea leaves with [2-14C]xanthineresulted in the release of very large amounts of I4CO2 withmost of the remaining radioactivity being associated withresidual [2-14C] xanthine and allantoin (Table 3, Fig. 4).Catabolism of [2-14C]xanthine to I4CO2 was slightly lowerin young leaves, which unlike their mature and agedcounterparts, incorporated small amounts of label intocaffeine, theophylline and theobromine. Inclusion of 5 mMallopurinol in the incubation medium resulted in a signifi-cant reduction in the production of 14CO2 and [14C]allan-

5 10 15 20 25

Retention time (mins)

Fig. 3 Reversed-phase HPLC-RC analysis of aliquots of metha-nol extracts from young (A), mature (B) and aged (C) leaves ofC. sinensis following incubation with 36.7 kBq [8-l4C]theophyI-line for 24 h. AH, allantoin; X, xanthine; 3-mX, 3-methylxan-thine; Tb, theobromine; Tp, theophylline; Cf, caffeine. Samplesize: A-400 Bq, B-280 Bq, C-290 Bq.

toin and a much larger pool of unmetabolised [2-l4C]xan-thine. In young leaves treated with allopurinol, the reducedactivity of the purine catabolism pathway was also associat-

Table 2 Summary of the metabolism of [2-14C]theobromine (36.7 kBq) by young, mature and aged leaves of Camelliasinensis in the presence and absence of 5 mM allopurinol for 24 h

Leaves

Young

Mature

Aged

Treatment

ControlAllopurinol

ControlAllopurinol

ControlAllopurinol

Tb

47.3±0.548.1 ±0.1

17.2±0.422.1 ±2.2

15.7±0.926.3±0.1

Distributior

3-mX

11.6±0.417.5±0.9

8.4±0.410.9±4.5

8.3±0.212.5±0.4

l of recovered radioactivity (% of

X

3.1±0.111.4±1.1

1.7±0.552.5±3.3

3.7±1.45O.7±3.3

All

5.0±0.91.0±0.3

1.4±0.21.6±0.1

2.7±0.21.9±0.1

CO2

1O.8±1.54.6±2.6

67.8±0.83.4±0.3

68.2±3.16.5±2.6

total ±SE)

Tb

4.0±0.15.5±0.9

1.9±0.14.3±0.4

0.6±0.20.9±0.1

Cf

18.2±0.311.9±0.1

1.7±0.11.3±0.5

0.4±0.10.8 + 0.2

Total radioactivityrecovered

(kBq±S.E.)

4.00±0.023.90±0.03

6.26±0.365.48±0.27

6.59±0.075.16±0.06

Total radioactivity recovered presented as kBq per 50 mg fresh weight (young leaves) and per 80 mg fresh weight (mature and agedleaves)±S.E. (n = 3)). Levels of residual [8-14C]theophylline (Tp) and radiolabelled metabolites, 3-methylxanthine (3-mX), xanthine (X),allantoin (All), CO2, theobromine (Tb) and caffeine (Cf) are expressed as a percentage of the total radioactivity recovered at the end ofthe 24 h incubation period. n.d. = not detected.



g•5a

(A)

/ n \\D )

All

IiAl!

X

Xl

3-mX Tp

Tp

I CfI

5 10 15 20 25

Retention time (mins)

30

Fig. 4 Reversed-phase HPLC-RC analysis of aliquots of metha-nol extracts from young leaves of C. sinensis following incubationwith 36.7 kBq [2-l4C]xanthine (A) and 36.7 kBq [214C]xanthine inthe presence of 5 mM allopurinol (B) for 24 h. AH, allantoin; X,xanthine; 3-mX, 3-methylxanthine; Tb, theobromine; Tp, theo-phylline; Cf, caffeine. Sample size: A-370 Bq, B-450 Bq.

ed with a very large increase in the radioactivity incorporat-ed into 3-methylxanthine, theophylline, theobromine andcaffeine, with these compounds representing more than40% of the recovered radioactivity compared to 1.4% incontrol leaf segments (Table 3, Fig. 4). From the amountsof total radioactivity recovered, it can be seen that the up-take of [2-14C]xanthine by young leaves was much higherthan in mature and aged leaves and that this uptake wasinhibited, to varying degrees, by the presence of 5 mMallopurinol in the incubation medium (Table 3). This con-trasts with the uptake of [2-14C]theobromine, [8-l4C]-caffeine (Table 1), and [8-l4C]theophylline (Table 2), whichwere not affected to any great extent by either leaf age orallopurinol.

Discussion

The data obtained in this investigation, demonstratethat, as in C. arabica leaves (Ashihara et al. 1996a, b), theo-bromine is the immediate precursor of caffeine in C. sinen-sis. A previous investigation, in which [l4C]adenine wasused as a substrate, indicated that caffeine biosynthesis ismost active in young tea leaves (Ashihara and Kubota1986). It was unexpected, therefore, that in the presentstudy, the highest rate of [l4C]caffeine production from [2-l4C]theobromine was observed in mature leaves (Table 1).This may genuinely reflect that the conversion of theobro-mine to caffeine is more rapid in mature leaves, but becauseof a rate limiting step earlier in the pathway the overall ca-pacity for caffeine biosynthesis is highest in young leaves.Alternatively, the seemingly greater rates of conversion of[2-14C]theobromine to [14C]caffeine could be due to dilu-tion of the exogenously supplied radiolabelled theobro-mine by endogenous theobromine, which in tea is presentat higher concentrations in young leaves than in matureleaves (Ashihara and Kubota 1986).

Caffeine accumulates in tea leaves in concentrations ofca. 2-5% of the dry weight of the leaf (Takeda 1994, Ashi-hara et al. 1995) and the data presented in this paper dem-onstrate that this is because caffeine is degraded very slow-ly, as a consequence of its conversion to theophylline beingvery effectively blocked (Table 1, Fig. 2). In aged andmature leaves and, to a lesser extent in young leaves, [8-14C]theophylline is demethylated rapidly via 3-methylxan-thine to xanthine, which enters the purine catabolism path-way and is broken down to CO2 and NH3. However, mostnotably in young leaves, [8-14C]theophylline is also incor-porated, in sizable amounts, into theobromine and caffeine(Table 2, Fig 3).

In mature and aged C. sinensis leaves, [2-l4C]xanthineacts almost exclusively as a substrate for the purine ca-

Table 3 Summary of the metabolism of [2-14C]xanthine (36.7 kBq by young, mature and aged leaves of Camellia sinen-sis in the presence and absence of 5 mM allopurinol for 24 h

Leaves

Young

Mature

Aged

Treatment

ControlAllopurinol

ControlAllopurinol

ControlAllopurinol

X

2.9±1.525.3±0.3

3.0±0.162.6±3.4

4.8±1.583.0±0.7

Distribution

All

27.3±13.73.2±

11.0±7.7±

9.8±n.d.

0.9

0.40.4

3.1

of recovered radioactivity

CO

68.3±27.9±

85.9±25.4±

85.4±17.0±

2

16.16.5

0.54.1

4.60.7

3-mX

0.6±0.4 0.12.6±1.1 10.

n.d.4.3±0.2

n.d.n.d.

(%of

Tp

3±0.22±1.4

n.d.n.d.

n.d.n.d.

total ±SE)

Tb

0.1±0.07.0±1.3

n.d.n.d.

n.d.n.d.

Cf

0.4±0.213.8±1.6

n.d.n.d.

n.d.n.d.

Total radioactivityrecovered

(kBq±S.E.)

7.96±1.262.51±0.14

4.66±0.852.82±0.55

3.16±0.072.00±0.34

Total radioactivity recovered presented as kBq per 50 mg fresh weight (young leaves) and per 80 mg fresh weight (mature and agedleaves)±S.E. (n = 3)). Levels of residual [2-'"C]xanthine (X) and radiolabelled metabolites, allantoin (All), CO2, 3-methylxanthine (3-mX), theophylline (Tp) theobromine (Tb) and caffeine (Cf) are expressed as a percentage of the total radioactivity recovered at the endof the 24 h incubation period. n.d. = not detected.



tabolism pathway and, when this route is partially block-ed by allopurinol treatment, it is not further metabolised,to any extent, to alternative products. [14C]Allantoin levelsand I4CC>2 output indicate that the purine catabolism path-way is also active in young leaves incubated with [2-14C]xan-thine. However, young leaves also convert [2-uC]xanthineinto small, but readily detectable, quantities of 3-methyl-xanthine, theophylline, theobromine and caffeine and, inthe presence of allopurinol, there is a substantial increasein the incorporation of label into these purine alkaloids(Table 3, Fig. 4).

The data obtained in this study indicate that thecatabolism and salvage pathways illustrated in Figure 5operate in leaves of C. sinensis. Theobromine is convertedto caffeine which accumulates as its further catabolism totheophylline is blocked in young, mature and aged leaves.The main fate of [8-I4C]theophylline incubated with ma-ture and old leaves, and to a lesser extent young leaves,is conversion to xanthine, via 3-methylxanthine, and entryinto the purine catabolism pathway. However in [8-14C]-theophylline feeds to young leaves, a significant amountof label is salvaged for the synthesis of caffeine via a 3-methylxanthine —»• theobromine —»• caffeine pathway. Traceamounts of [2-14C]xanthine are also salvaged for caffeinebiosynthesis, and this is increased when purine catabolismis blocked by allopurinol. Salvage of xanthine occurs as aconsequence of its conversion to 3-methylxanthine which ismetabolised to caffeine via theobromine. Interestingly, the[2-l4C]xanthine feeds to young leaves show that 3-methyl-

xanthine, as well as yielding theobromine, is also convertedvia 1-methylation to theophylline. Supporting evidence forthe operation of the pathways illustrated in Figure 5 comesfrom in vitro studies with N-methyltransferase activityfrom young leaves of C. sinensis which have shown that (i)xanthine is metabolized to 3-methylxanthine (Negishi et al.1985), (ii) 3-methylxanthine is converted to theophylline,theobromine and caffeine (Kato et al. 1996) and (iii) theo-phylline does not act as a methyl acceptor and thereforedoes not undergo direct conversion to caffeine (Kato et al.1996).

Caffeine homeostasis is similar in leaves of C. sinensisand C. arabica in that caffeine accumulates because its con-version to theophylline is the rate limiting step in the purinealkaloid catabolism pathway (Ashihara et al. 1996b). Inboth species conversion of [8-14C]theophylline —*• 3-methyl-xanthine -* xanthine links theophylline to the purinealkaloid catabolism pathway which results in breakdown toCO2 and NH3. In young tea leaves there is detectablesalvage of 3-methylxanthine and xanthine and resynthesisof caffeine via theobromine. However, there is no evidencefor the operation of similar pathways in coffee leaves. In-stead, young, aged and mature C. arabica leaves, treatedwith 5 mM allopurinol, convert xanthine to 7-methylxan-thine which does not appear to be further metabolised toany extent (Ashihara et al. 1996b).

In general, the caffeine content of C. sinensis leaves at2-5% dry weight, (Takeda 1994) is higher than the \-2% ofcaffeine found in C. arabica leaves (Mazzafera et al. 1991).

O CH3II f

CH3

Theobromine

CH3

Caffeine

CH3

Theophylline

CH3

3-Methylxan thine

HNHN

Xandiine

Purine Catabolism

Pathway

Fig. 5 Purine alkaloid catabolism and salvage pathways operating in leaves of C. sinensis. Arrow with two vertical bars represents ablocked conversion. Double headed arrows indicate reversible conversions.



It is possible that this may be a consequence of the salvageof purine alkaloid catabolites for caflFeine synthesis whichoccurs in tea but not in coffee leaves. The physiological sig-nificance of the accumulation of caffeine in young tealeaves remains to be established, although it has been pro-posed that caffeine may act as a chemical defence that pro-tects young soft tissues from predators, such as insect lar-vae (see Harborne 1993).

This work was supported in part by a Grant-in-Aid for Scien-tific Research (No. 0845255) from the Ministry of Education,Science, Sports and Culture of Japan to H.A., UK-Japan travelgrants from the British Council to H.A. and A.C. and a JapaneseSociety for the Promotion of Science Fellowship for Priority AreaResearch in Japan (Grant RC10506) to F.M.G.

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(Received November 13, 1996; Accepted January 21, 1997)


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Metabolism of Caffeine and Related Purine Alkaloids in Leaves of Tea