Plant Foods for Human Nutrition51: 231–243, 1997.c 1997Kluwer Academic Publishers. Printed in the Netherlands.

Recent advances in caffeine and theobromine toxicities:a review

M. U. ETENG1�, E. U. EYONG, E. O. AKPANYUNG,M. A. AGIANG and C. Y. AREMUDepartment of Biochemistry, College of Medical Sciences, University of Calabar, Calabar,Nigeria; 1Department of Chemistry and Biochemistry, University of Uyo, Uyo, Nigeria(� author for correspondence)

Received 17 October 1996; accepted in revised form 8 October 1997

Abstract. Caffeine and theobromine are purine alkaloids widely consumed as stimulants andsnacks in coffee and cocoa based foods and most often as part of ingredients in drugs. Manhas enjoyed a long history of consumption of caffeine and theobromine. Recent interest inthese two alkoloids, however, is centered on their potential reproductive toxicities. Caffeineand theobromine are now known to cross the placental and blood brain barrier thus potentiallyinducing fetal malformation by affecting the expression of genes vital in development. Thedeveloping fetus may not have developed enzymes for detoxification of these methylxanthinealkaloids via demethylation. There is a need, therefore, to protect the conceptus against ‘insults’from teratogens of this nature. Apart from its reproductive toxicity, the presence of caffeine andtheobromine in cocoa could limit its potentials as a nourishing food. This is an issue that needsto be addressed by nutritionists and the food industry at large. This paper discusses the naturalsources, consumption and uses, toxicity and the major advances in the reproductive toxicologyof caffeine and theobromine. The biosynthesis of these compounds in plants, metabolism inmammalian systems and the involvement of cytochrome P450 are reviewed and summarized.Evidence in favor of the toxicity of these compounds in experimental animals is presentedwith emphasis on the implications of these findings in humans. The paper concludes with acall for caution in the use of caffeine and theobromine pending further and more elaborateinvestigations.

Key words: Biosynthesis, Caffeine, Theobromine, Toxicity

Introduction

Caffeine (3.7 dihydro-1, 3.7 trimethyl-1H-purine-2,6 dione) commonly called1,3,7-trimethylxanthine and theobromine (3,7 dehydro- 3,7 dimethyl-IH-purine-2,6-dione) also known as 3,7-dimethylxanthine are both purine alka-loids. They are widely distributed in nature with theobromine occurring ina variety of plants such asCommelia thea(tea leaves),Theobroma cacao(cocoa),Cola acuminata(cola),Paullinia cupana, Ilex aquifoliumand otherIlex species including theacea, sterculiacea, spindacea. On the other hand, caf-feine is a component of coffee beans (Coffee arabica), tea leaves (Commeliathea), Cola accuminataand other plants including the rubiacea, sterculiacea

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and theacea [1]. Of these sources, coffee beans and tea leaves are the richest incaffeine (1–2%). Although theobromine is a metabolite of caffeine, as muchas 1.5–3% of theobromine is found in cocoa beans [2-5].

Caffeine is widely consumed in analgesics and other over the counterdrugs. In the western world, particularly the USA, it is the most widely usedpsychoactive drug with an estimated average consumption of 200 mg/day,equivalent to 2–3 cups of brewed coffee per day [6]. Although the applicationof its counterpart theobromine, in both verterinary and human medicine arerather limited, theobromine is still being employed medicinally as a diuretic[1] and may still be an ingredient in many prescription and over the countermedication.

Leonard et al. [7] showed that through the mediation of the central nervoussystem both methylxanthines affect almost every physiological system of thebody. The probable biochemical basis being the ability of methylxanthines toinhibit phosphodiesterase breakdown of cAMP leading to the accumulationof the latter.

Caffeine and theobromine produce central stimulation because of theireffects on the brain cortex. Thus, both alkaloids are distinctly classified ascentral nervous system stimulants.

The widest use of caffeine and theobromine is perhaps as snacks (coke,cola, chocolate and cocoa beverages). Less commonly known of these drinksare extracts made from colanuts,Cola veraorCola acuminataby West Africannatives; guarana,Paullinia cupanain Brazil; and mat́e, Ilex paraguayensisor Paraguay tea in Paraguay. These drinks are widely consumed and theirpopularity in the public diet has aroused considerable medical interest becauseof the stimulatory effects of caffeine and theobromine on the brain, heart,gastric secretion and urine flow.

There are various reports in the literature about the toxicities of caffeineand theobromine in foods. For instance, theobromine is established to betoxic in many laboratory species including dog [8], rat [9–11] and rabbit [12].Caffeine has also been demonstrated to be toxic and teratogenic in rodentsand laboratory species [13].

A recent human study demonstrated that exposure to caffeine was relatedto poorer neuromuscular development and significant increases in breechpresentation of fetuses [14]. Interest and major advances recorded on thereproductive toxicology of caffeine and theobromine is borne out of theirphysicochemical characteristics which allows both compounds to penetratethe placental barrier with ease [15] and may have effect on the fetus during itsdevelopment. This review was, therefore, undertaken with the aim of definingthe state of current knowledge on the toxicity of caffeine and theobromine. Aclear picture of developments on the subject will assist in understanding past

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Figure 1. Possible scheme for the biosynthesis of caffeine and theobromine [16–19].

achievements, current state of knowledge on caffeine and theobromine andareas for further research.

Biosynthesis of caffeine and theobromine in plants

The biosynthesis of caffeine, theobromine and theophylline commences frominosinic acid according to the scheme depicted in Figure 1. The inosinic acid isoxidized by the enzyme inosine-5-phosphate dehydrogenase to give xanthylicacid. Subsequent reactions leading to the synthesis of caffeine, theobromineand theophylline are catalyzed by methylases with 5-adenosyl methionine asa cofactor [16–19].

In cocoa bean, N-7 methylation may be faster, producing theobrominewith minute amounts of caffeine. This may explain the high ratio of theo-

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bromine to caffeine [20] obtainable in cocoa bean, compared with the lowratio, accounting for the high proportion of caffeine in coffee and tea.

Consumption levels

Theobromine is widely taken in the form of chocolate and cocoa beverageswhile caffeine is consumed in coffee drinks. The average daily consumptionof caffeine is estimated at 200–300 mg/day which is equivalent to 2–3 cupsof brewed coffee per day [6]. This range accomodates the earlier estimateput at approximately 227 mg/day or 2–3 cups of coffee per day by Graham[21]. Although cocoa may supply 230–280 mg of theobromine per cup, thereis a dearth of data on the average daily consumption levels or intakes oftheobromine in the diet. Should 2–3 cups of cocoa beverage be consumed perday as is the case for coffee, then about 460–690 mg of theobromine will betaken daily in the diet. This would be based on the fact that 1.5–3.0 mg % oftheobromine is present in cocoa bean compared with 1–2 mg % of caffeinein coffee bean.

Absorption and distribution

Caffeine is well absorbed from gastrointestinal routes. The absorption oforal doses, nevertheless, quickly approaches that from the intravenous route.Ingested caffeine induces gastric emptying through the stimulation of bothinternal gastric myenteric and submucous nerves. This is followed by thedirect absorption of caffeine from the stomach into the blood stream. Thecentral nervous system is also stimulated by caffeine via the cholinergicautonomous nerve supply to the gastrointestinal tract [22].

Studies on the absorption and distribution of theobromine are rather limit-ed. Delbeke et al. [23] found that theobromine is well absorbed and distributedto body tissues and also excreted rapidly. For instance, theobromine couldnot be detected in the plama upon feeding a meal containing 38.4 mg oftheobromine twice daily to female horses for 2.5 days. This suggests thattheobromine was rapidly cleared and a peak excretion rate of 500 ng/ml ofurine was attained between 2–12 hours after the last administration. A thresh-old value of 2 ng/ml for theobromine has been reported in horse urine [24].However, Pollard et al. [25] have indicated that animal models such as horsesand ewes are not suitable in the clinical context since they display significantdifferences in theobromine elimination rates.

In man the disposition of theobromine [26] and caffeine [27] followsfirst order kinetics. Stanley et al. [26] report that after a single oral dose of

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10 mg/kg, approximately 50% of theobromine and its metabolites are excretedwithin the first 8–12 hours.

The undissociated form of the molecule which is soluble in the lipoidalgastric membrane is the form in which caffeine and theobromine are absorbedand uniformly distributed to tissues. Transport across the placental membraneinto the fetus has been identified for theobromine and caffeine in both humansand rats [28].

Wilkerson & Pollard [3], have reported the deposition of caffeine and itsmetabolites, theobromine, theophylline and paraxanthine in fetal rat brain.The report further indicates the percentage plasma protein binding for caf-feine (25–30%), theophylline 50–60%), and theobromine (15–25%). Proteinbinding occurs partly through the methyl group at N-1 which is lacking intheobromine. Anion formation also increases binding. Thus, caffeine with ahigh lipid solubility and low protein binding penetrates the brain most rapidly.

Metabolic degradative pathways of caffeine and theobromine

Caffeine and theobromine, which are N-methylated compounds are degradedby demethylation. When administered in a dose of 10 mg/kg body weight to20 day old fetal rats, caffeine is demethylated to yield primary metabolitessuch as theobromine, theophylline and paraxanthine [2]. The demethylationstep (N-1, N-3 and N-7) is inhibited by pregnendiol at a concentration of5 mg/kg body weight. In rats, the N-1 demethylation of caffeine which yieldstheobromine is the most important pathway of caffeine metabolism, andaccording to Bienvenu et al. [4], constitutes 51% of total dimethylxanthines.

Dietary theobromine enters the pathway of caffeine metabolism at thispoint and is further demethylated to 3-methylxantine and 7-methylxanthine,which are subsequently oxidized to their corresponding uric acids (7-methyluric and 3-methyl uric acids) (Figure 2). A fraction of theobromine is directlyoxidized to 3,7-dimethyl uric acid, which undergoes further biotransforma-tion.

In the presence of cellular thiol group (GSH), the imidazole ring of 3,7-dimethyl uric acid is cleaved open and the resulting intermediate simultane-ously reduced to yield 6-amino 5-(N-methyl formyl amino)-1-methyl uracil.The other primary metabolite of caffeine (theophylline) is broken down to1-methyl and 1,3-dimethyl uric acid. A fraction of caffeine is also directlyconverted to 1, 3,7-trimethyl uric acid by oxidation. The uric acids and uracilare the final end products of caffeine and theobromine biodegradation. Themethyl groups produced in the process are available for a transmethylationreaction to methionine, which also was the donor of methyl groups during thebiosynthesis [29].

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Figure 2. Metabolic biotransformation of theobromine and caffeine [29].

Various intermediates of the pathway have been isolated from the urineof humans and experimental animals fed caffeine and theobromine rich diets[30, 31]. The pathway of caffeine and theobromine degradation is presentedin Figure 2.

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Involvement of cytochrome P450 in the metabolic biotransformation ofcaffeine and theobromine

The enzyme xanthine oxidase converts free xanthines to uric acids but themethylated xanthines like caffeine, theobromine and theophylline are notmetabolised by this enzyme. This indicates that other enzymes are involvedin the metabolism of caffeine and theobromine. The capacity of humancythochrome P450 monoxygenase to metabolize caffeine yielding trimethyluric acids, paraxanthine and minor amounts of theobromine has been demon-strated [32].

Cytochrome P450 monoxygenase is also a critical enzyme mediating theconversion of theobromine into its ultimate end products [29]. A hypothesizedoxidized intermediate of theobromine is believed to exist from which the finalproduct 6 amino-5-(N-methyl formyl amino)-1-methyl uracil (6AMMU) isgenerated via 3,7-dimethyl uric acid (3,7 DMU). The data of Lelo et al.[29], support the existence of such an intermediate, whose formation is therate limiting step. Evidence in support of the involvement of cytochromeP450 in caffeine and theobromine biodegradation has come from studieswith inducers and inhibitors of this critical enzyme. Lelo et al. [29], haveshown that addition of cytochrome P450 inducers such as cholanthrene andphenobarbitone to liver microsomal fraction increases the formation of 3,7-DMU and (6AMMU) by approximately 1.6 fold and 12 fold, respectively.

On the other hand, addition of inhibitors of cytochrome P450, such ascimetidine, metyrapone and SKF 525A block the conversion of theobromineinto 3,7-DMU and (6AMMU). Thus, cytochrome P450 monoxygenase isinvolved in the metabolism of caffeine and theobromine to their ultimateend products. More importantly, the toxication and detoxication of manyxenobiotics is known to be caused by cytochrome P450 [33].

Evidence for the toxicity of caffeine and theobromine in experimentalanimals

Various studies of theobromine toxicity in laboratory species are reported inthe literature. The earliest observations of theobromine toxicity were madefollowing preliminary trials with cocoa and its byproducts as feed for live-stock in the wake of the 1939–45 wars. For instance, Temperton & Dudley[34], feeding ground undecorticated cocoa beans which contain 1.9 mg %theobromine to chickens at levels of 10, 20 and 30% of the cocoa mash eitherdry or wet, found that death occurred in all groups fed cocoa beans. Furtherattempts to identify the cause of death proved that the poisonous effect inpoultry of feeding cocoa waste was due to the presence of theobromine.

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The reports of Temperton & Dudley [34], instigated further research, andexperimental trials with laying hens were conducted. Black & Barron [35],found that feeding cocoa meal, which contained 1.7 mg % of theobromine, ata level of 15 or 30% in the ration of laying hens resulted in high mortality, lossof weight and poor egg production. More recently, theobromine poisoninghas been documented in dogs [8].

In dairy cows, theobromine intake via cocoa shell fed at a rate of 1.04 kgdaily for one week, resulted in decreased milk production [36]. However,feeding detheobromized cocoa shells to friesian cows in the second and thirdmonths of lactation at 20 and 30% of the ration for successive periods of 14and 19 days showed that there was no effect on milk yield, pulse rate or liverweight of the cows [37].

Although no symptoms were observed in pigs upon feeding cocoa mealcontaining 2.8 mg % theobromine at a level of 5% in the ration for 168 days,higher levels of about 7.5 mg % and above had adverse effects [38]. Owusu-Domfeh et al. [39], have reported that mice fed diets containing 26 and 31% ofcocoa meal and cocoa bean shell, respectively, died after about a week on thetest diet. Manifestation of poorer growth, low protein efficiency and nitrogenretention was observed in rats [40]. Elevated amounts of plasma triglycerideand LDL-cholesteral have been observed upon intraperitoneal administrationof theobromine rich extracts in rats [41], a factor which may contribute toatherogenesis.

The reproductive toxicity of theobromine has received the attention ofinvestigators in recent times. Acute structural toxicity on the testis and thymustissue leading to fatality in rabbits, rats and mice [9, 11–12]. There was alsoa time and dose dependent mortality demonstrated in mature rabbits whichwere fed a commercial diet containing 1.0 and 1.5 mg % theobromine for120 days. The accompanying testicular alteration ranged from vacuolationof spermatids and spermatocytes to multinucleated cell formation, extensivetubular cell degeneration and oligospermia or aspermia. Thus, theobromine istoxic in virtually all species of animals ranging from houseflies [42], chickens[35], dogs [8, 36], mice [39], rats [9, 11], rabbits [12], and humans [43].

Caffeine is a known teratogen causing acute structural teratogenesis in ani-mals [13, 44]. Since caffeine, theobromine and theophylline could be presentas a result of biotransformation, their embryopathic and teratogenic activitieswere studied. Fujji [44], reported that sublethal doses of these compoundsadministered intraperitoneally to mice on days 9 and 12 of gestation, resultin a high frequency of malformed fetuses with mainly skeletal defects (cleftpalate).

Recent work on caffeine has been directed at its reproductive toxicity.It has, therefore, become particularly important to document the effects of

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caffeine consumption on the reproductive capacities of parents and their off-springs because caffeine is one of the most widely used neuroactive substancesconsumed and because a majority of women still continue its consumptionduring their pregnancies. Caffeine and its metabolites are transferred freelyacross the placenta [15, 28] and the fetus may lack or have a diminishedability to demethylate caffeine and its immediate metabolites, theobromine,theophylline and paraxanthine. The fetus is, therefore, in danger of exposureto these toxicants which may adversely affect its growth and development.Studies by Wilkinson & Pollard [45] have demonstrated that caffeine in adose of 25 mg/kg body weight administered by oral gavage to pregnant ratson days 8–9 of gestation caused delayed neural tube closure in rat embryos;also development of the heart, eyes and limbs were reduced confirming earlierreports on significant fetal and post natal mortality [46].

Acute studies following a single oral maternal dose of caffeine (5 or25 mg/kg body weight) have shown that caffeine and its primary metabolites,theobromine, theophylline and paraxanthine, are deposited in the fetal brain[3]. Thus, the methylxanthines may impair brain differentiation indirectlyby nutrient deprivation through a caffeine induced placental vasoconstrictionand/or directly by virtue of its chemical characteristic at any period of timeduring pregnancy. The reason is that the brain is still developing during lategestation and early neonatal life and is, therefore, particularly vulnerable toadverse effects of xenobiotics that freely cross the placenta or are passed tooffsprings during lactation [3].

Sexual differentiation of fetal gonads is another aspect also affected. Caf-feine administered to rats at 30 mg/kg body weight per day during pregnancyaffected some aspects of normal sexual differentiation of fetal gonads. Inmales, significant inhibition of interstitial tissue and Leydig cell differentia-tion subsequently leading to decreased testosterone biosynthesis and vacuo-lation of granulose cells of females has been reported [46, 47]. The observedstructural toxicity of theobromine on the testis as indicated earlier, is similarto the effect of caffeine on male gonads. Because caffeine is degraded tobiologically active metabolites including theobromine, the observed toxicityof caffeine on the testis may be due to its theobromine metabolite rather thancaffeine itself.

It has been suggested that the action of caffeine may be as result of adirect toxic effect on cellular function thus affecting the sperm (germ cell)or growing fetus. Indirect effect may result through changes in maternal orpaternal physiological function mediated by an altered endocrine environment[48].

Since pregnancy requires coordinated physiologic adaptation that createsan environment for optimum development of the fetus, an altered endocrine

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milieu may adversely affect the development of the fetus. The alteration of theendocrine environment has been demonstrated. For instance, a single acuteadministration of caffeine (30 or 60 mg/kg body weight) to 85 days old malerats produced a profound endocrine response characterized by a significantincrease in plasma levels of corticosterone, progesterone and testosterone[48].

From the various recent studies on the reproductive toxicity of caffeine, itis evident that administration of caffeine during pregnancy affects the normaldifferentiation of fetal ovaries and testis resulting in significant fetal andpost natal growth retardation and an increase in post natal mortality [46, 47]and impaired brain differentiation resulting in delayed closure of the neuraltube [47]. Furthermore, consumption of caffeine during gestation results inchanges in the levels of brain chemicals such as DNA, Zinc, cAMP, protein,alkaline phosphatase and several behavioral indices such as motor activityand emotionality [49–51].

It is not only the fetus that is affected, the effect of caffeine consumptionon the mother are quite remarkable. For instance, as a result of an alterationin endocrine response characterized by significant increases in plasma levelsof corticosterone/progesterone, testosterone and, at a higher dose, Na+, caf-feine consumption has been linked with possible spontaneous abortion, fetalresorption, premature birth and fetal distress in females [48, 52].

Toxicity in man

Caffeine and theobromine are widely consumed by man in coffee, tea, cola,beverages, cocoa and chocolate as well as preservatives, analgesics, diureticsand other pharmacological preparations. The average daily intake of caffeineby moderate to heavy drinkers is estimated at 463 mg/day or 5–6 cups ofbrewed coffee [5, 53]. While there are several reports on the toxic effects ofcaffeine, theobromine and theophylline in animals, data on the toxic effectsof these methylxanthines in humans have not been extensively documented.Only three human fatalities from caffeine have been reported and the lowesttoxic dose was 2–3 g or 57 mg/kg body weight [54]. A recent human studyhas demonstrated that prenatal exposure to caffeine was related to poorerneuromuscular development and a significant increase in breech presentationof fetuses [14].

Theobromine constitutes the largest percentage of the biologically activemetabolites of caffeine. The levels of theobromine in the plasma of humansmight be quite high and hence capable of inducing human toxicity followingthe dual or combined exposure of man to theobromine directly in cocoa dietsand indirectly through biotransformation of ingested caffeine in vivo to form

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theobromine. This is an area where more research data will be beneficial toconsumers of methylxanthines worldwide.

Conclusion

Cocoa based foods are consumed world wide and the cocoa bean has beenshown to be very nutritious, containing substantial amounts of amino acidsexcept methionine and arginine. Vitamins, minerals and fat are also presentin a high proportion. The high level of fat contributes to the high gross energycontent of the cocoa bean. It was on account of its nutritional qualities thatit was described in Mexican theology as a ‘devine food’ or ‘food of thegods’. Despite its high nutritional value, however, the presence of caffeineand theobromine alkaloids may limit its potential as a nourishing food.

Theobromine is a biologically active metabolite of caffeine. The averagedaily intake of both organic molecules could be high hence the need forserious concern over reports in the literature on their reproductive toxicities.Pregnant women are especially vulnerable in view of the demonstrated abil-ity of caffeine and theobromine to alter the endocrine environment and tocross the placenta. The developing fetus and children are also susceptiblebecause the detoxifying mechanisms have not yet attained optimal levels inthese individuals. The potential hazards of caffeine and theobromine to thedeveloping fetus include long term physiological and neurological handicapsinduced by impaired fetal differentiation. Thus, it is the conclusion of thesereviewers that caution should be exercised in the consumption of coffee andcocoa products pending the outcome of further investigations.

References

1. Southon IW, Bukingham J (1989) Dictionary of alkaloids. London/New York: Cham andHall.

2. Kokwaro GO, Szandt IS, Glaziev AP, Ward SA, Edward B (1993) Metabolism of caffeineand theophylline in rats with malaria and endotoxin induced fever. Xenobiotica 23: 1391–1397.

3. Wilkinson JM, Pollard I (1993) Accumulation of theophylline, theobromine and parax-anthine in fetal rat brain following a single oral dose of caffeine. Brain Res 92: 193–199.

4. Bienvenu T, Pons G, Rey E, Thiroux G, Olive G (1993) Effect of pregnandiol on caffeinemetabolism in female rats. Eur J Drug Metab Pharmak 8: 181–185.

5. Aremu CY, Agiang MA, Ayatse JOI (1995) Nutrient and antinutrient profiles of raw andfermented cocoa beans. Plant Food Hum Nutr 48: 217–223.

6. Narod SA, Desanjose S, Victoria C (1991) Coffee during pregnancy: A reproductivehazard? Am J Obstet Gynecol 164: 1109–1114.

7. Leonard TK, Waison RR, Mohs MS (1987) The effect of Caffeine on various bodysystems: A review. J Am Diet Assoc 87: 1048–1053.

8. Strachan TR, Bennet A (1994) Theobromine poisoning in dogs. Vet Res 134: 284.

qual3265.tex; 7/04/1998; 10:54; v.7; p.11

242

9. Wang Y, Waller DD, Hilkim AP, Russel LD (1992) Reproductive toxicity of theobromineand cocoa extracts in male rats. Toxicol 6: 347–353.

10. Agiang MA (1992) Evaluation of the nutritive value of bread baked from compositeflour of wheat and fermented cocoa beans. MSc Thesis, University of Calabar, Calabar,Nigeria.

11. Wang Y, Waller DP (1994) Theobromine toxicity on sertoli cells and comparison withcocoa extracts in male rats. Toxicol Letter 70: 155–164.

12. Soffietti MG, Nebbia C, Valenza F, Amedeo S, Re G (1989) Effects of theobromine inmature and immature rabbits. J Comp Pathol 100: 47–58.

13. Legator MS, Zimmeving S (1979) Review of genetic effects of caffeine. J Env Sc andHealth 13: 135–138.

14. Barr HM, Streissguth P (1991) Caffeine use during pregnancy and child outcome: A7year prospective study. Neurotoxicol Teratol 13: 441–448.

15. Kimmel CA, Kimmel GL, White C, Grasto TE, Young JF, Nelson GJ (1984) Blood flowchanges and conceptual development in pregnant rats in response to caffeine. Fund andApplied Toxicol 4: 240–247.

16. Robinson T (1968) The Biochemistry of alkaloids. Weinzeller A, Springer GF,Wittman HG (eds) Molecular biology: biochemistry and biophysics, pp. 1–141. NewYork: Springer.

17. White A, Handler P, Smith EL, Hill RL, Lehman IR (1978) Principles of biochemistry,6th ed, pp 756–762. Japan: Kasaido Publishing Co.

18. Kumar H, Singh HN (1976) Plant metabolism. New Delhi, India: Affiliated East-WestPress.

19. Beever L (1976) Nitrogen metabolism in plants. London: Edward Arnold.20. Kreiseer WR, Martin J (1978) Variation in theobromine and caffeine contents of various

chocolate liquor. J Assoc Anal Chem 61: 1–18.21. Grayham DM (1978) Caffeine: its identity, dietary sources, in take and biological effects.

Nutr Rev 36: 97–120.22. Truitt EB (1954) The Xanthines. In: Dipalma JR (ed) Drills pharmacology in medicine,

4th ed, pp 533–556.23. Delbeke FT, Dabeckere M (1991) Urinary excretion of theobromine in horses given

contaminated pelleted food. Vet Res Commun 15: 107–116.24. Haywood PE, Teale P, Moss MS (1990) The excretion of theobromine in thorough bred

race horses after feeding compounded cubes containing husk: establishment of thresholdvalues in horse Urine. Equine Vet J 22: 224–226.

25. Pollard I, Williamson S, Downing J, Scaramuzzi R (1996). Phamaco-kinetics of caffeinein the oestrogen implanted ovariectomised ewe. Pharmacol Ther 19: 113–117.

26. Stanley MJ, Maurice JA, Barry HO, Elliot SV (1983) Theobromine kinetics and metabolicdisposition. Clin Pharmacol Ther 34: 546–555.

27. Bonati M, Latini R, Gallatti F, Young JF, Tognoni G, Gerattini S (1982) Caffeine dispo-sition after oral doses. Clin Pharmacol Ther 32: 98–106.

28. Abdi FB, Pollard I, Wilkinson JM (1993) Placental transfer and foetal disposition ofcaffeine and its metabolites in 20 day pregnant rat: A function of dose. Xenobiotica 23:449–456.

29. Lelo A, Birkett DJ, Miners JO (1990) Mechanism of formation of 6 amino-5-(N-methylformyl amino)-l-methyl uracil and 3,7-dimethyl uric acid from theobromine in rats invitro: Involvement of cytochrome P450 and cellular thiol. Xenobiotica 20: 823–835.

30. Cornish HH, Christman AA (1956) Identification of two metabolites in human urine.Acta Pharmacol Toxicol 40: 529–536.

31. Vangennig AH, DeBree PK, Vanderheiden C, Wadman SK (1973) Urinary excretion of3-methylxanthines and related compounds in children. Clin Chem Acta 45: 119–127.

32. Tanaka E, Ishikawa A, Yamamoto Y, Osada A, Tsuji K, Fukao K, Iwasaki Y (1993)Comparison of hepatic drug oxidising activity after simultaneous administration of twoprobe drugs caffeine and trimethedone to human subjects. Pharmacol Toxicol 72: 31–33.

qual3265.tex; 7/04/1998; 10:54; v.7; p.12

243

33. Klinger W (1982) Biotransformation of drugs and other xenobiotics during post nataldevelopment. Pharmac Ther 16: 377–380.

34. Temperton H, Dudley FJ (1943) Toxicity of alkaloids. Harpes Adams Util Poultry J 2:28–29.

35. Black JG, Barron NS (1943) The poisoning of life stock by cocoa products. Vet Res 55:166–167.

36. Weniger JH, Funk K, Brossea F (1956) Feeding experiments with theobromised andresidues from manufacturers of cocoa. Nutr Abst Rev 27: Abstract No 1176.

37. Bonadone T, Colonghills S, Curfo G (1963) Test of tolerance for meals of detheobromisedcocoa husk in diet of pigs and lactational cows. Nutr Abst Rev 33: Abstract No 7102.

38. Braude R (1943) Toxic effect of feeding cocoa meal to pigs. Vet J 29: 302–307.39. Owusu-Domfeh K, Christensen DA, Owen BP (1970) Nutritional value of some Ghanaian

feed stuff. Can J Anim Sci 50: 114–118.40. Reynolds MS, Beyer RA (1950) Effect of cocoa on protein utilization. J Am Diet Assoc

26: 590–591.41. Eteng MU, Eyong EU, Ebong PE (1996) Influence of theobromine rich extracts of cocoa

on plasma lipid profile in female albino Wistar rats. Biosc Res Commun (in press).42. Srinivasau AP, Kesava C (1979) Housefly test in toxicological studies. Indian J Exp Biol

17: 319–320.43. Stavric B (1986) Methylxanthines – Theobromine, Paraxanthine and combined effect of

methylxanthine toxicity to humans. Food Chem Toxicol 26: 725–733.44. Fujii T (1977) Comparison of teratogenic action of adenine and methylxanthine com-

pounds in mice. J Kansai Med Univ 28: 901–918.45. Wilkinson JM, Pollard I (1994) In utero exposure to caffeine causes delayed neural tube

closure in rat embryo. Teratogen carcinogen Mutagen 14: 205–211.46. Pollard I, Jabbour H, Mehrabani PA (1987) Effects of caffeine administration during

pregnancy on fetal development and subsequent function in adult rat: Prolonged effecton a second generation. J Toxicol Env Health 22: 1–5.

47. Pollard I, Wilkinson S, Magre S (1990) Influence of caffeine administration duringpregnancy on early differentiation of fetal rat ovaries and testes. J Dev Physiol 13:59–65.

48. Pollard I (1988) Increase in Plasma concentration of steroids in the rat after the admin-istration of caffeine: Comparison with plasma disposition of caffeine. J Endocrinol 119:275–280.

49. Concannon IT, Braughler JM, Schechter MD (1983) Pre and post natal effects of caf-feine on brain biogenic amines, cyclic nucleotides and behaviour in developing rats. JPharmacol Exp Ther 226: 673–679.

50. Huges RN, Beveridge IJ (1991) Behavioural effects of exposure to caffeine duringgestation, lactation or both. Neurotoxicol Teratol 13: 641–647.

51. Yazdani M, Joseph F, Grant S, Harhman A, Nakamato T (1990) Various levels of mater-nal caffeine ingestion affect biochemical parameters of fetal rat brain differently. DevPharmacol Ther 14: 52–61.

52. Weathersbee PS, Olsen LK, Lodge JR (1977) Caffeine and pregnancy. Postgraduate Med62: 64–69.

53. James JE (1991) Caffeine and health. San Diego: Academic Press.54. Khodaserich AP (1956) Fatal caffeine poisoning (cases from practice). Famacol Toks

Kol 19, suppl 62.

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