The Effects of Caffeine and Caffeine-containing Beverages on the Disposition of Brain Serotonin in Ratsf

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1 Agric. Biol Chem., 51 (12), , The Effects of Caffeine and Caffeine-containing Beverages on the Disposition of Brain Serotonin in Ratsf Hidehiko Yokogoshi,* Shouichi Tani and Nobuyuki Amano Laboratory of Nutritional Biochemistry, Department of Agricultural Nagoya University, Nagoya 464, Japan Chemistry, Received June 12, 1987 Caffeine and caffeine-containing beverages (instant coffee, black tea, green tea, or oolong tea) caused a significant decrease in serum tryptophan, and significant increases in brain tryptophan, serotonin, and 5-hydroxyindole acetic acid over those in rats fed a control diet. Adenosine supplementation partially counteracted the increase of brain serotonin caused by caffeine. These results are interpreted as indicating that caffeine-containing beverages may have some nutritional and behavioral effects. Caffeine, 1,3,7-trimethylxanthine, is one of the most widely consumed pharmacologically active substances. We usually ingest the caffeine as a constituent of beverages. Caffeine is also present in several medications including analgesics, appetite suppressants, and central stimulants. Until now, many investigators have demonstrated relationships between the consumption of caffeine-containing beverages and the induce of cardiovascular disease,1 ~4) because caffeine has a hypercholesterolemic effect.5~7) In our previous paper,8) the hypercholesterolemic effect of caffeine contained in beverages and xanthine derivatives was shown, but the mechanisms of this action are still unclear. Caffeine also inhibits the cyclic 3',5'-adenosine monophosphate (CAMP)-degrading enzyme, phosphodiesterase,9'10) and is also a competitive antagonist at extracellular adenosine receptors.n) Adenosine administration to animals can produce many physiological actions (sedation, bradycardia, hypotension, hypothermia, and sympathetic stimulations), and these effects are generally opposite to those produced by caffeine. Berkowitz and Spector reported that caffeine administration increased the concentration of serotonin and 5-hydroxyindole acetic acid (5HIAA) in brain.12) In this paper, the effects of caffeine contained in beverages, pure caffeine, and adenosine on brain 5-hydroxyindoles were investigated. MATERIALS AND METHODS Young male rats*1 of the Wistar strain (about 100g) were fed on the test diets for 1 or 4 d, ad libitum, and then decapitated on the morning at 10 : 00. Their whole brains were immediately removed, dissected, frozen on dry ice, and stored at -70 C until assayed. Blood was collected from the cervical wound, and serum samples were stored at -20 C until assayed. The composition of the 20% casein diet is shown in Table I. Test diets were prepared by the addition of 10% dry coffee,*2 10% decaffeinated coffee,*3 10% decaffeinated coffee plus f Supported by a research grant from the Ministry of Education, Science and Culture of Japan. * Present address for correspondence: Laboratory of Nutritional Biochemistry, School of Food and Nutritional Sciences, The University of Shizuoka, Shizuoka 422, Japan. *J Shizuoka Laboratory Animal Center, Hamamatsu, Japan. *2 Nescafe Gold blend, Nestle S. A. Vevey, Switzerland. *3 Decaffeinated Nescafe Gold blend, Nestle S. A. Vevey, Switzerland.

2 3282 H. Yokogoshi, S. Tani and N. Amano Table I. Composition of the Basal Diet Vitamin mixture (AIN-76á") produced by Ninon Nosan Kogyo K.K., Yokohama, Japan. Mineral mixture (AIN-76á") produced by Nihon Nosan Kogyo K.K., Yokohama, Japan. 0.3% caffeine,*4 6.6% black tea,*5 10% green tea,*6 10% oolong tea,*7 0.3% caffeine, 0.3% caffeine and adenosine,*8 and 0.3% adenosine to the basal diet. These beverages contained about 0.35% caffeine. The room temperature was kept at 24 Cwith a 12-hr cycle of light (between 08 : 00 and 20:00 hr) and dark. All rats were individually housed and were weighed daily during the experimental period. Tryptophan,13) serotonin,14) and 5HIAA14) were assayed fluorimetrically. Serum CAMPconcentrations were measured by radioimmunoassay using anti-succinyl CAMP serum.*9 The statistical significances of differences between values were evaluated by analysis of variance and Duncan's multiple range test.15) RESULTS Effects of caffeine-containing beverages on serum and brain levels of tryptophan and brain 5-hydroxyindoles in rats Changes in serum and brain levels of tryptophan and brain level of 5-hydroxyindoles caused after one day of feeding were the same as observed after 4 days of feeding of diets. Therefore in this paper, the results obtained with the rats fed on the experimental diets for one day were shown. The concentrations of serumand brain tryptophan are shown in Figs. 1A and B, respectively, and the directions of their changes (decrease or increase) in each group are opposite. When rats were fed on the coffee-supplemented diet, serum tryptophan levels decreased, but brain tryptophan increased significantly over that of rats fed on the control diet. On the decaffeinated-coffee-supplemented diet, the levels of tryptophan in serum and brain remained at the same level as those in the control groups. However supplementation with either decaffeinated coffee or caffeine decreased serum tryptophan and, in the opposite way, increased brain tryptophan significantly. Supplementation with other caffeine-containing beverages (black tea, green tea, and oolong tea) decreased serum tryptophan and increased brain tryptophan the same as with the coffee-supplemented diet. The concentrations of brain serotonin (Fig. 1C) and 5HIAA (Fig. ID) were reflected in the levels of brain tryptophan (Fig. IB), and the manners of their changes were same as those in brain tryptophan. That is, when rats were fed the caffeine-containing beverages (coffee, black tea, green tea, or oolong tea), brain serotonin and 5HIAAlevels increased significantly, but in the supplementation with decaffeinated coffee, the levels of brain 5-hydroxyindoles were not changed. The food intakes of rats fed on the caffeine-containing diet for Id, were decreased, but within the following a few days, the levels of food intake were recovered. Then we calculated the correlations between food intake (low or high) and brain serotonin levels during the experimental periods, but did not obtain a good correlation. Therefore, the quality of diet, that is dietary composition, rather than the levels of food intake affected the levels of brain 5- hydroxyindoles. Caffeine: 1,3,7-trimethylxanthine, Katayama Chemical Co., Ltd., Osaka, Japan. Freeze-dried instant tea, AGFCo., Ltd., Tokyo, Japan. Instant green tea, Koyamaen, Kyoto, Japan. Freeze-dried instant oolong tea, AGFCo., Ltd., Tokyo, Japan. Adenosine; Katayama Chemical Co., Ltd., Osaka, Japan. CAMP-(125I)-assay system; Amersham Japan Ltd., Tokyo, Japan.

3 Caffeine and Brain Serotonin 3283 Serum Tryptophan ( n moles/ ml) Brain Tryptophan ( n moles/g ) "a] I å e 20%Casein -J- ab 20%Casein -L c 10% Coffee cd 10% Coffee -I- b 10% Decaffeinated coffee ab 10% Decaffeinated coffee - - c 10% Decaffeinated coffee % Caffeine cd 10% Decaffeinated coffee + 0.3% Caffeine b 6.6% Black tea be 6.6% Black tea -I- b I I ab 10% Green tea C 10% Green tea 10%Oolong tea d 10%Oolong tea à"à"a 0.3% Caffeine -- cd 0i3%Caffeine -L-8 Brain serotonin ( ng/g) Brain 5-hydroxyindole acetic acid ( ng/g ) « à" ~*~ ' 5] I ' ""^ ' d 20%Casein - 20%Casein "" C 10% Coffee { 3 10% Coffee j- b 10% Decaffeinated coffee 4- C 10% Decaffeinated coffee - " c 10% Decaffeinated coffee + 0.3% Caffeine -L-3 10% Decaffeinated coffee + 0.3% Caffeine -j- b 6.6% Black tea 4-a 6.6% Black tea 4- ab 10% Green tea a % Green tea ^^ -» 10%Oolongtea " a 10%Oolongtea --J 0.3% Caffeine * 0. 3% Caffeine -J Fig. 1. Effects of Caffeine and Caffeine-containing Beverages on Serum Tryptophan (A), Brain Tryptophan (B), Serotonin (C), and 5-Hydroxyindole Acetic Acid (D). Vertical bars indicate standard errors. Different letters (a ~d) represent significant differences (p < 0.05). Effects of caffeine and/or adenosine on brain tryptophan, serotonin, 5HIAA, and serum CAMPin rats Whencaffeine was added to the casein diet, the levels of brain tryptophan, serotonin, and 5HIAA were significantly increased (Figs. 2A, B and C, respectively). Adenosine administration induced a significant increase of brain tryptophan, but the levels of brain serotonin were decreased significantly, and 5HIAAlevels

4 3284 H. Yokogoshi, S. Tani and N. Amano D 1] å o( BI ' aujsouapv % 'O + 9ui9^B3 %e*o "L- auisouapv % *0 + auia^eo % *o I L ' * i *I amsouapv % *0 y I amsouapv % *o ujaseo %02 -u!asb3 %03 I I "a u < < 1X I O t ^ V Ck - g 'S "S å 2 g u ' "( 6/611") muoiojas ujejg * (juj/a ouiu) dlajv^ mnjas "< ' ' ' ' ' I [u ' ' ' ' ' " ~ ro-- -^~^^ aujsouapy % 'O +å auia^eo % *0 -- aujsouspv % *0 + auia^eo % 'O aujsouapv % "0 -" 9U!SOU8PV% *0 auj8^e3 %e*o 8u!aiiBD % 'O u - UJ8SB3 %QZ -- LH9SB0 %0^ ( 6/S9 om u ) uendcndajj_ u Bjg ( 6/6u } ppe 3,;a3B aiopuiaxojpaq-g uiejg < I I! I 1 PQ e S ^ o <d es.1 ^ s s ^_> i s < E o g g -s 1 1 & I à"b 1 S 1 wg5 "å &1 s, a< 3 > s

were not changed. In the case of both caffeine and adenosine administration, brain tryptophan and 5HIAAlevels were increased like those in the caffeine-eating group.

5 were not changed. In the case of both caffeine and adenosine administration, brain tryptophan and 5HIAAlevels were increased like those in the caffeine-eating group. However, in brain serotonin concentrations, additional adenosine partially reduced the increase of brain serotonin caused by caffeine (Fig. 2B). The concentrations of serum CAMPwere significantly increased by the administration of caffeine or adenosine in the control diet (Fig. 2D). Administration of caffeine and adenosine together caused an additive increase of serum CAMP levels. DISCUSSION Caffeine is knownto increase serum cholesterol, microsomal mixed function oxidase activity, and ascorbic acid levels in tissues and urine.8'16'17) Caffeine-containing beverages also induce hypercholesterolemia, and enhance excretion of urinary ascorbic acid.8'17) However, the mechanisms of these phenomena are still unclear. On the other hand, caffeine is also known to elevate CAMPby an inhibition of the catabolism of CAMPby phosphodiesterase,9'10) and to prevent the release of brain serotonin or increase serotonin synthesis.12) In this paper, we have focused on whether caffeine-containing beverages affects brain serotonin levels. When rats were fed caffeinecontaining beverages (instant coffee, black tea, green tea, or oolong tea), brain levels of tryptophan, serotonin, and 5HIAAwere significantly increased (Fig. 1). Decaffeinated coffee did not increase brain 5-hydroxyindoles, but decaffeinated coffee with added caffeine increased the brain 5-hydroxyindoles over those of the control group. Weknowthat an average American consumes 2 to 3mg caffeine/kg of body weight daily,18) and many people consume more than 10 mg/kg/day from coffee, tea, or some soft drinks. In Japan, we also consumecaffeine mainly from green tea. In this paper, the experimental level of 10% of coffee in diet is equivalent to about 20 to 25 cups of coffee per day (because, if we assume that energy requirements are 40kcal (for loog of Caffeine and Brain Serotonin 3285 rat) and 2000kcal (for 50kg of subject), and 1 g of diet is about 4kcal, instant coffee intake is about 50g per subject. If we require 2g of instant coffee powder per one cup of coffee, 50 g of instant coffee powder corresponds to 25 cups of coffee). We usually consume caffeine from some kind of beverages (not only coffee). Therefore, the intake of caffeine-containing beverages may produce some behavioral effects. On the other hand, the hypothesis that the physiologically most important effect of caffeine involves competitive antagonism at extracellular adenosine receptors has received considerable attention.19) Here we showedthe hypothetical opposite actions between caffeine and adenosine on brain serotonin levels, in that adenosine administration induced a significant additional decrease of brain serotonin, adenosine supplementation and the partially prevented the increase of brain serotonin levels caused by caffeine. The levels of serum CAMPwere significantly increased by the addition of caffeine or adenosine to the control diet. This result supports the hypothesis that caffeine acts in vivo mainly by inhibiting the phosphodiesterase activity, and adenosine is a potent stimulator of adenylate cyclase in many tissues including brain. Therefore, it was considered that the addition of both caffeine and adenosine caused the additive increase of serum CAMPlevels. From our observations, it can be said that caffeine-containing beverages cause some significant neurochemical changes. The mechanism of the hypercholesterolemic action of caffeine is still unclear. Further extensive investigations are needed. REFERENCES 1) T. R. Dawber, W. B. Kannel and T. Gordon, New Engl. J. Med., 291, 871 (1974). 2) J. I. Mann and M. Thorogood. Lancet ii, 1975, ) C. H. Hennekens, M. E. Drolette, M. J. Jesse, J. E. Davies and G. B. Hutchison, 633 (1976). New Engl. J. Med., 294, 4) A. B. Nichols, C. Ravenscroft, D. E. Lamphier and

6 3286 H. Yokogoshi, S. Tani and N. Amano L. D. Ostrander, /. Am. Med. Ass., 236, 1948 (1976). 5) D. J. Naismith, P. A. Akinyanju and J. Yudkin, /. Nutr., 97, 375 (1969). 6) P. Akinyanju and J. Yudkin, Nature, 214, 426 (1967). 7) R. Fears, Br. J. Nutr., 39, 363 (1978). 8) H. Yokogoshi, S. Mochizuki, M. Takahata, S. Quazi and A. Yoshida, Nutr. Rep. Int., 28, 805 (1983). 9) R. W. Butcher and E. W. Sutherland, J. Biol. Chem., 237, 1244 (1962). 10) W. Y. Cheung, Biochemistry, 6, 1079 (1967). ll) B. B. Fredholm, Trends Pharmacol. ScL, 1, 129 (1980). 12) B. A. Berkowitz and S. Spector, Eur. J. Pharm., 16, 322 (1971). 13) W. D. DencklaandH. K. Dewey, J. Lab. Clin. Med., 69, 160 (1967). 14) J. H. Thompson, Ch. A. Spezia Experientia, 26, 327 (1970). and M. Agnulo, 15) D. B. Duncan, Biometrics, 13, 164 (1957). 16) N. Kato and A. Yoshida, Agric. Biol. Chern., 43, 191 (1979). 17) D. J. Naismith, P. A. Akinyanju and J. Yudkin, J. Nutr., 97, 375 (1969). 18) FASEB: SCOGS-89, Federation of the American Society for Experimental Biology, Bethesda, Maryland. 19) S. H. Snyder,J.J.Katims,Z.Annau, R. BransandJ. W. Daly, Proc. Natl. Acad. Sci. U.S.A., 78, 3260 (1981).

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