Naunyn-Schmiedeberg's Arch. Pharmacol. 314, 47-50 (1980) Naunyn-Schmiedeberg's

Archives of Pharmacology �9 by Springer-Verlag 1980

Ethanol-Induced Increase in Brain Concentrations of Administered Neutral Amino Acids

Tomas Eriksson and Arvid Carlsson

Department of Pharmacology, University of G6teborg, Box 33031, S-400 33 G6teborg, Sweden

Summary. Ethanol 2 g kg-1 i.p. to rat increased the concentrations in the brain of administered large neutral amino acids (tyrosine, tryptophan, 5-hydroxy- tryptophan and c~-methyldopa). We have previously found a similar effect of ethanol on administered L- Dopa, resulting in increased brain/plasma ratios of dopa. Since large neutral amino acids are known to compete with each other for the carrier-mediated transport into the brain we suggest that the increased concentrations of the administered amino acids in the brain are at least p a r t y the consequence of the ethanol- induced decrease in plasma amino acids observed previously.

Key words: Ethanol - Blood-brain barrier - Carrier transport - Tyrosine - Tryptophan - 5-Hydroxy- tryptophan - e-Methyldopa.

Introduction

The flux of circulating substances through the blood- brain barrier occurs either through the lipid face or via a carrier mechanism (Oldendorf 1975). Recently the existence of eight different and independent carrier systems have been demonstrated (Pardridge and Oldendorf 1977). One of these is a saturable carrier system for large neutral amino acids. Thus tyrosine, tryptophan, phenylalanine, leucine, isoleucine, valine and also exogenously administered L-Dopa are assumed to be transported into the brain by the same system (Oldendorf 1975) and to compete with each other for its carrier. The relation between the levels of these plasma amino acids rather than the actual concentration of a certain amino acid seems to determine how much of each of the different amino acids that will be transport-

Send offprint requests to Tomas Eriksson at the above address

ed into the brain (Fernstrom and Wurtman 1972; Wurtman and Fernstrom 1976).

Alterations in the total plasma amino acid pool might thus be expected to influence the transport into the brain of exogenously administered amino acids. The concentration of amino acids in plasma varies considerably, depending upon food consumption (Fernstrom and Wurtman 1971 ; Cohen and Wurtman 1976) but also on a daily rhythm independent of food intake (Wurtman et al. 1968).

We have recently demonstrated that acutely admin- istered ethanol causes a rapid decrease in almost every amino acid occurring in plasma in rat (Eriksson et al. 1980) and in man (to be published).

In a preliminary report (Eriksson et al. 1979) we have shown that ethanol increases the passage through the blood-brain barrier of exogenously administered n- Dopa. This increased passage was suggested to be at least partly due to a decrease in the plasma amino acids competing with L-Dopa for the transport into the brain. The present study was undertaken to investigate whether this effect of ethanol involves other neutral amino acids as well.

Methods

Male Sprague Dawley rats (about 200 g) were purchased from Anticimex, Sollentuna, Sweden. Before use the animals were housed for at least one week in a room maintained on a 14/10 h light/dark cycle. They had free access to food and water.

Ethanol (20 % w/v) 2 g kg 1 or an equivalent volume of saline was injected intraperitoneally. Twenty minutes later all rats were injected intraperitoneally with 100 mg kg- 1 of tyrosine, tryptophan, c~-methyldopa or 5-hydroxytryptophan (5-HTP). For solubility re- asons the tyrosine was administered in the form of its ethyl ester in an amount equivalent to 100 mg kg- 1 (I 36 mg kg- t). The ester binding is split very quickly in the organism.

Forty minutes after the last injection the rats were killed by decapitation. About 5 ml blood was collected in a tube containing 0.5 ml of a 1% EDTA solution. The brains were immediately taken out and frozen on dry ice. The brains were homogenized and deproteinized and the plasma samples deproteinized (Bertler et al.

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48 Naunyn-Schmiedeberg's Arch. Pharmacol. 314 (1980)

1958). The brains and the plasma samples were purified on a strong cation exchange column (Dowex 50 W • 4) (Atack and Magnusson 1978; Kehr et al. t972). Spectrophotofluorimetric analyses of noradrenaline (Bertler et al. 1958), tyros• Waalkes and Udenfriend 1957), tryptophan (B~dard et al. 1972), 5-HTP, serotonin (5-HT), 5- HIAA (Atack and Lindqvist 1973), c~-methyldopa (Kehr et al. 1972) and c~-methyldopamine (Carlsson and Lindqvist 1962) were carried out.

Statistical significances were assessed by Student's t-test.

Results

Table 1 shows the effect of ethanol on the brain and plasma concentrations of tryptophan, 5-HT, 5-HIAA and tyrosine, respectively, after an intraperitoneal injection of tryptophan. Tryptophan was significantly increased in the brains of the ethanol-treated rats while the concentrations of 5-HT and 5-HIAA did not differ between the groups. There was no significant change in

the concentration of plasma tryptophan or 5-HT in the ethanol group compared to the controls but 5-HIAA in plasma showed a significant 29 ~ decrease. Tyros• in plasma was decreased in the ethanol group.

After the e-methyldopa injection both a-methyl- dopa and ~-methyldopamine but also tyrosine in the brain were increased by ethanol (Table 2). In this case there was also a significant 24 ~ increase of plasma e-methyldopa in the ethanol group. Tryptophan in plasma was decreased in the ethanol group.

Tyros• but not dopamine or noradrenaline was increased in the brain by ethanol after a tyrosine injection (Table 3). Tyros• and noradrenaline in plasma were not altered by ethanol in this experiment. Tryptophan in plasma was decreased to 83 ~ in the ethanol group but this decrease was not significant.

The levels of 5-HTP and 5-HIAA in the brain were increased by ethanol after an injection of 5-HTP

Table 1. Effect of ethanol on rat brain and plasma concentrations of tryptophan, 5-HT, 5-HIAA and tyrosine after the administration of tryptophan. Ethanol 2 g kg- 1 (20 ~ w/v) or an equivalent volume of saline to the control rats was given intraperitoneally followed 20 rain later by tryptophan 100 mg kg-1 to all rats. The animals were killed 40 min after the last injection. Shown are the means _+ S.E.M. in nmol g-1. Statistical significances were calculated by Student's t-test. (n = 6)

Tryptophan 5-HT 5-HIAA Tyrosine

Brain Controls 145 _+ 13.4 1.7 +_ 0.10 0.81 • 0.055 67.9 _+ 4.64 Ethanol treated 195 +_ 9.1 1.7 _+ 0.07 0.81 +_ 0.017 60.7 + 2.98 Ethanol treated

~o of controls 134 98 97 90 P < 0.025 N.S. N,S. N.S.

Plasma Controls 368 _+ 26.5 1.9 _+ 0.18 1.7 • 0.021 75.6 • 3.09 Ethanol treated 419 + 13.4 1.5 + 0.19 1.2 + 0.01 56.8 + 2.70 Ethanol treated

of controls 114 81 71 76 P < N.S. N.S. 0.025 0.005

Table 2. Effect of ethanol on rat brain and plasma concentrations of tyrosine, e-methyldopa, c~-methyldopamine and tryptophan after the administration of c~-methyldopa. Ethanol 2 g kg- 1 (20 ~ w/v) or an equivalent volume of saline to the control rats was given intraperitoneally followed 20 min later by ~-methyldopa 100 mg kg- 1 to all rats. The animals were kilted 40 mN after the last injection. Shown are the means _+ S.E.M. in nmol g - t. Statistical significances were calculated by Student's t-test. (n = 6)

c~-Methyldopa c~-Methyldopamine Tryptophan Tyrosine

Brain Controls 57.8 + 3.46 1~6 +0.06 19_+ 0.4 60.7_+ 1.93 Ethanol treated 97.5 _+ 9.28 2.0 _+ 0.10 21 +_ 0.7 75.6 +_ 1.60 Ethanol treated

of controls 169 127 108 125 p < 0.005 0.005 0.05 0.001

Plasma Controls 251.4 +_ 13.87 0.14 + 0.024 86 • 1.7 69.5 • 2.20 Ethanol treated 311.5 • 12.21 0.17 + 0.042 74 • 3.6 77.3 • 4.14 Ethanol treated

of controls 124 121 86 111 P < 0.01 N.S. 0.025 N.S.

T. Eriksson and A. Carlsson: Ethanol and Brain Uptake of Amino Acids 49

Table 3. Effect of ethanol on rat brain and plasma concentrations of tyrosine, dopamine, noradrenaline and tryptophan after the administration of tyrosine. Ethanol 2 g kg- * (20 ~ w/v) or an equivalent volume of saline to the control rats was given intraperitoneally followed 20 min later by tyrosine ethyl ester 136 mg kg- 1 to all rats. The animals were killed 40 rain after the last injection. Shown are the means • S.E.M. in nmol g- 1. Statistical significance were calculated by Student's t-test. (n = 6)

Tyros• Dopamine Noradrenaline Tryptophan

Bra#1 Controls 234• 8.5 3.8k0AI 2.4 _+0.09 19_+0.9 Ethanol treated 303 + 2L 1 3.7 • 0.07 2.4 • 0.08 18 • 0.6 Ethanol treated

of controls 130 97 99 94 P < 0.025 N.S. N.S. N.S.

Plasma Controls 201 • 8.5 Not detectable 0.088 _+ 0.0177 74 + 3.8 Ethanol treated 227 i 8.9 Not detectable 0.095 + 0.0059 62 +_ 4.2 Ethanol treated

% of controls 113 107 83 P< N.S. N.S. N.S.

Table 4. Effect of ethanol on rat brain concentrations of 5-HTP, 5-HT, 5-HIAA and tryptophan after the administration of 5-HTP. Ethanol 2 g kg ~ (20 ~o w/v) or an equivalent volume of saline to the control rats was given intraperitoneaiiy followed 20 min later by 5-HTP 100 mg kg to all rats. The animals were killed 40 min after the last injection. Shown are the means • S.E.M. in nmol g-1. Statistical significances were calculated by Student's t-test (n - 6)

5 HTP 5-HT 5-HIAA Tryptopban

Controls 24.1 • 2.81 3.9 _+ 0.20 14.9 • 1.19 33 +_ 1.3 Ethanol treated 35.0 • 3.50 4.5 • 0.28 19.1 • 0.89 37 _+ 2.1 Ethanol treated

% of controls 146 115 128 112 P< 0.05 N.S. 0.025 N.S.

(Table 4), 5 -HT too was raised to 115 % c o m p a r e d to the cont ro ls b u t this difference was no t significant. B lood p l a sma was no t ana lysed in this exper iment .

The initial values for the ana lysed animals and amino acids vary f rom one ba tch o f rats to another . This has to be taken into account when c o m p a r i n g the different exper iments with each other.

Discussion

In a p rev ious s tudy (Er iksson et aI. 1979) we in- ves t iga ted in detai l the effects o f e thano l on b ra in concen t ra t ions o f d o p a and d o p a m i n e after adminis-

t r a t ion of L-Dopa . We found tha t the concen t ra t ions of bo th d o p a and d o p a m i n e in b ra in were increased by e thanol . This increase was s ignif icant a l r eady 10 min after the L - D o p a inject ion and r ema ined for at least 80 min. The b ra in d o p a / p l a s m a d o p a ra t ios were also s ignif icant ly increased ind ica t ing an effect o f e thano l on the d i s t r ibu t ion o f d o p a between p l a s m a and brain. In every exper iment in the present s tudy we have found tha t e thanol causes an increased concen t r a t i on o f the admin i s te red amino acid in the b ra in c o m p a r e d to

p lasma, indica t ing a s imilar change in d i s t r ibu t ion as in the exper iments with L-Dopa.

This effect o f e thanol on the concen t ra t ion adminis- tered amino acids in the bra in might be o f clinical impor tance . L -Dopa is widely used in Pa rk inson ' s disease. ~ - M e t h y l d o p a is a wel l -known ant• sive d rug and its effect is cons idered to be med ia t ed via central mechanisms (Henning 1975). T r y p t o p h a n ap- pears to be effective in the t r ea tment o f some forms o f menta l depression. T r y p t o p h a n , 5 -HTP and tyrosine are all p recursors o f neuro t ransmi t t e r s involved in i m p o r t a n t cerebral functions. The poss ib le in terac t ion be tween e thanol and these amino acids should thus be considered. This view is suppo r t ed by the f inding tha t an acute oral dose of e thanol to m a n causes a decrease in mos t p l a s m a amino acids (see In t roduc t ion) , com- b ined with the fact tha t large neut ra l amino acids compete with each o ther for the t r anspo r t into the brain.

The f inding tha t 5 - H I A A in p l a s m a decreases in the e thano l - t r ea t ed g roup in the exper iment in which t r y p t o p h a n was given (Table 1) c anno t be expla ined fi 'om the d a t a avai lable thus far.

50 Naunyn-Schmiedeberg's Arch. Pharmacol. 314 (1980)

The ethanol-induced increase in brain tyrosine in the experiment in which the rats were given ~- methyldopa (Table 2) could be due to either a facili- tated transport of tyrosine into the brain or a decrease in tyrosine metabolism. Mandel and Aunis (1974) reported that noradrenaline inhibits the enzyme ty- rosine aminotransferase, which catalyzes the major pathway of tyrosine metabolism. Other catechols, such as e.g. c~-methyldopa or its amine metabolites might exert a similar effect on this enzyme, the inhibition being more pronounced when more 0~-methyldopa or ~- methyldopamine is present in the brain, as observed after ethanol treatment. This possible inhibiting effect on the enzyme might as well explain the slight, not significant, increase in plasma tyrosine in the ethanol- treated group in this experiment. The significant in- crease in plasma c~-methyldopa in the same experiment could not be explained at present. In a similar experi- ment (Eriksson et al. 1979) in which the amino acid L- Dopa was given together with ethanol we noticed a transient increase in plasma L-Dopa concentration compared to animals treated with L-Dopa alone. The possible connection between these effects deserves further study.

Acknowledgements. This work was supported by the Swedish Medical Research Council (project No. 155) and by the Faculty of Medicine, University of G6teborg. We wish to thank Mrs. Birgitta Holmgren and Mrs. Gerd Leonsson for their competent technical assistance.

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Received February 8/Accepted July 8, 1980