Dose-response decrease in plasma tryptophan and in brain tryptophan and serotonin after tryptophan-free amino acid mixtures in rats

Life Sciences, Vol. 44, pp. 971-976 Printed in the U.S.A.

Pergamon Press

DOSE-RESPONSE DECREASE IN PLASMA TRYPTOPHAN AND IN BRAIN TRYPTOPHAN AND SEROTONIN AFTER TRYPTOPHAN-FREE AMINO ACID MIXTURES IN RATS

Egidio A. MoJa

(I), Piervitto Cipolla (2), naniele Castoldi and Odoardo Tofanetti (2)

(2)

(I) Chair of Psychobiology, Medical School, University of Modena, 41100 Modena, Italy; (2) Research Department, Boehringer Biochemia Robin, Milan, Italy. (Received in final form February 6, 1989) Summary Rats fasted 15 hours were treated p.o. with increasing amounts (660 and 1320 mg/kg body weight) of a mixture containing a fixed proportion of seven essential amino acids (L-phenylalanine 13.6%, L-leucine 6.0%, L-isoleucine 12.1%, L-methionine 12.1%, L-lysine 30.3%, L-threonine I0.6%, L-valine 15.2%) and lacking tryptophan. The mixtures produced a dose-response decrease of free (by 34% after the lower dose and by 58% after the higher dose of the mixture) and total (by !0 and 31%) plasma tryptophan and of brain tryptophan (by 38 and 65%), serotonin (by 17 and 41%) and 5-hydroxyindole acetic acid (by 21 and 49%). The mechanisms of these changes are discussed. A specific and non toxic method to deplete brain serotonln was described in rats by Gessa et al. (I). These authors showed that the acute administration of an amino acid mixture lacking tryptophan (TRY) caused the rapid fall of plasma TRY. This effect was associated with a marked decrease of brain TRY, serotonin (5-HT) and 5-hydroxyindole acetic acid (5-HIAA). The decrease of TRY was interpreted as due to the rapid removal of endogenous TRY from tissues to allow protein synthesis induced by the amino acid diet (I, 2; see also Discussion). Due to its simplicity and lack of toxicity, the technique of acutely administering TRY-free mixtures was used in the study of a variety of behaviors in which 5-HT was thought to play a role. In particular, it was shown to induce modifications of sexual activity (3, 4) and sleep (5) in laboratory animals and of sleep (6), mood (7) and food intake (8) in humans. In all these studies single doses of TRY-free mixtures were used. Recently we showed in humans that the decrease of blood TRY after TRY-free amino acid diets is proportional to the amount of amino acids administered

(9). Here

we

report

that

also in rats the decrease

0024-3205/89 $3.00 + .00 Copyright (c) 1989 Pergamon Press plc

of blood TRY

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after TRY-free mixtures is proportional to the amount of administered amino acids and that this decrease is paralleled by a decrease in brain TRY, 5-HT and 5-HIAA. Materials

and Methods

Experiments were carried out with male Wistar rats weighing 230-250 g. They were housed four to a cage in a room with a ]2 hrs light (08:00-20:00) 12 hrs dark cycle and an ambient temperature of 20 C. The animals had free access to water and food; this was removed 15 hrs before treatments, which were given at 9:00 A.M. Two hours after the treatments, the rats were anesthetized with ether and blood was collected from abdominal aorta and immediately processed for determination of free and total plasma TRY. After the blood collection, animals were rapidly sacrificed, their brains were quickly removed, frozen in liquid nitrogen and stored at -70 C until assayed. Experimental treatments. Four different groups of seven rats were treated per garage with distilled water (I0 ml/kg body weight) or with one of three amino acid mixtures in distilled water (I0 ml/kg b.w.). The first group received distilled water. The second group received the following TRY-free mixture (IT-): L-phenylalanine 90 mg/kg b.w., L-leucine 40 mg/kg b.w. , L-isoleucine 80 mg/kg b.w., L-methionine 80 mg/kg b.w., L-lysine 200 mg/kg b.w., L-threonine 70 mg/kg b.w., L-valine I00 mg/kg b.w. The third group received a TRY-free mixture (2T-) which contained a double amount of the same amino acids: L-phenylalanine 180 mg/kg b.w., L-leucine 80 mg/kg b.w., L-isoleucine 160 mg/kg b.w., L-methionine 160 mg/kg b.w., L-lysine 400 mg/kg b.w., L-threonine 140 mg/kg b.w., L-valine 200 mg/kg b.w. The fourth group received a mixture (IT+) which had the same composition of IT- but contained L-TRY 27 mg/kg b.w. The same eight essential amino acids were recently used as an experimental mixture in humans (q). In IT+, each amino acid was given at a dose corresponding to one quarter the amount present in the diet which the animal normally consumed in 24 hrs (I). Chemical @nalysis. Immediately after each blood withdrawal, free and total plasma TRY were measured by HPLC method of Krstulovic et al. (I0). Free plasma TRY was separated from bound TRY by centrifugation through Amicon Centrifree Micropartltion System. Brain TRY, 5-qT and 5-qlAA were measured by HPLC method of Mefford

(11). and data analysis. On each of the five quantified les, a on--~-way analysis of variance was applied and multiple comparisons were performed using the method of Scheffe' (]2). Results Effects

of various

treatments

are reported

in Table I.

Statistical analysis indicated an highly significant effect of the treatments on total plasma TRY (F=7.23; d.f.=3; p<0.O05), free plasma TRY (F=18.05; d.f.=~; p=O.O001), brain TRY (F=55.19; d.f.=3; p=O.0001), brain 5-HT (F=14.90; d.f.=3; p=0.0001) and brain 5-HIAA (F=27.60; p=O.O001). Multiple comparisons for each parameter gave the following results: I) for the variable "total plasma TRY", the Scheffe's test indicated a significant difference

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973

(p<0.05) between IT+ and 2T-; 2~ for the variable "free plasma TRY", there was a significant difference between IT+ and IT-, between IT+ and 2Tand between water and 2T-; 3) for the variables "brain TRY" and "brain 5-HIAA", the test indicated that all the comparisons were significant but the one between water and IT+; 4) for the variable "brain 5-HT", there was a significant difference between 2T- and each other treatment. TABLE 1 Changes in plasma TRY and in brain TRY, 5-HT and 5-HIAA induced by the administration of different amounts of a tryptophan-free amino acid mixture. Treatments : Total plasma TRY (mcg/ml) Free plasma TRY (mcg/ml) Brain TRY (mcg/g wet weight) Brain 5-HT (mcg/g wet weight) Brain 5-H!AA (mcg/g wet weight)

water

IT-

16.13_+2.04 14.50+4.26

2T-

1T+

11.20+2.11

18.65+3.32

2.85_+0.42

1.88+0.45

1.19+0.32

4.06+1.39

4.16_+0.36

2.58+0.47

1.44+_0.31

4.14+0.66

0.58.+0.0q

0.4~_+0.05

0.34_+0.08

0.54+-0.05

0.33+0.04

0.26+-0.03

0.17_+0.02

0.33_+0.05

Rats fasting 15 hours were given by gavage different amino acid mixtures in distilled water (I0 ml/kg body weight) and were killed 2 hours after treatments. Each value is the mean + s.d. of seven animals. IT- = amino acid mixture devoid of TRY (see Methods); 2T- = the same mixture as IT- but twice its amount; IT+ = the same composition as IT- but containing TRY.

Discussion. Statistical analysis and inspection of Table I suggest that the TRY-free mixtures caused a decrease in each of the five parameters studied which was proportional to the amount of amino acids administered. The IT+ diet caused an increase in free and total plasma TRY but no change in brain TRY, 5-HT and 5-HIAA. TRY, unique among natural amino acids, is present in plasma in a free pool and in an albumin-bound pool (13). Our data show that after IT- and 2T- free TRY was reduced by 34.0 and 58.2 per cent respectively; total plasma TRY was reduced by ]0.1 and 30.6 per cent respectively. Therefore the ratio of free to total TRY decreased from 17.7% (control values) to 12.9% (after IT-) and to 10.6% (after 2T-). In fed rats the ratio of free to total TRY is around 10% (20). In fasted animals the ratio of free to total TRY is more elevated because starvation can increase free fatty acids (NEFA) (20) and because NEFA can displace TRY from its binding sites in plasma (13). In agreement with these data, in control animals we found an elevated level of the ratio of free to total TRY (17.7%). The possible mechanism to explain the decrease of blood TRY after TRY-free mixtures was investigated by Gessa et al.

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(I, 2). They concluded that because TRY-free mixtures, like any mixture of essential amino acids, can promote synthesis of new proteins, endogenous TRY is removed from tissues and blood to allow new protein synthesis. In agreement with this hypothesis, it was shown that the protein synthesis inhibitor cycloheximlde blocked the decrease of blood TRY after a TRY-free diet (2). The data that we reported are consistent with this hypothesis because, on its basis, one would expect a dose-response increase of synthesis of new protein after the administration of increasing amount of amino acids and, therefore, a dose-response decrease of endogenous TRY. The decrease of the ratio of free to total TRY after the ITand the 2T- mixtures suggests that the induced protein synthesis utilized more free than bound TRY. There is evidence, however, that TRY-free mixtures are less efficacious than complete amino acid mixtures in inducing new protein synthesis: both Wunner et el. (14) and Sidransky et el. (15) have found a low rate of polysome formation and a low incorporation of amino acids from the liver of rodents previously fasted then acutely fed a TRY-free mixture. The IT+ mixture caused an increase in free (42.5%) and total plasma TRY (15.6%). This increase was expected because the diet provided new TRY and because there is no feed-back mechanism to maintain plasma TRY level within narrow ranges after consumption of meals of varying composition in TRY

(1~, 17). The problem of how brain TRY content is controlled has been an area of debate. Fernstrom and Wurtman (18) suggested that in physiological conditions brain TRY is controlled by the ratio of the concentration of total plasma TRY to that of other neutral amino acids (i.e., tyrosine, phenylalanine, leuclne, isoleucine and reline) that compete with TRY for the same carrier system that transports them across the blood brain barrier. In contrast with this suggestion, Knott and Curzon (Ig), Tagliamonte et el. (20) and Biggio et al. (21) concluded that free plasma TRY level is a better measure for TRY available for transport into brain. In the present experiment we found that brain TRY was reduced by 37.g per cent after IT- and by 65.4 per cent after 2T-. As it was discussed in the previous paragraph, IT- and 2T- reduced free plasma TRY by 34.0 and 58.2 per cent respectively and total plasma TRY by I0.I and 30.6 per cent respectively. These data suggest that in our experimental conditions brain TRY uptake and brain TRY levels correlated better with the size of the free TRY pool than with the size of the total TRY pool. The IT+ mixture ~ncreased free and total plasma TRY by 42.5 and 15.6 per cent respectively but did not change brain TRY. It is possible to explain this discrepancy observing that the IT+ diet added new TRY but added also a significant amount of competing amino acids that blocked the brain uptake. After IT- and 2T- mixtures, levels of brain 5-HT were reduced by 17.2 and 41.3 per cent respectively; brain 5-HIAA levels were reduced by 21.2 and 48.5 per cent respectively. The IT+ mixture did not change brain 5-HT nor brain 5-HIAA levels. It is well established that TRY availability in the brain limits the synthesis of 5-HT and of its metabolite, 5-HIAA (22). The rate limiting step in 5-HT synthesis is considered to be the hydroxylation of TRY by TRY hydroxylase (22). In presence of a reduction of brain TRY, the decrease of brain 5-HT and 5-HIAA was therefore expected. It must be noted, however, that the decreases

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of brain TRY (37.9 per cent after IT- and 65.4 per cent after 2T-, as discussed in the previous paragraph) were more pronounced than the decreases of brain 5-HT. This difference is consistent with the data of Neckers et al. (23) that found that treatments that decreased brain TRY levels also increased the activity (Vmax) of TRY hydroxylase as measured in vlvo. In the present paper we showed that increasing amounts of TRY-free mixtures induced a dose-response decrease of blood TRY and of brain TRY and 5-HT. Recently we found that the administration of increasing amounts of TRY-free mixtures caused a dose-related decrease of blood TRY in man (9). The above evidence strongly suggests that also in man increasing amounts of TRY-free mixtures cause a dose-relate decrease of brain TRY and 5-HT. As we mentioned in the Introduction, the acute administration of mixtures devoid of TRY was used to manipulate human and animal behavior. In particular, such diets increased stage 4 sleep (6) and produced a rapid lowering of mood (7) in humans and enhanced sexual activity (3, 4) and decreased REM sleep in rats (5). These behavioral changes were interpreted as due to the decrease in the concentration of brain TRY and 5-HT. All these studies utilized only single doses of m~xtures devoid of TRY. Our results might imply the possibility to modulate the behavioral changes reported after TRY-free mixtures in humans and in animals according to dose-response curves. Acknowledgements ~

thank Dr. A. Petroccione

for help

in statistical

analysis.

References I. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13.

14.

G.L. GESSA, G. BIGGIO,

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(1966).

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15. H. SIDRANSKY, D.S.R. SARMA, M. BONGIORNO and E. WERNEY, J. Biol. Chem., 2 4 3 : 1 1 2 3 - 1 1 3 2 (1968). 16. J.D. FERNSTRO , ~ . J . ~TMAN, B. HAMMARSTROM-WIKLUND, W.M. RAND, H.N. MUNRO and C.S. DAVIDSON, Am. J. Clin. Nutr., 32: 1912-1922 (1979). 17. R.J. WURTMAN, F. HEFTI and E. MELAMED, Pharmacol. Rev., 32: 315-335 (1981). 18. J.D. FERNSTROM and R.J. WI~TMAN, Science, 1 7 8 : 4 1 4 - 4 1 6 (1972). 19. P.J. KNOTT and G. CURZON, Nature, 239: 452---~ (1972). 20. A. TAGLIAMONTE, G. BIGGIO, L. VARGIU and G.L. GESSA. Life Sci., 1 2 : 2 7 7 - 2 8 7 (1973). 21. G. BIG~-I~, F. FADDA, P. FANNI, A. TAGLIAMONTE and G.L.GESSA, Life Sci., 1 4 : 1 3 2 1 - 1 3 2 9 (1974). 22. E. JEQUIER, D.S. ROBINSON, W. LOVENBER and A. SJOERDSMA, Biochem. Pharmacol., 1 8 : I 0 7 1 - I 0 ~ 1 (1969). 23. L.M. NECKERS, G. BIGGI~, E. MOJA AND J.L. MEEK, J. Pharmacol. Exp. Ther., 2 0 1 : 1 1 0 - 1 1 6 (1977).