PROGRESS

IN ENDOCRINOLOGY

AND METABOLISM

Effects of the Diet on Brain Neurotransmitters John D. Fernstrom The synthesis of neurotransmitters in mammalian brain responds rapidly to changes in precursor availability. Serotonin synthesis depends largely on the brain concentrations of l-tryptophan, its precursor amino acid. This relationship appears to be physiologic: when brain tryptophan levels vary because of insulin secretion or meal ingestion, corresponding alterations occur in the rate of serotonin formation. The ability of any food to modify brain tryptophan (and serotonin) depends on how its ingestion changes the serum concentrations of not only tryptophan, but also several other large neutral amino acids that compete with tryptophan for uptake into the brain. Such precursorinduced changes in brain serotonin appear to be functionally important: animals having a reduced level of brain serotonin (caused by the chronic ingestion of a naturally tryptophan-poor diet, such as corn) demonstrate a heightened sensitivity to painful stimuli; this pain sensitivity can be acutely restored to normal values by a single injection of L-tryptophan, which rapidly elevates brain serotonin. The synthesis of catecholamines (e.g., dopamine, norepinephrine) in the brain also varies with the availability of the precursor amino acid L-tyrosine. Single injectionsof this amino acid increase brain tyrosine levels and accelerate brain cate-

chol synthesis, while injections of a competing neutral amino acid (e.g., leucine, tryptophan) reduce brain tyrosine and its rate of conversion to dopa. The rate of catecholamine synthesis, however, appears to be influenced less by precursor levels than is serotonin formation: tymsine hydroxylase, which catalyzes the ratelimiting step in catecholamine synthesis, responds strongly to end-product inhibition and to other controls that reflect variations in neuronal activity. The synthesis of acetylcholine in brain responds to substrate (choline) availability much like serotonin synthesis. Short-term alterations in brain choline levels are mirrored by similar changes in brain acetylcholine concentration. Variations in the daily dietary intake of choline also modify brain choline and acetylcholine. The relationship between choline availability and acetylchoiine synthesis has already found a clinical application: choline has been used successfully in the treatment of tardive dyskinesia, a disorder of the central nervous system thought to reflect a deficiency in cholinergic transmission. These relationships between precursor availability from the periphery and brain neurotransmitter synthesis may ultimately provide the brain with information about peripheral metabolic state.

N

EUROPHARMACOLOGISTS HAVE FOR YEARS NOTED correlations between the behavioral and physiologic effects of drugs, and the changes these agents produce in the brain concentrations of particular neurotransmitters. When a drug both changes the level of a transmitter and alters a

From the Laboratory of Brain and Metabolism, Massachusetts Institute of Technology, Cambridge, Receivedforpublic&on April 27, 1976.

Department Mass.

of Nutrition

and

Food

Science,

Supported in part by grants from the National Science Foundation, the Alfred P. Sloan Foundation, the Grant Foundation. and (to Dr. R. J. Wurtman) the John A. Hartford Foundation, the National Aeronautics and Space Administration (NGR 22009672). and the National institute of Arthritis and Metabolic Diseases (AM 14228). Reprint reyuesrs should be addressed fo Dr. John D. Fernstrom, Laborarory of Brain and Merabolism, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Mass. 02139. $a1976 by Grune dt Stratton, inc. Metabolism, Vol. 26, No. 2 (February), 1977

207

208

JOHN

D. FERNSTROM

behavior or physiologic function, the suggestion has been, in general, that the neurons containing the transmitter contribute somehow to that brain function. For example, the injection of p-chlorophenylalanine reduces brain serotonin synthesis, lowers brain serotonin levels, and disrupts normal sleep patterns;‘,* these findings have been interpreted as supporting a role for serotonergic neurons in the control of sleep. Similarly, the effects of cy-methyl-p-tyrosinei.e., suppression of brain catecholamine synthesis and levels and stimulation of prolactin secretion-have been taken as evidence that catecholaminergic (probably dopaminergic) neurons normally inhibit the release of the pituitary hormone.3 All such studies assume that if more or less transmitter is present within the brain, it is because more or less is contained within nerve endings to be released when the neuron depolarizes. Such variations in the amounts of neurotransmitter that are released will, presumably, cause parallel changes in the amount of information flowing across the synapse (i.e., in the number of postsynaptic receptors that are activated when the neuron fires). Ultimately, such alterations should modify the net impulse traffic through brain networks containing the affected neurons, and should presumably be reflected as changes in the behavioral or physiologic brain outputs under their influence. These assumptions underlie drug therapy in the following clinical situations: (1) the use of L-dopa in the treatment of Parkinson’s disease; (2) the administration of choline to improve the symptoms of tardive dyskinesiat (3) the use of monoamine oxidase inhibitors in the treatment of depression. If this series of assumptions is correct, it seems reasonable to anticipate that other treatments influencing brain neurotransmitter synthesis and levels may also modify the amount of transmitter released and, ultimately, alter brain outputs linked to neurons utilizing the affected transmitter. One such “treatment” occurs naturally: each time a person consumes food, changes occur in the availability to the brain of several compounds that are precursors for the neurotransmitters. As will be discussed in this review, eating causes large fluctuations in the uptake of tryptophan into the brain; such fluctuations rapidly modify brain tryptophan concentrations and, thereby, the rate at which the essential amino acid is converted to the neurotransmitter serotonin. A similar but less responsive relationship holds between tyrosine availability and catecholamine synthesis. In addition, brain acetylcholine levels appear to be as dependent on brain choline as serotonin concentrations are on brain tryptophan. The functional significance of these (and perhaps other, as yet undefined) relationships between precursors in serum and brain and brain neurotransmitters may be to link peripheral metabolic state to the activity of particular neurons and, ultimately, to brain functions. One might anticipate that the particular functions most likely to be coupled to serum precursor levels would be those related to eating; e.g., whether to eat or sleep, how much total protein or how many calories to consume,5 and when to secrete hormones that are under brain control, but which have major metabolic effects in the body. Already, administration of L-tryptophan has been shown to modify sleep,6 pituitary prolactin secretion,’ and pain sensitivity8-all brain outputs thought to be influenced by serotonergic neurons. Studies are currently in progress to de-

BRAIN

209

NEUROTRANSMITTERS

termine whether the diet, by changing brain tryptophan and serotonin, normally affects these particular brain outputs; studies will also assess whether the dietary manipulation of other transmitters can similarly modify behaviors and physiologic functions. SEROTONIN

The Eflects of Single Meals on Brain Tryptophan and Serotonin The rate at which the mammalian brain synthesizes serotonin (5HT) normally varies with the availability of the precursor amino acid, L-tryptophan. This relationship derives from two biochemical properties of the 5HT synthetic pathway (Fig. 1): (1) Tryptophan hydroxylase is the enzyme that catalyzes the initial reaction in the formation of serotonin; its K, (50 PM) is within the range of tryptophan concentrations normally found in the brain (20-70 PU.M).~-” Consequently, the enzyme should be unsaturated at normal brain tryptophan levels, and the formation of Shydroxytryptophan (SHTP), the intermediate in the pathway, probably proceeds at rates that are conspicuously submaximal. As a result, a rise or fall in brain tryptophan level should either increase or decrease the degree of saturation of the enzyme and elicit a parallel change in the rate of tryptophan hydroxylation. (2) Tryptophan hydroxylase catalyzes the rate-limiting step in serotonin formation. The conversion of SHTP to 5HT is mediated by the ubiquitous enzyme, aromatic L-amino acid decarboxylase,12 and appears to be quite rapid; the amounts of SHTP that normally accumulate in the brain are relatively small. Consequently, SHTP is probably converted to 5HT as rapidly as it is formed, and the variations in tryptophan hydroxylation, induced by modifying brain tryptophan concentrations, rapidly influence the over-all rate of serotonin synthesis. Perhaps the most straightforward evidence that a change in brain tryptophan levels leads quickly to an alteration in serotonin synthesis (and levels) derives from studies in which animals received a single injection of the amino acid. Such a treatment, which rapidly increases serum and brain tryptophan concentrations, increases brain 5HT concentrations9,‘3 and stimulates the rate of 5HT formation,‘4 as estimated in vivo by the method of Carlsson et a1.15 These increases have been localized both to brain regions known to contain serotonergic nerve terminals and to the brainstem, which contains the cell bodies of 5HT neurons. These findings suggest that tryptophan-induced changes in brain 5HT probably occur in the functionally important pool of the amine in the brain: i.e., the one involved in synaptic transmission. Such modifications in the

in the brain. Th, tryptophan hydroxylase; AAAD, aromatic Fig. 1. Serotonin biosynthesis amino acid decarboxylase; MAO, monoamine oxidase; and ADH, aldehyde dehydrogenase.

L-

210

JOHN

D. FERNSTROM

5HT content of nerve terminals may influence the amount of neurotransmitter released when 5HT neurons depolarize. In making these observations, we were particularly intrigued by the apparent link between serotonin synthesis and the brain concentration of an amino acid that cannot be synthesized in the brain (or in the body). This finding suggests that all of the tryptophan in the brain utilized in the formation of 5HT must ultimately derive from the circulation. Because 5HT synthesis seemed to respond so sensitively to a change in brain tryptophan, we conjectured that brain tryptophan levels and serotonin synthesis must be responsive to alterations in serum tryptophan concentrations, and that any physiologic event that modified serum tryptophan would also influence the rate of serotonin synthesis in brain. To test this hypothesis, we applied a standard paradigm for modifying serum amino acid concentrations: injection of the hormone insulin. Our intent was to determine whether injection of insulin modified serum tryptophan levels and, if so, whether it elicited parallel changes in brain tryptophan and 5HT levels. Fasting rats were thus given an intraperitoneal injection of the hormone (2 units/kg) and were killed 2 hr later. In response to this treatment, serum tryptophan concentrations increased 40”,/,-SOP{ over control values.“j Although this response was completely unanticipated (we expected the hormone to decrease the serum tryptophan concentrations), the insulin-induced increase in serum tryptophan nonetheless provided an opportunity for determining whether brain tryptophan and 5HT would mirror the changes in serum tryptophan. They did: brain tryptophan and 5HT concentrations exceeded control values by about 409~ and ZOO,,!-25O,a,respectively, among rats injected with insulin.” Because of these striking effects of injected insulin, we sought to confirm the results using a more physiologic paradigm, one in which the rat secretes its own insulin. Consequently, we fasted rats overnight and, the next morning, allowed them to consume a largely carbohydrate, nonprotein diet for 2 hr before they were killed. Each rat ingested about 10 g of food during this period. Carbohydrate ingestion caused serum tryptophan, brain tryptophan, and brain 5HT concentrations to increase significantly; the magnitudes of the increases in brain tryptophan and 5HT were surprisingly similar to those observed after injection of insulin.” The carbohydrate-induced increase in brain 5HT reflects an acceleration in the rate of serotonin synthesis,14 as measured in vivo by the method of Carlsson et al.i5 Moreover, when we examined the effects in individual brain regions, we found that increases occurred in both 5HT level and synthesis in brain regions known to contain either serotonergic nerve terminals (spinal cord, cerebral cortex) or cell bodies (the brainstem).14 These results supported the hypothesis that physiologic events which modify serum tryptophan levels also alter brain tryptophan concentrations and ultimately influence serotonin formation. The insulin studies provided further evidence that brain tryptophan levels simply mirror the tryptophan concentrations in serum. Insulin release normally accompanies the ingestion of almost any food.

BRAIN

NEUROTRANSMITTERS

211

Therefore, from the results of the carbohydrate experiment, we suspected that the act of eating constituted a major, indirect stimulus of serotonin synthesis in the brain. We anticipated, for example, that the consumption of a meal containing both protein and carbohydrates would elicit an even greater surge in serotonin synthesis: serum tryptophan should rise to higher levels than those observed after rats ingested only carbohydrate, because amino acids would be included in the diet. Consequently, brain tryptophan levels would also rise, providing an enhanced stimulus to serotonin formation. We easily tested this hypothesis by first allowing fasting rats to consume a carbohydrate diet to which protein (18%) had been added and then killing them 2 hr later. As anticipated, serum tryptophan concentrations were substantially greater than the values observed in animals consuming carbohydrate alone. However, no increase in brain tryptophan occurred in rats ingesting the diet containing protein.18 This finding clearly indicates that the hypothesis that brain tryptophan simply reflects serum tryptophan concentrations is incorrect. The ultimate solution of this paradox requires the recognition of two facts: (1) other amino acids, in addition to tryptophan, enter the circulation when protein is consumed; (2) tryptophan uptake into brain is not normally a simple process of diffusion. Instead, tryptophan and the other amino acids are transported into brain by “carriers,” which are charge- and size-specific (e.g., see Blasberg and Lajthalg). Tryptophan, a large neutral amino acid, appears to be transported into brain by the same mechanism that transports the aromatic amino acids (tyrosine and phenylalanine) and the branched-chain amino acids (leucine, isoleucine, and valine). In addition, within a transport group, individual amino acids compete with each other for uptake. For example, a treatment that raises serum tyrosine levels decreases tryptophan uptake into brain; likewise, a treatment that increases the serum concentration of tryptophan or of leucine blocks tyrosine access to the brain.20 The access of tryptophan to the brain thus seems to depend not only on serum tryptophan levels, but also on the serum concentrations of the other large neutral amino acids that compete with it for uptake. Competition for brain uptake among amino acids provides the basis for a new hypothesis, one that could readily account for the effects on brain tryptophan resulting from the ingestion of either the carbohydrate or the carbohydrate-protein diet. When the rat ingests carbohydrate, insulin is secreted and brain tryptophan levels rise because of the concomitant increase in serum tryptophan and the decrease in the serum concentrations of the other large neutral amino acids. Brain tryptophan does not increase after the consumption of both carbohydrate and protein (in the form of casein); however, the serum concentrations of the competing neutral amino acids increase at least as much as tryptophan due to the presence of these amino acids in the dietary protein. The revised hypothesis was tested in another diet experiment: groups of rats were fasted overnight and then presented with one of three diets 2 hr before they were killed. The first diet contained, in addition to carbohydrate and fat, a mixture of amino acids that approximated the amino acid content of the 18% casein diet. The second diet was identical, except that it lacked tyrosine, phenylalanine, leucine, isoleucine, and valine. Ingestion of the complete amino acid diet produced results very similar to those seen in rats that consumed the 182,

212

JOHN

Table 1. The Effect of Ingesting Various Amino Acid-containing on Brain Concentrations

of Tryptophan,

Tryptophan Treatment

Fasted Complete amino acid diet Incomplete amino acid diet Groups of eight male Sprague-Dawley

kJ/s)

D. FERNSTROM

Diets

SHT, and 5HIAA* 5HT

5HIAA

(w/e)

(w/d

4.5 l 0.2

660 i

10

390 f 20

5.8 & 0.2t

670 f 20

450 * 20

870 i

680 f

14.6 +z 0.6t

20t

20t

rats were fasted overnight; they then either received one of two

diets or continued to fast for 2 hr before being killed. The complete amino acid diet contained all of the amino acids present in on 18% casein diet. The incomplete amino acid diet contained all the amino acids present in the complete diet, except for the branched-chain and aromatic amino acids. Data are presented as means f SE. *Data from Fernstrom and Wurtman.” tp < 0.001 compared to fasted values.

casein diet. Compared with fasting controls, brain tryptophan increased only slightly, without significant alterations in brain 5HT or 5-hydroxyindoleacetic acid (SHIAA), the principal metabolite of serotonin. However, when rats ingested the diet lucking the large neutral amino acids, brain tryptophan, 5HT, and SHIAA all increased dramatically** (Table 1). When rats ingested a third diet, which was similar to the first but lacked the acidic amino acids aspartate and glutamate, the results were identical to those obtained in animals ingesting the complete diet, thus confirming that the acidic amino acids are transported into the brain by a carrier different from that for tryptophan.22 Analyses of the sera from the rats in these studies confirmed the anticipated alterations in amino acid patterns, as based on the composition of each diet12’ ingestion of the diet lacking the large neutral amino acids (but containing tryptophan) increased serum tryptophan and decreased the serum concentrations of the aromatic and branched-chain amino acids (an effect of insulin). Ingestion of the complete amino acid diet or the diet lacking aspartate and glutamate raised both serum tryptophan and the serum level of its competitors. Hence, these data strongly suggest that the competition among large neutral amino acids in serum for uptake into the brain is an important determinant of brain tryptophan and 5HT concentrations after food ingestion. Brain tryptophan appears to vary directly with serum tryptophan concentrations and inversely with the serum levels of the competing neutral amino acids. A more “manageable” expression of these relationships is that brain tryptophan levels vary directly with the serum ratio of tryptophan, divided by the sum of the concentrations of the other large neutral amino acids (Fig. 2). The ability of any diet to modify brain tryptophan thus depends on how it affects this serum ratio. For example, when carbohydrate is consumed, serum tryptophan increases, the serum concentrations of the other large neutral amino acids decrease, and the serum ratio increases. Consequently, brain tryptophan, .5HT, and SHIAA are all increased. When the diet contains protein (18% or 24x), the change in the serum ratio and thus in brain tryptophan and 5-hydroxyindole levels is insignificant, because of the substantial dietary input of large neutral amino acids.‘7J8 An indirect confirmation of the physiologic importance of neutral amino acid competition (and of the utility of the serum ratio as an expression of it)

BRAIN

213

NEUROTRANSMITTERS

Fig. 2. Proposed model to describe diet-induced changes in brain serotonin concentration in the rat. The ratio of total tryptophan to the combined levels of tyrosine, phenylalanine, Ieucine, isoleucine, and valine in serum is thought to predict the tryptophan level in brain. The three branched-chain amino acids may be more important suppressors of tryptophan uptake into the brain than the aromatic amino acids. (From Fernstrom and Wurtmanz’)

Brain 5-HIAA

in predicting brain tryptophan concentrations was obtained when we subsequently observed that the postprandial brain level of almost any of the large neutral amino acids correlated surprisingly well with its serum ratio (i.e., with the serum concentration of the amino acid of interest in the numerator of the ratio). In many instances, changes in the brain concentrations of an amino acid could easily be dissociated from the concurrent alterations in its serum level, but not in its serum ratio. Figure 3, for example, shows the results for brain

I * 0.94

t’ Serum Oiitary Protein Canfont

20

1

Phrnylalaninr TtTtLtltV

Serum Phonylalaninr

Serum Ratio

nM/ml Fasting 0% I8 % 40%

Brain Ph6wlakmim

8712

0.llf0.01

nM/g 39 +- I

63 *2 93 f2

0.17 f 0.01 0.12 t 0.01

67 f I 46 f2

I49 f4

008t0.01

39 *2

Fig. 3. Correlation between brain phenylalanine concentrations and the serum ratio of phenylalanine to the sum of five competing amino acids in individual rats consuming ditferent diets. T + T + 1 + I + V = tryptophan + tyrosine + leucine + isoleucine + valine. Groups of six male Sprague-Dawley tats were fasted overnight and the next morning given free accessfor 2 hr to diets containing 0%, 18%, or 40% casein. Data are presented as the means f standard errors of the means.

214

JOHN

D. FERNSTROM

phenylalanine from a diet study in which groups of fasting rats were killed 2 hr after they began to consume diets containing different amounts of protein. Each rat’s individual values of brain phenylalanine and its serum ratio are plotted; these same data are tabulated beneath the figure, along with the values for serum phenylalanine. Clearly, the correlation is very good. Moreover, in the group of rats ingesting the 0% protein diet, changes in brain phenylalanine were dissociated from serum phenylalanine, but not from the serum ratio: compared to fasting values, brain phenylalanine and the serum ratio increased, but serum phenylalanine concentrations actually decreased. This same effect was observed with tyrosine but not with the branched-chain amino acidP (the serum levels, ratios, and brain levels of these amino acids all fell after the rats consumed the 0% protein diet).* Lack of Correlation Between Serum-Free Tryptophan Levels

Tryptophan and Brain

Tryptophan, unlike other amino acids in serum, is distributed unevenly between a free and albumin-bound pool: 80’4-90% of this amine attaches loosely to the serum protein, while the remainder circulates in the free form.24 Several investigators have suggested that tryptophan uptake into brain depends solely on serum-free tryptophan levels. Knott and Curzon25 and Tagliamonte et al.26 found, for example, that in rats starved for 24 hr, both serum-free and brain tryptophan concentrations were increased, but total tryptophan (free plus albumin-bound) concentrations were either unchanged or decreased. In drug studies, the administration of agents that dissociate tryptophan from albumin (e.g., acetylsalicylic acid) have been shown to increase brain tryptophan;27,28 Curzon and his associates have also provided some evidence that the increase in brain (or CSF) tryptophan that accompanies fulminant hepatic failure in experimental animals and humans often occurs in association with similar increments in serum-free, but apparently not total tryptophan concentrations.2g During the past 3 yr, my associates and I have performed a variety of studies to assess whether the relationship between serum-free tryptophan and brain tryptophan has a physiologic basis. Our results, summarized below, demonstrate that it does not. (1) Administration of insulin or carbohydrate ingestion acutely elevates serum total tryptophan and brain tryptophan levels but depresses serum-free tryptophan 1evels.g,30 (2) When groups of fasting rats consume one of several protein-containing diets that differ in fat content, substantial changes rapidly occur in serum-free tryptophan concentrations, the degree of change depending on the fat content of the meal: however, no changes occur in brain tryptophan.3’ This effect derives from the fact that circulating albumin normally binds a variety of compounds besides tryptophan, notably nonesterified fatty acids (NEFA), which compete with tryptophan molecules for binding.24 Hence by varying the fat content of a single meal, the investigator can produce widely differing serum NEFA concentrations in several groups of animals. In the study of *Fernstrom

JD, Hirsch

MJ, Failer DV: submitted

for publication.

215

BRAIN NEUROTRANSMITTERS

Madras et al.,3’ the serum NEFA levels among different diet groups ranged between 0.3 and 1.7 mEq/l of serum 2 hr after the food was presented to the rats. In the same animals, the serum-free tryptophan levels paralleled the NEFA concentrations, as anticipated, and varied over a threefold range. Because all of the diets included casein (20%), we also expected, and found, no change in brain tryptophan after the ingestion of any of these diets; this finding further confirmed our results, discussed previously, regarding the importance of amino acid competition in the transport of tryptophan into the brain. (3) The ingestion, by groups of fasting rats, of skim milk, whole milk, or light cream-all natural dietary fluids that contain similar amounts of protein, but enormously different amounts of fat (0.03:,, 1.83,, and 18.0?;, respectively) -elicits changes in serum NEFA, free and total tryptophan, and brain tryptophan that are remarkably similar to those changes observed with the experimental diets.32 Serum NEFA and free tryptophan levels are significantly higher, for example, in rats consuming light cream than in those ingesting skim milk; serum total tryptophan and brain tryptophan levels, however, do not differ significantly between these two dietary groups. (4) Correlation analyses between serum-free tryptophan (or the serum ratio of free tryptophan to the sum of its competitors) and brain tryptophan yield values of the correlation coefficient r that are substantially smaller than those obtained when the correlations are made between serum total tryptophan (or the serum ratio of total tryptophan to the sum of its competitors) and brain tryptophan (Figs. 4 and 5)” These results were obtained by feeding groups of fasting rats different diets for 2 hr before they were killed, as in the above paradigms. In this study, however, a diet was included that had previously been shown to decrease both serum total, and brain tryptophan soon after its ingestion-i.e., a carbohydrate diet containing only the aromatic and branchedchain amino acids.” The ingestion of this diet, to which 40”/, fat had been added, elicited substantial reductions in concentrations of both total serum tryptophan and brain tryptophan relative to fasting control values, but actually increased serum-free tryptophan concentrations.* These studies thus dispute the published results of Perez-Cruet et al.,34 who fed single meals to human subjects and observed the subsequent change in CSF tryptophan, which correlated with the serum ratio of free tryptophan to its competitors, but did not correlate with the ratio of total serum tryptophan to its competitors. Perhaps these findings are really not incompatible with our results, if one recalculates their data in micromoles. This unit seems more appropriate since it emphasizes numbers of molecules rather than molecular weights. The branched-chain amino acids all have molecular weights substantially smaller than those of the aromatic amino acids; the recalculation therefore places more emphasis on the serum changes in the branched-chain amino acids than on the aromatics. From the values of Perez-Cruet et al.,34 a recalculation based on moles yields a decrease in the ratio with total serum tryptophan (12”~) which more nearly approximates the decline in CSF tryptophan (20”/,) than does the recalculated serum ratio of free tryptophan to its competitors (8”;). *Fernstrom

JD. Hirsch

MJ: submitted

for publication.

JOHN

216

I

,\

I

I

I

I

I

I

F

5 5

40-

5 I

30-

-40

.

a.

8 t

l

.

.

..

. .

* .

5’

l

0.

.

.

8. .

2 L

.

?

r

r =

I

I

I

I

10203040 SERUM

FREE

-IO

5

TRYPTOPHAN

6

0.07

=0.23

m

F E

l8 *

l

l

II

I 60 SERUM

5

-20

0.’

8. IO

30

:..

l

20”

-

5 5

E .*

& t-

D. FERNSTROM

I 80

I 100

TOTAL

I 120

I

TRYPTOPHAN

(d/ml)

(nM/ml)

Correlations between brain tryptophan and serum free or total tryptophan concentraFig. 4. tions in individual rats consuming single meals. Groups of fasting rats were killed 2 hr after they were given free access to one of the following diets: carbohydrates + 0% fat; carbohydrates + 40% fat; carbohydrates + large neutral amino acids + 0% fat; and carbohydrates t large neutral amino acids t 40% fat.

This calculation contrasts with their values, expressed in micrograms, for these ratios: the ratio using total tryptophan did not change; that using free tryptophan decreased by 5O%.34 Diet, Brain Serotonin, and the Control of Brain Functions

The ability of the diet to modify rapidly the amount of serotonin in brain, particularly in regions containing 5HT nerve terminals, suggests that food ingestion may ultimately influence the amount of neurotransmitter released when 5HT neurons depolarize and consequently affect, either directly or tangentially, brain functions thought to be controlled by neuronal networks including 5HT neurons. At present, it is not possible to cite evidence directly affirming such effects of the diet; data do show, however, that single injections of L-tryptophan acutely affect brain outputs linked to the normal functioning of serotonergic neurons. Pain sensitivity, for example, responds to changes in brain 5HT: the

sEW

Fig. 5. Correlations between brain neutral amino acid mtios in individual experimental protocol.

TOTAL TRYPTWHAN T+P+L+ I+V

tryptophan and the serum (free or total) tryptophanl rats consuming single meals. See legend to fig. 4 for

BRAIN

NEUROTRANSMITTERS

217

administration of p-chlorophenylalanine, a drug that reduces 5HT, decreases the threshold to painful stimuli in experimental animals, an effect that can rapidly be reversed by an injection of 5 HTP, the immediate but noncirculating precursor of 5HT.35 Such increases in sensitivity to pain can also be reduced by injection of tryptophan: weanling rats that consume tryptophan-poor corn diets for several weeks develop substantial reductions in brain tryptophan and 5HT concentrations36 and a heightened sensitivity to painful stimuli;’ a single injection of L-tryptophan can reverse the increase in sensitivity to pain and simultaneously restore brain serotonin concentrations toward normal levels.8337 Administration of tryptophan has also been shown to modify sleep6 as well as the secretion of prolactin and gonadotropins’ in humans. Both of these body functions have been shown, through the use of other experimental systems, to be influenced by serotonergic neurons. CATECHOLAMINES

The catecholamine neurotransmitters, dopamine and norepinephrine, are synthesized from a common precursor, the amino acid tyrosine. The reaction sequence involves the hydroxylation of tyrosine to dihydroxyphenylalanine (dopa) and, subsequently, its decarboxylation to form dopamine (DA). In neurons that normally utilize norepinephrine as a neurotransmitter, dopamine undergoes one further transformation, P-hydroxylation, to form norepinephrine (NE) (Fig. 6). The hydroxylation of tyrosine appears to be the rate-limiting step in catecholamine formation; the enzyme catalyzing this reaction, tyrosine hydroxylase, is thus thought to be the focal point for the mechanisms controlling the over-all rate of catecholamine synthesis. In general, it has been widely held that end-product inhibition and the flux of nerve impulses through catecholamine neurons are the most important controls on DA and NE synthesis. Tyrosine availability has not been considered an additional mechanism of control, in part because injections of the amino acid failed to increase brain catecholamine concentrations. 38 It would be somewhat surprising, however, were tyrosine levels in brain not an influence on catecholamine synthesis: tyrosine hydroxylase, like tryptophan hydroxylase, appears not to be fully saturated with substrate in vivo, and plasma and brain tyrosine levels normally vary over a broad dynamic range, rising considerably, for example, after the ingestion of almost any food.23 We suspected that the failure of brain catecholamine levels to rise after tyrosine injection might not constitute a valid proof that catecholamine syn-

0e; HCH

HCH

HCH

HC-OH

H$ -OH

HC-COOH

Hb-COOH

H&H

HCH

H$H

IjH,

k

I;H,

AH,

HN tn,

Tyrosme

DOPA

Dopmnine

Ncfepineptwine

Epinephrine

Fig. 6. Catecholamine biosynthesis in the brain. TH, tyrosine hydroxylase; AAAD, aromatic l-amino acid decarboxylase; DBH, dopamine-P-hydroxylase; PNMT, phenylethanolamine-Nmethyltmnsferase.

218

JOHN

Table 2. Effect of Administration on the Accumulation

of Tyrosine or Tryptophan

D0pCl

Number of

(wglg)

(w/g)

Rot Broinr

Control

14.7

250

28

Tyrosine

26.6t

2833

20

Tryptophon

12.17

1707

received

tryptophan *Data tp

FERNSTROM

of Tyrosine and Dopa in Rat Brain*

Tyrosine Treatment

Rats

D.

from

(50

the mg/kg

Wurtmon

decorboxylose i.p.)

or diluent;

inhibitor they

were

R04-4602 killed

(800 1 hr after

mg/kg

10 i.p.)

and,

after

15

min,

tyrosine

or

the first injection.

et ol.39

< 0.01

compared

to control

values.

$p < 0.05

compared

to control

values.

thesis is not stimulated by increased availability of tyrosine. Relatively large quantities of DA and NE with slow turnover rates are present within storage pools in the brain and may obscure a tyrosine-induced increase in brain catecholamine synthesis. To explore this relationship, we chose as our method for estimating brain catecholamine synthesis the short-term accumulation of dopa after inhibition of the decarboxylase enzyme (aromatic L-amino acid decarboxylase, the enzyme catalyzing the conversion of dopa to DA) with R04-4602 (800 mg/kg i.p.). Groups of rats were injected with an amino acid to raise or lower brain tyrosine levels 15 min after they received the drug; they were then killed 45 min later (Table 2). In animals given a dose of tyrosine that increased brain tyrosine concentrations by SOY0(50 mg/kg), dopa accumulated 1.5?,/,faster than it did in rats receiving diluent. 39 In contrast, an injection of tryptophan, another neutral amino acid that lowered brain tyrosine levels by 18%, reduced dopa accumulation to 68% of control levels (Table 2; p < 0.001). When other amino acids were tested, only those that decreased brain tyrosine suppressed brain dopa synthesis (i.e., the other large neutral amino acids, including the unnatural amino acid p-chlorophenylalanine). In using centrally acting decarboxylase inhibitors to estimate catechol synthesis, one assumes that any changes in dopa formation among experimental groups will not cause parallel alterations in catecholamine synthesis or release, since the inhibitor presumably blocks the transformation of dopa to amines. However, if one estimates catechol (or catecholamine) synthesis by following the rise either in brain catecholamine levels (after treatment with a monoamine oxidase inhibitor) or in brain homovanillic acid levels (after administration of probenecid), the changes in catecholamine synthesis may elicit parallel changes in the release of DA and NE. If more transmitter is released, for example, preor postsynaptic receptors might become activated to a greater extent and ultimately cause feedback inhibition to tyrosine hydroxylase. This control loop would tend to return the net rate of catecholamine synthesis to normal. Preliminary experiments have focused on these latter paradigms and suggest that the naturally occurring variations in brain tyrosine levels in intact, untreated rats, similar to those occurring after ingestion of a high-protein meal, may be so well compensated by feedback changes in tyrosine hydroxylase activity as to have relatively little prolonged effect on catecholamine synthesis.40 If ongoing studies confirm this speculation, they will imply a fundamental difference between the normal functions of serotonergic and catecholaminergic neurons.

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ACETYLCHOLINE

of choline Recent studies by Cohen and Wurtman 41 indicate that availability influences acetylcholine synthesis in brain. The following evidence suggests that the synthesis of this transmitter in brain might normally be subject to variations in brain choline levels, and that these levels might, in turn, be subject to changes in serum choline levels: (1) Choline acetyltransferase, the enzyme that catalyzes the synthesis of acetylcholine (ACh) from choline and acetylCoA, appears to be unsaturated with its substrates in vivo: rat brain choline acetyltransferase has a K, of 400 1LM for choline and 18 PM for acetylCoA;42 rat brain choline concentrations are approximately 37 pM4’,43,44 and acetylCoA levels range from 7 to 11 P~.45,46 Substrate concentrations are thus conspicuously less than the respective K, values of the enzyme. (2) The brain apparently does not have the enzymatic capacity for synthederive sizing choline, .47 thus all of the choline in the brain must ultimately from the circulation.4’ (3) The system for transporting choline into the brain does not appear to become saturated even at very high serum choline concentrations.4g From these considerations, my associates Edith Cohen and Richard Wurtman suspected that an elevation in the circulating choline levels might elicit an increase in brain choline concentrations and accelerate ACh synthesis (which might ultimately appear as a rise in brain ACh levels). By using both microwave radiation to kill the animals (because it inactivates acetylcholinesterase at the moment of death) and a sensitive method to assay choline and ACh,” they found that elevation of levels of serum choline (either by injecting choline chloride or administering it in the food or drinking water) does indeed increase ACh synthesis and levels in brain.4’,5’ When rats were injected with a relatively small dose of choline chloride (60 mg/kg, or approximately 33”, of the rat’s normal daily choline intake), serum and brain choline concentrations increased rapidly and were followed 40 min later by a significant increase in brain ACh (22”,; Fig. 7). The dose of choline and the brain ACh level 40 min after injection appeared to be linearly related with dosages less than 60 mg/kg; no further increments in brain ACh occurred with greater dosages, although brain choline concentrations continued to increase.4’ The increase in brain ACh that follows choline injection has been recently confirmed by Haubrich et al.,” who used a choline dose of 100 mg/kg. Brain ACh concentrations have also been shown to increase after infusion of choline intravenously.52.53 Because a significant fraction of the choline in the serum and hence, the brain derives from the diet,54 it was suspected that changes in dietary choline levels might influence blood choline and ultimately, brain choline and ACh concentrations. This notion seems to be supported by the earlier finding of Nagler et a1.55 that brain ACh levels were depressed in weanling rats after 5 days of choline deficiency. In initial studies, Cohen and Wurtman4’ gave groups of rats 11 days of free access to a choline-deficient diet and to distilled water containing choline chloride in differing amounts (0, 1.5, or 15 mg/ml). The amount of choline actually ingested averaged 0, 23, and 171 mg/day in the three different groups. (For purposes of comparison, animals eating Charles River Rat, Mouse, and Hamster Maintenance Formula [0.183”; choline] normally con-

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Fig. 7. lime course of the response of serum and brain choline and brain ACh to a single injection of choline. For measurement of brain choline and ACh, groups of male Sprague-Dawley rats received choline chloride (60 mg/kg, i.p.) and were killed by microwave irradiation of the head after various intervals. For estimation of serum choline concentrations, other groups of rats were similarly injected and killed by decapitation after the same intervals. Vertical bars represent standard errors of the mean; open circles without vertical bars indicate the range of values for serum choline levels at each time point. *p < 0.01; **p < 0.001, compared to control rats injected with diluent. (From Cohen and Wurtman” )

sume 18-27 mg of choline/day.) In rats drinking the high-choline water, both brain choline and ACh levels were significantly elevated, compared to the group that had consumed a normal amount of choline (about 23 mg/day).4’ In similar experiments, Cohen and Wurtman” gave groups of rats free access to diets containing differing amounts of choline, and 11 days later assayed different brain regions for their content of choline and ACh. Concentration of ACh in the caudate nucleus, a portion of the brain particularly rich in cholinergic nerve terminals, was 28”,/,greater in rats consuming 20 mg of choline/day and 45% greater in animals ingesting 129 mg of choline/day than in rats consuming no choline. Choline and ACh levels were also significantly elevated in other brain regions in rats consuming the high-choline diet. This observation-that administration of choline raises brain ACh levelssuggests a potentially useful technique for modifying the functional activity of cholinergic neurons, provided that intraneuronal ACh content influences the amount of transmitter released when these neurons depolarize. If administration of choline elevates ACh levels and subsequently, cholinergic transmission in the human brain, this pharmacologic technique might offer a convenient and fairly selective treatment for disease states thought to involve insufficient transmission across cholinergic synapses. Such diseases might include extrapyramidal motor disorders (e.g., Huntington’s chorea and tardive dyskinesia56,57)and a variety of affective disorders and psychoses.5*-6’ In a recent case report based on the studies of Cohen and Wurtman4’ Davis et al.4 described a patient with tardive dyskinesia who responded favorably to large oral doses of choline. Thus, the possible relationship between precursor control of brain ACh concentrations and cholinergic transmission has already generated studies aimed at determining the potential therapeutic use of choline. The physiologic and evolutionary significance of having ACh synthesis coupled to the vagaries of the diet, however, remains to be clarified.

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5-hydroxyindoles in various regions of the rat central nervous system. J Neurochem 25:825-~ 829, 1975 15. Carlsson A, Kehr W, Lindqvist M, Magnussen T, Atack CV: Regulation of monoamine metabolism in the central nervous system. Pharmacol Rev 24:371-384, 1972 16. Fernstrom JD, Wurtman RJ: Elevation of plasma tryptophan by insulin in the rat. Metabolism 21:337-342, 1972 17. Fernstrom JD. Wurtman RJ: Brain serotonin content: increase following ingestion of carbohydrate diet. Science 174:1023-1025. 1971 18. Fernstrom JD. Larin F. Wurtman RJ: Correlations between brain tryptophan and plasma neutral amino acid levels following food consumption in the rat. Life Sci 13:517.-524, 1973 19. Blasberg R, Lajtha A: Substrate specificity of steady-state amino acid transport in mouse brain slices. Arch Biochem Biophys 112: 361-377, 1965 20. Guroff G, Udenfriend S: Studies on aromatic amino acid uptake by rat brain in vivo. J Biol Chem 237:803-806, 1962 21. Fernstrom JD, Wurtman RJ: Brain serotonin content: physiological regulation by plasma neutral amino acids. Science 178:414416. 1972 22. Fernstrom JD, Wurtman RJ: Nutrition and the brain. Sci Am 230:84-91. 1974 23. Fernstrom JD, Faller DV: Modifications in the brain concentrations of individual amino acids following the ingestion of a single meal by rats. Fed Proc 34:243, 1975 24. McMenamy RH, Oncley JL: Specific binding of tryptophan to serum albumin. J Biol Chem 233:1436-1447, 1958 25. Knott PJ, Curzon G: Free tryptophan in plasma and brain tryptophan metabolism. Nature 239~452-453, 1972 26. Tagliamonte A, Biggio G, Vargiu L, Gessa GL: Free tryptophan in serum controls brain tryptophan levels and serotonin synthesis. Life Sci 12:277--287, 1973 27. Guerinot F, Poitou P, Bohuon C: Serotonin synthesis in the rat brain after acetylsalicyclic acid administration. J Neurochem 22: 19lll92, 1974 28. Curzon G, Knott PJ: Effects on plasma and brain tryptophan in the rat of drugs and hormones that influence the concentration of unesterified fatty acid in the plasma. Br J Pharmacol50: 197-204, 1974 29. Curzon G, Knott PJ, Murray-Lyon IM, Record CO. Williams R: Disturbed tryptophan

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metabolism in hepatic coma. Lancet 1:10921093, 197s 30. Madras BK, Cohen EL, Fernstrom JD, Larin F, Munro HN. Wurtman RJ: Dietary carbohydrate increases brain tryptophan and decreases serum-free tryptophan. Nature 244: 34-35, 1973 31. Madras BK, Cohen EL, Messing R, Munro HN, Wurtman RJ: Relevance of free tryptophan in serum to tissue tryptophan concentrations. Metabolism 23: 1107-l 116, 1974 32. Fernstrom JD, Hirsch MJ, Madras BK, Sudarsky L: Effects of skim milk, whole milk, and light cream on serum tryptophan binding and brain tryptophan concentrations in rats. J Nutr 105: 1359-I 362, 1975 33. Fernstrom JD, Failer DV, Shabshelowitz H: Acute reduction in brain serotonin and SHIAA following food consumption: correlation with ratio of serum tryptophan to the sum of competing neutral amino acids. J Neural Transm 36:113~121,1975 34. Perez-Cruet J, Chase TN. Murphy DL: Dietary regulation of brain tryptophan metabolism by plasma ratio of free tryptophan and neutral amino acids in humans. Nature 248: 6933695. I974 35. Tenen SS: The effects of p-chlorophenylalanine, a serotonin depletor, on avoidance acquisition, pain sensitivity, and related behaviors in the rat. Psychopharmacologia 10:204-219, 1967 36. Fernstrom JD, Wurtman RJ: Effect of chronic corn consumption on serotonin content of rat brain. Nature [New Biol] 23462264, 1971 37. Fernstrom JD, Hirsch MJ: Rapid repletion of brain serotonin in malnourished, cornfed rats following t_-tryptophan injection. Life Sci 17:455-464. 1975 38. Dairman W: Catecholamine concentrations and the activity of tyrosine hydroxylase after an increase in the concentration of tyrosine in rat tissues. Br J Pharmacol 443077310, 1972 39. Wurtman RJ. Larin F, Mostafapour S, Fernstrom JD: Brain catechol synthesis: control by brain tyrosine concentration. Science 185: 183-184, 1974 40. Scally MC, Wurtman RJ: Relation between tyrosine levels and homovanillic acid accumulation. Trans Am Sot Neurochem 7:76, 1976 41. Cohen EL, Wurtman RJ: Brain acetylcholine: increase after systemic choline administration. Life Sci 16: 1095-l 102. 1975 42. White HL, Wu JC: Kinetics of choline acetyltransferases (EC 2.3. I .6) from human and

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other mammalian central and peripheral nervous tissues. J Neurochem 20:297-307. 1973 43. Stavinoha WB, Weintraub ST: Choline content of rat brain. Science l83:964-965, 1974 44. Schmidt DE. Speth RC: Simultaneous analysis of choline and acetylchoiine levels in rat brain by pyrrolysis gas chromatography. Anal Biochem 67:353-357, 1975 45. Sollenberg J: Determination of acetyl coenzyme A, in Heilbronn E. Winter A (eds): Drugs and Cholinergic Mechanisms in the CNS. Stockholm, Forsvarets Forskningsanstalt, 1970, pp 27-32 46. Shea PA, Aprison MH: The simultaneous measurement of acetyl-CoA, acetylcholine, and choline in the same tissue extract from rat brain by a radio-enzymatic method. Trans Am Sot Neurochem 6:362, 1975 47. Ansell GB, Spanner S: The origin and metabolism of brain choline, in Waser PC (ed): Cholinergic Mechanisms. New York, Raven Press, 1975. pp 92-149 48. Schuberth J, Jenden DJ: Transport of choline from plasma to cerebrospinal fluid in the rabbit with reference to the origin of choline and to acetylcholine metabolism in brain. Brain Res 84:245-256, 1975 49. Freeman JJ, Choi RL, Jenden DJ: Plasma choline: its turnover and exchange with brain choline. J Neurochem 24:729-734. 1975 50. Shea PA, Aprison MH: An enzymatic method for measuring picamole quantities of acetylcholine and choline in CNS tissue. Anal Biochem 56:165-177, 1973 51. Cohen EL, Wurtman RJ: Brain acetylcholine: control by dietary choline. Science 191: 561-562. 1976 52. Haubrich DR, Wang PFL. Clody DE, Wedeking PW: Increase in rat brain acetylcholine induced by choline or deanol. Life Sci 17: 975-980, 1975 53. Racagni G, Trabucchi M. Cheney DL: Steady-state concentrations of choline and acetylcholine in rat brain parts during a constant rate infusion of deuterated choline. Naunyn Schmiedebergs Arch Pharmacol 290: 99-105, 1975 54. Hanin I, Schuberth J: Labelling of acetylcholine in the brain of mice fed on a diet containing deuterium labelled choline: studies utilizing gas chromatography-mass spectrometry. J Neurochem 23:819-824, 1974 55. Nagler AL, Dettbarn WD, Seifter E, Levenson SM: Tissue levels of acetylcholine and acetylcholinesterase in weanling rats subjected to acute choline deficiency. J Nutr 94: 13-19, 1968 56. Klawans HL, Rubovits R: Central cho-

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linergic-anticholinergic antagonism in Huntington’s chorea. Neurology 22: 107-I 16, I972 57. Klawans HL, Rubovits R: Effect of cholinergic and anticholinergic agents on tardive dyskinesia. J Neurol Neurosurg Psychiatry 27: 941~947.1914 58. Janowsky DS. El-Yousef MK, Davis JM, Sekerke HJ: A cholinergic-adrenergic hypothesis of mania and depression. Lancet 2:632-635, 1972 59. Janowsky

DS, El-Yousef

MK, Davis JM.

Sekerke HJ: Parasympathetic suppression manic symptoms by physostigmine. Arch Psychiatry 28:5422547, 1973

of Gen

60. Janowsky DS, El-Yousef MK, Davis JM: Acetylcholine and depression. Psychosom Med 361248-256, 1974 61. Friedhoff AJ, Alpert M: A dopaminergiccholinergic mechanism in production of psychotic symptoms. Biol Psychiatry 6: 165- 169. 1973