AMERICAN
JOURNAL
OF PHYSIOLOGY
Vol. 229, No. 1, July
1975.
Printed in U.S. A.
Monosodium
glutamate
in the neonatal LEWIS
monkey
D. STEGINK, W. ANN M. C. BRUMMEL
AND Departments Iowa 52242;
metabolism
REYNOLDS,
L. J. FILER, JR., ROY M. PITKIN,
DAVID
P. BOAZ,
Biochemistry, Obstetrics and Gynecology, University of Iowa College of Medicine, and Department of Anatomy, University of Ih’nois Medical Center, Chicago, Illinois 60680
Iowa Gity,
of Pediatrics,
W. ANN REYNOLDS, L. J# FILER, JR., Roy P. BOAZ, AND M. C. BRUMMEL. Monosodium in the neonatal monkey. Am. j. Physiol. 229(l) 246-250. Z975.-Monosodium glutamate (MSG) administered by gastric tube to 10 infant monkeys at doses of 1-4 g/kg produced rapid increases in plasma glutamate (17- to 33-fold) and aspartate (50- to go-fold) levels. The degree of elevation was proportional to the dose administered. Levels of other amino acids were unaffected. Two of the monkeys exhibited high fasting glutamate levels and abnormal glutamate tolerance curves. Despite this apparent decreased ability to metabolize glutamate, neither these animals nor any of the others for whom morphologic studies have been previously reported demonstrated neurotoxicity. Studies using 14C-labeled glutamate indicated conversion of administered glutamate to two ninhydrin-negative compounds identified as glucose and lactate, as well as to aspartate. LEWIS
STEGINK,
ID.,
MATERIALS
h/ll. PITKIN, DAVID glutamate metabolism
aspartate;
ORAL
:
neurotoxicity
OR
SUBCUTANEOUS
ADMINISTRATION
Of large
QuantitieS
of glutamate
to the newborn mouse results in a variety of neurotoxic effects, the most marked of which is a massive hypothalamic neuronal necrosis (17). Similar studies in neonatal primates are contradictory. Although the initial report by Olney and Sharpe (20) indicated extensive glutamate-induced neuronal necrosis in a single premature primate injected with 2.7 g glutamate/kg body wt, other investigators (1, 16, 24, 34) were unable to confirm these findings, despite the ability of at least some of them (1, 12, 34) to reproduce the lesion in mice. In subsequent studies Olney et al. (19, 21) reported that the size of lesion was dose related paralleling blood glutamate concentrations and suggested that the failure of other research groups to observe glutamate-induced neuronal necrosis in the neonatal primate may have resulted from vomiting of the administered glutamate with resultant failure to produce marked elevation of blood glutamate levels. The present study reports plasma levels of glutamate and other amino acids in infant monkeys, most of whom were included in our previously reported morphologic study (24), demonstrating an absence of neuronal lesions after glutamate administration.
AND
METHODS
Infants of three closely related macaque species (M. mulatta, hf. arctoides, and 1M. irus), ranging in age from 30 min to 17 days, were studied. Ten animals were given monosodium glutamate (MSG) dissolved in distilled water (50 G/cwt/vol solution) in doses of 1, 2, or 4 g/kg, and one control animal was given only distilled water. The age, weight, dosage level, and fasting plasma glutamate concentration for each monkey are listed in Table 1. Administration was by gastric tube. The animals were tranquilized with phencyclidine hydrochloride (Sernylan, Parke-Davis) 0.5 mg/kg and kept on a heating pad during the period of study. Heparinized blood samples of 1 ml each were obtained from a catheter placed in the inferior vena cava (through a femoral vein) prior to dosing and at hourly intervals for 6 h. After centrifugation, plasma with sulfodeproteinized immediately samples were salicyclic acid and either analyzed promptly or stored at - 70°C. These conditions prevented conversion of glutamine to glutamate and pyrrolidone carboxylate, as well as loss of cystine (5, 22). At the conclusion of each experiment, cerebrospinal fluid was obtained by ventricular tap and prepared by the method of Dickinson and Hamilton (4). Amino acid analyses were done on Technicon NC-1 amino acid analyzers using the Efron buffer system (7). For seven of the animals identified in Table 1, morphologic studies by light and electron microscopic examination of the hypothalamic area have been reported previously (24). The results of morphologic studies of brain tissue from two additional animals are reported in this paper. These brains, embedded in plastic, were serially sectioned through the entire ventral hypothalamic area at 1 ,um. Methods of perfusion, sectioning, and staining have been described previously (12, 24), The monosodium-L-glutamate given assayed at greater than 99.9 76 pure by amino acid analysis, and no amino acids other than glutamate were found. In order to examine the metabolic pathways of glutamate, three of the animals listed in Table 1 (MJ, MI, and ME) were given, in addition to 1 g/kg MSG, 10 @i of [3, 4-14C]glutamate (sp act 200 mCi/pmol). Simultaneous radioactivity and amino acid analyses were done by previously described methods (28) permitting detection of both ninhydrin-positive and -negative metabolites derived In order to obtain sufficient plasma for from glutamate.
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GLUTAMATE TABLE
1.
METABOLISM
MW MG MJ MC* MF* IMP* MA* MI MO” MB” ME
* Animal previously
Age, days
arctozdes mulatta mulatta mulatta mulatta irus mulatta mulatta irus mulatta irus
0.02 1 1 2 4 6 7 8 9 14 17
in which (24).
morphologic
Weight, g
MSG Load, g/kg
Fasting Plasma Glutamate Levels, bmoI/dI)
550 510 370 450 430 300 445 490 310 480
1 4 1 1 2 4 2
62 77 20 9 15 9 9
1
15
0 4
6 11
319
1
8
studies
have
been
GLUTAMATE
reported
RESULTS
Figure 1 illustrates plasma glutamate and aspartate levels following glutamate loads of l-4 g/kg to eight infant monkeys whose fasting plasma glutamate concentration was 20 pmol/dl or less (average 12 pmol/dl). In these animals, glutamate increased 17- to 33-fold and aspartate 50- to go-fold from fasting values. Maximal elevations were attained 1-2 h after gastric administration and were proportional to the dose of glutamate given Plasma levels of amino acids other than glutamate and aspartate, including those which could potentially be derived from glutamate or aspartate, were essentially unchanged. The normal fasting glutamate level in adult monkeys has been reported to be 5-10 pmol/dl (23). Two of our infant monkeys (MH and MG) had values substantially above this level. Glutamine levels in these animals were normal, indicating that the elevated glutamate was not due to degradation of glutamine. These two animals, who were two of the youngest studied, appeared to have an .
impaired ability to metabolize glutamate. In addition to markedly elevated fasting glutamate levels (62 and 77 pmol/dl), both demonstrated abnormal glutamate tolerance curves, as illustrated in Fig. 2, in which MSG loading produced plasma glutamate levels which rose higher (by 1.5 and 3 times) and returned to normal more slowly, compared with other animals receiving an equivalent dosage. The data for aspartate levels in Fig. 2 further suggest a delay or lag in conversion of glutamate to aspartate. For example, at 1 h in monkey MH, glutamate levels were twice wr. as great and aspartate levels one-fourth as much as
7I
identification of the ninhydrin-negative metabolites, two additional adult monkeys (not included in Table 1) were given 1 g/kg MSG with 100 $Zi [3 ,4J4C]glutamate by stomach tube and exanguinated at the previously determined time-of-peak concentration of the respective compounds. Samples of deproteinized plasma were applied to an amino acid analyzer column and the eluate collected in Z-ml fractions. The radioactivity profile obtained in this manner was identical to that found with the simultaneous radioactivity-amino acid analysis technique. The fractional collections were then desalted with high-voltage electrophoresis (33), a method giving satisfactory separation of ninhydrin-negative acidic metabolites. All of the infant monkeys included in the study were observed continuously during the 6 h after dosing. One (MP) vomited an estimated 1.5 ml of clear fluid 7 min after dosing and was redosed with 1 ml of MSG solution immediately ; there was no subsequent vomiting, Four (MA, ME, MG, and MH) vomited small amounts (estimated volume less than 1 ml) of a frothy, bile-colored fluid at 35, 40, 55, and 70 min after dosing. Since considerable time had elapsed and the quantity vomited was small, these animals were not redosed.
s
247
MONKEY
leuels
Species
M. M. M. M. M. M. M. M. M. M. M.
NEONATAL
Animalsstudiedand initial fasting
plasma glutamate Animal
IN THE
0
2
4
6 HOURS
1. Plasma glutamate and aspartate levels with time followmg LISG loading: mean value from 4 animals administered 1 g/kg body wt (A); mean value from 2 animals administered 2 g/kg body wt (x) ; mean value from 2 animals administered 4 g/kg body wt (0); value in control animal administered water (e). Standard deviation not more than 2070 of mean. FIG.
- A i300 0 0 - 200 K w CT) W 1.
h-
--
1
1
-0
1
I
2
1
4
012345G HOURS
I
I
6,
2 34 HOURS
56
FIG, 2. Plasma glutamate and aspartate levels in neonatal monkeys administered A : 1 g hfSG per kg body wt-monkey MH (x); other mean + SD (0); control (A). I?; animals adneonatal monkeys, ministered 4 g MSE per kg body wt : monkey MG (o), other animals
(a> .
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248
STEGINK
means of the other animals receiving an equivalent dose of 1 g/kg. While administration of large doses of MSG resulted in marked elevation of plasma glutamate levels, none of the animals in the present study who received MSG and was studied by light and electron microscopy of the brain (24) showed evidence of either the massive or small focal lesions reported by Olney et al. (21). Representative sections of the hypothalamic area from treated and control animals are illustrated in Fig. 3.
ET
AL.
Administration of MSG (1 g/kg) with added tracer [3,4-14C]glutamate (10 FCi) to three infant monkeys permitted determination of the major glutamate-derived metabolites. Figure 4 illustrates the results of these studies involving simultaneous radioactivity-amino acid analysis. As expected, the major portion of radioactivity remained in association with glutamate. However, there was also significant incorporation of label into aspartate, glutamine, and ornithine as well as two ninhydrin-negative compounds. The two ninhydrin-negative metabolites were isolated from plasma by ion-exchange chromatography and high-voltage electrophoresis, characterized as single compounds, and identified as glucose and lactate by chemical and enzymatic methods described in detail in a previous publication (29). As is apparent in Fig. 4, glucose was the major metabolite. Radioactivity in other compounds representing potential metabolites of glutamate was also examined. No detectable radioactivity was found in succinate, pyrrolidone carboxylate, malate, citrate, or oxaloacetate. In view of the demonstration in the mouse that glutamate-induced brain damage occurs in a distribution suggesting the influx of a deleterious agent from the cerebrospinal fluid (12), we examined the cerebrospinal fluid of one monkey given MSG (1 g/kg) with added tracer [3,414C]glutamate (10 PCi). Radioactivity and amino acid composition of plasma and cerebrospinal fluid were measured 2 h after dosing. The results of this study are listed in Table 2. Comparison of incorporation of label in plasma with that found in the cerebrospinal fluid indicates that neither glutamate nor aspartate entered spinal fluid at the time of anticipated maximal plasma levels of glutamate and aspartate. Only labeled glutamine appeared at approximately the same level in both plasma and spinal fluid. In direct contrast to the amino acids, glucose and lactate
1-
_,-,-O-Q-; Lwfl_,-A-A-A-Aor” , , , , ,
,
,
,
f
2
1
a-@
asp..,
lactade
‘9
l-
FID. 3. A: section through
ventral hypothalamic region of a control infant monkey. Ventral aspect of third ventricle may be seen and below it neurons comprising infundibular nucleus (X90). B: section through ventral hypothalamic region of an infant rhesus monkey which received 4 g/kg of hlSG by stomach tube 6 h prior to brain perfusion. There is no evidence of dendritic or neuronal degeneration and dilation. This section is slightly caudal to A (X90).
/
A’
&AN ,lLA’ ,,,,, 0
1
2
A’
A’@
\a II
1
2
ziO”Ros FIG. 4. Playma distribution of radioactivity in 3 infant monkeys administered monosodium glutamate in water (1 g/kg body wt, 10 PCiL-[3,4-Wlglutamate). Mean values shown, SD did not exceed 20% of mean.
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GLUTAMATE
METABOLISM
IN
THE
NEONATAL
TABLE 2. Comparison of quantity and distribution of label in plasma and spinal fluid fallowing monosodium glutamate loading of an infant monkey Plasma
Compound
Cerebrospinal
Aspartate Glutamine Glutamate Alanine Ornithine Arginine Glucose Lactate
638 (3.3)” 195 (1.0) 10,440 (55.0) 100 (0.5) 19 (0.1) 0 5,715 (30.0) 1,854 (9.7)
0 112 (2.0)* 90 (1.5) 5 (0.1) 0 0 (76.9) 4,677 1,186 (19.5)
Total
18,961
6,080
counts
* Percent
di&ibution
levels were similar reflecting their rapid
(100) of counts
in plasma equilibration
is given
249
MONKEY
Fluid
(100)
in parentheses.
and cerebrospinal fluid, between compartments.
DISCUSSION
The principal finding of the present study is that monosodium glutamate administration produced marked elevations of plasma glutamate and aspartate levels in infant monkeys previously reported (24) to lack any evidence of neuronal damage. Thus, these data indicate that our failure to observe neurotoxic effects of MSG administration in primates was not due to vomiting or other factors resulting in a lack of elevation of plasma glutamate. Failure to observe neuronal necrosis in the infant monkey despite enormous elevations in plasma glutamate concentration is striking in view of the ease with which the lesion is produced in the infant mouse, Neuronal damage to the newborn mouse has been reported (18) under conditions which result in elevation of plasma glutamate concentration to 50 to 80 pmol/dl (30), levels approximately onefourth of those found in the present study. From these in response to observations it appears that neurotoxicity glutamate loading differs in mouse and primate. The reasons for these differences in sensitivity to glutamate are not clear. Neither glutamate nor aspartate is increased in concentration in the spinal fluid of the infant monkey, despite elevated blood levels of these amino acids. These data may be of particular importance if the hypothesis advanced by Lemkey-Johnston and Reynolds (12) is correct. These authors suggest that in the mouse there is an influx of a neurotoxic agent from cerebrospinal fluid following a glutamate load* Thus, the hypothalamus of the primate may be spared because of the failure of glutamate or aspartate to enter the cerebrospinal fluid. Most investigators have found little if any net transfer of glutamate across the blood-brain barrier in a variety of adult species, including mouse (26), rat (15, 26), and dog (9). However, it is conceivable that glutamate may be able to enter the
spinal fluid of the infant mouse. It is also possible that a glutamate metabolite could be the neurotoxic agent for the infant mouse. Preliminary studies indicate a totally different pattern of ninhydrin-negative metabolites present in the mouse following a glutamate load (25). This includes at least two major metabolites not seen in the primate. Lemkey-Johnston et al. (11) have recently found large cerebral and small hypothalamic lesions in the infant mouse following oral administration of NaCl equimolar to &g/kg doses of glutamate. These new findings implicate hyperosmolarity as a potential causative factor in the production of central nervous system lesions following glutamate administration. The unusual glutamate tolerance curves observed for the two infant monkeys whose fasting plasma glutamate levels were elevated are of interest. It is possible that the elevated fasting levels and abnormal glutamate tolerance in the two animals, who were the youngest studied, represent an unusual maturational lag in an enzyme system for Maturational lags in enzyme glutamate metabolism. systems metabolizing amino acids are known to occur in the human infant, particularly those of low birth weight Specific examples of delayed maturation al induction include: the delay in enzyme systems catabolizing tyrosine, resulting in the transient tyrosinemia of the premature (3, 6); the lag in the development of a part of the phenylalain a transient hypernine hydroxylase system, resulting phenylalanemia (2, 3 1) ; and the lag in cystathionase (32), which makes cysteine an essential amino acid for the human fetus and premature infant (27). On the other hand, maturational studies on the monkey (10) and human (5, 8, 14), including the low-birth-weight infant (6, 13), have failed to indicate an increase in plasma glutamate level during the neonatal period. The present study suggests that the pathway of glutamate metabolism in the infant primate is as follows: ingested glutamate is rapidly removed from portal bIood by the liver where, once inside the liver cell, it enters the Mitochondrial glutamate is converted to mitochondria. cr-ketoglutarate and other tricarboxylic acid cycle components, principally malate and oxalacetate+ Oxalacetate remains within the mitochondria and is transaminated to aspartate, which is then released into the peripheral blood. Malate diffuses into the cytoplasm to be converted to phosphoenolpyruvate and thence to glucose and through pyruvate to lactate. Thus, in the peripheral blood, glutamate and aspartate levels are increased markedly with lessened increase in concentration of glucose and lactate. These studies were supported in part by Grants-In-Aid from the Gerber Products Company and the International Minerals and Chemical Corporation. R. M. Pitkin is an Academic Career Development Awardee in Nutrition, National Institute of Arthritis, hfetabolism, and Digestive Diseases, KO7 Ahl 70694. Received
for publication
4 February
1974.
REFERENCES 1, ABRAHAM, R., W. DOUGHERTS, The response of the hypothalamus glutamate in mice and monkeys
L. GOLBERG, AND F. COULSTON. to high doses of monosodium Cytochemistry and ultrastruc-
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G.
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STEGINK Phenylalaninemia, produce elevation
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N., V. BUTLER, AND W. A. REYNOLDS. Brain damage in neonatal mice following high dosages of monosodium glutamate (MSG), NaCI and sucrose. Anat. Record 178 : 401, 1974 LEMKEY-JOHNSTON, N., AND W. A. REYNOI,DS. Nature and extent of brain lesions in mice related to ingestion of monosodium glutamate: a light and electron microscope study* J. Neuropathol. Exptl. Neural. 33 : 74-97, 1974. LINBLAD, B. S. The plasma aminogram in “small for dates” newborn infants. In : Metabolic Processes in the Foetus and Newborn Infant, edited by J. H. P. Jonxis, I-I. K. A. Visser, and J. A. Troelstra. Leiden : Stenfert-Kroese, 1971, p. 11 l-l 26. LTNBLAD, B. S., AND A. BALDESTEN. The normal venous plasma free amino acid levels of non-pregnant women and of mother and child during delivery. Acta Paediat. &and. 56 : 37-48, 1967. MCLAUGHLAN, J. M., F. J. NOEL, H. G. BOTTING, AND J. E. KNIPPEL. Blood and brain levels of glutamic acid in young rats given monosodium glutamate. Ahtr. Rept. Intern. 1 : 13 l-l 38, 1970. NEWMAN, A. J., R. HEYWOOD, A. K. PALMER, D. H. BARRY, F. P. EDWARDS, AND A. N. WORDEN. The administration of monosodium L-glutamate to neonatal and pregnant primates. Toxicol0,gy 1 : 197-204, 1973. OLNEY, J. W. Brain lesions, obesity and other disturbances in mice treated with monosodium glutamate. Science 164 : 719721, 1969. OLNEY, J.
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1973. OLNEY,
J* W., AND L. G. SHARPE. Brain lesions in an infant rhesus monkey treated with monosodium glutamate. Science 166 : 386-388, 1969. 21. OLNEY, J* W., L. G. SHARPE, AND R. D. FEIGIN. Glutamateinduced brain damage in infant primates. J. Neuropathol. Exptl, Neurol. 31 : 464-488, 1972. 22. PERRY, 71‘. L., AND S. HANSEN. Technical pitfalls leading to errors in the quantitation of plasma amino acids, C/in. Chim. Acta 25: 53-58, 1969. 23. PETERS, J. H., B. J. BERRIDGE, JR., W. R. CHAO, J. G. CUMMINGS, AND S. C. LIN. Amino acid patterns in the plasma of old and new world primates. Camp. Biochem. Physiol. 39B : 639-647, 20.
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24. REYNOLDS, W. A., N. LEMKEY-JOHNSTON, L. J. FILER, JR., AND R. M. PITKIN. Monosodium glutamate: absence of hypothalamic lesions after ingestion by newborn primates. Scienca 172 : 134213444, 1971. 25. REYNOLDS, W. A., N, LEMKEY-JOHNSTON, R. M. PXTKIN, L. D. STEGINK, 1;. J- FILER, JR., AND A, BINGEL. Metabolism of monosodium glutamate: contrasts in rodent and primate. Intern. Congr. Nutr.
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JOURNAL
OF PHYSIOLOGY
Vol. 229, No. 1, July
1975.
Printed in U.S. A.
Monosodium
glutamate
in the neonatal LEWIS
monkey
D. STEGINK, W. ANN M. C. BRUMMEL
AND Departments Iowa 52242;
metabolism
REYNOLDS,
L. J. FILER, JR., ROY M. PITKIN,
DAVID
P. BOAZ,
Biochemistry, Obstetrics and Gynecology, University of Iowa College of Medicine, and Department of Anatomy, University of Ih’nois Medical Center, Chicago, Illinois 60680
Iowa Gity,
of Pediatrics,
W. ANN REYNOLDS, L. J# FILER, JR., Roy P. BOAZ, AND M. C. BRUMMEL. Monosodium in the neonatal monkey. Am. j. Physiol. 229(l) 246-250. Z975.-Monosodium glutamate (MSG) administered by gastric tube to 10 infant monkeys at doses of 1-4 g/kg produced rapid increases in plasma glutamate (17- to 33-fold) and aspartate (50- to go-fold) levels. The degree of elevation was proportional to the dose administered. Levels of other amino acids were unaffected. Two of the monkeys exhibited high fasting glutamate levels and abnormal glutamate tolerance curves. Despite this apparent decreased ability to metabolize glutamate, neither these animals nor any of the others for whom morphologic studies have been previously reported demonstrated neurotoxicity. Studies using 14C-labeled glutamate indicated conversion of administered glutamate to two ninhydrin-negative compounds identified as glucose and lactate, as well as to aspartate. LEWIS
STEGINK,
ID.,
MATERIALS
h/ll. PITKIN, DAVID glutamate metabolism
aspartate;
ORAL
:
neurotoxicity
OR
SUBCUTANEOUS
ADMINISTRATION
Of large
QuantitieS
of glutamate
to the newborn mouse results in a variety of neurotoxic effects, the most marked of which is a massive hypothalamic neuronal necrosis (17). Similar studies in neonatal primates are contradictory. Although the initial report by Olney and Sharpe (20) indicated extensive glutamate-induced neuronal necrosis in a single premature primate injected with 2.7 g glutamate/kg body wt, other investigators (1, 16, 24, 34) were unable to confirm these findings, despite the ability of at least some of them (1, 12, 34) to reproduce the lesion in mice. In subsequent studies Olney et al. (19, 21) reported that the size of lesion was dose related paralleling blood glutamate concentrations and suggested that the failure of other research groups to observe glutamate-induced neuronal necrosis in the neonatal primate may have resulted from vomiting of the administered glutamate with resultant failure to produce marked elevation of blood glutamate levels. The present study reports plasma levels of glutamate and other amino acids in infant monkeys, most of whom were included in our previously reported morphologic study (24), demonstrating an absence of neuronal lesions after glutamate administration.
AND
METHODS
Infants of three closely related macaque species (M. mulatta, hf. arctoides, and 1M. irus), ranging in age from 30 min to 17 days, were studied. Ten animals were given monosodium glutamate (MSG) dissolved in distilled water (50 G/cwt/vol solution) in doses of 1, 2, or 4 g/kg, and one control animal was given only distilled water. The age, weight, dosage level, and fasting plasma glutamate concentration for each monkey are listed in Table 1. Administration was by gastric tube. The animals were tranquilized with phencyclidine hydrochloride (Sernylan, Parke-Davis) 0.5 mg/kg and kept on a heating pad during the period of study. Heparinized blood samples of 1 ml each were obtained from a catheter placed in the inferior vena cava (through a femoral vein) prior to dosing and at hourly intervals for 6 h. After centrifugation, plasma with sulfodeproteinized immediately samples were salicyclic acid and either analyzed promptly or stored at - 70°C. These conditions prevented conversion of glutamine to glutamate and pyrrolidone carboxylate, as well as loss of cystine (5, 22). At the conclusion of each experiment, cerebrospinal fluid was obtained by ventricular tap and prepared by the method of Dickinson and Hamilton (4). Amino acid analyses were done on Technicon NC-1 amino acid analyzers using the Efron buffer system (7). For seven of the animals identified in Table 1, morphologic studies by light and electron microscopic examination of the hypothalamic area have been reported previously (24). The results of morphologic studies of brain tissue from two additional animals are reported in this paper. These brains, embedded in plastic, were serially sectioned through the entire ventral hypothalamic area at 1 ,um. Methods of perfusion, sectioning, and staining have been described previously (12, 24), The monosodium-L-glutamate given assayed at greater than 99.9 76 pure by amino acid analysis, and no amino acids other than glutamate were found. In order to examine the metabolic pathways of glutamate, three of the animals listed in Table 1 (MJ, MI, and ME) were given, in addition to 1 g/kg MSG, 10 @i of [3, 4-14C]glutamate (sp act 200 mCi/pmol). Simultaneous radioactivity and amino acid analyses were done by previously described methods (28) permitting detection of both ninhydrin-positive and -negative metabolites derived In order to obtain sufficient plasma for from glutamate.
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GLUTAMATE TABLE
1.
METABOLISM
MW MG MJ MC* MF* IMP* MA* MI MO” MB” ME
* Animal previously
Age, days
arctozdes mulatta mulatta mulatta mulatta irus mulatta mulatta irus mulatta irus
0.02 1 1 2 4 6 7 8 9 14 17
in which (24).
morphologic
Weight, g
MSG Load, g/kg
Fasting Plasma Glutamate Levels, bmoI/dI)
550 510 370 450 430 300 445 490 310 480
1 4 1 1 2 4 2
62 77 20 9 15 9 9
1
15
0 4
6 11
319
1
8
studies
have
been
GLUTAMATE
reported
RESULTS
Figure 1 illustrates plasma glutamate and aspartate levels following glutamate loads of l-4 g/kg to eight infant monkeys whose fasting plasma glutamate concentration was 20 pmol/dl or less (average 12 pmol/dl). In these animals, glutamate increased 17- to 33-fold and aspartate 50- to go-fold from fasting values. Maximal elevations were attained 1-2 h after gastric administration and were proportional to the dose of glutamate given Plasma levels of amino acids other than glutamate and aspartate, including those which could potentially be derived from glutamate or aspartate, were essentially unchanged. The normal fasting glutamate level in adult monkeys has been reported to be 5-10 pmol/dl (23). Two of our infant monkeys (MH and MG) had values substantially above this level. Glutamine levels in these animals were normal, indicating that the elevated glutamate was not due to degradation of glutamine. These two animals, who were two of the youngest studied, appeared to have an .
impaired ability to metabolize glutamate. In addition to markedly elevated fasting glutamate levels (62 and 77 pmol/dl), both demonstrated abnormal glutamate tolerance curves, as illustrated in Fig. 2, in which MSG loading produced plasma glutamate levels which rose higher (by 1.5 and 3 times) and returned to normal more slowly, compared with other animals receiving an equivalent dosage. The data for aspartate levels in Fig. 2 further suggest a delay or lag in conversion of glutamate to aspartate. For example, at 1 h in monkey MH, glutamate levels were twice wr. as great and aspartate levels one-fourth as much as
7I
identification of the ninhydrin-negative metabolites, two additional adult monkeys (not included in Table 1) were given 1 g/kg MSG with 100 $Zi [3 ,4J4C]glutamate by stomach tube and exanguinated at the previously determined time-of-peak concentration of the respective compounds. Samples of deproteinized plasma were applied to an amino acid analyzer column and the eluate collected in Z-ml fractions. The radioactivity profile obtained in this manner was identical to that found with the simultaneous radioactivity-amino acid analysis technique. The fractional collections were then desalted with high-voltage electrophoresis (33), a method giving satisfactory separation of ninhydrin-negative acidic metabolites. All of the infant monkeys included in the study were observed continuously during the 6 h after dosing. One (MP) vomited an estimated 1.5 ml of clear fluid 7 min after dosing and was redosed with 1 ml of MSG solution immediately ; there was no subsequent vomiting, Four (MA, ME, MG, and MH) vomited small amounts (estimated volume less than 1 ml) of a frothy, bile-colored fluid at 35, 40, 55, and 70 min after dosing. Since considerable time had elapsed and the quantity vomited was small, these animals were not redosed.
s
247
MONKEY
leuels
Species
M. M. M. M. M. M. M. M. M. M. M.
NEONATAL
Animalsstudiedand initial fasting
plasma glutamate Animal
IN THE
0
2
4
6 HOURS
1. Plasma glutamate and aspartate levels with time followmg LISG loading: mean value from 4 animals administered 1 g/kg body wt (A); mean value from 2 animals administered 2 g/kg body wt (x) ; mean value from 2 animals administered 4 g/kg body wt (0); value in control animal administered water (e). Standard deviation not more than 2070 of mean. FIG.
- A i300 0 0 - 200 K w CT) W 1.
h-
--
1
1
-0
1
I
2
1
4
012345G HOURS
I
I
6,
2 34 HOURS
56
FIG, 2. Plasma glutamate and aspartate levels in neonatal monkeys administered A : 1 g hfSG per kg body wt-monkey MH (x); other mean + SD (0); control (A). I?; animals adneonatal monkeys, ministered 4 g MSE per kg body wt : monkey MG (o), other animals
(a> .
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248
STEGINK
means of the other animals receiving an equivalent dose of 1 g/kg. While administration of large doses of MSG resulted in marked elevation of plasma glutamate levels, none of the animals in the present study who received MSG and was studied by light and electron microscopy of the brain (24) showed evidence of either the massive or small focal lesions reported by Olney et al. (21). Representative sections of the hypothalamic area from treated and control animals are illustrated in Fig. 3.
ET
AL.
Administration of MSG (1 g/kg) with added tracer [3,4-14C]glutamate (10 FCi) to three infant monkeys permitted determination of the major glutamate-derived metabolites. Figure 4 illustrates the results of these studies involving simultaneous radioactivity-amino acid analysis. As expected, the major portion of radioactivity remained in association with glutamate. However, there was also significant incorporation of label into aspartate, glutamine, and ornithine as well as two ninhydrin-negative compounds. The two ninhydrin-negative metabolites were isolated from plasma by ion-exchange chromatography and high-voltage electrophoresis, characterized as single compounds, and identified as glucose and lactate by chemical and enzymatic methods described in detail in a previous publication (29). As is apparent in Fig. 4, glucose was the major metabolite. Radioactivity in other compounds representing potential metabolites of glutamate was also examined. No detectable radioactivity was found in succinate, pyrrolidone carboxylate, malate, citrate, or oxaloacetate. In view of the demonstration in the mouse that glutamate-induced brain damage occurs in a distribution suggesting the influx of a deleterious agent from the cerebrospinal fluid (12), we examined the cerebrospinal fluid of one monkey given MSG (1 g/kg) with added tracer [3,414C]glutamate (10 PCi). Radioactivity and amino acid composition of plasma and cerebrospinal fluid were measured 2 h after dosing. The results of this study are listed in Table 2. Comparison of incorporation of label in plasma with that found in the cerebrospinal fluid indicates that neither glutamate nor aspartate entered spinal fluid at the time of anticipated maximal plasma levels of glutamate and aspartate. Only labeled glutamine appeared at approximately the same level in both plasma and spinal fluid. In direct contrast to the amino acids, glucose and lactate
1-
_,-,-O-Q-; Lwfl_,-A-A-A-Aor” , , , , ,
,
,
,
f
2
1
a-@
asp..,
lactade
‘9
l-
FID. 3. A: section through
ventral hypothalamic region of a control infant monkey. Ventral aspect of third ventricle may be seen and below it neurons comprising infundibular nucleus (X90). B: section through ventral hypothalamic region of an infant rhesus monkey which received 4 g/kg of hlSG by stomach tube 6 h prior to brain perfusion. There is no evidence of dendritic or neuronal degeneration and dilation. This section is slightly caudal to A (X90).
/
A’
&AN ,lLA’ ,,,,, 0
1
2
A’
A’@
\a II
1
2
ziO”Ros FIG. 4. Playma distribution of radioactivity in 3 infant monkeys administered monosodium glutamate in water (1 g/kg body wt, 10 PCiL-[3,4-Wlglutamate). Mean values shown, SD did not exceed 20% of mean.
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GLUTAMATE
METABOLISM
IN
THE
NEONATAL
TABLE 2. Comparison of quantity and distribution of label in plasma and spinal fluid fallowing monosodium glutamate loading of an infant monkey Plasma
Compound
Cerebrospinal
Aspartate Glutamine Glutamate Alanine Ornithine Arginine Glucose Lactate
638 (3.3)” 195 (1.0) 10,440 (55.0) 100 (0.5) 19 (0.1) 0 5,715 (30.0) 1,854 (9.7)
0 112 (2.0)* 90 (1.5) 5 (0.1) 0 0 (76.9) 4,677 1,186 (19.5)
Total
18,961
6,080
counts
* Percent
di&ibution
levels were similar reflecting their rapid
(100) of counts
in plasma equilibration
is given
249
MONKEY
Fluid
(100)
in parentheses.
and cerebrospinal fluid, between compartments.
DISCUSSION
The principal finding of the present study is that monosodium glutamate administration produced marked elevations of plasma glutamate and aspartate levels in infant monkeys previously reported (24) to lack any evidence of neuronal damage. Thus, these data indicate that our failure to observe neurotoxic effects of MSG administration in primates was not due to vomiting or other factors resulting in a lack of elevation of plasma glutamate. Failure to observe neuronal necrosis in the infant monkey despite enormous elevations in plasma glutamate concentration is striking in view of the ease with which the lesion is produced in the infant mouse, Neuronal damage to the newborn mouse has been reported (18) under conditions which result in elevation of plasma glutamate concentration to 50 to 80 pmol/dl (30), levels approximately onefourth of those found in the present study. From these in response to observations it appears that neurotoxicity glutamate loading differs in mouse and primate. The reasons for these differences in sensitivity to glutamate are not clear. Neither glutamate nor aspartate is increased in concentration in the spinal fluid of the infant monkey, despite elevated blood levels of these amino acids. These data may be of particular importance if the hypothesis advanced by Lemkey-Johnston and Reynolds (12) is correct. These authors suggest that in the mouse there is an influx of a neurotoxic agent from cerebrospinal fluid following a glutamate load* Thus, the hypothalamus of the primate may be spared because of the failure of glutamate or aspartate to enter the cerebrospinal fluid. Most investigators have found little if any net transfer of glutamate across the blood-brain barrier in a variety of adult species, including mouse (26), rat (15, 26), and dog (9). However, it is conceivable that glutamate may be able to enter the
spinal fluid of the infant mouse. It is also possible that a glutamate metabolite could be the neurotoxic agent for the infant mouse. Preliminary studies indicate a totally different pattern of ninhydrin-negative metabolites present in the mouse following a glutamate load (25). This includes at least two major metabolites not seen in the primate. Lemkey-Johnston et al. (11) have recently found large cerebral and small hypothalamic lesions in the infant mouse following oral administration of NaCl equimolar to &g/kg doses of glutamate. These new findings implicate hyperosmolarity as a potential causative factor in the production of central nervous system lesions following glutamate administration. The unusual glutamate tolerance curves observed for the two infant monkeys whose fasting plasma glutamate levels were elevated are of interest. It is possible that the elevated fasting levels and abnormal glutamate tolerance in the two animals, who were the youngest studied, represent an unusual maturational lag in an enzyme system for Maturational lags in enzyme glutamate metabolism. systems metabolizing amino acids are known to occur in the human infant, particularly those of low birth weight Specific examples of delayed maturation al induction include: the delay in enzyme systems catabolizing tyrosine, resulting in the transient tyrosinemia of the premature (3, 6); the lag in the development of a part of the phenylalain a transient hypernine hydroxylase system, resulting phenylalanemia (2, 3 1) ; and the lag in cystathionase (32), which makes cysteine an essential amino acid for the human fetus and premature infant (27). On the other hand, maturational studies on the monkey (10) and human (5, 8, 14), including the low-birth-weight infant (6, 13), have failed to indicate an increase in plasma glutamate level during the neonatal period. The present study suggests that the pathway of glutamate metabolism in the infant primate is as follows: ingested glutamate is rapidly removed from portal bIood by the liver where, once inside the liver cell, it enters the Mitochondrial glutamate is converted to mitochondria. cr-ketoglutarate and other tricarboxylic acid cycle components, principally malate and oxalacetate+ Oxalacetate remains within the mitochondria and is transaminated to aspartate, which is then released into the peripheral blood. Malate diffuses into the cytoplasm to be converted to phosphoenolpyruvate and thence to glucose and through pyruvate to lactate. Thus, in the peripheral blood, glutamate and aspartate levels are increased markedly with lessened increase in concentration of glucose and lactate. These studies were supported in part by Grants-In-Aid from the Gerber Products Company and the International Minerals and Chemical Corporation. R. M. Pitkin is an Academic Career Development Awardee in Nutrition, National Institute of Arthritis, hfetabolism, and Digestive Diseases, KO7 Ahl 70694. Received
for publication
4 February
1974.
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