Kinetics of monosodium glutamate in relation to its neurotoxicity

123

Toxicology Letters, l(l977) 123-130 o ElsevierjNorth-Holland Biomedical Press

KINETICS OF MONOSODIUM NEUROTOXICITY

A. BIZZI, E. VENERONI,

GLUTAMATE

IN RELATION TO ITS

M. SALMONA and S. GARA’M’INI

Zstituto di Ricerche Farmacologiche

“‘Mario Negri”, Via Eritrea, 62, 20157 Milan (Italy)

(Received June 27th, 1977) (Accepted July 13th, 1977)

SUMMARY

Glutamic acid (GA) plasma levels were measured after oral administration of monosodium glutamate (MSG) to laboratory animals, in relation to several variables. Adult mice and rats show similar GA peak plasma levels, area under the curve (AUC) and apparent half-life (TX), while in guinea pigs the values for all these kinetic parameters are higher. Newborn rats and, to a lesser extent, newborn mice show a larger AUC and T% than the adults, while the opposite was observed in guinea pigs. Within a given animal species the peak level and AUC, but not the TX, increase with the dose of MSG and with the concentration at which the dose is administered. In human volunteers oral administration of MSG (60 mg/kg) results in higher plasma peak levels and AUC when MSG is given at 8% as compared to 2% solution. Mouse brain and guinea pig brain GA levels are not affected by oral MSG until the GA plasma level exceeds the basal plasma concentrations by a factor of about 20. These data are utilized to discuss the relevance for man of the neurotoxic effects induced in rodents by MSG.

INTRODUCTION

Toxicological observations made in laboratory animals need considerable interpretation as regards the extent to which they can be extrapolated to man. Since all compounds, even those considered most “innocuous”, may show some form of toxicity in suitable experimental conditions, attention has always centered on the dose at which toxicity is observed. However, the dose may not be the best parameter to extrapolate results from animals to man, since it is now well documented that different animal species absorb, Abbreviations: AUC, area under the curve; GA, glutamic acid; MSG, monosodium glutamate.

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distribute, metabolize and excrete chemicals each in their own characteristic manner. Therefore it is becoming more realistic to consider as a suitable parameter for extrapolation not the dose but the plasma or tissue concentration at which a chemical shows toxic effects. What should be ideal would be to know the concentration of chemicals and/or their toxic metabolites at the exact sites where toxic effects occur, but it is more practical, with a view to comparison with man, to establish the blood concentrations at which chemicals produce toxic effects [ 11. These principles are applied here to interpret the relevance of Olney’s findings on the neurotoxic effects of glutamic acid [g---14]. METHODS

CD1 mice, CD rats (Charles River, Italy) and albino guinea pigs of both sexes and different ages were used. Animals received food (Altromid pellets) and water ad libitum and were housed in plastic cages with sawdust bedding under standard conditions (temperature 22”C, relative humidity 60% and light and dark cycles of 12 h each). MSG was given orally, at various doses, volumes and concentrations as reported in the text. MSG was given to newborn animals through a plastic tube connected to a microsyringe. Groups of at least 4 animals were used. In the case of newborn mice the plasma from 2 animals was pooled so those groups contained 8 animals each. At set times (usually 0, 5, 30, 60, 90, 120, 240 and 480 min) after administration of the amino acid, animals were decapitated and their brains and plasma collected for analysis of GA, which was determined by an enzymatic method as described by Bernt and Bergmeyer [2]. Kinetic calculations were performed according to NONLIN [ 71, RESULTS

1. Basal levels during development Preliminary investigations indicated that the basal concentration of GA did not substantially change as a result of overnight fasting, so no reference will be made to the nutritional conditions of the control animals. Similarly, no significant differences were found between the plasma or brain GA levels in males and females. Table I indicates that the GA plasma levels in mice, rats and guinea pigs were not markedly different. Furthermore, they are not influenced by growth, the levels in newborns and adults being similar, with the possible exception of the high GA levels in plasma of newborn rats the 1st day of life. In mice and rats brain GA was low at birth and increased with age while in guinea pigs the brain levels were similar at and after birth. These findings are in agreement with the report that guinea pigs are born with a more mature brain than mice and rats [ 41. It should be recalled that human newborns are also considered relatively more mature in their brain myelinization than rats and mice.

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TABLE

I

LEVELS SPECIES Animal

OF GLUTAMIC AT DIFFERENT species

Mouse Mouse Rat Rat Rat Rat Guinea pig Guinea pig Guinea pig Guinea pig

ACID IN PLASMA AGES

Age (days)

Plasma ~moles/ml

7 Adults 1 I 15 Adults 1 7 15 Adults

0.15 0.17 0.25 0.20 0.17 0.15 0.19 0.17 0.23 0.20

aP < 0.01 compared Dunnett tests).

f * f * f * * + ? *

AND BRAIN

+ S.E.

Brain rmoles/g 4.42 8.21 3.21 4.83 6.50 8.05 8.01 8.04 9.15 7.56

0.01 0.01 0.02 0.01 0.01 0.02 0.01 0.01 0.01 0.01

OF THREE

+ 2 * r f + * + + f

to values in adults of the same species

ANIMAL

f S.E. 0.18* 0.21 0.14a 0.14a 0.06a 0.25 0.19 0.15 0.41 0.15

(Student’s

t and

2. Kinetics of glutamic acid in new horns and adults Oral administration of a standard dose of MSG (1 g/kg) to all three species tested (mice, rats and guinea pigs) led to plasma GA levels over 10 times the basal values. However, as summarized in Table II, there are quantitative differences which depend on the animal species and on age. For rats and to a lesser extent, for mice too, the plasma GA levels expressed as AUC are higher for newborns than adults, whereas for guinea pigs the opposite holds true. This confirms the importance of maturity at birth as a factor in the ability to dispose GA. TABLE

II

KINETIC PARAMETERS FOR GLUTAMIC ACID IN NEWBORN AND ADULT RAT AND GUINEA PIGS AFTER ORAL ADMINISTRATION OF 1 g/kg OF MONOSODIUM GLUTAMATE AT A CONCENTRATION OF 10% w/v Animal species

Mouse Mouse Rat Rat Guinea Guinea AUC,

Age (days)

pig pig

7 Adults 7 Adults 7 Adults

Plasma peak level (kmoles/ml 2.10 2.08 2.26 1.91 1.91 2.28

+ S.E.)

+ 0.06 5 0.02 f 0.36 +_0.09 + 0.02 f 0.03

plasma area under the curve; T%, apparent

Plasma AUC (fimoles/ml 315 309 1.085 288 321 487 plasma half-life.

X min)

MICE,

Plasma T% (min) 111 98 231 88 101 173

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Data not reported here in detail indicate that GA levels are similar in arterial (aorta) and venous (jugular vein) plasma; the ratio between erythrocyte and plasma levels is about 1 in both the arterial and venous compartments, either in basal conditions or after oral administration of MSG. Table III shows that the plasma AUC, but not the TX, increases linearly for newborn mice as a function of the oral MSG dose. The concentration at which MSG is given is also an important factor in the kinetics of GA. At equal doses the MSG concentration determines the volume in which it is given and hence the time required to administer the dose orally to newborn animals. Table IV confirms that the plasma peak rises for newborn mice and rats as the concentration at which MSG is given increases. The same dose of MSG (1 g/kg) given to rats leads to a five-fold increase in the plasma AUC when the concentration increases from 2 to 10%. Similarly in mice, giving an oral dose of 0.5 g/kg at concentrations from 2 to 20%, the AUC increases by a factor of about 2.5. Similar results were obtained in adult animals. TABLE

III

KINETIC PARAMETERS ORAL ADMINISTRATION Animal species

Mouse Mouse Mouse

OF PLASMA GLUTAMIC ACID IN ‘I-DAY-OLD OF DIFFERENT DOSES OF MONOSODIUM

Dose of MSG

Plasma peak level

Plasma AUC

(g/kg

&moles/ml + S.E.)

(pmoles/ml

0.72 f 0.04 1.07 f 0.02 2.10 t 0.05

103 199 375

oral)

0.25 0.50 1.00

x min)

MICE AFTER GLUTAMATb Plasma T% (min) 79 71 77

____~ aThe concentration

TABLE

was constant

(10% w/v).

IV

KINETIC PARAMETERS OF PLASMA GLUTAMIC ACID IN 7-DAY-OLD MICE AND RATS AFTER ORAL ADMINISTRATION OF MONOSODIUM GLUTAMATE AT DIFFERENT CONCENTRATIONS Animal species

Mouse Mouse Mouse Mouse Rat Rat Rat

Dose

Concentration

(g/kg)

% (w/v)

0.5 0.5 0.5 0.5 1.0 1.0 1.0

2 5 10 20 2 5 10

Plasma peak level (Mmoles/ml 0.82 0.86 1.08 1.43 0.97 2.26 2.89

i: ?; f + f * +

0.03 0.04 0.02 0.02 0.05 0.36 0.11

* S.E.)

Plasma AUC (rmoleslml 126 147 200 318 275 1085 1391

Plasma T% x min)

(min) 88 117 71 106 183 237 241

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3. Levels of glutamic acid in the brain Throughout the variety of conditions described in the previous paragraph, the levels of brain GA remained unchanged in both mice and rats, adults and newborns, even when GA plasma levels were up to 15 times the basal concentrations. Rat brain GA concentration significantly increased, by about 20%, only when the oral dose of MSG was raised to 2 g/kg (20% w/v) and peak plasma GA exceeded the basal level by about 19 times. The fact that the level of brain GA was not changed even by doses of MSG which can induce brain damage [ 121 does not exclude the possibility - as suggested by Perez and Olney [16] - that GA may increase in a small brain area such as the arcuate nucleus of the hypothalamus where the amino acid has been seen to produce toxic effects. However, recent data [ 51 indicate that in adult rats GA does not substantially increase in the retina, another tissue where it causes damage [3]. 4. Plasma glutamic acid levels in man The levels of plasma GA in man are somewhat lower than in the rodents examined in this study. An oral dose of MSG (60 mg/kg) to overnight fasted volunteers (both sexes, age 20-30 years) induces a measurable increase of plasma GA. When MSG is given at a concentration of 8% the increase is greater than at 2%, the AUC with the higher concentration being about 2.5 times that with the lower (see Table V). A single dose of 60 mg/kg would be relatively high for any food enriched with MSG. The 8% concentration is also a limit because the volunteers showed various adverse gastrointestinal reactions. Higher concentrations would probably result in vomiting. The adult levels of plasma GA could not be compared with human infant levels for ethical reasons. However, the scant data avail-

TABLE

V

PLASMA LEVELS OF GLUTAMIC ACID IN FOUR HUMAN VOLUNTEERS AFTER ORAL ADMINISTRATION OF MONOSODIUM GLUTAMATE AT THE DOSE OF 60 mg/kg AND CONCENTRATIONS OF 2 OR 8% (w/v) Time after administration (min)

Plasma levels (rmoles/ml 2%

8%

0 5 10 15 30 60 90 120

0.09 + 0.01 0.11 * 0.01 0.15 + 0.02 0.17 + 0.03 0.27 + 0.01 0.12 * 0.01 0.11 f 0.01 0.09 * 0.01

0.07 + 0.01 0.20 f 0.02 0.22 f 0.02 0.24 f 0.03 0.31 + 0.06 0.20 ?r 0.04 0.19 * 0.01 0.13 * 0.03

AUC (Mmoles/mlX

min)

6.7

+ S.E.) of MSG

-

16.9

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able [18] suggest that infants probably do not differ from adults when the dose of MSG does not exceed 60 mg/kg. DISCUSSION

1. Significance of kinetic data for the toxicity of glutamic acid The present data might help us interpret the incidence of hypothalamic lesions observed in newborn animals [ 9,111. The discussion will be restricted to mice since this species is the most sensitive to the toxicity of MSG and has therefore been widely tested. Table VI summarizes the percentage of mice with hypothalamic lesions, according to several authors who used various doses and concentrations of MSG. The toxicological data compared with the plasma AUC obtained under similar conditions show that the percentage of lesions is related more to the plasma AUC than to the dose of MSG administered. For instance, the same dose of MSG (0.5 g/kg) can give 50% lesions if used at a 20% concentration or no lesions when employed at 2 or 5%. The total exposure to MSG, therefore, expressed in this study by the AUC value, may be a better index of toxicity than the administered dose. Within the limits of the present results, it looks as though plasma exposure to MSG over about 150 @g/ml X min may result in hypothalamic damage.

TABLE

VI

CORRELATIONS BETWEEN HYPOTHALAMIC LESIONS AND PLASMA AREA UNDER THE CURVE (AUC) IN NEWBORN MICE AFTER ADMINISTRATION OF MONOSODIUM GLUTAMATE AT VARIOUS DOSES AND CONCENTRATIONS Dose (g/kg,

Concentration oral)

0% w/v),

Hypothalamic lesions (a)

Plasma AUC (rmoleslml

0.25 0.50 0.50 0.50 0.50 0.75 0.75 1.00 1.00

10 2 5 10 20 20 2 2 5

Ob 0a 0a 20a 50b 75b 40a 50a 80qc

103 126 147 200 318 -

1.00 1.00

10 20

1OOb

375 -

aHeywood et al., personal communication. bOlney [lo]. c Lemkey-Johnston and Reynolds [ 61.

X min)

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2. Relevance of glutamic acid neurotoxicity

in man

Necrotic lesions of the hypothalamus have been found as a result of the administration of MSG to newborn animals [9,11-141. The mouse appears to be the most sensitive species, followed by rats and guinea pigs (Worden, personal communication). At variance with initial observations [ 141 in a variety of experimental conditions, monkeys are apparently insensitive to this toxic effect of MSG [8,17,19]. When any particular toxic effect differs among various animal species, in the absence of other criteria the safest attitude is to take the most sensitive species as initial basis for extrapolation to man. If this is done for MSG it may be calculated according to Olney and Ho [12] that a safe oral dose of MSG for mice is around 250 mg/kg. This is only about four times the largest single intake (60 mg/kg) of free MSG likely with food according to western habits. The safety margin thus appears to be very low and certainly not acceptable. However, analysis of the experimental conditions used to establish the neurotoxic effect of MSG shows that the effect of a given dose is related to the concentration at which it is administered. It may thus be possible to establish that a dose of 500 mg/kg at 5% is not neurotoxic for newborn mice and, if so, the safety ratio for man can be raised to a factor of about 8. However if plasma levels, not doses, are considered, it is evident that the higher the concentration of MSG administered, the greater the total plasma exposure (AUC) to MSG at equal doses. When the safety ratio is calculated from the plasma AUC in newborn mice after a dose of 500 mg/kg at 5% against the AUC in man after a dose of 60 mg/kg at 2%, the factor becomes about 22. This is obviously much more reassuring in considering possible neurotoxicity of MSG in man. In conclusion, therefore, although Olney’s findings are extremely interesting from a toxicological point of view, it is fortunate for man that they were obtained by selecting a number of factors (animal species, age, maturity, dose and concentration of MSG) which all tend to induce high and long-lasting plasma levels of MSG. REFERENCES 1 S.B. de C Baker and D.M. Foulkes, Blood concentrations of compounds in animal toxicity tests, in D.S. Davies and B.N. Prichard (Eds.), Biological Effects of Drugs in Relation to Their Plasma Concentrations, McMillan, London, 1973, p. 41. 2 E. Bernt and H.U. Bergmeyer, UV assay with glutamate dehydrogenase and NAD, in H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Vol. 4, Academic Press, New York, 1974, p. 1704. 3 A.I. Cohen, An electron microscopic study of the modification by monosodium glutamat of the retinas of normal and “rodless” mice, Am. J. Anat., 120 (1967) 319. 4 J. Folch-Pi, Composition of the brain in relation to maturation, in H. Waelsch (Ed.), Biochemistry of the Developing Nervous System, Academic Press, New York, 1955, p. 121. 5 J. Liebschutz, L. Airoldi, G.N. Chinn, R.J. Wurtman and M.J. Brownstein, Regional distribution of endogenous and parenteral glutamate, glutamine, aspartate in rat brain, Biochem. Pharmacol., (1977) in press.

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6 N. Lemkey-Johnston and W.A. Reynolds, Incidence and extent of brain lesions in mice following ingestion of monosodium glutamate (MSG), Anat. Rec., 172 (1972) 354. 7 C.M. Metzler, NONLIN. A computer program for parameter estimation in nonlinear situations, Upjohn Co., Kalamazoo, Mich., 1969. 8 A.J. Newman, R. Heywood, A.K. Palmer, D.H. Barry, F.P. Edwards and A.N. Worden, The administration of monosodium i-glutamate to neonatal and pregnant rhesus monkeys, Toxicology, 1 (1973) 197. 9 J.W. Olney, Brain lesions, obesity, and other disturbances in mice treated with monosodium glutamate, Science, 164 (1969) 719. 10 J.W. Olney, Glutamate-induced retinal degeneration in neonatal mice. Electron microscopy of the acutely evolving lesion, J. Neuropath. Exp. Neurol., 28 (1969) 455. 11 J.W. Olney, Glutamate-induced neuronal necrosis in the infant hypothalamus. An electron microscopic study, J. Neuropath. Exp. Neurol., 30 (1971) 75. 12 J.W. Olney and O.L. Ho, Brain damage in infant mice following oral intake of glutamate, aspartate or cysteine, Nature (London), 227 (1970) 609. 13 J.W. Olney, O.L. Ho and V. Rhee, Cytotoxic effects of acidic and sulphur containing amino/acids on the infant mouse central nervous system, Exp. Brain Res., 14 (1971) 61. 14 J.W. Olney, L.G. Sharpe and R.D. Feigin, Glutamate-induced brain damage in infant primates, J. Neuropath. Exp. Neurol., 31 (1972) 464. 15 B.L. Oser, K.M. Morgareidge and S. Carson, Monosodium glutamate studies in four species of neonatal and infant animals, Food Cosmet. Toxicol., 13 (1975) 7. 16 V. J. Perez and J.W. Olney, Accumulation of glutamic acid in the arcuate nucleus of the hypothalamus of the infant mouse following subcutaneous administration of monosodium glutamate, J. Neurochem., 19 (1972) 1777. 17 W.A. Reynolds, N. Lemkey-Johnston, L.J. Filer Jr. and R.M. Pitkin, Monosodium glutamate: Absence of hypothalamic lesions after ingestion by newborn primates, Science, 172 (1971) 1342. 18 L.D. Stegink and G.L. Baker, Infusion of protein hydrolysates in the newborn infant: Plasma amino acid concentrations, J. Pediat., 78 (1971) 595. 19 C. Wenn, C.H. Kenneth and S.N. Gershoff, Effects of dietary supplementation of monosodium glutamate on infant monkeys, weanling rats and suckling mice, Am. J. Clin. Nutr., 26 (1973) 803.