THE AMMONIA AND GLUTAMINE CONTENT OF THE BRAIN

BY DEREK RICHTER AND REX M. C. DAWSON

(From the Neuropsychiatric Research Centre, Whitchurch Hospital, Cardiff, England)

(Received for publication, July 29, 1948)

The values given in the literature for the ammonia content of the brain show wide variation (l-3). Most of these values are of doubtful relia- bility, since the methods of estimation have not generally taken into ac- count the presence of labile acid amides such as glutamine, which are now known to occur in the brain and which easily liberate ammonia on treat- ment with alkalies under the conditions commonly used for the estimation of ammonia (4,5). Interest in the brain ammonia level centers around the fact that ammonia is a powerful cerebral irritant; the administration of ammonium salts causes convulsions and it has long been thought that ammonia may play a part in the precipitation of epileptic seizures (3-6). It would appear that the brain ammonia level is closely related to the glutamic acid-glutaminase system, which can remove ammonia by combi- nation with glutamic acid to form glutamine (7). The report that glutamic acid inhibits ammonium chloride convulsions (8) may be significant in this connection, since it is known to reduce the incidence of attacks of petit ma1 in epileptics (9).

The present paper reports an attempt to obtain reliable values for the ammonia content of rat brain with a method by which the free ammonia can be distinguished from the labile acid amide ammonia. At the same time a study has been made of the effect on the brain ammonia level of convulsant drugs and other factors which might affect it.

EXPERIMENTAL

Ammonia Estimation-Ammonia was estimated by Conway’s method (10) of microdiffusion analysis. The acid used was 0.0004 N HCl con- taining Tashiro’s indicator, prepared as described by him (1 I). The baryta solution :O.OOlS N) was delivered from an Agla micrometer syringe (12), which was capable of delivering as little as 0.0001 ml. or considerably less than the error inherent in detecting the indicator change at these dilutions. The stock baryta solution was kept in a rubber-capped bottle of the type used for keeping sterile solutions for injection, which gave a convenient method of refilling the syringe without contamination by COZ in the air; the solution was restandardized each day. Preliminary tests with pure crystalline glutamine showed that the rate of liberation of ammonia by

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hydrolysis was 0.001 mg. of a.mmonia per mg. of glutamine per hour in contact with 50 per cent-saturated K&O3 at 20” under the experimental conditions of the Conway method. The glutamine in the brain filtrate was determined separately and a correction was calculated on this basis for the brain ammonia figures. The magnitude of the correction was kept low by using a diffusion time of 1 hour. Glutamine is relatively stable at neutral pH at O”, but it was found to hydrolyze at an appreciable rate, 0.00042 mg. of NH3 per mg. of glutamine per hour being liberated at a room temperature of 18’ in contact with 10 per cent trichloroacetic acid, which was used at approximately this strength in the brain filtrates; the error due to this cause was reduced to a negligible figure by carrying out the ammonia est,imations without delay and keeping the solution at 0” with ice. An estimation on a standard solution of ammonium chloride was included in each series.

Glutamine Estimation-Glutamine was estimated by the method of Harris (13), which depends on estimating the ammonia liberated on hydrolysis under standard conditions with 10 per cent trichloroacetic acid at 70” for 75 minutes. Preliminary tests confirmed the observation of Harris that under these conditions a solution containing 50 mg. per cent of pure glu- tamine was hydrolyzed approximately 100 per cent; under the same con- ditions asparagine was hydrolyzed 11 per cent, acetamide 69 per cent, and nicotinamide 4 per cent. There was no appreciable liberation of ammo- nia from n-arginine, guanidine, guanine, adenine, creatine, glutamic acid, or metrazol, but urea liberated 0.002 mg. of NH8 per mg. under these condi- tions. Although crystalline glutamine has been isolated from horse brain (14), it was not known to what extent other labile acid amides might be present in the brain. To obtain further evidence on this point, measure- ments were made of the rates of hydrolysis of the labile acid amides of the brain filtrates and the figures were compared with the rates of hydrolysis of pure glutamine solutions under the same conditions. Four experiments in which a comparison was made of the amount of ammonia liberated from the brain filtrate and from an equivalent amount of pure glutamine on hydrolysis by 50 per cent-saturated K&O3 at 20” are summarized inTable I.

Hydrolysis curves for brain filtrates under acid conditions, carried out as described by Harris (13), also showed good agreement with the curves for pure glutamine, the specific reaction rate k being 7.7 X lob4 in each case. This gave evidence that the labile acid amide of the brain is mainly, if not wholly, glutamine, and the results are therefore expressed in terms of this substance.

Experimental Animals-In most of the work, young Wistar albino rats weighing 25 to 40 gm. were used. They were killed by being dropped into liquid air, which froze them solid in a few seconds and produced a rapid

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D. RICHTER AND R. M. C. DAWSON 1201

fixation of ,the metabolites in the brain. The frozen brain was rapidly removed, crushed to a powder in a cooled steel crusher, and transferred to a previously weighed centrifuge tube containing 4 ml. of 12 per cent tri- chloroacetic acid at 0’. The mixture was centrifuged and the estimations were carried out on the supernatant solution. An allowance was made in the calculations for the water content of the brain, which was taken as 80 per cent of the weight of the fresh tissue.

In the experiments in which figures for the blood ammonia content were required, larger rats of 200 to 300 gm. were used and were killed by decapi- tation. The heads were dropped into liquid air and treated in the same way as for the young rats, while blood samples of 0.5 to 1 ml. were pipetted from the carotid artery. The method of killing by decapitation was not entirely satisfactory, for it was found that the stimulus of decapitation was sufficient in itself to cause a rise in the brain ammonia content; but this

TABLE I Comparison of Ammonia Liberated from Brain Filtrate and from Equivalent Amount

of Pure Glutamine

Rat No. Brain ammonia Brain glutamine

mng. )CI cent tug. #Cf c.M hrs. n&g. )I?? cent mg. per Cm&l

0.59 89 2 0.16 0.18 0.59 89 5 0.47 0.45 0.54 91 2 0.18 0.18 0.59 91 5 0.45 0.46

Time of Ammonia from Ammonia from hydrolysis brain filtrate pure glutamino

-

was the best way in which a blood sample large enough for the ammonia determination could be obtained at the time of fixation of the brain. The rise in the brain ammonia after decapitation continued for about 5 seconds, but by allowing exactly 1 second (timed with a stop-watch) from the mo- ment of decapitation to the commencement of freezing in liquid air, fairly consistent figures could be obtained. In this way changes in the brain ammonia content due to other factors could be demonstrated. Immersion in liquid air must also act as a cerebral stimulus, for it takes several seconds for the brain to freeze right through,’ but there is evidence that freezing of the intact brain in liquid air is a less violent stimulus than decapitation (15, 16).

Results

The values obtained for the ammonia and glutamine content of the brain of normal rats are listed in Table II. The mean figure of 0.28 mg.

* Richter, D., and Dawson, R. M. C., Am. J. Physiol., in press.

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1202 BRAIN AMMONIA

per cent for the ammonia content was considerably lower than the value 2.2 mg. per cent of N previously reported by Biilow and Holmes (1) for the mouse brain. The lower figure obtained in the present work may be attributed partly to the improved method of fixing in liquid air and partly to the correction for ammonia present as glutamine. The mean figure of 79 mg. per cent of glutamine is close to the value of 64 mg. per cent re- ported by Hamilton (5) for the dog brain.

Emotional Excitement-The recent observation that the brain lactic acid content is increased in emotional excitement (17) suggested that there may also be a liberation of ammonia under these conditions. A series of nine rats was excited by removing their support and allowing them to drop from side to side in a glass beaker for 4 minutes. The method has been

TABLE II

Ammonia and Glutamine Content of Rat Brain

The rats were killed by immersion in liquid air.

Rat No. Weight Ammonia Glutamine

gm. mg. per cent 35 0.28 25 0.02 25 0.34 30 0.14 30 0.35 35 0.42 35 0.32 40 0.25 30 0.37

mg. per cent 62 73

86 81 85 88

/ Mean. . . . , . . , . .I 32 I

0.28 I

79

described more fully e1sewhere.l The results, which are listed in Table III, showed a mean ammonia content of 0.22 mg. per cent, which was not sig- nificantly different from the normal. There was also no change in the glutamine content.

Picrotoxin Convulsions-Rats killed after convulsions induced by injec- tion of picrotoxin gave a mean value of 0.47 mg. per cent of ammonia, which was 74 per cent above the normal. The difference was statistically significant (P < 0.05 when tested by Fisher’s t test). There was no sig- nificant change in the glutamine level.

E$ect of Decapitation-Rats killed by decapitation gave a higher am- monia content than those killed by immersion in liquid air; this was true for young rats of 30 to 40 gm. as well as for adult rats of 200 to 300 gm. (Table IV). The rise in the brain ammonia occurred in the first few seconds

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D. RICHTER AND R. M. C. DAWSON 1203

TABLE III

Effect of (a) Emotional Excitement and (b) Picrotoxin Convulsions on Ammonia and Glutamine Content of Rat Brain

The rats weighed 35 to 40 gm. (a) The rats were excited for 4 minutes. (5) Pic- rotoxin convulsions were produced by intraperitoneal injection of 0.25 ml. of a solution containing 3 mg. per ml. of picrotoxin in 10 per cent alcohol. All the rats were killed by immersion in liquid air.

Emotional excitement Picrotoxin convulsions

-7 -- Rat No.

- - Ammonia Glutamine Ammonia Glutamine Duration of

convulsions Rat No.

1 2 3 4 5 6 7 8 9

___- Mean....

I-

mg. per cent

0.23 0.26 0.20 0.14 0.22 0.22

0.27

mg. $5er cent

70 75

78 82 92

min. mg. )‘37 cent mg. ger cent

8 0.57 72 10 0.44 93 5 0.39 68 5 0.46 65 3 0.52 62 3.5 0.60 88 3 0.31 89

10 11 12 13 14 15 16

0.22 79 0.47 77

TABLE IV

Effect of Decapitation on Ammonia Content of Blood and Brain in (a) Young Rats and (b) Adult Rats

Freezing of the heads in liquid air commenced 1 second after decapitation. (a) The young rats weighed 30 to 40 gm. (b) The adult rats weighed 200 to 300 gm.

Young rats Adult rats

Brain ammonia 7 Blood ammonia r . -

_-

Blood ammonia Brain ammonia

mg. per cent

0.54

0.35 0.50 0.60 0.53

0.29 _--__

Rat No.

9

10

11 12 13 14 15 16

-__-

Rat No.

1 2 3 4 5 6 7 8

I-

1

mg. #er cent

0.35 0.23

0.19 0.19

0.32

0.57 0.58 0.53 0.46 0.33 0.33

mg. per ceni

0.19 0.19

0.13

Mean...... 0.47 I 0.26 I I 0.47 / 0.17

after decapitation. Analyses carried out on a series of six rats examined from 4 to 30 seconds after decapitation gave figures ranging from 0.76 to

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1.05 mg. per cent of ammonia with a mean of 0.93 mg. per cent, but by reducing the interval between decapitation and fixing in liquid air lower values were obtained (Table V). With an interval of 1 second between decapitation and freezing in liquid air a mean value of 0.47 mg. per cent of ammonia was obtained and this was adopted as a standard procedure in the experiments with adult rats, which were always killed by decapita- tion.

TABLE V

Ammonia Content of Rat Brain (a) after Anoxia in Viva and (b) at Various Times after Decapitation

In series (a) young rats of 30 to 40 gm. were rendered anoxic by keeping them for 1 minute in nitrogen containing 5 per cent COz; they were killed by decapitation and the heads were frozen after 1 second. The brain ammonia figures should be com- pared with the mean of 0.47 mg. per cent for normal, decapitated rats. In series (5) the heads of rats of 200 to 300 gm. were transferred to liquid air at varying times after decapitation. The heads were kept at 37”.

I AXE&I

Brain Blood ammonia ammonia

nag. per cent

0.64 0.96 0.74 0.54 1.18

mg. per ten

0.46 0.80

0.65 0.16

Remarks

Strong convulsions Slight tremors Brief convulsion Preconvulsive

‘I

Change after decapitation

T Rat No.

6 7 8 9

10 11 12 13 14 15 16

----- -

Time after de- Brain capitation ammonia

sec. mg. per cent

3 0.29

1 0.33 16 0.62 2 0.86 3 0.77 4 0.76 4 1.05 5 0.92

10 0.94 15 0.92 30 1.01

Mean.. 0.81 0.52

Effect of Anoxia-Rats kept for 1 minute in a desiccator containing ni- trogen with 5 per cent COZ showed a marked increase in the brain ammonia content (Table V) ; this was observed in animals which had convulsions and also in rats which had not had convulsions but were in the precon- vulsive state. It is known that anoxia causes an increase in the blood am- monia level (18) and this may account for a part of the ammonia found in anoxia in the brain; but it is likely that the blood ammonia comes from the brain and other tissues rather than that the tissue ammonia comes from the blood.

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D. RICHTER AND R. M. C. DAWSON 1205

E$ect of Narcosis-Rats anesthetized for 30 minutes or longer with nem- butal showed a marked fall in the brain ammonia content, the mean level for young rats killed by immersion in liquid air being 0.06 mg. per cent of ammonia. The figures, which are listed in Table VI, give evidence that the brain ammonia depends on the duration of the narcosis, for two ani- mals anesthetized for shorter periods of 10 and 17 minutes showed no sig- nificant change from the normal. This may be the explanation of the

TABLE VI

Effect of Nembutal Narcosis on Ammonia and Glutamine Content of Rat Bruin

The brain ammonia figures should be compared with the mean of 0.47 mg. per cent for normal, decapitated rats and 0.28 mg. per cent for normal rats killed by immersion in liquid air.

Rat No. Weight of rat Method of killing Duration of narcosis

Brain ammonia

Brain glutamine

b-m. min. mg. ger ceni mg. per cent

1 200-300 Decapitation 30 0.15 86 2 200-300 “ 30 0.26 s4 3 200-300 ‘I 55 0.13 79 4 200-300 “ 65 0.23 83

Mean............................................... 0.19

5 35-40 Liquid air 30 0.05 6 35-40 “ “ 45 0.06 7 35-40 “ “ 70 0.15 8 35-40 “ “ 80 0.02 9 35-40 “ “ 90 0.01

Mean............................................... 0.06

10 11

negative findings of Btilow and Holmes (l), who found that narcosis had no effect on the brain ammonia level,

E$ect of Electrical Xtimulation-Electrical stimulation of the brain was carried out with stainless steel electrodes of 0.25 sq. cm. area, which were applied to the scalp 0.5 cm. posterior to the eyes. Contact was obtained by cutting the fur in this region with sharp scissors and applying electrode jelly. The current used was 50 cycles A.C. at 40 volts. Stimulation for 1 to 3 seconds produced a satisfactory convulsion after the usual latent period of about 10 seconds. The procedure was similar to that used in the “electroshock” treatment of psychiatric patients. In the experiments

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listed in Table VII the rats were sacrificed while undergoing strong con- vulsions and the figures showed a brain ammonia content above the normal.

Brain Ammonia in Preconvulsive State-The experiments on stimulation by anoxia showed a rise in the brain ammonia level in the preconvulsive state, before the onset of convulsions, which suggested that the rise in brain ammonia might be associated with the increased cerebral irritability rather than with the actual convulsions. The further experiments given in Table VIII confirmed this impression, showing a clear rise in the brain

TABLE VII Ammonia Content of Rat Brain during Convulsions Induced (a) by Electrical

Stimulation and (b) by Injection of Ammonium Chloride

The rats were killed by immersion in liquid air or decapitation on the first strong convulsion. The heads of decapitated rats were transferred to liquid air in 1 second. The ammonia content of controls killed by liquid air was 0.27 and killed by decapita- tion 0.47 mg. per cent. Ammonium chloride convulsions were induced by intra- peritoneal injection of 1.2 ml. of 20 per cent NH&l.

Convulsions

Electric shock

1 a $ky Method of killing

I

m.

35-40 Liquid air 35-40 “ “

200-300 Decapitation 200300 “ 200-300 “ 200-300, “

I

sec.

2 3 1 1 3 3

Meandecapitatedrats only..........................

Ammonium chloride

i

WT. per cent

0.40 0.49 0.58 0.75 0.78 0.94

Brain Brain .mmonia g lutamine

w. )e7 cent

86 76 79 82

0.76 _

8.0 70 8.3 9.5 93 9.8 9.2 101 10.0

Blood ammonia

*eY%nt

1.42 0.69

ammonia level in animals sacrificed 1 second after electrical stimulation and therefore several seconds before the onset of convulsions. Estima- tions of blood ammonia carried out on blood from the carotid arteries of rats killed by decapitation showed a rise in the blood ammonia during electri- cally induced convulsions; this was to be expected, since it is known that ammonia is liberated in the muscles and enters the blood during severe muscular activity (18). Determinations of blood ammonia on rats in the preconvulsive state gave values which were generally in the normal range, but in a few cases the figures were high; this might be due to the diffusion into the blood of ammonia liberated in the muscles through the tonic mus-

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D. RICHTER AND R. M. C. DAWSON 1207

cular spasm produced by the electrical stimulation. The wide disparity between the ammonia levels in the blood and the brain observed in these and in other experiments gave evidence that the brain ammonia was formed in the brain itself and could not be attributed as a rule to ammonia entering the brain from the blood.

Since the brain ammonia content was raised in the preconvulsive state, it appeared of interest to find out how far the cerebral irritability in this

TABLE VIII

Ammonia Content of Rat Blood and Brain in Preconvulsive State after (a) Picro- toxin Administration and (b) Electrical Stimulation

The rats were killed before the onset of convulsions. Other conditions as in Table VII; picrotoxin dose, 1 ml. of 3 per cent solution.

Picrotoxin

Electric shock

Rat No.

sm. 200-300 200-300

35-40 3540 3540 35-40

Method of killing

Decapitation “

Liquid air “ “ “ “ ‘< “

sec.

1

1 I 1

Mean, rats killed by liquid air only.. . . . 0.49

7 200-300 8 200-300 9 200-300

10 2OG300 11 200-300 12 200-300 13 200-300 14 266-300

-

-

Decapitation “ “ “ “ “ “ “

Mean......................................................

Brain Blood ammonia ammonia

g. per cm

0.65 0.49 0.40 0.73 0.44 0.41

g. per cent

0.11 0.42

0.71 0.83 1.04 1.10 0.92 0.92 1.10 0.93

___-

0.94 0.38

0.38 0.13 0.22 0.93 0.32 0.70 0.17 0.20

state could be attributed to the toxic action of the free ammonia. It was found that, when ammonium chloride was administered by intraperitoneal injection, convulsions occurred at the time when the brain ammonia level was approximately 9 mg. per cent, or about 10 times the level in the pre- convulsive state (Table VII). The brain glutamine content appeared to be raised after ammonium chloride administration, but accurate figures could not be obtained in this series, owing to the large amount of free ammonia present. In contrast to the figures on amonia, the brain glu-

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1208 BRAIN AMMONIA

tamine values showed little variation with the various experimental pro- cedures which were tried.

DISCUSSION

It was found that the ammonia content of the rat brain, analyzed after rapid fixation by freezing in liquid air, was not constant but depended on the state of activity of the brain at the time of fixation. Values obtained on anesthetized animals were significantly lower than those for normal controls, while much higher values were obtained in animals taken during convulsions produced by convulsant drugs or by other methods of stimu- lation. The brain ammonia content during electrically induced convulsions was more than eight times that found in anesthetized animals. Unless it is believed that these changes occurred during the brief period of freezing with liquid air, it must be concluded that they represent changes oc- curring in viva. This view is supported by the work of Tashiro (ll), who showed that free ammonia is liberated by the isolated frog nerve and that the amount liberated is increased on stimulation. The observations on the frog nerve were confirmed by Winterstein and Hirshberg (19).

The present work showed that the brain contains some source which can liberate up to about 1 mg. per cent of ammonia within 5 seconds upon stimulation. The source of this ammonia is not yet clear, but the brain glutamine content showed little variation, which made it unlikely that glu- tamine was the source. It might come from the deamination of nucleo- tides; this has generally been regarded as a relatively slow process (20), but recent work has shown that the simultaneous dephosphorylation and deamination of adenosine diphosphate can proceed with considerable ra- pidity in muscle preparations (21). It is noteworthy that the liberation of ammonia did not run parallel to the formation of lactic acid in the brain by glycolysis. The brain ammonia and lactic acid are both increased by convulsants and decreased by anesthetics, but there was no liberation of ammonia corresponding to the rise in lactic acid in the brain in emotional excitement.l The rapid liberation of ammonia after decapitation came to an end in a few seconds, while the lactic acid formation by postmortem glycolysis continues for a much longer time. Again, the fall in the ammonia content of the brain in anesthesia appeared to follow some time after the decrease in the lactic acid level.

The rise in the brain ammonia content in electrical convulsions was not due to the convulsions themselves, for it could be shown that the liberation of ammonia in the brain occurred in the preconvulsive stage of cerebral irritability before the start of the convulsions. This raised the question of whether the convulsions could be attributed directly to the toxic action of the ammonia. This suggestion was previously made for epileptic con-

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D. RICHTER AND R. M. C. DAWSON 1209

vulsions by Riebeling (3) and Brtihl (22), who reported that the ammonia content of the cerebrospinal fluid was increased in epileptics after seizures. Richter, Dawson, and Rees2 were unable to confirm that observation but that did not disprove their hypothesis, for it is not likely that the cere- brospinal fluid would give an accurate reflection of the rapid changes oc- curring in the brain. The view that epileptic seizures may be attributable to the toxic action of ammonia is attractive, as it can explain a number of the experimentally observed facts; these include (a) the precipitation of seizures by anoxia, which has been shown to liberate ammonia in the brain, (b) the autocatalytic spread of the discharge, as ammonia liberated at one point stimulates the neighboring regions of the brain, and (c) the specific effect of glutamic acid in inhibiting certain types of seizures, in which the rate of detoxication of ammonia might be the limiting factor.

Further experiments designed to test this hypothesis showed that the ammonia liberated in the rat brain on stimulation reached about a tenth of the concentration required to produce convulsions in a normal animal. The figure for the convulsant level was obtained by injecting ammonium chloride and determining the brain ammonia content after the convulsions had started. Under these conditions the ammonia would be fairly evenly distributed throughout the vascular bed and substance of the brain, while it might be expected t’hat the ammonia liberated on stimulation would be formed mainly in the metabolically active regions, where local concentra- tions higher than the average for the whole brain would be found. The apparent discrepancy between the ammonia level after stimulation and the convulsant level may therefore be less in fact than these experiments would appear to indicate. The present investigation gives evidence that am- monia is liberated in the brain on stimulation and shows that the brain ammonia concentrat,ion can approach the toxic range; it suggests that in conditions such as epilepsy, in which the brain is abnormally irritable, the toxic action of ammonia may play a significant part.

SUMMARY

Values are reported for the ammonia and glutamine content of the rat brain. The normal ammonia content, determined by a method which avoided the error due to the decomposition of labile acid amides, was 0.28 mg. per cent and the glutamine content was 79 mg. per cent. A study of the factors affecting the brain ammonia level showed that it was decreased by prolonged nembutal narcosis and markedly increased by direct stimu- lation of the brain or by procedures which increase cerebral irritability. The ammonia level was increased to 0.47 mg. per cent by picrotoxin ad-

2 Richter, D., Dawson, R. M. C., and Rees, L., J. Merit. SC., in press.

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1210 BRAIN AMMONIA

ministration and 0.81 mg. per cent by anoxia. Electrical stimulation caused a rapid increase in the ammonia level in 1 to 2 seconds and it was also raised by the stimulus of decapitation. The brain ammonia was not affected by emotional excitement and the glutamine content was not sig- nificantly affected by any of the factors which were tested. Injection of ammonium chloride in the rat caused convulsions when the brain ammonia level had risen to 9 mg. per cent. The significance of these data in relation to the mechanism of epileptic seizures is discussed.

The authors thank the Medical Research Council for a personal grant to one of us (R. M. C. D.) and for a grant for expenses; they thank the Rockefeller Foundation and the Royal Society for grants for equipment.

BIBLIOGRAPHY

1. Btilow, M., and Holmes, E. G., Biochem. Z., 245, 459 (1932). 2. Schwarz, H., and Dibold, H., Biochem. Z., 251, 187 (1932). 3. Riebeling, C., K&n. Wochschr., 10, 554 (1931); 13, 1422 (1934). 4. Harris, M. M., J. Nerv. and iwent. Dis., 102, 466 (1945). 5. Hamilton, P. B.,, J. Biol. Chem., 158, 397 (1945). 6. Brain, R., Proc. Roy. Sot. Med., 20, 861 (1927). 7. Krebs, H. A., Biochem. J., 29, 1951 (1935). 8. Sapirstein, M. R., Proc. Sot. Exp. Biol. and Med., 52, 334 (1943). 9. Price, J. C., Waelsch, H., and Putnam, T. J., J. Am. Med. Assn., 122,1153 (1943).

10. Conway, E. J., Micro-diffusion analysis and volumetric error, London (1939). 11. Tashiro, S., Am. J. Physiol., 60,519 (1922). 12. Trevan, J. W., Biochem. J., 19, 1111 (1925). 13. Harris, M. M., J. Clin. Invest., 22, 569 (1943). 14. Ferdman, D. L., Frenkel, S. R., and Silakova, L. I., Biokhimiya, 7, 43 (1943). 15. Avery, B. F., Kerr, S. E., and Ghantus, M., J. BioZ. Chem., 110, 637 (1935). 16. Kerr, S. E., J. BioZ. Chem., 110,625 (1935). 17. Richter, D., and Dawson, R. M. C., Nature, 161, 205 (1948). 18. Stanojevic, L., Die physiologische Bedeutung und klinische Bedeutung des

Blutammoniaks, Leipzig (1938). 19. Winterstein, H., and Hirshberg, E., Biochem. Z., 156, 138 (1925). 20. Kerr, S. E., J. BioZ. Chem., 145, 647 (1942). 21. Szent-Gybrgyi, 8., Chemistry of muscular contraction, New York, 50 (1947). 22. Briihl, H. H., Z. Kinderh., 59, 446 (1938).

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Derek Richter and Rex M. C. DawsonCONTENT OF THE BRAIN

THE AMMONIA AND GLUTAMINE

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