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Concentrations of free amino acids in plasma, erythrocytes and cerebrospinal fluid in coma
Resuscitation, 4, 131-137
Concentrations of free amino acids in plasma, erythrocytes and cerebrospinal fluid in coma A. BONDOLI, R. RANIERI,
S. I. MAGALINI,
E. SCRASCIA and D. CAMAIONI
Istituto di Anestesiologia e Rianimazione, Universita Cattolica de1 Sacro &ore, Facolth di Medicina e Chirurgia, Via Della Pineta Sacchetti 644, 00168 Roma, Italy
The concentrations of free amino acids were measured in plasma and erythrocytes in 15 patients, and in cerebrospinal fluid in eight patients, who were in coma. Although there was a variety of causes of coma, there seemed to be a characteristic pattern of the concentrations of amino acids for coma, and the plasma/cerebrospinal fluid ratios also showed constant patterns. Introduction In recent years measurements of amino acids in plasma and cerebrospinal fluid have contributed greatly to the understanding of metabolic disorders (Allan, Cusworth, Dent & Wilson, 1958; Perry &Jones, 1961;Crome,Tymms& Woolf, 1962; Efron, 1965; Dickinson & Hamilton, 1966; Fischer, Pomeroy & Henry, 1968; Van Sande, Mardens, Adriaenssens & Lowenthal, 1970; Gjessing, Gjesdahl & Sjaastad, 1972; Plum, 1974). With the exception of a study of hepatic coma (Gilon, Szeinberg, Tauman & Bodonyi, 1959), very little work has been published on the amino acids in body fluids in coma. Materials and methods Subjects Blood and cerebrospinal fluid were examined from a group of 15 patients in coma due to the various diseases indicated in Table 1, and from three other patients without major neurological disease used as control subjects. Plasma and ery throcy tees Blood samples (10 ml) were collected in heparinized tubes and the plasma was separated by centrifugation at 1000 rev./min. The plasma was deproteinized by the addition of 10 mg of crystalline sulphosalicylic acid/ml. The supernatant fluid obtained by centrifugation at 3000 rev./min for 30 min was filtered through Whatman no.1 filter paper, and stored at -2O’C. Protein-free supernatant (0.5 ml) was used for the analyses. Erythrocytes, which had been thoroughly washed so that the washings contained no detectable protein, were lysed by the addition of an equal volume of distilled water. The haemolysate was deproteinized by the addition of 15 mg of crystalline sulphosalicylic acid/ml, and centrifuged 131
132
A. BONDOLI AND OTHERS
“:,”
e
L...
methionine
isoleucine
leucine
phenylalanine
lysine
histidine
“.
..
. .
.
01-1
I
h-d
i
I
.%a
I*
MD**k
a
1
TI
.
.
I *El
_r:;y
arginine
”
,
,
200
300
I
0
100
Concn. bmd/l)
Pig.1. Concentrations of free essential amino acids in the plasma. In this and in Figs.2-4, each point is a value from an individual patient and the bar represents the range of values from the normal subjects.
at 3000 rev./min for 30 min. The supematant was filtered through Whatman no.1 filter paper and stored at -20°C. Protein-free supernatant (1 ml) was used for the analyses. Cerebrospinal fluid
Cerebrospinal fluid was obtained by lumbar puncture from eight patients. The proteins were removed by mixing 2 ml of the fluid with 20 mg of crystalline sulphosalicylic acid, allowing it to stand for 10 min, and then centrifuging it at 3000 rev./min for 30 min. The supernatant was filtered through Whatman no.1 paper and stored at -20°C. Proteinfree supematant (1 ml) was used for the analysis. The amino acid concentrations were determined quantitatively by the procedures of Stein & Moore (1954) and Spa&man, Stein & Moore (1958) with a 3A-27 Ammo Acid Autoanalyzer (Carlo-Erba Ltd).
AMINO ACIDS AND COMA
0 Fig.2.
100
200 Concn. ~mollll
133
300
Concentrations of free essential amino acids in erythrocytes. See also the legend to Fig.l.
Results In the control patients, the concentrations of the essential alpha-amino acids, which are indicated on all the Figures by bars, were similar to published values (see below). In the samples from the patients in coma, the concentrations of these amino acids were lower than normal; they differed by a mean value of 25% in plasma, 23% in erythrocytes and 17% in cerebrospinal fluid; the mean values for non-essential amino acids were 52% lower in plasma, 90% lower in cerebrospinal fluid and 1.9% lower in erythrocytes. The concentrations of threonine, valine, leucine, lysine, histidine and arginine were decreased. Methionine and isoleucine were close to the normal range, and phenylalanine was consistently increased (F&l). All the amino acids were markedly decreased in the erythrocytes, and some of them were present in traces only (Fig.2). In cerebrospinal fluid the concentrations of threonine, valine, leicine, lysine, histidine and arginine were lower than normal, but the phenylalanine and methionine were markedly increased (Fig.3). Results of measurements of non-essential amino acids are given in Fig.4.
134
A. BONDOLI AND OTHERS
threonine
val i ne
methionine
1
1 .
**a*
I
I
pa
-
isoleucine
leucine
.
phenylalanine
lysine
histidine
arginine
. i 0
10
20
30
40 Concn.
Fig.3. Fig. 1.
Concentrations
60
60
70
80
90
100
Cmolll)
of free essential amino acids in cerebrospinal fluid. See also the legend to
Discussion The concentrations of the essential alpha-amino acids found in the control subjects were similar to those reported in the literature (Stein & Moore, 1954; Perry 8c Jones, 1961; Altman, 1961; Dickinson & Hamilton, 1966; Fischer, Pomeroy & Henry, 1968; Van Sande et al., 1970; Gjessing et al., 1972; Plum, 1974). A preliminary hypothesis may be proposed to explain the changes of ammo acids observed in coma. In coma, the energy from normal reserves was insufficient, and amino acids shifted from the intracelhrlar pool within the erythrocytes. In respect of the amino acids, the changes in the cerebrospinal fluid paralleled those in the plasma only qualitatively (Fig.3). Dickinson & Hamilton (1966) suggested that there was differential and selective distribution in a dynamic way of amino acids across the blood-brain barrier. This can be seen in the present experiments when comparing the plasma/cerebrospinal fluid ratios between normal and comatose patients. They were lower for threonine, methionine and phenylahmine, similar for leucine, and high for valine, isoleucine, lysine,
AMINO ACIDS AND COMA
taurine
glutamic acid
glycine
alanine
tyrosine
aspartic acid
ornithine
citrulline
tarrine glutamic
acid
giycine tyrosine
Concn.
Concentrations of free non-essential See also the legend to Fig. 1.
Pig.4.
bmolll)
amino acids in (a) plasma and (b) cerebrospinal
fluid.
histidine and arginine (Table 2). These ratios can be explained as a change in protein metabolism and/or in the permeability of the blood-brain barrier, as has been demonstrated in some metabolic and neurological diseases (Van Sande et al., 1970). It has recently been proposed that the y-glutamyl cycle is involved in the transport of some amino acids across the blood-brain barrier (Orlowski & Meister, 1970, 1973; Orlowski, Sessa & Green, 1974). This hypothesis is supported by the observations that methionine, a good substrate for the transpeptidase, is rapidly taken up by the brain (Battistin, Grynbaum & Lajtha, 197 1). The high methionine concentration found in the cerebrospinal fluid of all our patients in coma, suggested damage to the blood-brain barrier that may be correlated with an enzymatic deficiency in the r_glutamyl cycle. In the group of patients studied, the plasma and cerebrospinal fluid concentrations of some nonessential amino acids were also changed significantly (Fig.4). The lower concentrations of glutamic acid in cerebrospinal fluid might be related to an increased synthesis of -r-aminobutyric acid in brain tissue, due to an increase of the energy
136
A. BONDOLI
AND OTHERS
Table 1. Causes of coma in the 15 patients studied. plasma, (b) erythrocytes and (c) cerebrospinal fluid. Patient
no.
Sex
Measurements
Age (years)
1 (a, b) 2 (a, b, c) 3 (a, b, c) 4 (a, b, c) 5 (a, b, c) 6 (a, b) 7 (a, b) 8 (a, b) 9 (a, b, c) 10 (a, b, c) 11 (a, b) 12 (a, b, c) 13 (a, b)
F M M F F 1: F M M M M M 1:
46 II 58 49 49 58 2 74 72 65 55 60 34
14 (a, b, c)
M
65
15 (a, b)
F
16
Table 2. Plasma/cerebrospinal subjects and patients in coma.
fluid (CSF) concentration
were made as indicated
in (a)
Causes of coma __
______..__
-~
Cerebral oedema Ictus cerebri Ictus cerebri Acute encephalitis Brain tumour Portal hypertension Hypoxia Hypercapnia Hypercapnia Hypercapnia Hypercapnia Hypercapnia Hepatorenal syndrome Poisoning by nicotine, mineral oil and benzdiazepines Barbiturate poisoning
(nmol/l)
ratios
of amino
acids in normal
-Plasma/CSF Amino acids
ratio
Normal
Coma -__
Threonine Valine Methionine Isoleucine Leucine Phenylalanine Lysine Histidine Arginine
7.3 5.0 8.5 12.2 10.8 6.8 6.0 6.3 4.9
3.6 10.8 3.0 39.4 10.7 3.0 8.0 7.1 10.0 _____
needs. ydminobutyric acid is involved, through succinic acid, with the cirtric acid cycle in the brain. In addition, the low arginine concentration observed in the cerebrospinal fluid suggested a shift in metabolism towards the 7-guanidinobutyric acid cycle, that is an even more potent synaptic inhibitor. A possible interpretation for the high plasma and cerebrospinal fluid concentrations of phenylalanine may lie in a reduction of cerebral and systemic adrenergic activity. In conclusion, in all patients in coma of different aetiologies deviations of ammo acids from the normal values have been found. These preliminary observations need further evaluation, not only to contribute to a better.understanding of some of the pathological process involved in this neurological condition, but also to provide new means of treatment.
AMINO ACIDS AND COMA
137
References Allan, J. D., Cusworth, D. C., Dent, C. E. &Wilson, V. K. (1958) A disease, probably hereditary, characterized by severe deficiency and a constant gross abnormality of amino acid metabolism. Lancer, i, 182-187. Altman, P. L. (1961) In: Blood and Other Body Fluids. p.73. Ed. Dittmer, D. S. Federation of American Societies for Experimental Biology, Washington D.C. Battistin, L., Grynbaum, A.& Lajtha, A. (1971) The uptake of various amino acids by the mouse brain in vivo. Brain Research, 29,85-89. Crome, L.,Tymms, V. & Woolf, L. I. (1962) A chemical investigation of the defect of myelination in phenylketonuria. J. Neural. Neurosurg. Psych&. 25,143-147. Dickinson, J. C. & Hamilton, P. B. (1966) The free amino acids of human spinal fluid determined by ion-exchange chromatography. J. Neurochem. 13, 1179-1187. Efron, M. L. (1965) Aminoaciduria. New Eng. J. Med. 272, 1058-1067. Fischer, R. G., Pomeroy. J. & Henry. J. P. (1968) The free amino acids in adult human cerebrosoinal L fluid. A& Neural.-kandinav. k,6191630.’ Gilon, E., Szeinberg, A., Tauman, G. & Bodonyi, E. (1959) Glutamine estimation in cerebrospinal fluid in cases of liver cirrhosis and hepatic coma. J. Lab. Clin. Med. 53, 714-719. Gjessing, L. R., Gjesdahl, P. & Sjaastad, 0. (1972) The free amino acids in human cerebrospinal fluid. J. Neurochem. 19,1807-1808. Orlowski, M. & Meister, A. (1970) The rghrtamyl cycle: a possible transport system for amino acids. Proc. Nat. Acad. Sci. 67, 1248-1255. Orlowski, M. & Meister, A. (1973) T-Glutamyl cyclotransferase. Distribution, isozymic forms and specificity. J. Biol. Chem. 248,2836-2844. Orlowski, M., Sessa, G. & Green, J. P. (1974) rGlutamy1 transpeptidase in brain capillaries: possible site of blood-brain barrier for amino acids. Science, 184,66-68. Perry, T. L. & Jones, R. T. (1961) The amino acids content of human cerebrospinal fluid in normal individuals and in mental defectives. J. Clin. Invest. 40, 1363-1372. Plum, C. M. (1974) Free amino acid levels in the cerebrospinal fluid of normal humans and their variation in cases of epilepsy and Spielmeyer-Vogt-Batten disease. J. Neurochem. 23,595600. Spa&man, D. H., Stein, W. H. & Moore, S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Analyt. Chem. 30,1190-1206. Stem, W. H. & Moore, S. (1954) The free amino acids of human blood plasma. J. Biol. Chem. 211, 915-92s. Van Sande, M., Mardens, Y., Adriaenssens, K. & Lower&al, A. (1970) The free amino acids in human cerebrospinal fluid. J. Neurochem. 17, 125-135.
Concentrations of free amino acids in plasma, erythrocytes and cerebrospinal fluid in coma A. BONDOLI, R. RANIERI,
S. I. MAGALINI,
E. SCRASCIA and D. CAMAIONI
Istituto di Anestesiologia e Rianimazione, Universita Cattolica de1 Sacro &ore, Facolth di Medicina e Chirurgia, Via Della Pineta Sacchetti 644, 00168 Roma, Italy
The concentrations of free amino acids were measured in plasma and erythrocytes in 15 patients, and in cerebrospinal fluid in eight patients, who were in coma. Although there was a variety of causes of coma, there seemed to be a characteristic pattern of the concentrations of amino acids for coma, and the plasma/cerebrospinal fluid ratios also showed constant patterns. Introduction In recent years measurements of amino acids in plasma and cerebrospinal fluid have contributed greatly to the understanding of metabolic disorders (Allan, Cusworth, Dent & Wilson, 1958; Perry &Jones, 1961;Crome,Tymms& Woolf, 1962; Efron, 1965; Dickinson & Hamilton, 1966; Fischer, Pomeroy & Henry, 1968; Van Sande, Mardens, Adriaenssens & Lowenthal, 1970; Gjessing, Gjesdahl & Sjaastad, 1972; Plum, 1974). With the exception of a study of hepatic coma (Gilon, Szeinberg, Tauman & Bodonyi, 1959), very little work has been published on the amino acids in body fluids in coma. Materials and methods Subjects Blood and cerebrospinal fluid were examined from a group of 15 patients in coma due to the various diseases indicated in Table 1, and from three other patients without major neurological disease used as control subjects. Plasma and ery throcy tees Blood samples (10 ml) were collected in heparinized tubes and the plasma was separated by centrifugation at 1000 rev./min. The plasma was deproteinized by the addition of 10 mg of crystalline sulphosalicylic acid/ml. The supernatant fluid obtained by centrifugation at 3000 rev./min for 30 min was filtered through Whatman no.1 filter paper, and stored at -2O’C. Protein-free supernatant (0.5 ml) was used for the analyses. Erythrocytes, which had been thoroughly washed so that the washings contained no detectable protein, were lysed by the addition of an equal volume of distilled water. The haemolysate was deproteinized by the addition of 15 mg of crystalline sulphosalicylic acid/ml, and centrifuged 131
132
A. BONDOLI AND OTHERS
“:,”
e
L...
methionine
isoleucine
leucine
phenylalanine
lysine
histidine
“.
..
. .
.
01-1
I
h-d
i
I
.%a
I*
MD**k
a
1
TI
.
.
I *El
_r:;y
arginine
”
,
,
200
300
I
0
100
Concn. bmd/l)
Pig.1. Concentrations of free essential amino acids in the plasma. In this and in Figs.2-4, each point is a value from an individual patient and the bar represents the range of values from the normal subjects.
at 3000 rev./min for 30 min. The supematant was filtered through Whatman no.1 filter paper and stored at -20°C. Protein-free supernatant (1 ml) was used for the analyses. Cerebrospinal fluid
Cerebrospinal fluid was obtained by lumbar puncture from eight patients. The proteins were removed by mixing 2 ml of the fluid with 20 mg of crystalline sulphosalicylic acid, allowing it to stand for 10 min, and then centrifuging it at 3000 rev./min for 30 min. The supernatant was filtered through Whatman no.1 paper and stored at -20°C. Proteinfree supematant (1 ml) was used for the analysis. The amino acid concentrations were determined quantitatively by the procedures of Stein & Moore (1954) and Spa&man, Stein & Moore (1958) with a 3A-27 Ammo Acid Autoanalyzer (Carlo-Erba Ltd).
AMINO ACIDS AND COMA
0 Fig.2.
100
200 Concn. ~mollll
133
300
Concentrations of free essential amino acids in erythrocytes. See also the legend to Fig.l.
Results In the control patients, the concentrations of the essential alpha-amino acids, which are indicated on all the Figures by bars, were similar to published values (see below). In the samples from the patients in coma, the concentrations of these amino acids were lower than normal; they differed by a mean value of 25% in plasma, 23% in erythrocytes and 17% in cerebrospinal fluid; the mean values for non-essential amino acids were 52% lower in plasma, 90% lower in cerebrospinal fluid and 1.9% lower in erythrocytes. The concentrations of threonine, valine, leucine, lysine, histidine and arginine were decreased. Methionine and isoleucine were close to the normal range, and phenylalanine was consistently increased (F&l). All the amino acids were markedly decreased in the erythrocytes, and some of them were present in traces only (Fig.2). In cerebrospinal fluid the concentrations of threonine, valine, leicine, lysine, histidine and arginine were lower than normal, but the phenylalanine and methionine were markedly increased (Fig.3). Results of measurements of non-essential amino acids are given in Fig.4.
134
A. BONDOLI AND OTHERS
threonine
val i ne
methionine
1
1 .
**a*
I
I
pa
-
isoleucine
leucine
.
phenylalanine
lysine
histidine
arginine
. i 0
10
20
30
40 Concn.
Fig.3. Fig. 1.
Concentrations
60
60
70
80
90
100
Cmolll)
of free essential amino acids in cerebrospinal fluid. See also the legend to
Discussion The concentrations of the essential alpha-amino acids found in the control subjects were similar to those reported in the literature (Stein & Moore, 1954; Perry 8c Jones, 1961; Altman, 1961; Dickinson & Hamilton, 1966; Fischer, Pomeroy & Henry, 1968; Van Sande et al., 1970; Gjessing et al., 1972; Plum, 1974). A preliminary hypothesis may be proposed to explain the changes of ammo acids observed in coma. In coma, the energy from normal reserves was insufficient, and amino acids shifted from the intracelhrlar pool within the erythrocytes. In respect of the amino acids, the changes in the cerebrospinal fluid paralleled those in the plasma only qualitatively (Fig.3). Dickinson & Hamilton (1966) suggested that there was differential and selective distribution in a dynamic way of amino acids across the blood-brain barrier. This can be seen in the present experiments when comparing the plasma/cerebrospinal fluid ratios between normal and comatose patients. They were lower for threonine, methionine and phenylahmine, similar for leucine, and high for valine, isoleucine, lysine,
AMINO ACIDS AND COMA
taurine
glutamic acid
glycine
alanine
tyrosine
aspartic acid
ornithine
citrulline
tarrine glutamic
acid
giycine tyrosine
Concn.
Concentrations of free non-essential See also the legend to Fig. 1.
Pig.4.
bmolll)
amino acids in (a) plasma and (b) cerebrospinal
fluid.
histidine and arginine (Table 2). These ratios can be explained as a change in protein metabolism and/or in the permeability of the blood-brain barrier, as has been demonstrated in some metabolic and neurological diseases (Van Sande et al., 1970). It has recently been proposed that the y-glutamyl cycle is involved in the transport of some amino acids across the blood-brain barrier (Orlowski & Meister, 1970, 1973; Orlowski, Sessa & Green, 1974). This hypothesis is supported by the observations that methionine, a good substrate for the transpeptidase, is rapidly taken up by the brain (Battistin, Grynbaum & Lajtha, 197 1). The high methionine concentration found in the cerebrospinal fluid of all our patients in coma, suggested damage to the blood-brain barrier that may be correlated with an enzymatic deficiency in the r_glutamyl cycle. In the group of patients studied, the plasma and cerebrospinal fluid concentrations of some nonessential amino acids were also changed significantly (Fig.4). The lower concentrations of glutamic acid in cerebrospinal fluid might be related to an increased synthesis of -r-aminobutyric acid in brain tissue, due to an increase of the energy
136
A. BONDOLI
AND OTHERS
Table 1. Causes of coma in the 15 patients studied. plasma, (b) erythrocytes and (c) cerebrospinal fluid. Patient
no.
Sex
Measurements
Age (years)
1 (a, b) 2 (a, b, c) 3 (a, b, c) 4 (a, b, c) 5 (a, b, c) 6 (a, b) 7 (a, b) 8 (a, b) 9 (a, b, c) 10 (a, b, c) 11 (a, b) 12 (a, b, c) 13 (a, b)
F M M F F 1: F M M M M M 1:
46 II 58 49 49 58 2 74 72 65 55 60 34
14 (a, b, c)
M
65
15 (a, b)
F
16
Table 2. Plasma/cerebrospinal subjects and patients in coma.
fluid (CSF) concentration
were made as indicated
in (a)
Causes of coma __
______..__
-~
Cerebral oedema Ictus cerebri Ictus cerebri Acute encephalitis Brain tumour Portal hypertension Hypoxia Hypercapnia Hypercapnia Hypercapnia Hypercapnia Hypercapnia Hepatorenal syndrome Poisoning by nicotine, mineral oil and benzdiazepines Barbiturate poisoning
(nmol/l)
ratios
of amino
acids in normal
-Plasma/CSF Amino acids
ratio
Normal
Coma -__
Threonine Valine Methionine Isoleucine Leucine Phenylalanine Lysine Histidine Arginine
7.3 5.0 8.5 12.2 10.8 6.8 6.0 6.3 4.9
3.6 10.8 3.0 39.4 10.7 3.0 8.0 7.1 10.0 _____
needs. ydminobutyric acid is involved, through succinic acid, with the cirtric acid cycle in the brain. In addition, the low arginine concentration observed in the cerebrospinal fluid suggested a shift in metabolism towards the 7-guanidinobutyric acid cycle, that is an even more potent synaptic inhibitor. A possible interpretation for the high plasma and cerebrospinal fluid concentrations of phenylalanine may lie in a reduction of cerebral and systemic adrenergic activity. In conclusion, in all patients in coma of different aetiologies deviations of ammo acids from the normal values have been found. These preliminary observations need further evaluation, not only to contribute to a better.understanding of some of the pathological process involved in this neurological condition, but also to provide new means of treatment.
AMINO ACIDS AND COMA
137
References Allan, J. D., Cusworth, D. C., Dent, C. E. &Wilson, V. K. (1958) A disease, probably hereditary, characterized by severe deficiency and a constant gross abnormality of amino acid metabolism. Lancer, i, 182-187. Altman, P. L. (1961) In: Blood and Other Body Fluids. p.73. Ed. Dittmer, D. S. Federation of American Societies for Experimental Biology, Washington D.C. Battistin, L., Grynbaum, A.& Lajtha, A. (1971) The uptake of various amino acids by the mouse brain in vivo. Brain Research, 29,85-89. Crome, L.,Tymms, V. & Woolf, L. I. (1962) A chemical investigation of the defect of myelination in phenylketonuria. J. Neural. Neurosurg. Psych&. 25,143-147. Dickinson, J. C. & Hamilton, P. B. (1966) The free amino acids of human spinal fluid determined by ion-exchange chromatography. J. Neurochem. 13, 1179-1187. Efron, M. L. (1965) Aminoaciduria. New Eng. J. Med. 272, 1058-1067. Fischer, R. G., Pomeroy. J. & Henry. J. P. (1968) The free amino acids in adult human cerebrosoinal L fluid. A& Neural.-kandinav. k,6191630.’ Gilon, E., Szeinberg, A., Tauman, G. & Bodonyi, E. (1959) Glutamine estimation in cerebrospinal fluid in cases of liver cirrhosis and hepatic coma. J. Lab. Clin. Med. 53, 714-719. Gjessing, L. R., Gjesdahl, P. & Sjaastad, 0. (1972) The free amino acids in human cerebrospinal fluid. J. Neurochem. 19,1807-1808. Orlowski, M. & Meister, A. (1970) The rghrtamyl cycle: a possible transport system for amino acids. Proc. Nat. Acad. Sci. 67, 1248-1255. Orlowski, M. & Meister, A. (1973) T-Glutamyl cyclotransferase. Distribution, isozymic forms and specificity. J. Biol. Chem. 248,2836-2844. Orlowski, M., Sessa, G. & Green, J. P. (1974) rGlutamy1 transpeptidase in brain capillaries: possible site of blood-brain barrier for amino acids. Science, 184,66-68. Perry, T. L. & Jones, R. T. (1961) The amino acids content of human cerebrospinal fluid in normal individuals and in mental defectives. J. Clin. Invest. 40, 1363-1372. Plum, C. M. (1974) Free amino acid levels in the cerebrospinal fluid of normal humans and their variation in cases of epilepsy and Spielmeyer-Vogt-Batten disease. J. Neurochem. 23,595600. Spa&man, D. H., Stein, W. H. & Moore, S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Analyt. Chem. 30,1190-1206. Stem, W. H. & Moore, S. (1954) The free amino acids of human blood plasma. J. Biol. Chem. 211, 915-92s. Van Sande, M., Mardens, Y., Adriaenssens, K. & Lower&al, A. (1970) The free amino acids in human cerebrospinal fluid. J. Neurochem. 17, 125-135.