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Assessment of reliability and biological significance of glutamate levels in cerebrospinal fluid.

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in antegrade memory and learning was associated with
limitations in making associations when confronted
with multiple new pieces of information and from semantically related intrusive and perseverative errors
that interfered with the process of recall. Testing also
supported a deficit more in retrieval than in encoding
and consolidation. This was also found in the patients
of Damasio and co-workers 131 and of Irle and colleagues 151. S.C.'s response to bromocriptine suggests
that these functions are partly mediated by dopaminergic pathways and point to the potential iatrogenic complications that might accompany the use of dopamine
blockers in agitated patients with similar lesions.
No generalizations can be made about the efficacy
of bromocriptine or lack of efficacy of the other agents
tried in S.C. for patients with medial forebrain lesions
and antegrade amnesia. However, a repeated-measures
design 122) using outcome tests with equivalent forms
is a practical approach for testing drug interventions.
We thank Rod Little, PhD, for assistance with the statistical analysis.
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Assessment of Reliability
and Biological Significance
of Glutamate Levels in
Carlo Ferrarese, MD, PhD, Nicoletta Pecora, P h D ,
M~~~~F ~ , MD,
~ ~lldebrando
~ M
~ ~l , l
and Lodovico ~
~MD ~
The published information on glutamate levels in cerebrospinal fluid (CSF) and modifications in neurological
disorders is controversial. In the present study, we demonstrated a metabolic instability of glutamate in untreated CSF and a spurious elevation of its levels by the
current methods of CSF acidification. These findings
may explain the discrepancies observed with different
methods of CSF processing and analysis. We suggest a
method of inactivating CSF enzymes that yields stable
glutamate levels under different storage conditions. Use
of such a method may be necessary for clinical studies.
Ferrarese C, Pecora N, Frigo M, Appollonio I,
Frattola L. Assessment of reliability and biological
significance of glutamate levels i n cerebrospinal
fluid. A n n Neurol 1993;31:316-319
Recent experimental evidence suggests a role for glutamate and other excitatory amino acids in the pathoFrom the Department of Neurology, University of Milan, San Gerardo Hospita', Monza7 Italy.
Received Apr 20, 1992, and in revised form Jul 13, Aug 20, and
Sep 16. Accepted for publication Sep 22, 1992.
Address correspondence to Prof Ferrarese, Department of Neurology, University of Milan, Ospedale San Gerardo, Via Donizetti, 106,
20052 Monza, Italy.
316 Copyright 0 1993 by t h e American Neurological Association
Table 1. Glutamate h e i s in Control CSF According t o Various Studies
Glutamate Level
(pmol/ml, k SD)
Plum C7)
Lakke and Teelken [lo}
McGale et a1 [23]
Iijima et al {24}
Smith et a1 [ 11)
J Neurochem
J Neurochem
Tohoku J Exp Med
J Neurol Neurosurg
Anal Biochem
Acta Neurol Scand
Acta Neurol Scand
11,200 2 7,200
15,800 +- 15,000
26,100 2 18,900
3,000 2 900
28,300 2 10,300
Spink et a1 122)
Araki et al [8]
Lundqvist et al [21J
Pitkinen et a1 [9}
Perry et al [ 141
Rothstein et al [l5}
Rothstein et a1 (171
J Neural Transm
Ann Neurol
Ann Neurol
Ann Neurol
genesis of acute ischemic neuronal damage 111 and in
epileptic phenomena C2}. Attempts to provide a biochemical explanation for acute and chronic neurological disorders have been based o n measurements of
excitatory amino acids and receptor levels in tissues at
autopsy 13-51 or of amino acid levels in cerebrospinal
fluid (CSF). However, brain levels of glutamate may
not be an index of its function as a neurotransmitter,
as most of the amino acid has a metabolic role [b}
and the significance of this substance in CSF is still
controversial. Varying glutamate levels have been
found in the CSF in controls and in neurological disorders. Increased, decreased, and unchanged levels of
glutamate have been observed in the CSF of epileptic
patients C7-91. A defect of amino acid transport from
the CSF to the plasma has been hypothesized in Parkinson’s disease and in other extrapyramidal disorders
[lo]. Reduced CSF levels of glutamate have been
found in Alzheimer’s dementia Cll], in which a degeneration of glutamatergic pathways has been described
[12]. T h e neurodegenerative disorder most extensively
studied in recent years is amyotrophic lateral sclerosis.
A systemic defect in glutamate metabolism was initially
proposed [13J but subsequent analyses of CSF levels of
glutamate have produced conflicting results [ 14- 171.
Discrepancies have been explained as the effects of
different methods of analysis o r processing and storage
of CSF samples 1181. T h e best method of collection
and storage of CSF samples still is uncertain, because
modifications of these procedures have yielded up to
100-fold differences of CSF glutamate levels (Table 1).
Since handling procedures can bias the results, we
tried several strategies to optimize the method of glutamate measurement in CSF. W e used an improved highperformance liquid chromatography (HPLC) technique with which amino acids can be determined in
picomolar quantities [ 191, and measured glutamate lev-
480 2
1,471 -t
700 2
2,700 -t
183 2
200 2
2,900 ?
350 2
els under various conditions of CSF storage and processing.
Materials and Methods
CSF was collected from patients hospitalized for lumbar disk
herniation or peripheral neuropathies, without signs of central nervous system involvement. For each CSF sample, aliquots were processed in the following ways: (1) Native
(untreated) CSF was incubated at room temperature for various times and subsequently deproteinized and analyzed by
HPLC; (2) other aliquots were collected in perchloric acid,
incubated, and analyzed as above; and (3) other aliquots were
collected in perchloric acid and immediately neutralized with
K2C03,and underwent the same incubation procedure. Exogenous L-glutamate was added to some aliquots and incubated as above, to analyze its recovery in CSF. Finally, CSF
aliquots processed in the different ways were also stored at
- 80°C for different lengths of time. Before analysis, all CSF
aliquots were deproteinized with perchloric acid and potassium carbonate (K2C03)and filtered using Millipore filters
(0.45-pm size exclusion) (Millipore Corp, Bedford, MA),
and 300 pl of CSF was derivatized with the same volume of
derivatizing solution (10 ml of 0.4 M borate buffer, pH 9.5,
containing 50 pl of 0.5 mg/ml o-phthaldialdehyde [OPA]
dissolved in methanol and 5 pl of 2-mercaptoethanol). Fifteen microliters of 5 pM a-aminoadipic acid was employed
as internal standard both in CSF samples and in the amino
acid standard solution (200 pI = 100 pmol of each amino
acid). CSF and amino acid standard solution were injected
after 2 minutes of derivatization. The elution of amino acids
from a C,, reverse-phase column (Waters 30 cm x 4.9 mm;
flow rate, 1.5 mlimin) was obtained by a multistep gradient
of two solvents (solvent A, 0.1 M sodium acetate buffer, pH
7.2; solvent B, methanol and tetrahydrofuran, 97 : 3 vol/vol).
Fluorimetric detection was carried out with excitation and
emission wavelengths of 254 and 418 nm (Shimadzu RF 535,
Kyoto, Japan), respectively, and analysis of chromatographic
peaks was performed with a Shimadzu C-R3A integrator.
In addition to glutamate, this analysis clearly identified the
Brief Communication: Ferrarese et al: Glutamate Levels in CSF 317
Table 2. Time Course of Changes in Glutamate Lmel
in CSF Treated in Different Ways and Incubated
at Room Temperaturt?
AcidTime (min) Untreated CSF Treated CSF
24 hr
t 40
* 20b
* 25b
t lob
t 20
+- 30
Neutral CSF
240 t 30
30b 220 ? 20
50' 280 -+ 30
* 50'
t 50'
t 40'
t 60'
250 t 30
220 t 10
206 ? 30
240 t 30
240 ? 50
"Values are expressed as picomoles per milliliter and are the mean
t standard error of the mean of three different samples.
bp 5 0.05 versus levels at to (Student's t test).
' p 5 0.01 versus levels at to(Student's t test).
aspartate peak and, after 1 : 100 CSF dilution, peaks of glutamine and y-aminobutyric acid (GABA).
When native (untreated) CSF was left at room temperature, glutamate levels fell rapidly. At 30 minutes, glutamate levels were only 50% of the original values; the
decrease continued for about 1 hour and was followed
by a late and progressive increase of the amino acid
levels (+250@ after 24 hours) (Table 2). Exogenous
glutamate added to untreated CSF disappeared within
2 hours (Fig). When CSF aliquots were collected in
perchloric acid and incubated at room temperature, a
time-dependent increase of glutamate levels was observed (see Table 2). The rate of glutamate formation
was calculated as 2 nmol/ml/hr. When CSF aliquots
were collected in perchloric acid and immediately neutralized with K,CO,, glutamate levels were unchanged
after different periods of incubation at room temperature (see Table 2). Exogenous glutamate added to acidtreated CSF aliquots was recovered unchanged after
CSF aliquots treated in the different ways and immediately frozen at -80°C contained different levels of
glutamate after 1 month: Untreated CSF had lower
levels ( - 30%), acid-treated CSF had higher levels
( + loo%), while acid-treated and neutralized CSF had
glutamate levels similar to the original values.
NOchange in CSF levels of aspartate, glutamine, and
GABA were detected after the different incubation
and storage procedures; only a small and not significant
decrease of glutamine levels was observed in acidtreated CSF aliquots (data not shown).
The pattern of time-related glutamate changes in untreated CSF suggests that two types of enzymatic pro318 Annals of Neurology Vol 33 No 3 March 1993
time (min)
Metabolic degradation of endogenous and exogenous glutamate
in human CSF, demonstrated by high-perfrmance liquid chromatography elution profiles. (A) CSF injected immediately after
collection. Peaks of aspartate (asp) and glutamate (glu) are resolved between two larger, unidentified peaks. ( B , Same CSF left
untreated at room temperature for 1 hour before injection. Glutamate levels are reduced by 1209%.(Ci Exogenous L-glutamate
(300 pmollml) was added to CSF just before injection. (Dj
L-Glutamate (300 pmollml) was added to untreated CSF 1
hour before injection. Levels of the amino acid were reduced by
cesses could occur in native CSF: (1 fast degradation
of free glutamate and (2) slow formation of new glutamate from glutamine or proteins. This interpretation
is supported by previous findings of different enzymes
involved in glutamate metabolism in CSF C20). Metabolism on the amino acid appears to be very rapid at
room temperature, but may occur also in frozen samples since glutamate levels decreased with the time of
storage of untreated CSF samples. From these observations it appears that results obtained from stored untreated CSF may be compared only if the storage time
is similar for the different groups of patients and if the
CSF is not left untreated at room temperature. These
conditions have probably not been observed in previous clinical studies, and could explain the different glutamate levels reported.
Different procedures have been employed to inactivate the enzymes in CS F sulfosalicylic acid {lo],
perchloric acid r2 11, and freeze-thawing cycles [22).
However, sulfosalicylic acid- and perchloric acidinduced increases of glutamate have previously been
observed and linked to acid hydrolysis of glutamine
f18-21). Our finding of elevated glutamate levels in
perchloric acid-treated CSF confirms this interpretation. The slight (and not significant) decrease of glutamine that we observed in acid-treated CSF samples
may be largely responsible for the glutamate rise, since
levels of glutamine in CSF are three orders of a magnitude higher than glutamate levels.
Thus, two different factors may explain the discrepancies in glutamate levels reported in the literature: (1)
metabolic instability of glutamate in the CSF, with the
possibility of intrathecal or in vitro glutamate formation
and/or degradation, according to activation of different
enzymes, and (2) artifactual in vitro increase of glutamate caused by the addition of acids to CSF to inactivate enzymes.
From our study, we believe that the only possibility
of obtaining glutamate levels stable over time is to inactivate the enzymes with acid immediately and neutralize the acidified CSF at once. CSF samples treated in
this way present glutamate levels stable in different
storage conditions. Thus, this method of collection,
processing, storage, and analysis of CSF is proposed to
avoid artifactual changes of glutamate levels resulting
from in vitro modifications of the amino acid.
As a corollary of our study, it appears that the functional interpretation of CSF levels of glutamate must
be very cautious. Although various studies demonstrated the existence of a blood-CSF barrier to amino
acids and suggested that CSF glutamate concentrations
should reflect its function within the central nervous
system f23-261, different processes such as neuronal
release and transport, glial uptake, diffusion barriers,
sequestration in distinct metabolic pools, and degradation may be responsible for the modifications of glutamate levels in the CSF. Only extensive studies of such
processes will reveal the physiological significance of
changes of glutamate levels in the CSF, which until
now are the only clinically available indices of glutamatergic functions in patients.
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Brief Communication: Ferrarese e t al: Glutamate Levels in CSF 319
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