Glutamate levels in cerebrospinal fluid in amyotrophic lateral sclerosis: a reappraisal using a new HPLC method with coulometric detection in a large cohort of patients

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Abstract

Glutamate is involved in the degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). However, the aetiology of ALS appears heterogeneous, leading to the possibility that patient subgroups with different pathophysiology may exist. The concentration of glutamate in cerebrospinal fluid (CSF) is measured using a new HPLC method with coulometric detection in a large cohort of ALS patients and controls: 377 ALS patients, 88 neurological patients and 18 normal controls. In ALS patients, and only in these subjects, the existence of two groups was observed, one with normal glutamate concentrations and one (40.8% of ALS patients) with high glutamate concentrations. High glutamate concentrations were correlated with a spinal onset of the disease, more impaired limb function and a higher rate of muscle deterioration. These results suggest that elevations of CSF glutamate concentrations could reflect the intensity of cell insult in the spinal cord. It remains to be determined if the group of patients with high CSF glutamate concentrations represents a specific subgroup of patients in terms of mechanism of disease, or only in terms of the spatial extent of motor neuron insult.

Introduction

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a progressive degeneration of motor neurons both in spinal cord or brainstem and in cerebral cortex. Most authors consider the disease to be multifactorial in origin, with several molecular mechanisms contributing to motor neuron degeneration [1]. This heterogeneity raises the question of heterogeneity of patients, and by the way of the heterogeneity of the response to a given treatment. However, in the principal therapeutic trials performed during the last decade, no clinical evidence has appeared that could resolve this issue [2].

The possible involvement of glutamate in the pathogenesis of the disease was suggested in view of an increased level of this amino acid in plasma [3] and firmly assessed by the discovery by Rothstein et al. [4] that there was an increase in glutamate levels in cerebrospinal fluid (CSF) of ALS patients. Using better methods, this group published 1 year later their revised but consistent results confirming this hypothesis [5]. The effect of riluzole, a drug with antiglutamate properties that extends survival in ALS patients [6], [7], has been used to support the probable implication of glutamate in motor neuron injury in ALS.

Following the initial publications from the Rothstein group, conflicting results were obtained from different laboratories [8], [9], [10], [11], [12], [13], [14] due in part to technical factors [15] and in part to the small number of patients analyzed.

Only one publication by Shaw et al. [16] comparing 37 ALS patients to 35 neurological controls reported a significant elevation of CSF glutamate concentrations only in part of ALS patients, suggesting that the ALS patients population could be heterogeneous. However, in the opinion of these authors, the relatively small number of patients included, the heterogeneity of the findings, and the lack of stabilization of CSF glutamate by acidification with perchloric acid and its subsequent neutralization with potassium carbonate [17] do question the validity of their results.

We have therefore undertaken an analysis of CSF glutamate concentrations with a new High Pressure Liquid Chromatographic Method (HPLC) using coulometric detection in a very large sample of 377 ALS patients, compared to a control group of both normal and neurological controls. Individual concentrations of glutamate in the CSF of these ALS patients were correlated with clinical parameters.

Section snippets

Cerebrospinal fluid sampling

Cerebrospinal fluid (CSF) was obtained after a minimal period of 12 h (maximum 18 h) of fasting and resting. Patients were punctured in a sitting position, and only one attempt was made to introduce the needle. The initial 500 μl of CSF were set aside since in a preliminary study in 10 patients (data not shown), we found a 2- to 10-fold higher amount of glutamate in this sample as compared to the rest, in which the concentration of glutamate was stable. CSF for glutamate determination was

Results

In the overall population, the mean age was 60.4±13.6 years. The overall mean CSF concentration of glutamate was 0.31±0.16 μM/l. No correlation was observed between CSF glutamate concentrations and age (p=0.237) or gender (p=0.23).

In the overall controls population (including all normal and neurological controls), the mean age was 55.5±16.1 years (range: 20–87) and the mean CSF concentration of glutamate was 0.201±0.101 μM/l (range: 0.046–0.635 μM/l) (Table 1). In the three categories

Discussion

Using a new HPLC method with coulometric detection, we determined the concentration of glutamate in CSF in a large cohort of ALS patients. Our study has shown a significant increase in CSF glutamate concentrations in approximately one-half of these patients (40.8%). This appears to be specific for this disease since such a pattern was not observed in the other neurological controls, including other neurodegenerative disorders, such as idiopathic Parkinson's disease, parkinsonism and AD. These

Acknowledgements

We are grateful to Bernadette Balagny for skillful technical assistance, to Philippe Julien for providing normal controls CSF and to Adam Doble for reviewing the manuscript.

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