LETTERS
Excitatory Amino Acids in Amyotrophic Lateral-Sclerosis David C. Spink, PhD,” and David L. Martin, PhD? In a recent issue of Annals, a series of articles was published 11-41 concerning possible alterations of excitatory amino acid metabolism in amyotrophic lateral sclerosis (ALS). Plaitakis C 11 presented an hypothesis whereby a generalized defect in glutamate metabolism causes elevated levels of this excitatory neurotransmitter and neuronal cell loss by an excitotoxic mechanism. Apparent evidence in support of this model was presented by Rothstein and coworkers {41, who reported elevated levels of glutamate and aspartate in cerebrospinal fluid (CSF) of patients with ALS. Perry and colleagues {31, however, did not observe elevated concentrations of glutamate and aspartate in the CSF of ALS patients, and reported CSF concentrations of aspartate and glutamate for their control group that were more than an order of magnitude lower than those reported for a similar control group in the study by Rothstein and coworkers E4f.Young {23 offered some salient comments and suggestions but was unable to resolve the discrepancy, particularly because both groups used nearly identical methods of analysis. Both groups deproteinized CSF samples with sulfosalicylic acid and analyzed the supernatants on amino acid analyzers (ion-exchange chromatography and post-column color development with ninhydrin). Perry and coworkers [3] have greatly refined the technique over the years, and their estimates of glutamate and aspartate in CSF have steadily fallen. Several factors leading to the overestimation of glutamate and aspartate in CSF have been identified by Perry and coworkers and by others {5-81. The most important sources of error are the hydrolysis of glutamine, asparagine, and possibly peptides, either by enzymes present in the CSF or by incautious use of sulfosalicylic acid used to deproteinite the specimens, and the lack of chromatographic resolution of glutamate and aspartate from acidic peptides and other interfering components in the CSF. With their current techniques, Perry and coworkers {3] do not have adequate sensitivity to measure aspartate in CSF and can barely detect glutamate. In recent years, high-performance liquid chromatography (HPLC) following derivatization with o-phthalaldehyde has become a widely used method for amino acid analysis because it offers high-resolution chromatography, flexibility in the use of gradient methods, and the high sensitivity of fluorescence detection. The technique has been validated for the analysis of glutamate and aspartate in CSF, particularly with regard to chromatographic resolution, quantitative measurement of glutamate and aspartate added to CSF, and removal of protein from the specimens [6-81. Values for the concentrations of glutamate and aspartate in CSF from groups of neurologically normal patients as determined by the HPLC method 16-81 are in agreement with those reported by Perry and coworkers [3] (mean values 50.5 pM for both glutamate and aspartate) rather than the higher values reported by Rothstein and colleagues [4].The HPLC technique may be useful for resolving the disagreement a b m t rhe levels of 110
glutamate and aspartate in the CSF of ALS patients. It appears unlikely that the values reported by Rothstein and colleagues 141 are accurate estimates of glutamate and aspartare in CSF. W e d o not mean to imply that their findings are insignificant in understanding the pathobiology of ALS, but the meaning of their measurements will require the identification and determination of the origin of the compounds that were measured.
Vadswortb Center for Laboratories and Kesearcb New York State Department of Health Albany, NY fDepartment of Environmental Health and Toxicology School of Public Health State University of New York at Albany Albany, NY
References 1. Plaitakis A. Glutamate dysfunction and selective motor neuron degeneration in amyotrophic lateral sclerosis: a hypothesis. Ann
Neurol 1790;28:3-8 2. Young AB. What’s the excitement about excitatory amino acids in amyotrophic lateral sclerosis? Ann Neurol 1990;28:9-11 3. Perry TL, Krieger C, Hansen S, Eisen A. Amyotrophic lateral sclerosis: amino acid levels in plasma and cerebrospinal fluid. Ann Neurol 1990;28:12-17 4. Rothstein JD, Tsai G, Kuncl RW, et al. Abnormal excitatory
5.
6.
7.
8.
amino acid metabolism in amyotrophic lateral sclerosis. Ann Neurol 1990;28:18-25 Ferraro TN, Hare TA. Triple-columnion-exchange physiological amino acid analysis with fluorescent detection: baseline characterization of human cerebrospinal fluid. Anal Biochem 1984; 143:82-94 Spink DC, Swann JW, Snead OC, et al. Analysis of aspartate and glutamate in human cerebrospinal fluid by high-perfurmance liquid chromatography with automated precolumn derivatization. Anal Biochem 1986;158:79-86 Cmldsmith RF,Earl JW, Cunningham AM. Determination of yaminobutyricacid and other amino acids in cerebrospinal fluid of pediatric patients by reversed-phase liquid chromatography. Clin Chem 1987;33:1736-1740 Alfredsson G, Wiesel FA, Lindberg M. Glutamate and glutamine in cerebrospinal fluid and serum from healthy volunteersanalytical aspects. J Chromatog 1988;424:378-384
Surgical Treatment for Infantile Spasms? Richard A. Hrachovy, MD, James D. Frost, Jr, MD, Daniel G. Glaze, MD, and Peter Kellaway, PhD Chugani and associates [I] describe localized positron emission tomography (PET) scan and electroencephalographic changes in 5 infants with “intractable epilepsy.” Four of the 5 infants subsequently underwent surgical removal of the cortical focus for treatment of their seizures. W e found the article interesting, but the lack of sufficient data concerning the patients makes assessment of its significance difficult. For example, the authors imply initially that the PET scai studies, as well as the surgical procedures, were performed on infants with infantile spasms. However, as Ttated in the Methods section, 2 of the 5 patients in the cudy were no
Copyright 0 1991 by the American Neurological Association
Excitatory Amino Acids in Amyotrophic Lateral-Sclerosis David C. Spink, PhD,” and David L. Martin, PhD? In a recent issue of Annals, a series of articles was published 11-41 concerning possible alterations of excitatory amino acid metabolism in amyotrophic lateral sclerosis (ALS). Plaitakis C 11 presented an hypothesis whereby a generalized defect in glutamate metabolism causes elevated levels of this excitatory neurotransmitter and neuronal cell loss by an excitotoxic mechanism. Apparent evidence in support of this model was presented by Rothstein and coworkers {41, who reported elevated levels of glutamate and aspartate in cerebrospinal fluid (CSF) of patients with ALS. Perry and colleagues {31, however, did not observe elevated concentrations of glutamate and aspartate in the CSF of ALS patients, and reported CSF concentrations of aspartate and glutamate for their control group that were more than an order of magnitude lower than those reported for a similar control group in the study by Rothstein and coworkers E4f.Young {23 offered some salient comments and suggestions but was unable to resolve the discrepancy, particularly because both groups used nearly identical methods of analysis. Both groups deproteinized CSF samples with sulfosalicylic acid and analyzed the supernatants on amino acid analyzers (ion-exchange chromatography and post-column color development with ninhydrin). Perry and coworkers [3] have greatly refined the technique over the years, and their estimates of glutamate and aspartate in CSF have steadily fallen. Several factors leading to the overestimation of glutamate and aspartate in CSF have been identified by Perry and coworkers and by others {5-81. The most important sources of error are the hydrolysis of glutamine, asparagine, and possibly peptides, either by enzymes present in the CSF or by incautious use of sulfosalicylic acid used to deproteinite the specimens, and the lack of chromatographic resolution of glutamate and aspartate from acidic peptides and other interfering components in the CSF. With their current techniques, Perry and coworkers {3] do not have adequate sensitivity to measure aspartate in CSF and can barely detect glutamate. In recent years, high-performance liquid chromatography (HPLC) following derivatization with o-phthalaldehyde has become a widely used method for amino acid analysis because it offers high-resolution chromatography, flexibility in the use of gradient methods, and the high sensitivity of fluorescence detection. The technique has been validated for the analysis of glutamate and aspartate in CSF, particularly with regard to chromatographic resolution, quantitative measurement of glutamate and aspartate added to CSF, and removal of protein from the specimens [6-81. Values for the concentrations of glutamate and aspartate in CSF from groups of neurologically normal patients as determined by the HPLC method 16-81 are in agreement with those reported by Perry and coworkers [3] (mean values 50.5 pM for both glutamate and aspartate) rather than the higher values reported by Rothstein and colleagues [4].The HPLC technique may be useful for resolving the disagreement a b m t rhe levels of 110
glutamate and aspartate in the CSF of ALS patients. It appears unlikely that the values reported by Rothstein and colleagues 141 are accurate estimates of glutamate and aspartare in CSF. W e d o not mean to imply that their findings are insignificant in understanding the pathobiology of ALS, but the meaning of their measurements will require the identification and determination of the origin of the compounds that were measured.
Vadswortb Center for Laboratories and Kesearcb New York State Department of Health Albany, NY fDepartment of Environmental Health and Toxicology School of Public Health State University of New York at Albany Albany, NY
References 1. Plaitakis A. Glutamate dysfunction and selective motor neuron degeneration in amyotrophic lateral sclerosis: a hypothesis. Ann
Neurol 1790;28:3-8 2. Young AB. What’s the excitement about excitatory amino acids in amyotrophic lateral sclerosis? Ann Neurol 1990;28:9-11 3. Perry TL, Krieger C, Hansen S, Eisen A. Amyotrophic lateral sclerosis: amino acid levels in plasma and cerebrospinal fluid. Ann Neurol 1990;28:12-17 4. Rothstein JD, Tsai G, Kuncl RW, et al. Abnormal excitatory
5.
6.
7.
8.
amino acid metabolism in amyotrophic lateral sclerosis. Ann Neurol 1990;28:18-25 Ferraro TN, Hare TA. Triple-columnion-exchange physiological amino acid analysis with fluorescent detection: baseline characterization of human cerebrospinal fluid. Anal Biochem 1984; 143:82-94 Spink DC, Swann JW, Snead OC, et al. Analysis of aspartate and glutamate in human cerebrospinal fluid by high-perfurmance liquid chromatography with automated precolumn derivatization. Anal Biochem 1986;158:79-86 Cmldsmith RF,Earl JW, Cunningham AM. Determination of yaminobutyricacid and other amino acids in cerebrospinal fluid of pediatric patients by reversed-phase liquid chromatography. Clin Chem 1987;33:1736-1740 Alfredsson G, Wiesel FA, Lindberg M. Glutamate and glutamine in cerebrospinal fluid and serum from healthy volunteersanalytical aspects. J Chromatog 1988;424:378-384
Surgical Treatment for Infantile Spasms? Richard A. Hrachovy, MD, James D. Frost, Jr, MD, Daniel G. Glaze, MD, and Peter Kellaway, PhD Chugani and associates [I] describe localized positron emission tomography (PET) scan and electroencephalographic changes in 5 infants with “intractable epilepsy.” Four of the 5 infants subsequently underwent surgical removal of the cortical focus for treatment of their seizures. W e found the article interesting, but the lack of sufficient data concerning the patients makes assessment of its significance difficult. For example, the authors imply initially that the PET scai studies, as well as the surgical procedures, were performed on infants with infantile spasms. However, as Ttated in the Methods section, 2 of the 5 patients in the cudy were no
Copyright 0 1991 by the American Neurological Association
