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Editorials |
Division of Clinical Neuroscience, Chiba University Center for Forensic Mental Health, Chiba, Japan
Address correspondence to the author at: Division of Clinical Neuroscience Chiba University Center for Forensic Mental Health 1-8-1 Inohana Chiba 260-8670, Japan Fax +81-423-226-2150 E-mail hashimoto{at}faculty.chiba-u.jp
It was long believed that only the L-isomer of amino acids existed in mammals, and D–amino acids were regarded as laboratory artifacts and categorized as "unnatural" isomers. This term was widely used in textbooks of biochemistry. D–amino acids were known to be prominent in bacteria, and there were occasional reports of D–amino acids found in invertebrates (1). Hans Krebs accidentally discovered in kidney tissue an enzyme, D–amino acid oxidase (DAAO)1 , which recognized unnatural D–amino acids (but not their L-counterparts) (2). DAAO was found to degrade D–amino acids produced by bacteria from foods in the gut.
With the advance of chromatographic analysis techniques, small amounts of D–amino acids can now be measured in lower and higher animals, plants, and foods. By the use of 2-dimensional thin-layer chromatography and HPLC, Nagata et al. (3) found free D–amino acids, including D-serine, in kidney and blood of mutant mice lacking DAAO. Subsequently, Hashimoto and colleagues (4) demonstrated that D-serine was present in rat brain at high concentrations that were up to one-third those of L-serine, and that D-serine is heterogeneously distributed throughout rat brain with a pattern resembling that of the N-methyl D-aspartate (NMDA) subtype of glutamate receptors. Glutamate cannot activate the NMDA receptor in the absence of the coagonist glycine, and D-serine is up to 3 times more potent than glycine at the glycine site of the NMDA receptors. D-serine is likely to be the predominant endogenous ligand for the NMDA receptors in most areas of the brain. Although glycine serves this purpose in some sites, in most parts of the brain D-serine distribution matches that of NMDA receptors more than does glycine (5). D-Serine is produced by serine racemase from L-serine in the brain, and D-serine is metabolized by DAAO (5).
Column-switching HPLC with fluorescence detection (6) and HPLC with ultraviolet-visible detection (7) have been used for quantification of D- and L-serine. The former method includes precolumn fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and separation of the derivatives on a reversed-phase column and then on Sumichiral OA-2500 (S) Pirkle-type chiral columns (6). In this issue of Clinical Chemistry, Fuchs et al. (8) report 2 novel mass-spectrometric techniques for separation and quantification of D- and L-serine. To enable simultaneous determination of D-serine, L-serine, and glycine in small volumes of biological fluids, these authors developed stable isotope dilution assays using GC-MS and LC-MS. The GC-MS system they used is a nonchiral derivatization with chiral (Chirasil-L-val column) separation, and the LC-MS system is a chiral derivatization with Marfeys reagent and LC-MS analysis. Quantification limits for D-serine, L-serine, and glycine in cerebrospinal fluid (CSF) from human sample donors were 0.14, 0.44, and 0.14 µmol/L (GC-MS) and 0.20, 0.41, and 0.14 µmol/L (LC-MS), respectively. The concentrations of D-serine and L-serine in human CSF were also consistent with those previously reported by other groups (9)(10). Sample preparation time was 60 min for the LC-MS system, whereas it was approximately 6–8 h for the GC-MS system. The relatively short sample preparation time indicates that the LC-MS system is superior to the GC-MS system in its potential to be automated and used to perform high-throughput analysis.
The ability to measure CSF amino acids raises the question of whether CSF D-serine, L-serine, and glycine have enough potential clinical utility to warrant ongoing study. In patients with 3-phosphoglycerate dehydrogenase deficiency (OMIM 606879), a rare disorder of L-serine biosynthesis, CSF concentrations of D-serine, L-serine, and glycine are much lower than those in controls (11). In these patients postnatal L-serine supplementation normalized CSF D-serine, L-serine, and glycine as well as the clinical phenotype. Interestingly, in neonates whose mothers had undergone prenatal L-serine treatment, CSF concentrations of these amino acids at birth were near reference interval concentrations and the clinical phenotype was normal. Thus, it seems that L-serine biosynthesis, leading to D-serine synthesis and NMDA-receptor activation, is crucial for early neuronal development in humans (11). In the neonatal cerebellum of rodents, D-serine concentrations are high and peak at the time of granule cell migration, a finding that suggests that the principal role of D-serine in the developing cerebellum is to serve as a coagonist for NMDA receptor–dependent granule cell migration (12). Taken together, these findings suggest a pivotal role of both isomers of serine in normal and aberrant human brain development.
Alterations in blood or CSF concentrations of D- and L-serine have been found in patients with schizophrenia (9)(10)(13)(14), 3-phosphoglycerate-dehydrogenase deficiency, (11), and chronic pain(7). Furthermore, we reported increased glutamine:glutamate ratios in CSF of first-episode and drug-naive schizophrenic patients, suggesting that a dysfunction in the glutamate-glutamine cycle between neurons and glia may play a role in the pathophysiology of schizophrenia (15). Thus the work from multiple groups has provided evidence that serine enantiomers play important roles in neurological and psychological diseases.
In their current report Fuchs et al. confirm their previous finding (11) that in humans CSF concentrations of D-serine, L-serine, and glycine decrease with age. This process was most pronounced for D-serine concentrations, which decreased 4.9-fold (GC-MS) or 9.0-fold (LC-MS) during the first 3 years of life and remained constant thereafter (8). The age-dependent changes in CSF amino acids illustrate the potential need for differing interpretations of neonatal, prenatal, adolescent, and adult CSF concentrations. Fortunately the novel methods reported here (8) enable such differentiation and should be valuable tools for continued evaluation of the role of amino acids, including D-serine and L-serine, in investigating both normal brain function and the pathophysiology of various neuropsychiatric disorders.
Acknowledgments
Grant/Funding Support: None declared.
Financial Disclosures: None declared.
Footnotes
1 Nonstandard abbreviations: DAAO, D–amino acid oxidase; NMDA, N-methyl D- aspartate; CSF, cerebrospinal fluid. 
References
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