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HPLC Methods for Determination of d-Aspartate and N-methyl-d-Aspartate

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Unnatural Amino Acids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 794))

Abstract

d-Amino acids are stereoisomers or optical isomers of naturally occurring l-amino acids and thus possess the same chemical structure, but may differ in their biological/physiological properties. Until a half century ago, d-amino acids had been considered to be unnatural substances found only in microorganisms. However, improvements in analytical instruments and methods have revealed that d-amino acids are present in invertebrates and vertebrates, including humans, and that they possess important physiological functions. d-Aspartate (d-Asp) and its methylated form N-methyl-d-aspartate (NMDA) possess neuroendocrine properties in many species. Several methods have been developed for determination of d- and l-enantiomers of amino acids by high performance liquid chromatography (HPLC). We report here improved HPLC methods for the specific determination of d-Asp and NMDA in biological tissues.

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References

  1. D’Aniello A, Giuditta A (1977) Identification of D-aspartic acid in the brain of Octopus vulgaris. J Neurochem 29, 1053–1057.

    Google Scholar 

  2. Hashimoto A, Oka T (1997) Free d-aspartate and d-serine in the mammalian brain and periphery. Prog Neurobiol 52, 325–353.

    Article  PubMed  CAS  Google Scholar 

  3. D’Aniello A, DiFiore M M, Fisher G (1998) Occurrence of d-aspartic acid in animal tissues and its role in the nervous and endocrine systems. Trends Comp Biochem Physiol 4, 1–24.

    Google Scholar 

  4. Spinelli P, Brown E et al. (2006) D-Aspartic acid in the nervous system of Aplysia limacina: Possible role in neurotransmission. J Cell Physiol 206, 672–681.

    Article  PubMed  CAS  Google Scholar 

  5. D’Aniello A, DiFiore M M, Fisher G H et al. (2000) Occurrence of d-aspartic acid and N-methyl-d-aspartic acid in rat neuroendocrine tissues and their role in the modulation of luteinizing hormone and growth hormone release. FASEB J 14, 699–714.

    PubMed  Google Scholar 

  6. Fisher G H, D’Aniello A et al. (1991) Free d-aspartate and d-alanine in normal and Alzheimer brain. Brain Res Bull 26, 983–985.

    Article  PubMed  CAS  Google Scholar 

  7. D’Aniello A (2007) D-Aspartic acid: An endogenous amino acid with an important neuroendocrine role. Brain Res Rev 53, 215–234.

    Article  PubMed  Google Scholar 

  8. Watkins J C, Evans R H (1981) Excitatory amino acid transmitters. Ann Rev Pharmacol Toxicol 21, 165–204.

    Article  CAS  Google Scholar 

  9. Monaghan D T, Cotman C W (1986) Identification and properties of N-methyl-d-aspartate receptors in rat brain synaptic plasma membranes. Proc Natl Acad Sci USA 83, 176–179.

    Article  Google Scholar 

  10. Mondadori C, Weiskrantz L et al. (1989) NMDA receptor antagonists can enhance or impair learning performance in animals. Exp Brain Res 75, 449–456.

    Article  PubMed  CAS  Google Scholar 

  11. Aswad D W (1984) Determination of d- and l-aspartate in amino acid mixtures by high performance liquid chromatography after derivatization with a chiral adduct of o-phthaldialdehyde. Anal Biochem 137, 405–407.

    Article  PubMed  CAS  Google Scholar 

  12. Nimura N, Kinoshita T (1986) o-Phthaldialdehyde-N-acetyl-l-cysteine as a chiral derivatization reagent for liquid chromatographic optical resolution of amino acid enantiomers and its application to conventional amino acid analysis. J Chromatog 352, 169–177.

    Article  CAS  Google Scholar 

  13. Nishikawa T, Oka T et al. (1992) Determination of free amino acid enantiomers in rat brain and serum by HPLC after derivatization with N-tert.-butoxycarbonyl-L-cysteine and o-phthaldialdehyde. J Chromatog 582, 41–48.

    Article  Google Scholar 

  14. Brüchner H, Haasmann S et al. (1994) Liquid chromatographic determination of d- and l-amino acids by derivatization with o-phthaldialdehyde and chiral thiols. J Chromatog 666, 259–273.

    Article  Google Scholar 

  15. Todoroki N, Shibata K et al. (1999) Determination of N-methyl-d-aspartic acid in tissues of bivalves by HPLC. J Chromatog B 728, 41–47.

    Article  CAS  Google Scholar 

  16. D’Aniello A, De Simone A et al. (2002) A specific high-performance liquid chromatography method to determine N-methyl-d-aspartic acid in biological tissues. Anal Biochem 308, 42–51.

    Article  PubMed  Google Scholar 

  17. Skine M, Fukuda H, Nimura N et al. (2002). Automated column-switching high-performance liquid chromatography system for quantifying N-methyl-d- and -L-aspartate. Anal Biochem 310, 114–121.

    Article  Google Scholar 

  18. Szókán G, Mezö G, Hudecz F (1988) Application of Marfey’s reagent in racemization studies of amino acids and peptides. J Chromatog 444, 115–122.

    Article  Google Scholar 

  19. Galindo E, Tsesarskaia M, Fisher G et al. (2009) An Improved HPLC Method for Determination and Quantification of D- and L-Aspartic Acid. In: Konno, Brueckner, D’Aniello, Fisher, Fujii, Homma (eds), D-Amino acids: practical methods and protocols, Volume 1: Analytical methods for D-amino acids. Nova Science Publishers, New York. pp. 43–48.

    Google Scholar 

  20. Tsesarskaia M, Galindo E, Fisher G, Szókán G (2009) A Sensitive One Step HPLC Method for Simultaneous Determination of N-Methyl-(D and L)-Aspartate, N-Methyl-(D and L)-Glutamate and (D and L)-Aspartate in Biological Tissues. In: Konno, Brueckner, D’Aniello, Fisher, Fujii, Homma (eds), D-Amino acids: practical methods and protocols, Analytical methods for D-amino acids. Nova Science Publishers, New York, pp 25–31.

    Google Scholar 

  21. Tsesarskaia M, Galindo E, Szókán G, Fisher G (2009) HPLC determination of acidic d-amino acids and their N-methyl derivatives in biological tissues. Biomed Chromatog 23, 581–587.

    Article  CAS  Google Scholar 

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Acknowledgments

We wish to acknowledge the Barry University students who helped develop the experimental procedures described here: Erika Galindo, Susana Lopez, and Collins Boston. Separate reports of these methods have previously been published in references (19–21).

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Correspondence to George H. Fisher .

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Fisher, G.H., Tsesarskaia, M. (2012). HPLC Methods for Determination of d-Aspartate and N-methyl-d-Aspartate. In: Pollegioni, L., Servi, S. (eds) Unnatural Amino Acids. Methods in Molecular Biology, vol 794. Humana Press. https://doi.org/10.1007/978-1-61779-331-8_16

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  • DOI: https://doi.org/10.1007/978-1-61779-331-8_16

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-330-1

  • Online ISBN: 978-1-61779-331-8

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