Determination of D-Aspartic Acid and D-Glutamic Acid in Midbrain of Parkinson's Disease Mouse by Reversed Phase High Performance Liquid Chromatography

A reversed high performance liquid chromatography (HPLC) method was established for the determination of the trace amounts of D-aspartic acid (D-Asp) and D-glutamic acid (D-Glu) in biological samples. Amino acids enantiomers were precolumn-derivatized with o-phthalaldehyde (OPA) and N-isobutyryl-L-cysteine. The separation of two amino acids enantiomers was achieved within 40 min on a Shim-pack VP-ODS column with acetate-methanol-acetonitrile mixture solution as mobile phase using a gradient elution program. The column eluent was monitored using fluorescence detection. The calibration curve was linear in the range of 1.0 × 10−8–1.0 × 10−6 M for both D-Asp and D-Glu. The detection limits of D-Asp and D-Glu were 2.0 × 10−9 M and 1.0 × 10−9 M, respectively, when the ratio of signal to noise was 3. The levels of D-Asp and D-Glu in the midbrains of Parkinson's disease (PD) mouse were detected by the presented method. When compared to controls, the levels of D-Asp and D-Glu showed different changes; the former showed no significant difference while the latter did. The result suggested that the biosynthesis and the transportation of endogenous D-Asp were not influenced by neurotoxicity 1-methyl-4-phenyl-1,2,3,6-tetrahydrophridine and still maintained the original dynamic state of homeostasis, but the D-Glu may have excited toxicity similar to L-enantiomer, and may participate in PD pathogenesis.