A specific enzymatic high-performance liquid chromatography method to determine N-methyl-D-aspartic acid in biological tissues.
Recently we demonstrated that N-methyl-D-aspartic acid (NMDA) is present as an endogenous compound in the nervous tissues and endocrine glands of the rat where it plays a role in the regulation of the luteinizing hormone, growth hormone, and prolactin (FASEB J. 14 (2000) 699; Endocrinology 141 (2000) 3861). Based on the prediction that NMDA could have future importance in neuroendocrinology, we have devised an improved method for the specific and routine determination of NMDA in biological tissue. This method is based on the detection by HPLC of methylamine (CH(3)NH(2)) which comes from the oxidation of NMDA by D-aspartate oxidase, an enzyme which specifically oxidizes NMDA, yielding CH(3)NH(2) as one of the oxidative products of the reaction. The sensitivity of the method permits the accurate determination of NMDA in the supernatant of a tissue homogenate at levels of about 5-10 picomol/assay. However, for those tissues in which the concentration of NMDA is less than 1nmol/g, the sample must be further purified by treatment with o-phthaldialdehyde in order to separate the NMDA from the other amino acids and amino compounds and then concentrated and analyzed by HPLC. Using this method we have conducted a comparative study in order to measure the amount of NMDA in neuroendocrine and other tissues of various animal phyla from mollusks to mammals.
D'Aniello
Antimo
A
Laboratory of Neurobiology, Zoological Station of Naples, Villa Comunale, Italy. daniello@alpha.szn.it
De Simone
Antonella
A
Spinelli
Patrizia
P
D'Aniello
Salvatore
S
Branno
Margherita
M
Aniello
Francesco
F
Rios
Jeannette
J
Tsesarskaja
Mara
M
Fisher
George
G
D000594:Amino Acid Oxidoreductases / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D050804:D-Aspartate Oxidase; D005625:Frontal Lobe / Q000737:chemistry / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000737:chemistry / Q000378:metabolism; D008099:Liver / Q000737:chemistry / Q000378:metabolism; D008297:Male; D008744:Methylamines / Q000032:analysis; D016202:N-Methylaspartate / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D009490:Neurosecretory Systems / Q000737:chemistry* / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013737:Testis / Q000737:chemistry / Q000378:metabolism
D008744:Methylamines; D016202:N-Methylaspartate; C027451:methylamine; D000594:Amino Acid Oxidoreductases; D050804:D-Aspartate Oxidase