Fluorescence determination of N‐acetylaspartic acid in the rat cerebrum homogenate using high‐performance liquid chromatography with pre‐column fluorescence derivatization

Abstract

N-acetyl-L-aspartic acid (NAA) is an endogenous compound, and its brain concentration is suggested to be altered in neurological disorders. In the present study, a fluorescence determination method for NAA was developed by employing reversed-phase high-performance liquid chromatography (HPLC) with pre-column fluorescence derivatization using 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole (DBD-ED). Using methylsuccinic acid as the internal standard, a linear calibration curve for NAA was constructed in the range 125-1000 microM (n=3). The detection limit on the column was approximately 5.0 fmol (signal-to-noise ratio 3). The proposed HPLC method was applied to determine NAA in the rat cerebrum homogenate. Cerebrum NAA was successfully determined using 10 microL of the homogenate, and the validation data for the proposed HPLC method demonstrated satisfactory results. Intra- and inter-day precision and accuracy were within 1.1-7.0 and -8.1-6.3%, respectively. The concentration of NAA in the male rat cerebrum (13 weeks old) was 84 +/-4.6 nmol/mg protein (n = 3) [corrected].

Proteomics-driven Antigen Discovery for Development of Vaccines Against Gonorrhea

“…Bioinformatic Analysis-Subcellular localization of identified proteins was assessed as described previously (21,35,37) using PSORTb 3.0.2 (39), SOSUIGramN (40), and CELLO 2.5 (41), and majority-votes strategy was used for protein localization assignment. In cases where these three software systems predicted different subcellular localizations for a specific protein, the protein was assigned to a group of "unknown subcellular localization."…”

Simultaneous determination of N-acetylaspartylglutamate and N-acetylaspartate in rat brain homogenate using high-performance liquid chromatography with pre-column fluorescence derivatization

“…After filtration using the Millex-LG ® (0.20 m membrane, Nihon Millipore K.K., Tokyo, Japan), 20 L of the final solution was injected into our HPLC system. For the confirmation of the chemical structure of the derivatives, the HPLC-mass spectrometer (MS) was used , and the condition was the same as our previous paper [17].…”

“…The protein concentration of the rat brain homogenate was assayed using a commercial kit bicinchoninic acid (BCA TM ) protein assay (Pierce Biotechnology Inc., Rockford, IL, USA), as previously reported [17]. Statistical analyses were performed by Student's unpaired t-test and a p value of <0.05 was regarded as significant difference.…”

“…The fluorescence derivatization procedure was performed according to our previous paper with minor modifications [17]. Each 10 L of NAAG, NAA, and 0.20 mM MSA (I.S., in CH 3 CN) was mixed vigorously with 470 L of MeOH, following which, 50 L of the mixture was evaporated in vacuo.…”

“…In our previous paper [17], we developed a sensitive determination method of NAA by using high-performance liquid chromatography. A fluorescence derivatization of NAA with a fluorescence reagent, 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole (DBD-ED) [18], was employed to increase the detection sensitivity.…”