Short communication

Received 25 February 2011, Revised 18 March 2011, Accepted: 21 March 2011 Published online in Wiley Online Library: 6 May 2011

(wileyonlinelibrary.com) DOI 10.1002/bmc.1639

Determination of Nacetylaspartic acid concentration in the mouse brain using HPLC with uorescence detection
Ziyu Songa, Dan Gea, Kana Ishiia, Hiroshi Yamadaa, Kazuya Toriumib, Hiroyuki Watanabeb, Toshitaka Nabeshimab and Takeshi Fukushimaa*
ABSTRACT: The concentration of brain Nacetylaspartic acid (NAA) in mice was determined by highperformance liquid chromatography (HPLC) using uorescence detection after precolumn derivatization with 4N,Ndimethylaminosulfonyl7 N(2aminoethyl)amino2,1,3benzoxadiazole (DBDED). Six different brain parts, namely, the prefrontal cortex, olfactory bulb, nucleus accumbens, striatum, cerebellum and hippocampus, of male C57BL6/J mice, were investigated. The NAA concentration (nmol/mg protein) was highest in the olfactory bulb (58.2 4.0, n = 8) and lowest in the hippocampus (42.8 1.6, n = 8). The proposed HPLC method with uorescence detection was successfully used to determine the NAA concentration in each investigated brain area. Copyright 2011 John Wiley & Sons, Ltd. Keywords: NAcetylLaspartic acid; DBDED; mouse brain; uorescence detection; HPLC

Introduction
NAcetylaspartic acid (NacetylLaspartic acid; NAA) is an Nacetylated derivative of Laspartic acid that is present in the vertebrate brain (Tallan et al., 1956; Moffett et al., 2007). NAA has been proposed to function as a neuronal osmolyte involved in uid balance in the brain, a source of acetate for lipid and myelin synthesis in oligodendrocytes, a contributor to energy production in neuronal mitochondria (Baslow, 2003) and a precursor to Nacetylaspartylglutamate (NAAG), a selective agonist at the type 3 metabotropic glutamate receptor (mGluR3) (Neale et al., 2000). In the brain, NAA is considered to be a neuronal marker that is present predominantly in neuronal cell bodies. Decit of NAA correlates with psychopathy and can therefore be used as an indicator of the severity of neurologic disorders (Schuff et al., 2006; Rigotti et al., 2007). Recently, decit of NAA, determined using HPLC, was observed in the postmortem hippocampus, parahippocampal gyrus, amygdale, caudate nucleus and putamen of subjects with psychiatric illnesses, namely, schizophrenia, bipolar disorder and major depression (Reynolds and Reynolds, 2011). Reversedphase HPLC with UV detection at 210215 nm is typically employed to determine brain NAA concentration (Tavazzi et al., 2000; Battistuta et al., 2001; Harte et al., 2005; Reynolds and Reynolds, 2011). Using UV absorbance for NAA detection, however, limits detection sensitivity. A more sensitive method is required for precise determination of NAA concentrations in isolated brain sections of experimental rodents. Previously, we developed an HPLCuorescence detection method for NAA using precolumn derivatization with 4N, Ndimethylaminosulfonyl7N(2aminoethyl)amino2,1, 3benzoxadiazole (DBDED) (Fukushima et al., 2008; Arai et al., 2008; Fig. 1). This HPLC method was used to determine NAA

concentration in rat brain homogenate. Mice are frequently used in neuropharmacological research, particularly transgenic mice expressing abnormal behaviors and biochemical alterations. Therefore, it is also necessary to determine NAA concentrations in mouse brain samples. In the present study, we attempted to determine the concentration of NAA in six isolated brain regions, namely, the prefrontal cortex, olfactory bulb, nucleus accumbens, striatum, cerebellum and hippocampus, of male C57BL6/J mice using the HPLCuorescence detection method.

Materials and methods

Chemicals NAA, ()methylsuccinic acid (MSA) and formic acid (HCO2H) were obtained from Wako Pure Chemical Industries Ltd (Osaka, Japan). DBDED, 1ethylN,Ndimethylaminocarbodiimide (EDC) and N,Ndimethylaminopyridine (DMAP) were purchased from

* Correspondence to: Takeshi Fukushima, Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Toho University, 221 Miyama, Funabashishi, Chiba 2748510, Japan. Email: tfukushima@phar.tohou.ac.jp
a

Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Toho University, 221 Miyama, Funabashishi, Chiba 2748510, Japan Department of Chemical Pharmacology, Meijo University Graduate School of Pharmaceutical Sciences, Nagoya 4688503, Japan Abbreviations used: Cel, cerebellum; DBDED, 4N,Ndimethylaminosulfonyl 7N(2aminoethyl)amino2,1,3benzoxadiazole; Hip, hippocampus; MSA, () methylsuccinic acid; NAA, Nacetylaspartic acid; Nac, nucleus accumbens; NAAG, NacetylLaspartylLglutamate; Olb, olfactory bulb; Pre, prefrontal cortex; Str, striatum.

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Figure 1. Fluorescence derivatization scheme of NAA and MSA with DBDED.

Tokyo Chemical Industry Co. Ltd (Tokyo, Japan). Dimethylformamide (DMF) was supplied by Nacalai Tesque Inc. (Kyoto, Japan). Bovine serum albumin (BSA) was purchased from Sigma (St Louis, MO, USA). Coomassie Blue was purchased from BioRad Laboratories Inc. (CA, USA). Water was puried using a MilliQ Labo system (Nihon Millipore Co. Ltd., Tokyo, Japan). Acetonitrile (CH3CN) and methanol (MeOH) of special grade were obtained from Kanto Kagaku Co. Ltd (Tokyo, Japan) and Wako Pure Chemical Industries (Osaka, Japan), respectively. Precolumn uorescence derivatization NAA (100, 50, 25, and 12.5 M) was prepared in H2O, and 200 M MSA, used as an internal standard (IS), was dissolved in CH3CN. A 10 L aliquot of each concentration of NAA was mixed with the IS and evaporated in vacuo. The residues obtained were dissolved in 50 L DMF, after which the following solutions were added serially: 50 L of 80 mM DBDED in CH3CN, 50 L of 200 mM DMAP in CH3CN, and 50 L of 200 mM EDC in MeOH. Solutions were mixed vigorously after each addition. After reacting at 60C for 30 min, the reaction mixtures were diluted 10fold using 0.1% HCOOH in H2OCH3CN (50:50). Subsequently, a 50 L aliquot of each solution was loaded onto a solid phase extraction (SPE) cartridge bearing a membrane coated with sulfonated poly(styrenedivinylbenzene) polymer (SCX, MonoSpin; GL Sciences, Tokyo, Japan) and initialized with 50 L of 0.1% HCO2H in H2OCH3CN (50:50). Finally, 10 L of each solution was injected into the HPLC system. HPLCuorescence detection A Shimadzu LC 2010C HPLC system (Shimadzu Corporation, Kyoto, Japan) was used with a Hitachi L7485 uorescence detector (Hitachi Ltd, Tokyo, Japan) and a Shimadzu CR8A recorder (Shimadzu Corporation, Kyoto, Japan). A Cadenza CD C18 separation column (250 4.6 mm; i.d., 3 m; Imtakt Co. Ltd, Kyoto, Japan) was used along with a TSK guardgel ODS80Ts guard column (3.2 1.5 mm; i.d., 5 mm; Tosoh Co. Ltd). The mobile phases A and B used in the study were 0.1% HCO2H in

H2O and 0.1% HCO2H in CH3CN, respectively. The mobile phase was eluted according to the following program: 025 min, B% = 34 (isocratic); 2535 min, B% = 3442 (linear gradient). The ow rate was constantly maintained at 0.8 mL/min. The column temperature was set at 40C, and the detection wavelength was set at 562 nm with 438 nm of excitation, as reported previously (Fukushima et al., 2008; Arai et al., 2008). Animal experiments Male C57BL6/J mice were purchased from Japan SLC Inc. (Shizuoka, Japan) and housed in an environmentally controlled room for at least 1 week prior to use. All animal care and experimentation were performed in accordance with the guidelines for the care and use of laboratory animals set by Meijo University. The mice were decapitated without anesthesia, and the brains were removed and separated into the required parts: olfactory bulb, hippocampus, prefrontal cortex, nucleus accumbens, striatum and cerebellum according to a mouse brain atlas (Franklin and Paxinos, 1997). The isolated brain regions were immediately homogenized using an Astrason Ultrasonic Processor XL (Misonix) with 10 vols of phosphatebuffered saline (PBS) under icecooled conditions, and then centrifuged at 3000 g for 10 min at 4C. The resulting supernatant uid was stored at 80C until use. To a 20 L aliquot of each sample, 40 L CH3CN and 40 L MeOH were added; the aliquot was then centrifuged at 600 g for 10 min at 4C. A 20 L sample of this supernatant was then added to a transparent tube. Subsequently, 10 L of 200 M MSA in CH3CN was added, and the procedure for precolumn uorescence derivatization was followed. Precision and recovery A prefrontal cortex (PFC) sample was used for the validation study. After pretreatment, a sample of PFC supernatant was added to a transparent tube. Subsequently, 10 L of 200 M MSA in CH3CN and 10 L of 100 M NAA in H2O were added, and the

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Determination of mouse brain Nacetylaspartic acid procedure for precolumn uorescence derivatization was followed (n = 4). Precision was expressed as relative standard deviation (RSD, %), and recovery (%) was calculated using the following equation: concentration of NAA standard spiked with PFC sample concentration of PFC sample=concentration of NAA standard (1) 100 Determination of protein concentration in each mouse brain section using the Bradford method The Bradford method was adopted to determine protein concentrations in this study. Mouse brain homogenate after centrifugation was diluted 10fold with H2O, and 10 L aliquots of the diluted solutions were added to each well of a 96well microtiter plate (n = 3). Then, 150 L of H2O and 40 L of BioRad Protein Assay Dye Reagent Concentrate (BioRad Laboratories Inc., Hercules, CA, USA) were added to each well and mixed. An absorbance of 595 nm was measured immediately using a microplate reader (Multiskan FC; Thermo Fisher Scientic Inc., Waltham, MA, USA). As a blank, 10 L of H2O was used following the same procedure used for the diluted homogenate solutions. By subtracting the blank value from the value found for each homogenate solution, an absorbance based on protein content was found and used to calculate the concentration of protein in each sample. Final protein concentration was determined using a calibration curve constructed using 1 mg/mL of BSA in H2O as a standard. LCMS/MS HPLCmass spectrometric (MS) detection was carried out using Xevo TQS Tandem Quadrupole Mass Spectrometer (Waters Corporation, Milford, MA). An Acquity UPLC system equipped with an Acquity UPLC BEH C18 separation column (1.7 m, 2.1 100 mm) was used. The mobile phases A and B were 0.05% AcOH in H2O and 0.05% AcOH in CH3CN, respectively. The gradient program was as follows: 04 min, B% = 1540; 46 min, B% = 40; 6.017 min, B% = 5595. The ow rate and column temperature were 0.4 mL/min and 40C, respectively. For analyses using the LCMS/MS system, the positiveion mode was selected. The capillary voltage, desolvation gas and cone gas were 1.5 kV, 1100 L/h (650C) and 150 L/h, respectively.

Results and discussion

The proposed HPLC method was used to determine the NAA concentration in brain homogenate of male C57BL6/J mice, which are frequently used as experimental models for various diseases. Figure 2(b, c) shows representative chromatograms of the NAA standard and the prefrontal cortex brain sample, respectively. Distinct uorescence peaks for NAA and MSA (IS) were clearly distinguished in the chromatogram. NAA in the mouse brain sample was easily detected using the proposed HPLC method. Figure 3 shows the MS/MS spectra of the NAA standard derivatized with DBDED (DBDNAA). A protonated molecular ion [M + H] + of DBDNAA (C26H35N11O9S2) was found at m/z 710, indicating that two carboxyl groups of NAA were fully tagged with DBDED. This result was consistent with our previous ndings (Fukushima et al., 2008). The product ion was found at m/z 425, indicating that one moiety of DBDED ( C10H14N5O3S) was cleaved from DBDNAA. Thereby, the uorescence peak observed in Fig. 2 (c) was identied as DBDNAA (m/z 710 m/z 425) using the LCMS/MS system.

Figure 2. Chromatograms of the reagent blank (a), the NAA standard (with IS) (b), and a sample from the PFC (c).

Figure 3. LCMS/MS spectra of the NAA standard derivatized with DBDED, showing the product ion (upper) and the precursor ion (lower).

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Z. Song et al. The calibration curves, constructed using the peak area ratios of NAA to IS plotted against NAA concentration, demonstrated good linearity within the concentration range of 25200 M with a correlation coefcient of 0.995 (n = 3). The detection limit of NAA was approximately 5.0 fmol on the column (signaltonoise ratio, 3). Most previous methods for the determination of NAA in biological samples have employed HPLC with UV detection (210215 nm). For the selective determination of NAA from a biological specimen, many of these methods required a pretreatment procedure whereby the crude extracts were separated using an anion exchangetype cartridge (Harte et al., 2005; Reynolds and Reynolds, 2011). In addition, an ionpair reagent dissolved in the mobile phase was necessary to retain NAA on the ODS column (Tavazzi et al., 2000). Using these HPLC methods, it could be difcult to determine the concentration of NAA in a small section of mouse brain. In contrast, NAA concentrations in the nucleus accumbens, prefrontal cortex and striatum, with wet weights of approximately 7.5, 10, and 13 mg, respectively, could be satisfactorily determined using the proposed HPLC uorescence detection method. Intra and interday precisions were 0.45 and 7.24% (n = 4), respectively, and the intra and interday recoveries were 93.64 0.36 and 91.49 4.84% (n = 4), respectively. Figure 4 shows the NAA concentration (nmol/mg protein, n = 8) in the six isolated brain regions. It was possible to detect NAA in each of the mouse brain regions, namely, the olfactory bulb (58.2 4.0), prefrontal cortex (52.8 3.54), nucleus accumbens (51.1 4.29), striatum (48.0 2.72), cerebellum (45.8 2.98) and hippocampus (42.8 1.75). The NAA concentration (nmol/ mg protein) was highest in the olfactory bulb and lowest in the hippocampus, and the NAA concentration tended to decrease when moving from the anterior to posterior areas of the mouse brain. A signicant difference in NAA concentration was observed between the olfactory bulb and the hippocampus (p < 0.05, n = 8, Bonferroni test). This NAA distribution could be related to the varied distribution of the biosynthetic enzyme necessary for NAA production. Many biochemical studies have examined the synthetic enzyme for NAA biosynthesis, namely aspartate Nacetyl transferase (Madhavarao et al., 2003; Ariyannur et al., 2008). An Nacetyltransferase8 like protein, NAT8L, was found to produce [14C]NAA through the reaction of L[14C]aspartic acid with acetyl coenzyme A in NAT8Loverexpressing HEK293T cells (Wiame et al., 2010). In the rat brain, NAT8L and NAA are colocalized in the neocortical pyramidal neurons, dentate gyrus granule cells, supraoptic nucleus neurons and hypothalamic neurons of rat forebrain tissue, as revealed by immunohistochemical localization studies (Ariyannur et al., 2010). Therefore, NAT8L is thought to synthesize NAA within neurons. NAA is then converted to NAAG, an endogenous agonist of the metabotropic glutamate receptor 3(mGluR3), by NAAG synthetase in neuronal cells (Becker et al., 2010). Interestingly, NAT8L was found to be identical to the protein shati (Niwa et al., 2007), which is upregulated by methamphetamine. In addition, aspartate Nacetyl transferase activity in SHSY5Y human neuroblastoma cells increases approximately 2fold after addition of methamphetamine (Ariyannur et al., 2010). Therefore, alterations of shati expression induced by an exogenous substance, e.g. methamphetamine, may affect NAA concentration in the brain. The precise mechanism by which NAA concentration is diminished in brains affected by psychiatric diseases is not fully understood. Future research will need to employ not only in vitro but also in vivo experiments using transgenic mice to further understand the relationship between NAA and psychiatric disease. In conclusion, the proposed HPLC method with precolumn uorescence derivatization with DBDED was successfully used for the determination of NAA concentration in the homogenate samples of six different parts of the mouse brain. This method can be used in future research to determine NAA concentrations in various brain regions of drugtreated or transgenic mice.

Acknowledgment
The authors gratefully thank Professor R. Takahashi, Dr K. Odera and Ms S. Zhao of Toho University for their kind advice on determination of protein concentration by Multiskan FC. The authors also thank Mr N. Sato, of Nihon Waters K.K., for his kind support regarding measurement by LCMS/MS. This study was nancially supported in part by a GrantinAid for Scientic Research (no. 22590147) from the Ministry of Education, Culture, Sports, Science and Technology.

References
Arai K, Fukushima T, Tomiya M, Mitsuhashi S, Sasaki T and Toyooka T. Simultaneous determination of Nacetylaspartylglutamate and Nacetylaspartate in rat brain homogenate using highperformance liquid chromatography with precolumn uoescence dervatization. Journal of Chromatography B 2008; 875: 358362. Ariyannur PS, Madhavarao CN and Namboodiri AMA. Nacetylaspartate synthesis in the brain: Mitochondria vs. microsomes. Brain Research 2008; 1227: 3441. Ariyannur PS, Moffett JR, Manickam P, Pattabiraman N, Arun P, Nitta A, Nabeshima T, Madhavarao CN and Namboodiri AMA. Methamphetamineinduced neuronal protein NAT8L is the NAA biosynthetic enzyme: Implications for specialized acetyl coenzyme A metabolism in the CNS. Brain Research 2010; 1335: 113. Baslow MH. Nacetylaspartate in the vertebrate brain: metabolism and function. Neurochemical Research 2003; 28: 941953. Battistuta J, Bjartmar C and Trapp BD. Postmortem degradation of Nacetyl aspartate and Nacetyl aspartylglutamate: an HPLC analysis of different rat CNS regions. Neurochemical Research 2001; 26: 695702. Becker I, Lodder J, Gieselmann V and Eckhardt M. Molecular characterization of Nacetylaspartylglutamate synthetase. Journal of Biological Chemistry 2010; 285: 2915629164. Franklin KBJ and Paxinos G. The Mouse Brain in Stereotaxic Coordinates. Academic Press: San Diego, CA, 1997. Fukushima T, Arai K, Tomiya M, Mitsuhashi S, Sasaki T, Santa T, Imai K and Toyooka T. Fluorescence determination of Nacetylaspartic acid in the rat cerebrum homogenate using highperformance liquid

Figure 4. Concentrations of NAA (nmol/mg protein) in six different parts of mouse brain (n = 8). Olb, olfactory bulb; Pre, prefrontal cortex; Nac, nucleus accumbens; Str, striatum; Cel, cerebellum; Hip, hippocampus. Data are expressed as mean SE.

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chromatography with precolumn uorescence derivatization. Biomedical Chromatography 2008; 22: 100105. Harte MK, Bachus SB and Reynolds GP. Increased Nacetylaspartate in rat striatum following longterm administration of haloperidol. Schizophrenia Research 2005; 75: 303308. Madhavarao CN, Chinopoulos C, Chandrasekaran K and Namboodiri AMA. Characterization of the Nacetylaspartate biosynthetic enzyme from rat brain. Journal of Neurochemistry 2003; 86: 824835. Moffett J, Ross B, Arun P, Madhavarao CN and Namboodiri AMA. NAcetylaspartate in the CNS: From neurodiagnostics to neurobiology. ProgressinNeurobiology2007;81: 89131. Neale JH, Bzdega T and Wroblewska B. NAcetylaspartylglutamate: the most abundant peptide neurotransmitter in the mammalian central nervous system. Journal of Neurochemistry 2000; 75: 443452. Niwa M, Nitta A, Mizoguchi H, Ito Y, Noda Y, Nagai T and Nabeshima T. A novel molecule shati is involved in methamphetamineinduced hyperlocomotion, sensitization, and conditioned place preference. Journal of Neuroscience 2007; 27: 76047615. Reynolds LM and Reynolds GP. Differential regional Nacetylaspartate decits in postmortem brain in schizophrenia, bipolar disorder and major depressive disorder. Journal of Psychiatric Research 2011; 45: 5459. Rigotti DJ, Inglese M and Gonen O. Wholebrain Nacetylaspartate as a surrogate marker of neuronal damage in diffuse neurologic disorders. American Journal of Neuroradiology 2007; 28: 18431849. Schuff N, Meyerhoff DJ, Mueller S, Chao L, Sacrey T, Laxer K and Weiner MW. NAcetylaspartate as a marker of neuronal injury in neurodegenerative disease. Advances in Experimental Medicine and Biology 2006; 576: 241363. Tallan HH, Moore S and Stein WH. NAcetylLaspartic acid in brain. Journal of Biological Chemistry 1956; 219: 257264. Tavazzi B, Vagnozzi R, Di Pierro D, Amorini AM, Fazzina G, Signoretti S, Marmarou A, Caruso I and Lazzarino G. Ionpairing highperformance liquid chromatographic method for the detection of Nacetylaspartate and Nacetylglutamate in cerebral tissue extracts. Analytical Biochemistry 2000; 277: 104108. Wiame E, Tyteca D, Pierrot N, Collard F, Amyere M, Noel G, Desmedt J, Nassogne MC, Vikkula M, Octave JN, Vincent MF, Courtoy PJ, Boltshauser E and Van Schaftingen E. Molecular identication of aspartate Nacetyltransferase and its mutation in hypoacetylaspartia. Biochemical Journal 2010; 425: 127136.

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