Takeshi Fukushima

Regional distribution and postnatal changes of d-amino acids in rat brain

Regional distribution of D-amino acids in rat brain was studied by the modified highly sensitive analytical method which was previously developed. The method includes fluorogenic derivatization of each amino acid, isolation of each amino acid by reverse-phase HPLC, followed by enantiomeric separation with Pirkle-type chiral stationary phases. D-Amino acid contents were determined in the cerebrum, cerebellum, hippocampus, medulla oblongata, pituitary gland and pineal gland. D-Aspartic acid was observed in the pineal gland (3524 +/- 263 nmol/g, data are for male rats of 6 weeks of age) and the pituitary gland (80.5 +/- 9.0 nmol/g). D-Serine was found in various regions of the brain except for the cerebellum and medulla oblongata. D-Alanine was observed exclusively in the pituitary gland (25.9 +/- 4.4 nmol/g), whereas D-leucine was found in the pineal gland (3.4 +/- 0.4 nmol/g) and the hippocampus (1.6 +/- 0.07 nmol/g). No other D-amino acids were detected in the brain. The contents of D-aspartic acid in the pituitary gland and D-serine in the pineal gland were higher in female rats. In contrast the contents of D-alanine in the pituitary gland and D-leucine in the pineal gland and the hippocampus were higher in males. Postnatal changes of D-aspartic acid and D-leucine in the pineal gland and D-alanine in the pituitary gland were also investigated. The results described in this paper suggested that distinct regulatory mechanisms exist for individual D-amino acids in the corresponding region of rat brain.

ANALYTICAL CHEMISTRY AND BIOCHEMISTRY OF D-AMINO ACIDS

The methodologies for the analysis of D-amino acids in biological materials have been reviewed, including the use of enzymes, gas and liquid chromatography with chiral stationary phases and diastereomer derivatization with chiral reagents followed by GC or HPLC separation. The distribution of D-amino acids in the body, their origin, metabolism and possible roles in human diseases are discussed.

Recent progress in derivatization methods for LC and CE analysis.

The derivatization procedure with a suitable fluorescence or chemiluminescence reagent is performed for the purpose of increasing the detection sensitivity and selectivity, in high-performance liquid chromatography (HPLC) and/or capillary electrophoresis (CE). In this article, recent derivatization methods and their applications to biosamples are described. In HPLC, femto mol order of mass detection limits are obtained by derivatization. Regarding the fluorescence reagents, the use of water-soluble reagents has been effective to avoid an undesired adsorption in the process of determination of peptides. In CE, the advantages of having extremely low mass detection limits (ranging from atto to yocto mol level) and requiring only a very short analysis time (less than a few minutes) are made possible by using laser-induced fluorescence or near infra-red detections.

BIOSYNTHESIS OF D-ASPARTATE IN MAMMALIAN CELLS

In this communication, we demonstrate that d‐aspartate (d‐Asp) is synthesized in pheochromocytoma cells (PC12). To our knowledge this is the first report of biosynthesis of d‐Asp in mammalian cells. Synthesis of d‐Asp was demonstrated by its time‐dependent accumulation in the cell culture, and by the fact that this accumulation was proportional to the number of inoculated cells. d‐Asp in PC12 cells was identified by (i) co‐elution with authentic d‐Asp on two different HPLC columns, an octadesyl silica column and a Pirkle‐type chiral column, (ii) reversed elution order of d‐Asp and l‐Asp on another Pirkle‐type chiral column with an opposite configuration, and (iii) sensitivity to d‐Asp oxidase. In the cells the amount of d‐Asp was approx. 12–14% of total Asp and no other investigated d‐amino acid was detected. The amount of d‐Asp did not increase during the culture of mouse 3T3 fibroblasts and human neuroblastoma NB‐1 cells. Immunocytochemical staining with anti‐d‐Asp antiserum demonstrated that d‐Asp synthesized is present in the cytoplasm of the cells.

Liquid chromatographic-atmospheric pressure chemical ionization mass spectrometric determination of anandamide and its analogs in rat brain and peripheral tissues

A simple and selective method for the determination of anandamide (arachidonoylethanolamide), an endogenous cannabinoid receptor ligand, and its analogs with liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS) was developed. The calibration curve for standard anandamide was linear over the range 625 fmol-125 pmol per injection (r = 0.999) with a precision of 1.0% (C.V.) at 25 pmol. The detection limit attained was 200 fmol per injection at a signal-to-noise ratio of 2. Anandamide and its analogs were extracted from rat brain and peripheral tissues according to the method of Folch, and the recovery of anandamide from rat brain homogenates was 67.0-72.6%. The method was applied to their determination in rat brain and peripheral tissues.

Emergence of D-aspartic acid in the differentiating neurons of the rat central nervous system.

The rat embryonic brain was probed with anti-d-aspartic acid (d-Asp) antiserum at different stages of development. At gestational day (E) 12, weak immunoreactivity (IR) of d-Asp was apparent at the hindbrain, midbrain and caudal forebrain, whereas it became more intense and extended over the whole brain at E20. However, IR markedly decreased after parturition. In the region of the immature forebrain at an early stage of development (E12), IR was mainly a characteristic of the cytoplasm of the neuronal cells, while in the more mature hindbrain it was localized in the axonal zone. In the more differentiated forebrain at a later stage of development (E18), the IR became restricted to zones which mainly consisted of axons and processes. Consequently, in the rat central nervous system, d-Asp first emerges during embryonic development as a feature of the cytoplasm and thereafter spreads into the axonal regions of neuronal cells, before disappearing almost completely after parturition.

Possible role of d-serine in the pathophysiology of Alzheimer's disease

Several lines of evidence suggest that D-serine may function as an endogenous agonist of the glycine site on the N-methyl-D-aspartate (NMDA) receptor that has been implicated in the pathophysiology of Alzheimers disease (AD). The purpose of the study was to determine whether serum levels of D- and L-serine in patients with AD are altered as compared with normal controls. Serum levels of D- and L-serine in patients of AD and age- and gender-matched normal controls were determined using a high-performance liquid chromatography (HPLC). Serum levels of D-serine in the patients with AD were slightly (z=-1.77, p=0.078) lower than those of normal controls. In contrast, serum levels of L-serine in the patients were slightly (z=-1.73, p=0.083) higher than those of controls. In addition, the percentage (%) of D-serine in the total (L+D) serine in the patients was significantly (z=-2.36, p=0.018) lower than that of controls. The present study suggests that the reduced activity of serine racemase, an enzyme catalyzing the formation of D-serine from L-serine may play a role in the pathophysiology of AD.

Sensitive determination of anandamide in rat brain utilizing a coupled-column HPLC with fluorimetric detection.

A fluorimetric determination method for N-arachidonoylethanolamine (anandamide) was developed using a precolumn fluorescence derivatization followed by coupled-column high-performance liquid chromatography (HPLC). Anandamide extracted from the rat brain tissue was derivatized with 4-N-chloroformylmethyl-N-methylamino-7-N, N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl), purified by a solid-phase extraction (Emporetrade mark), and assayed by the coupled-column HPLC. The HPLC consisted of phenyl (100 x 4.6 mm i.d. ) and octadecylsilica columns (250 x 4.6 mm i.d.), both connected by a six-port valve. The concentration of anandamide in rat brain was 3. 37 +/- 0.73 pmol/g with 6.47 and 3.57% of intra- and inter-day precisions, respectively. Using this method, we investigated the alteration of anandamide concentration in rat brain 30 min after administration of anandamide (2 mg/kg, i.p.) to rats pretreated with or without phenylmethylsulfonyl fluoride (PMSF; 30 mg/kg, i.p.), an inhibitor of amidohydrolase. In rats pretreated with PMSF, the brain concentration of anandamide was approx. 16-fold higher than that of rats without PMSF (p < 0.01).

Determination of fluoxetine enantiomers in rat plasma by pre-column fluorescence derivatization and column-switching high-performance liquid chromatography

A column-switching HPLC method employing both octadecylsilica (ODS) and chiral columns with fluorescence detection was developed for the determination of enantiomer of fluoxetine (FLX), an antidepressant drug, in rat plasma. Racemic FLX was derivatized with a fluorescent reagent, 4-(N-chloroformylmethyl-N-methyl)amino-7-nitro-2,1,3-benzoxadiazole (NBD-COCl) or 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl) and the enantiomeric separation of the resultant derivatives was examined on an amylose-based chiral column (CHIRALPAK AD-RH) in the reversed-phase mode. The derivative with NBD-COCl (NBD-FLX) showed a sufficient separation factor (a) and resolution (Rs) compared with that with DBD-COCl. Thus, FLX was derivatized with NBD-COCl and the resultant NBD-FLX was first quantified on the ODS column and then introduced to the CHIRALPAK AD-RH column via a six-port switching valve to examine the enantiomeric ratio. The intra- and inter-day accuracy (97.6-112.7%) and precision (1.47-10.60%) were satisfactory in the range 10-1000 nM FLX and the limit of quantification was approximately 10 nM. The absolute recoveries of FLX with hexane from rat plasma were in the range 87.5-92.2% (n = 3). The method was applied to determine FLX enantiomers in the plasma of rats administered FLX orally, and it was shown that the R-isomer was eliminated faster than the S-isomer.

Determination of aspartic acid enantiomers in bio-samples by capillary electrophoresis

Enantiomeric separation and detection of D,L-aspartic acid (Asp) derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) by capillary electrophoresis (CE) using modified cyclodextrins as chiral selectors was studied. Heptakis(2,3, 6-tri-O-methyl)-beta-cyclodextrin(TM-beta-CD) was most effective for enantiomeric separation of NBD-D,L-Asp with optimum conditions of 30 mM TM-beta-CD in 50 mM phosphate buffer (pH 4.0) and the limit of detection (LOD) attained was 100 nM for each enantiomer. The method proposed in the present study was convenient for both D- and L-Asp determination since the other amino compounds migrated differently and D-Asp in bio-samples such as rat pineal gland and foods was determined with a simple sample pretreatment and a short analysis run time.