Development of a Fully-automated On-line Oxidation Column-switching HPLC System for the Determination of Endogenous Melatonin in Human Clinical Samples.

A fully-automated on-line oxidation column-switching HPLC system has been developed for the determination of endogenous melatonin in human plasma and saliva. This HPLC system consists of four processes. In the first step, melatonin is fractionated using a reversed-phase C4 column (Proteonavi, 75 mm × 1.0 mm i.d.). In the second step, the obtained melatonin fraction is on-line collected, and oxidized to a highly-fluorescent compound, N-[(6-methoxy-4-oxo-1,4-dihydroquinolin-3-yl)methyl]acetamide (6-MOQMA), by mixing with hydrogen peroxide under alkaline conditions. In the third step, the produced 6-MOQMA is concentrated, and the oxidation reagents are removed using an alkaline resistive reversed-phase column, Asahipak ODP (35 mm × 1.0 mm i.d.). In the final fourth step, the 6-MOQMA is determined by a microbore-ODS column packed with ultrafine particles (CAPCELL PAK C18 IF, 250 mm × 1.0 mm i.d.). The limit of detection of melatonin using this system is about 200 amol/injection, and the determination of endogenous melatonin in a small volume of human physiological fluids, such as 100 µL of plasma and 300 µL of saliva, was successfully accomplished.

D-serine regulates cerebellar LTD and motor coordination through the δ2 glutamate receptor.

D-serine (D-Ser) is an endogenous co-agonist for NMDA receptors and regulates neurotransmission and synaptic plasticity in the forebrain. D-Ser is also found in the cerebellum during the early postnatal period. Although D-Ser binds to the δ2 glutamate receptor (GluD2, Grid2) in vitro, its physiological significance has remained unclear. Here we show that D-Ser serves as an endogenous ligand for GluD2 to regulate long-term depression (LTD) at synapses between parallel fibers and Purkinje cells in the immature cerebellum. D-Ser was released mainly from Bergmann glia after the burst stimulation of parallel fibers in immature, but not mature, cerebellum. D-Ser rapidly induced endocytosis of AMPA receptors and mutually occluded LTD in wild-type, but not Grid2-null, Purkinje cells. Moreover, mice expressing mutant GluD2 in which the binding site for D-Ser was disrupted showed impaired LTD and motor dyscoordination during development. These results indicate that glial D-Ser regulates synaptic plasticity and cerebellar functions by interacting with GluD2.

Simultaneous determination of D-aspartic acid and D-glutamic acid in rat tissues and physiological fluids using a multi-loop two-dimensional HPLC procedure.

For a metabolomics study focusing on the analysis of aspartic and glutamic acid enantiomers, a fully automated two-dimensional HPLC system employing a microbore-ODS column and a narrowbore-enantioselective column was developed. By using this system, a detailed distribution of D-Asp and D-Glu besides L-Asp and L-Glu in mammals was elucidated. For the total analysis concept, the amino acids were first pre-column derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to be sensitively and fluorometrically detected. For the non-stereoselective separation of the analytes in the first dimension a monolithic ODS column (750 mm × 0.53 mm i.d.) was adopted, and a self-packed narrowbore-Pirkle type enantioselective column (Sumichiral OA-2500S, 250 mm × 1.5 mm i.d.) was selected for the second dimension. In the rat plasma, RSD values for intra-day and inter-day precision were less than 6.8%, and the accuracy ranged between 96.1% and 105.8%. The values of LOQ of D-Asp and D-Glu were 5 fmol/injection (0.625 nmol/g tissue). The present method was successfully applied to the simultaneous determination of free aspartic acid and glutamic acid enantiomers in 7 brain areas, 11 peripheral tissues, plasma and urine of Wistar rats. Biologically significant D-Asp values were found in various tissue samples whereas for D-Glu the values were very low possibly indicating less significance.

Simultaneous two-dimensional HPLC determination of free D-serine and D-alanine in the brain and periphery of mutant rats lacking D-amino-acid oxidase.

A fully automated two-dimensional HPLC system combining a microbore-ODS column and a narrowbore-enantioselective column was designed and validated, and the amounts of D-serine (D-Ser) and D-alanine (D-Ala) in various tissues and physiological fluids of Long-Evans agouti/SENDAI (LEA/Sen) rats lacking D-amino-acid oxidase (DAO) were determined. Intra- and inter-day precision was less than 4.3% and accuracy ranged between 99.9 and 104%. LEA/Sen rats were reported to lack DAO in their kidneys and expected to be a novel mutant animal lacking DAO, however, the amounts of D-amino acids in the LEA/Sen rats have not been investigated. In the present study, the intrinsic amounts of D-Ser and D-Ala, which are neuromodulators of the N-methyl-D-aspartate (NMDA) receptors, were determined in seven brain tissues, four peripheral tissues, plasma and urine of the LEA/Sen rats and compared to those of the control (Wistar and SD) rats having normal DAO activity. The levels of D-Ser in the tissues and physiological fluids of the LEA/Sen rats were significantly higher than those of the Wistar and SD rats except for the frontal brain regions. Concerning D-Ala, the amounts in the tissues and physiological fluids of the LEA/Sen rats were drastically increased compared to those of the Wistar and SD rats. These results indicate that the intrinsic amounts of D-Ser and D-Ala in the tissues of rats are regulated by DAO, and that LEA/Sen rats would be useful for the study of NMDA receptor-related diseases in which DAO is implicated.

Fluorescence properties of 2-aryl substituted indoles.

The fluorescence properties of 2-phenylindole, 2-naphthylindole and 2-anthracenylindole were investigated. 2-Anthracenylindole was newly synthesized by Suzuki-Miyaura's coupling. The fluorescence quantum yield of 2-phenylindole was the highest and the fluorescence emission maximum wavelength of 2-anthracenylindole was the longest. The ab initio quantum chemical calculation of the 2-anthracenylindole showed that the HOMO and LUMO of 2-anthracenylindole were localized in the anthracene moiety.

Brain-specific Phgdh deletion reveals a pivotal role for L-serine biosynthesis in controlling the level of D-serine, an N-methyl-D-aspartate receptor co-agonist, in adult brain.

In mammalian brain, D-serine is synthesized from L-serine by serine racemase, and it functions as an obligatory co-agonist at the glycine modulatory site of N-methyl-D-aspartate (NMDA)-selective glutamate receptors. Although diminution in D-serine level has been implicated in NMDA receptor hypofunction, which is thought to occur in schizophrenia, the source of the precursor L-serine and its role in D-serine metabolism in adult brain have yet to be determined. We investigated whether L-serine synthesized in brain via the phosphorylated pathway is essential for D-serine synthesis by generating mice with a conditional deletion of D-3-phosphoglycerate dehydrogenase (Phgdh; EC 1.1.1.95). This enzyme catalyzes the first step in L-serine synthesis via the phosphorylated pathway. HPLC analysis of serine enantiomers demonstrated that both L- and D-serine levels were markedly decreased in the cerebral cortex and hippocampus of conditional knock-out mice, whereas the serine deficiency did not alter protein expression levels of serine racemase and NMDA receptor subunits in these regions. The present study provides definitive proof that L-serine-synthesized endogenously via the phosphorylated pathway is a key rate-limiting factor for maintaining steady-state levels of D-serine in adult brain. Furthermore, NMDA-evoked transcription of Arc, an immediate early gene, was diminished in the hippocampus of conditional knock-out mice. Thus, this study demonstrates that in mature neuronal circuits L-serine availability determines the rate of D-serine synthesis in the forebrain and controls NMDA receptor function at least in the hippocampus.

Fluorescence and chemiluminescence properties of indolylmaleimides: experimental and theoretical studies.

Various indolylmaleimides (IMs) were synthesized, and their fluorescence (FL) and chemiluminescence (CL) were measured. The substitution at the 2-position of the indole ring and the 3- or 4-position of the maleimide moiety caused an obvious change in the FL and CL of the IMs. An almost on-off switching of the FL of the IMs was observed. The intramolecular charge transfer from the indole moiety to the maleimide moiety occurred in 3-(1H-3-indolyl)-2,5-dihydro-1H-2,5-pyrroledione. In the FL of the IMs, CASPT2 calculations showed deprotonation of the NH group of the indole ring and the maleimide moiety at the excited state. The C[double bond, length as m-dash]C bond in the maleimide moiety was needed for strong CL in the IMs without substitution at the 2-position of the indole ring. The relationships between the FL or CL properties and the structures of the IMs were clarified. These results provide significant information on the rational design of IMs as FL and CL probes.

Mutant mice and rats lacking D-amino acid oxidase.

D-amino acid oxidase (DAO) catalyzes oxidative deamination of D-amino acids. Since D-amino acids are considered to be rare in eukaryotes, physiological function of this enzyme has been enigmatic for a long time. Mutant mice lacking DAO were found, and their strain was established. The urine of the mutant mice contained large amounts of D-amino acids. D-Amino acids were also present in their organs and blood. The origin of these D-amino acids was pursued. The results indicate that one of the physiological functions of DAO is the metabolism of D-amino acids of internal and external origin. A large amount of D-serine is shown to exist in the brain of mammals. It binds to the coagonist-binding site of N-methyl-D-aspartate (NMDA) subtype of glutamate receptors and enhances the neurotransmission. DAO metabolizes this D-serine and, therefore, modulates neurotransmission. Mutant mice displayed phenotypes resulting from the enhanced NMDA receptor function. Recent studies have shown that DAO is associated with schizophrenia. Mutant mice were resistant to the drugs which act on NMDA receptors and elicit schizophrenia-like symptoms. Recently, mutant rats lacking DAO have also been found. They were free from D-serine-induced nephrotoxicity, indicating involvement of DAO in this toxicity. The mutant mice and rats lacking DAO would be useful for the elucidation of the physiological functions of DAO and the etiology of neuronal diseases associated with DAO.

6,7-Difluoro-1,4-dihydro-1-methyl-4-oxo-3-quinolinecarboxylic acid, a newly designed fluorescence enhancement-type derivatizing reagent for amino compounds.

A novel fluorescence enhancement-type derivatizing reagent for amino compounds, 6,7-difluoro-1,4-dihydro-1-methyl-4-oxo-3-quinolinecarboxylic acid (FMQC), was developed. FMQC reacts with aliphatic primary amino compounds to afford strong fluorescent derivatives having high photo-and thermo-stabilities. The FMQC derivatives of amino compounds showed 12-159 times higher fluorescence quantum efficiencies than those of FMQC in aqueous and polar organic media. Additionally, the absorption and fluorescence emission wavelength of the derivatives are red-shifted from those of FMQC. These differences in the fluorescence properties between FMQC and the fluorescent derivative enabled the simple and highly sensitive determination of amino compounds without removing any excess unreacted FMQC by using a simple spectrofluorometer as well as HPLC.

Chemiluminescence enhancement of 1,2-di[3,4,5-tri(3,4,5-trihydroxybenzoyloxy)benzoyloxy] benzene in the presence of quaternary ammonium ions.

The chemiluminescence intensity of 1,2-di[3,4,5-tri(3,4,5-trihydroxybenzoyloxy)benzoyloxy] benzene increased in the presence of quaternary ammonium ions, such as acetylcholine chloride, choline chloride or benzyltrimethylammonium chloride. The complex of 1,2-di[3,4,5-tri(3,4,5-trihydroxybenzoyloxy)benzoyloxy] benzene with acetylcholine chloride, choline chloride or benzyltrimethylammonium chloride was investigated by (1)H-NMR spectroscopy. The structure of the complex formed from 1,2-di[3,4,5-tri(3,4,5-trihydroxybenzoyloxy)benzoyloxy] benzene with choline chloride was described by an ab initio quantum chemical calculation.

Simultaneous determination of hydrophilic amino acid enantiomers in mammalian tissues and physiological fluids applying a fully automated micro-two-dimensional high-performance liquid chromatographic concept.

A validated two-dimensional HPLC system combining a microbore-monolithic ODS column and a narrowbore-enantioselective column has been established for a sensitive and simultaneous analysis of hydrophilic amino acid enantiomers (His, Asn, Ser, Gln, Arg, Asp, allo-Thr, Glu and Thr) and the non-chiral amino acid, Gly, in biological samples. To accomplish this goal, the amino acids were first tagged with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to the respective fluorescent NBD derivatives which were separated in the first dimension by a micro-reversed-phase column. The automatically collected fractions of the target peaks were then transferred to the second dimension consisting of a Pirkle type enantioselective column generating separation factors higher than 1.13 for all the enantiomeric target analytes. The system was validated using standard amino acids and a rat plasma sample, and analytically satisfactory calibration and precision results were obtained. The present 2D-HPLC system enables the fully automated determination of hydrophilic amino acid enantiomers in mammalian samples. The d-isomers of all the investigated 9 amino acids were found in rat urine but at various enantiomeric ratios.

Analysis of small amounts of D-amino acids and the study of their physiological functions in mammals.

D-amino acids, the enantiomers of L-amino acids, are candidates to be novel physiologically active substances and the biomarkers in mammals. However, the amounts of D-amino acids in the tissues and physiological fluids are extremely small in most cases, and sensitive and selective analytical methods are needed for their determination. In the present manuscript, we review the analytical technologies including our recent advances for the determinations of small amounts of D-amino acids in mammals and the applications to clarify their physiological meanings.

Theoretical study of photophysical properties of bisindolylmaleimide derivatives.

The photophysical properties of two bisindolylmaleimide derivatives, 3,4-bis(3-indolyl)-1-H-pyrrole-2,5-dione (arcyriarubin A) and indolo[2,3-a]pyrrolo[3,4-c] carbazole-5,7-(6 H)-dione (arcyriaflavin A), are investigated by using ab initio molecular orbital (MO) and multireference perturbation theory. These compounds are suggested to exist as monovalent anions deprotonated from an indole NH group in aprotic polar solvents. The analysis of MOs shows that the electronic structures of the S(1) and S(2) states are described by the single- or double-electron excitation between the naturally localized MOs on an indole moiety and on the maleimide part. This indicates that the intramolecular charge transfer (ICT) transfer may occur by photoexcitation. The minimum-energy structure of the arcyriarubin A anion is twisted; the dihedral angles between the indole and maleimide rings are 83.4 degrees and 20.2 degrees for the S(1) and S(0) states, respectively. The analysis of the minimum energy path along the coordinate of the twist angle is performed to explore the emission process from the S(1) state. It has been shown that the magnitude of the Stokes shift increases with increasing the twist angle, but the oscillator strength decreases. It has been suggested that the experimentally observed fluorescence arises on the way toward the energy minimum of the S(1) state. The Stokes-shifted emission of arcyriaflavin A is contributed by the S(1)-S(0) electronic relaxation after the excitation in the S(2) state.

Determination of D-serine and D-alanine in the tissues and physiological fluids of mice with various D-amino-acid oxidase activities using two-dimensional high-performance liquid chromatography with fluorescence detection.

Two-dimensional-HPLC procedures have been established for the sensitive and selective determination of D-serine (D-Ser) and D-alanine (D-Ala), and their amounts in the tissues and physiological fluids of mice with various D-amino-acid oxidase (DAO) activities have been demonstrated. These two D-amino acids are modulators of the N-methyl-D-aspartate receptor mediated neurotransmission, and the alterations in their amounts following the changes in the DAO activity are matters of interest. After pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), the D-amino acids were determined by the 2D-HPLC system with fluorescence detectors. As the first dimension, a microbore-monolithic-ODS column (750 mm x 0.53 mm I.D.) was adopted and a self-packed narrowbore-Pirkle type enantioselective column (Sumichiral OA-2500S, 250 mm x 1.5 mm I.D.) was selected for the second dimension. The lower limits of quantitation of D-Ser and D-Ala were 500 amol, and the within-day and day-to-day precisions were less than 6.8%. Using these methods, the amounts of D-Ser and D-Ala in 6 brain tissues, 4 peripheral tissues, serum and urine of mice having various DAO activities were determined; the amounts of these D-amino acids were drastically increased with a lowering of the DAO activity except for the cases of D-Ser in the frontal brain regions. The present micro-2D-HPLC procedures are powerful tools for the determination of small amounts of D-Ser and D-Ala in mammalian samples, and the obtained results would be useful for developing novel drugs that modulate the DAO activity, such as DAO inhibitors, against neuronal diseases.

Simple and rapid genotyping of D-amino acid oxidase gene recognizing a crucial variant in the ddY strain using microchip electrophoresis.

A rapid genotyping method of D-amino acid oxidase (DAO), an enzyme that catalyzes the oxidative degradation of most of the D-amino acids in mammals, has been established. This method employs a one-step PCR, restriction enzyme digestion and rapid microchip electrophoresis (MCE), and the DAO genotype of the living individual mice was definitely determined within a day by clearly separating the 95 and 107 bp Hpa II digested DNA fragments. For verification of the method, the DAO activity in the kidney of individual mice was also determined, and the obtained values completely matched the estimated genotypes (DAO(+/+), DAO(+/-), and DAO(-/-)). The intrinsic amounts of D-Pro in the serum and kidney of mice with three DAO genotypes were compared for the first time, and demonstrated that the D-amino acid amounts in the DAO(+/+) mice (1.93 +/- 0.66 nmol/mL serum, not detectable in the kidney) and DAO(+/-) mice (1.50 +/- 0.24 nmol/mL serum, not detectable in the kidney) were almost the same. The present method should be a powerful tool to establish various pathologic-model animals under the complete care of their intrinsic DAO activity, which are useful for the screening of D-amino acids having physiological activity and/or diagnostic values.

Enantioselective visualization of D-alanine in rat anterior pituitary gland: localization to ACTH-secreting cells.

The cellular localization of D: -alanine (D: -Ala) in the rat pituitary gland, the tissue containing the highest amount of D: -Ala, has been clarified for the first time by enantioselective visualization of D: -Ala using our own established mouse monoclonal antibody against D: -Ala. D: -Ala immunopositive cells were present predominantly in the anterior lobe, while no intense staining was observed in the intermediate and posterior lobes. The anterior pituitary gland contains five types of cells secreting specific hormones (growth hormone, adrenocorticotropic hormone (ACTH), gonadotropic hormone, prolactin, and thyroid-stimulating hormone), and the double staining results indicated that D: -Ala is localized to the ACTH-secreting cells. The localization of D: -Ala is clearly different from that of D: -aspartic acid (D: -Asp), which is observed in the prolactin cells. Considered together with our previous findings that D: -Ala is localized to the insulin-secreting beta-cells in the pancreas, and both ACTH and insulin are typical regulatory hormones of blood glucose, D: -Ala is suggested to have some functional relationships to blood glucose level regulation in mammals.

Chemiluminescence change of polyphenol dendrimers with different core molecules.

Second generation polyphenol dendrimers (PDs) with different core molecules were synthesized, and their chemiluminescence (CL) was measured by reacting the PDs with H2O2 under alkaline conditions. All of the PDs showed a strong CL, more than 120-fold greater than that of gallic acid. Various CL intensities of the PDs were obtained using different core molecules in the PDs. The distance between each dendron in the PD structure is crucial in the PD CL intensity.

Automated and simultaneous two-dimensional micro-high-performance liquid chromatographic determination of proline and hydroxyproline enantiomers in mammals.

A fully automated 2D-HPLC system employing a microbore-ODS column and a narrowbore-enantioselective column has been developed for the simultaneous enantiomer determination of proline, trans-4-hydroxyproline and cis-4-hydroxyproline in mammals. As a first dimension, a monolithic ODS column of 0.53 mm i.d. showed 3-6 times larger theoretical plate numbers than those of particle-packed ODS columns, and the best enantioseparations were obtained by a Chiralpak QN-2-AX column of 1.5 mm i.d. in the second dimension (separation factors: 1.44-1.83). The R.S.D. values for within-day and dayto-day precisions were less than 5.8%, and the lower limits of quantitation for the D-enantiomers were 1 fmol. The present method was successfully applied to the determination of proline and hydroxyproline enantiomers in the serum and collagen-rich skin tissue. Small amounts of D-proline were found both in the serum (1.57 +/- 0.19 nmol/mL) and in the skin (0.093 +/- 0.015 nmol/mg protein), while the amounts of D-hydroxyproline were smaller than the lower limit of quantitation.

Structural and photophysical properties of novel dual fluorescent compounds, 1-aryl-substituted 6-methoxy-4-quinolones.

Dual fluorescent compounds are useful platforms for the development of ratiometric probes and sensors. We have developed a new series of dual fluorescent compounds, 1-aryl-substituted 6-methoxy-4-quinolones, and investigated their structural and photophysical properties. The X-ray crystallographic analysis and ab initio quantum chemical calculations revealed that the developed compounds exhibited 60-75 degrees twisted structures. The dual fluorescence of the compounds were observed in polar solvents, and the ratiometric fluorescence responses to alterations in the acidity and temperature were obtained.

Bisindolylmaleimides with large Stokes shift and long-lasting chemiluminescence properties.

Various bisindolylmaleimides have fluorescence emission maxima wavelengths longer than 500 nm, large Stokes shifts longer than 200 nm, different fluorescence emission wavelengths at an excitation wavelength of 365 nm, and a long-lasting chemiluminescence. The expansion of the pi-conjugation, the pi-bond electronic structure, and oxidation of the C=C bond at the 2,3-position of the maleimide moiety are crucial for producing these fluorescence and chemiluminescence properties.