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Biomedical subjects

Toshimasa Toyo'oka

Publications and source records attributed to Toshimasa Toyo'oka.

At least 19 recordsLinked to original sources
PubMed2006

Hair analysis of histamine and several metabolites in C3H/HeNCrj mice by ultra performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry (UPLC-ESI-TOF-MS): influence of hair cycle and age.

BACKGROUND: According to a previous study, the concentration of HA in the hair of SD rats was similar in each rat and the variation in HA concentration was not so great. However, the concentration in human hair was fairly different in each person. As possible reasons for the higher variation in human hair, the differences in hair cycles and age in each person may be considerable. Based on this idea, the studies using C3H/HeNCrj mice who can synchronize their hair cycle were performed for resolution of the influence of hair cycle and age. METHODS: The effects of hair cycle and age on the concentration of histamine (HA) and several metabolites, i.e., 1-methylhistamine (MHA), imidazole-4-acetic acid (IAA), and 1-methyl-4-imidazole-acetic acid (MIAA), in C3H/HeNCrj mice hair were investigated by ultra-performance liquid chromatography (UPLC) with electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). HA and the metabolites were labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) and 4-(N,N-dimethylaminosulfonyl)-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ). The resulting derivatives were separated by UPLC and determined with ESI-TOF-MS. RESULTS: A good linearity was achieved from the calibration curves, obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS), i.e., histamine-alpha,alpha,beta,beta-d4 (HA-d4) or 4-imidazolecarboxylic acid (ICA), against the injected amounts of each compound. The detection limits of HA, MHA, IAA, and MIAA on mass chromatograms were 0.21, 1.0, 0.17, and 0.11 pmol, respectively. The concentrations of HA and the metabolites in the hair shafts and hair root of C3H/HeNCrj mice were determined by this method. The concentration of HA in the hair shaft was relatively higher in the telogen phase. In contrast, the HA content in the anagen phase was increased only in the hair root of old mice. CONCLUSION: HA appears to possess some effect on hair growth, although the exact reason was not obvious. The HA metabolites, i.e., MHA, MIAA and IAA, were also determined the same as HA; however, the difference in the metabolite concentrations between the hair cycle and age was not clear in both hair shaft and hair root. Such studies of the effect of hair cycle and age on these concentrations are the first report. This analytical technique may be applicable to the determination of various biological compounds in hair.

Age Factors↗
PubMed2006

Determination of kynurenic acid in human serum and its correlation with the concentration of certain amino acids.

BACKGROUND: Kynurenic acid (KYNA)--a tryptophan metabolite--elicits antagonistic activity against glutaminergic and cholinergic receptors; it has been suggested to have some relationship with neurological disorders. Considering this, serum KYNA may be an important marker in clinical diagnosis. We determined serum KYNA concentration and elucidate its correlation with several amino acids in human serum. METHODS: KYNA and amino acids concentrations in human serum of healthy subjects [n=35 (21 males and 14 females)] were determined by HPLC with fluorescence detection; thus, the correlation between KYNA concentration and that of several amino acids was examined in these subjects. RESULTS: Of the amino acids examined in this study, a significant negative correlation was observed between KYNA and glutamine (Gln) concentrations (r=-0.452, p<0.01) in the healthy subjects, particularly males (r=-0.687, p<0.01), and age-related changes were not observed. In addition to Gln, Gly and Ala concentrations showed a significant negative correlation with KYNA concentration in the serum of male subjects (r=-0.440 and -0.456, respectively, p<0.05). CONCLUSION: The significant correlation between KYNA and Gln concentrations in vivo may support the previous finding that kynurenine aminotransferase I (KAT I), responsible for the biosynthesis of KYNA, was identical to Gln transaminase K (GTK), which catalyses the transamination of Gln to 2-oxoglutamic acid. Both KYNA and Gln concentrations in vivo might be influenced due to altered KAT I/GTK activity.

Adolescent↗
PubMed2006

Rapid determination of histamine and its metabolites in mice hair by ultra-performance liquid chromatography with time-of-flight mass spectrometry.

The rapid determination of histamine (HA) and several metabolites, i.e., 1-methylhistamine (MHA), imidazole-4-acetic acid (IAA), and 1-methyl-4-imidazole-acetic acid (MIAA), in mice hairs was performed by ultra-performance liquid chromatography with time-of-flight mass spectrometry (UPLC-TOF-MS). HA and MHA, having a primary amino group (NH(2)) in their structures, were first labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) at 60 degrees C for 45 min in the mixture of 0.1M borax (pH 9.3) and acetonitrile (CH(3)CN). On the other hand, 4-(N,N-dimethylaminosulfonyl)-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ) was used for the labeling of a carboxylic acid group (COOH) in IAA and MIAA in the presence of 2,2'-dipyridyl disulfide (DPDS) and triphenylphosphine (TPP). The reaction with DBD-PZ was completed at 50 degrees C after 2h. The resulting derivatives of HA and the metabolites were perfectly separated using an ACQUITY UPLCtrade mark BEH C(18) column (1.7 microm, 100 x 2.1mm, i.d.) with the mixture of 20 mM HCOONH(4) and CH(3)CN (8:2). The structures of HA and the metabolites were identified from the protonated-molecular ions [M+H](+) and the de-protonated-molecular ions [M-H](-) of authentic compounds, obtained from TOF-MS measurement. A good linearity was achieved from the calibration curves, obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS), i.e., histamine-alpha,alpha,beta,beta-d(4) (HA-d(4)) or 4-imidazolecarboxylic acid (ICA), against the injected amounts of each compound (1.0-25 pmol, r(2)=0.998). The detection limits of HA and the metabolites were less than 1 pmol. The proposed method was applied to the determination in the hair shafts of C3H mice. The average concentrations of HA, MHA, IAA and MIAA in 1mg of the hair shafts were 16.3 pmol (n=7), 21.6 pmol (n=3), 6.6 pmol (n=3) and 7.1 pmol (n=3), respectively. Because the proposed method provides good mass accuracy and the trace detection of HA and several metabolites in hair, this analytical technique seems to be applicable for the determination of various biological compounds in hair.

Animals↗
PubMed2006

Simultaneous analysis of cationic, anionic, and neutral compounds using monolithic CEC columns.

A new capillary electrochromatography (CEC) column for the simultaneous analysis of cationic, neutral, and anionic compounds using CEC-ESI-MS is described. Three different silica monolith columns were prepared by changing the poly(ethylene glycol) (PEG) contents for comparison of the separation property of these columns. Different separation programs were used for the simultaneous separation of different charged compounds under the same conditions. The column prepared with 80 mg of PEG separated typical compounds within 15 min using 1 M formic acid as the electrolyte. The analytes migrated in the order of cationic, neutral, and anionic compounds, which means that the migration order was mainly determined by the electrophoresis. The hydrodynamic flow by pressure from the inlet side was significant for a stable analysis to be achieved. The effect of the composition of the sheath liquid was also examined. All analytes (14 amino acids, thiourea, urea, citric acid, and ATP) were detectable when 1% acetic acid in 50% (v/v) methanol was used as the sheath liquid.

Journal Article↗
PubMed2006

Cationic amylopectin derivatives as additives for analysis of proteins in capillary electrophoresis.

Positively charged amylopectin, which is a major constituent of cationic starch, was used to modify the inner surface of fused-silica capillaries by addition to the running solution, which was subsequently employed in CE. Capillaries filled with cationic amylopectin derivatives were shown to generate a stable reversed EOF in the investigated range of pH 4-8. Among the additives studied, quaternary ammonium amylopectin derivatives with high amino and low hydroxypropyl groups showed fast electroosmotic mobility and very effectively suppressed the adsorption of proteins. The run-to-run and batch-to-batch repeatability of the procedures were satisfactory with RSDs of 0.5% and 2.4%, respectively. A basic protein, alpha-chymotrypsinogen, migrated within 6 min and the theoretical plate number of it reached 560 000 plates/m.

Amylopectin↗
PubMed2005

Creation of a P450 array toward high-throughput analysis.

The rapid metabolism testing of many new chemical entities enables unsuitable candidates to be eliminated from consideration at an early stage of the drug discovery process. We have developed a P450 array toward high-throughput analysis of P450-mediated metabolic reaction. The microsomes containing expressed human P450 enzymes were immobilized on the microassay plate using sol-gel chemistry. A thin-film hydrogel containing microsomes was fabricated using aqueous silicate as a starting material. The TEM image clearly showed that the nanoclusters derived from the silicate formed branched chains, and microsomes were entrapped in the silica network. The different P450 isozymes were immobilized on the microassay plate, and the metabolites by each isozyme were visualized as fluorescent images, which creates opportunity for the inhibitor assays. This method offers several advantages over use of conventional enzyme preparations, including increased storage stability, ease of product isolation from the incubation mixture, and the ability to recover and reuse the enzyme. Because this methodology enabled the development of assay system using P450 that is unstable and involves other enzymes for its function, it can be applicable to various screening assays that require complicated reactions involving many biological components.

Cytochrome P-450 Enzyme System↗
PubMed2005

Cationic starch derivatives as dynamic coating additives for protein analysis in capillary electrophoresis.

Positively charged starch derivatives were used to modify the inner surface of fused-silica capillaries by addition to running buffer, which were subsequently employed in capillary electrophoresis (CE). Capillaries coated with the cationic starch derivatives were shown to generate a stable, reversed electroosmotic flow (EOF) in the investigated pH range of 3-9. The presented coating procedure was fast, based on a simple rinsing protocol where the polymer created a physically adsorbed, cationic polymer layer. Among the additives studied, a quaternary ammonium starch derivative showed a fast EOF mobility and effectively suppressed the adsorption of proteins. The intra- and inter-day reproducibility of the coating referring to the EOF mobility were satisfactory with relative standard deviation (RSD) of 0.27 and 1.67%, respectively. The coating enabled separation of some protein mixtures including basic proteins within l3 min with efficiencies up to 280,000 plates/m. In addition, this cationic starch derivative possessed a good solubility (about 100mg/mL), and it does not significantly contribute to the background adsorption in the UV region of 190-400 nm.

Cations↗
PubMed2005

Resolution of N-linked oligosaccharides in glycoproteins based upon transglycosylation reaction by CE-TOF-MS.

The resolution of asparagine-type oligosaccharides in glycoproteins was carried out by combination of the transglycosylation reaction and CE-TOF-MS. The oligosaccharides enzymatically transferred to a fluorescent acceptor (NDA-Asn-GlcNAc) with Endo-M. The resulting fluorescent-oligosaccharides were separated by CE and detected by TOF-MS. Disialo-Asn was successfully identified by the proposed procedure. Application to oligosaccharides in ovalbumin was also described in this communication.

Acetylglucosamine↗
PubMed2005

Monolithic bioreactor immobilizing trypsin for high-throughput analysis.

A miniaturized trypsin reactor was prepared by coating a trypsin-containing gel on a porous silica monolith. The trypsin-encapsulated gel was prepared by the sol-gel method. The sol-gel reaction was optimized so that the sol solution containing trypsin forms a thin film on the sol-gel monolith. The trypsin was encapsulated into the gel matrix without losing its activity. The silica monolith was fabricated to fit into a 96-well microtiter plate well and could then be easily removed. The trypsin-immobilized monolith was reacted in the 96-well microtiter plate. After the reaction, the monolith was removed, and the enzymatic activity was measured. The large surface area of the monolith enabled the immobilized trypsin to achieve a high catalytic turnover rate. Furthermore, the kinetic parameter of the immobilized trypsin indicates the absence of diffusional limitations. The durability and repeatability of the fabricated trypsin-coated monolith was tested and found to be satisfactory. The encapsulated trypsin exhibits an increased stability even after continuous use compared with that in free solution. Furthermore, this on-plate bioreactor was applicable to the digestion of protein with multiple cleavage sites.

Bioreactors↗
PubMed2005

Synthesis of fluorescent label, DBD-beta-proline, and the resolution efficiency for chiral amines by reversed-phase chromatography.

DBD-d(and l)-beta-proline, new fluorescent chiral derivatization reagents, were synthesized from the reaction of 4-(N,N-dimethylaminosulfonyl)-7- fl uoro-2,1,3-benzoxadiazole (DBD-F) with beta-proline. The racemic mixture synthesized was separated by a chiral stationary phase (CSP) column, Chiralpak AD-H, with n-hexane-EtOH-TFA-diethylamine (70:30:0.1:0.1) as the mobile phase. The dl-forms were decided according to the results obtained from a circular dichroism (CD) detector after separation by the CSP column. The fractionated enantiomers reacted with chiral amine to produce a couple of diastereomers. The labeling proceeded in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and pyridine as the activation reagents. The reaction conditions were mild and no racemization occurred during the diastereomer formation. The resulting diastereomers fluoresced at around 570 nm (excitation at around 460 nm). Good linearity of the calibration curves was obtained in the range 1-75 pmol and the detection limits on chromatogram were less than 1 pmol. The separability of the diastereomers was compared with the diastereomers derived from DBD-d(or l)-proline. The resolution values (Rs) obtained from the diastereomers of three chiral amines with DBD-d(or l)-beta-proline were higher than those derived from DBD-d(or l)-proline, e.g. dl-phenylalanine methylester (dl-PAME), 2.23 vs 1.37; (R)(S)-1-phenylethylamine [(R)(S)-PEA], 2.09 vs 1.13; and (R)(S)-1-(1-naphthyl)ethylamines [(R)(S)-NEA], 5.19 vs 1.23. The results suggest that the position of COOH group on pyrrolidine moiety in the structures is one of the important factors for the efficient separation of a couple of the diastereomers.

Amines↗
PubMed2005

Analysis of amino acids and proteins using a poly(methyl methacrylate) microfluidic system.

Plastic microchips are very promising analytical devices for the high-speed analysis of biological compounds. However, due to its hydrophobicity, their surface strongly interacts with nonpolar analytes or species containing hydrophobic domains, resulting in a significant uncontrolled adsorption on the channel walls. This paper describes the migration of fluorescence-labeled amino acids and proteins using the poly(methyl methacrylate) microchip. A cationic starch derivative significantly decreases the adsorption of analytes on the channel walls. The migration time of the analytes was related to their molecular weight and net charge or pI of the analytes. FITC-BSA migrated within 2 min, and the theoretical plate number of the peak reached 480,000 plates/m. Furthermore, proteins with a wide range of pI values and molecular weights migrated within 1 min using the microchip.

Amino Acids↗
PubMed2004

Cationic starch derivatives as dynamic coating additives for analysis of amino acids and peptides using poly(methyl methacrylate) microfluidic devices.

Plastic microchips are very promising analytical devices because they are less fragile and are suitable for mass production. However, due to their hydrophobicity, the surface strongly interacts with nonpolar analytes or species containing hydrophobic domains, resulting in significant uncontrolled adsorption on channel walls. This paper describes the poly(methyl methacrylate) surface treatment by dynamic coating additives that considerably decreases adsorption of analytes to channel walls. Among the additives studied, quaternary ammonium starch derivatives suppressed the adsorption of fluorescently labeled amino acids and peptides most effectively. The effect was valid over the wide pH range from 2.5 to 8.0. Using a 10 mM phosphate buffer (pH 7.0) with 3% (w/v) quaternary ammonium starch as the running buffer, Asp and Glu, respectively, migrated at 54.6 and 57.6 s with efficiencies of 380 000 and 370 000 plates/m. In addition, this cationic starch derivative was found to possess good solubility and low viscosity.

Amino Acids↗
PubMed2004

Hair analysis of histamine after fluorescence labeling by column-switching reversed-phase liquid chromatography with electrospray ionization mass spectrometry and application to human hair.

Sensitive determination of histamine (HA) in hair was carried out by column-switching reversed-phase high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS). HA was labeled with excess amounts of 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) at 60 degrees C for 30 min in a mixture of 0.1 M borax (pH 9.3) and acetonitrile (CH(3)CN). The resulting DBD-HA derivative was roughly separated by a Mightysil RP-18 GP (100 x 2mm i.d., 3 microm) with an acidic mobile phase containing 0.1% trifluoroacetic acid. DBD-HA in the fraction flowing due to a position change in the six-port column-switching valve was then completely separated by a Wakopak Navi C30 (150 x 2mm i.d., 5 microm) with 20 mM AcONH(4)-CH(3)CN (8:2). The mass spectrometer was operated in the selected reaction monitoring (SRM) mode for the product ion (m/z 292) obtained from MS-MS measurement using the protonated molecular ion [M+H](+) (m/z 337) as the precursor ion. Good linearity was achieved from the calibration curve obtained by plotting peak area ratios of the internal standard (HA-d(4)) against the injected amounts of HA (1.66-16.6 pmol, r(2)=0.999). The coefficients of variation, at 1.66- and 16.6-pmol injections, were 5.6 and 3.7%, respectively (n=6). Furthermore, the detection limit was 0.167 pmol. The efficiency of the recommended procedure was identified from the determination in the rat hair root after intraperitoneal administration of HA. The proposed method was applied to HA determination in the hair shaft of Dark Agouti rats and healthy volunteers. The variations in the concentrations in 1mg of hair shaft were 0.80-1.84 pmol (mean+/-SD=1.33+/-0.33, n=12) in rats and 0.94-72.3 pmol (17.2+/-21.5, n=16) in humans. The determination of HA in the plasma of rats and humans was also performed successfully by this method. Because the proposed method provides good precision and trace detection of HA in hair, the analytical technique seems to be applicable for the determination of various biogenic amines in hair.

Animals↗
PubMed2004

Screening of inhibitors of uridine diphosphate glucuronosyltransferase with a miniaturized on-line drug-metabolism system.

Inhibition of uridine diphosphate glucuronosyltransferase (UGT), a major drug-metabolyzing enzyme, has been studied using an on-line drug-metabolism system integrated into capillary electrophoresis. Microsomes isolated from rat liver were encapsulated in tetramethoxysilane (TMOS)-based silica matrices within a capillary in a single step under mild conditions. This microsome-immobilized capillary column allows both the metabolism of drugs and determination of the metabolites in a single capillary simultaneously, just by injecting the substrate-coenzyme mixture onto the column. Glucuronidation of acetaminophen, a widely used pharmaceutical analgesic and antipyretic agent, was investigated using this system. The glucuronidation was inhibited by 4-nitrophenol (4NP) or probenecid that was injected onto a column along with the substrate-coenzyme mixture. On the other hand, valproate did not inhibit the metabolizing reaction. The extents of inhibition using encapsulated UGT were almost the same as those obtained using free UGT. On the other hand, this electrophoretic enzyme-inhibitor assay in microfabricated devices consumes 10(4) less sample and 10(3) less microsome per experiment compared to the conventional reaction schemes. These results demonstrate that this on-line system can circumvent laborious procedures for the isolation and determination of drug metabolites from the reaction mixtures required in the conventional schemes and can provide an attractive alternative technique for the analysis of drug interactions in the metabolic pathways.

Animals↗
PubMed2004

Silica sol-gel/organic hybrid material for protein encapsulated column of capillary electrochromatography.

A new-type of sol-gel/organic hybrid composite material using gelatin or chitosan with tetramethoxysilane was developed for the bovine serum albumin (BSA)-encapsulated monolithic column for capillary electrochromatography (CEC). The composite monolith was used to immobilize BSA in a fused-silica capillary. The addition of gelatin and chitosan to the alkoxysilane enabled the enantioseparation of Trp. A very small amount of these polymers were effective for the enantioseparation. Especially, the monolithic column prepared from chitosan with tetramethoxysilane showed a high enantioselectivity for Trp enantiomers and the value (alpha' = t2/t1, t1: fast eluted enantiomer, t2: second eluted enantiomer) reached 1.15 on CEC mode. Furthermore, the composite materials exhibited a higher stability compared to the silica sol-gel column. These results showed that the sol-gel/organic hybrid composite was useful as a monolithic matrix for the BSA-encapsulated column for CEC.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2004

Integration of on-line protein digestion, peptide separation, and protein identification using pepsin-coated photopolymerized sol-gel columns and capillary electrophoresis/mass spectrometry.

A miniaturized pepsin reactor was prepared inside a fused-silica capillary (i.d. 75 microm) by coating a pepsin-containing gel on a photopolymerized porous silica monolith. The pepsin-encapsulated film was prepared by a sol-gel method. The sol-gel reaction was optimized so that the sol solution containing pepsin forms a thin film on the photopolymerized sol-gel (PSG) monolith that was initially fabricated at the inlet of the capillary. Pepsin was encapsulated into the gel matrix without losing its activity. The large surface area of the PSG monolith enabled the immobilized pepsin to achieve a high catalytic turnover rate, and the porous nature of the PSG promotes penetration of large molecular proteins into the column. The immobilized pepsin-digested peptides and proteins, and the resulting mixture of peptide fragments, could be directly separated in the portion of the capillary where no PSG monolith exists. The durability and repeatability of the fabricated pepsin-coated column was tested and found to be satisfactory. An acidic solution consisting of 0.5 M formic acid was used as the running buffer, because it suppresses the adsorption of proteins or peptides on the inner surface of the capillary as well as enables direct connection of the output of the capillary electrophoresis column to a mass spectrometer. The on-line digestion of insulin chain beta and lysozyme provides identification of the proteolytic peptides. Recovery was achieved for 100% of the insulin chain beta amino acid sequence and 73% of the lysozyme amino acid sequence.

Amino Acid Sequence↗
PubMed2004

Simultaneous determination of D- and L-serine in rat brain microdialysis sample using a column-switching HPLC with fluorimetric detection.

Both D- and L-serine in rat brain microdialysis sample were simultaneously determined by pre-column fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), separation of the derivatives on ODS column, TSKgel ODS-80TsQA, followed by Pirkle type chiral columns, Sumichiral OA-2500 (S), which gave a sufficient enantiomeric separation of NBD-D-serine and NBD-L-serine, and fluorimetric detection at a wavelength of 540 nm with an excitation wavelength of 470 nm. The peaks of NBD-D-serine and NBD-L-serine in the rat brain microdialysis sample were clearly found, and the validation study showed satisfactory results; the precision and accuracy were within 5.14 and 109%, respectively. Using the proposed HPLC method, the time-course profile of D-serine concentration in rat prefrontal cortex following intraperitoneal administration of D-serine was investigated. As a consequence, D-serine appeared to be rapidly distributed in the brain, and then decreased gradually with time in the extracellular fluid of the rat prefrontal cortex. The proposed HPLC method will be useful for in vivo studies on D-serine, which acts as a coagonist for N-methyl-D-aspartate receptor, to the extracellular fluid of rat brain.

4-Chloro-7-nitrobenzofurazan↗