Biomedical subjects

Masaru Kato

Publications and source records attributed to Masaru Kato.

At least 37 records · Page 2Linked to original sources

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↗

PubMed2003

An enzyme-immobilization method for integration of biofunctions on a microchip using a water-soluble amphiphilic phospholipid polymer having a reacting group.

A water-soluble phospholipid polymer having an active ester group in the side chain, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl polyethyleneglycol methacrylate (MEONP)(PMBN), was used for the immobilization of an enzyme on a plastic microchip. The MPC polymers with BMA units were adsorbed onto the poly(methyl methacrylate)(PMMA) microchip, and the active ester group in the MEONP unit reacted with the amino groups of the proteolytic enzyme, trypsin. Trypsin was immobilized on the sample reservoir, and catalyzed the hydrolysis of the fluorescently labeled ArgOEt to Arg. The consequent separation of product from the substrate, and their detection, were integrated on the microchip and this meant that all procedures from the enzymatic activity to product detection were completed in less than three minutes.

Adsorption↗

PubMed2003

On-line preconcentration prior to on-column derivatization monolith octadecasiloxane capillary electrochromatography for the determination of biogenic amines.

To expand the applications of the on-line preconcentration technique with capillary electrochromatography (CEC) to biogenic amines that have no specific chromophore or fluorophore in their molecules, a method of on-line preconcentration prior to on-column derivatization CEC is presented. A monolithic ODS capillary column (20 cm effective length x 75 microm i.d.) for CEC was fabricated using a thermal sol-gel reaction of tetraethyl orthosilicate to capture ODS particles (5-microm particle diameter) in a capillary tube. A standard model biogenic amine solution consisting of histamine, methylhistamine, and serotonin was electrokinetically injected from the anodic site of the capillary column with 5 kV, and these amines were effectively concentrated at the inlet site of the capillary column by a field-amplified sample stacking, a gradient effect mode, or both. This preconcentration occurred whenever the several types of solvent for reconstitution of the amines, e.g., water (noneluting solvent or low-conductivity solvent), 0.9% sodium chloride (noneluting solvent or high-conductivity solvent), or 60% acetonitrile in 10 mM borate buffer (pH 10) (eluting solvent) were employed. After concentration, the amines were subsequently derivatized, separated, and detected during CEC with an optimum CEC run buffer solution containing 60% acetonitrile in 5 mM o-phthalaldehyde/2-mercaptoethanol-10 mM borate buffer (pH 10) when 5 kV was continuously applied. Using the present system, equipped with a fluorescence detector instead of a UV/visible detector, the detection sensitivity for amines reached a 0.1 microM level, which increased sensitivity by a factor of 10(3) times greater than that of normal on-column derivatization CEC.

Biogenic Amines↗

PubMed2003

Rapid analysis of amino acids in Japanese green tea by microchip electrophoresis using plastic microchip and fluorescence detection.

Microchip electrophoresis for the short-time analysis of amino acids in Japanese green tea was developed. The amino acids in Japanese green tea were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The derivatives were filtered and directly analyzed by electrophoresis on a plastic microchip with a 31-mm long separation channel with fluorescence detection. Amino acid analysis of Japanese green tea was improved by removing polyphenols using a polyvinylpolypyrrolidone pretreatment. Elution profiles of NBD-amino acids were examined under different running buffer conditions, and the sodium dodecyl sulphate in the running buffer exhibited a dramatically high-separation efficiency of amino acids by inhibiting their adsorption on the channel walls. Under the optimized conditions (5 mM phosphate buffer (pH 5.5) containing 0.05 mM sodium dodecylsulfate as running buffer), the main amino acids contained in Japanese green tea were well separated within 2 min, and theanine (1475 mg/100 g tea leaf), Arg (408 mg/100 g tea leaf) and Gln (217 mg/100 g tea leaf) were detected in Japanese green tea.

Acetonitriles↗

PubMed2003

Determination of glutamine and serine in rat cerebrospinal fluid using capillary electrochromatography with a modified photopolymerized sol-gel monolithic column.

Capillary electrochromatographic separations of amino acid mixtures were studied using two modified porous photopolymerized sol-gel monolithic columns. One was modified with dimethyloctadecylchlorosilane (DMOS), and the other was modified with DMOS, followed by chlorotrimethylsilane to end-cap residual silanol groups. Prior to separation, amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole using as a mobile phase 50 mM phosphate (pH 2.5), water, and acetonitrile in the ratio of 1:1:8. Five derivatized amino acids (Asn, Phe, Ala, Ile, and Leu) were separated within 7 min. Theoretical plate numbers varied between 58700 and 105000/m. This separation method with the end-capped monolithic column was applied to rat cerebrospinal fluid. The dominant amino acid found was Gln at a concentration of 420 microM along with small quantities of Ser (54 microM).

Animals↗

PubMed2003

Investigation of structure and enantioselectivity of BSA-encapsulated sol-gel columns prepared for capillary electrochromatography.

We have developed a protein-encapsulation technique using sol-gels for the preparation of monolithic capillary columns for capillary electrochromatography. Due to the silica-based matrix used, this hydrogel generates the appropriate electroosmotic flow. Electroosmotic mobility varied according to the gels fabricated under the different gelation conditions and using different starting materials. Using attenuated total reflectance (ATR)-FT-IR, the residual silanol groups in each hydrogels could be measured without drying procedures and it was found that electroosmotic mobility decreased with a reduction in the residual silanol groups. Enantiomeric separation of D,L-Trp was evaluated using bovine serum albumin (BSA)-encapsulated column. Preparatory conditions for BSA-encapsulated columns also influenced the retention time and enantioselectivity of D,L-Trp. The gels composed of clusters with the diameter of around 1 microm. According to ATR-FT-IR study, BSA maintained its structure after encapsulation in the gel.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed2003

Creation of an on-chip enzyme reactor by encapsulating trypsin in sol-gel on a plastic microchip.

Trypsin-encapsulated sol-gel was fabricated in situ onto a plastic microchip to form an on-chip bioreactor that integrates tryptic digestion, separation, and detection. Trypsin-encapsulated sol-gel, which is derived from alkoxysilane, was fabricated within a sample reservoir (SR) of the chip. Fluorescently labeled ArgOEt and bradykinin were digested within the SR followed by electrophoretic separation on the same chip. The plastic microchip, which is made from poly(methyl methacrylate), generated enough electroosmotic flow that substrates and products could be satisfactorily separated. The sol-gel in the SR did not alter the separation efficiency of each peak. With the present device, the analytical time was significantly shortened compared to conventional tryptic reaction schemes. This on-chip microreactor was applicable to the digestion of protein with multiple cleavage sites and separation of digest fragments. Furthermore, the encapsulated trypsin exhibits increased stability, even after continuous use, compared with that in free solution.

Drug Compounding↗

PubMed2003

Determination of hypnotic benzodiazepines (alprazolam, estazolam, and midazolam) and their metabolites in rat hair and plasma by reversed-phase liquid-chromatography with electrospray ionization mass spectrometry.

Sensitive determination of benzodiazepines i.e., alprazolam (ALP), estazolam (EST), and midazolam (MDZ), and their metabolites, was carried out by reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS). The chromatography separations were achieved using a semi-micro HPLC column (3 microm particle size; 100 x 2.0 mm, i.d.) with acetonitrile-water containing 1% acetic acid as eluent. The mass spectrometer was operated in selected-ion monitoring mode at protonated-molecular ions [M+H](+) of parent drugs and the metabolites. The proposed procedure was applied to the determination in hair shaft of Dark Agouti rats after intraperitoneal (i.p.) administration of benzodiazepines twice a day for 5 days. Various metabolites together with parent drugs were identified in the hair shaft, 1-hydroxyalprazolam (1-HA) and 4-hydroxyalprazolam (4-HA) from ALP administration; 8-chloro-6-phenyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine-4-one (K-EST) from EST administration; 1-hydroxymidazolam (1-HM) and 4-hydroxymidazolam (4-HM) from MDZ administration. A few unknown metabolites were also detected in the hair samples. These structures were elucidated with acetylation using acetic anhydride and pyridine. The time course studies of parent drugs and the metabolites in both hair root and plasma were also carried out after single i.p. administration of benzodiazepines. The results suggested that the concentrations of parent drugs and the metabolites in the hair samples were mainly dependent upon those in the plasma.

Alprazolam↗

PubMed2003

Investigation of chromatographic performances and binding characteristics of BSA-encapsulated capillary column prepared by the sol-gel method.

We have developed a novel protein-encapsulation technique using the sol-gel method for the preparation of monolithic capillary columns for capillary electrochromatography. Bovine serum albumin (BSA) was encapsulated in tetramethoxysilane-based hydrogel and the enantioselectivity was evaluated. The present work examined the effect of various factors such as running buffer pH and concentration or organic modifier on the chromatographic performances as well as binding characteristics of BSA for D,L-tryptophan (Trp). The retention and enantioseparation dramatically changed depending on the buffer pH and concentrations. These factors influenced EOF and especially binding characteristics of BSA for L-Trp. Under the optimized conditions, the BSA-encapsulated columns revealed the adequate repeatability concerning column-to-column and run-to-run.

Animals↗

PubMed2003

Chronic angle closure glaucoma secondary to frail zonular fibres and spherophakia.

PURPOSE: We describe a 39-year-old Japanese woman with chronic angle closure glaucoma secondary to spherophakia and frail zonular fibres. The patient was 143 cm in height with short fingers and had no family history of eye problems. High intraocular pressure, total optic disc cupping and severe visual field constriction were found in the right eye. METHODS: The patient was treated successfully with trabeculectomy in the right eye and laser iridotomy in the left eye. CONCLUSION: The clinical findings imply that this was a borderline case of Weil-Marchesani syndrome.

Adult↗

PubMed2003

Electrophysiologic evaluation of retinal pigment epithelial damage induced by photic exposure.

PURPOSE: To evaluate the retinal pigment epithelial damage induced by light exposure. METHODS: One eye of 20 rabbits was exposed to xenon light for 2 hours at an irradiance of 140 mW/cm2 at the surface of the cornea. The contralateral eye served as a control. Forty-eight hours after the light exposure, corneal direct-coupled electroretinograms and the 7% NaHCO3 (bicarbonate) responses of the standing potential were recorded. RESULTS: The amplitudes of the a-, b-, and c-waves of the electroretinograms were significantly reduced in the light-exposed eyes, with the c-waves more reduced than the a- and b-waves. The bicarbonate response was also significantly reduced in the irradiated eyes. CONCLUSIONS: The decrease of the bicarbonate response of the standing potential indicated that significant functional damage of the retinal pigment epithelium was induced by the light exposure.

Animals↗

PubMed2003

Reversible labeling of tyrosine residue in peptide using 4-fluoro-7-nitro-2,1,3-benzoxadiazole and N-acetyl-L-cysteine.

The reversible labeling of tyrosine (Tyr)-containing peptide, which involves detection and recovery, is described in this paper. The phenolic-OH in Tyr structure reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) under a mild condition at room temperature in alkaline medium. The resulting derivative absorbed at around 280 nm and 380 nm. However, the fluorescence was very weak. The NBD moiety in the derivative was efficiently removed by the treatment of N-acetyl-L-cysteine (N-AcCys), and the original peptide before the labeling was completely recovered. The proposed procedure was successfully applied to the reversible labeling of N-terminal amine-blocked peptides, i.e., N-AcTyr-Val-Gly, Z-Glu-Tyr, Z-Phe-Tyr, N-Formyl-Met-Leu-Tyr, and N-AcArg-Pro-Pro-Gly-Phe-Ser-Pro-Tyr-Arg. Although the proposed method could not recover the N-terminal amine-free peptides without blocking, the selective detection and the recovery of Tyr-containing peptide fragments were possible by the combination with enzyme digestion. The reversible labeling of Tyr-containing peptide was demonstrated with [Tyr8]-bradykinin as a model for high-molecular-mass peptides and proteins. The peptide fragments containing NBD-O-Tyr moiety, obtained after the digestion, were easily discriminated from various peptides with the monitoring of UV and FL, because the target peptide did not fluoresce, but absorbed at both 280 nm and 380 nm. The peptide fragment containing Tyr was finally recovered from the de-labeling reaction with N-AcCys. The proposed method hence provides a novel technique for the reversible labeling of Tyr-containing peptides, which will enable the selective detection and the recovery of the original peptide.

4-Chloro-7-nitrobenzofurazan↗

PubMed2002

On-line drug-metabolism system using microsomes encapsulated in a capillary by the sol-gel method and integrated into capillary electrophoresis.

A novel microsome-encapsulation technique using the sol-gel method was developed for the on-line drug-metabolism analytical system integrated into capillary electrophoresis. This analytical system allows both the metabolism of drugs and the determination of the metabolites in a single capillary simultaneously. Microsomes isolated from rat liver were encapsulated in tetramethoxysilane-based silica matrices within a capillary in a single step under mild conditions. The availability of this system was evaluated using UDP-glucuronyltransferase, which is one of the most important microsomal enzymes. 4-Nitrophenol and testosterone, which were metabolized by the different isoforms of UDP-glucuronyltransferase, were used as substrates. The resultant monolithic reactor showed enzymatic activity at the same level as that of the soluble form. The following separation of the unreacted substrates and metabolites in the same capillary also showed high selectivity. Furthermore, the sample amount required for one analysis decreased more than 3 orders of magnitude from conventional reaction schemes in free solution. This on-line system could largely simplify the laborious procedures which were needed in conventional analytical schemes.

Animals↗

PubMed2002

On-line trypsin-encapsulated enzyme reactor by the sol-gel method integrated into capillary electrophoresis.

A novel trypsin-encapsulation technique using the sol-gel method was developed for the preparation of an on-line enzyme reactor integrated into capillary electrophoresis. Trypsin was encapsulated in tetramethoxysilane-based hydrogel, and its enzymatic activity was evaluated using alpha-N-benzoyl-L-arginine ethyl ester and two peptides (bradykinin and [Ter8]-bradykinin). The enzyme encapsulation was carried out in a single step under mild conditions within a capillary, and 1.5-cm gel was formed at the inlet of the capillary. The resultant monolithic reactor showed excellent enzymatic activity, which was approximately 700 times higher than that in free solution, without stopping the flow. Separation of the unreacted substrates and products in the same capillary also showed high selectivity, and sample size in this system decreased 3 orders of magnitude from conventional tryptic reaction schemes. The encapsulated trypsin maintains its substrate specificity even in a sol-gel matrix. Furthermore, the encapsulated trypsin exhibits increased stability even after continuous use compared to that in free solution.

Electrophoresis, Capillary↗

PubMed2002

Effect of preparatory conditions on the performance of photopolymerized sol-gel monoliths for capillary electrochromatography.

We prepared different photopolymerized sol-gel (PSG) columns by varying the amount of monomer (methacryloxypropyltrimethoxysilane), porogen (toluene) and catalyst (hydrochloric acid) in the reaction solution containing a photoinitiator (Irgacure 1800). The effects of these variations on the chromatographic behavior of the PSG columns were studied. All of the columns studied exhibited reversed-phase character. The concentration of hydrochloric acid was important for the rigidity of the columns, although it did not affect the separation property. The ratio of monomer solution to porogen was a critical factor in controlling the through-pore size and the surface area of PSG, which were found to significantly affect the separation properties, such as permeability, theoretical plate number, retention time, and separation efficiency, of a mixture of test analytes-thiourea, benzene, and naphthalene. There was no change in the retention order for the test analytes. Short separation times were achieved on PSG columns made from a 10% monomer stock solution and 90% porogen with 1 M hydrochloric acid. Mixtures of polycyclic aromatic hydrocarbons and alkylbenzenes were separated with theoretical plate numbers greater than 100 000 plates/m.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed2002

A protein-encapsulation technique by the sol-gel method for the preparation of monolithic columns for capillary electrochromatography.

A novel protein-encapsulation technique using sol-gels was developed for the preparation of monolithic capillary columns for capillary electrochromatography. Two chiral compounds, bovine serum albumin (BSA) and ovomucoid (OVM) from chicken egg white, were encapsulated in tetramethoxysilane-based hydrogel and their chiral selectivity was evaluated for the separation of some selected enantiomers (tryptophan, benzoin, eperisone, chlorpheniramine). The protein encapsulation was carried out within a capillary in a single step under mild conditions. The resultant monolithic columns showed adequate chromatographic performance, including mechanical strength, penetration of pressurized flow, and chiral separation. Two different proteins, BSA and OVM, were successfully encapsulated into the gel matrixes by changing the alkoxysilane compositions of the gel. Run-to-run repeatability was quite satisfactory. The consecutive analysis of the neutral compound, benzoin, by the OVM-encapsulated column showed good repeatability in the retention time (RSD = 1.23% for the first peak, N = 10). Under optimized conditions, the theoretical plate number for the first peak of benzoin reached 72,000 plates/m.

Benzoin↗

PubMed2002

Analysis of moricizine block of sodium current in isolated guinea-pig atrial myocytes. Atrioventricular difference of moricizine block.

The effects of moricizine on Na+ channel currents (INa) were investigated in guinea-pig atrial myocytes and its effects on INa in ventricular myocytes and on cloned hH1 current were compared using the whole-cell, patch-clamp technique. Moricizine induced the tonic block of INa with the apparent dissociation constant (Kd,app) of 6.3 microM at -100 mV and 99.3 microM at -140 mV. Moricizine at 30 microM shifted the h infinity curve to the hyperpolarizing direction by 8.6 +/- 2.4 mV. Moricizine also produced the phasic block of INa, which was enhanced with the increase in the duration of train pulses, and was more prominent with a holding potential (HP) of -100 mV than with an HP of -140 mV. The onset block of INa induced by moricizine during depolarization to -20 mV was continuously increased with increasing the pulse duration, and was enhanced at the less negative HP. The slower component of recovery of the moricizine-induced INa block was relatively slow, with a time constant of 4.2 +/- 2.0 s at -100 mV and 3.0 +/- 1.2 s at -140 mV. Since moricizine induced the tonic block of ventricular INa with Kd,app of 3.1 +/- 0.8 microM at HP = -100 mV and 30.2 +/- 6.8 microM at HP = -140 mV, and cloned hH1 with Kd,app of 3.0 +/- 0.5 microM at HP = -100 mV and 22.0 +/- 3.2 microM at HP = -140 mV, respectively, either ventricular INa or cloned hH1 had significantly higher sensitivity to moricizine than atrial INa. The h infinity curve of ventricular INa was shifted by 10.5 +/- 3.5 mV by 3 microM moricizine and that of hH1 was shifted by 5.0 +/- 2.3 mV by 30 microM moricizine. From the modulated receptor theory, we have estimated the dissociation constants for the resting and inactivated state to be 99.3 and 1.2 microM in atrial myocytes, 30 and 0.17 microM in ventricular myocytes, and 22 and 0.2 microM in cloned hH1, respectively. We conclude that moricizine has a higher affinity for the inactivated Na+ channel than for the resting state channel in atrial myocytes, and moricizine showed the significant atrioventricular difference of moricizine block on INa. Moricizine would exert an antiarrhythmic action on atrial myocytes, as well as on ventricular myocytes, by blocking Na+ channels with a high affinity to the inactivated state and a slow dissociation kinetics.

Animals↗