It has been a desire to develop orally effective therapeutic agents that restore the liver functi... more It has been a desire to develop orally effective therapeutic agents that restore the liver function in chronic injury. Here we demonstrated that trans-4-L-hydroxyprolyl-L-serine (JBP923) and cyclo-trans-4-L-hydroxyprolyl-L-serine (JBP485), which was previously isolated from hydrolysate of human placenta, exhibit potent antihepatitis activity after their oral administration. The increase in bilirubin concentration and activities of liver cytosolic enzymes in serum caused by alpha-naphthylisothiocyanate intoxication in rats were significantly countered both after i.v. and oral administration of these dipeptides, whereas glycyrrhizin, which has been used in the treatment of chronic hepatitis, is active only after its i.v. administration. Antihepatitis activity of dipeptides results, at least partially, from their direct effect on hepatocytes because glutamic-oxaloacetic transaminase and lactate dehydrogenase activities in the medium of hepatotoxin-exposed primary cultured hepatocytes were reduced by these compounds. When comparing the plasma concentration-time profile of JBP923 after its i.v., oral, and portal vein injection, it is suggested that JBP923 is almost completely absorbed from gastrointestinal lumen, and hepatic first-pass removal is minor. JBP923 inhibited the proton-dependent transport of glycylsarcosine in brush-border membrane vesicles, suggesting that peptide transport system(s) may recognize JBP923. Thus, these dipeptides are potent antihepatitis reagents that are still active after oral administration and may be useful for clinical applications.
Archives of Biochemistry and Biophysics, Aug 1, 2002
In a previous report (FEBS Lett. 434 (1998) 231), we demonstrated for the first time that D-aspar... more In a previous report (FEBS Lett. 434 (1998) 231), we demonstrated for the first time that D-aspartate (D-Asp) is synthesized in rat pheochromocytoma 12 (PC12) cells. This unique amino acid is believed to act as a novel messenger in mammalian cell regulation. However, the dynamics of D-Asp homeostasis in mammalian cells is yet to be elucidated. In this communication, we demonstrate that D-Asp is also synthesized in MPT1 cells (a subclone of PC12 cells) and that the D-and L-Asp levels in cells are regulated by cell density of the culture. Our data show that D-Asp levels increase, while in contrast, L-Asp levels decrease as a function of increased cell density. Conversely, in PC12 cells, which do not express the glutamate transporter involved in the incorporation of D-and L-Asp into cells, L-Asp levels decrease upon cell density increase while D-Asp concentrations remain almost unchanged. The results indicate that the biochemical behaviors of D-and L-Asp in mammalian cells are distinct and that the cellular levels of these stereoisomers appear to be under different control mechanisms.
Six oxalate esters of 2-nitro-4-alkoxycarbonylphenol and 2-alkoxycarbonyl-4-nitrophenol were synt... more Six oxalate esters of 2-nitro-4-alkoxycarbonylphenol and 2-alkoxycarbonyl-4-nitrophenol were synthesised for use in the peroxyoxalate chemiluminescence reactions that take place via several steps with 4-hydroxy-3-nitrobenzoic acid and 5-nitrosalicylic acid as starting ...
Progress in Neuro-psychopharmacology & Biological Psychiatry, Mar 1, 2004
Several lines of evidence suggest that d-serine may function as an endogenous agonist of the glyc... more 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 Alzheimer's 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 = À 1.77, p = 0.078) lower than those of normal controls. In contrast, serum levels of L-serine in the patients were slightly (z = À 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 = À 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.
Dopamine (DA), norepinephrine (NE) and 3,4-dihydroxybenzylamine (DHBA) are converted to highly fl... more Dopamine (DA), norepinephrine (NE) and 3,4-dihydroxybenzylamine (DHBA) are converted to highly fluorescent cyanobenz[f]isoindole (CBI) derivatives by reacting these amines with naphthalene-2,3-dicarboxaldehyde in the presence of cyanide ion. Femtomole amounts of these CBI derivatives separated by reverse-phase high performance liquid chromatography can be detected by a post-column chemiluminescence system utilizing bis(2,4-dinitrophenyI) oxalate and hydrogen peroxide. Linear detection response was observed between 1 to 600 fmol, giving a signal to noise ratio of approximately 15 at the 1 fmol level. An assay procedure was developed for the determination of DA and NE in 20 p L of urine sample using DHBA as the internal standard.
Effects of the Substituent Groups at the 4-and 7-Positions on the Fluorescence Characteristics of... more Effects of the Substituent Groups at the 4-and 7-Positions on the Fluorescence Characteristics of Benzofurazan Compounds.-Study of the fluorescence intensity, maximum excitation wavelength and maximum emission wavelength of seventy title compounds, e.g. (I), reveals a relationship between the substituents at position 4 and 7 of the benzofurazan skeleton and the fluorescence characteristics. Hammett substituent constants are adopted as parameters for the electronic effects of the substituent groups.
A sensitive determination method for a non‐fluorescent anti‐arrhythmic drug, mexiletine, in rat p... more A sensitive determination method for a non‐fluorescent anti‐arrhythmic drug, mexiletine, in rat plasma is presented utilizing a HPLC peroxyoxalate chemiluminescence (PO‐CL) detection system. After an internal standard (4‐methylmexiletine, 4.35 pmol) and 0.1 N sodium hydroxide solution were added to 5 μL rat plasma, the solution was poured onto an Extrelut 1 column. Both mexiletine and the internal standard were eluted with diethy ether and then the eluate was evaporated to dryness. The residue was dissolved in 0.2 M borate buffer (pH 8.5) and mixed with dansyl chloride (75 nmol) in acetronitrile. After standing of 90 min at room temperature, 0.5 N HCI was added to the reaction mixture to stop the reaction and a 2/45 aliquot of the mixture was subjected to a HPLC PO‐CL detection system using bis(4‐nitro‐2(3,6,9‐trioxadecyloxycarbonyl)phenyl)oxalate (TDPO) and hydrogen peroxide. The calibration curve for mexiletine in rat plasma was linear over the range 20–100 ng/mL plasma (20.6–103 fmol/injection). The detection limit (S/N = 2) was 1.0 fmol over the whole procedure. The method was applied to the measurement of the time courses of plasma mexiletine concentration after oral administration of the drug [25 mg (115.9 μmol)/kg] to rats.
This article elucidates the relationships between the chemical structure and the fluorescence cha... more This article elucidates the relationships between the chemical structure and the fluorescence characteristics (fluorescence intensity, maximum excitation wavelength and maximum emission wavelength) of 4,7-disubstituted benzofurazan compounds with computer calculation. Our previous study using Hammett substituent constants of the substituent groups at the 4- and the 7-positions suggested that the total of electron densities on the benzofurazan skeleton and the dipole moment directed from the 4- to the 7-position might affect the fluorescence characteristics of these compounds. The sum of atomic charges on the benzofurazan skeleton, the HOMO energy and the LUMO energy were selected as parameters to reflect the total of electron densities on the benzofurazan skeleton and were obtained with the AM1 and PM3 calculations. The dipole moment directed from the 4- to the 7-position was also calculated with two hamiltonians. The LUMO energy and the dipole moment from the 4- to the 7-position obtained with the PM3 calculation is most closely related with the fluorescence characteristics. Using these parameters, the fluorescent 4,7-disubstituted benzofurazan compounds were classified into two groups, and the maximum excitation and emission wavelengths were different between these two groups. These relationships indicated that the fluorescence characteristics of 4,7-disubstituted benzofurazan compounds were determined by the total of electron densities on the benzofurazan skeleton and the dipole moment directed from the 4- to the 7-position. Furthermore, we predicted the fluorescence characteristics of four 4,7-disubstituted benzofurazan compounds based on the relationships obtained, and confirmed that the prediction agreed with the measured data.
The effects of various factors (organic solvent, type of reservoir, temperature, water content an... more The effects of various factors (organic solvent, type of reservoir, temperature, water content and concentration of hydrogen peroxide) were investigated on the stability of a chemilumigenic reagent, bis(2,4,6-trichlorophenyl) oxalate (TCPO), for high-performance liquid chromatography. In a mixture with hydrogen peroxide, TCPO was most stable when dissolved in acetonitrile and kept in a polyethylene bottle or a high quality glass (borosilicate glass free from metals) flask. The stability of TCPO was also increased by lowering the temperature, the water content and the concentration of hydrogen peroxide in the chemilumigenic reagent solution. Under these conditions, TCPO can be used in a mixed acetonitrile solution with hydrogen peroxide for HPLC-chemiluminescence detection.
Benzofurazan derivatization reagents, 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminopenty... more Benzofurazan derivatization reagents, 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminopentylamino)-2,1,3-benzoxadiazole (DAABD-AP) and 4-[2-(N,N-dimethylamino) ethylaminosulfonyl]-7-(2-aminobutylamino)-2,1,3-benzoxadiazole (DAABD-AB), for short-chain carboxylic acids in liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) were synthesized. These reagents reacted with short chain carboxylic acids in the presence of the condensation reagents at 60C for 60 min. The generated derivatives were separated on the reversed-phase column and detected by ESI-MS/MS with the detection limits of 0.1-0.12 pmol on column. Upon collision-induced dissociation, a single and intense product ion at m/z 151 was observed. These results indicated that DAABD-AP and DAABD-AB are suitable as the derivatization reagents in LC/ESI-MS/MS analysis.
Benzotriazole UV stabilizers are emerging compounds used in personal care products and can enter ... more Benzotriazole UV stabilizers are emerging compounds used in personal care products and can enter surface water after passing through wastewater treatment plants without being removed. Because these analytes are strongly hydrophobic, there is an environmental risk of accumulation in solid matrices and magnification through the trophic chain. In this work, a method based on stir bar sorption extraction with liquid desorption is presented for the extraction of benzotriazole UV stabilizers from water samples. Stir bar sorptive extraction was combined with ultra-high performance LC with MS/MS detection. All important factors affecting the stir bar sorptive extraction procedure are discussed, and the optimized method was applied to seawater and wastewater samples from Gran Canaria Island, providing good selectivity and sensitivity with LODs and limits of quantification in the range of 18.4-55.1 and 61.5-184 ng/L, respectively. Recoveries between 68.4-92.2% were achieved for the more polar compounds, whereas the recoveries were lower for the two less polar compounds, most likely due to their strong absorption into the polydimethylsiloxane stir bar phase that does not allows the complete desorption. The repeatability studies gave RSDs of between 6.45 and 12.6% for all compounds in the real samples.
Publisher Summary This chapter describes an attempt to separate a proper peptide fragment by mean... more Publisher Summary This chapter describes an attempt to separate a proper peptide fragment by means of chemical reactivity of the residual substituent of amino acids with newly synthesized fluorogenic reagents. The chapter describes a sensitive amino-acid-analyzer-employed chemiluminescent technology and an efficient method for peptide analysis with two-dimentional phase separation. It also discusses a simple method for purification of tryptophan- or methionine-containing peptides. In a study described in the chapter, 2-carboxy-1-hydroxy-4-naphthylmethyl-dimethylsulfonium chloride, which is a fluorogenic Koshland-type reagent (FKR) with chelating ability, was synthesized. Lysozyme and carboxymethylated lysozyme were used as model proteins. The experiments presented in the chapter reveal the selective isolation of half-cystine containing peptides by means of the specific reactivity and fluorogenic properties of 4-(aminosulfonyl)-7-fluoro-2,l,3-benzoxadiazole(ABD-F). The reagent may be useful as one of the preferable differentiation reagents in thiol groups in a protein structure.
A method for the determination of S-D-lactoylglutathione (SLG), an intermediate metabolite of the... more A method for the determination of S-D-lactoylglutathione (SLG), an intermediate metabolite of the glyoxalase system, in rat blood is described. After hemolysis and deproteinization of 30 l of rat blood, SLG in the blood was determined by a column-switching HPLC system with precolumn Xuorescence derivatization with a Xuorogenic reagent, 4-Xuoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). Calibration curves for the determination of SLG showed a good linearity (r 2 1 0.999) over the range of 20-100 pmol SLG spiked in rat blood samples. The accuracy was in the range of 97-104% (20-100 pmol SLG spiked in rat blood sample, N D 4). The detection limit was 8.12 fmol, and the quantitation limit was 27.07 fmol, respectively. The intra-and interday coeYcients of variance were 4.63% (N D 5) and 9.98% (N D 5), respectively. The concentrations of SLG in whole blood from male Wistar-Kyoto rats (12 weeks old) were 3.48 § 0.78 M (N D 4, mean § SE). In streptozotocin-induced diabetic rats, the concentration of SLG was signiWcantly increased, approx 5-fold, compared with normal rats, suggesting that the metabolic Xux of the glyoxalase system increases in red blood cells during hyperglycemia.
Abstract A facile and sensitive method for the determination of the fluorescent drugs dipyridamol... more Abstract A facile and sensitive method for the determination of the fluorescent drugs dipyridamole (coronary vasodilator) and benzydamine hydrochloride (anti-inflammatory) in rat plasma is presented. The method consists of the addition of an internal standard (DNS-...
It has been a desire to develop orally effective therapeutic agents that restore the liver functi... more It has been a desire to develop orally effective therapeutic agents that restore the liver function in chronic injury. Here we demonstrated that trans-4-L-hydroxyprolyl-L-serine (JBP923) and cyclo-trans-4-L-hydroxyprolyl-L-serine (JBP485), which was previously isolated from hydrolysate of human placenta, exhibit potent antihepatitis activity after their oral administration. The increase in bilirubin concentration and activities of liver cytosolic enzymes in serum caused by alpha-naphthylisothiocyanate intoxication in rats were significantly countered both after i.v. and oral administration of these dipeptides, whereas glycyrrhizin, which has been used in the treatment of chronic hepatitis, is active only after its i.v. administration. Antihepatitis activity of dipeptides results, at least partially, from their direct effect on hepatocytes because glutamic-oxaloacetic transaminase and lactate dehydrogenase activities in the medium of hepatotoxin-exposed primary cultured hepatocytes were reduced by these compounds. When comparing the plasma concentration-time profile of JBP923 after its i.v., oral, and portal vein injection, it is suggested that JBP923 is almost completely absorbed from gastrointestinal lumen, and hepatic first-pass removal is minor. JBP923 inhibited the proton-dependent transport of glycylsarcosine in brush-border membrane vesicles, suggesting that peptide transport system(s) may recognize JBP923. Thus, these dipeptides are potent antihepatitis reagents that are still active after oral administration and may be useful for clinical applications.
Archives of Biochemistry and Biophysics, Aug 1, 2002
In a previous report (FEBS Lett. 434 (1998) 231), we demonstrated for the first time that D-aspar... more In a previous report (FEBS Lett. 434 (1998) 231), we demonstrated for the first time that D-aspartate (D-Asp) is synthesized in rat pheochromocytoma 12 (PC12) cells. This unique amino acid is believed to act as a novel messenger in mammalian cell regulation. However, the dynamics of D-Asp homeostasis in mammalian cells is yet to be elucidated. In this communication, we demonstrate that D-Asp is also synthesized in MPT1 cells (a subclone of PC12 cells) and that the D-and L-Asp levels in cells are regulated by cell density of the culture. Our data show that D-Asp levels increase, while in contrast, L-Asp levels decrease as a function of increased cell density. Conversely, in PC12 cells, which do not express the glutamate transporter involved in the incorporation of D-and L-Asp into cells, L-Asp levels decrease upon cell density increase while D-Asp concentrations remain almost unchanged. The results indicate that the biochemical behaviors of D-and L-Asp in mammalian cells are distinct and that the cellular levels of these stereoisomers appear to be under different control mechanisms.
Six oxalate esters of 2-nitro-4-alkoxycarbonylphenol and 2-alkoxycarbonyl-4-nitrophenol were synt... more Six oxalate esters of 2-nitro-4-alkoxycarbonylphenol and 2-alkoxycarbonyl-4-nitrophenol were synthesised for use in the peroxyoxalate chemiluminescence reactions that take place via several steps with 4-hydroxy-3-nitrobenzoic acid and 5-nitrosalicylic acid as starting ...
Progress in Neuro-psychopharmacology & Biological Psychiatry, Mar 1, 2004
Several lines of evidence suggest that d-serine may function as an endogenous agonist of the glyc... more 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 Alzheimer's 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 = À 1.77, p = 0.078) lower than those of normal controls. In contrast, serum levels of L-serine in the patients were slightly (z = À 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 = À 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.
Dopamine (DA), norepinephrine (NE) and 3,4-dihydroxybenzylamine (DHBA) are converted to highly fl... more Dopamine (DA), norepinephrine (NE) and 3,4-dihydroxybenzylamine (DHBA) are converted to highly fluorescent cyanobenz[f]isoindole (CBI) derivatives by reacting these amines with naphthalene-2,3-dicarboxaldehyde in the presence of cyanide ion. Femtomole amounts of these CBI derivatives separated by reverse-phase high performance liquid chromatography can be detected by a post-column chemiluminescence system utilizing bis(2,4-dinitrophenyI) oxalate and hydrogen peroxide. Linear detection response was observed between 1 to 600 fmol, giving a signal to noise ratio of approximately 15 at the 1 fmol level. An assay procedure was developed for the determination of DA and NE in 20 p L of urine sample using DHBA as the internal standard.
Effects of the Substituent Groups at the 4-and 7-Positions on the Fluorescence Characteristics of... more Effects of the Substituent Groups at the 4-and 7-Positions on the Fluorescence Characteristics of Benzofurazan Compounds.-Study of the fluorescence intensity, maximum excitation wavelength and maximum emission wavelength of seventy title compounds, e.g. (I), reveals a relationship between the substituents at position 4 and 7 of the benzofurazan skeleton and the fluorescence characteristics. Hammett substituent constants are adopted as parameters for the electronic effects of the substituent groups.
A sensitive determination method for a non‐fluorescent anti‐arrhythmic drug, mexiletine, in rat p... more A sensitive determination method for a non‐fluorescent anti‐arrhythmic drug, mexiletine, in rat plasma is presented utilizing a HPLC peroxyoxalate chemiluminescence (PO‐CL) detection system. After an internal standard (4‐methylmexiletine, 4.35 pmol) and 0.1 N sodium hydroxide solution were added to 5 μL rat plasma, the solution was poured onto an Extrelut 1 column. Both mexiletine and the internal standard were eluted with diethy ether and then the eluate was evaporated to dryness. The residue was dissolved in 0.2 M borate buffer (pH 8.5) and mixed with dansyl chloride (75 nmol) in acetronitrile. After standing of 90 min at room temperature, 0.5 N HCI was added to the reaction mixture to stop the reaction and a 2/45 aliquot of the mixture was subjected to a HPLC PO‐CL detection system using bis(4‐nitro‐2(3,6,9‐trioxadecyloxycarbonyl)phenyl)oxalate (TDPO) and hydrogen peroxide. The calibration curve for mexiletine in rat plasma was linear over the range 20–100 ng/mL plasma (20.6–103 fmol/injection). The detection limit (S/N = 2) was 1.0 fmol over the whole procedure. The method was applied to the measurement of the time courses of plasma mexiletine concentration after oral administration of the drug [25 mg (115.9 μmol)/kg] to rats.
This article elucidates the relationships between the chemical structure and the fluorescence cha... more This article elucidates the relationships between the chemical structure and the fluorescence characteristics (fluorescence intensity, maximum excitation wavelength and maximum emission wavelength) of 4,7-disubstituted benzofurazan compounds with computer calculation. Our previous study using Hammett substituent constants of the substituent groups at the 4- and the 7-positions suggested that the total of electron densities on the benzofurazan skeleton and the dipole moment directed from the 4- to the 7-position might affect the fluorescence characteristics of these compounds. The sum of atomic charges on the benzofurazan skeleton, the HOMO energy and the LUMO energy were selected as parameters to reflect the total of electron densities on the benzofurazan skeleton and were obtained with the AM1 and PM3 calculations. The dipole moment directed from the 4- to the 7-position was also calculated with two hamiltonians. The LUMO energy and the dipole moment from the 4- to the 7-position obtained with the PM3 calculation is most closely related with the fluorescence characteristics. Using these parameters, the fluorescent 4,7-disubstituted benzofurazan compounds were classified into two groups, and the maximum excitation and emission wavelengths were different between these two groups. These relationships indicated that the fluorescence characteristics of 4,7-disubstituted benzofurazan compounds were determined by the total of electron densities on the benzofurazan skeleton and the dipole moment directed from the 4- to the 7-position. Furthermore, we predicted the fluorescence characteristics of four 4,7-disubstituted benzofurazan compounds based on the relationships obtained, and confirmed that the prediction agreed with the measured data.
The effects of various factors (organic solvent, type of reservoir, temperature, water content an... more The effects of various factors (organic solvent, type of reservoir, temperature, water content and concentration of hydrogen peroxide) were investigated on the stability of a chemilumigenic reagent, bis(2,4,6-trichlorophenyl) oxalate (TCPO), for high-performance liquid chromatography. In a mixture with hydrogen peroxide, TCPO was most stable when dissolved in acetonitrile and kept in a polyethylene bottle or a high quality glass (borosilicate glass free from metals) flask. The stability of TCPO was also increased by lowering the temperature, the water content and the concentration of hydrogen peroxide in the chemilumigenic reagent solution. Under these conditions, TCPO can be used in a mixed acetonitrile solution with hydrogen peroxide for HPLC-chemiluminescence detection.
Benzofurazan derivatization reagents, 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminopenty... more Benzofurazan derivatization reagents, 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminopentylamino)-2,1,3-benzoxadiazole (DAABD-AP) and 4-[2-(N,N-dimethylamino) ethylaminosulfonyl]-7-(2-aminobutylamino)-2,1,3-benzoxadiazole (DAABD-AB), for short-chain carboxylic acids in liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) were synthesized. These reagents reacted with short chain carboxylic acids in the presence of the condensation reagents at 60C for 60 min. The generated derivatives were separated on the reversed-phase column and detected by ESI-MS/MS with the detection limits of 0.1-0.12 pmol on column. Upon collision-induced dissociation, a single and intense product ion at m/z 151 was observed. These results indicated that DAABD-AP and DAABD-AB are suitable as the derivatization reagents in LC/ESI-MS/MS analysis.
Benzotriazole UV stabilizers are emerging compounds used in personal care products and can enter ... more Benzotriazole UV stabilizers are emerging compounds used in personal care products and can enter surface water after passing through wastewater treatment plants without being removed. Because these analytes are strongly hydrophobic, there is an environmental risk of accumulation in solid matrices and magnification through the trophic chain. In this work, a method based on stir bar sorption extraction with liquid desorption is presented for the extraction of benzotriazole UV stabilizers from water samples. Stir bar sorptive extraction was combined with ultra-high performance LC with MS/MS detection. All important factors affecting the stir bar sorptive extraction procedure are discussed, and the optimized method was applied to seawater and wastewater samples from Gran Canaria Island, providing good selectivity and sensitivity with LODs and limits of quantification in the range of 18.4-55.1 and 61.5-184 ng/L, respectively. Recoveries between 68.4-92.2% were achieved for the more polar compounds, whereas the recoveries were lower for the two less polar compounds, most likely due to their strong absorption into the polydimethylsiloxane stir bar phase that does not allows the complete desorption. The repeatability studies gave RSDs of between 6.45 and 12.6% for all compounds in the real samples.
Publisher Summary This chapter describes an attempt to separate a proper peptide fragment by mean... more Publisher Summary This chapter describes an attempt to separate a proper peptide fragment by means of chemical reactivity of the residual substituent of amino acids with newly synthesized fluorogenic reagents. The chapter describes a sensitive amino-acid-analyzer-employed chemiluminescent technology and an efficient method for peptide analysis with two-dimentional phase separation. It also discusses a simple method for purification of tryptophan- or methionine-containing peptides. In a study described in the chapter, 2-carboxy-1-hydroxy-4-naphthylmethyl-dimethylsulfonium chloride, which is a fluorogenic Koshland-type reagent (FKR) with chelating ability, was synthesized. Lysozyme and carboxymethylated lysozyme were used as model proteins. The experiments presented in the chapter reveal the selective isolation of half-cystine containing peptides by means of the specific reactivity and fluorogenic properties of 4-(aminosulfonyl)-7-fluoro-2,l,3-benzoxadiazole(ABD-F). The reagent may be useful as one of the preferable differentiation reagents in thiol groups in a protein structure.
A method for the determination of S-D-lactoylglutathione (SLG), an intermediate metabolite of the... more A method for the determination of S-D-lactoylglutathione (SLG), an intermediate metabolite of the glyoxalase system, in rat blood is described. After hemolysis and deproteinization of 30 l of rat blood, SLG in the blood was determined by a column-switching HPLC system with precolumn Xuorescence derivatization with a Xuorogenic reagent, 4-Xuoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). Calibration curves for the determination of SLG showed a good linearity (r 2 1 0.999) over the range of 20-100 pmol SLG spiked in rat blood samples. The accuracy was in the range of 97-104% (20-100 pmol SLG spiked in rat blood sample, N D 4). The detection limit was 8.12 fmol, and the quantitation limit was 27.07 fmol, respectively. The intra-and interday coeYcients of variance were 4.63% (N D 5) and 9.98% (N D 5), respectively. The concentrations of SLG in whole blood from male Wistar-Kyoto rats (12 weeks old) were 3.48 § 0.78 M (N D 4, mean § SE). In streptozotocin-induced diabetic rats, the concentration of SLG was signiWcantly increased, approx 5-fold, compared with normal rats, suggesting that the metabolic Xux of the glyoxalase system increases in red blood cells during hyperglycemia.
Abstract A facile and sensitive method for the determination of the fluorescent drugs dipyridamol... more Abstract A facile and sensitive method for the determination of the fluorescent drugs dipyridamole (coronary vasodilator) and benzydamine hydrochloride (anti-inflammatory) in rat plasma is presented. The method consists of the addition of an internal standard (DNS-...
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Papers by Kazuhiro Imai