Kiyohito Sato

Integrated pulsed amperometric detection of glufosinate, bialaphos and glyphosate at gold electrodes in anion-exchange chromatography.

A rapid and practical method for direct detection of the herbicides (glufosinate, bialaphos and glyphosate) in anion-exchange chromatography has been developed with integrated pulsed amperometric detection (IPAD). The electrochemical behavior of these herbicides showed catalytic currents based on the oxidation of amines in their structures. Waveform in IPAD was similar to that for amino acids, which exhibited adsorption/desorption catalytic features at gold electrode surface in alkaline solution. Under optimized conditions, detection limits (signal-to-noise ratio of 3) for glufosinate, bialaphos and glyphosate were 20, 65 and 50 ng ml(-1), respectively, with correlation coefficients of 0.995, 0.997 and 0.996 over concentration ranges of 0.1-45, 0.3-32 and 0.1-50 microg ml(-1), respectively. The relative standard deviations (n=5) were 1.7-3.0%. The present method was successfully applied to the determination of glyphosate in urine and serum.

Simultaneous determination of antidepressants by non-aqueous capillary electrophoresis-time of flight mass spectrometry.

Simultaneous determination of 20 antidepressants in plasma samples was carried out by non-aqueous capillary electrophoresis with time of flight mass spectrometry via electrospray ionization, where a mixture of 60 mM ammonium acetate and 1M acetic acid in acetonitrile, and water, as well as methanol (100:1:0.5, v/v/v) was selected as the background electrolyte. By using time of flight mass spectrometry, accurate mass information was obtained and the background noise was dramatically decreased, thus causing a great improvement in qualitative ability. As for the plasma sample, solid phase extraction with Oasis HLB was used. The limits of detection and quantification were in the range of 0.5-1 and 1-5 ng/ml, respectively. The sensitivity of the present method was found better, i.e. approximately 10-60 folds compared to that using photo diode array detectors because the analyte peak could be clearly distinguished from the background derived from the plasma. The present method was found very useful and practical as regards to routine analysis of plasma samples.

Nickel–titanium alloy electrodes for stable amperometric detection of underivatized amino acids in anion-exchange chromatography

Nickel-titanium (Ni-Ti) alloy electrode was used as an electrochemical detector for the analysis of underivatized amino acids in flow systems. In strong alkaline solution, an oxide film on the Ni-Ti alloy electrode surface exhibited a high catalytic activity toward the oxidation of amino acids. Cyclic voltammetry experiments confirmed that electrogenerated Ni(III)O(OH) functioned as the key redox mediator associated with the oxidation of the amine group in amino acids. The electrochemical behavior of the Ni-Ti electrode in alkaline medium was very similar to the Ni electrode. However, the oxide film was found to be much stable on Ni-Ti than on Ni. Consequently, the Ni-Ti alloy electrode exhibited an excellent stability for constant-potential amperometric detection of amino acids in flow systems. For example, the relative standard deviation (R.S.D.) for the repetitive 100 injections of 50 muM (1.2 nmol) glycine over 10 h was less than 1%. It was postulated that the presence of Ti in the alloy stabilizes the microstructure of oxide layer on the electrode surface. The sensitivities of amino acids at the electrode were different, depending on their chemical structures. The detection limits obtained in a range from 0.9 pmol for arginine to 90.2 pmol for leucine and isoleucine. The Ni-Ti alloy electrodes have been demonstrated to be very suitable for the amperometric detection of underivatized amino acids in anion-exchange chromatography.

Analysis of wool fiber by alkali-catalyzed pyrolysis gas chromatography

Alkali-catalyzed pyrolysis gas chromatography (PyGC) has been used to identify minute samples of wool fiber. The wool sample to which aqueous sodium hydroxide was added was pyrolyzed in a Curie-point pyrolyzer attached to a gas chromatograph or a gas chromatograph-mass spectrometer. The addition of an aqueous solution of sodium hydroxide increased the production of specific volatile pyrolysis products from the constitutive amino acid residues of wool protein, i.e. acetaldehyde from alanine or proline, isobutyronitrile from valine, 2-methylbutyronitrile from isoleucine, isovaleronitrile from leucine and toluene from phenylalanine. Compared with conventional non-catalyzed PyGC, the alkali-catalyzed PyGC was found to greatly improve the detection limit of wool fiber and make it possible to analyze very minute samples. The alkali-catalyzed PyGC presented here has been shown to be applicable to minute thermally-denatured samples of wool fiber which cannot be identified successfully by morphological inspection using a microscope or by using Fourier-transform infrared microspectroscopy. Furthermore, the present PyGC method was successfully used for several protein samples and was shown to be useful for analysis of proteins other than wool fibers by using different special pyrograms reflecting different amino acid compositions.

Sibutramine provokes apoptosis of aortic endothelial cells through altered production of reactive oxygen and nitrogen species.

Abstract Overdose administration of sibutramine, a serotonin‐noradrenalin reuptake inhibitor, is considered to elicit severe side effects including hypertension, whose pathogenic mechanism remains unclear. Here, we found that 48‐h incubation with >10 &mgr;M sibutramine provokes apoptosis of human aortic endothelial (HAE) cells. Treatment with the lethal concentration of sibutramine facilitated production of reactive oxygen species (ROS), altered expression of endoplasmic reticulum stress response genes (heat shock protein 70 and C/EBP homologous protein), and inactivated 26S proteasome‐based proteolysis. The treatment also decreased cellular level of nitric oxide (NO) through lowering of expression and activity of endothelial NO synthase. These results suggest that ROS production and depletion of NO are crucial events in the apoptotic mechanism and may be linked to the pathogenesis of vasoconstriction elicited by the drug. Compared to sibutramine, its metabolites (N‐desmethylsibutramine and N‐didesmethylsibutramine) were much less cytotoxic to HAE cells, which hardly metabolized sibutramine. In contrast, both the drug and metabolites showed low cytotoxicity to hepatic HepG2 cells with high metabolic potency and expression of cytochrome P450 (CYP) 3A4. The cytotoxicity of sibutramine to HepG2 and Chang Liver cells was remarkably augmented by inhibition and knockdown of CYP3A4. This study also suggests an inverse relationship between sibutramine cytotoxicity and CYP3A4‐mediated metabolism into the N‐desmethyl metabolites. Graphical abstract Figure. No Caption available. HighlightsTreatment with sibutramine, an anorexiant, induces endothelial cell apoptosis.The apoptotic mechanism includes induction of ROS and NO depletion.There is an inverse relationship between sibutramine cytotoxicity and its metabolism.

α-Pyrrolidinononanophenone provokes apoptosis of neuronal cells through alterations in antioxidant properties

In this study, we found that exposure to α-pyrrolidinononanophenone (α-PNP), a highly lipophilic synthetic cathinone, provokes apoptosis of human neuronal SK-N-SH cells. The drug sensitivity of the cells (50% lethal concentration of 12μM) was similar to those of aortic endothelial and smooth muscle cells, and was higher than those of cells derived from colon, liver, lung and kidney, suggesting that α-PNP overdose and abuse cause serious damage in central nervous and vascular systems. SK-N-SH cell treatment with lethal concentrations (20 and 50μM) of α-PNP facilitated the reactive oxygen species (ROS) production. The treatment also prompted elevation of Bax/Bcl-2 ratio, lowering of mitochondrial membrane potential, release of cytochrome-c into cytosol, and resultant activation of caspase-9 and caspase-3. The apoptotic events (caspase-3 activation and DNA fragmentation) were abolished by pretreatment with antioxidants, N-acetyl-l-cysteine and polyethyleneglycol-conjugated catalase. These results suggest that ROS production, mitochondrial dysfunction and caspase activation are potential events in the mechanism underlying the α-PNP-triggered neuronal cell apoptosis. Intriguingly, the α-PNP treatment of SK-N-SH cells was found to promote formation of 4-hydroxynonenal, a reactive aldehyde generated from lipid peroxidation. The α-PNP treatment also decreased cellular levels of total and reduced glutathiones, expression of γ-glutamylcysteine synthetase mRNA and glutathione reductase activity. Furthermore, the α-PNP treatment resulted in both decrease in proteasomal activities and increase in expression of autophagy-related factors, which were significantly prevented by pretreating with N-acetyl-l-cysteine. Therefore, the ROS formation by α-PNP treatment may be ascribable to the decrease in glutathione level through its consumption during 4-hydroxynonenal detoxification and dysfunction of both de novo synthesis and regeneration of glutathione, in addition to impairments in proteasomal and autophagic systems that degrade cellular oxidized components.

Enhancement of Endothelial Barrier Permeability by Mitragynine

Persistent inhalation of mitragynine (MG), a major alkaloid in the leaves of Mitragyna speciosa, causes various systemic adverse effects such as seizure, diarrhea and arthralgias, but its toxicity to endothelial cells and effects on barrier function of the cells are poorly understood. In this study, we compared toxicities of MG and mitraphylline, another constituent of the leaves, against human aortic endothelial (HAE), bronchial BEAS-2B, neuronal SK-N-SH, hepatic HepG2, kidney HEK293, gastric MKN45, colon DLD1, lung A549, breast MCF7 and prostate LNCaP cells, and found that MG, but not mitraphylline, shows higher toxicity to HAE cells compared to the other cells. Forty-eight-hours incubation of HAE cells with a high concentration of MG (60 µM) provoked apoptotic cell death, which was probably due to signaling through enhanced reactive oxygen species (ROS) generation and resultant caspase activation. Treatment of the cells with MG at sublethal concentrations less than 20 µM significantly lowered transendothelial electrical resistance and elevated paracellular permeability, without affecting the cell viability. In addition, the MG-elicited lowering of the resistance was abolished by a ROS inhibitor N-acetyl-L-cysteine and augmented by H2O2 and 9,10-phenanthrenequinone, which generates ROS through its redox cycle. These results suggest the contribution of ROS generation to the increase in endothelial barrier permeability.

Sibutramine facilitates apoptosis and contraction of aortic smooth muscle cells through elevating production of reactive oxygen species

ABSTRACT Sibutramine had been prescribed as an oral anorexiant that reduces dietary intake, but was withdrawn from the market due to frequent occurrence of severe cardiovascular events including hypertension. To elucidate the pathogenic mechanism of hypertension, we here investigated whether sibutramine facilitates damage and contraction of human aortic smooth muscle (HASM) cells or not. Treatment with sibutramine provoked HASM cell apoptosis, which was attributed to production of reactive oxygen species and mitochondria dysfunction. In addition, the drug treatment of the cell promoted calcium influx, phosphorylation of myosin light chain and contraction, which were abrogated by pretreating the cells with antioxidant and nitric oxide (NO) donor. Thus, the drug‐evoked contraction is likely due to a preceding disturbance of balance between the drug‐elicited reactive oxygen species production and exogenous NO supply. Compared to sibutramine, its N‐desmethyl and N‐didesmethyl metabolites exhibited much less toxicity and contraction against HASM cells, in which sibutramine was hardly demethylated. The low metabolic capacity of the cells may also be pertinent to the damage and contraction elicited by sibutramine. Taken together, our data suggest that sibutramine facilitates apoptosis and contraction of aortic smooth muscle cells through elevating production of reactive oxygen species and decreasing exogenous NO supply, leading to pathogenesis of hypertension. Graphical abstract Symbol. No caption available.

Rapid Analysis by Capillary Gas Chromatography of Thinners and the Related Organic Solvents.

The inhalation of thinners and the related organic solvents (e.g., toluene, methanol, ethyl acetate etc.) expected as an anesthetic and stimulus, is still a social problem in connection with juvenile delinquency. For the purpose of a rapid identification of the abused solvents, 38 pure organic solvents and 18 commercial thinner samples were analyzed by a flame ionization detector (FID)-gas chromatograph with a DBTM-1, SUPELCOWAXTM 10 or DBTM-17 capillary column, respectively. In the cases of the DBTM-1 and SUPELCOWAXTM 10 columns, most organic solvents were successfully separated from each other under the gas chromatographic conditions as follows, a 30 m×0.53 mm i.d. thick-film (1.5 μm and 1.0 μm) fused-silica capillary column was used, and the column temperature was kept at 60°C for 1 min and then raised to 120°C and 150°C at 5°C/min. The total analysis times were 13 and 19 min. Especially, the DBTM-1 column was suitable for the forensic analysis of thinners, because methanol, ethylacetate and toluene prohibited by the law were succesfully separated from others. The column was also appropriate to identify lacquer thinner and industrial toluene. This method is more efficient, and less time-consuming than the other methods so far reported.