Toshiko Okuyama

Determination of glyphosate and its metabolite, (aminomethyl)phosphonic acid, in serum using capillary electrophoresis

Capillary electrophoresis has been used to separate and quantitate glyphosate and its major metabolite, (aminomethyl)phosphonic acid (AMPA), in serum. The two compounds, after derivatization with p-toluenesulphonyl chloride, were clearly separated with 0.1 M boric acid-sodium hydroxide buffer (pH 9.6) containing 10% methanol. The separation was completed within 15 min at an applied potential of 30 kV. Calibration curves for the assay were linear over both the lower (0.5-10 micrograms/ml) and the higher (10-100 micrograms/ml) concentration ranges. The within-run and day-to-day coefficients of variation of peak area were 1.4-4.4 and 4.4-8.5%, respectively, for glyphosate and 1.8-2.9 and 1.8-2.9%, respectively, for AMPA. The within-run and day-to-day precisions of the migration time for both compounds were less than 1.8% and less than 2.5%, respectively. The detection limit of both derivatives was 0.1 microgram/ml in spiked sera, and the recoveries of glyphosate and AMPA were 87.9-88.8 and 78.4-86.9%, respectively. In this study, the reproducibility and the effect of pH changes on the electropherograms were especially examined.

Application of capillary electrophoresis to the simultaneous screening and quantitation of benzodiazepines

Capillary electrophoresis (CE) is an attractive approach for the analysis of drugs in body fluids. We made a simultaneous analysis of nitrazepam, diazepam, estazolam, bromazepam, triazolam and flurazepam using CE with on-column detection at 200 nm. We obtained the best electropherograms under a condition of 5 mM phosphate-borate (pH 8.5) containing 50 mM SDS and 15% methanol. We examined the effect of the sample solvent matrix on the electropherograms obtained, indicating that increasing the methanol content in the sample solvent or the injection volume above a certain threshold limit decreased the resolution. We then focused on application of the CE to the analysis of the drugs in spiked serum, being appropriate for an analysis within 25 min. Linearity, the detection limit, accuracy and reproducibility were established using this method. The calibration curve was linear up to 1 mg/l of serum concentration. The lower limit of detection was 5 pg per injection and 0.025 mg/l of the serum concentration for all the compounds except for flurazepam, for which they were 40 pg/injection and 0.2 mg/l. The detection limits obtained allowed toxicological and pharmacological determinations for nitrazepam, diazepam, estazolam and bromazepam, but not for triazolam and flurazepam. Only toxic blood levels for the latter two benzodiazepines could be quantified by this method. We concluded that the CE could at least be applicable to simultaneous screening for toxic levels of benzodiazepines. We suggest that this technique may offer criminal toxicologists a rapid, simple and adaptable approach for the estimation of many other drugs in body fluids.

Determination of malonaldehyde in oxidized biological materials by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method was used to determine the level of malonaldehyde (MA) in materials containing unsaturated fatty acids and rat liver microsomes peroxidized in vitro. The detection limit was 8.3 pmol for fatty acid samples and 25 pmol for microsomal samples. The method was specific to MA and the relative standard deviation was 4.34-5.14%. The recovery of MA was about 100%. In general, the MA values in oxidized materials obtained by the proposed HPLC method were lower than those obtained by the thiobarbituric acid method, although similar results were obtained with both methods for microsomal samples oxidized by NADPH. The effect of temperature on the HPLC results was investigated and it was found that the MA values obtained by derivatization at 25 degrees C, followed by separation using HPLC, reflected the situation of the peroxidation more accurately.

The role of nitric oxide in paraquat-induced cytotoxicity in the human A549 lung carcinoma cell line

Paraquat (PQ) is a well-known pneumotoxicant that exerts its toxic effect by elevating intracellular levels of superoxide. In addition, production of pro-inflammatory cytokines has possibly been linked to PQ-induced inflammatory processes through reactive oxygen species (ROSs) and nitric oxide (NO). However, the role of NO in PQ-induced cell injury has been controversial. To explore this problem, we examined the effect of NO on A549 cells by exposing them to the exogenous NO donor NOC18 or to cytokines; tumor necrosis factor-α, interleukin-1 β and interferon-γ, as well as PQ. Although the exogenous NO donor on its own had no effect on the release of lactate dehydrogenase (LDH), remarkable release was observed when the cells were exposed to high concentrations of NOC18 and PQ. This cellular damage caused by 1 mM NOC18 plus 0.2 mM PQ was ascertained by phase contrast microscopy. On the other hand, NO derived from 25–50 μM NOC18 added into the medium improved the MTT reduction activity of mitochondria, suggesting a beneficial effect of NO on the cells. Incubation of A549 cells with cytokines increased in inducible NO synthase (iNOS) expression and nitrite accumulation, resulting in LDH release. PQ further potentiated this release. The increase in nitrite levels could be completely prevented by NOS inhibitors, while the leakage of LDH was not attenuated by the inhibition of NO production with them. On the other hand, ROS scavenging enzymes, superoxide dismutase and catalase, inhibited the leakage of LDH, whereas they had no effect on the increase in the nitrite level. These results indicate that superoxide, not NO, played a key role in the cellular damage caused by PQ/cytokines. Our in vitro models demonstrate that NO has both beneficial and deleterious actions, depending on the concentrations produced and model system used.

Quantitation of the hydroxyl radical adducts of salicylic acid by micellar electrokinetic capillary chromatography: oxidizing species formed by a Fenton reaction.

There has been controversy concerning the products formed by a Fenton reaction. We determined the hydroxyl radical (•OH) generated in a Fenton reaction system with no iron chelator using micellar electrokinetic capillary chromatography (MECC). The hydroxyl radical generated in this Fenton system attacked salicylic acid to produce major products of 2,3- and 2,5-dihydroxybenzoic acid (DHB), 2,3-DHB being prominent. Hydroxyl radical scavengers, such as mannitol, ethanol, thiourea and a ferric chelator, Desferal, significantly diminished the peaks for DHBs, showing production of •OH. We compared the MECC method with the electron paramagnetic resonance (EPR) spin trapping technique. The quantity of DHBs obtained by MECC increased dose-dependently up to 1 μM Fe2+ at a fixed concentration of H2O2, whereas that of the spin adduct by EPR showed a bell-shaped curve. This quantitation of •OH adducts by MECC supports the proposal that the oxidizing species formed by a Fenton reaction with no chelator is •OH. The EPR spin trapping method appears to be erroneous, particularly when iron is present at a higher concentration than hydrogen peroxide. The application of this method to the paraquat effect in vitro is demonstrated, and the possibility for analysis of •OH in vivo is also discussed.

High-performance liquid chromatographic determination of glyphosate and (aminomethyl)phosphonic acid in human serum after conversion into p-toluenesulphonyl derivatives

We have developed a simple, highly sensitive and fast assay method for determining glyphosate and its major metabolite, (aminomethyl)phosphonic acid (AMPA), in serum by high-performance liquid chromatography with ultraviolet detection. Both compounds were successfully extracted with an anion-exchange resin column and allowed to react with p-toluenesulphonyl chloride. The detection limits were 0.3 microgram/ml for glyphosate and 0.2 microgram/ml for AMPA. Recoveries of glyphosate and AMPA spiked to serum were ca. 75% and ca. 88%, respectively. We are convinced that this procedure, in practice, allows medical examiners to analyse both compounds in the serum of poisoned patients within a short time.

Effect of paraquat on the malondialdehyde level in rat liver microsomes (in vitro).

Toxicosis due to paraquat, a redox cycling xenobiotic, is still a subject of much debate. In the present study on lipid peroxidation, paraquat had a biphasic effect on the malondialdehyde (MDA) level in rat liver microsomes; stimulation at the initial stage (within 10 min) and depression at the later stage. Although paraquat increased the initial rate of NADPH oxidation dose-dependently, the rate was not necessarily parallel with the increase in the MDA level. The MDA level increased linearly up to 0.1 mM paraquat added, but then it attained a plateau. The stimulation obtained by paraquat within 10 min was absolutely dependent on exogenous Fe2+ ion and NADPH, and the stimulation was entirely SOD sensitive, while the irondriven increase in MDA was 20% sensitive. Thus, there were different mechanisms between iron-driven lipid peroxidation and paraquat-modified peroxidation. Catalase increased the level, but mannnitol, a scavenger of OH, had no effect. EPR spectra showed that superoxide was formed dose-dependently up to 0.1 mM paraquat and that it attained a plateau at the same as MDA level described above. From these results, we concluded that paraquat stimulates lipid peroxidation through a mechanism dependent on the superoxide complex involving Fe2+ ion.

Free Malondialdehyde Levels in the Urine of Rats Intoxicated with Paraquat.

We examined the excretion of free malondialdehyde (MDA) in the urine of rats to which a herbicide, Gramoxone, had been orally administered. The herbicide was administered for 2 days at a dose of 60 mg paraquat/kg body weight/day. As a result, the concentration of free MDA decreased following the intake of Gramoxone. The total amount of free MDA increased temporarily, but then it decreased significantly to below normal values. Rats that died during this experimental period did not excrete any free MDA. In the surviving animals, the MDA concentration in serum and lung microsomes decreased, while that in liver microsomes increased slightly after intake of the poison. Although the cause of the decrease in the urinary free MDA level remains unclear, the marked changes may provide valuable information regarding a toxic mechanism of paraquat intake.

Changes in mRNAs of inducible nitric oxide synthase and interleukin-1β in the liver, kidney and lung tissues of rats acutely exposed to paraquat

Nitric oxide (NO) reacts with superoxide to form the potent oxidant peroxynitrite, which causes serious cell damage. Interleukin-1 beta (IL-1 beta) is known to be a strong activator of NO production via induction of inducible nitric oxide synthase (iNOS). Since paraquat (PQ) undergoes redox cycling in vivo, resulting in a constant generation of superoxide, peroxynitrite may be a pathogenetic factor in the oxidative cell damage. In this study, we have investigated whether mRNAs of iNOS and IL-1 beta are affected in rat liver, kidney and lung tissues by exposure to non-lethal and lethal doses of PQ. Suppression and then marked stimulation of the iNOS mRNA were observed in the liver tissues of rats exposed to a lethal dose of PQ, while the kidney and lung tissues showed little changes. We also detected nitrotyrosine in liver tissues of rats exposed to a lethal dose by immunohistochemistry, suggesting the simultaneous generation of NO and superoxide in liver injury during acute lethal PQ poisoning. On the other hand, the IL-1 beta mRNA in the liver tissues decreased throughout the experiments, suggesting that this cytokine is not responsible for stimulation of the iNOS gene. IL-1 beta mRNA in lung tissues in the non-lethal group showed an increase, with maximum levels at 16-24 h, while little changes were observed in iNOS mRNA in this organ. These data suggest that acute lethal poisoning and non-lethal poisoning by PQ undergo different mechanisms of action of NO and IL-1 beta systems; the former is due, at least in part, to an increase in NO production, while the latter is due to stimulation of IL-1 beta and/or other cytokines.

Simultaneous determination of nitrazepam and its metabolites in urine by micellar electrokinetic capillary chromatography

We applied micellar electrokinetic capillary chromatography to simultaneous separation and determination of nitrazepam and its major metabolites, 7-aminonitrazepam and 7-acetamidonitrazepam, in spiked urine. Prior to electrophoresis, the three compounds were successfully extracted from the spiked urine with commercial disposable solid-phase cartridges. The optimum running buffer for the separation was prepared by combining 85 parts of 60 mM sodium dodecyl sulphate-6 mM phosphate-borate, adjusted to pH 8.5, with 15 parts of methanol. The separation order, completed within 25 min, was 7-aminonitrazepam > 7-acetamidonitrazepam > nitrazepam, at an applied potential of 20 kV. We obtained reproducible electropherograms in successive repetitions, and few other peaks or interferences appeared in the electropherogram. The detection limits of the three compounds were 50-100 pg (0.1-0.2 microgram/ml of analyte in spiked urine), and the recoveries were 78.9-100.8% for 1 microgram/ml and 84.1-100.3% for 5 micrograms/ml. The application of this method to forensic or clinical samples is demonstrated.