Kunihiko Kurosaki

Determination of dextromethorphan in human plasma using pipette tip solid-phase extraction and gas chromatography-mass spectrometry.

Dextromethorphan was extracted from human plasma samples (100xa0μL) using MonoTip C18 tips, which are packed with C18-bonded monolithic silica gel that is attached to the inside of the tip. The samples, which contained dextromethorphan and trimeprazine as an internal standard (IS), were mixed with 200xa0μL of distilled water and 50xa0μL of 1xa0mol/L glycine–sodium hydroxide buffer (pHxa010). The mixture was extracted to the C18 phase of the tip by 20 sequential aspirating/dispensing cycles using a manual micropipettor. The analytes retained on the C18 phase were then eluted with methanol by five sequential aspirating/dispensing cycles. The eluate was injected directly into a gas chromatograph and detected by a mass spectrometer with selected ion monitoring in positive electron ionization mode. An Equity-5 fused silica capillary column (30xa0mu2009×u20090.32xa0mm i.d., film thickness 0.5xa0μm) gave adequate separation of the dextromethorphan, IS, and impurities. The recoveries of dextromethorphan and the IS spiked into plasma were >87.4%. The regression equation for dextromethorphan showed excellent linearity from 2.5 to 320xa0ng/mL of plasma, and the limit of detection was 1.25xa0ng/mL of plasma. The intraday and interday coefficients of variation were less than 10.5% and 14.7%, respectively. The accuracy ranged from 91.9% to 107%. The validated method was successfully used to quantify the plasma concentration of dextromethorphan in a human subject after oral administration of the drug.

Genetic Study of the Paleolithic and Neolithic Southeast Asians

AbstractDNA samples were extracted from six prehistoric human remains, found on the Malay Peninsula, dating to the Paleolithic and the Neolithic periods. Nucleotide sequences of mitochondrial DNA were determined by the polymerase chain reaction-direct sequencing method. A phylogenetic tree between prehistoric and present humans was constructed based on the nucleotide sequence data. Mitochondrial DNA phylogenetic relationships and ethnoarchaeological evidence suggest that there is a continuity beetween the pre-Neolithic humans and the present Semang and that the Neolithic humans in this area might be an ancestral group of the Senoi.

Overexpression and gene amplification of EGFR, HER2, and HER3 in biliary tract carcinomas, and the possibility for therapy with the HER2-targeting antibody pertuzumab

BackgroundPertuzumab is a humanized monoclonal antibody that binds to HER2 at an epitope that prevents HER2 from dimerizing with ligand-activated HER-family receptors. To assess the potential of pertuzumab as a new therapy, the expression status of HER family members was determined in biliary tract carcinoma (BTC), and the antitumor activity of pertuzumab was investigated by assessing the inhibition of BTC cell growth.MethodsThe expression status of HER family members in 113 archival specimens of BTC was analyzed by using immunohistochemistry and fluorescence in situ hybridization. Using ten BTC cell lines, heregulin-alpha (HRG-α) stimulated cell proliferation and its inhibition by pertuzumab was tested in vitro. The phosphorylated HER family proteins and their respective downstream molecules were analyzed. In vivo antitumor activity of pertuzumab was evaluated in a xenograft model.ResultsProtein overexpression of HER2 and/or HER3 was observed in 23–34xa0% of the specimens and gene amplification in 17–27xa0%. Seven of the ten cell lines showed HER2 and/or HER3 protein overexpression and gene amplification, and HRG-α stimulated cell proliferation was observed in four of the ten cell lines. In a BTC cell line co-overexpressing HER2 and HER3, pertuzumab potently inhibited the HRG-α stimulated cell proliferation in a dose-dependent manner, and completely blocked the phosphorylation of HER3. Suppression of downstream pathway molecules including p-AKT was also observed. Pertuzumab inhibited the in vivo growth of subcutaneous tumors, and increased the number of apoptotic cancer cells.ConclusionsPertuzumab exerts potent antitumor activity in BTC cells co-overexpressing HER2 and HER3. Pertuzumab provides a new therapeutic option against BTC.

Involvement of extracellular ascorbate and iron in hydroxyl radical generation in rat striatum in carbon monoxide poisoning.

Carbon monoxide (CO) poisoning stimulated generation in rat striatum of toxic hydroxyl radicals (*OH), which might participate in the CO-induced neuronal injury. Since an increase in extracellular ascorbate (AA) stimulated *OH generation in the presence of endogenous metals, including iron, in rat striatum in vivo, we examined the role of extracellular AA in *OH generation due to CO poisoning in the present study. The CO-induced *OH generation in the striatum was strongly suppressed by intrastriatal administration of active, but not inactivated, AA oxidase, which degrades extracellular AA. In addition, CO poisoning caused a significant increase in extracellular AA in rat striatum, suggesting a role of extracellular AA in the CO-induced *OH generation. However, the time-course of changes in extracellular AA could not be completely superimposed on that of the CO-induced *OH generation. On the other hand, the CO-induced *OH generation was completely suppressed by an iron chelator, deferoxamine. These findings suggest that *OH generation in rat striatum due to CO poisoning may involve both extracellular AA and chelatable iron.

A new method for quantitative determination of dimemorfan in human plasma using monolithic silica solid-phase extraction tips

Dimemorfan was extracted from human plasma samples (100 μL) using MonoTip C(18) tips, which were packed with a C(18)-bonded monolithic silica gel attached to the inside of the tip. The samples, which contained dimemorfan and trimeprazine as an internal standard (IS), were mixed with 300 μL of distilled water and 50 μL of 1M glycine-sodium hydroxide buffer (pH 10). The mixture was extracted onto the C(18) phase of the tip by 20 sequential aspirating/dispensing cycles using a manual micropipettor. The analytes retained on the C(18) phase were then eluted with methanol by five sequential aspirating/dispensing cycles. The eluate was injected directly into a gas chromatograph and detected by a mass spectrometer with selected ion monitoring in positive electron ionization mode. An Equity-5 fused silica capillary column (30 m × 0.32 mm i.d., film thickness 0.25 μm) gave adequate separation of the dimemorfan, IS, and impurities. The recoveries of dimemorfan and the IS spiked into plasma were ≥83%. The regression equation for dimemorfan showed excellent linearity from 0.25 to 32.0 ng/100 μL of plasma, and the limit of detection was 0.125 ng/100 μL of plasma. The maximum intra-day and inter-day relative standard deviations were 13%, while accuracy ranged from 88% to 105%. Dimemorfan was stable for at least 12 h at 4°C, 4 weeks at -80°C, and three freeze-thaw cycles in plasma. This new method is expected to have application as a pretreatment for the rapid, simple, and quantitative determination of dimemorfan in plasma samples.

Existence of a threshold for hydroxyl radical generation independent of hypoxia in rat striatum during carbon monoxide poisoning.

We examined the role of hypoxia in the carbon monoxide (CO)-induced generation of the hydroxyl radical (•OH) in the striatum, which could contribute to brain damage due to CO poisoning. Exposure of free-moving rats to 1,000 and 3,000xa0ppm CO or 8 and 5% O2 for 40xa0min caused concentration-dependent hypoxic conditions in terms of carboxyhemoglobin (COHb), deoxyhemoglobin, oxyhemoglobin, and O2 contents in arterial blood. The hypoxic conditions seemed comparable between 3,000xa0ppm CO and 5% O2, although alterations of pH and partial O2 pressure (PO2) were complex and concentration independent. In the striatum, CO and low O2 decreased tissue PO2 levels in a concentration-dependent and concentration-independent manner, respectively, but levels at the end of exposure were comparable among all groups. This was also the case for the increase in striatal blood flow. Although the increases in extracellular glutamate (excitatory), taurine (inhibitory), and alanine (non-neurotransmitter), in the striatum in response to CO and low O2 were complex, 3,000xa0ppm CO and 5% O2 had comparable effects. Thus, 3,000xa0ppm CO and 5% O2 seemed to induce comparable hypoxic conditions. Nevertheless, the former more strongly stimulated •OH generation in the striatum than the latter. In addition, in contrast to low O2 which caused a concentration-dependent increase in •OH, 1,000xa0ppm CO had no effect. The findings suggest that striatal •OH generation due to CO poisoning may be independent of hypoxia per se and that a threshold might exist.

Sudden death caused by tension pneumothorax after rupture of a thoracic aortic aneurysm. Case report.

A rare case of fatal tension pneumothorax is reported. An aged Japanese man with marked subcutaneous emphysema of the neck was found collapsed in a betting office. He was ascertained to have left tension pneumothorax, based on radiographic examinations carried out before his death. At autopsy, severe pneumomediastinum was observed, and the descending thoracic aorta with a ruptured dissecting aneurysm was closely adhered to the left lung pleura. The hemorrhage spread into the pulmonary parenchyma and finally spouted out from the surface of the lung apex. Because the blood loss itself was not fatal in quantity, it is concluded that the patient died of tension pneumothorax caused by a lung penetration from the rupture of an aortic aneurysm.

Gene expression in rat striatum following carbon monoxide poisoning

Carbon monoxide (CO) poisoning causes brain damage, which is attenuated by treatment with hydrogen [1], [2], a scavenger selective to hydroxyl radical (•OH) [3]. This suggests a role of •OH in brain damage due to CO poisoning. Studies have shown strong enhancement of •OH production in rat striatum by severe CO poisoning with a blood carboxyhemoglobin (COHb) level > 70% due to 3000 ppm CO, but not less severe CO poisoning with a blood COHb level at approximately 50% due to 1000 ppm CO [4]. Interestingly, 5% O2 causes hypoxia comparable with that by 3000 ppm CO and produces much less •OH than 3000 ppm CO does [4]. In addition, cAMP production in parallel with •OH production [5] might contribute to •OH production [6]. It is likely that mechanisms other than hypoxia contribute to brain damage due to CO poisoning [7]. To search for the mechanisms, we examined the effects of 1000 ppm CO, 3000 ppm CO and 5% O2 on gene expression in rat striatum. All array data have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE94780.

Analysis of quazepam and its metabolites in human urine by gas chromatography-mass spectrometry: application to a forensic case.

A sensitive method for the simultaneous determination of quazepam and two of its metabolites, 2-oxoquazepam and 3-hydroxy-2-oxoquazepam, in human urine was developed using gas chromatography-mass spectrometry (GC/MS) with an Rtx-5MS capillary column. The quazepam and its metabolites were extracted from human urine using a simple solid-phase extraction Oasis(®) HLB cartridge column, and the 3-hydroxy-2-oxoquazepam was derivatised using BSTFA/1%TMCS and pyridine at 60 °C for 30 min. The mass spectrometric detection of the analytes was performed in the full scan mode, m/z 60-480, and selected ion monitoring (SIM) mode, m/z 386, for quazepam; m/z 342, for 2-oxoquazepam; m/z 429, for 3-hydroxy-2-oxoquazepam-TMS; and m/z 284, for alprazolam-d5 (internal standard), by electron ionization. The calibration curves of quazepam and its metabolites in urine showed good linearity in the concentration range of 2.5-500 ng/0.2 ml of urine. The average recoveries of quazepam and its metabolites from 0.2 ml of urine containing 500 ng and 50 ng of each drug were 71-83% and 88-90%, respectively. The limits of detection of quazepam, 2-oxoquazepam and 3-hydroxy-2-quazepam in urine by the selected ion monitoring mode were 0.096-0.37 ng/ml. This method would be applicable to other forensic biological materials containing low concentrations of quazepam and its metabolites.

Right ventricular free wall dissection as a rupture tract in left ventricular rupture during acute myocardial infarction

Three rare cases of cardiac rupture with right ventricular wall dissection during acute myocardial infarction (AMI) were reported. The cases comprised 2% among our 148 previously reported postinfarction cardiac ruptures with sudden death. The dissections occurred in hearts with biventricular inferior wall AMI and developed between the superficial layers and the deeper layers of inferior wall of the right ventricle. All had an endocardial tear at the basal septum where it meets the inferior free wall of the left ventricle, and had an epicardial tear on the middle inferior wall of the right ventricle. Based on the evidence of the ages of the thrombi of the rupture tracts, delayed epicardial rupture was found besides that soon after the right ventricular dissection.