Tatsuo Shinozuka

Expression of paraoxonase isoform did not confer protection from acute sarin poisoning in the Tokyo subway terrorist attack

Abstract We previously reported the polymorphism of the high density lipoprotein-associated enzyme paraoxonase (PON1), in the 10 sarin poisoning victims in the Tokyo subway terrorist attack. Arg192 PON1, which has low sarin hydrolysing activity, was found to be more common in the Japanese population than in people of other races. However, from our analyses seven of the victims expressed the PON1 phenotype with high sarin hydrolysing activity and three with low sarin hydrolysing activity. These results indicate that the main factor contributing to the tragedy of the Tokyo subway terrorist attack was the high toxicity of sarin rather than the race-dependent genetic difference in the Arg192 PON1 polymorphism.

Preferential formation of the hydroperoxide of linoleic acid in choline glycerophospholipids in human erythrocytes membrane during peroxidation with an azo initiator.

The formation of phospholipid hydroperoxides was monitored in human red blood cell (RBC) membranes that had been peroxidized with an azo initiator. Peroxidation of RBC membranes caused a profound decrease in the amount of polyunsaturated fatty acids and concomitantly hydroperoxides, as primary products of peroxidation, appeared in the phospholipids. Hydroperoxides were predominantly generated in choline glycerophospholipid (CGP), while the extent of formation of ethanolamine glycerophospholipid (EGP) hydroperoxides was low and their presence was transient. Hydroxy and hydroperoxy moieties in CGP were identified as 9-hydroxy and 13-hydroxy octadecanoic acid, derived from linoleic acid, by gas chromatography-mass spectrometric analysis. No consistent generation of hydroperoxide from arachidonic acid was evident in CGP. The CGP-hydroperoxide accounted for approximately 76% of linoleic acid consumed during peroxidation of RBC membranes. The prominent generation of phospholipid hydroperoxides was observed in the linoleic acid-rich membranes from rabbit RBC, indicating that the level of linoleic acid in phospholipids determines, in part, the extent of formation of phospholipid hydroperoxides. Aldehydic phospholipids, as secondary products of peroxidation, were detected in oxidized membranes. EGP was the most prominent aldehydic phospholipid, while negligible amounts of aldehydic CGP were formed. This study indicates that the process of oxidation of individual phospholipids clearly differs among phospholipids and depends on the structure of each.

Binding of lectins to "young" and "old" human erythrocytes.

Summary“Old” human erythrocytes showed a 21.2% decrease in cell surface area and a 2% decrease in the number of WGA receptor sites, but a 27% increase in the distribution density of the WGA (lectin) receptor site, when compared with “young” human erythrocytes. For a list of lectin abbreviations, see Materials and methods). Both “young” and “old” erythrocytes exhibited very weak binding activity for 125I-labeled PNA, but there was no difference in binding activity for PNA between “young” erythrocytes and “old” ones. Compared with “young” erythrocytes, decreases in the number and distribution density of receptor sites for five lectins including LPA, Con A, RCA-II, SBA and BPA on the cell surface were observed in aged erythrocytes. “Old” erythrocytes also showed a decrease in the number of PHA-E receptor sites, while the distribution density of the same receptor site remained unchanged. In view of these and other observations, it is thought that human erythrocyte aging is accompanied by elimination of some glycoconjugates which have affinity for six lectins, LPA, Con A, RCA-II, PHA-E, SBA and BPA, whereas no WGA receptor-containing glycoconjugates are released from erythrocyte membranes. Elimination of the glycoconjugates results in shrinkage of erythrocytes to reduce their cell surface areas.

Comparative study on the main membrane-surface sialoglycopeptides released from young and old human erythrocytes with trypsin

1. The amount of sialoglycopeptide (SGP) mixture released from young (Y) erythrocytes by trypsin treatment was significantly larger than that from old (O) ones. 2. The two main membrane-surface sialoglycopeptides (Y-SGP and O-SGP) were isolated in yields of about 45% and 42% from the mixtures by a combination of Sephadex G-50 superfine gel filtration with DEAE-cellulose chromatography, respectively. Molecular weights of 12,000 and 12,100, respectively, were found for the Y-SGP and the O-SGP. 3. The Y-SGP showed a greater proportion of sialic acid than the O-SGP. The contents of galactose, N-acetylglucosamine and N-acetylgalactosamine of the Y-SGP were significantly lower than those of the O-SGP. 4. Both the SGPs exhibited the same reactivities against the MN reagents, but the reactivities against the lectins of Limulus polyphemus and Phaseolus vulgaris (E) were stronger in the Y-SGP than in the O-SGP. Neither the Y-SGP nor the O-SGP showed the reactivity to Arachis hypogaea (anti-T) lectin.

A case of homicidal choking mistaken for suicide.

This report presents an autopsy case of the homicidal choking of an adult, who died as a result of tissue paper being thrust into his mouth. An in-patient (a 29-year-old Japanese man) at a mental hospital was choked to death by another male in-patient, his roommate, who thrust a large amount of tissue paper into his mouth after rendering him slightly unconscious by cervical compression. At the time of discovery, this mental patient was believed to have committed suicide by inserting tissue paper into his own mouth. Autopsy findings and re-investigation of the case revealed that the subject had been murdered. This is a very rare case of choking used as a method of homicide.

Coronary Aneurysms in a Young Adult: Report of a Case Suspected of Kawasaki Disease

An autopsy case of coronary aneurysm was reported. The patient, a 23-year-old Japanese man, suddenly fell unconscious during a karate match and died an hour later. The autopsy revealed coronary aneurysms, one of which was accompanied by thrombosis. Histologically, the aneurysmal walls showed marked scar formation and intimal thickening and the state was regarded as healed arteritis. By taking a precise history from his parents. Kawasaki disease was thought to be the probable cause of the coronary aneurysms.

Sensitive liquid chromatography/tandem mass spectrometry method for the simultaneous determination of nine local anesthetic drugs.

A high-performance liquid chromatography-tandem mass spectrometry (LC/MS/MS) with electrospray ionization (ESI) procedure for the simultaneous determination of nine local anesthetic drugs (procaine, mepivacaine, lidocaine, ropivacaine, oxybuprocaine, tetracaine, bupivacaine, T-caine and dibucaine) in human serum is described. The chromatographic separation was performed on a Mightysil-RP-18 GP II column (2.0mm×150mm, particle size 5μm). The mobile phase consisted of 10mM acetic ammonium buffer (pH 5.4) and acetonitrile and was delivered at a flow rate of 0.20mL/min. The triple quadrupole mass spectrometer was operated in positive ion mode, and multiple reaction monitoring was used for drug quantification. Solid-phase extraction of the nine local anesthetic drugs added to the human serum was performed with an Oasis(®) HLB extraction cartridges column. The method was linear for the investigated drugs over the concentration range of 10-100ng/mL. The recoveries of these drugs were in the range of 81.4-144%. The standard deviation (SD) values for all analytes were <0.10 for both intraday and interday accuracy and precision. The selectivity, accuracy and precision of this method are satisfactory for clinical and forensic applications. The sensitive and selective method offers the opportunity for the simultaneous screening and quantification, for clinical and forensic purposes, of almost all local anesthetics available in Japan.

Changes in En(a-) human red blood cell membranes during in vivo ageing

The human red blood cells with phenotype En(a-) were characterized by the lack of MN antigens. The red blood cells with phenotype En(a-) which were found in a Japanese family were tested to clarify the changes in membrane surfaces of the red blood cells during in vivo ageing. The contents of sialic acid, glucose, mannose, galactose, fucose, N-acetylglucosamine and N-acetylgalactosamine of the red blood cell membranes obtained from the old red blood cells with phenotype En(a-) were significantly lower than those of the young red blood cell membranes. Neither the young nor the old red blood cells with phenotype En(a-) showed the agglutination with Arachis hypogaea (PNA) which was capable of binding to T agglutinogen. It is presumed that En(a-) red blood cells are not exposed to sialidase in vivo. In comparison with the young En(a-) red blood cell membranes, the number and the distribution density of lectin receptor sites on the old ones for Limulus polyphemus (LPA), Canavalia ensiformis (Con A), Triticum vulgaris (WGA) and Bauhinia purpurea (BPA) were significantly lower. It is thought that En(a-) red blood cell ageing is accompanied by elimination of some sialoglycoconjugates which have affinity for LPA, Con A, WGA and BPA, whereas En(a-) red blood cells lack glycophorin A.

Rapid and simple analysis of oxazolobenzodiazepine drugs in sera by wide-bore capillary gas chromatography with nitrogen-phosphorus detection using on-column methylation

A rapid and simple method for determination of oxazolobenzodiazepines (oxazolam, haloxazolam, mexazolam, cloxazolam, and flutazolam) in sera was developed by gas chromatography (GC) using nitrogen-phosphorus detection (NPD) and a methyl silicone fused-silica wide-bore capillary (DB-1) column. Underivatized oxazolobenzodiazepine drugs gave relatively low sensitivity, because of decomposition during their passage through the column. On the other hand, on-column methylation of the oxazolobenzodiazepines with 0.4 mM trimethylanilium hydroxide in methanolic solution resulted in higher sensitivity; detection was possible at levels at least two to eight times lower than those without derivatization. The average recoveries of five oxazolobenzodiazepines from 0.5-ml volumes of sera containing 1.0 or 0.1 μg of each drug were 88–101% and 66–106%, respectively. The limits of detection of methylated oxazolobenzodiazepines by wide-bore capillary GC-NPD were 50–100pg on column. This method could be used for determination of the drugs in actual serum specimens with concentrations as low as 25–70ng/ml.

Sensitive Liquid Chromatography/Tandem Mass Spectrometry Method for the Simultaneous Determination of Risperidone, Olanzapine, Quetiapine, Clozapine, Ziprasidone, Perospirone, Aripiprazole and Blonanserin in Human Serum

The sensitive method for the simultaneous determination of eight atypical antipsychotic drugs (risperidone, olanzapine, quetiapine, clozapine, ziprasidone, perospirone, aripiprazole and blonanserin) in human serum has been developed based on a high-performance liquid chromatography-tandem mass spectrometry (LC/MS/MS) with electrospray ionization (ESI). The chromatographic separation was performed on a Mightysil-RP-18 MS column (2.0 mm × 150 mm, particle size 3 μm). The mobile phase consisted of 10 mM formic ammonium buffer (pH 6.0) and acetonitrile and was delivered at a flow rate of 0.20 mL/min. The triple quadrupole mass spectrometer was operated in positive ion mode, and multiple reaction monitoring was used for drug quantification. Solid-phase extraction of the eight antipsychotic drugs added to the human serum was performed with an Oasis®HLB extraction cartridges column. The method was linear for the investigated drugs over the concentration range of 20 - 100 ng/mL. The recoveries of these drugs were in the range of 81.3% - 140%. The intraday and inter day standard deviation (SD) values for all analytes were <0.10. Therefore, the selectivity, accuracy and precision of this method are sufficient for clinical and forensic studies. This simultaneous screening method will be also applicable to analyze other atypical antipsychotic drugs.