Forensic analysis of eleven cyclic antidepressants in human biological samples using a new reversed-phase chromatographic column of 2 microm porous microspherical silica gel.

A high-performance liquid chromatographic method has been developed for the forensic analysis of eleven frequently used cyclic antidepressant drugs (ADSs) (amitriptyline, amoxapine, clomipramine, desipramine, dosulepine, doxepin, imipramine, maprotiline, melitracen, mianserine and nortriptyline) using a recently developed reversed-phase column with 2 microm particles for the analysis of biological samples. The separation was carried out using two different C8 reversed-phase columns (column 1: 100 mm X 4.6 mm I.D., particle size 2 microm, TSK gel Super-Octyl; column 2: 100 mm X 4.6 mm I.D., particle size 5 microm, Hypersil MOS-C8) for comparison. The mobile phase was composed of methanol-20 mM KH2PO4 (pH 7) (60:40, v/v) and the flow-rate was 0.6 ml/min for both columns. The absorbance of the eluent was monitored at 254 nm. When the eleven drugs were determined, the sensitivity with the 2 microm particles was about five times greater than with the 5 microm particles. Retention times on column 1 were shorter than those on column 2. These results show that the new ODS column packing with a particle size of 2 microm gives higher sensitivity and a shorter analysis time than the conventional ODS column packing when applied to the analysis of biological samples.

Sudden death due to diabetic coma in insulin-department diabetes mellitus: an autopsy report.

Sudden death caused by the acute onset of diabetic coma is reported. A 15-year-old female had been suffering from insulin-dependent diabetes mellitus for the prior 8 years and had a fever and vomiting for the past few days. On the 4th day, after the onset of fever and vomiting, she died suddenly, and was autopsied to clarify the cause of death. Macroscopic examination revealed that the pancreas was atrophic (40 g) whereas the liver was markedly enlarged (2,740 g). Histological findings were: 1) The islets of Langerhans were decreased in size and number. They were not positive for aldehyde-fuchsin staining, 2) There were severe fatty changes in the liver cells. The retained blood in the left ventricle was analyzed: glucose, 1,016 mg/dl; acetone, 345 mg/l; acetoacetate, 5.91 mmol/l: D-3-hydroxybutyrate, 4.17 mmol/l; hemoglobin A1c, 10.2%; fructosamine, 416 mumol/l; total serum cholesterol, 220 mg/dl; triglycerides, 205 mg/dl; free fatty acid, 8.0 mEq/l; urea nitrogen, 40 mg/dl. Although the biochemical estimation of the glucose and ketone levels in post-mortem body fluids was recognized as being unreliable, many of these values were far elevated in comparison with those of normal individuals. Thus, we concluded that the cause of death was diabetic ketoacidosis. We also discuss the diagnostic problems of postmortem blood chemistry.

[An autopsy case of pulmonary thromboembolism: a delayed complication of a victim by traffic accident].

A 35-year-old male had fallen down from his motor bike caused a fracture of the medial condyle of the right tibia and the acromion of the left scapula. He had received screw fixed operation for his right tibia and had been in the hospital for one month. He died suddenly in the hospital and was autopsied to find out the cause of death. On external examination, it was found that affected leg was bruised and swollened. Partially organized thrombosis in the posterior tibial vein was revealed and bilateral pulmonary arteries were packed with reddish massive thromboemboli. The causes of the posterior tibial vein thrombosis were intimal damage and decreased venous flow, attributable to bed rest for long days and external fixation of the lower extremity. We presumed that direct cause of death was a large thromboemboi which were migrated from posterior tibial vein and lodged at the bifurcation of the pulmonary artery causing obstruction of pulmonary blood flow. Finally we opened the direct cause of death was pulmonary thromboembolism due to leg injury. Clinical instability may hinder the diagnostic elevation and delay therapeutic intervention, it is the most often happened in sudden death cases within the hospital. The importance of early diagnosis and early prevention is underscoped, because the symptoms of pulmonary thromboembolism are not always produced. To prevent fatal pulmonary thromboembolism in trauma, prophylactic use of low-dose heparin therapy and/or the use of oral anticoaglant (warfarin) have been effective and recommended in USA and Western Europe. The routine use of these therapy has not yet been spread in Japan, because the incidence is still apparently lower than in USA and Western Europe as well as the risk of hemorrhage. If the victims of road traffic accident receive the prophylactic use of low-dose heparin and/or oral anticoaglant (warfarin), it might be an effective way to reduce mortality.

Forensic analysis of 10 barbiturates in human biological samples using a new reversed-phase chromatographic column packed with 2-micrometre porous microspherical silica-gel.

A high-performance liquid chromatographic method has been developed for the forensic analysis of 10 frequently used barbiturates (BARs) (allobarbital, amobarbital, barbital, cyclobarbital, hexobarbital, metharbital, pentobarbital, phenobarbital, secobarbital and thiopental) using a recently developed reversed-phase column packed with 2-micron particles. The results show that the new ODS column packing gives higher sensitivity and a shorter analysis time than the conventional ODS column packing when applied to the analysis of biological samples.

An electrocution death of an infant who had received an electric shock from an uncovered oval shaped lamp switch in his mouth while in a hospital.

A male infant aged one year and nine months was found dead on a bed after admission to hospital with suspected pneumonia. The patient apparently put an uncovered oval shaped lamp switch (pendant switch) into his mouth and died of electric shock after contacting the exposed wires of the switch (100 V, 60 Hz alternating current). There were extensive first- to fourth-degree burns on the inner surface of the both lips. Because the histological findings were consistent with electric burns and the burns showed vital reactions, electric shock was judged to be the cause of death. The pendant switch is normally a very convenient piece of bedside equipment for inpatients. However, when the patient is an infant who naturally puts all the objects into the mouth, such a switch should be placed out of reach, and it should be certain that the cap is not loose.

Rapid and clear detection of ABO genotypes by simultaneous PCR-RFLP method.

We reported a new approach of ABO genotyping by a polymerase chain reaction and restriction fragment length polymorphism method. Instead of amplifying the loci containing the positions of nucleotides 258 and 700 of cDNA of the A transferase separately, we successfully amplified these 2 loci together in one reaction mixture using 2 sets of primers. The amplified DNA products were digested at the same time with restriction enzymes Kpn I and Alu I. The digested DNA products were then separated by electrophoresis on polyacrylamide gel. In addition, we evaluated the influence of various amplification parameters (concentration of template DNA, primers, Taq DNA polymerase, MgCl2, and number of cycles). In particular, high Mg2+ concentration (3.5 mM) made effective amplification of this locus without producing any unspecific band. By using that optimized condition for PCR, together with a simultaneous approach, our study proved to be time saving, more economic, and convenient in interpreting the results.

Determination of ester-type local anesthetic drugs (procaine, tetracaine, and T-caine) in human serum by wide-bore capillary gas chromatography with nitrogen-phosphorus detection.

A sensitive method for simultaneous determination of ester-type local anesthetic drugs (procaine, tetracaine, and T-caine) has been developed using wide-bore capillary gas chromatography with nitrogen-phosphorus detection (GC-NPD). The extraction procedure, the experimental conditions for heptafluorobutyryl (HFB) derivative formation, and the percentage of the ester-type local anesthetic drugs from the human serum are described. The HFB derivatives of ester-type local anesthetic drugs showed sensitivity of approximately 2-3 fold higher than that without derivatization. The detection limits of HFB derivatives of the ester-type local anesthetic drugs were approximately 60-70 pg on column. Recoveries from the human serum were 85-94%. This method could be used to determine concentrations as low as 24-28 ng/mliters of the ester-type local anesthetic drugs.

Genetic polymorphism of the 3' VNTR region of the human dopaminergic function gene DAT1 (human dopamine transporter gene) in the Mongolian population.

The hypervariable region of the dopamine transporter gene (DAT1) was amplified from samples in the Mongolian population. This region includes a variable number of tandem repeats of a 40-bp core unit in the 3' untranslated region of DAT1. Vandenbergh et al. (1992) reported variability in the number of repeats of this 3' flanking region ranging from 3 to 11 times in white and black populations. We examined polymorphism at the DAT1 locus in 78 native Mongolian subjects. We found alleles with 7 to 13 repeats, which is different from the findings of Vandenbergh et al. (1992). The allele distribution of the Mongolian population is similar to that in the Japanese population, reported previously by Nakatome et al. (1995). Chi-square analysis showed a significant lack of homogeneity between our findings in Mongolian subjects and those reported previously in white and black populations. The DAT1 locus was estimated to have a heterozygosity index of 14.1%, and the polymorphic information content was calculated to be 0.16.

Simultaneous determination of twelve benzodiazepines in human serum using a new reversed-phase chromatographic column on a 2-microns porous microspherical silica gel.

A high-performance liquid chromatographic method has been developed for the simultaneous analysis of twelve frequently used benzodiazepines (BZPs) (bromazepam, clonazepam, chlordiazepoxide, estazolam, etizolam, flutazoram, haloxazolam, lorazepam, nitrazepam, oxazolam, triazolam and diazepam, internal standard) by using commercially available 2 or 5 microns particle size reversed-phase columns and a microflow cell-equipped ultraviolet detector. The separation was achieved using a C18 reversed-phase column (condition 1: 100 x 4.6 mm I.D., particle size 2 microns, TSK gel Super-ODS: conditon 2: 100 x 4.6 mm I.D., particle size 5 microns, Hypersil ODS-C18). The mobile phase was composed of methanol-5 mM NaH2PO4 (pH 6) (45:55, v/v), and the flow-rate was 0.65 ml/min (condition 1 and 2). The absorbance of the eluent was monitored at 254 nm. Retention times under condition 1 were shorter than those of condition 2. When the twelve benzodiazepines were determined, sensitivity and limits of quantification were about four to ten times better under condition 1 than under condition 2. The rate of recovery and linearity in condition 1 were approximately the same as those in condition 2. These results show that a new ODS filler with a particle size of 2 microns was more sensitive, provided better separation and was more rapid than that with conventional ODS filler.

Detection and analysis of the human D4 dopamine receptor gene (DRD4) sequence variant in the Japanese and Mongolian population.

A PCR-based Sma I restriction fragment length polymorphism (RFLP) in the human D4 dopamine receptor (DRD4) gene was investigated in 200 subjects each of Japanese and Mongolian populations. When compared to allele frequencies for Caucasians previously reported by Petronis et al., Japanese and Mongolian populations showed a decreased frequency of the allele which could not be digested with Sma I. The difference in allele distributions between these two ethnically defined populations (Japanese and Mongolians, and Caucasians) was statistically significant (p<0.05). This RFLP was suggested to be a racial difference between Asian and European populations. However, since the frequency of the non-digested allele was extremely low (1%) in the Asian populations, we cannot exclude the possibility that this allele represents a mutation. Sma I PCR-RFLP typing would not only be one of genetic markers, but might also be a specific marker for searching some neuropsychiatric abnormalities.

Direct cardiotoxic effects of cocaine and cocaethylene on isolated cardiomyocytes.

We investigated the cardiotoxic effects of cocaine and cocaethylene on the Ca2+ flux responsible for excitation-contraction coupling in isolated ventricular rat myocytes. We simultaneously measured intracellular Ca2+ transients and cell length in isolated cardiac myocytes loaded with a fluorescent Ca2+ indicator, indo-1, during electrical field stimulation at 1 Hz. The cell length was estimated by video dimension analysis. We also measured the activities of Ca2+ ATPase and Ca2+ release channels of cardiac sarcoplasmic reticulum membrane vesicles. Both cocaine and cocaethylene produced significant decreases in both peak intracellular Ca2+ and the cell-contraction rate in a dose-dependent manner. The K0.5 for the reduction of peak intracellular Ca2+ was 157.5 microM for cocaine, but 90.0 microM for cocaethylene. Both cocaethylene and cocaine inhibited neither Ca2+ ATPase nor Ca2+ release channel activity. These results demonstrate that cocaethylene has a more potent direct negative inotropic action on cardiomyocytes, without preventing Ca2+ flux through the cardiac sarcoplasmic reticulum membrane.

Amplification of DAT1 (human dopamine transporter gene) 3' variable region in the Japanese population.

Amplification of the human dopamine transporter gene (DAT1) was performed by the polymerase chain reaction, and polymorphism of the VNTR locus was analyzed in a population sample of 176 randomly selected Japanese individuals. Genomic DNAs were extracted from whole blood, and the 3' untranslated region of the human DAT1 gene was amplified. The amplification products were distinguished by agarose gel electrophoresis, and visualized directly by ethidium bromide staining. In this way, four alleles were visualized directly and designated A, B, C, and D in decreasing order according to size. The relative frequencies of DAT1 allels A through D were 0.0171, 0.0625, 0.9119, and 0.0085, respectively. Evidence of significant deviations from the Hardy-Weinberg equilibrium was observed from these Japanese population data. The DAT1 locus was estimated to have a heterozygosity index of 13% and polymorphic information content of 0.16.

Cardiac lesions and their reversibility after long term administration of methamphetamine.

In order to clarify the effect of methamphetamine (MA) on myocardium, histological, immunohistochemical and electron microscopic changes in the myocardium of rats were examined following daily intraperitoneal administration of MA at a dose of 1 mg per kg body weight for 4, 8, and 12 weeks before sacrifice. Normal saline (NS) was similarly injected for the same period before sacrifice to constitute a control group. Light microscopic changes found in the myocardium of the MA-treated group included atrophy, hypertrophy, patchy cellular infiltration, eosinophilic degeneration and disarray, edema myolysis, fibrosis, and the appearance of vacuoles. Ultrastructurally, nuclei and normal mitochondria had various shapes and there were dilated T tubules and sarcoplasmic reticulum, the accumulation of glycogen granules and fat droplets. Intra- and extra-cellular edema and intramyocytic vacuoles were often found. Withdrawal of MA at the twelfth week in another group of rats evidenced gradual recovery of the myocardial changes, commencing at 3 weeks after withdrawal. Optimism is therefore generated about the possibility of the affected hearts in MA-abuse patients returning towards the normal state if they give up the drug.

[Deaths due to heat waves during the summer of 1994 in Osaka Prefecture, Japan].

The summer of 1994 was unusually hot and dry in Japan, setting a record for high temperature and low rainfall in many areas. The daily record of temperature of July and August which were announced by Osaka District Meteorological Observatory exceeded the past ones. The daily recorded temperature of 75 consecutive days in Osaka city was above 30 degrees C with the maximum 39.1 degrees C on 18 August 1994 and 38 degrees C on 16 July 1994. The number of unusual deaths (except traffic accidents) in Osaka Prefecture was 655 and 733 in the month of July and August respectively which exceeded the number of death for the corresponding period of the last 5 consecutive years by 1.5 folds, which was directly proportionate to the change of temperatures. This was due to the illness of the people concerned. We investigated the age, sex and rectal temperature during the inquest and compared them with those of the same period of 1992, which we kept as control. On comparing age and sex of the dead bodies, the cases of 1994 were older than those of 1992, which was due to increased number of death of female aged 70 or more. On comparing the rectal temperatures of those who were found indoors within 8 hours after death, the average temperature of 1994 was also higher than that of 1992. Between the age groups of 75 or more and 74 or younger, the rectal temperature of the former is significantly higher than those of later age group. While regarding the room temperature of the high rectal temperature (39 degrees C < or =) group, no significant difference was observed between the age groups of 75 or more and 74 or younger. In general, the number of unusual deaths in the summer of 1994 in Osaka was more compared to those of previous years. The analysis of the recorded rectal temperatures demonstrated that, 75 or older age group has a tendency to hyperthermia. Since the aging society of Japan is gradually increasing, preventive measures will be necessary when forecasting heat wave to reduce the morbidity and mortality of the aged people.

A comparison of coronary arteries from Japanese and NZ subjects.

This paper describes a comparison of the anterior descending branch of the left coronary artery from 198 Japanese subjects of ages less than 60 yrs, with 301 New Zealand individuals of similar ages. The object of the study was to determine whether there were structural differences present which could be partially responsible for the low incidence of atherosclerosis in Japanese as well as the known low blood lipid levels. It was found that the internal elastic lamina of Japanese coronary arteries was less well formed at birth than that of NZ subjects. Intimal thickening was greater in Japanese coronary arteries from birth to the end of the first decade, but increased less rapidly with age, and was only about half as great as that of NZ vessels in the older age groups. The thickened intima of Japanese arteries was more uniform round the circumference of the vessel, the luminal surface was better formed with more stainable elastin present subjacent to the endothelial cells, and there was less evidence of mural thrombosis. NZ arteries showed pronounced eccentricity of the intima, more extensive lipid deposits, a poorly defined luminal surface, and frequent evidence of mural thrombosis.

Detection of D1S80 (pMCT118) locus polymorphism using semi-nested polymerase chain reaction in skeletal remains.

We evaluated the usefulness of a semi-nested polymerase chain reaction (PCR) method for detecting D1S80 (pMCT118) locus polymorphisms of DNA extracted from old skeletal remains. The semi-nested PCR has been applied to the amplification of D1S80 nucleic acid sequences. For amplification of the locus D1S80, a pair of oligonucleotide primers have been used widely as described by Kasai et al. We have designed another set of primers for semi-nested PCR. This method resulted in D1S80-VNTR detection from low-titered DNA isolated from old skeletal remains. The first and second step PCR achieved amplification from as little as 10 ng and 10 pg of template DNA, respectively. Specificity and sensitivity of the amplification products was markedly improved by semi-nested PCR. In DNA extracted from biological samples, this method took about 5 hours to amplify the target DNA and 3 hours for electrophoretic separation. We demonstrated that this semi-nested PCR method was superior in sensitivity to conventional 1-step standard amplification for VNTR typing of the D1S80 locus.

Mixed cavernous angioma and glioma (angioglioma) in the hypothalamus--case report.

A 54-year-old female presented with a huge mixed cavernous angioma and astrocytoma in the hypothalamus manifesting as headache, visual field defect, gait disturbance, and convulsion. Radiological studies revealed a huge suprasellar tumor encasing all the major cerebral vessels. Craniotomy disclosed a hemorrhagic tumor poorly demarcated from the surrounding brain which was partially removed. Histological examination of the operative specimen revealed cavernous angioma with low grade glioma in the periphery. The residual tumor responded to radiation therapy remarkably well. An autopsy conducted 3 years later revealed a small hypothalamic astrocytoma with abundant vasculature.