Choei Wakasugi

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.

Forensic analysis of eleven cyclic antidepressants in human biological samples using a new reversed-phase chromatographic column of 2 μm 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.

Simultaneous determination of twelve benzodiazepines in human serum using a new reversed-phase chromatographic column on a 2-μm 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.

Fluidity of Cadaveric Blood After Sudden Death: Part Iii

In rats, fibrinolytic activity and acidosis increased rapidly after death. Postmortem fibrinolysis and the pH, base excess (BE), and [HCO3-] levels were affected by the method of sacrifice: the lower the pH, BE, and [HCO3-] levels, the higher the fibrinolytic activity. Conversely, in experiments using the vascular perfusion technique, low pH, BE, and [HCO3-] levels of the perfusate induced abundant release of plasminogen activator from the vascular wall.

Myocardial lesions induced after trauma and treatment

In order to clarify the effect of trauma and treatment as stresses on myocardia, we examined histological changes of myocardia in victims who received various kinds of traumata and treatments. We also undertook a histochemical study for calmodulin, which we found useful in the diagnosis of early ischemia. Those who died shortly after stab wounds, traffic accident or head injuries, showed mild cardiac lesions such as contraction bands or fragmentation and mild diffusion of calmodulin, a marker for necrosis. A case with hemorrhagic shock after a traffic accident, involving intense resuscitation for 2 h, showed severe cardiac lesions such as contraction bands, hydropic change and subendocardial hemorrhage along with severe diffusion of calmodulin. In most of the instant death cases after falls, severe contraction band necrosis and severe calmodulin diffusion were observed. Myocardia of victims, who died several days after head injuries or traffic accidents, generally demonstrated distinct diffusion of calmodulin as compared to the mild and non-specific lesions detected by hematoxylin-eosin (H&E) staining. In cases of long-term survival in a state of brain death, calmodulin staining was very low, which was not always associated with the severity of the lesions on H&E staining. In cases with intensive or extended treatment, it appeared to be difficult to determine the cause-effect relationship between trauma and cardiac lesions or to distinguish the lesions due to extrinsic factors from those of disease. In some cases, calmodulin intensely stained the areas with hydropic appearance or hypereosinophilia, which may be related to calcium overload.

Fluidity of cadaveric blood after sudden death: Part II: Mechanism of release of plasminogen activator from blood vessels

Experiments by the vascular pcrfusion technique in isolated hind leg of the dog showed that after addition of various vasoactive drugs to the perfusate, fibrinolytic activity (plasminogen activator level) of the effluent was elevated. The effects of the drugs were completely inhibited only by the specific blocker of their receptor on the vascular wall. High K+ level of the perfusate also induced abundant release of plasminogen activator from the vascular wall

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.

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.

Postmortem changes in drug-metabolizing enzymes of rat liver microsome

Postmortem changes in the drug-metabolizing enzymes in rat liver microsome were studied. Parameters investigated were: microsomal protein, NADPH-cytochrome P-450 reductase activity, NADH-cytochrome b5 reductase activity, cytochrome b5 content, cytochrome P-450 content, aminopyrine N-demethylase activity, aniline p-hydroxylase activity, p-nitroanisole O-demethylase activity, uridine diphosphate (UDP)-glucuronyl transferase activity and glutathione S-transferase activity. Nearly all the parameters based on microsomal protein decreased during autolysis and the time-dependent decrement ratios of the parameters changed by various amounts. Cytochrome b5 content decreased more rapidly than that of other components. By 36 h post mortem, levels of cytochrome b5 were not detectable. By 48 h post mortem, NADPH-cytochrome P-450 reductase activity decreased to 91%, NADH-cytochrome b5 reductase activity decreased to 94%, and cytochrome P-450 content decreased to 92% of relative activities. By 48 h post mortem, aminopyrine N-demethylase activity decreased to 87%, aniline p-hydroxylase activity decreased to 98% and p-nitroanisole O-demethylase activity decreased to 75% of relative activities. The activity of p-nitroanisole O-demethylase appeared to be more stable than that of aminopyrine N-demethylase or aniline p-hydroxylase. These results demonstrate that there are multiple forms of isozymes of the cytochrome P-450-linked monooxygenase system. Hepatic transferases showed decrease patterns different to those of monooxygenases, so UDP-glucuronyl transferase activity of approximately 32% of relative activity was detected at 48 h post mortem. Thus, UDP-glucuronyl transferase activity appeared to be more stable than the cytochrome P-450-linked monooxygenases. These results show that these activities and components would be useful as markers of postmortem time. The causes of the variety of instability of these enzyme systems are discussed.

Rupture of a giant splenic artery aneurysm. Report of an autopsy case.

We describe an autopsy case of a 61-year-old woman with von Recklinghausens disease, who died suddenly following intraperitoneal hemorrhage due to the rupture of a giant splenic artery aneurysm. The aneurysm measured 16 x 13 x 5.5 cm--much larger than those in most previous reports. The pancreatic body, which was pressed by the aneurysm, was widely atrophic. In general, splenic artery aneurysms are more frequent in pregnant women or patients with portal hypertension. The pathogenesis of this aneurysm is presumed to be arterial dysplasia, focal arterial inflammation, or portal hypertension, unlike other aneurysms due to arteriosclerosis or syphilis. Since the patient had not been pregnant and had not had liver cirrhosis or arteriosclerosis, the pathogenic factor could not be determined in this case. The relationship between the genesis of the aneurysm and von Recklinghausens disease was not clear either.