Temperature and time after urine collection affect the detection of phenethylamine, a substance prohibited in sports.

2-Phenylethylamine (phenethylamine) and its derivatives are stimulant drugs, which are prohibited in sports because of their potential performance-enhancing properties. If phenethylamine is detected in an athlete's urine, the athlete may be subjected to serious sanctions, such as disqualification for both domestic and international competitions. Given the serious consequences athletes face for phenethylamine detection, great care should be taken to avoid false positive tests. In forensic medicine, it is widely known that phenethylamine is produced by putrefactive bacteria in autopsy urine samples; it is possible that this process could also occur in an athlete's urine sample without proper storage. In this study, human urine samples were stored at -20, 4, or 22°C for 14 days, and phenethylamine in the samples was quantitatively analyzed using ultra-high-performance liquid chromatography-tandem mass spectrometry. No phenethylamine was detected in urine samples stored at -20°C throughout 14-day period. Nevertheless, phenethylamine was detected after 6 days in these samples stored at 4°C and after only 1 day in samples stored at 22°C. Additionally, the concentration of phenethylamine in these samples increased each day after detection. These results suggest that urine samples should be stored immediately at -20°C after collection when testing athletes for phenethylamine, especially if the sample must be stored for extended period before testing.

A Fatal Case of Poisoning with Fentanyl Transdermal Patches in Japan.

Fentanyl transdermal patches have been used to treat cancer- and noncancer-related chronic pain. However, its inappropriate or illegal application may cause fatal poisoning. We herein present the case of a Japanese woman in her 40s who was found dead with seven 25-µg/h fentanyl transdermal patches on her body. We established a detailed toxicological analysis procedure to quantify fentanyl, and its metabolite norfentanyl, and other drugs (acetaminophen, allylisopropylacetylurea, celecoxib, estazolam, promethazine, and sertraline) in human whole blood by ultra-high-performance liquid chromatography-tandem mass spectrometry. The measured fentanyl and norfentanyl concentrations in the femoral and cardiac blood were 0.051 and 0.072 µg/mL and 0.033 and 0.076 µg/mL, respectively. The decedent's fentanyl concentrations were consistent with previously reported postmortem blood levels for fatal cases of poisoning by fentanyl transdermal patches. Based on the decedent's case history, autopsy findings, and toxicological analyses, the cause of death was identified as intoxication with transdermal fentanyl.

Forensic analysis using ultra-high-performance liquid chromatography-tandem mass spectrometry with solid-phase extraction of α-solanine and α-chaconine in whole blood.

PURPOSE: The potato glycoalkaloids (PGAs), α-solanine and α-chaconine can exert adverse effects on human health when consumed in excess. This study aimed to investigate the optimal extraction method for the quantitative analysis of PGAs in whole blood by using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and to apply this validated method to postmortem blood. METHODS: A total of 200 µL of human whole blood was prepared for PGA extraction. For validation, a solid-phase extraction (SPE) using Oasis® PRiME HLB, in which extraction could be performed in three simple steps (sample loading, washing, and elution) was used, with no need for both conditioning and equilibration of columns for sample preparation. RESULTS: In this method, the limit of detection and the lower limit of quantification (LLOQ) of both α-solanine and α-chaconine were 1 and 2 µg/L, respectively. The calibration curves of the two compounds were obtained with good linearity in the range of 2-100 µg/L. The recovery rates at the LLOQ of α-solanine and α-chaconine were ≥ 91.8% and ≥ 85.9%, respectively. The validation data (intra- and inter-day combined) for accuracy ranged from 93.5 to 106.6% for α-solanine and from 93.9 to 107.7% for α-chaconine. This validated method was successfully applied to one forensic autopsy case, and the concentrations of α-solanine and α-chaconine in the postmortem cardiac blood were 45.1 and 35.5 µg/L, respectively. CONCLUSIONS: This validated UHPLC-MS/MS with SPE for quantitative analysis of PGAs could be useful in forensic toxicology.

Fatal Poisoning with Both Dichlorvos and Phenthoate.

Organophosphates are widely used as pesticides. However, organophosphates are occasionally orally ingested to commit suicide. In this case, a man in his late 80s committed suicide by ingesting both dichlorvos and phenthoate. Autopsy findings revealed a characteristic volatile odor from his mouth, stomach, lungs, liver, and kidneys. The esophageal mucosa was denatured and had lost elasticity. Serum cholinesterase activity was 9 IU/L. Toxicological analyses performed using high-performance liquid chromatography-tandem mass spectrometry revealed that dichlorvos concentrations in the left and right cardiac blood samples were 11.6 and 4.6 µg/mL, respectively. Phenthoate concentrations in the left and right cardiac blood samples were 5.8 and 0.51 µg/mL, respectively. The total amounts of dichlorvos and phenthoate in the stomach were 7.35 and 4.55 g, respectively. The case history, autopsy findings, and toxicological analyses indicated that the cause of death was acute fatal poisoning after oral ingestion of both dichlorvos and phenthoate.

Myoglobinemia markers with potential applications in forensic sample analysis: lipid markers in myoglobinemia for postmortem blood.

The crush syndrome, in which rhabdomyolysis and trauma occur as a result of heat stroke and drug intoxication, can lead to myoglobinemia. This condition can be diagnosed by measuring myoglobin (Mb) levels in blood and urine. However, postmortem Mb levels are unreliable indicators, since blood Mb concentration drastically increases within a very short time after death and urine cannot always be obtained at dissection; this makes it difficult to diagnose myoglobinemia in a corpse. To address this issue, in this study, we used a lipidomics approach to identify markers that can be used to detect myoglobinemia in postmortem blood samples. We found that increases in levels of fatty acid oxides such as stearic, oleic, linoleic, and arachidonic acid and decreases in levels of plasmalogens and phosphatidylethanolamine in the blood were associated with high Mb level. These results demonstrate that postmortem samples are amenable to lipidomics analysis and provide a set of markers other than Mb that can be used for postmortem diagnosis of myoglobinemia.

Evaluations of lipid peroxidation and inflammation in short-term glycerol-induced acute kidney injury in rats.

Rhabdomyolysis is characterised by acute kidney injury (AKI) resulting from skeletal muscle injury. Lipid peroxidation-mediated oxidant injury and pro-inflammatory cytokine-mediated inflammatory response play critical roles in the pathogenesis of rhabdomyolysis-induced AKI. The present study aimed to investigate the short-term effects of both lipid peroxidation and inflammatory responses on rhabdomyolysis-induced AKI in a rat model of glycerol-induced rhabdomyolysis. Rhabdomyolysis was induced by the intramuscular injection of 50% glycerol in saline (10 mL/kg) into the hind limbs of rats. Rats were killed 1 or 3 hours after glycerol injection. Time-dependent increases in serum biochemical parameters, including blood urea nitrogen, creatinine, lactate dehydrogenase and creatine phosphokinase levels, were observed 1 hour after glycerol injection. In kidneys, glycerol injection resulted in histopathological changes such as renal tubular injury and renal tubular myoglobin deposition. Levels of Nε-(hexanoyl)lysine-modified, 4-hydroxy-2-nonenal-modified, and nitrotyrosine-modified proteins in rat kidneys were unaltered at 1 hour after glycerol injection, but increased significantly at 3 hours. Increases in renal nitric oxide production and the expression levels of inducible nitric oxide synthase, interleukin-6 and tumour necrosis factor-α in the renal parenchyma were observed at 1 hour after glycerol injection and plateaued at 3 hours. Our findings suggest that the pro-inflammatory cytokine-mediated inflammatory response may cause rhabdomyolysis-induced AKI very shortly after glycerol injection, and lipid peroxidation-mediated oxidant injury may promote the development of these pathophysiological processes.

Postmortem redistribution mechanism of donepezil in the rat.

Population aging is rapidly advancing in numerous parts of the world and, accordingly, the prevalence of Alzheimer's disease (AD) is rising. The safety of donepezil (DPZ), which is used for AD treatment, has been established in clinical trials. However, some studies have indicated that DPZ may be associated with severe cardiac side effects, and excessive doses may induce toxicity-related symptoms or death. Therefore, the measurement of blood DPZ levels is important for the postmortem investigation of related causes of death. However, postmortem drug concentrations in the blood may not always reflect those obtained antemortem because of the postmortem redistribution (PMR) of drugs. Therefore, the aim of this study was to investigate the potential PMR of DPZ using a rat model. The DPZ concentration was measured using a validated HPLC/Q-TOF-MS system in cardiac and peripheral blood, and in the brain, lungs, myocardium, liver, and thigh muscle at different postmortem intervals (0, 1, 3, 6, 12, and 24h). Overall, the DPZ tissue to peripheral blood ratio decreased throughout the postmortem period. Furthermore, the DPZ concentration increased in the peripheral and cardiac blood but decreased in both of the lungs, postmortem. Furthermore, the blood pH was significantly lowered. We used a perfusion approach to examine the rat lung and heart to further investigate the relationship between the pH and DPZ release from the lungs. The outflow concentrations when the inflow pH changed from 7.4 to 5.5 were approximately 2-fold higher than the inflow pH fixed 7.4. These findings suggest that the antemortem accumulated DPZ in the lungs is released into the pulmonary blood owing to postmortem acidification of blood, and subsequently flows into the cardiac blood, leading to the observed increase in concentration. Although we could not determine the underlying mechanism, we confirmed that PMR occurs similarly in the cardiac and peripheral blood.

An unusual autopsy case of lethal hypothermia exacerbated by body lice-induced severe anemia.

Pediculus humanus humanus (known as body lice) are commonly found in the folds of clothes, and can cause skin disorders when they feed on human blood, resulting in an itching sensation. Body lice are known as vectors of infectious diseases, including typhus, recurrent fever, and trench fever. An infestation with blood-sucking body lice induces severe cutaneous pruritus, and this skin disorder is known as "vagabond's disease." A body lice infestation is sometimes complicated with iron deficiency anemia. In the present case, a man in his late 70s died of lethal hypothermia in the outdoors during the winter season. The case history and autopsy findings revealed that the cause of the lethal hypothermia was iron deficiency anemia, which was associated with a prolonged infestation of blood-sucking body lice. Also, he had vagabond's disease because the skin on his body was abnormal and highly pigmented. This is an unusual autopsy case since the body lice contributed to the cause of the death.

Pulmonary Macrophages Attenuate Hypoxic Pulmonary Vasoconstriction via ß3AR/iNOS Pathway in Rats Exposed to Chronic Intermittent Hypoxia.

Chronic intermittent hypoxia (IH) induces activation of the sympathoadrenal system, which plays a pivotal role in attenuating hypoxic pulmonary vasoconstriction (HPV) via central ß1-adrenergic receptors (AR) (brain) and peripheral ß2AR (pulmonary arteries). Prolonged hypercatecholemia has been shown to upregulate ß3AR. However, the relationship between IH and ß3AR in the modification of HPV is unknown. It has been observed that chronic stimulation of ß3AR upregulates inducible nitric oxide synthase (iNOS) in cardiomyocytes and that IH exposure causes expression of iNOS in RAW264.7 macrophages. iNOS has been shown to have the ability to dilate pulmonary vessels. Hence, we hypothesized that chronic IH activates ß3AR/iNOS signaling in pulmonary macrophages, leading to the promotion of NO secretion and attenuated HPV. Sprague-Dawley rats were exposed to IH (3-min periods of 4-21% O2) for 8 h/d for 6 weeks. The urinary catecholamine concentrations of IH rats were high compared with those of controls, indicating activation of the sympathoadrenal system following chronic IH. Interestingly, chronic IH induced the migration of circulating monocytes into the lungs and the predominant increase in the number of pro-inflammatory pulmonary macrophages. In these macrophages, both ß3AR and iNOS were upregulated and stimulation of the ß3AR/iNOS pathway in vitro caused them to promote NO secretion. Furthermore, in vivo synchrotron radiation microangiography showed that HPV was significantly attenuated in IH rats and the attenuated HPV was fully restored by blockade of ß3AR/iNOS pathway or depletion of pulmonary macrophages. These results suggest that circulating monocyte-derived pulmonary macrophages attenuate HPV via activation of ß3AR/iNOS signaling in chronic IH.

The study of forensic toxicology should not be neglected in Japanese universities.

Forensic toxicology is aimed at identifying the relationship between drugs or poison and the cause of death or crime. In the authors' toxicology laboratory at Chiba University, the authors analyze almost every body for drugs and poisons. A simple inspection kit was used in an attempt to ascertain drug abuse. A mass spectrometer is used to perform highly accurate screening. When a poison is detected, quantitative analyses are required. A recent topic of interest is new psychoactive substances (NPS). Although NPS-related deaths may be decreasing, use of NPS as a cause of death is difficult to ascertain. Forensic institutes have recently begun to perform drug and poison tests on corpses. However, this approach presents several problems, as are discussed here. The hope is that highly accurate analyses of drugs and poisons will be performed throughout the country.

An unusual autopsy case of cytokine storm-derived influenza-associated encephalopathy without typical histopathological findings: autopsy case report.

Cytokine storm-derived influenza-associated encephalopathy is a severe complication, affecting not only the brain but also multiple systemic organs including the heart and lungs. Hundreds of Japanese children are afflicted by influenza-associated encephalopathy every year. Influenza-associated encephalopathy can be diagnosed by pathological changes, such as advanced brain edema and disruption of astrocytic projections, which is known as clasmatodendrosis. In the present case, despite the absence of significant histopathological findings in the brain, the diagnosis of influenza-associated encephalopathy was made on the basis of autopsy findings such as brain swelling, pathological findings including diffuse alveolar damage, and increase in the concentrations of interleukin-6 in both the serum and cerebrospinal fluid. In this case, the interval from high fever to death was approximately 7 hours and may have been too short for histopathological features to develop. This is an unusual autopsy case of cytokine storm-derived influenza-associated encephalopathy without typical histopathological findings.

Pulmonary arterial hypertension in rats due to age-related arginase activation in intermittent hypoxia.

Pulmonary arterial hypertension (PAH) is prevalent in patients with obstructive sleep apnea syndrome (OSAS). Aging induces arginase activation and reduces nitric oxide (NO) production in the arteries. Intermittent hypoxia (IH), conferred by cycles of brief hypoxia and normoxia, contributes to OSAS pathogenesis. Here, we studied the role of arginase and aging in the pathogenesis of PAH in adult (9-mo-old) and young (2-mo-old) male Sprague-Dawley rats subjected to IH or normoxia for 4 weeks and analyzed them with a pressure-volume catheter inserted into the right ventricle (RV) and by pulsed Doppler echocardiography. Western blot analysis was conducted on arginase, NO synthase isoforms, and nitrotyrosine. IH induced PAH, as shown by increased RV systolic pressure and RV hypertrophy, in adult rats but not in young rats. IH increased expression levels of arginase I and II proteins in the adult rats. IH also increased arginase I expression in the pulmonary artery endothelium and arginase II in the pulmonary artery adventitia. Furthermore, IH reduced pulmonary levels of nitrate and nitrite but increased nitrotyrosine levels in adult rats. An arginase inhibitor (N(ω)-hydroxy-nor-1-arginine) prevented IH-induced PAH and normalized nitrite and nitrate levels in adult rats. IH induced arginase up-regulation and PAH in adult rats, but not in young rats, through reduced NO production. Our findings suggest that arginase inhibition prevents or reverses PAH.

Lethal pulmonary air embolism caused by the removal of a double-lumen hemodialysis catheter.

Pulmonary air embolisms due to the removal of a central venous catheter are rare, but catheter removal is known to be a high risk factor for air embolism. In particular, the removal of a large catheter, such as a double-lumen hemodialysis catheter, can allow a large amount of air to enter into the bloodstream, which often results in sudden death. So, during catheter removal, special care should be taken to prevent air from entering blood vessels, for example, to ensure that the patient's head is tilted downward, that they have inhaled and are holding their breath, and that a covering gauze and inert ointment have been applied to the exit site. We report a lethal case of pulmonary air embolism caused by the removal of a double-lumen catheter from the right internal jugular vein of a patient who was sitting up and had not been instructed to hold their breath.

ß2-Adrenergic receptor-dependent attenuation of hypoxic pulmonary vasoconstriction prevents progression of pulmonary arterial hypertension in intermittent hypoxic rats.

In sleep apnea syndrome (SAS), intermittent hypoxia (IH) induces repeated episodes of hypoxic pulmonary vasoconstriction (HPV) during sleep, which presumably contribute to pulmonary arterial hypertension (PAH). However, the prevalence of PAH was low and severity is mostly mild in SAS patients, and mild or no right ventricular hypertrophy (RVH) was reported in IH-exposed animals. The question then arises as to why PAH is not a universal finding in SAS if repeated hypoxia of sufficient duration causes cycling HPV. In the present study, rats underwent IH at a rate of 3 min cycles of 4-21% O2 for 8 h/d for 6 w. Assessment of diameter changes in small pulmonary arteries in response to acute hypoxia and drugs were performed using synchrotron radiation microangiography on anesthetized rats. In IH-rats, neither PAH nor RVH was observed and HPV was strongly reversed. Nadolol (a hydrophilic ß(1, 2)-blocker) augmented the attenuated HPV to almost the same level as that in N-rats, but atenolol (a hydrophilic ß1-blocker) had no effect on the HPV in IH. These ß-blockers had almost no effect on the HPV in N-rats. Chronic administration of nadolol during 6 weeks of IH exposure induced PAH and RVH in IH-rats, but did not in N-rats. Meanwhile, atenolol had no effect on morphometric and hemodynamic changes in N and IH-rats. Protein expression of the ß1-adrenergic receptor (AR) was down-regulated while that of ß2AR was preserved in pulmonary arteries of IH-rats. Phosphorylation of p85 (chief component of phosphoinositide 3-kinase (PI3K)), protein kinase B (Akt), and endothelial nitric oxide synthase (eNOS) were abrogated by chronic administration of nadolol in the lung tissue of IH-rats. We conclude that IH-derived activation of ß2AR in the pulmonary arteries attenuates the HPV, thereby preventing progression of IH-induced PAH. This protective effect may depend on the ß2AR-Gi mediated PI3K/Akt/eNOS signaling pathway.

Hyperstimulation of macropinocytosis leads to lysosomal dysfunction during exposure to methamphetamine in SH-SY5Y cells.

Although various cytotoxic effects on neuronal cells caused by methamphetamine (METH) have been investigated, the cellular and molecular mechanisms of METH-induced neurotoxicity remain to be elucidated. We previously reported that METH-induced cytomorphological effects on retinoic acid (RA)-differentiated SH-SY5Y human neuroblastoma cells involved macropinocytosis, which is an actin-dependent endocytic pathway. We also noted that hyperstimulation of this process might play an important role in the cytotoxicity of METH in neuronal cells. In this study, we investigated the molecular mechanisms as well as subsequent outcomes of macropinocytosis during METH treatment. It was found that macropinosomes formed upon exposure to METH were colocalized with constitutively active GFP-Ras (G12V) and GFP-Rac1 (Q61L). Furthermore, both Ras inhibitor, farnesylthiosalicylic acid (FTS), and Rac1 inhibitor, EHT1864, significantly inhibited the formation of macropinosomes, suggesting the involvement of these molecules. The expressions of lysosome-associated membrane proteins (lamps) gradually increased by METH in a time-dependent manner. In contrast, the proteolytic activation of cathepsin L, a marker of lysosomal function, was markedly suppressed by METH. METH-induced dysfunction of lysosomal enzyme as well as cell death was significantly attenuated by nocodazole, a microtube interfering reagent that inhibits the transport of vesicles, including macropinosome, to lysosomes. Our results indicate that METH has cytotoxic effects, at least in part, by inhibiting normal lysosomal function through Ras- and Rac1-mediated macropinocytosis in RA-differentiated SH-SY5Y cells.

Critical roles of Rho-associated kinase in membrane blebbing and mitochondrial pathway of apoptosis caused by 1-butanol.

Alcohols are widely used as industrial solvents and chemical intermediates but can cause serious damage to human health. Nevertheless, few studies have addressed the molecular mechanisms underlying the cytotoxicity of industrial alcohols, with the notable exception of ethanol. The goal of our current study is to elucidate the molecular mechanism of cytotoxicity caused by primary alcohols containing longer carbon chains than ethanol. We find that 1-butanol induces morphological changes in H9c2 cardiomyoblastoma including nuclear condensation and membrane blebbing, both of which are features of apoptotic response. Moreover, a decrease in the mitochondrial membrane potential, the cytosolic release of cytochrome c, and the activation of caspase 9 and 3 was observed, thus revealing the activation of the mitochondrial apoptotic pathway by 1-butanol. The addition of Y-27632, a specific inhibitor of Rho-associated kinase (ROCK), suppressed the membrane blebbing and mitochondrial apoptotic pathway. In comparison z-VAD-fmk, a pan-caspase inhibitor, did not inhibit membrane blebbing but did prevent cell death following exposure to 1-butanol. These results indicate that mitochondrial pathway of apoptosis and membrane blebbing are parallel phenomena that occur downstream of ROCK. This kinase thus plays an essential role in 1-butanol cytotoxicity and subsequent cell death in H9c2 cells.

A case in which a bone fragment caused by a bullet made a second channel in addition to the bullet channel.

A 64-year-old male was found dead in his house with his face covered with blood and a 38-caliber revolver between his legs. He had been suffering from type 2 diabetes mellitus and aftereffects of cerebral infarction. Autopsy revealed a normal round contact wound in the left lateral cervical region. A bullet from the firearm had entered through the left lateral cervical region and traveled to the outer right sternocleidomastoid muscle. This also triggered another wound from the fifth cervical vertebra to the muscle tissue near the right cartilage thyroid. At the end of this channel, there were three bone fragments. Here, we report this interesting case with two channels caused by a bullet and by a resulting bone fragment. We also discuss the characteristics of an ear lobe injury found on the victim and show how this injury and blood and skin on the revolver were used as clues to determine the posture at the time of the shot.

Cytoplasmic vacuolization during exposure to drugs and other substances.

Cytoplasmic vacuolization upon exposure to a variety of chemicals and bioactive substances has been extensively reported. Nearly 30 years have passed since the description by Nobel Laureate Christian de Duve of the mechanism underlying the lysosomal accumulation of lipophilic weak bases referred to these substances as lysosomotropic agents. It has now been revealed, however, that vacuolization occurs upon exposure to compounds other than lipophilic weak bases. Vacuolization of organelles/vesicles other than acidic compartments has also now been reported. In this mini-review, we provide an overview of the origin, mechanism, and possible outcomes of cellular vacuolization during exposure to substances with lysosomotropic as well as other properties.

Autopsy findings of a patient with rapidly progressive massive ascites caused by alcoholic cirrhosis.

A 54-year-old man, who lived alone, was hospitalized due to rapid deterioration of the general condition over a three-week period caused by alcoholic cirrhosis. One month after he left hospital, he was found dead in his house by his friend. Three days before he was found dead, he had met his friend and seemed to be in poor condition. Autopsy was conducted by a medical examiner to clarify the cause of death. Externally, signs of severe jaundice were apparent over the whole body, along with extensive abdominal swelling and edema of the extremities. Autopsy findings demonstrated that the abdominal cavity contained an amount of massive turbid and slight pale reddish brown ascites (23 l). There were no findings of severe peritoneal inflammation. The liver (650 g) was elastic hard and had a micro-nodular surface, which showed severe atrophy. Microscopic examination of the liver showed clear pseudolobule with severe fibrosis in the stroma. There were no significant changes in the heart or brain. The stomach was empty and only a slight amount of intestinal contents. There was no ethanol detected in the blood or urine. The direct cause of his death was circulatory dysfunction due to massive accumulation of the ascites. The reasons for the massive ascites accumulation over 20 l in this case were (1) that he had no serious complications other than ascites; and (2) he did not have any medical treatment just before his death.