Azumi Kuse

Rapid determination of glyphosate, glufosinate, bialaphos, and their major metabolites in serum by liquid chromatography―tandem mass spectrometry using hydrophilic interaction chromatography

We developed a simple and rapid method for the simultaneous determination of phosphorus-containing amino acid herbicides (glyphosate, glufosinate, bialaphos) and their major metabolites, aminomethylphosphonic acid (AMPA) and 3-methylphosphinicopropionic acid (MPPA), in human serum. Serum samples were filtrated through an ultrafiltration membrane to remove proteins. The filtrate was then washed with chloroform, and injected into a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. Chromatographic separation was achieved on a hydrophilic interaction chromatography (HILIC) column. Determination of the target herbicides and metabolites was successfully carried out without derivatization or solid phase extraction (SPE) cartridge clean-up. The recoveries of these compounds, added to human serum at 0.2μg/mL, ranged from 94% to 108%, and the relative standard deviations (RSDs) were within 5.9%. The limits of detection (LODs) were 0.01μg/mL for MPPA, 0.02μg/mL for AMPA, 0.03μg/mL for both glyphosate and glufosinate, and 0.07μg/mL for bialaphos, respectively.

Estimates of exposure to cold before death from immunohistochemical expression patterns of HSP70 in glomerular podocytes

Environmental factors such as outside temperature at the time of death are very important for forensic diagnoses and police investigations. In particular, death in a cold environment is associated with factors of forensic interest, including hypothermia, drowning in cold water, or postmortem body movement by a suspect. Hypothermia raises a special problem because of the difficulty of evaluation during autopsy. We describe here a unique method of estimating antemortem environmental temperature, involving the immunohistochemical analysis of HSP70 expression patterns in glomerular podocytes. Using this method, we found that HSP70 was present in glomerular podocytes at autopsy and that HSP70 was highly expressed, mainly in the nucleus of podocytes, in deaths associated with exposure to cold. Interestingly, this expression pattern was specific to death in a cold environment, including hypothermia and drowning in cold water. Analysis of the pattern of HSP70 expression in glomeruli may therefore be very useful in forensic diagnosis, for determining whether the antemortem environmental temperature was low. Moreover, immunohistochemical and real-time PCR assays of the molecular mechanism of HSP70 and HSF1 expression in glomeruli following exposure to cold indicated that HSP70 was rapidly translocated to the nucleus of podocytes following exposure to cold, but without new protein synthesis.

Rupture of massive coronary artery aneurysm resulting in cardiac tamponade

Coronary artery aneurysm is a fairly uncommon clinical entity, which is defined by a characteristic dilatation that exceeds 1.5 times the width of normal adjacent coronary artery segments. In the present report, we describe a case of rupture of a massive coronary artery aneurysm. A man in his 40s was found dead in his bed. The pericardial cavity contained 270mL of blood with 428.2g of coagulation. Two true aneurysms of the right coronary artery were identified. A proximal aneurysm, adjacent to the right auricle, had ruptured on the right. A distal unruptured aneurysm was identified 5.1cm distal to the proximal ruptured aneurysm. Atherosclerosis of the coronary arteries and aorta was severe. The heart weighed 799.1g and showed concentric ventricular hypertrophy, myocardial thinning, and patchy fibrosis. Histological analysis showed that both aneurysms were purely atherosclerotic true aneurysms without considerable inflammation. The cause of death was determined as cardiac tamponade due to rupture of a giant coronary atherosclerotic aneurysm.

An autopsy case of cardiac tamponade caused by a ruptured ventricular aneurysm associated with acute myocarditis

We report an autopsy case of hemopericardium caused by rupture of a ventricular aneurysm associated with acute myocarditis in an infant boy aged 2 years and 10 months. Three days before his death, the patient developed fever. On the day of death, he described an urge to defecate and attempted to do so in an upright position. While straining to defecate without success for a prolonged period, he stopped breathing and collapsed. On autopsy, his heart weighed 91.7 g and cardiac tamponade was evident, the pericardial cavity being filled with 140 mL of blood that had come from a 1.5-cm-long rupture in a 2.7×1.5 cm ventricular aneurysm in the posterior left ventricular wall. Patchy grayish-white discoloration was noted in the myocardium. Histologically, CD3-positive T lymphocytic infiltration accompanied by pronounced macrophage infiltration was observed in the myocardium. Hemorrhagic necrosis was detected in the area of the ventricular aneurysm. Staining for matrix metalloproteinase (MMP) expression revealed abundant MMP-2, MMP-7, and MMP-9. Polymerase chain reaction to detect viruses failed to identify any specific causative viruses in the myocardium. In this case of lymphocytic (viral) and histiocytic myocarditis with pronounced macrophage infiltration and upregulation of MMP expression, myocardial remodeling and associated wall weakening had resulted in formation and rupture of an aneurysm.

Postmortem detection of antibiotic-specific immunoglobulin E in the case of anaphylactic death

An allergic anaphylactic reaction is a potentially fatal cascade consisting of an initial sensitization, antigen exposure, crosslinking of immunoglobulin E (IgE) specific for the antigen, activation of mast cells, and release of bioactive substances. Postmortem diagnosis of anaphylaxis is challenging because of the limited availability of antemortem history and minimal macroscopic evidence at autopsy. The elevated activity of a neutral proteinase, such as tryptase, can be a surrogate marker for the activation and degranulation of mast cells. However, it does not directly indicate the involvement of antigen-specific IgE, which has an important role in IgE-mediated allergic anaphylaxis. In the present study, we examined blood from a case of death following infusion of the antibiotic ceftriaxone (CTRX), with a control case for comparison. The aim was to detect IgE specific for CTRX. A pull-down assay with N-hydroxysuccinimide-activated sepharose identified IgE specific for CTRX only in the serum obtained from the case of CTRX exposure, and not in the control case. The specificity of IgE was confirmed by adsorption to an excess of CTRX, which resulted in the signal for IgE disappearing in the pull-down assay. This antigen-specific IgE is a key molecule in the IgE-mediated allergic anaphylaxis and seldom investigated in postmortem examinations. Its detection can provide support for the postmortem diagnosis of allergic anaphylaxis, especially when combined with an antemortem history of allergen exposure and elevated neutral proteinase levels in serum.

Autopsy case of undiagnosed gangliocytoma in the medulla oblongata complicated with cerebral palsy.

A Japanese man in his 30s who had congenital cerebral palsy was found unresponsive in bed. His death was confirmed after resuscitation attempts. He had a history of occasional falling (despite the use of walking sticks and a wheelchair) owing to a slowly progressive gait disturbance, and had a medical examination without full neurological re-examination. Autopsy revealed gangliocytoma in the medulla oblongata, which was diagnosed as the cause of death. Although gangliocytoma is a well-differentiated benign tumor, the almost total replacement of the medulla oblongata by the tumor cells was assumed to result in ataxia via the olivocerebellar tract and secondary cerebellar atrophy, followed by central hypoventilation and death of the patient. The symptoms caused by gangliocytoma may be overlooked owing to long-standing cerebral palsy.

Unexpected neonatal death caused by interrupted aortic arch, an uncommon congenital great vessel malformation: An autopsy case report

A 21-day-old Japanese male infant became inactive and then collapsed unexpectedly. On autopsy, there was no connection between the ascending and descending thoracic aorta. The site of interruption was the aortic isthmus. The heart showed hypertrophy and exhibited intracardiac malformations, including subaortic valve stenosis resulting from posterior deviation of the ventricular outlet septum and ventricular septal defect. The cause of death was diagnosed as prolonged physiological closure of the ductus arteriosus complicated by interrupted aortic arch and followed by assumed ductal shock.

An autopsy case of a ruptured pseudoaneurysm of the ascending aorta complicated by previous cardiac surgery for ventricular septal defect.

A man in his thirties was found dead in bed. He had undergone repair of a ventricular septal defect in his infancy and had a 2-month history of antemortem chest pain. On autopsy, a ruptured saccular aneurysm of the ascending aorta was identified, and the right thoracic cavity was found to contain coagulated blood. The oval ostium of the aneurysm was smoothly endothelialized and a black suture was found near its edge. Histological analysis revealed a defect of the intimal and medial layers in the wall of the aneurysm and hypertensive changes in several organs. The cause of death was presumed to be the rupture of a pseudoaneurysm at the aortic cannulation site after a long postoperative period. In the present case, preexisting hypertension was suspected as the cause of the formation and rupture of the pseudoaneurysm. In case of unexpected death, it is important to examine the past medical history even after a long postoperative period.

Immunohistochemical Characterization of Sudden Death While Bathing:Evaluating the Expression of SP-A, HSP70 and c-Fos Following Drowningand Thermal Stress

Sudden death while bathing is a major issue in elderly Japanese individuals. Despite its growing incidence, the exact processes underlying sudden death while bathing remain unknown. This study was designed to establish reliable diagnostic markers for drowning while bathing, as well as to assess the pathological conditions associated with sudden death while bathing, by analyzing the immunohistochemical intensity and localization of surfactant protein-A (SP-A), heat shock protein 70 (HSP70) and c-Fos protein in the lungs and central nervous system, including the cerebral cortex and hippocampus. We found that a distinct pattern of immunohistochemical expression of SP-A, HSP70 and c-Fos in the lung can be utilized to diagnose drowning. In most of these individuals, c-Fos protein was absent from the central nervous system, providing insight into patterns of ischemia in the central nervous system, or a loss in consciousness while bathing. We also found that rapid hypothermia or a cold stimulus increased c-Fos expression in the hippocampus (CA4). Furthermore, our results suggest that HSP70 expression in the hippocampus (CA4) can be used to estimate the length of time between the initial occurrence of stress and death.

Lipid hydroperoxides in human plasma after ethanol consumption

Oxidative stress contributes to the pathogenesis of alcoholic liver disease. The purpose of this study is to estimate the amount of oxidative stress that is present when healthy humans consume moderate amounts of ethanol. Blood was collected from healthy volunteers before, 1 h, and 3 h after drinking 400 ml of Japanese rice wine at the rate of 100 ml per 5 min. The aldehyde dehydrogenase 2 genotype and the concentrations of blood ethanol, total lipid hydroperoxides (LOOH), and cholesterol hydroperoxides were determined. The plasma LOOH was found to have significantly increased 1h after drinking. Cholesterol hydroperoxides were not detected in plasma, either before or after drinking. There was no relationship between the LOOH and the ethanol concentration. We showed that one-shot of moderate ethanol consumption temporarily increases the plasma LOOH in healthy volunteers but excessive plasma LOOH compounds were eliminated within a short time.