Correlations between blood volatile hydrocarbon concentrations in different types of fire-related deaths.

Analysis of volatile hydrocarbons in blood from fire-related deaths provides useful information such as whether the victim inhaled smoke from the fire before death or whether an accelerant was used in the fire. In this study, we used headspace gas chromatography-mass spectrometry to quantify volatile hydrocarbons in post-mortem heart blood from 121 fire victims. The cases were classified into the following four groups according to the detected volatile hydrocarbons: construction fires without accelerants, kerosene fires, gasoline fires, and a group with no fire-related hydrocarbons detected (other fires). We investigated the relationships between blood concentrations of carboxyhemoglobin (COHb) and volatile hydrocarbons, and between various volatile hydrocarbons. The mean COHb concentrations were higher in the construction fire group than in the kerosene and gasoline fire groups. In the construction fire group, there was a high correlation coefficient between the concentrations of benzene and COHb and relatively high coefficient correlations between the concentrations of benzene and toluene, benzene and xylene, toluene and styrene, and ethylbenzene and styrene. Our results indicate that the relationships between benzene, xylene, and toluene concentrations could be used to distinguish between deaths in construction fires, kerosene fires, and gasoline fires.

High blood mirtazapine concentration in a newborn - A case of suspected postpartum infanticide.

We report a sudden death of an infant due to mirtazapine poisoning. A 15-day-old newborn boy was found dead when he was sleeping beside his mother who had suffered from panic disorder for approximately 1 year. After giving birth, she complained of palpitations and shaky hands, and was prescribed mirtazapine. The deceased newborn weighed 3,282 g and his height was 55 cm. There were no autopsy findings related to the death. The mirtazapine concentration as quantitated by liquid chromatography-tandem mass spectrometry analysis was 620 ng/mL in right heart blood, and was approximately 10 times higher than the therapeutic level in adults. Because transfer of mirtazapine into breast milk is low, mirtazapine was likely administered intentionally to the newborn. Based on the newborn's immature renal, liver, and blood-brain barrier function, the cause of death was attributed to mirtazapine poisoning. Poison-related homicide in the infant is rare. We report the first case of intentional mirtazapine poisoning case in a newborn.

Effects of arterial hemorrhage speed on the blood coagulation/fibrinolysis system and hemodynamics in rats.

: The effects of rapid hemorrhage on coagulopathy have been reported. However, the effects of different hemorrhage speeds on the blood coagulation/fibrinolysis system have not been investigated. This study aimed to compare different hemorrhage speeds for clarifying their effects on the coagulation/fibrinolysis system and circulation disorders in rats. Male Sprague-Dawley rats (301-396 g) were randomly assigned to five groups depending on hemorrhage speed and length of procedure: first, rapid (1.4 ml/min, 30-min bleeding); second, rapid-L (1.4 ml/min, 30-min bleeding and observation until 6 h); third, slow (0.1 ml/min, intermittently, 6-h bleeding); fourth, control (30-min observation); and fifth, control-L (6-h observation). Hemorrhage was induced by withdrawing blood until 40% of the estimated blood volume from the femoral artery. We measured vital signs, hematology, general chemistry, blood gas status, coagulation parameters, fibrinolytic markers [tissue-type plasminogen activator and plasminogen activator inhibitor one (PAI-1)], vascular endothelial damage (syndecan-1), and liver PAI-1 mRNA expression. Rapid hemorrhage induced elevation of lactate and syndecan-1 levels and prolonged prothrombin time and activated partial thromboplastin time in the rapid group. In contrast, slow hemorrhage did not induce these changes. Hemorrhage speed had no effect on plasma tissue-type plasminogen activator and hematology. Plasma PAI-1 levels were significantly increased in the rapid-L group, while liver PAI-1 mRNA levels were increased in the slow group. This study shows changes in the circulatory and fibrinolysis systems, depending on the hemorrhage speed. Hemorrhage might promote production of PAI-1, while tissue hypoxia due to rapid hemorrhage might promote release of PAI-1.

Cerebrospinal fluid neurotransmitter levels and central nervous system depression in a rat drug overdose model.

A neuropsychiatric drug overdose impairs physiological function via central nervous system (CNS) depression. In drug-related deaths, only the drug concentration can currently provide information regarding CNS depression in victims. In this study, using a drug overdose model, we investigated the ability of neurotransmitters in the cerebrospinal fluid (CSF) to serve as biomarkers for CNS depression. Four groups of rats were orally administered diazepam (200 mg/kg) and/or phenobarbital (100 mg/kg) or vehicle. In a hot plate test performed to assess physiological impairment, drug-administered animals showed prolongation of the response latency. Serum drug concentrations were also sufficient to observe the effect of drug overdose. The levels of benzoyl-derivatized neurotransmitters were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Noradrenaline, adrenaline, serotonin, melatonin, phosphoethanolamine, and histamine levels in the CSF decreased as the response latencies in the hot plate test increased. These reduced CSF neurotransmitter levels may represent physiological dysfunction through CNS depression.

Development of a fluvoxamine detection system using a Quenchbody, a novel fluorescent biosensor.

The misuse of psychotropic drugs intended for medical treatment represents a recent worldwide public health concern. Quenchbody (Q-body) is a novel fluoroimmunosensor that can detect an antigen immediately without additional reagents or washing steps. Here, we describe creating Q-bodies for the detection of the antidepressant fluvoxamine (FLV) and determining optimal conditions to achieve the highest fluorescence intensity (FI). We prepared five Q-bodies with the fluorophore labeled at either the N- or C- terminus and with different linker lengths. Fluorescence was measurable within minutes, indicating the interaction of Q-bodies with FLV. The normalized FI (FI ratio) of the N-terminus labeled Q-body increased approximately 1.5-fold upon FLV addition; Q-bodies labeled at the C-terminus did not significantly increase FI. Among the fluorescence dyes used in this study, Rhodamine 6G labeled Q-body showed the best FI ratio. EC50 values of the N-terminus labeled Q-bodies were similar (23.2-224nM) regardless of linker length or labeling dye. We examined whether the Q-body could be applicable to serum matrix instead of phosphate-buffered saline. The intact serum interfered strongly with the Q-body fluorescence. However, the FI ratios of the Q-body for FLV-spiked serum filtrate, for which proteins were removed by filtration, showed a dose-dependency for detecting FLV levels. Deproteinization, which does not interfere with Q-body fluorescence measurements, is likely necessary to detect serum FLV with high sensitivity. This study demonstrates the potential of Q-body probes as a tool towards developing creative immunoassay applications.

Liquid chromatography-tandem mass spectrometry detection of benzalkonium chloride (BZK) in a forensic autopsy case with survival for 18 days post BZK ingestion.

We report a forensic autopsy case of an elderly man who ingested unknown amount of germicidal disinfectant containing 50% benzalkonium chloride (BZK). He survived for 18 days after BZK ingestion and then died because of pneumonia. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to detect three BZK compounds (C12-BZK, C14-BZK and C16-BZK) in the blood. Extraction of BZK was carried out according to a modified QuEChERS method. Chromatographic separation was achieved on an ODS column and detection was performed in selected reaction monitoring mode. The accuracy and the precision were acceptable for quantitative analysis in the concentration range of 10-200 ng/mL for the three BZK compounds. BZK was detected in heart and femoral vein blood samples even 18 days after BZK ingestion. Taking into consideration clinical information during 18 days hospitalization and the autopsy findings, the cause of death was attributed to BZK poisoning. Several toxico-kinetic factors regarding absorption and excretion of BZK in the body were also discussed to elucidate the detection of BZK such a long time after ingestion.

A fatal poisoning case by intravenous injection of "bath salts" containing acetyl fentanyl and 4-methoxy PV8.

A man in his 30's was found at home, not breathing. He was admitted to an emergency hospital and the doctor confirmed his death. He had a history of methamphetamine abuse spanning several years, and while fresh needle marks were visible on his arm, the only other autopsy findings indicated an acute death. A small plastic bag containing a pale brown white powder, and a small amount of liquid in a syringe were found at the scene. The police forensic laboratory detected acetyl fentanyl and 4-methoxy PV8 (4-methoxy PHPP) in both the powder and the liquid. Scan analysis by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) identified acetyl fentanyl and 4-methoxy PV8 in the urine sample. Both drugs were quantitated simultaneously by liquid chromatography-tandem mass spectrometry (LC-MS/MS), using the selected reaction monitoring method. The concentration of acetyl fentanyl in the femoral vein blood, urine, and gastric contents were 153, 240, and 880ng/mL respectively, and the concentration of 4-methoxy PV8 in the femoral vein blood, urine, and gastric contents were 389, 245, and 500ng/mL respectively. Cause of death was attributed to acute poisoning by "bath salts" containing acetyl fentanyl and 4-methoxy PV8. Evidence indicated that self-administered intravenous injection was the most likely scenario, and that the deceased had been a habitual user of the "bath salt" drug for some time. Drugs detected in the gastric contents could be explained by the gastric secretion of basic drugs, or drug-containing bile entering the gastric contents.

Quantitative determination of n-butane metabolites in three cases of butane sniffing death.

Butane is an addictive volatile substance like toluene. We report three forensic autopsy cases of sudden death that occurred while sniffing n-butane and isobutane from portable gas cartridges. n-Butane and isobutane were detected in all three cases. In cases 1-3, n-butane concentrations in heart blood were 54.3, 25.5, and 30.7µg/mL, respectively. These concentrations were considered fatal according to the previous reports. In addition, n-butane metabolites (2-butanol and 2-butanone) were detected in cases 1 and 3 but not in case 2. Blood levels of 2-butanol and 2-butanone were 6.5 and 1.8µg/mL, respectively, in case 1, and 6.3 and 5.6µg/mL, respectively, in case 3. According to the police investigation, the decedent in case 1 had misused butane gas for more than 6 months in the period leading up to death. The decedent in case 3 also had a history of chronic misuse of butane gas. There was no history of chronic misuse of butane gas by the decedent in case 2. It was suspected that he attempted suicide via inhalation of butane gas using a plastic bag, leading to a rapid death. The presence or absence of n-butane metabolites might reflect the way of butane inhalation, such as the frequency and duration. Although additional experimental and case studies are necessary to establish the forensic applications of n-butane metabolite detection, it may be a useful method to understand the decedents' pattern of butane sniffing before death.

Detection of carbon monoxide poisoning that occurred before a house fire in three cases.

In our institutes, we perform a quantitative evaluation of volatile hydrocarbons in post-mortem blood in all fatal fire-related cases using headspace gas chromatography mass spectrometry. We previously reported that benzene concentrations in the blood were positively correlated with carbon monoxide-hemoglobin (CO-Hb) concentrations in fire-related deaths. Here, we present 3 cases in which benzene concentrations in the blood were not correlated with CO-Hb concentrations. A high CO-Hb concentration without a hydrocarbon component, such as benzene, indicates that the deceased inhaled carbon monoxide that was not related to the smoke from the fire. Comparing volatile hydrocarbons with CO-Hb concentrations can provide more information about the circumstances surrounding fire-related deaths. We are currently convinced that this is the best method to detect if carbon monoxide poisoning occurred before a house fire started.

Postmortem serum tenascin-C (TN-C) concentrations in forensic autopsy cases: A pilot study.

In forensic investigations, autopsy findings offer major clues for the diagnosis of the cause of death. Thus, various clinical biochemical markers are now being tested to complement conventional investigation in the field of forensic medicine. In this study, we focused on tenascin-C (TN-C), a glycoprotein present in the extracellular matrix and expressed in pathological states. We reviewed autopsy cases for a 4-year period (2006-2009) using autopsy records, and analyzed the blood serum concentrations of TN-C and C-reactive protein (CRP) in these cases (N=101). The TN-C levels were relatively higher in the postmortem serum samples than in the samples from healthy individuals, and in cases of head injury, both TN-C and CRP levels were high in the postmortem serum sample. Moreover, high TN-C levels were observed particularly in cases with a long survival period. These findings indicate that postmortem serum TN-C levels may represent a useful tool for identifying the cause of specific fatal traumas.

Quantitative evaluation of volatile hydrocarbons in post-mortem blood in forensic autopsy cases of fire-related deaths.

Volatile hydrocarbons in post-mortem blood from victims of fires were analyzed quantitatively by headspace gas chromatography mass spectrometry. The benzene and styrene concentrations in the blood were positively correlated with the carboxyhemoglobin (CO-Hb) concentration, which is evidence that the deceased inhaled the hydrocarbons and carbon monoxide simultaneously. By contrast, the concentrations of toluene and CO-Hb in the blood were not significantly correlated. This lack of correlation could be explained by two different sources of toluene, with low blood concentrations of toluene arising when the deceased inhaled smoke and high blood concentrations of toluene arising when the deceased inhaled petroleum vapor or other unknown vapors. The quantity of soot deposited in the respiratory tract was classified into four grades (-, 1+, 2+, 3+). The mean CO-Hb concentration in the 1+ soot group was significantly lower than those in the 2+ (p<0.05) and 3+ (p<0.01) soot groups. The blood CO-Hb concentrations in the 1+ soot group were all below 30%. Those indicated that the deceased aspirated smoke that contained both soot and carbon monoxide. The wide variation in CO-Hb concentrations for each soot classification could be caused by the different types of smoke produced by different materials. For example, petroleum combustion with a limited supply of oxygen, like in a compartment fire, may produce a large volume of dense black smoke that contains a large quantity of soot. Soot deposits in the airways and the blood CO-Hb concentration are basic and essential autopsy findings that are used to investigate fire-related deaths. The quantitative GC-MS analysis of blood volatile hydrocarbons can provide additional useful information on the cause of the fire and the circumstances surrounding the death. In combination, these three findings are useful for the reconstruction of cases.

Vertebral arterial injury due to fatal dog bites.

Dog bite-related injuries and fatalities are major public health problem worldwide. The authors present a case of a Japanese Tosa-dog bite-related fatality in an old woman who died from a vertebral arterial laceration with the C5 vertebral fracture. This was an uncommon type of injury that dog bites injured the vertebral artery only without damage to carotid artery. The identity of the Tosa-dog as an offending dog in this case was unsuccessful by a DNA analysis. Then the detection of the offending dog was made by comparisons of the dental casts of the dog with the victim's wounds. This case report describes making dental cast of the dog, and statistical data on dog-bite incidents in Japan.

Immunohistochemical demonstration of the distribution of chloroquine (CQ) and its metabolites in CQ-poisoned mice.

Chloroquine (CQ) distribution in tissues of acutely poisoned mice was demonstrated by immunohistochemistry using anti-CQ polyclonal antibodies (PAC). PAC recognized 4-amino-7-chloro-quinoline structure and sufficiently reacted with CQ and CQ's metabolite bisdesethyl-chloroquine. In the brain, CQ and its metabolites (CQs) localized in the region of the choroids plexus, indicating an important role in the blood-cerebrospinal barrier system. In the heart, most regions showed diffused positive staining, and relatively strong reaction was observed in Purkinje cells, indicating an important role in acute CQ toxicity. In the lungs, CQs were observed in the bronchial epithelium, type II pneumocytes, and on the surface of alveolar walls. It was suggested that CQs were excreted to the alveolar wall with surfactant phospholipids, which are produced by type II pneumocytes. In the liver, CQs were concentrated in the centrolobular area rather than in the periportal area, in agreement with CQ's metabolic pathway. In the kidneys, tubular cells were strongly stained compared to glomerular capsules, and the distal part of renal tubules was better stained than the proximal tubules. These findings suggested that CQs were predominantly excreted or reabsorbed through the distal tubules and the collecting duct. Distribution of CQs in tissues presented here were mostly consistent with the physico-chemical properties of CQ and its metabolites. However, the elucidation of CQs' localization in Purkinje cells remains open. Further experimental studies at the level of microorganella will be needed to clarify the present result.

[Two cases of diphenhydramine related suicide].

Two suicidal cases associated with ingestion of diphenhydramine (DPH) were reported. Case 1 is a typical DPH overdose case of a young man with the blood DPH concentration of 12.2 microg/ mL. Case 2 is a double suicide of a man and a woman. They ingested DPH and fell asleep in a vehicle which had a cooking clay charcoal stove. Their blood DPH concentrations were 0.4 and 0.7 microg/mL, which were high enough to make them sleep. Their cause of death, however, was carbon monoxide poisoning with blood CO-Hb concentration of 14 and 19%. DPH is a low toxic agent and is available as an OTC drug in Japan. Similar fatal cases can be expected to happen in Japan.

An accidental death due to Freon 22 (monochlorodifluoromethane) inhalation in a fishing vessel.

A case of accidental Freon 22 (monochlorodifluoromethane) poisoning in a fishing vessel is reported. Forensic autopsy revealed severe pulmonary edema and congestion (left lung; 576 g, right lung; 740 g). GC-MS analysis clearly showed that the deceased inhaled Freon 22 gas prior to his death. Freon 22 concentration was 169+/-7.0 microg/ml in the heart blood. The distribution pattern of Freon 22 in tissue samples was similar to that in previously reported cases. The brain had the highest concentration of Freon 22 followed by the spleen, liver, kidney and lung, respectively. Histopathologically, Oil red O staining of the liver showed many small, positive red areas in the cytosol, which have been reported in other cases of Freon 22 poisoning. However, Schmorl staining revealed that most areas of Oil red O positivity were lipofuscin granules. Lipofuscin in the liver, which closely relates to aging and other cell stresses, could have a relevance to Freon 22 exposure, but further experimental studies are needed to confirm it.

Estimation of early postmortem intervals by a multiple regression analysis using rectal temperature and non-temperature based postmortem changes.

Five general methods based on rectal temperature and a multiple regression analysis using rectal temperature and non-temperature based postmortem changes were applied to 212 postmortem cases of within 24h postmortem (PM) intervals. Non-temperature based postmortem changes of rigidity, hypostasis and corneal turbidity were numerically categorized and used with rectal temperatures as four statistical variables in the multiple regression analysis. The correlation coefficient values between true and calculated postmortem intervals were 0.78-0.82 in the five general methods based on rectal temperature. The multiple regression analysis produced a multiple correlation coefficient value of 0.89 and according to the error ranges of the PM intervals, 72% of the cases were estimated within the error of +/-1.0 h and 92% within +/-5.0 h. Although assessments of non-temperature based PM changes are mostly subjective and have a wide variation, the present study demonstrated a usefulness of non-temperature based PM changes in the estimation of PM intervals.

HPLC analysis of anti-malaria agent, chloroquine in blood and tissue from forensic autopsy cases in Tanzania.

HPLC analysis of anti-malaria agent, chloroquine (CQ) in blood and tissues with a simple HCl back extraction method was applied to three forensic autopsy cases in Dar es Salaam, Tanzania. CQ concentrations in femoral vein blood were 8.5, 48.4 and 43.8 microg/ml in three cases, respectively, which were high enough to attribute the cause of deaths to an acute CQ poisoning. There were great site dependent variations in blood CQ levels. The right heart blood samples were very high, which may be explained by incomplete distribution of the drug before death or postmortem diffusion from liver and its surrounding blood, as high CQ levels were remarkable in the liver. Suicidal and accidental CQ poisonings are very common and CQ is a very important chemical in the field of forensic toxicology in Tanzania.

Fatal intussusception in infancy: an experience in forensic autopsy.

Intussusception, although a common cause of pediatric surgical emergencies, is a rarely fatal condition. A 7-month-old infant who was discovered in her cot was unresponsive and pronounced dead after 2 h of uneventful cardiopulmonary resuscitation in an emergency hospital. Forensic autopsy which was performed in order to clarify the circumstances surrounding the death revealed intussusceptions at two sites of the ileum. Although morbidity and mortality rates from the condition have progressively declined in recent decades but avoidable deaths still occur as was experienced in the present case. The forensic pathology significance in this case was the occurrence of 'painless intussusception' whereby the affected child clinically exhibited no discomfort or characteristic features of acute abdomen until death. In summary, the present case has exhibited an uncommon fatal occurrence and demonstrated the importance of forensic autopsy in such unexpected sudden infant deaths.

Inhibitory activity of epigallocatechin gallate (EGCg) in paraquat-induced microsomal lipid peroxidation--a mechanism of protective effects of EGCg against paraquat toxicity.

Recently we have reported that epigallocatechin gallate (EGCg), a major component of Japanese green tea, significantly increased the survival rate of paraquat (Pq) poisoned mice. This paper describes two biochemical activities of EGCg, which relate to its protective effects against Pq toxicity. EGCg inhibited Pq-induced microsomal malondialdehyde (MDA) productions in rat liver microsome system containing 40 microM FeSO(4). Forty micromolar EGCg inhibited MDA production significantly. EGCg may inhibit the Pq-induced MDA production by at least two mechanisms. One may be iron-chelating activity as the inhibition disappeared when excess amounts of FeSO(4) were added to the reaction mixture, which indicated that EGCg reduced iron driven lipid peroxidation by pulling out available irons in the reaction mixture. The other is radical scavenging activity. EGCg scavenged DMPO-OOH spin adducts generated by the microsome-Pq system. The dose response curve of EGCg was similar to that obtained by ascorbic acid which is a typical water-soluble radical scavenger. Although ascorbic acid had a potential activity of scavenging superoxide radicals, it can not be recommended to use for the treatment of Pq poisoning, because ascorbic acid acts as a pro-oxidant in the presence of free transition metal ions by accelerating the Fenton reaction (Fe(2+)+H(2)O(2)-->Fe(3+)+OH(-)+OH*), which is responsible for lipid peroxidation. On the contrary, EGCg inhibited iron-driven lipid peroxidation presumably not only by chelating to Fe ions but also by scavenging superoxide radicals, which are responsible for the reduction of ferric (Fe(3+)) to ferrous (Fe(2+)) that catalyzes the Fenton reaction. Chelating and radical scavenging activity of EGCg can be expected simultaneously in the occurrence of Pq toxicity, which may explain the protective effects of EGCg against Pq toxicity.

[Ethanol concentrations in multi-site sampling blood in forensic autopsy cases--a retrospective analysis over a period of six years (1994-1999) in Kumamoto University].

Ethanol and n-propanol concentrations in forensic autopsy cases determined in Department of Forensic Medicine, Kumamoto University School of Medicine were reviewed retrospectively. Out of 388 autopsies in 6 years (1994-1999), ethanol was positive in 88 (22.7%) cases. Higher positive rates were observed in bleeding and burning cases compared to other cases. Histograms of the blood ethanol concentrations in all ethanol positive cases had two peaks at 0.1 mg/ml to 0.5 mg/ml and 1.5 mg/ml to 2.0 mg/ml ranges, which indicated that not only an intermediate but also a weak drunkenness level could be a risk factor of being involved in forensic fatalities. There were no differences in mean ethanol concentrations in the blood samples of the right, left and whole heart blood collected from each victim. The femoral blood, however, was slightly higher than those of heart blood. N-Propanol, an indicator for postmortem ethanol production, was detected in 14.7% of stomach contents samples as early as 6 to 12 hours of post mortem intervals, whereas it was not remarkable in urine and femoral vein blood.