Time-frequency analysis reveals an association between the specific nuclear magnetic resonance (NMR) signal properties of serum samples and arteriosclerotic lesion progression in a diabetes mouse model.

Diabetes causes arteriosclerosis, primarily due to persistent hyperglycemia, subsequently leading to various cardiovascular events. No method has been established for directly detecting and evaluating arteriosclerotic lesions from blood samples of diabetic patients, as the mechanism of arteriosclerotic lesion formation, which involves complex molecular biological processes, has not been elucidated. "NMR modal analysis" is a technology that enables visualization of specific nuclear magnetic resonance (NMR) signal properties of blood samples. We hypothesized that this technique could be used to identify changes in blood status associated with the progression of arteriosclerotic lesions in the context of diabetes. The study aimed to assess the possibility of early detection and evaluation of arteriosclerotic lesions by NMR modal analysis of serum samples from diabetes model mice. Diabetes model mice (BKS.Cg db/db) were bred in a clean room and fed a normal diet. Blood samples were collected and centrifuged. Carotid arteries were collected for histological examination by hematoxylin and eosin staining on weeks 10, 14, 18, 22, and 26. The serum was separated and subjected to NMR modal analysis and biochemical examination. Mice typically show hyperglycemia at an early stage (8 weeks old), and pathological findings of a previous study showed that more than half of mice had atheromatous plaques at 18 weeks old, and severe arteriosclerotic lesions were observed in almost all mice after 22 weeks. Partial least squares regression analysis was performed, which showed that the mice were clearly classified into two groups with positive and negative score values within 18 weeks of age. The findings of this study revealed that NMR modal properties of serum are associated with arteriosclerotic lesions. Thus, it may be worth exploring the possibility that the risk of cardiovascular events in diabetic patients could be assessed using serum samples.

Simultaneous Analysis of Zolpidem, Four Hydroxyzolpidems and Two Zolpidem Carboxylic Acids in Postmortem Urine Using Liquid Chromatography--Tandem Mass Spectrometry.

Zolpidem (ZOL) is a short-acting hypnotic that is sometimes used in drug-facilitated crimes such as sexual assaults, robbery and homicides. Therefore, it is important to understand the metabolism of ZOL. This study quantified ZOL and its metabolites, including two carboxylic acids (zolpidem phenyl-4-carboxylic acid [M1] and 6-carboxylic acid [M2]) and four hydroxyzolpidems (4-(hydroxymethyl)phenyl zolpidem [M3], 6-hydroxymethyl zolpidem [M4], 7-hydroxyzolpidem [7OH] and 8-hydroxyzolpidem [8OH]) in postmortem urine using liquid chromatography--triple quadrupole mass spectrometry. The concentration of M1 was highest in all cases, followed by total 7OH in five of six samples. The concentrations of M2 and total M4 were relatively high. Most of M4 and 8OH were excreted as conjugates, whereas up to 55% of 7OH was excreted in its free form. Peaks corresponding to zolpidem dihydrodiol (ZHDH), dihydro(hydroxy)zolpidem cysteine adduct (DHZCys) and zolpidem cysteine adduct (ZCys) were also detected in all the urine samples. ZDHD was excreted as conjugates, whereas almost all DHZCys and ZCys were in their free form.

Qualitative analysis of 7- and 8-hydroxyzolpidem and discovery of novel zolpidem metabolites in postmortem urine using liquid chromatography-tandem mass spectrometry.

PURPOSE: Zolpidem (ZOL) is a hypnotic sometimes used in drug-facilitated crimes. Understanding ZOL metabolism is important for proving ZOL intake. In this study, we synthesized standards of hydroxyzolpidems with a hydroxy group attached to the pyridine ring and analyzed them to prove their presence in postmortem urine. We also searched for novel ZOL metabolites in the urine sample using liquid chromatography-triple quadrupole mass spectrometry (LC-QqQMS) and liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QqTOFMS). METHODS: 7- and 8-Hydroxyzolpidem (7OHZ and 8OHZ, respectively) were synthesized and analyzed using LC-QqQMS. Retention times were compared between the synthetic standards and extracts of postmortem urine. To search for novel ZOL metabolites, first, the urine extract was analyzed with data-dependent acquisition, and the peaks showing the characteristic fragmentation pattern of ZOL were selected. Second, product ion spectra of these peaks at various collision energies were acquired and fragments that could be used for multiple reaction monitoring (MRM) were chosen. Finally, MRM parameters were optimized using the urine extract. These peaks were also analyzed using LC-QqTOFMS. RESULTS: The presence of 7OHZ and 8OHZ in urine was confirmed. The highest peak among hydroxyzolpidems was assigned to 7OHZ. The novel metabolites found were zolpidem dihydrodiol and its glucuronides, cysteine adducts of ZOL and dihydro(hydroxy)zolpidem, and glucuronides of hydroxyzolpidems. CONCLUSIONS: The presence of novel metabolites revealed new metabolic pathways, which involve formation of an epoxide on the pyridine ring as an intermediate.

Deaths in Bathtubs in Japan: Forensic and Clinical Implications.

In Japan, deaths in bathtubs or bathtub deaths are frequently investigated as unnatural deaths. About 19,000 bathtub deaths occur annually in Japan. This pattern of death has become a social issue in forensic pathology and emergency medicine and public health. It is assumed that the death of an adult by drowning in a bathtub cannot be avoided due to disturbance of consciousness. The PubMed database was used for literature search using the retrieval words, "bathing "OR" bathtub "AND" submersion "OR" drowning "OR" death "OR" cardiopulmonary arrest". From the epidemiological characteristics and pathophysiological findings of bath mortality in Japan, three etiologies of impaired consciousness have been proposed: acute ischemic heart failure, heatstroke, and blood pressure fluctuation. Moreover, other causes such as epilepsy and alcohol or drug intake cannot be ignored as potential risks for death in a bathtub. It is also important to note the possibility of suicide and, although extremely rare, homicide in a bathtub. Despite research, the exact causal relationship between bathtub bathing and death remains unclear. Further, the cause of death by postmortem investigation is not always easily determined. Hence, it is desirable to carry out a field survey of causes of death, including bathing conditions, and, wherever possible, a complete autopsy survey. An exclusion of critical cases such as crime-related death, suicide, drug poisoning, and carbon monoxide poisoning is optimal. Of the many hypotheses about the causes of bathtub mortality, the most consistent hypothesis will be medically inferred from the death history, case findings, and test results.

Determining the effect of water temperature on the T1 and T2 relaxation times of the lung tissue at 9.4 T MRI: A drowning mouse model.

Japanese individuals have a unique culture of soaking in a bathtub, and forensic pathologists have experienced fatal cases due to drowning. However, T1 and T2 relaxation times of a drowning lung are poorly documented. In the present study, we investigated the relationship between drowning water temperature and T1 and T2 relaxation times of drowning lung tissues at 9.4 T MRI (Bruker, BioSpec94/20USR). The mice used as animal drowning models were directly submerged in freshwater. Water temperature was set to 8 °C-10 °C (cold), 20 °C-22 °C (normal), 30 °C, and 45 °C. The regions of interest (ROIs) on the axial section of the third slice were set at the central and peripheral areas of each-the left and the right-lung. T1 relaxation times measured immediately after death differed by the presence or absence of soaking water, except in case of cold water temperature. In the drowning groups, T1 relaxation time showed a linear dependency on water temperature. By contrast, T2 relaxation time was almost constant regardless of the presence of drowning under the same temperature condition; when compared in the lung areas of the same individuals, the times were uniformly reduced in drowning models. To minimize the effects of hypostasis and decomposition, we performed measurements immediately after death and were able to determine the noticeable difference in drowning water temperature. These results may be useful for qualitative assessments of a drowning lung and may serve as a basis when imaging the human body during forensic autopsy cases.

Time-frequency analysis of serum with proton nuclear magnetic resonance for diagnosis of pancreatic cancer.

Although serum markers such as carcinoembryonic antigen (CEA) and carbohydrate antigen (CA19-9) have been widely used in screening for pancreatic cancer (PC), their sensitivity and specificity are unsatisfactory. Recently, a novel tool of analyzing serum using the short-time Fourier transform (STFT) of free induction decays (FIDs) obtained by 1H-NMR has been introduced. We for the first time evaluated the utility of this technology as a diagnostic tool for PC. Serum was obtained from PC patients before starting any treatments. Samples taken from individuals with benign diseases or donors for liver transplantation were obtained as controls. Serum samples from both groups underwent 1H-NMR and STFT of FIDs. STFT data were analyzed by partial least squares discriminant analysis (PLS-DA) to clarify whether differences were apparent between groups. As a result, PLS-DA score plots indicated that STFT of FIDs enabled effective classification of groups with and without PC. Additionally, in a subgroup of PC, long-term survivors (≥ 2 years) could be discriminated from short-term survivors (< 2 years), regardless of pathologic stage or CEA or CA19-9 levels. In conclusion, STFT of FIDs obtained from 1H-NMR have a potential to be a diagnostic and prognostic tool of PC.

Solitary Deaths in the Tokyo Metropolis and Labor Force Status: Characteristics of Unnatural Deaths at Home among Persons Living Alone.

OBJECTIVE: To identify associations of solitary death with social determinants of health, namely, labor force status and welfare status, in Tokyo in 2015. METHODS: We obtained data on solitary deaths in 2015 in the 23 special wards of Tokyo and calculated the incidence rate and postmortem interval of solitary death in relation to sex, age, and labor force status. RESULTS: Data for 3,972 solitary deaths (2,785 males, 1,187 females) were analyzed. The non-employed rate was 79.3% among males and 89.5% among females. The incidence rate was significantly higher among non-employed persons than among employed persons in both sexes. Moreover, with the exception of women 65 years or older, the postmortem interval was significantly longer among non-employed persons than among employed persons in both sexes. CONCLUSIONS: The incidence rates of solitary death were significantly higher among non-employed persons than among employed persons in both sexes, and the postmortem interval was significantly longer for non-employed persons.

Short-time Fourier Transform of Free Induction Decays for the Analysis of Serum Using Proton Nuclear Magnetic Resonance.

Proton nuclear magnetic resonance (NMR) is useful for the analysis of biological samples such as serum. Free induction decays (FIDs) are NMR signals that follow a radio-frequency pulse applied at the resonance frequency. Short-time Fourier transform (STFT) is a basic method for time-frequency analyses. The purpose of this study was to ascertain whether the STFT of FIDs enables the sensitive detection of changes and differences in serum properties. FIDs were obtained from serum collected from young, healthy, male volunteers ≤ 40 years of age and seniors ≥ 65 years of age. Temporal changes in the instantaneous amplitudes for the time-domain analysis, fast Fourier transform for frequency-domain analysis, and STFT were applied to the FIDs. The STFT-based spectrogram represented the complex frequency components that changed dynamically over time, indicating that the spectrogram enabled the visualization of the features of an FID. Furthermore, the results of a partial least-squares discriminant analysis demonstrated that the STFT was superior to the other two methods for discriminating between serum from younger and older subjects. In conclusion, the STFT of FIDs obtained from proton NMR measurements was useful for evaluating similarities and dissimilarities in the FIDs obtained from serum samples.

Association between sudden unexpected deaths in bathtubs and ambient temperature among elderly Japanese adults: A time-series regression study.

BACKGROUND: Sudden unexpected deaths in bathtubs among elderly Japanese adults occur predominantly during the cold season. This study investigated the relationship between these deaths and bathing day temperature among elderly adults in Tokyo. METHODS: Data for 1408 cases of bath-related deaths from January 1 to December 31, 2015 were obtained from the Tokyo Medical Examiner's Office. We excluded 409 cases for the following reasons: criminal death, injury-related death, suicide, intoxication, non-sudden death, not bathtub-related death, out-of-bathroom death, subject aged under 65 years, undetermined bathing date, institutional housing, and bathing not at subject's home. Ultimately, 999 cases were analyzed. Daily mean temperature data were collected. A time-series regression study was performed to estimate the influence of sex, age, and bathing day temperature. Monthly changes in the population bathing in a bathtub were considered in the model. RESULTS: The relative risk (RR) of sudden unexpected death in a bathtub was 1.381 for males (95% confidence interval [CI]: 1.218-1.564) compared to females. The RRs were 4.182 (95% CI: 3.523-4.986) and 9.382 (95% CI: 7.836-11.273) among those aged 75-84 years and ≥85 years, respectively, compared to among those aged 65-74 years. The RR increased to 1.092 (95% CI: 1.082-1.102) as the daily mean temperature decreased by 1 °C. CONCLUSION: Sudden unexpected death in a bathtub correlated with bathing day temperature among elderly Japanese adults, and extremely low temperature, male sex, and older age increased the risk of such death. Our findings provide insight into preventing sudden unexpected deaths in bathtubs.

Pattern recognition analysis of proton nuclear magnetic resonance spectra of postmortem cerebrospinal fluid from rats with drug-induced seizure or coma.

Cerebrospinal fluid (CSF) is routinely subjected to gross evaluation in postmortem investigations; however, its use in chemical evaluations has not been fully realized. Analysis of nuclear magnetic resonance (NMR) spectra with pattern recognition methods was applied to CSF samples. Rats were treated with pentylenetetrazol (PTZ) to induce seizure or pentobarbital (PB) to induce coma, and postmortem CSF was collected after CO2 gas euthanization. Pattern recognition analysis of the NMR data was performed on individual postmortem CSF samples. The aim of this study was to determine if pattern recognition analysis of NMR data could be used to classify the rats according to their drug treatment. The applicability of NMR data with pattern recognition analysis using postmortem CSF was also assessed. Partial Least Squares-Discriminant Analysis (PLS-DA) score plots indicated that the PTZ, PB, and NS (control) groups were clustered and clearly separated. PLS-DA correlation loading plots showed respective spectral and category variances of 41% and 42% for factor 1, and 17% and 27% for factor 2. Thus, factors 1 and 2 together described 58% (41%+17%) and 69% (42%+27%) of the variation, respectively. NMR study of postmortem CSF has the potential to be utilized as both a novel forensic neurochemistry method and in the clinical setting.

An Autopsy Case of a Pregnant Woman With Severe Placental and Fetal Damage From Domestic Violence.

We present an autopsy case of a pregnant woman who was a victim of domestic violence. The deceased showed injuries mainly to her head and abdomen. Postmortem examination revealed 1400 mL of abdominal hemorrhage, ablation of the perimetrium, placental avulsion, and intracranial hematoma. The cause of death was diagnosed as hemorrhagic shock. The uterus contained a fetus of 7 months' gestational age. Fetal autopsy revealed laceration of the lungs, laceration and avulsion of the liver, and 15 mL of hemoperitoneum. Both placental and fetal injuries suggested repeated severe attacks to the abdomen, such as those expected to result from kicking or hitting.

High-resolution 3D-MRI of postmortem brain specimens fixed by formalin and gadoteridol.

PURPOSE: The purpose of this investigation was to compare magnetization-prepared rapid gradient echo (MP-RAGE) images with T1-weighted images (T1WI) and T2-weighted images (T1W2) of postmortem brain tissue fixed by admixtures of formalin and gadoteridol. We additionally sought to explore the feasibility of using fixed brain magnetic resonance imaging (MRIs) in forensic practices. METHODS: Specimens included in the study were eight whole brains that had been removed during forensic autopsy. Brain specimens were randomly divided into three groups and MRIs were performed either (A) the day of autopsy (n=2) on unfixed tissue, (B) after immersion fixation in 20% formalin (n=3), or (C) after immersion fixation in 20% formalin mixed with 4 mL/L ProHance® (gadoteridol) (n=3). T1WI, T2WI, and MP-RAGE images of all group samples were acquired with a 3T clinical MR scanner. Gray and white matter contrasts of the cortex and basal nucleus in every fixation group and image sequence were then visually compared. RESULTS: Gray/white matter contrasts of the cortex were good in all images obtained by MP-RAGE, and T1WIs of specimens fixed by formalin and gadoteridol-mixed formalin. Additionally, gray/white matter contrast in the basal nucleus was sufficient in the MP-RAGE sequence of specimens fixed by gadoteridol-mixed formalin. CONCLUSIONS: MRI of brains immersion-fixed in formalin and gadolinium could serve as a promising tool for neuropathological assessment in forensic practices.

An unusual case of suicide by handcrafted shotgun and slug.

Injuries by handcrafted firearms are uncommon, and those by handcrafted shotguns are very rare. We present an unusual case of suicide using a handcrafted shotgun and slug. A 73-year-old male, who was a glass-grinding artisan, was found dead at a workshop on the premises of his residence. Autopsy showed a contact shotgun wound to the forehead, which perforated the cranium and caused extensive damage to the brain. The handcrafted shotgun was found in the proximity of the body. The shotgun consisted of metal pipes fixed to the vise and did not take the form of a conventional gun. The recovered slug was also found to be manufactured manually. It appeared that the handcrafted shotgun was only intended for committing suicide using his ballistics knowledge, skills, and resources.

An autopsy case of otogenic intracranial abscess and meningitis with Bezold's abscess: evaluation of inflammatory bone destruction by postmortem cone-beam CT.

The deceased was an unidentified young male found unconscious on a walkway. On autopsy, outer and inner fistulae of the left temporal bone, subcutaneous abscess in the left side of the neck and head, and an intracranial abscess were noted. A portion of the left temporal bone was removed and scanned by cone-beam computed tomography (CT) (normally used for dentistry applications) to evaluate the lesion. The cone-beam CT image revealed roughening of the bone wall and hypolucency of the mastoid air cells, consistent with an inflammatory bone lesion. According to autopsy and imaging findings, the cause of death was diagnosed as intracranial abscess with Bezold's abscess secondary to left mastoiditis as a complication of otitis media. Although determining the histopathology of bone specimens is time-consuming and costly work, we believe that use of cone-beam CT for hard tissue specimens can be useful in forensic practice.

A case of lethal soft tissue injuries due to assault.

A 42-year-old male had been assaulted by his family over the two previous days and went into a deep coma. When the emergency technician arrived, the patient was in a state of cardiopulmonary arrest. On arrival, his electrocardiogram showed asystole. His body showed swelling with subcutaneous hemorrhage, suggesting multiple contusional wounds. Serum biochemistry evaluation revealed blood urea nitrogen of 80 mg/dL, creatinine of 5.99 mg/dL, creatine phosphokinase of 10,094 IU/L, and potassium of 11.0 mEq/L. Advanced cardiopulmonary resuscitation failed to obtain a return of spontaneous circulation. Laboratory findings revealed rhabdomyolysis, renal failure, and hyperkalemia. Autopsy did not indicate the direct cause of death to be traumatic organ injuries. Because trauma was not the direct reason of death, we speculated that the patient died of hyperkalemia induced by multiple contusional soft tissue injuries, following rhabdomyolysis, hemolysis, and acute renal failure. The physician should maintain a high index of suspicion for hyperkalemia induced by rhabdomyolysis and acute renal failure, especially in patients presenting with symptoms of multiple soft tissue injuries with massive subcutaneous hemorrhaging.

Measurement of temperature changes in cooling dead rats using magnetic resonance thermometry.

Magnetic resonance imaging thermometry has been introduced as a technique for measurement of temperature changes in cooling dead rats. Rat pelvic magnetic resonance images were acquired sequentially more than 2h after euthanasia by halothane overdose. A series of temperature difference maps in cooling dead rats was obtained with calculating imaging phase changes induced by the water proton frequency shift caused by temperature changes. Different cooling processes were monitored by the temperature difference maps in the rats. Magnetic resonance imaging thermometry applied in the study of laboratory animals could theoretically reproduce a variety of causes of death with different environmental conditions. Outcomes from experimental animal studies could be translated into a temperature-based time of death estimation in forensics.

[Statistical investigation on cases associated with medical implication in administrative autopsy within the 23 wards of Tokyo: the role of the medical examiner system in investigation of cases associated with medical implication].

Actual circumstances of administrative autopsies which proved connections between medical implication and death had not been very clear in the past. Therefore, using the records of administrative autopsies performed from 2003 to 2005 at Tokyo Medical Examiner's Office, this study looked into the cases in which a certain level of connection between medical implication and death was proved or suspected. This study dealt with 877 cases. The largest age group among the male was the one between 65 and 74, and the number of female cases increased as the age increased. The percentage of the studied cases among all the administrative autopsy cases has become larger after 2004. As regarding medical departments for implication, "internal medicine" had the largest number of the cases, and "unknown", "psychiatry", and "emergency" followed in order. 30 percent were being hospitalized during the final medical consultations, and the percentage went up to just over 60 when the deaths during and on the day after the final consultations were added to the figure. Regarding the causes of death, the great majority was natural death, and the other causes were 'unknown', 'fall', and 'asphyxia'. Also, about 80 percent of the natural deaths were caused by circulatory, gastroenteric and respiratory diseases. Contradictions between clinical and forensic diagnoses were found in approximately 10 percent of the cases. In any case, medical examiners are to diagnose the causes of deaths by autopsy, not to evaluate the quality and safety of medical treatment. But if the quality and safety of medical treatment could be improved through the diagnoses of administrative autopsy, the medical examiner system would function practically as a part of administration for health and welfare. However, as it handles not only deaths by medical treatment but also all the other types of unnatural deaths, the system can also deal with other social problems, for which the correct causes of death must be diagnosed initially. It is socially very unhealthy to focus only on death associated with medical implication, as other types of unnatural death could be seen as relatively less important. Therefore, it is considered that the medical examiner system is effective for investigation of a variety of unnatural death as well as cases associated with medical implication.

Gas dispersal potential of bedding as a cause for sudden infant death.

We assessed the gas dispersal potential of bedding articles used by 14 infants diagnosed with sudden unexpected infant death at autopsy. Of these cases, eight exhibited FiCO(2) values greater than 10% within 2.5 min, six of which were found prone and two supine. The results demonstrated that these eight beddings had a high rebreathing potential if they covered the babies' faces. We did not, however, take into account in our model the large tissue stores of CO(2). As some bicarbonate pools will delay or suppress the increase of FiCO(2), the time-FiCO(2) graphs of this study are not true for living infants. This model, however, demonstrated the potential gas dispersal ability of bedding. The higher the FiCO(2) values, the more dangerous the situation for rebreathing infants. In addition, FiO(2) in the potential space around the model's face can be estimated mathematically using FiCO(2) values. The FiO(2) graph pattern for each bedding item corresponded roughly to the inverse of the FiCO(2) time course. The FiO(2) of the above eight cases decreased by 8.5% within 2.5 min. Recent studies using living infants placed prone to sleep reported that some babies exhibited larger decreases in FiO(2) than increases observed in FiCO(2). While the decrease of FiO(2) in our model is still theoretical, CO(2) accumulation and O(2) deprivation are closely related. If a striking O(2) deficiency occurs in a short period, babies can lose consciousness before an arousal response is evoked and all infants could be influenced by the poor gas dispersal of bedding; the main cause of sudden death in infancy would thus be asphyxia. When the bedding is soft, the potential for trapping CO(2) seems to be high; however, it is impossible to assess it by appearance alone. We sought to provide some objective indices for the assessment of respiratory compromise in relation to bedding using our model. When a baby is found unresponsive with his/her face covered with poor gas dispersal bedding, we should consider the possibility of asphyxia.

Adult autopsy case with marked myositis ossificans: association with repetitive physical assault and battery.

A man in his thirties was found dead in his room. The autopsy findings were as follows: multiple bruises and subdermal edema, small sized stab wounds, a number of skin scars, new and old rib fractures, mild liver contusion, and marked myositis ossificans traumatica (MOT) at both thighs. In particular, the MOT was closely associated with repetitive physical assault and battery. The deceased did not have any lethal injuries of internal organs or great vessels, and the cause of death was diagnosed as traumatic hypovolemic shock. The deceased seemed to have accepted as fate his "specific situation" in which he would be subjected to continued physical violence from his cohabiter, a woman in her thirties.

Sudden collapse after minor head injury in an elderly man; association with cardiocerebral decompensation and fat embolism syndrome.

An 86-year-old man suffering from right hemiparesis and dementia fell from a stretcher and sustained laceration and bruising to his right eyebrow. He was brought to an emergency room, and his wounds were treated. Shortly after discharge, his respiratory and mental status dramatically declined. Despite supportive care, he died about three hours after re-admission. Autopsy revealed a minor laceration to the eyebrow with minor intracranial injuries, mild cardiomegaly (370 g) with right atrial dilatation, pericardial effusion (170 ml) and marked pulmonary edema. Microscopically, fatty droplets were observed in the lung capillaries, the glomeruli and the intracerebral vessels. Although the extent of the original injuries seemed insufficient to cause systemic fat embolism syndrome (FES), the patient's decreased cardiac reserves and stress associated with this event likely contributed to his death consistent with the physiochemical model of FES pathogenesis.