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.

Effect of Ethanol on Gene Expression in Beating Neonatal Rat Cardiomyocytes: Further Research with Ingenuity Pathway Analysis Software.

BACKGROUND: There have been no comprehensive studies of changes in heart gene expression due to ethanol exposure. Therefore, we attempted to determine gene expression in cultured neonatal rat cardiomyocytes exposed to ethanol (0, 10, 50, 100 mM) for 24 h. METHODS: The total RNA extract of beating cardiomyocytes was evaluated by DNA microarray analysis, and fold changes (FCs) in differential gene expression of ethanol-exposed cardiomyocytes were analyzed against the control using Ingenuity Pathway Analysis (IPA) software. RESULTS: The 1,394 genes with an |FC| of ≥1.8 were uploaded to IPA. IPA predicted 23 canonical pathways working in the ethanol groups. Three canonical pathways related to ethanol degradation- "Ethanol Degradation IV", "Oxidative Ethanol Degradation III", and "Ethanol Degradation II" -were inhibited in the ethanol groups. IPA predicted "ethanol" as an upregulated upstream regulator of the network with 22 downstream members for the 100 mM ethanol group only. Three members (NTRK2, TGFB3, and TLR8) were activated in all groups. Certain cellular functions were predicted to be changed dose-dependently. "Myocarditis" was dose-dependently inhibited, whereas "Cell death of heart cells" was dose-dependently activated. Several functions were inhibited in 50 mM ethanol only, eg, "Failure of heart" was enhanced in 50 mM ethanol only. Certain functions were activated in 100 mM ethanol only. "Cardiac fibrosis" was not predicted in any ethanol group. CONCLUSIONS: IPA predicted that ethanol-induced activation or inhibition of canonical pathways and functions of cardiomyocyte depended on ethanol concentration, and 3 networks related to heart functions for cardiomyocytes exposed to 3 concentrations of ethanol.

Cultured Neonatal Rat Cardiomyocytes Continue Beating Through Upregulation of CTGF Gene Expression.

BACKGROUND: Some cultured neonatal rat cardiomyocytes continue spontaneous beating even in serum-free medium. The present study explored the cause and genes responsible for this phenomenon. METHODS: Ingenuity Pathway Analysis (IPA) software was used to analyze fold changes in gene expression in beating neonatal rat cardiomyocytes, as compared with non-beating cardiomyocytes, which were obtained from DNA microarray data of total RNA extracts of cardiomyocytes. To confirm the involvement of the 8 genes selected by IPA prediction, cellular protein abundances were determined by Western blot. The gene expression of connective tissue growth factor (CTGF) was substantially higher in beating cardiomyocytes than in non-beating cardiomyocytes; thus, CTGF protein content released from cardiomyocytes into the culture medium was examined. RESULTS: IPA showed that the "Apelin Cardiac Fibroblast Signaling Pathway" was significantly inhibited and that microtubule dynamics and cytoskeleton organization were significantly activated. Each fluctuation in the cellular abundances of the 8 proteins in beating cardiomyocytes, as compared with non-beating cardiomyocytes, was primarily in the same direction as that of gene expression. However, the cellular CTGF protein abundance as well as CTGF content released into the medium did not substantially differ between beating and non-beating cardiomyocytes. CONCLUSIONS: The present results suggest that the large increase in CTGF gene expression in beating cardiomyocytes is not a cause but a result of beating, which may provide a putative pathway for controlling beating. Beating is sustained by developed cardiomyofibrils and directly upregulates CTGF gene expression, which is not followed by CTGF protein synthesis.

Roles of Two Major Alcohol Dehydrogenases, ADH1 (Class I) and ADH3 (Class III), in the Adaptive Enhancement of Alcohol Metabolism Induced by Chronic Alcohol Consumption in Mice.

AIMS: It is still unclear which enzymes contribute to the adaptive enhancement of alcohol metabolism by chronic alcohol consumption (CAC). ADH1 (Class I) has the lowest Km for ethanol and the highest sensitivity for 4-methylpyrazole (4MP) among ADH isozymes, while ADH3 (Class III) has the highest Km and the lowest sensitivity. We investigated how these two major ADHs relate to the adaptive enhancement of alcohol metabolism. METHODS: Male mice with different ADH genotypes (WT, Adh1-/- and Adh3-/-) were subjected to CAC experiment using a 10% ethanol solution for 1 month. Alcohol elimination rate (AER) was measured after ethanol injection at a 4.0 g/kg dose. 4MP-sensitive and -insensitive AERs were measured by the simultaneous administration of 4MP at a dose of 0.5 mmol/kg in order to estimate ADH1 and non-ADH1 pathways. RESULTS: AER was enhanced by CAC in all ADH genotypes, especially more than twofold in Adh1-/- mice, with increasing ADH1 and/or ADH3 liver contents, but not CYP2E1 content. 4MP-sensitive AER was also increased by CAC in WT and Adh3-/- strains, which was greater in Adh3-/- than in WT mice. The sensitive AER was increased even in Adh1-/- mice probably due to the increase in ADH3, which is semi-sensitive for 4MP. 4MP-insensitive AER was also increased in WT and Adh1-/- by CAC, but not in Adh3-/- mice. CONCLUSION: ADH1 contributes to the enhancement of alcohol metabolism by CAC, particularly in the absence of ADH3. ADH3 also contributes to the enhancement as a non-ADH1 pathway, especially in the absence of ADH1.

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.

The Contribution of Alcohol Dehydrogenase 3 to the Development of Alcoholic Osteoporosis in Mice.

BACKGROUND: Alcohol dehydrogenase 3 (ADH3) plays major roles not only in alcohol metabolism but also in nitric oxide metabolism as S-nitrosoglutathione reductase (GSNOR). ADH3/GSNOR regulates both adipogenesis and osteogenesis through the denitrosylation of peroxisome proliferator-activated receptor γ. The current study investigated the contribution of ADH3 to the development of alcoholic osteoporosis in chronic alcohol consumption (CAC). METHODS: Nine-week-old male mice of different ADH genotypes [wild-type (WT) and Adh3-/-] were administered a 10% ethanol solution for 12 months. The femurs were evaluated by histochemical staining and computed tomography-based bone densitometry. The mRNA levels of ADH3 were evaluated in the WT mice by reverse transcription-quantitative polymerase chain reaction. RESULTS: The Adh3-/- control mice exhibited increased activities of both osteoblasts and osteoclasts and lower bone masses than the WT control mice. CAC exhibited no remarkable change in osteoblastic and osteoclastic activities, but decreased bone masses were observed in WT mice despite an increase in the mRNA levels of ADH3. Conversely, bone masses in the Adh3-/- control mice were not reduced after CAC. CONCLUSIONS: The Adh3-/- control mice exhibited a high turnover of osteoporosis since osteoclastogenesis dominated osteoblastogenesis; however, bone resorption was not enhanced after CAC. In comparison, CAC lead to alcoholic osteoporosis in WT mice, accompanied by increased mRNA levels of ADH3. Hence, ADH3 can prevent osteoporosis development in normal ADH genotypes with no alcohol ingestion. However, ADH3 contributes to the development of alcoholic osteoporosis under CAC by participating in alcohol metabolism, increasing metabolic toxicity, and lowering GSNO reducing activity.

Metabolic pharmacokinetics of early chronic alcohol consumption mediated by liver alcohol dehydrogenases 1 and 3 in mice.

BACKGROUND AND AIM: Alcohol dehydrogenases (ADHs) 1 and 3 are responsible for systemic alcohol metabolism. The current study investigated the contribution of liver ADH1 and ADH3 to the metabolic pharmacokinetics of chronic alcohol consumption (CAC). METHODS: The 9-week-old male mice of different ADH genotypes (wild-type [WT], Adh1-/- , and Adh3-/- ) were administered with 10% ethanol solution for 1 month, followed by acute ethanol administration (4.0 g/kg). The alcohol elimination rate (AER), area under the blood alcohol concentration curve (AUC), and the maximum blood alcohol concentration (Cmax ) were calculated. The liver content, activity, and mRNA levels of ADH were evaluated. RESULTS: Chronic alcohol consumption increased the AER and reduced the AUC in all ADH genotypes. The increased ADH1 content was correlated with AER in WT mice but not in the Adh3-/- mice. Similarly, the increased ADH3 content was also correlated with AER in both WT and Adh1-/- mice. The Cmax was significantly higher in Adh3-/- control mice than in WT control mice. It decreased in the Adh1-/- mice by CAC along with an increase in the ADH3 content. CONCLUSIONS: Alcohol dehydrogenases 1 and 3 would accomplish the pharmacokinetic adaptation to CAC in the early period. ADH1 contributes to the metabolic pharmacokinetics of CAC with a decrease in AUC in conjunction with an increase of AER by increasing the enzyme content in the presence of ADH3. ADH3 also contributes to a decrease in AUC in conjunction with not only an increase in AER but also a decrease in Cmax by increasing the enzyme content.

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.

Real-time PCR assay for the detection of picoplankton DNA distribution in the tissues of drowned rabbits.

The detection of plankton DNA is one of the important methods for the diagnosis of drowning from postmortem tissues. This study investigated the quantities of picoplankton (Cyanobacteria) DNA in the lung, liver, kidney tissues and blood of drowned and non-drowned rabbits, and the sensitivity of detection of picoplankton DNA by polymerase chain reaction (PCR) detect for the diagnosis of death from drowning. For this purpose, the DNA of the 16S ribosomal RNA gene of picoplankton was quantitatively assayed from the tissues of drowned and non-drowned rabbits immersed in water after death. Each of the liver, kidney and lung tissues and blood were obtained from drowned and non-drowned rabbits. Picoplankton DNA in the tissues was extracted using the DNeasy® Blood & Tissue kit to determine the yield of picoplankton DNA from each tissue. TaqMan real-time PCR was performed for quantitative analysis of picoplankton DNA. Target DNA was detected in the liver, kidney and lung samples obtained from the drowned rabbits, while no picoplankton DNA was detected in the non-drowned rabbit tissues (except in lung samples). The results verified that direct PCR for the detection of picoplankton DNA is useful for the diagnosis of drowning. Although we observed seasonal changes in the quantity of picoplankton in river water, we were able to detect DNA from various organs of drowned bodies during the season when picoplankton were not the most abundant.

Possibility of visualization of gastrothorax based on unenhanced postmortem computed tomography/PMCT.

We present a fatal case of a gastrothorax due to an acute gastric volvulus resulting from a Bochdalek hernia. A 5-year-old boy without previous medical history was brought to our institution in a state of cardiopulmonary arrest and was subsequently pronounced dead. Postmortem computed tomography (PMCT) of the torso showed abdominal organs involving the lower section of the esophagus up to the entire stomach, the left side of the transverse colon, the entire spleen, and the tail of the pancreas herniated into the left thoracic cavity. The stomach was markedly expanded and a mesentero-axial (rotation along the short axis) volvulus was observed, displacing mediastinal structures to the right side and depressing the diaphragmatic contour. A PMCT of the thorax at the lung window setting revealed displacement of bilateral lungs. The bilateral lungs were severely atelectatic and congested. The PMCT findings mentioned above were consistent with the autopsy findings. PMCT can provide useful information for the diagnosis in cases we initially cannot predict any significant changes, for example, organ displacement.

Ethanol Dose- and Time-dependently Increases α and ß Subunits of Mitochondrial ATP Synthase of Cultured Neonatal Rat Cardiomyocytes.

Mitochondria are target subcellular organelles of ethanol. In this study, the effects of ethanol on protein composition was examined with 2-dimensional electrophoresis of protein extracts from cultured neonatal rat cardiomyocytes exposed to 100 mM ethanol for 24 hours. A putative ß subunit of mitochondrial ATP synthase was increased, which was confirmed by Western blot. The cellular protein abundances in the α and ß subunits of ATP synthase increased in dose (0, 10, 50, and 100 mM) - and time (0.5 hour and 24 hours) -dependent manners. The DNA microarray analysis of total RNA extract demonstrated that gene expression of the corresponding messenger RNAs of these subunit proteins did not significantly alter due to 24-hour ethanol exposure. Therefore, protein expression of these nuclear-encoded mitochondrial proteins may be regulated at the translational, rather than the transcriptional, level. Alternatively, degradation of these subunit proteins might be decreased. Additionally, cellular ATP content of cardiomyocytes scarcely decreased following 24-hour exposure to any examined concentrations of ethanol. Previous studies, together with this study, have demonstrated that protein abundance of the α subunit or ß subunit or both subunits of ATP synthase after ethanol exposure or dysfunctional conditions might differ according to tissue: significant increases in heart but decreases in liver and brain. Thus, it is suggested that the abundance of subunit proteins of mitochondrial ATP synthase in the ethanol-exposed heart, being different from that in the liver and brain, should increase dose-dependently through either translational upregulation or decreased degradation or both to maintain ATP production, as the heart requires much more energy than other tissues for continuing sustained contractions.

Structural analysis of quazepam metabolites in bile by ion trap time-of-flight mass spectrometry.

Quazepam (QZP) is a long-acting benzodiazepine-type hypnotic. We searched for novel QZP metabolites in bile and determined their structures by liquid chromatography-ion trap time-of-flight mass spectrometry (LC-IT-TOF MS). The metabolites were extracted with ethyl acetate after ß-glucuronidase treatment. First, a single MS spectrum was acquired. Second, MS(n) spectra were acquired for peaks that consisted of ions with the isotope pattern corresponding to molecules bearing one chlorine atom. The novel QZP metabolites found in this study were hydroxyquazepam, hydroxy-methoxyquazepam, hydroxy-oxoquazepam, and hydroxy-methoxy-oxoquazepam, which have the hydroxy and methoxy groups on the fluorophenyl group, and dihydroxy-oxoquazepam and dihydroxy-methoxy-oxoquazepam, which have one hydroxy group at the 3-position of the seven-membered ring and the other hydroxy group and the methoxy group on the fluorophenyl group. We demonstrated that LC-IT-TOF MS was a useful tool for determining the structure of the metabolites. However, the exact locations of the hydroxy and methoxy groups on the fluorophenyl group could not be identified.

Forensic toxicological analyses of drugs in tissues in formalin solutions and in fixatives.

Forensic toxicological drug analyses of human specimens are usually performed immediately after autopsy or on frozen preserved tissues. Occasionally, cases require analysis of drugs from tissues fixed in formalin solution. To improve the estimation of the level of drug in tissues following formalin fixation, we studied drug concentrations in human tissues, liver and kidney, that were collected from a drug-positive autopsy case. Parts of tissues were preserved in formalin solution for 1, 3, 6 and 13 months. Tissues obtained before and after preservation, along with tissue-exposed fixatives, were assayed using gas chromatography-mass spectrometry; all of the samples were assayed for the presence of drugs and changes in the drug concentrations both before and after preservation in formalin. Concentrations of assayed drugs decreased upon fixation in formalin; levels of these drugs did not necessarily show further decreases during subsequent storage in fixative, up to 13 months. Distinct trends in drug levels were found in liver and kidney. In liver, the levels of chlorpromazine, levomepromazine, and promethazine decreased to 23-39% at 1 month after preservation; all 3 of these drugs were detected at all tested time points of preservation. Bromazepam was not detected at 13 months after preservation. Milnacipran was the most unstable after preservation in formalin solution among all of the assayed drugs. In kidney, all assayed drugs exhibited reduced stability during preservation compared to levels in liver. Methamphetamine and methylenedioxymethamphetamine were not detected in any time points of tissues. The proportions of the drugs that remained within the tissues differed between liver and kidney. Also, S-oxide compounds of chlorpromazine and levomepromazine, which were not observed before preservation, were detected in fixed liver tissues and their fixatives at 3, 6 and 13 months of preservation. These results suggest that analyses in formalin-fixed tissues need to include analysis of various organ-tissues and their fixatives at multiple time points for the duration of preservation. These analyses should include detection of chemical degradation/denaturation products, such as S-oxides of chlorpromazine and levomepromazine.

Pericardial tear as a consequence of cardiopulmonary resuscitation (CPR) involving chest compression: a report of two postmortem cases of acute type A aortic dissection with hemopericardium.

We present two cases of a pericardial tear as a consequence of cardiopulmonary resuscitation involving chest compressions in fatal acute type A aortic dissection (AoD) with hemopericardium. For each case, postmortem computed tomography revealed a hematoma in the false lumen of the ascending aorta with a slight hemopericardium and a large left hemothorax, as well as focal pericardial dimpling and discontinuity around the left ventricle. At autopsy, we confirmed a convex lens-shape gaping pericardial tear at the left posterolateral site of the pericardium and a massive volume of bloody fluid in the left thoracic cavity. It has been hypothesized that the pericardium ruptured due to chest compressions during resuscitation in these cases of acute type A AoD with hemopericardium and that intrapericardial blood leakage through the pericardial tear resulted in a hemothorax.

Visualization of different characteristics of cerebrospinal fluid with acute encephalopathy and febrile seizures using pattern recognition analysis of 1H NMR.

BACKGROUND: In acute encephalopathy, deterioration of the condition can be rapid, and early intervention is essential to prevent progression of the disease. However, in the acute period, differentiating acute encephalopathy from febrile seizures is difficult. Thus, an early diagnostic marker has been sought to enable early intervention. Proton nuclear magnetic resonance ((1)H NMR) spectroscopy is used to study the chemical characteristics of biological fluids such as cerebrospinal fluid (CSF). The purpose of this study was to ascertain if pattern recognition of (1)H NMR spectra could differentiate CSF obtained from patients with acute encephalopathy and febrile seizures. METHODS: CSF was obtained from patients with acute encephalopathy (n = 4), complex febrile seizures (n = 9), and simple febrile seizures (n = 9). RESULTS: NMR spectra of CSF did not visually differ across the three groups. Spectral data were analyzed by partial least squares discriminant analysis and visualized by plotting the partial least squares scores of each sample. The three patient groups clustered separately on the plots. CONCLUSION: In this preliminary study, we were able to visualize different characteristics of CSF obtained from patients with acute encephalopathy and simple and complex febrile seizures using pattern recognition analysis of (1)H NMR data.

Pattern recognition analysis of proton nuclear magnetic resonance spectra of extracts of intestinal epithelial cells under oxidative stress.

BACKGROUND: Mesenteric ischemia-reperfusion induces gut mucosal damage. Intestinal mucosal wounds are repaired by epithelial restitution. Although many different molecular mechanisms have been shown to affect cell metabolism under oxidative conditions, these molecular mechanisms and metabolic phenotypes are not well understood. Nuclear magnetic resonance (NMR) spectroscopic data can be used to study metabolic phenotypes in biological systems. Pattern recognition with multivariate analysis is one chemometric technique. The purpose of this study was to visualize, using a chemometric technique to interpret NMR data, different degrees of oxidant injury in rat small intestine (IEC-6) cells exposed to H2O2. METHODS: Oxidant stress was induced by H2O2 in IEC-6 cells. Cell restitution and viability were assessed at different H2O2 concentrations and time points. Cells were harvested for pattern recognition analysis of (1)H-NMR data. RESULTS: Cell viability and restitution were significantly suppressed by H2O2 in a dose-dependent manner compared with control. Each class was clearly separated into clusters by partial least squares discriminant analysis, and class variance was greater than 90% from 2 factors. CONCLUSION: Pattern recognition of NMR spectral data using a chemometric technique clearly visualized the differences of oxidant injury in IEC-6 cells under oxidant stress.

Quantitative analysis of quazepam and its metabolites in human blood, urine, and bile by liquid chromatography-tandem mass spectrometry.

Quazepam (QZP), which is a long-acting benzodiazepine-type hypnotic, and its 4 metabolites, 2-oxoquazepam, N-desalkyl-2-oxoquazepam (DOQ), 3-hydroxy-2-oxoquazepam (HOQ), and 3-hydroxy-N-desalkyl-2-oxoquazepam, in human blood, urine, and bile were quantitatively analyzed by liquid chromatography-tandem mass spectrometry. The analytes were extracted from blood by protein precipitation followed by solid phase extraction, and from urine and bile by liquid-liquid extraction and cleanup using a PSA solid phase extraction cartridge. This method was applied to a medico-legal autopsy case, in which the deceased had been prescribed QZP approximately 3 weeks before his death. In blood, the concentrations of free DOQ (160±7 ng/mL for heart blood and 181±12 ng/mL for femoral blood) were the highest of all the analytes and in agreement with the concentration at a steady state. This indicates that the deceased consecutively received QZP for at least several days until the concentrations reached approximately the same level as that in the steady state. An extremely high concentration of total HOQ (the sum of conjugated and free HOQ) in bile was also found (56,200±1900 ng/mL). This accumulation of HOQ in bile is probably due to enterohepatic circulation. This study demonstrates that the combination of the concentrations of QZP and its metabolites in biological matrices can provide more information about the amount and frequency of QZP administration.