Left Heart Blood Research Articles

Sex-specific Genetic Determinants of Right Ventricular Structure and Function.

While sex differences in right heart phenotypes have been observed, the molecular drivers remain unknown. To provide biological insights into sex differences in the structure and function of the right ventricle (RV) using common genetic variation. RV phenotypes were obtained from cardiac magnetic resonance imaging in 18,156 women and 16,171 men from the UK Biobank. Observational analyses and sex-stratified genome-wide association studies were performed. Candidate female-specific loci were evaluated against invasively measured cardiac performance in 479 female patients with idiopathic or heritable pulmonary arterial hypertension (PAH), recruited to the UK NIHR BioResource Rare Diseases study. Sex was associated with differences in RV volumes and ejection fraction in models adjusting for left heart counterparts, blood pressure, lung function and sex hormone levels. Six genome-wide significant loci (13%) revealed heterogeneity of allelic effects between women and men, and significant sex-by-genotype interaction. These included two sex-specific candidate loci present in women only: a locus for RV ejection fraction in BMPR1A and a locus for RV end-systolic volume near DMRT2. Epigenetic data in RV tissue indicate that variation at the BMPR1A locus likely alters transcriptional regulation. In female patients with PAH, a variant located in the promoter of BMPR1A was significantly associated with cardiac index (effect size 0.16 l/min/m2), despite similar RV afterload. BMPR1A has emerged as a biologically plausible candidate gene for female-specific genetic determination of RV function, showing associations with cardiac performance under chronically increased afterload in female patients with PAH.

Association of Thyroid Dysfunction in Individuals ≥ 65 Years of Age With Subclinical Cardiac Abnormalities.

The relationship between thyroid dysfunction and measures of myocardial disease in older individuals remains to be defined. To evaluate the impact of thyroid dysfunction on structure and function of the left heart chambers and blood markers of cardiac disease. Cross-sectional analysis of the Cardiovascular Health Study, a community-based cohort of older individuals recruited from 4 urban areas in the United States. Of 3163 participants studied, 2477 were euthyroid, 465 had subclinical hypothyroidism (SCH), 47 overt hypothyroidism (OH), 45 endogenous (endo) subclinical hyperthyroidism (endo-SCT), and 129 had exogenous (exo) SCT due to thyroid hormone supplementation. Participants underwent clinical evaluation, blood sampling and biomarker measurement, 2-dimensional and speckle tracking echocardiography for assessment of left heart myocardial deformation, circulating biomarkers of diastolic overload (NT-proBNP), fibrosis (sST2, gal-3), and cardiomyocyte injury (hs-cTnT). SCH was associated with higher NT-proBNP (beta = 0.17, P = .004), whereas OH was associated with higher hs-cTnT (beta = 0.29, P = .005). There were also suggestive associations of SCH with higher sST2, as well as endo-SCT with higher gal-3 and lower (worse) left atrial reservoir strain. Left ventricular longitudinal strain and end diastolic strain rate did not differ significantly from euthyroid participants in SCH, OH, or exo-SCT. In this free-living elderly cohort, subclinical and overt hypothyroidism were associated with abnormalities of blood biomarkers consistent with diastolic overload and myocardial necrosis respectively, whereas subclinical hyperthyroidism tended to be associated with myocardial fibrosis and decreased left atrial strain. Our findings could represent stage B heart failure and illuminate distinct aspects of the pathobiology of heart disease related to thyroid gland dysfunction with potential clinical implications.

Changes in Left Heart Geometry, Function, and Blood Serum Biomarkers in Patients with Obstructive Sleep Apnea after Treatment with Continuous Positive Airway Pressure.

Background: Cardiovascular remodeling is essential in patients with obstructive sleep apnea (OSA), and continuous positive airway pressure (CPAP) therapy could improve these processes. Two-dimensional (2D) speckle-tracking (ST) echocardiography is a useful method for subclinical biventricular dysfunction diagnosis and thus might help as an earlier treatment for OSA patients. It is still not clear which blood serum biomarkers could be used to assess CPAP treatment efficacy. Objectives: To evaluate left heart geometry, function, deformation parameters, and blood serum biomarker (galectin-3, sST2, endothelin-1) levels in patients with OSA, as well as to assess changes after short-term CPAP treatment. Materials and Methods: Thirty-four patients diagnosed with moderate or severe OSA, as well as thirteen patients as a control group, were included in the study. All the subjects were obese (body mass index (BMI) > 30 kg/m2). Transthoracic 2D ST echocardiography was performed before and after 3 months of treatment with CPAP; for the control group, at baseline only. Peripheral blood samples for the testing of biomarkers were collected at the time of study enrolment before the initiation of CPAP therapy and after 3 months of CPAP treatment (blood samples were taken just for OSA group patients). Results: The left ventricle (LV) end-diastolic diameter and volume, as well as LV ejection fraction (EF), did not differ between groups, but an increased LV end-systolic volume and a reduced LV global longitudinal strain (GLS) were found in the OSA group patients (p = 0.015 and p = 0.035, respectively). Indexed by height, higher LV MMi in OSA patients (p = 0.007) and a higher prevalence of LV diastolic dysfunction (p = 0.023) were found in this group of patients. Although left atrium (LA) volume did not differ between groups, OSA group patients had significantly lower LA reservoir strain (p < 0.001). Conventional RV longitudinal and global function parameters (S′, fractional area change (FAC)) did not differ between groups; however, RV GLS was reduced in OSA patients (p = 0.026). OSA patients had a significantly higher right atrium (RA) diameter and mean pulmonary artery pressure (PAP) (p < 0.05). Galectin-3 and sST2 concentrations significantly decreased after 3 months of CPAP treatment. Conclusions: OSA is associated with the left heart remodeling process—increased LV myocardial mass index, LV diastolic dysfunction, reduced LV and RV longitudinal strain, and reduced LA reservoir function. A short-term, 3-months CPAP treatment improves LV global longitudinal strain and LA reservoir function and positively affects blood serum biomarkers. This new indexing system for LV myocardial mass by height helps to identify myocardial structural changes in obese patients with OSA.

Development of an LC-MS/MS method for quantification of 3-chloro-L-tyrosine as a candidate marker of chlorine poisoning.

A simple and sensitive liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) method for the determination of 3-chloro-L-tyrosine (Cl-Tyr) was developed and validated. For sample preparation, 50μL of the body fluids or tissue extracts were processed by protein precipitation followed by the derivatization with dansyl chloride. The calibration curve was linear over the concentration range of 2.0-200ng/mL blood or 4.0-400ng/g tissue. Our method allowed the reproducible and accurate quantification. That is, the intra- and inter-assay coefficients of variation were below 7.73 and 6.94%, respectively in both the blood and lung. We applied the developed method to the analysis of Cl-Tyr in the human autopsy samples, which were suspected of chlorine poisoning, and detected 55.2ng/mL and 206.6ng/g Cl-Tyr in left heart blood and lung, respectively. Furthermore, in more than 20 autopsy samples, which were obtained from other causes of death including burn, drowning, hanging, internal disease, trauma and drug poisoning, Cl-Tyr was almost not detected in their both body fluids and organ tissues. In conclusion, the data here reported demonstrate that the LC/ESI-MS/MS method allows the Cl-Tyr in the autopsy samples and that chlorine exposure strongly affects its level, providing a basis for novel identification tool of chlorine poisoning.

Sensitive SQUID Bio-Magnetometry for Determination and Differentiation of Biogenic Iron and Iron Oxide Nanoparticles in the Biological Samples.

This study aimed to develop the method for determination of the ultra-small superparamagnetic iron oxide nanoparticle (USPION)-originated iron (UOI) in the tissues of rats on the basis of the magnetic characteristics (MC) in the liver, left heart ventricle (LHV), kidneys, aorta and blood of Wistar-Kyoto (WKY). Rats were treated intravenously by USPIONs dispersed in saline (transmission electron microscope (TEM) mean size ~30 nm, hydrodynamic size ~51 nm, nominal iron content 1 mg Fe/mL) at the low iron dose of 1 mg/kg. MC in the form of the mass magnetisation (M) versus the magnetic field (H) curves and temperature dependences of M (determined using the SQUID magnetometer), histochemical determination of iron (by Perl’s method) and USPION-induced superoxide production (by lucigenin-enhanced chemiluminescence) were investigated 100 min post-infusion. USPIONs significantly elevated superoxide production in the liver, LHV, kidney and aorta vs. the control group. Histochemical staining confirmed the presence of iron in all solid biological samples, however, this method was not suitable to unequivocally confirm the presence of UOI. We improved the SQUID magnetometric method and sample preparation to allow the determination of UOI by measurements of the MC of the tissues at 300 K in solid and liquid samples. The presence of the UOI was confirmed in all the tissues investigated in USPIONs-treated rats. The greatest levels were found in blood and lower amounts in the aorta, liver, LHV and kidneys. In conclusion, we have improved SQUID-magnetometric method to make it suitable for detection of low amounts of UOI in blood and tissues of rats.

Numerical Investigation of Centrifugal Blood Pump Cavitation Characteristics with Variable Speed

In this paper, the cavitation characteristics of centrifugal blood pumps under variable speeds were studied by using ANSYS-CFX and MATLAB software. The study proposed a multi-scale model of the “centrifugal blood pump—left heart blood circulation”, and analyzed the cavitation characteristics of the centrifugal blood pump. The results showed that the cavitation in the impeller first appeared near the hub at the inlet of the impeller. As the inlet pressure decreased, the cavitation gradually strengthened and the bubbles gradually developed in the outlet of the impeller. The cavitation intensity increased with the increase of impeller speed. The curve of the variable speeds of the centrifugal blood pump in the optimal auxiliary state was obtained, which could effectively improve the aortic pressure and flow. In variable speeds, due to the high aortic flow and pressure during the ejection period, the sharp increases in speeds led to cavitation. The results could provide a guidance for the optimal design of the centrifugal blood pump.

Green tea extract reduces insulin level and folliculogenesis in insulin-resistant PCOS rats model

Objectives: Polycystic ovary syndrome (PCOS) is an endocrine disorder with still unknown pathophysiology. Insulin resistance is one of major factors that affect the pathogenesis of PCOS (69%). Green tea has compounds that can be used to improve the condition of insulin resistance, so that folliculogenesis may recur in the case of PCOS.Materials and Methods: The subjects in this study were rats which were divided into 5 groups, consisting of 2 groups serving as a control group (negative and positive) and 3 groups as the treatment group. Each group consisted of 7 experimental animals. Negative control group was given with distilled water for 14 days. Positive control group was given with injections of testosterone propionate 1 mg/100 g BW intramuscularly in the thigh for 28 days to obtain a model of insulin-resistant PCOS and the distilled water was used as therapy. The treatment groups were given wth injections of testosterone propionate 1 mg/100gBW intra-muscularly for 28 days and then treated with green tea extract in doses of each 200 mg/kg, 400 mg/kg, and 800 mg/kg for 14 days. Rats were dissected on day 42 for harvesting the right and left ovary and heart blood sample was taken for examining insulin levels.Results: The results showed that administration of green tea extract on K5 can reduce insulin levels and increase folliculogenesis significantly (significance p &lt;0.05).Conclusion: Green tea extract can reduce insulin levels and increase folliculogenesis in rats. PCOS - Insulin resistance at a dose of 800 mg/kg.

Evaluation of the distribution of nicotine intravenous injection: an adult autopsy case report with a review of literature

We reported the first comprehensive autopsy case of death due to intravenous injection of nicotine. We examined the distribution of nicotine in the body tissues and fluid and exposed the pathophysiology of nicotine poisoning. A 19-year-old woman was rushed to the hospital in cardiorespiratory arrest and was confirmed dead upon arrival. Liquid nicotine, hydrogen peroxide water, and a syringe were found in the hotel room where she stayed. On autopsy, nicotine concentration was the highest (15,023 μg/mg) in the tissue around the injection mark on the right upper arm. Among the body fluids, the intraperitoneal fluid had the highest, whereas the pericardial fluid had the lowest (0.736 μg/mL) nicotine concentration. Among the organs, the brain had the highest (11.637 μg/mg), whereas the fat tissue had the lowest (1.307 μg/mg) nicotine concentration. The concentration of cotinine, which is the metabolite of nicotine, was the highest in the tissue around the injection mark on the right arm (5.495 μg/mg) and was almost the same among the other body fluids and organs. The respective concentrations of nicotine and cotinine were 1.529 μg/mL and 0.019 μg/mL in the left heart blood and 3.157 μg/mL and 0.002 μg/mL in right heart blood. In this case, the nicotine concentrations in blood reached the lethal level. The distributions of nicotine and cotinine, as indicated by the intravenous injection, were related to the distribution of organs that metabolize nicotine and the distribution of nicotinic acetylcholine receptors.

Predictive equation for post-mortem interval using spectrophotometric values of post-mortem lividity: A pilot study

The colour of post-mortem lividity and control skin of 86 cadavers was measured spectrophotometrically to obtain L* (value), a* (chroma) and b* (hue) values. In addition, left heart blood (n = 58), right heart blood (n = 57) and blood from the femoral vein (n = 21) were measured. Using these data, we analysed the relationship between post-mortem lividity, control skin and blood colours. L* of post-mortem lividity (L*p) and control skin (L*c) were strongly correlated ( r = 0.64). a* and b* of post-mortem lividity (a*p and b*p) significantly increased with an increasing post-mortem interval (PMI) but r2 values were low (0.11 and 0.070, respectively). Predictive equations for post-mortem lividity colour (L*p, a*p and b*p) were developed using control skin colour (L*c, a*c and b*c) and autopsy findings for the first time. The predictive equation for L*p explained almost 65% of the observed L*p. We created predictive equations for PMI with and without blood colour values, and the most accurate equation, which did not use blood colour values, made it possible to estimate PMI within ± 10.29 h. Further study of these equations will help us to understand the factors that affect post-mortem lividity colour and increase the accuracy of equations for predicting post-mortem lividity colour and PMI.

Dexamethasone improves pulmonary hemodynamics in COPD-patients going to altitude: A randomized trial

BackgroundChronic obstructive pulmonary disease (COPD) may predispose to symptomatic pulmonary hypertension at high altitude. We investigated hemodynamic changes in lowlanders with COPD ascending rapidly to 3100 m and evaluated whether preventive dexamethasone treatment would mitigate the altitude-induced increase in pulmonary artery pressure. MethodsIn this placebo-controlled, double-blind trial, non-hypercapnic COPD patients living <800 m, were randomized to receive either dexamethasone (8 mg/day) or placebo tablets one day before ascent from 760 m and during a 3-day-stay at 3100 m. Echocardiography was performed at 760 m and after the first night at 3100 m. The trans-tricuspid pressure gradient (RV/RA, main outcome), cardiac output (Q) by velocity-time integral of left ventricular outflow, indices of right and left heart function, blood gases and pulse-oximetry (SpO2) were compared between groups. Results95 patients, 79 men, mean ± SD age 57 ± 8y FEV1 89 ± 21% pred, SpO2 95 ± 2% were included in the analysis. In 52 patients receiving dexamethasone, RV/RA, Q and SpO2 at 760 and 3100 m were 19 ± 5 mm Hg and 26 ± 7 mm Hg, 4.9 ± 0.7 and 5.7 ± 1.1 l/min, SpO2 95 ± 2% and 90 ± 3% (P < 0.05 all changes). In 43 patients receiving placebo the corresponding values were 20 ± 4 mm Hg and 31 ± 9 mm Hg, 4.7 ± 0.9 l/min and 95 ± 3% and 89 ± 3% (P < 0.05 all changes) between group differences of altitude-induced changes were (mean, 95% CI): RV/RA −4.8 (−7.7 to −1.8) mm Hg, Q 0.13 (−0.3 to 0.6) l/min and SpO2 1 (−1 to 2) %. ConclusionsIn lowlanders with COPD travelling to 3100 m preventive dexamethasone treatment mitigates the altitude-induced rise in RV/RA potentially along with a reduced pulmonary vascular resistance and improved oxygenation.

Fatal zolpidem poisoning due to its intravenous self-injection: Postmortem distribution/redistribution of zolpidem and its predominant metabolite zolpidem phenyl-4-carboxylic acid in body fluids and solid tissues in an autopsy case

We experienced a curious fatal case, in which a male in his 20s self-administered zolpidem intravenously. The victim was found dead lying on floor of his apartment room, with a tourniquet band and new injection marks on his right forearm. Nearby the body, a medical disposal syringe containing small-volume solution dissolving crushed zolpidem tablets was found. The postmortem interval was estimated at about two days.The direct cause of his death was judged as asphyxia due to the aspiration of stomach contents into the trachea and bronchi. The specimens dealt with were body fluids and solid tissues including femoral vein blood, right and left heart blood, pericardial fluid, urine, bile, stomach contents, the brain, lung, heart muscle, liver, spleen, kidney, pancreas and skeletal muscle. For the extractions of zolpidem, zolpidem phenyl-4-carboxylic acid, deuterated internal standards zolpidem-d7 and zolpidem phenyl-4-carboxylic acid-d4, a modified QuEChERS method was used, followed by the analysis by liquid chromatography–tandem mass spectrometry. Because this study included various kinds of human matrices with quite different properties, the standard addition method was most preferable to overcome the matrix effects and recovery rates, and also did not need to use blank human matrices for validation experiments. The concentration of zolpidem and its phenyl-4-carboxylic acid metabolite in various specimens tested were generally extreme higher than those of reported fatal cases, supporting that the victim had died of intravenous zolpidem injection. The concentrations of zolpidem in femoral vein blood and right and left heart blood specimens in the present case were 9.55, 28.5 and 46.9μg/mL, respectively, which far exceeded estimated fatal levels. The present study also showed the postmortem distribution/redistribution of zolpidem and its phenyl-4-carboxylic acid metabolite in 15 body fluid and solid tissue specimens including stomach contents. Although a number of published literatures dealt with zolpidem poisoning cases due to oral ingestion of the drug, this is the first report on fatal intravenous zolpidem injection case and postmortem distribution of zolpidem and its predominant metabolite.

G-protein receptor kinases 2, 5 and 6 redundantly modulate Smoothened-GATA transcriptional crosstalk in fetal mouse hearts

G-protein receptor kinases (GRKs) regulate adult hearts by modulating inotropic, chronotropic and hypertrophic signaling of 7-transmembrane spanning neurohormone receptors. GRK-mediated desensitization and downregulation of β-adrenergic receptors has been implicated in adult heart failure; GRKs are therefore a promising therapeutic target. However, germ-line (but not cardiomyocyte-specific) GRK2 deletion provoked lethal fetal heart defects, suggesting an unexplained role for GRKs in heart development. Here we undertook to better understand the consequences of GRK deficiency on fetal heart development by creating mice and cultured murine embryonic fibroblasts (MEFs) having floxed GRK2 and GRK5 alleles on the GRK6 null background; simultaneous conditional deletion of these 3 GRK genes was achieved using Nkx2–5 Cre or adenoviral Cre, respectively. Phenotypes were related to GRK-modulated gene expression using whole-transcriptome RNA sequencing, RT-qPCR, and luciferase reporter assays. In cultured MEFs the atypical 7-transmembrane spanning protein and GRK2 substrate Smoothened (Smo) stimulated Gli-mediated transcriptional activity, which was interrupted by deleting GRK2/5/6. Mice with Nkx2–5 Cre mediated GRK2/5/6 ablation died between E15.5 and E16.5, whereas mice expressing any one of these 3 GRKs (i.e. GRK2/5, GRK2/6 or GRK5/6 deleted) were developmentally normal. GRK2/5/6 triple null mice at E14.5 exhibited left and right heart blood intermixing through single atrioventricular valves or large membranous ventricular septal defects. Hedgehog and GATA pathway gene expression promoted by Smo/Gli was suppressed in GRK2/5/6 deficient fetal hearts and MEFs. These data indicate that GRK2, GRK5 and GRK6 redundantly modulate Smo-GATA crosstalk in fetal mouse hearts, orchestrating transcriptional pathways previously linked to clinical and experimental atrioventricular canal defects. GRK modulation of Smo reflects convergence of conventional neurohormonal signaling and transcriptional regulation pathways, comprising an unanticipated mechanism for spatiotemporal orchestration of developmental gene expression in the heart.

Association between cytochrome P450 2D6 polymorphisms and body fluid methamphetamine concentrations in Japanese forensic autopsy cases

Methamphetamine (MA) is an illicit stimulant that affects the central nervous system. Cytochrome P450 2D6 (CYP2D6) plays an important role in MA metabolism. Numerous allelic variants confer substantial variation in CYP2D6 activity among individuals. In the present study, we examined the frequencies of CYP2D6 alleles, including CYP2D6*1, *2, *4, *5, *10, *14A, *14B, *18, and *36, and multiplication, in 82 forensic autopsy cases of MA abusers and 567 autopsy cases in which MA was not detected (controls). Ultrarapid metabolizer (UM), extensive metabolizer (EM), intermediate metabolizer (IM), and poor metabolizer (PM) phenotypes were predicted from CYP2D6 genotypes. Of MA abusers, 64 subjects were predicted to be EM, 17 were IM, and 1 was UM. No MA abuser had the predicted PM phenotype. No significant differences in CYP2D6 phenotype frequencies were found between MA abusers and controls. MA and amphetamine (AMP) concentrations were measured in the right heart blood, left heart blood, peripheral external iliac blood, urine, pericardial fluid, and bone marrow of MA abusers. MA concentrations in urine and bone marrow were significantly higher in IM than in EM. AMP concentration was not associated with CYP2D6 phenotype in any body fluid. These results suggest that the MA concentration in body fluids is influenced by CYP2D6 phenotypes in the Japanese population.

Morphological dilemma: Anomalous pulmonary venous confluence or cor triatriatum—does it matter?

Cardiac variant of total anomalous pulmonary venous return is a rare entity, whereby all the pulmonary veins drain directly to the right atrium or coronary sinus. The effective left heart blood flow channels through a small stretched patent foramen ovale and can often be confused with a variant of cor triatriatum. Cor triatriatum is a rare congenital cardiac anomaly where pulmonary veins drain to a persistent chamber above the left atrium with a membrane separating these two. There persists either a small aperture directly from the true to the accessory left atrium or none at all. Where there is no such aperture, it is often physiologically akin to the cardiac variant of total anomalous pulmonary venous return described above. Such morphological differentiation is often challenging in a clinical situation, but the effective treatment remains the same. It involves removal of the common wall between the two chambers and baffling the pulmonary veins effectively to the left atrium. We describe such a case where the pulmonary venous return is to the right atrium, managed recently in our centre, and discuss the morphological differences between these two.

Thyroglobulin Level in Postmortem Blood Samples as a Diagnostic Tool for Cause of Death

There were many conflicting results about the significance of postmortem thyroglobulin (Tg) level as a biomarker in forensic diagnosis. This work aimed to assess the relation between postmortem blood Tg level and the cause of death. Sixty cadavers received at Cairo Department of Forensic Medicine (Zenhum Morgue), Egypt, were classified into two groups; 1st group included 30 cadavers died by different types of asphyxia, and 2nd group included 30 cadavers died by causes other than asphyxia. In all cases, right heart blood (RHB) and left heart blood (LHB) Tg levels were measured using chemiluminescence immunometric assay and thyroid tissues were histopathlogically examined. Thyroglobulin level was high in 76.6% of 1st group cases and in 83.3% of 2nd group cases. In fatal traumatic brain injury cases, there was a significant positive correlation between Tg level and survival period. The mean value of RHB Tg levels was higher than that of LHB Tg levels in both studied groups while Tg was high in all cases with skin discoloration as a sign of putrefaction regardless of the cause of death. In conclusion, thyroglobulin level is unreliable as a diagnostic tool for cause of death because it may be affected by postmortem changes.

Postmortem redistribution of methamphetamine and amphetamine in blood specimens from various blood vessels and in the specimens from pericardial fluid, bile, stomach contents and various solid tissues collected from a human cadaver

We recently encountered an autopsy case in which methamphetamine (MA) was involved. The postmortem interval was estimated to be as long as 6–7 days. We collected the blood specimens from 9 locations of the heart and blood vessels, and specimens from pericardial fluid, bile, stomach contents and 9 solid tissues (in total 21 specimens), which were subjected to analysis of MA and the metabolite amphetamine by liquid chromatography–tandem mass spectrometry after a modified QuEChERS extraction. The highest concentration of MA was found for the stomach contents, suggesting the oral ingestion of MA. Among the blood specimens, their MA concentrations varied from 268 to 911 ng/mL. The concentration of MA in the left heart blood was higher than that in the right heart blood. In spite of this, the MA concentration in the pulmonary vein was much lower than that in the left heart blood. The comparison of MA concentrations in venous blood vessels showed that MA concentration was highest in the vena cava, followed by the iliac vein, and was lowest in the femoral vein, suggesting that MA concentration is decreased by diffusion across the thin walls of venous blood vessels during the postmortem interval. The partial loss of a xenobiotic by diffusion via the thin wall of the femoral vein should be taken into consideration especially when the postmortem interval is relatively long.

An objective approach using three indexes for determining fatal hypothermia due to cold exposure; statistical analysis of oxyhemoglobin saturation data.

Analysis of oxyhemoglobin (O2-Hb) saturation levels in the left and right heart blood is useful in the assessment of exposure to cold surroundings before death. We quantified conventional subjective visual evaluation of O2-Hb saturation levels and developed useful diagnostic criteria for fatal hypothermia: O2-Hb saturation in the left heart blood (L-O2Hb) was ⩾36%, the O2-Hb saturation gap between the left and right heart blood (L-R gap) was ⩾13%, and the O2-Hb saturation ratio of the left to right heart blood (L/R ratio) was ⩾1.8. When we used L-O2Hb of ⩾36% as a basic criterion and applied a further criterion of an L-R gap of ⩾13% or an L/R ratio of ⩾1.8, these criteria registered a sensitivity level of ⩾86% and specificity level of ⩾93% for the diagnosis of fatal hypothermia. This method can be useful for determining fatal hypothermia in connection with conventional autopsy findings, as well as histological and biochemical markers.

Postmortem distribution of flunitrazepam and its metabolite 7-aminoflunitrazepam in body fluids and solid tissues in an autopsy case: Usefulness of bile for their detection

We experienced an autopsy case of a woman in her 70s, in which the direct cause of her death was judged as asphyxia due to the occlusion of food in the trachea. The postmortem interval was estimated at about 2days. The specimens dealt with were femoral vein blood, right heart blood, left heart blood, bile, brain, lung, heart muscle, liver, spleen, kidney, pancreas, skeletal muscle, and adipose tissue. By tentative drug screening, we found a high concentration of 7-aminoflunitrazepam in the femoral vein blood, which lead us to examine the postmortem distribution of flunitrazepam and its metabolite 7-aminoflunitrazepam in her body fluids and solid tissues. The extraction of flunitrazepam, 7-aminoflunitrazepam and internal standard nimetazepam was performed by a modified QuEChERS method, followed by the analysis by liquid chromatography-tandem mass spectrometry. Because this study included various kinds of human matrices with quite different properties, we used the standard additional method to overcome the matrix effects. The concentration of 7-aminoflunitrazepam were generally much higher than those of the parent drug flunitrazepam for most specimens except for the adipose tissue, showing that flunitrazepam is readily metabolized to its 7-amino metabolite after absorption into the body both antemortem and postmortem. The outstandingly highest concentration of 7-animoflunitrazepam was found in the bile, followed by the kidney, pancreas, left heart blood, spleen and liver. Although a majority of flunitrazepam was converted to 7-aminoflunitrazepam, the flunitrazepam concentration was highest in the pancreas, followed by the spleen, bile, left heart blood, and brain. In contrast to the results on synthetic cannabinoids, the levels of flunitrazepam and 7-animoflunitrazepam in the adipose tissue were relatively low. The present study showed that the bile may be a useful specimen for detection of unchanged benzodiazepines/their metabolites to be collected at autopsy.

Identification and quantitation of 5-fluoro-ADB, one of the most dangerous synthetic cannabinoids, in the stomach contents and solid tissues of a human cadaver and in some herbal products

Since late September 2014, there have been approximately 10 cases in Japan where people have died from inhaling smoke from herbal blends containing a newly emerged synthetic cannabinoid. Although the drug was tentatively identified by some drug-monitoring institutions as 5-fluoro-ADB, to our knowledge, its data have not been published in any scientific context. We recently encountered an autopsy case in which 5-fluoro-ADB was involved. The deceased was a 34-year-old man who was found dead in his room. The postmortem interval was estimated at 35–40 h. The direct cause of the death was asphyxia due to aspiration of stomach contents into the trachea, which likely took place during vomiting under low-consciousness conditions provoked by inhalation of the 5-fluoro-ADB smoke. The cadaver was subjected to autopsy at our department. Femoral vein blood, right heart blood, left heart blood, urine, stomach contents, and nine solid tissues including the adipose tissue were collected and frozen until analysis. The extraction of 5-fluoro-ADB and internal standard 5-fluoro-AMB was performed using a modified QuEChERS method plus filtration through Captiva ND Lipids cartridges, followed by liquid chromatography–tandem mass spectrometry (LC–MS–MS) analysis. Because this study dealt with various kinds of human matrices, we used the standard addition method for quantitation to overcome the matrix effects. The levels of 5-fluoro-ADB in the cadaver specimens were generally low; it could not be detected from blood or urine specimens. The levels of 5-fluoro-ADB in solid tissues were 1.17–7.95 ng/g. Because the highest levels were found for the adipose tissue and heart muscle, the final extracts of the adipose tissue and/or heart muscle were concentrated 10- and 200-fold to obtain product ion mass spectra of 5-fluoro-ADB using LC–MS–MS and its mass spectrum by gas chromatography–mass spectrometry, respectively. Both spectra completely coincided with those obtained from the reference standard 5-fluoro-ADB, confirming that the target compound was 5-fluoro-ADB. The quantitative results obtained by selected reaction monitoring of LC–MS–MS showed the highest level, at 7.95 ng/g, in the adipose tissue, followed by stomach contents, brain, heart muscle, pancreas, and spleen. For the lung, liver, kidney, and skeletal muscle, levels were below the quantitation limit (about 0.5 ng/g), although very small peaks above the detection limit (about 0.1 ng/g) could be observed for all of the above solid tissues. The low levels of 5-fluoro-ADB in the solid tissues were likely as a result of only a small amount of 5-fluoro-ADB incorporated into the body via the lungs due to the short period from the beginning of smoking the herb to the fatal asphyxia resulting from aspiration of a massive amount of stomach contents into the trachea under low-consciousness conditions. In addition, we measured the content of 5-fluoro-ADB in three packages, all of which were opened, that were found under a pillow near the deceased; their levels of 5-fluoro-ADB were 49.2 mg/g, 12.2 μg/g, and 0.77 μg/g. To our knowledge, this is the first reported identification and quantitation of 5-fluoro-ADB in human specimens and herbal products.

Postmortem distribution of AB-CHMINACA, 5-fluoro-AMB, and diphenidine in body fluids and solid tissues in a fatal poisoning case: usefulness of adipose tissue for detection of the drugs in unchanged forms

We encountered an autopsy case in which the cause of death was judged as poisoning by multiple drugs, including AB-CHMINACA, 5-fluoro-AMB, and diphenidine. The deceased was a 30-year-old man. The postmortem interval to autopsy was estimated to be 3.5 days. Femoral vein blood, right heart blood, left heart blood, urine, and eight solid tissues including adipose tissue were collected and frozen until analysis. Extraction of the three drugs, and internal standards phencyclidine and 5-fluoro-AB-PINACA was performed by a modified QuEChERS method, followed by analysis by liquid chromatography–tandem mass spectrometry. Because this study dealt with various kinds of human matrices, we used the standard addition method to overcome the matrix effects. Quantitation of all three compounds was only achieved for the adipose tissue, whereas the levels of 5-fluoro-AMB were below the lower limit of quantitation (about 1 ng/g or ml) in all other samples. AB-CHMINACA was quantitated for all solid tissues, but not for all body fluid specimens. Diphenidine showed high concentrations in all specimens; it was highest in the adipose tissue (11,100 ± 1,120 ng/g), an order of magnitude lower in other solid tissue specimens, and two orders of magnitude lower in body fluid samples. The results suggest that adipose tissue is the best specimen for detection of lipophilic drugs, such as AB-CHMINACA, 5-fluoro-AMB, and diphenidine, in their unchanged forms. In this poisoning case, diphenidine appeared to have played the major role in the cause of death, because the concentrations of diphenidine were much higher than those of the synthetic cannabinoids in all specimens tested. To our knowledge, this is the first report to document the presence of AB-CHMINACA, 5-fluoro-AMB, and diphenidine in actual postmortem specimens in a fatal poisoning case.