Postmortem redistribution of ketamine in ocular matrices: A study of forensic relevance

The present study evaluates the postmortem redistribution of ketamine in ocular matrices, such as vitreous humor, aqueous humor, and ocular tissues in an animal model. To understand the redistribution of ketamine and its metabolite (norketamine) in the ocular matrices, an in vivo study was performed in rabbits. The rabbits were divided into two groups: perimortem and postmortem. The postmortem samples were collected at 17 h after the administration of ketamine (40 mg/kg) intravenously. For a better understanding of the metabolism of ketamine in eyes, an ex vivo study was conducted in goat eyes after administration of ketamine intravitreally. The samples were analyzed by LC-MS/MS and the levels of ketamine and norketamine in these matrices were compared with that of whole blood and plasma. The results of the in vivo study showed a decrease in ketamine levels in whole blood and plasma while an increase in ocular matrices at postmortem. Though, in most cases, this increase/decrease was statistically insignificant. Moreover, there was an increase of norketamine level in ocular matrices. Ex vivo study also shows the presence of norketamine in ocular matrices of goat eyes. The presence of norketamine in goat eyes may be indicative of the metabolism of ketamine in the eyes.     

Ethyl sulfate in blood shows the potential to distinguish alcoholic death and postmortem alcohol instillation

Alcohol is often found in the blood of the deceased. To cover up the true cause of victim’s death, postmortem instillation of alcohol occurs in some criminal cases. Explaining the finding of alcohol is extremely vital in forensic practice. This study aims to evaluate whether ethyl glucuronide (EtG) and ethyl sulfate (EtS) in blood and vitreous humor (VH) can be used to distinguish alcoholic death and postmortem alcohol instillation. Saline or 12.6 g/kg ethanol (antemortem alcohol poisoning group) was introduced into rabbits’ stomachs 2 h before sacrificed. Same amount of ethanol was introduced into rabbits’ stomachs at 0 h, 0.5 h, 1 h and 2 h after death in four subgroups of postmortem alcohol instillation group, respectively. Cardiac blood and VH were collected at 10 min, 4 h, 10 h and 24 h after death in blank and antemortem alcohol poisoning group, and after instillation of alcohol in postmortem alcohol instillation group. Blood was also collected at 34 h. Ethanol and EtG levels in blood and VH and EtS in VH in antemortem alcohol poisoning group were overlapped with those in postmortem alcohol instillation group. The contents of EtG and EtS in blood in antemortem alcohol poisoning group (mean ≥ 7.833 μg/mL for EtG and ≥ 19.990 μg/mL for EtS) were much higher than those in postmortem alcohol instillation group (mean ≤ 0.118 μg/mL for EtG and ≤ 0.091 μg/mL for EtS), but apparent decomposition was observed in EtG, which might lead to misinterpretation. Blood EtS showed better stability and could be used to distinguish alcoholic death and postmortem alcohol instillation.     

Postmortem vitreous chemistry – An evaluation of sodium,potassium and chloride levels in estimation of time since death (during the first 36 h after death)

Estimation of time since death is a paramount medico-legal issue in any postmortem examination. The present study is intended to study the correlation between postmortem interval and vitreous humor chemistry for sodium, potassium, and chlorides. The study is aimed to find male–female differences and differences between right and left eyes in vitreous chemistry. The vitreous humor samples were collected in 114 autopsies conducted in the study center and analyzed biochemically. All the cases where exact time of death was known and where the time since death ranged between 0 and 36 h were included in the study. Data obtained was analyzed statistically using spss version 11.0. The present research did not find a significant correlation between vitreous chemistry and postmortem interval. The differences in vitreous sodium, potassium, chloride levels and the sodium potassium ratio among males and females and between right and left eyes were not found to be statistically significant.     

Measurement of digitalis-glycoside levels in ocular tissues:

Summary Prompted by animal studies reporting the accumulation of digitalis-glycosides in ocular tissues, we investigated whether measurement of digoxin levels in human ocular tissues can improve the postmortem diagnosis of lethal digoxin intoxication. Digoxin was measured in the vitreous humor and choroid-retina of patients who had received in-patient treatment with digoxin prior to death (therapeutic group) and in a single case of suicidal intoxication. The results were compared with the digoxin levels in the femoral vein blood, myocardium, kidney and liver, and evaluated in light of the medical history of each patient. In the therapeutic group the mean digoxin level was higher in the choroid-retina than in other tissues and body fluids. The range of variation in levels in the choroid-retina following therapeutic doses was comparable to that in the other tissues. An extremely high level of digoxin was present in the choroid-retina in the case of suicidal intoxication. In all cases, levels in the vitreous humor were very low compared to those in the choroid-retina. Hence, it is unlikely that significant distortion of choroid-retinal levels occurs due to postmortem diffusion of digoxin into the vitreous body. Our results indicate that measurement of digoxin levels in the choroid-retina can aid the postmortem diagnosis of lethal digoxin intoxication.This study contains results from the dissertation of P. Harding     

Implication of High-mobility group box-1 and skin post mortem changes in estimation of time passed since death: Animal and human study

Determination of postmortem interval (PMI) is one of the goals of the forensic autopsy. The study aimed to correlate the postmortem skin changes and High-mobility group box-1 (HMGB1) alterations in serum and skin immunohistochemical staining with time since death. We used animal and human specimens; forty adult male albino rats were dissected to obtain samples at PMI (0, 3, 6, 12, 24 h); forty human medicolegal autopsy cases with a known time of death (within the first 24 h PMI). Cases were classified into 5 groups according to the PMI: I (0 h); II (≤3h); III (4 to 6); IV (7 to 12); V (13 to 24) hour intervals after death; blood and full-thickness skin samples were collected from both models. Results showed a significant time-dependent elevation in serum HMGB1 levels along with its overexpression in immunohistochemically stained skin tissue. Also, the degree of histopathological changes in epidermis, dermis, and hypodermis progressively increased with PMI in both models. The timetable of postmortem skin histological changes, serum HMGB1 concentration, and immunoexpression for HMGB1 proteins in skin tissues has a profile that could serve as actual and simply convenient parameters for accurate determination of postmortem intervals in both models. HMGB1 displayed a pivotal role in the estimation of PMI at the examined periods.     

Postmortem redistribution of THC in the pig

To improve the knowledge of the postmortem redistribution of Δ⁹-tetrahydrocannabinol (THC), an animal model using the Large White pig has been developed, whereby 15 pigs received an intravenous injection of THC (200 µg/kg body weight) and were euthanized 2 h after administration. An autopsy was performed on three pigs immediately after being euthanized while the others were stored in supine position at ambient temperature for 6, 15, 24, or 48 h. THC concentration in blood from the vena cava decreased after death whereas left or right cardiac blood concentrations increased. No blood specimens collected from different sites of the carcasses adequately reflected the perimortem THC concentrations. The highest concentrations of THC at anytime were observed in lung tissue, and brain tissue seemed to present the most stable concentrations over time. This study can assist toxicologists in determining which specimens can, most appropriately, be used for interpretation of cannabinoid concentrations in postmortem specimens.     

Ocular disposition of diazepam in rabbits: understanding its level in vitreous humour for forensic applications

The disadvantages associated with routine toxicological samples, such as blood samples, have encouraged the forensic toxicologist to use vitreous humour as an alternative matrix. However, its use is still limited. The reason for this is the lack of a systematic study in this domain. Moreover, the phenomena of drug disposition in ocular tissues are mostly unexplored. This paper presents a controlled study to investigate the distribution of diazepam in post-mortem blood, vitreous humour and ocular tissues using rabbit as an animal model. This study reveals that diazepam levels in vitreous and aqueous humour increase with their decrease in whole blood at post-mortem. A further study on the distribution of diazepam in ocular tissues reveals that the anterior and posterior segment tissues might be responsible for the increase in diazepam levels in aqueous humour and vitreous humour respectively. The present study strengthens the possibility of using vitreous humour as a complementary sample to blood in forensic toxicological investigations.     

Estimation of blood and urine levels of eight metals and essential trace elements collected from living Subjects compared to urine,cardiac and femoral postmortem blood,and other postmortem samples: A forensic toxicology study

Along with the regular toxicology testing, different samples collected during the autopsy might be subjected to metal level estimation to investigate the cause of death in some cases. Utilizing a scientific procedure on postmortem specimens is crucial for interpreting forensic toxicological analytical results. Even modest procedural errors made by incompetent forensic toxicologists and chemists who lack proper specialized training and knowledge can alter the scientific conclusions and hence the legal verdict.The current work studies an overview of eight metals and element levels in living and deceased human bodies. It could be a substantial contribution to establishing normal or so-called "reference" metal levels under antemortem and postmortem situations, hence aiding in identifying reliable future interpretations of results produced by numerous researchers in the same field.Aim of the workThe current work aimed to study the concentration of eight metals in the blood samples (cardiac and femoral), urine, and other samples (Spleen, liver, and renal tissues) collected from human cadavers at different postmortem intervals in addition to blood and urine samples collected from the living population as a contribution to establishing normal or so-called "reference" metal levels under antemortem and postmortem situations.Subjects and methodsPostmortem autopsy blood samples (cardiac and femoral), urine, and other samples (Spleen, liver, and renal tissues) were collected from 400 deceased subjects. These samples were analyzed for the estimation of the eight metals under research, namely, Arsenic (As), Selenium (Se), Silver (Ag), Cadmium (Cd), Antimony (Sb), Mercury (Hg), Zinc (Zn) and Lead (Pb). In addition, blood and urine samples from 400 living volunteer subjects were analyzed for the same eight elements under study.ResultsIn the postmortem group, the mean metal levels in cases with absent, early, and advanced putrefaction simultaneously in μg/L were 2.45 ± 3.30, 3.25 ± 5.18, and 3.81 ± 1.95 for As. For Se, the results were 10.74 ± 4.21, 10.54 ± 5.28, and 9.96 ± 4.14. 4.04 ± 1.74, 3.48 ± 1.32, and 3.74 ± 0.91 were the results for Ag. For Cd, they were 8.35 ± 3.91, 12.15 ± 3.05, and 24.51 ± 31.25 with P < 0.0001**. 1.48 ± 1.85, 1.61 ± 1.85, and 1.62 ± 1.74 were the same results for Sb; 6.07 ± 2.44, 5.22 ± 2.17, and 5.39 ± 1.82 for Hg. 395 ± 79.8, 553 ± 51.7, and 704 ± 97.2 for Zn with a P-value <0.005*. As for lead, the results were 15.61 ± 24.19, 14.76 ± 23.05, and 24.61 ± 52.72. As the postmortem interval increased, Cd and Zn levels increased (p < 0.0001, <0.005* simultaneously).     

LC-ESI-MS/MS on an ion trap for the determination of LSD, <Emphasis Type="Italic">iso</Emphasis>-LSD, <Emphasis Type="Italic">nor</Emphasis>-LSD and 2-oxo-3-hydroxy-LSD in blood,urine and vitreous humor

A method has been developed for the simultaneous determination of lysergic acid diethylamide (LSD), its epimer iso-LSD, and its main metabolites nor-LSD and 2-oxo-3-hydroxy LSD in blood, urine, and, for the first time, vitreous humor samples. The method is based on liquid/liquid extraction and liquid chromatography-multiple mass spectrometry detection in an ion trap mass spectrometer, in positive ion electrospray ionization conditions. Five microliter of sample are injected and analysis time is 12 min. The method is specific, selective and sensitive, and achieves limits of quantification of 20 pg/ml for both LSD and nor-LSD in blood, urine, and vitreous humor. No significant interfering substance or ion suppression was identified for LSD, iso-LSD, and nor-LSD. The interassay reproducibilities for LSD at 20 pg/ml and 2 ng/ml in urine were 8.3 and 5.6%, respectively. Within-run precision using control samples at 20 pg/ml and 2 ng/ml was 6.9 and 3.9%. Mean recoveries of two concentrations spiked into drug free samples were in the range 60–107% in blood, 50–105% in urine, and 65–105% in vitreous humor. The method was successfully applied to the forensic determination of postmortem LSD levels in the biological fluids of a multi drug abuser; for the first time, LSD could be detected in vitreous humor.     

Perimortale Artefakte

Background

In routine postmortem examination of corpses doctors are often confronted with findings the origin of which is not immediately clear. Some of these findings result from interventions by lay rescuers, paramedics, rescue personnel, morticians and previous postmortem examinations. The origins of these external and internal changes of the body are often not recognized at first glance.

Material and methods

Based on personal experience and literature searches perimortem artefacts were compiled and evaluated in terms of their importance for the external examination and the autopsy. The objective was not a complete collection but a selection of typical perimortem changes and differential diagnoses.     

曲伐沙星滴眼液预防家兔实验性金黄色葡萄球菌性眼内炎

目的评价曲伐沙星眼局部滴眼预防兔实验性金葡菌性眼内炎的有效性。方法构建兔细菌性眼内炎模型,按给药方案于造模前、后滴眼给药,对兔眼内炎进行观察、评分,并进行病理组织观察和房水、玻璃体细菌培养。结果曲伐沙星和左氧氟沙星组临床评价指标均优于空白对照组,曲伐沙星和左氧氟沙星组之间无显著性差异。曲伐沙星和左氧氟沙星均可有效降低房水组织中的荷菌数,且两者是等效的。但相比于空白组,曲伐沙星和左氧氟沙星均不能有效降低玻璃体组织细菌感染率和细菌数。结论曲伐沙星眼局部给药用于预防金葡菌性眼内炎方面具有与左氧氟沙星相似的疗效,提示其在眼局部应用中具有潜在的临床价值。     

Postmortem changes in tissue concentrations of triazolam and diazepam in rats

The aim of this study was to investigate the postmortem changes of concentrations of triazolam and diazepam in the rat model and to confirm the factor causing the phenomenon. We administered triazolam or diazepam orally to rats and then sacrificed them 1 h after administration. Abdominal tissues including stomach content and small intestine, thigh muscle and heart blood were collected at 0, 12, 24 h after death, and postmortem changes of the two drug concentrations were compared. Drug concentrations in the samples were analyzed by a selected ion monitoring of gas chromatography/mass spectrometry. Gastrointestinal postmortem concentrations of triazolam and diazepam decreased. On the other hand, the drug concentrations in the liver and kidney increased markedly, and those in lung and heart blood increased slightly during postmortem periods by diffusion from the gastrointestinal tract. The patterns of both drug concentration changes were similar. However, the extent of the triazolam increase tended to be larger than that of diazepam. This difference may be accounted for by lower triazolam concentrations before death. The postmortem drug concentrations of thigh muscle did not change when administered with triazolam and diazepam. For both triazolam and diazepam, a significant correlation was observed in the drug concentration between the small intestine and the kidney at 24 h after death, indicating that major cause of the change in the drug concentration in the kidney was diffusion from the small intestine up to 24 h after death. The results in this study indicate that diazepam as well as triazolam diffuses from the gastrointestinal tract into the surrounding tissues after death and suggest that the diffusion is influenced by their pharmaceutical properties before death.     

Endogenous gamma-hydroxybutyric acid levels in postmortem blood

The goal of this study was to determine how the postmortem interval and duration of storage of blood at 4 degrees C affect endogenous gamma-hydroxybutyric acid (GHB) levels in blood. Forty-three autopsy cases of non-users of GHB were involved. The postmortem interval ranged from 8 to 132 h. Blood samples were collected and stored without any preservatives at 4 degrees C for 1 day up to 15 months until analysis. In some cases, samples were also stored at -20 degrees C for 10 days to 7 months to determine GHB levels at autopsy. Blood GHB concentrations were measured by headspace gas chromatography after GHB was converted to gamma-butyrolactone. Blood GHB concentrations ranged from 0 to 43.0 microg/ml and averaged 9.80 microg/ml. A positive correlation was observed between concentration and postmortem interval (r = 0.571) but no correlation was found between concentration and storage interval at 4 degrees C. In 14 blood samples stored at -20 and 4 degrees C for 10 days, GHB concentrations were 4.55+/-3.88 and 6.06+/-4.27 microg/ml, respectively. In another eight blood samples stored at -20 and 4 degrees C for 1-7 months, GHB concentrations were 3.77+/-2.76 and 5.49+/-2.97 microg/ml, respectively. A large portion of endogenous GHB detected in blood of corpses may be produced during the interval between death and autopsy, rather than during storage of blood at 4 degrees C until analysis. In an additional experiment, it was suggested that glycolysis by bacteria may enhance endogenous GHB production.     

Application of Lithium Assay kit LS for quantification of lithium in whole blood and urine

A commercially available kit for the quantitation of lithium, the Lithium Assay kit LS, was originally developed to measure lithium in serum or plasma using a conventional microplate reader. We investigated whether use of the kit could be extended to quantify lithium in whole blood and urine samples collected at autopsy. The calibration curve for whole blood showed good linearity ranging from 0.5 to 20 µg/mL with a coefficient of determination of 0.998 when samples were pretreated with methanol followed by acetonitrile. Moreover, for urine, we obtained excellent linearity with a coefficient of determination of 0.999 without any pretreatment. The accuracies and precisions were 106.3–174.7% and 1.9–18.1% for whole blood and 83.3–118.8% and 5.7–33.8% for urine. The values in the lower concentration range (0.5–1 µg/mL) were not satisfactory, whereas those in the higher range (2–20 µg/mL) were acceptable. The Lithium Assay kit LS was successfully applied to the measurement of lithium in whole blood and urine samples collected at autopsies. This method appears to be useful for forensic toxicological investigations because of its simplicity and speed.     

The significance of post-mortem vitreous calcium concentration in forensic practice

Calcium, as one of the main extracellular ions, maintains a key role in numerous biologic functions. For forensic purposes, it was analyzed mostly for estimation of postmortem interval (PMI). We have designed our experiment with the concept that the repetitive withdrawal of vitreous humor (VH) might clarify the postmortem metabolism of calcium in greater detail to estimate the PMI. Accordingly, 248 samples of VH from 31 autopsy cases were evaluated over three years; samples (0,1 mL of VH) were taken and analyzed at equal time intervals after death—every three hours until 24 h after death. Each sample was centrifuged and analyzed using the ARCHITECT C SYSTEM 8000. Moreover, functional relationship between PMI and calcium concentration was established: PMI (hours) = [Ca²⁺] × 13.696–7.843. Although the concentration of calcium in VH in the analyzed group increases with time, the coefficient of variation for the regression (CVreg = 46.8%) indicates that this correlation is not so strong, meaning that the level of predictiveness of calcium for estimation of time since death is poor when is not used in combination with other relevant substances.     

Evaluation of postmortem measurement of NT-proBNP as a marker for cardiac function

Clinical biomarkers of cardiac function could also be monitored postmortem. Among the natriuretic peptides, the aminoterminal portion of pro-brain natriuretic peptide (NT-proBNP) appears to be a more reliable postmortem tool than the BNP, owing to its longer half-life and greater stability. In living persons, NT-proBNP is considered to be a marker of heart failure, and its level rises after cardiac ischemia. The goal of this study was first to evaluate the postmortem stability of NT-proBNP, then to measure the NT-proBNP levels in postmortem cases of heart failure related to coronary ischemia. The goal of this study was also to evaluate the correlations between different specimens collected at autopsy (e.g. blood, serum, vitreous humor and pericardial fluid). The study included 96 cases, which were classified into 4 groups according to the autopsy and histological findings. The NT-proBNP levels were significantly higher in individuals who had suffered from chronic cardiac ischemia, with or without acute coronary events, than in either control cases or those who had suffered from acute thromboembolism or acute rupture of a plaque without chronic cardiac ischemia. The highest levels were registered in individuals who had suffered from acute coronary thromboembolism in association with chronic coronary ischemia. Good correlations in the NT-proBNP levels for the different specimens were observed between samples of femoral blood, serum, and pericardial fluid. Our data indicated that postmortem measurements of NT-proBNP are reliable and compatible with clinical findings.     

Vitreous humor fructosamine concentrations in the autopsy diagnosis of diabetes mellitus

In clinical practice, biochemical markers, particularly serum glucose levels are used to diagnose diabetes mellitus. However, at autopsy this marker is of no value due to the substantial and capricious fluctuations in glucose levels after death. The aim of this study was to evaluate the usefulness of the postmortem determination of fructosamine in vitreous humor for confirming the presence of antemortem hyperglycemia. This was a study of 92 cadavers with a mean age of 60.05 years (SD 17.73) and a mean postmortem interval of 17.02 h (SD 9.76, range 2–58 h). Cases were assigned to two diagnostic groups according to the antemortem diagnosis of diabetes mellitus based on the patients’ medical records. In vitreous humor statistically significant differences were found in glucose and fructosamine concentrations between the two diagnostic groups, the highest values being obtained in the group of subjects with a previous diagnosis of diabetes mellitus. Received: 5 August 1998 / Received in revised form: 21 January 1999     

Course of exogenous ethanol in the first hours after death – two experimental approaches

Post-mortem redistribution may contribute to changes in blood alcohol concentration, rendering questionable results in a court of law. Two experimental approaches using Wistar rats and human plasma were developed to improve the reliability of femoral blood in post-mortem alcohol analyses. First, rats were administrated with ethanol (0.6 g/kg, p.o) and 30 min later were euthanized by exposure to CO₂. After perimortem cardiac blood sampling, samples of blood, liver and lung were collected 6, 12 and 24 h post-mortem in order to measure alcohol concentration, water content and haematocrit. Plasma from human corpses obtained from central and peripheral blood was supplemented with ethanol and afterwards incubated at 37?°C. The animal model revealed a significant decrease of both blood alcohol concentration and water content. Moreover, significant differences between central and peripheral sites were observed in human blood autolysis markers, revealing an exacerbation of haemolysis, which in turn affected alcohol stability by the oxidation of alcohol to acetaldehyde. Our study confirmed that both dehydration and autolysis of blood exacerbate alcohol diminution in the central blood in the first six hours after death. This study reinforces the suitability of femoral blood over central blood for ethanol analyses.