Post-mortem changes in calmodulin binding proteins in muscle and lung

Estimation of post-mortem interval (PMI) remains an elusive issue in forensic investigations. In this study, we examined the possible use of calmodulin (CaM) binding proteins (CaMBPs) as indicators of PMI. Whole CaMBP populations from homogenized rat lung and rat skeletal muscle removed at 0, 24, 48 and 96 h post-mortem at 21 degrees C were detected by the calmodulin binding overlay technique (CaMBOT) using 35S-VU1-CaM and visualized by autoradiography. CaMBOT showed that, in both tissues, the CaMBP population remained relatively stable for up to 96 h post-mortem with the exception of a single approximately 200 kDa CaMBP that increased in 24 h post-mortem samples then showed decreasing amounts at subsequent times. Immunoblot analysis of the specific CaMBPs, Ca(2+)/CaM-dependent kinase II (CaMKII), calcineurin A (CNA), myristoylated alanine-rich C-kinase substrate (MARCKS) and inducible nitric oxide synthase (iNOS) were done on lung tissue samples. CaMKII levels did not change appreciably over the 96 h PMI examined. In contrast to iNOS levels, which varied from sample to sample, CNA and MARCKS showed predictable patterns of change: the level of MARCKS decreased steadily in the 0-96 h post-mortem lung samples while CNA underwent a shift in mobility on SDS-PAGE by 24 h post-mortem before slowly decreasing in amount. The stability of CaMKII levels over 96 h was also seen in skeletal muscle tissue while CNA showed variable levels at 0, 48 and 96 h with the presence of the rapidly migrating band at 24 h. These patterns of change in CaMBPs provide some insight into the post-mortem changes in calmodulin-mediated signaling components in lung and skeletal muscle and support the further study of CNA and CaMKII as potential markers for estimating short- and long-term PMIs.     

Postmortem toxicology of drugs of abuse

Conducting toxicology on post-mortem specimens provides a number of very significant challenges to the scientist. The range of additional specimens include tissues such as decomposing blood and other tissues, hair, muscle, fat, lung, and even larvae feeding on the host require special techniques to isolate a foreign substance and allow detection without interference from the matrix. A number of drugs of abuse are unstable in the post-mortem environment that requires careful consideration when trying to interpret their significance. Heroin, morphine glucuronides, cocaine and the benzodiazepines are particularly prone to degradation. Moreover, redistributive process can significantly alter the concentration of drugs, particularly those with a higher tissue concentration than the surrounding blood. The designer amphetamines, methadone and other potent opioids will increase their concentration in blood post-mortem. These processes together with the development of tolerance means that no concentration of a drug of abuse can be interpreted in isolation without a thorough examination of the relevant circumstances and after the conduct of a post-mortem to eliminate or corroborate relevant factors that could impact on the drug concentration and the possible effect of a substance on the body. This article reviews particular toxicological issues associated with the more common drugs of abuse such as the amphetamines, cannabinoids, cocaine, opioids and the benzodiazepines.     

Kinetics of ethanol degradation in forensic blood samples

To determine ethanol in human post-mortem blood samples is problematic, largely due to the inappropriate and variable methods of preserving and storing, which can cause decomposition and loss of alcohol concentration. In this study, four crucial parameters of sample conservation were studied: temperature (T), percentage of air chamber in container (%CA), ethanol concentration in blood and post-mortem time. Blood samples from post-mortem cases were stored under different conditions (ethanol levels were known in all cases); factorial design variables: (%CA) 0, 5, 20, 35, 65%; storage temperature: 25, 4 and -10 degrees C; in a total of 15 experiments. No preserving agent was used in samples. Quantification of ethanol in blood was carried out by gas chromatography with head-space FID detector. Initial ethanol concentration ranged from 0.50 to 4.30 g/L. The kinetics of degradation observed was pseudo-first-order. The parameter that characterised the kinetics of ethanol degradation (k(0)) ranged from (4 x 10(-4) and 5.0 x 10(-1) day(-1)), depending on storage conditions. A strong dependence between ethanol degradation and the content of the air chamber was observed and this dependence was found to be stronger than that between degradation and temperature; there was an experimental relation between (k(0)) and (%CA). Activation energy for different conditions, i.e. 0, 5, 20, 35 and 65 (%CA), were calculated and contour plots were made. A mathematical equation relating air chamber, temperature and ethanol concentration at a certain time was determined. This equation allowed estimation of initial concentrations of ethanol with minimal error. A good correlation between experimental data and data calculated with the equation was obtained (r(2) = 0.9998). The best storage conditions were: 0% CA and storage at -10 degrees C, obtaining an ethanol degradation of 0.01% after 15 days. However, 33% of ethanol degradation was obtained with 35% CA at 25 degrees C after 15 days. This equation is useful in forensic cases in which original concentration of ethanol has to be estimated under different sample storage conditions.     

The measurement and interpretation of morphine in blood

A method for the determination of morphine is post-mortem blood by HPLC with electrochemical detection is described. Blood morphine levels in post-mortem cases are reported and the importance of these in causing death is discussed. These post-mortem levels are compared further with morphine levels in the blood of patients receiving morphine as an analgesic.     

Post-mortem hypoxanthine levels in the vitreous humour an introductory report

Post-mortem hypoxanthine levels in vitreous humour were determined in 86 consecutive legal autopsy cases. In cases of sudden death caused by trauma or by myocardial infarction, levels ranging from 0 to 540 μmol/l were found. The mean value was about ten times higher than normal in vivo plasma levels. The hypoxanthine levels seem to be independent of time post-mortem, at least during the first 48 hours.It is known that augmentation of the hypoxanthine plasma, cerebrospinal fluid, and urine levels reflects tissue hypoxia. In the present material no elevation of hypoxanthine levels in the vitreous humour was found in cases of strangulation or suspension, while statistically significant elevation was found in cases of drug intoxication. It is concluded that this may reflect the effect of drug-induced prolonged tissue hypoxia caused by respiratory depression.     

Quantitative investigation of the amino acid levels in putrefying liver (author's transl)]

To elucidate the post-mortem proteolysis we investigated the behaviour of the free amino acids in rotting liver homogenates by means of two-dimensional thin-layer chromatography and reflection densitometry. The post-mortem alterations of the amino acid levels are characterized mostly by a two-phase increase with maxima at the end of the first and after the third week. A few amino acids showed only little changes hardly above the physiological intra-cellular levels. Anaerobic rat experiments and experiences with antibiotic treatment give rise to the supposition that increasing amino acid concentrations indicate a predominance of proteolytic activities over aminoacid catabolism. In the beginning autoenzymes are prevalent, while afterwards bacterial proteases probably predominate. The transient regression about the second and thrid week is interpreted as the effect of a temporarily intensified amino acid degradation by foreign enzymes (maximal bacterial growth).     

C-reactive protein (CRP), a comparison of pre- and post-mortem blood levels.

During autopsy of 26 inpatients with elevated CRP levels (14-536 mg/l) blood was taken from the femoral vessels and analysed for the content of CRP. The post-mortem CRP values were compared with the results of CRP analysis performed within the last 24h before death. This showed that the post-mortem values of CRP in average were reduced with 35% compared to the ante-mortem values. The decrease in CRP levels was not significantly influenced by the time interval from death until blood was drawn for analysis, at least up to 6 days. When blood taken from healthy individuals killed in accidents was analysed, no elevated CRP values were observed, indicating that there is negligible risk for false positive results. It is also shown that frozen blood can be used for CRP analysis if an immunometric kit assay based on whole blood is applied. The results demonstrate that an elevated level of CRP in post-mortem blood is a good marker for an ongoing inflammatory process prior to death. CRP analysis may therefore be a helpful tool in post-mortem examinations, especially in non-hospitalised individuals without medical records.     

Blood carbon monoxide and hydrogen cyanide concentrations in the fatalities of fire and non-fire associated civil aviation accidents, 1991-1998

To evaluate pathophysiological significance of post-mortem urinary myoglobin levels in determining the cause of death, we investigated 210 forensic autopsy cases, partially in comparison with serum levels. Post-mortem serum myoglobin levels were extraordinary high in most cases possibly due to post-mortem change. Urinary myoglobin levels did not correlate with the serum levels, showing possible post-mortem elevation in cases of a prolonged post-mortem period over 48h. A high (>1000 ng/ml), moderate (100-1000 ng/ml), slight (50-100 ng/ml) and not significant (<50 ng/ml) elevation of urinary myoglobin were observed in 26, 43, 31 and 110 cases, respectively. Half the highly elevated cases were those with a survival time over 24h. In cases of minor muscle injury such as head trauma, elevation of urinary myoglobin level was closely related to longer survival. In acute/subacute deaths with a post-mortem interval within 48h, a significant difference was observed in relation to the blood carboxyhemoglobin (COHb) levels of fire victims: myoglobinuria over 100 ng/ml was more frequently and markedly observed in cases with COHb below 60% than over 60%, suggesting muscle damage in fatal burns. Similar elevation was observed in heat stroke victims, and also in some cases of acute and subacute death from polytrauma, asphyxiation, drowning, electricity and spontaneous cerebral bleeding, but not in myocardial infarction. Thus, it was suggested that high post-mortem urinary myoglobin levels in acute and subacute death cases may be a possible indicator of antemortem massive skeletal muscle damage as well as exertional muscle hyperactivity or convulsive disorders associated with hypoxia.     

Diagnostic efficacy of biochemical markers in diagnosis post-mortem of ischaemic heart disease

In forensic medicine, there is a need for more sensitive biochemical markers for the post-mortem diagnosis of acute myocardial infarction. A study of the distribution of biochemical markers in different fluids is of great significance in post-mortem diagnosis, because their distribution depends on the location of tissue damage and release kinetics. The aim of this study is to compare the sensitivities and specificities of creatine kinase-MB (CK-MB), myoglobin and cTnI in serum and pericardial fluid for the post-mortem diagnosis of acute myocardial infarction (AMI). We studied 188 cadavers selected during 1 year from medicolegal autopsies. The groups were as follows: (1) myocardial infarction (n = 52); (2) asphyxia (n = 59); (3) multiple trauma (n = 41); (4) natural deaths excluding myocardial infarction (n = 36). We obtained statistically significant differences in pericardial fluid for all the biochemical markers, the highest levels being obtained in the group of cadavers who had died from myocardial infarction. A common factor is the high negative predictive value found in biochemical markers, which is contrary to the findings obtained in clinical practice, when the percentages of sensitivity are very high.     

Post-mortem analysis of apoptotic changes associated with human skin bruises

The estimation of the age of skin bruises is of importance in forensic medicine, especially in child abuse cases. Time-dependent changes in bruise colour and/or associated histological features have been used with a limited degree of success. An increased rate of apoptosis in the injured tissue has been considered as a novel time-dependent marker of cell death, by injury inflicted in a rat model. The object of the present study was to apply the TUNEL method of DNA end labelling to identify and enumerate apoptotic cells in bruised and normal skin in order to study the relationship of apoptotic cell density with the age of the bruise. A commercially available DNA end labelling kit, TUNEL method, was standardised, validated and used for this purpose. Twenty unselected post-mortem cases with bruises due to a variety of causes were studied. The apoptotic cells stained with TUNEL reaction were counted in 10 high power fields in the epidermis, as well as in the dermis of formaldehyde-fixed paraffin-embedded skin specimens. The mean positive cell densities (+/- 1 S.E.) were compared with respect to the age of the bruise. In the epidermis, the mean apoptotic cell count was statistically significantly greater in the bruised skin compared to normal skin in 2- to 6-day-old bruises; whilst in the dermis the same was true in 3- to 8-day-old bruises. The overall findings suggest that there is a quiescent period prior to the increase in the apoptotic cell activity that is seen following skin bruising. This is so provided the post-mortem skin samples were collected within a lapse of 6 days or less between the time of death and formalin fixation and paraffin embedding to avoid the bias made by the difference of length of post-mortem interval.     

Correlation of antemortem and postmortem digoxin levels.

Postmortem serum digoxin levels from any source routinely exceed antemortem values. Variation resulting from site of sampling gave a mean postmortem to antemortem ratio of 1.96 for heart, 1.63 for subclavian vein, and 1.42 for femoral vein samples. No correlation could be made between the postmortem interval and the increase in post-mortem serum values, irrespective of the site of sampling. A combination of femoral venous serum and vitreous humor values gave the best information for determining possible antemortem digoxin toxicity.     

Decapitation due to early post-mortem canine gnawing

A 53-year-old male was found dead in his home. The deceased's head, almost totally skeletonized, was lying at a distance of about 150 cm from the thorax inlet. The other occupant of the flat was a mongrel sheepdog. The autopsy conducted for the inquest established extensive damage to soft tissue in the head, neck and chest. The second to sixth cervical vertebrae were missing. The seventh cervical vertebra and the right first rib displayed bone lesions. The tissue injuries were attributed in the diagnosis to post-mortem canine gnawing. Cause of death was given as intermittent haemorrhaging of the gastro-intestinal tract from oesophageal varices in a status of hepatic cirrhosis. There was no indication that death had been caused by another party. About two days were estimated to have elapsed since death. Attention is drawn to this doubtless rare instance of total decapitation of the deceased with displacement of the head caused by a dog during the early post-mortem period.     

Post-mortem pregnancy: a proposed methodology for the resolution of conflicts over whether a brain dead pregnant woman should be maintained on life-sustaining treatment

In this article, the author examines conflicts over whether to maintain a brain dead pregnant woman on life-sustaining treatment. The author cautions that on the rare occasions when courts are confronted with such a conflict, they should employ a consistent methodology for resolution of the conflict and attempt to honor the wishes of the post-mortem mother and her family. The author draws on relevant areas of law to demonstrate the existence of a legal fiction that protects the interests of post-mortem pregnant women in refusing medical treatment. This article then proceeds to propose a methodology that enables courts to account for a post-mortem pregnant woman's interests, her family's interests, and the state's interests in resolving conflicts over whether to remove a post-mortem pregnant woman from life-sustaining treatment.     

Post-mortem drug redistribution--a toxicological nightmare

Detailed human case data is presented to illustrate the dramatic extent of the phenomenon of post-mortem drug redistribution. The data suggests that there is a post-mortem diffusion of drugs along a concentration gradient, from sites of high concentration in solid organs, into the blood with resultant artefactual elevation of drug levels in blood. Highest drug levels were found in central vessels such as pulmonary artery and vein, and lowest levels were found in peripheral vessels such as subclavian and femoral veins. In individual cases, in multiple blood samples obtained from ligated vessels, concentrations of doxepin and desmethyldoxepin ranged from 3.6 to 12.5 mg/l and 1.2 to 7.5 mg/l, respectively; amobartital, secobarbital and pentobarbital from 4.3 to 25.8 mg/l, 3.9 to 25.3 mg/l and 5.1 to 31.5 mg/l respectively; clomipramine and desmethylclomipramine from 4.0 to 21.5 mg/l and 1.7 to 8.1 mg/l, respectively and flurazepam 0.15 to 0.99 mg/l; imipramine and desipramine from 4.1 to 18.1 mg/l and 1.0 to 3.6 mg/l, respectively. We conclude that this poorly studied phenomenon creates major difficulties in interpretation and undermines the reference value of data bases where the site of origin of post-mortem blood samples is unknown.     

A study of ethyl glucuronide in post-mortem blood as a marker of ante-mortem ingestion of alcohol

The possibility of post-mortem production of ethanol makes correct interpretation of ethanol detection in forensic autopsy samples difficult. Even though the levels of ethanol formed post-mortem are generally low, this may be highly relevant in cases where intake of alcohol was forbidden, for instance for pilots, professional drivers and countries with low legal alcohol limits for driving. Different criteria are used to determine whether a finding of ethanol is of exogenous origin, but there is no marker for alcohol ingestion that has been studied in detail. In this study, we wanted to evaluate the sensitivity and specificity of ethyl glucuronide (EtG), a direct minor metabolite of ethanol, measured in blood, as a marker of ante-mortem alcohol ingestion. Forensic autopsy cases were divided into groups with and without ante-mortem alcohol ingestion, according to strict inclusion criteria. In 93 cases with information on ante-mortem alcohol ingestion, EtG was detected in blood in all cases, even when levels of ethanol were low. In another 53 cases where there were no indications of ante-mortem alcohol intake, EtG could not be detected in blood in a single case, also in 11 cases in which ethanol was detected and considered to be most probably formed post-mortem. In conclusion, blood EtG determination seems to be a reliable marker of ante-mortem ingestion of alcohol, and it could be considered in forensic autopsy cases when post-mortem formation of ethanol is questioned.     

Is zinc a reliable biochemical marker of chronic alcoholism in the overall context of a medico-legal autopsy?

The purpose of our investigation is to appreciate the usefulness of zinc determination in the human body with regard to the tracing of chronic alcoholism at forensic autopsies. The analysis of zinc was performed in vitreous humour and in skeletal muscle. Our results tend to prove that, in post-mortem material, zinc is not a suitable marker of chronic alcoholism. Furthermore one has to take into account the frequent contamination during collection and storage of the samples and also the post-mortem increase of zinc in the vitreous humour due to autolysis.     

Magnesium, potassium, sodium and calcium in post-mortem vitreous humour from humans

Levels of magnesium, potassium, sodium and calcium in post-mortem vitreous humour from human controls, fire fatalities and drowning victims have been determined. The effects of time-related internal changes, external environmental parameters and different causes of death are evaluated. Despite the positive correlation and marked increase of potassium and, to a lesser extent, of magnesium and calcium with the length of the post-mortem interval, individual biological variability severely limits the usefulness of predictions of post-mortem interval based on electrolyte metal data. At best, there is only a 2/3 chance of a prediction being within 12 h of the true value. Vitreous humour metal concentrations are affected by external influences, such as the elevated temperatures of fires which increase the rate of release of intracellular magnesium and potassium. In cases where drowning is suspected, establishment or exclusion of this cause of death is not possible on the basis of vitreous humour electrolyte metal data because of possible post-immersion diffusion across the permeable membrane of the eyeball. It appears, however, that magnesium in salt-water cases and sodium in fresh-water cases are related, albeit erratically, to the length of the immersion period.     

利用尸食性蚤蝇推断死亡时间的法医学研究进展

死亡时间的准确推断一直是法医学的难题。昆虫学方法已被认为是死亡时间推断的有效方法。尸食性蝇类发育生物学在死亡时间推断中有着重要地位。蚤蝇体形微小,在相对密闭的空间,可成为尸体上主要的甚至是唯一的昆虫证据。本文从尸食性蚤蝇的作用、种属鉴定以及日龄推断方面,综述了利用尸食性蚤蝇推断死亡时间的法医学研究进展。     

Headspace solid phase microextraction for the gas chromatographic analysis of methyl-parathion in post-mortem human samples. Application in a suicide case by intravenous injection

A simple and rapid procedure for the determination of methyl-parathion (m-p) in post-mortem biological samples was developed using headspace solid phase microextraction (SPME) and gas chromatography (GC) with nitrogen-phosphorous detection (NPD). Methyl-parathion was extracted on 85 microm polyacrylate SPME fiber. Salt addition, extraction temperature, and extraction time were optimized to enhance the sensitivity of the method. The linearity (y = 0.0473x - 0.0113, R2 = 0.9992) and the dynamic range (0.1-40 microg/ml) were found very satisfactory. The recoveries of methyl-parathion were found to be 46% in spiked human whole blood, 53% in spiked homogenized liver tissue, and 54% in spiked homogenized kidney tissue compared with samples prepared in water. The coefficients of variations for 2, 4, and 20 microg/ml of methyl-parathion in blood ranged from 0.9 to 5.1%, whereas the detection limit of the method was satisfactory (1 ng/ml in aqueous samples, 50 ng/ml in whole blood). The developed procedure was applied to post-mortem biological samples from a 21-year-old woman fatally poisoned (suicide) by intravenous injection of methyl-parathion. The intact insecticide was found in the post-mortem blood at a concentration of 24 microg/ml. No methyl-parathion was detected in the liver, kidneys, and gastric contents.     

A fatal diazinon poisoning

A case of fatal suicidal ingestion of diazinon insecticide is presented. Diazinon concentrations in post-mortem body fluids and tissues were determined using electron capture and flame ionization gas-liquid chromatography. The highest concentrations of diazinon were found in blood, stomach contents, bile and adipose tissue.