Rapid postmortem changes of rat striatum dopamine, serotonin, and their metabolites as monitored by brain microdialysis

Brain microdialysis was used to monitor changes in extracelluar dopamine (DA), serotonin (5-HT), and their metabolite levels in the rat striatum at death by cervical dislocation. Maximum respective 450-fold and 150-fold increases in the extracelluar output of DA and 5-HT were observed within the first 30 min of death. DA and 5-HT outputs remained elevated over the following 2 h at levels about 100-fold and 50-fold above pre-death values, respectively. In contrast with monoamine outputs, the outputs of the DA metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), and the 5-HT metabolite, 5-hydroxyindoleacetic acid (5-HIAA), rapidly decreased by 10% and 20%, respectively 1 h after death. 5-Hydroxytryptophol (5-HTOL) gradually decreased after death. Before death both the extracellular DOPAC/DA and 5-HIAA/5-HT ratios were about 400; after death these ratios dropped to 0.56 and 4.0, respectively at 30 min. These observations suggested that regulation of neurotransmitter releases through the neuronal membrane and metabolisms in the rat striatum were seriously disrupted at death. This finding may be helpful in the determination of death in the field of forensic medicine.     

Evaluation of Postmortem Cerebrospinal Fluid S100B Protein and Serotonin Levels: Comparison of Suicidal Versus Nonsuicidal Deaths in Konya,Turkey

Although suicide is a preventable public health problem, objective assays for suicide risk are limited. In this study, it was aimed to determine levels of S100B protein and serotonin as a marker for risk of suicide. S100B protein and serotonin levels were investigated with ELISA method in the cerebrospinal fluid (CSF) in medicolegal autopsy cases, including those of suicide cases (n = 32) and nonsuicide cases (n = 56). The CSF S100B levels were higher (9.3 ± 2.9 ng/mL vs. 5.4 ± 2.0 ng/mL), and serotonin levels were lower (10.4 ± 4.9 ng/mL vs. 19.0 ± 5.7 ng/mL) in suicide group than nonsuicide group (p < 0.05). There was no correlation between S100B protein and serotonin levels with gender, age groups, postmortem interval, and cause of death. It is concluded that both S100B protein and serotonin in CSF may be useful for determination of suicide risk.     

Postmortem changes in the levels of monoamine metabolites in human cerebrospinal fluid.

Four monoamine metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxy-4-hydroxyphenylglycol (MHPG) and 5-hydroxyindoleacetic acid (5-HIAA), were determined in the cerebrospinal fluid (CSF) of cadavers, whose causes of death had been suicidal hanging (SH) or ischemic heart failure (IHF). The concentration of DOPAC increased in parallel with the increment of the postmortem interval (PMI) (r = 0.626), whereas the concentrations of HVA, MHPG and 5-HIAA did not. The correlation coefficient was further increased by considering each cause of death separately: i.e., SH, r = 0.761; IHF, r = 0.705. These findings suggest the possible usefulness of the DOPAC level in CSF for estimating PMI.     

Utilizing the urinary 5-HTOL/5-HIAA ratio to determine ethanol origin in civil aviation accident victims

Specimens from fatal aviation accident victims are submitted to the FAA Civil Aerospace Medical Institute for toxicological analysis. During toxicological evaluations, ethanol analysis is performed on all cases. Care must be taken when interpreting a positive ethanol result due to the potential for postmortem ethanol formation. Several indicators of postmortem ethanol formation exist; however, none are completely reliable. The consumption of ethanol has been shown to alter the concentration of two major serotonin metabolites, 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindole-3-acetic acid (5-HIAA). While the 5-HTOL/5-HIAA ratio is normally very low, previous studies using living subjects have demonstrated that the urinary 5-HTOL/5-HIAA ratio is significantly elevated for 11-19 h after acute ethanol ingestion. Recently, our laboratory developed and validated an analytical method for the simultaneous determination of both 5-HTOL and 5-HIAA in forensic urine samples using a simple liquid/liquid extraction and LC/MS/MS and LC/MS/MS/MS. In this previous work a 15 pmol/nmol serotonin metabolite ratio cutoff was established in postmortem urine, below which it could be conclusively determined that no recent antemortem ethanol consumption had occurred. In the current study this newly validated analytical method was applied to five ethanol-positive aviation fatalities where the origin of the ethanol present could not previously be conclusively determined. In four of the five cases examined the detected ethanol was demonstrated to be present due to postmortem microbial formation, and not consumption, even though some indication of ethanol consumption may have been present.     

Postmortem serotonin (5-HT) concentrations in the cerebrospinal fluid of medicolegal cases

In a medicolegal study the postmortem serotonin (5-HT) cerebrospinal fluid (CSF) concentrations were determined in routine autopsies using a high performance liquid chromatographic procedure with electrochemical detection. There was no correlation between 5-HT concentrations and age, sex or blood alcohol concentration using a postmortem delay < or = 3 days. In suicides the suboccipital CSF concentrations were significantly decreased compared to the levels measured in the control group (8.55+/-5.99 ng/ml versus 20.15+/-13.56 ng/ml). Additionally, a decrease of 5-HT was found in the suboccipital CSF of opiate fatalities (15.56+/-13.52 ng/ml). The results support the hypothesis that decreased 5-HT concentrations in the CSF are characteristic in suicides. However, due to a rather broad overlapping of values between suicides and controls the results failed to define a possible cut-off level in the 5-HT CSF concentration to distinguish between a suicidal and a non-suicidal incident.     

Effects of ion channel blockers on rapid postmortem changes in extracellular dopamine and serotonin levels in the rat nucleus accumbens

In the present study, we used in vivo brain microdialysis to examine the effects of ion channel blockers tetrodotoxin (TTX), EGTA-free Ca²⁺ and verapamil on rapid postmortem changes in extracellular levels of doapmine (DA), serotonin (5-HT) and their metabolites dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) in the ACC of freely moving rats. Extracellular ACC DA levels decreased following the perfusion of the three ion channel blockers in freely moving rats, and then, at death by cervical dislocation, maximum respective 220-, 60- and 90-fold increases were observed in the extracellular output of DA in animals treated with EGTA, verapamil and TTX, respectively. Also, ACC 5-HT decreased following perfusion with the three blockers in the freely moving rats, and then maximum increases of 80-, 30- and 45-fold in the extracellular output of 5-HT were observed at death in animals treated with EGTA, verapamil and TTX, respectively, compared to the baseline. Cervical dislocation-induced rapid postmortem changes were inhibited markedly by perfusion with CSF containing the Ca²⁺ entry blocker verapamil. These observations suggested that rapid postmortem changes in ACC DA and 5-HT release were associated with the action of calcium ion channels and/or voltage gated channels in the CNS.     

Tissue distribution of tramadol and metabolites in an overdose fatality

Tramadol (Ultram) is a centrally acting, synthetic analgesic agent. Although it has some affinity for the opiate receptors, tramadol is believed to exert its analgesic effect by inhibiting the re-uptake of norepinephrine and serotonin. There are several published cases of tramadol's involvement in drug-related deaths and impairment. Reports of deaths involving tramadol alone with associated tissue concentrations are rare. This report documents a case in which tramadol overdose was identified as the cause of death. The following tramadol concentrations were found in various tissues: blood, 20 mg/L; urine, 110.2 mg/L; liver, 68.9 mg/kg; and kidney, 37.5 mg/kg. Tissue distributions of the two primary metabolites, N-desmethyl and O-desmethyl tramadol, are also reported. In each tissue or fluid except urine, the tramadol concentration was greater than either metabolite, consistent with other reports of drug-impaired drivers and postmortem cases. The O-desmethyl metabolite concentration was greater than the N-desmethyl metabolite concentration in all tissues; this is in contrast to other postmortem reports, in which the majority of cases report concentrations of O-desmethyl as less than those of N-desmethyl. This may be useful as an indicator of time lapse between ingestion and death.     

Comparative measurement of cocaine and its metabolites in blood, cerebrospinal fluid and the brain

This study investigated the transfer of cocaine and its metabolites from plasma into the cerebrospinal fluid. The concentration of cocaine and its metabolites in plasma and cerebrospinal fluid was determined by radioimmunoassay because this method only, the sum of the drug and metabolites restored. In sheeps a sublethal cocaine hydrochloride dose (2,4 mg/kg b. wt.) was administered intraarterial daily for up to 8 days. In the first hours after administration the concentration of cocaine in cerebrospinal fluid was low. It is supposed that a barrier against the transport of cocaine from blood into cerebrospinal fluid exists. After intrathecal administration a delay of transport could from CSF to blood not be seen.     

Distribution of morphine and morphine glucuronides in body tissue and fluids--postmortem findings in brief survival

An intoxication following administration of morphine, tramadol and atracurium in a suicide case is reported. The route of administration and the amount of the particular drug were known from the investigation of the death scene and the findings of the postmortem examination. Tramadol was present in the gastric contents as well as in blood, liver, kidney and brain samples, whereas the drug could not be detected in muscle. All body fluids and tissues investigated contained morphine as well as its 3- and 6-glucuronides with the exception of muscle tissue. The concentrations of morphine and its glucuronide metabolites were determined by LC/MS following solid phase extraction. Interestingly, the concentration of M6G in brain, liver and kidney were close to the concentration of M3G in the particular tissue. This phenomenon might be explained by a preferential hydrolysis of M3G or by a preferential formation of M6G postmortem. Measurement of morphine and M6G in femoral blood and cerebrospinal fluid may be a useful indicator in rapid deaths.     

Using DNA-barcoding to make the necrobiont beetle family Cholevidae accessible for forensic entomology

Although many cases of fatal hydrogen sulfide poisoning have been reported, in most of these cases, it resulted from the accidental inhalation of hydrogen sulfide gas. In recent years, we experienced 17 autopsy cases of fatal hydrogen sulfide poisoning due to the inhalation of intentionally generated hydrogen sulfide gas. In this study, the concentrations of sulfide and thiosulfate in blood, urine, cerebrospinal fluid and pleural effusion were examined using GC/MS. The sulfide concentrations were blood: 0.11-31.84, urine: 0.01-1.28, cerebrospinal fluid: 0.02-1.59 and pleural effusion: 2.00-8.59 (μg/ml), while the thiosulfate concentrations were blood: 0-0.648, urine: 0-2.669, cerebrospinal fluid: 0.004-0.314 and pleural effusion: 0.019-0.140 (μmol/ml). In previous reports, the blood concentration of thiosulfate was said to be higher than that of sulfide in hydrogen sulfide poisoning cases, although the latter was higher than the former in 8 of the 14 cases examined in this study. These results are believed to be strongly influenced by the atmospheric concentration of hydrogen sulfide the victims were exposed to and the time interval between exposure and death.     

A study on house fire victims: age, carboxyhemoglobin, hydrogen cyanide and hemolysis.

Correlation among age, concentrations of carboxyhemoglobin and hydrogen cyanide, oxygen density and hemolysis were studied in 120 house fire victims. Victims aged over 60 years comprised approximately 50% of the pooled subjects. Blood samples were mainly collected from the left ventricle, but sometimes from both the right and left ventricles. The concentration of carboxyhemoglobin ranged from 1-95%, of which 71 persons (59.7%) died with carboxyhemoglobin concentrations below 60%. Carboxyhemoglobin concentrations below 10% were found in 9 persons (7.5%). Most of these cases involved the elderly persons. In this paper, we report on the death of elderly victims as a result of low carboxyhemoglobin concentrations. A significant correlation of blood carboxyhemoglobin concentrations existed between the right and left ventricles. The concentration of carboxyhemoglobin in the left ventricle was significantly higher than that in the right. Two out of 31 victims whose hydrogen cyanide concentrations were determined, succumbed to hydrogen cyanide poisoning, having a high concentration of hydrogen cyanide and a low concentration of carboxyhemoglobin. On analysis, oxygen density was found to be low in 13 persons. A negative correlation was shown between carboxyhemoglobin concentration and hemolysis. Inasmuch as hemolysis may indicate the extent of heat dissociation, hemolysis should provide an index of carbon monoxide dissociation from carboxyhemoglobin. In the present study of victims, possible causes of death i.e., carbon monoxide gas poisoning, hydrogen cyanide poisoning, oxygen deprivation, burning, shock due to burns and others were estimated. The survival time for elderly victims was considered to be short.     

Acute intoxication with guaifenesin, diphenhydramine, and chlorpheniramine.

Mixed drug reactions are frequently encountered in emergency department overdose cases and also in fatal intoxications. Assessment of the relative contribution of each drug in producing adverse effects is often compounded by lack of case history and the paucity of cases reported in the literature. This report describes a fatal intoxication with three common over-the-counter medications: guaifenesin, diphenhydramine, and chlorpheniramine. A 48-year-old woman was found dead in the attic bedroom of her residence. Specimens obtained at autopsy for toxicologic analysis included heart blood, urine, bile, gastric contents, vitreous humor, and cerebrospinal fluid. The over-the-counter drugs were identified and quantitated by acid/neutral or basic liquid-liquid extraction followed by gas chromatographic analysis with nitrogen phosphorus detection. Concentrations of guaifenesin, diphenhydramine, and chlorpheniramine detected in the heart blood were 27.4, 8.8, and 0.2 mg/L, respectively. The cause of death was determined to be acute intoxication by the combined effects of guaifenesin, diphenhydramine, and chlorpheniramine, and the manner of death was determined to be suicide. To our knowledge, the blood guaifenesin concentration in this case is the highest reported concentration to date associated with an acute intoxication.     

Distribution of Venlafaxine,O‐desmethylvenlafaxine,and O‐desmethylvenlafaxine to Venlafaxine Ratio in Postmortem Human Brain Tissue

Venlafaxine (VEN) and its metabolite O‐desmethylvenlafaxine (ODV) inhibit reuptake of serotonin and norepinephrine. This study examines whether VEN is differentially distributed in postmortem brain and examines relationships between brain and femoral blood concentrations from donors prescribed VEN for treatment of depression. Using high‐pressure liquid chromatography‐ultraviolet detection, VEN and ODV concentrations were measured in temporal, occipital, and cerebellar cortex of six postmortem brains. The ODV/VEN ratio was calculated as a relative measure of drug metabolism within each region where higher ratios indicated a greater conversion of VEN to ODV. Compared to the other regions examined, the cerebellum showed decreased VEN (p = 0.056), ODV (p = 0.006), and ODV/VEN (p = 0.027) ratios. In parts per million, VEN was higher in temporal and occipital cortex, but not cerebellum, as compared to femoral blood concentration. These observations suggest that VEN and ODV are differentially distributed in the brain, and metabolism of VEN to ODV may vary across brain regions.     

Creatine phosphokinase in serum and cerebrospinal fluid, and microscopic findings in brain and heart in hypothermic rabbits

Signs of hypothermia injury were studied in rabbits cooled to a core temperature of 30 degrees C by immersion in ice water and thereafter rewarmed to 35 degrees C. Anaesthetized control rabbits were kept normothermic (37 degrees C) for a corresponding time (4 h). Creatine phosphokinase (CPK) activity increased 24 h after hypothermia to 20-fold in serum. In cerebrospinal fluid the activity was already significantly (5-fold) increased after hypothermia and was still as high at 24 h. Smaller increase was also found in the control normothermic rabbits both in serum (10-fold) and cerebrospinal fluid (2-fold). The values had returned to the initial level after 1 week. Small haemorrhages were observed in the brain at 24 h and slight scarring was seen in the myocardium of some rabbits which had lived 4 weeks following hypothermia. The results indicate that CPK can be a useful marker in the diagnostics of hypothermia death, especially in cerebrospinal fluid, which is less affected than blood by autolysis.     

Child and elderly victims in forensic autopsy during a recent 5 year period in the southern half of Osaka city and surrounding areas

To outline the recent features of child and elderly victims from the medico-legal perspective with special reference to abuse and neglect, a retrospective investigation of forensic autopsy cases over a 5 year period (1994-1998) in the southern half of Osaka city and surrounding areas (a population of 1.57 million) was undertaken. Among 646 autopsy cases, there were 53 child cases (under the age of 15 years, about 80% below 6) and 121 elderly cases (65 years old and above). Nearly half of the child deaths and more than half of the elderly deaths were described as accidental. Fire and traffic victims were much more frequent in the elderly. Child victims included those of neonaticide/infanticide (n=6), physical abuse (n=10), unintentional fatal infliction (n=2), neglect (n=2), mutual suicide (n=2), suicide (n=1) and murder (n=3). Child abuse and neglect were domestic maltreatment by the parents. In this series, there was a comparable number of fatalities due to maltreatment in the elderly (n=13) and in children, and non-domestic violence was more frequent in the elderly than domestic violence. Elderly females tended to be battered by their sons or grandsons in domestic violence cases, whereas males were predominantly attacked by younger males in non-domestic violence. The other elderly victims included those of self-neglect (n=2), murder (n=7) and suicide (n=9). Non-domestic homicide of the elderly occurred mainly in the center of the city, whereas domestic maltreatment of children and the elderly was sporadic, although somewhat more frequent in the peripheral zone of the city and the surrounding areas. The above profile of child and elderly abuse suggests a substantial influence of social and familial backgrounds.     

Validity testing of commercial urine cocaine metabolite assays: I. Assay detection times, individual excretion patterns, and kinetics after cocaine administration to humans

A validity study of eight commercial urine assays for detection of cocaine metabolite was performed on clinical specimens collected from human subjects who received single 20-mg intravenous doses of cocaine hydrochloride. The specimens were collected under controlled conditions and analyzed in random order under blind conditions. Benzoylecgonine concentration in each specimen also was determined by gas chromatography/mass spectrometry (GC/MS). Mean times of detection of the last positive specimen (greater than or equal to 300 ng/mL of benzoylecgonine equivalents) after cocaine administration varied among seven of the commercial tests from 16.9 to 52.9 h in the following ascending order: Toxi-Lab less than TDx = EMIT dau = EMIT st less than Abuscreen less than Coat-A-Count = Double Antibody. In contrast, a commercial spot test (KDI Quik Test) which was evaluated for detection of cocaine metabolite produced both false positives and false negatives for benzoylecgonine and was not considered to be a valid test for detection of cocaine metabolite. Half-lives of excretion of benzoylecgonine among four subjects varied from 5.9 to 7.9 h, and overall recovery of benzoylecgonine varied from 15.0 to 34.3% of the administered dose of cocaine.     

Jumping Together: A Fatal Suicide Pact

In suicide pact, two or more victims mutually agree and execute to end their lives together by predetermined method, preferably by hanging, drowning, gunshot, or poisoning. The victims are usually spouses, lovers, or friends, and the reasons behind such steps are various. In this reported suicide pact, husband–wife duo jumped from the terrace of a 12‐meter‐high building with their wrist bound to each other. Although they jumped together, the injury patterns were completely different. The man landed on head sustaining mainly craniocerebral injuries, and the wife landed on feet sustaining long bone injuries. They left a suicide note pointing out sudden demise of their only child as the reason for the suicide, and it was signed by both of them. Jumping from a height in suicide pact has not been reported in the scientific literature yet.     

Post-mortem analysis of formic acid disposition in acute methanol intoxication

Fifteen cases of fatal massive methanol intoxication have been investigated. Victims received either no treatment or ethanol therapeutic treatment. Methanol poisoning cases were classified in three groups according to survival time: more than 3 days (group 1), up to 3 days (group 2) and few hours (group 3). Body distribution of methanol and formic acid, as the main metabolite, was analyzed in blood and in different organs (brain, kidney, lung and liver). Relationships between formic acid concentration in the different tissues, survival time and type of treatment applied to victims were studied. Formic acid in blood and tissues was analyzed by head space gas chromatography (head space-GC) with FID detector, previous transformation in methyl formate, essentially as described by Abolin. Formic acid concentration was between 0.03 and 1.10g/l in the samples under study. A good correlation between blood and brain, but poor between blood and the remaining tissues was found. Obtained data suggested that the use of blood and brain could help to improve the analysis of formic acid intoxication. The best correlation among organs was found between lung and kidney for all groups (r(2)=0.91, 0.84 and 0.87, corresponding to groups 1, 2 and 3, respectively). Lethality index was defined as LI = (concentration of formic acid in blood in (g/l)/0.5) x 100, taking into account that 0.5g/l is the concentration reported by Mahieu in severe methanol poisoning. LI parameter was used to estimate formic acid incidence on the lethality of methanol poisoning cases. LI showed a good correlation with total formic acid concentration of the different tissues analyzed (r(2)=0.80). Furthermore, LI allowed us to discriminate between individuals that received therapeutic treatment and survived different periods. LI>100 indicated a severe intoxication and short survival time if the victim was assisted with ethanol therapy and hemodialysis was not applied. With regard to victims who received no therapeutic treatment and died in few hours, LI was in the range 40-100. LI was below 40 for individuals that survived more than 3 days and hemodialysis was not performed. Results showed the importance of performing formic acid analysis to diagnose severe methanol intoxication in post-mortem cases.     

Tissue distribution of mirtazapine and desmethylmirtazapine in a case of mirtazapine poisoning

An ingestion of an unknown quantity of mirtazapine in a suicide attempt leading to death is described. Sertraline and amitriptyline have been co-ingested. Because mirtazapine is reported to be relatively safe in overdose, body fluids and tissues were investigated for both mirtazapine and desmethylmirtazapine by high-pressure liquid chromatography/tandem mass spectrometry following liquid-liquid extraction. The limit of detection was sufficiently low to also apply the assay in pharmacokinetic studies. The levels of amitriptyline and nortriptyline were very low (38 and 19 ng/mL femoral venous blood) and the amount of sertraline in blood taken from the femoral vein (880 ng/mL) was considerably lower than those seen in overdosage. Accumulation of mirtazapine and N-desmethylmirtazapine was evident in fluids and tissues involved in enterohepatic circulation and excretion. The concentration determined in a brain sample suggests a contribution of the metabolite to the drug's pharmacodynamic activity. Based on literature data, significant adverse or synergistic effects among the drugs detected as well as adverse reactions such as a serotonin reaction appeared less probable. Mirtazapine exhibits alpha(1)-antagonistic properties on the cardiac-vascular system and may cause hyponatraemia. In the face of the cardiac findings at autopsy and the lack of an apparent cause of death, these effects of mirtazapine may have initiated a process leading to death.     

Suicide in children, youths and young adults

Suicides among children and youths are tragic events. The suicide cases of children, youths and young adults examined between 1995 and 2009 at the Institute of Legal Medicine of the Hanover Medical School were retrospectively analyzed. Altogether, 54 suicides among children, youths and young adults (37 males, 17 females; median age 17.5 years) were evaluated. The suicide victims were predominantly male (68.5%). The vast majority used "hard" methods such as hanging, jumping from a height or being run over by a train. In seven cases (13.0%) psychiatric disorders were known. Seven victims (13.0%) left a suicide note. Suicide was most frequently (40.7%) committed in the open air (40.7%). In addition, the evaluation shows that at the time of committing suicide more than 40% of the youths and adolescents, in whom a chemical toxicological examination was performed, were under the influence of alcohol, medical and/or illegal drugs.