Brain amino acid abnormalities in liver disease--a postmortem study

In a postmortem exploratory study, we examined whether specific amino acid abnormalities associated with liver diseases in vivo may also be detected in human brain samples obtained at clinical autopsies. The branched-chain amino acids (BCAA: valine, leucine, isoleucine) were decreased in the group of patients with liver diseases compared with the control group, whereas the aromatic amino acids (AAA: phenylalanine, tyrosine) were increased. However, the ranges overlapped significantly and were not statistically different. The molar ratio BCAA/AAA was determined to be 1.92 in the collection of patients with liver diseases compared with 2.27 in the control group. In patients with liver disease, ornithine concentrations in the brain appeared significantly decreased whereas glutamine was significantly increased. No significant difference was found in the brain concentrations of proline. Amino acid analysis may contribute to the understanding of pathophysiological mechanisms of liver disease, which are discussed, and may supplement the postmortem diagnosis.     

Postmortem vitreous humor beta-hydroxybutyrate: its utility for the postmortem interpretation of diabetes mellitus

Ketoacidotic coma is one of the most serious complications arising from diabetes mellitus, especially type I, and may be the cause of sudden death especially in diabetes type I. Since beta-hydroxybutyrate (beta-OHB) serum concentrations might provide more information on the severity of ketoacidosis, the aim of this study was to evaluate the concentrations of beta-OHB in vitreous humor and its correlation with other biochemical parameters during postmortem examination. We intended to ascertain the sensitivity and the specificity of these markers for diagnosing diabetes mellitus and the presence of ketoacidosis. This study involved 453 cadavers with a mean age of 57.6 years (S.D. 20.7) and a mean postmortem interval of 17.8 h (S.D. 9.6, range 2-61 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 biochemical marker concentrations between the two diagnostic groups, the highest values being obtained in the group of subjects with a previous diagnosis of diabetes mellitus. The measurement of beta-OHB in vitreous humor may be a useful alternative to using blood during postmortem analysis. The presence of high levels of beta-OHB may help interpret the cause of death in diabetics when the autopsy result is negative.     

Avian vitreous humor concentrations of inosine, hypoxanthine, xanthine, uric acid, uracil and uridine as influenced by age and sex: their relevance as indicators of ante-mortem hypoxia

An investigation was made to determine the effects of age and sex on postmortem concentrations (mumol/l) of inosine, hypoxanthine, xanthine, uric acid, uracil and uridine in the vitreous humor of chickens (Gallus domesticus). Five male and 5 female chickens were sampled each week from 0-10 weeks of age. Samples were collected at 0 and 24 h postmortem and analysed by HPLC. Hypoxanthine, uric acid and uridine were detected at both 0 and 24 h postmortem whereas inosine, xanthine and uracil were detected only at 24 h postmortem. Neither sex nor the sex X age interaction influenced the concentrations of the compounds investigated (P greater than 0.01). Age had a significant influence on the concentrations of all compounds at 24 h postmortem (P less than 0.0001). At 0 h postmortem, age significantly affected the levels of hypoxanthine and uridine (P less than 0.0001) but not uric acid (P = 0.014). All compounds occurred at higher concentrations at 24 than 0 h postmortem with the exception of uridine at 0 and 1 week of age. Previous studies using data confounded by age and postmortem sampling time have concluded that postmortem vitreous humor hypoxanthine concentration is a useful indicator of antemortem hypoxia. The results of this study cast doubt on that conclusion. Age and postmortem sampling time should be rigorously controlled when conducting quantitative analyses of these compounds in vitreous humor.     

Clinical and forensic examinations of glycemic marker 1,5-anhydroglucitol by means of high performance liquid chromatography tandem mass spectrometry

Postmortem diagnosis of diabetes and a diabetic coma can be difficult because of the lack of characteristic morphological findings. 1,5-Anhydroglucitol (1,5-AG), the 1-deoxy form of glucose, competes with glucose for reabsorption in the kidneys. Therefore, diabetics with a permanent hyperglycemia show significantly lower serum concentrations of 1,5-AG than non-diabetics. A liquid chromatography-mass spectrometric method for the determination of 1,5-AG in serum and postmortem blood was developed and validated according to international guidelines. Linearity was given between 1μg/ml and 50μg/ml. Recovery rates ranged between 70.8% and 89.8%, the limit of quantification of the procedure was 0.20μg/ml, limit of quantification was 0.55μg/ml. Serum of 199 diabetics and 116 non-diabetics and femoral blood of 31 diabetic and 27 non-diabetic deceased was measured. Average concentrations were significantly (p<0.001) higher in non-diabetics compared to diabetics ante and postmortem. Seven of the diabetics may have died because of a hyperglycemic coma indicated by a sum formula of Traub>450mg/dl. 1,5-AG average concentrations in these deceased were not significantly different to diabetics which did not die because of a diabetic coma. Concentrations of 1,5-AG give a hint for not well controlled diabetes antemortem and postmortem and can be assumed as an additional and alternative information postmortem to the measurement of HbA1c or fructosamine.     

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.     

Practical experiences with the determination of cadaver age by evaluation of bacterial metabolic products

In an experiment a corpse had been kept at room temperature (16 degrees-23 degrees C) for 1163 h. At regular intervals brain samples were taken and the content of free amino acids and related compounds was determined by column chromatography. It could be demonstrated that in a period of 4-20 days postmortem the age of the corpse could be calculated from the concentrations of alpha-aminobutyrate (ABU), gamma-aminobutyrate (GABA), and glutamic acid (GLU) in brain employing the previously [5] presented formula: (formula; see text) T is the postmortem time lapse (days). ABU, GABA, and GLU are the concentrations (mumol/g) wet tissue of the corresponding amino acids. During this postmortem interval there is nearly a linear correlation; from this interval a correct assignment between concentrations and time cannot be given. The determination method is in the range of about 15 degrees-25 degrees C (guarantees bacterial growth and metabolism) independent of ambient temperature. The brain samples (cortex or parts of the putrified brain mush) can be taken without any special precaution during normal autopsy. The results of the experiment (in Fig. 1) were supported by some practical cases where the time lapse since death was well (in Fig. 1) or reasonably (in Fig. 1) known. These results suggest that this method allows in many cases the determination of the age of a corpse found in a warm environment (approximately 15 degrees-25 degrees C) approximately 4-20 days after death.     

Forensic issues in cases of Diogenes syndrome

Diogenes syndrome is a syndrome described in the clinical literature in elderly individuals characterized by social isolation and extreme squalor. A number of typical features are found in the forensic evaluation of these deaths as the cases usually initiate medicolegal investigations due to the circumstances and the lack of recorded medical histories. Examinations of the death scenes are often difficult as victim's houses are in a state of disrepair, with filth and clutter, and pet dogs may resent the intrusion of strangers. Bodies are often filthy, with parasitic infestations, and are often putrefied due to the social isolation of the deceased and the delay in the finding of the corpse. Bodies may be traumatized from postmortem animal depredation by rodents or pets (eg, cats, dogs), and injuries such as bruises and lacerations may be present from falls associated with terminal illnesses or alcoholism. Blood or putrefactive fluids may be spread throughout the house by pets. Treatable medical conditions are often present in advanced stages, and features of hypothermia may be found. Attending police may suspect robbery due to disarray of the house and homicide due to apparent "bleeding" around the body from purging of putrefactive fluids, injuries from falls, or postmortem animal activity and "blood stains" throughout the house from antemortem injuries and/or fluid spread by animals. Finally, the identification of the deceased may be compromised by decay and/or postmortem animal activity. Thus, in addition to having typical clinical manifestations, such individuals appear to form a distinct subset of forensic cases having characteristic death scene and autopsy features and presenting particular difficulties in postmortem evaluations.     

Postmortem diagnosis of unsuspected diabetes mellitus established by determination of decedent's hemoglobin A1c level.

Although approximately 15.7 million Americans have diabetes mellitus, with the vast majority having type 2 diabetes, it is estimated that as many as 5.4 million are undiagnosed. The present case illustrates that undiagnosed diabetes can be a factor in otherwise unexplained deaths. A 39-year-old white male with no significant past medical history other than alcohol abuse was found deceased at his residence. The manner of death appeared to be natural, but no anatomic cause was found. Toxicological analysis revealed a blood ethanol level of 0.02 g/dL and was negative for drugs of abuse. Analysis of the vitreous fluid revealed a glucose level of 502 mg/dL. The blood glucose level was 499 mg/dL, and the hemoglobin A1c (HbA1c) level was 10.6%. Only trace urine ketones were detected, suggesting that the death was the result of hyperglycemic hyperosmolar non-ketosis (HHNK) from unsuspected diabetes. The postmortem HbA1c value serves as a definitive indicator of prolonged hyperglycemia. In order to aid the interpretation of the clinical data, this case is discussed in conjunction with a similar case of a known diabetic patient.     

Fatal Methemoglobinemia Due to Acute Inhalation of Methyl Nitrite in an Industrial Accident

Methyl nitrite is suggested to cause methemoglobinemia by generating methemoglobin, which may be lethal when the methemoglobin concentration exceeds 70%. However, intoxication with methyl nitrite is seldom reported compared with that with other nitrites. Here, we present an industrial accident involving methyl nitrite inhalation during its synthesis process that resulted in three fatalities and one survivor. The autopsy revealed conspicuous blue-gray discoloration in various parts of the body, including the skin, airway mucosa, vessels, brain, heart, and among other areas. The toxicological tests on the deceased showed methemoglobin concentrations in the blood over the lethal level and increased nitrite ion levels in the blood, gastric contents, liver, and lung tissue compared with those in control samples. The cause of death was determined to be methemoglobinemia-induced hypoxia due to methyl nitrite inhalation. This report provides evidence that in methyl nitrite intoxication, exposure duration has a significant influence on the postmortem changes and likelihood of a fatal outcome may be related to the age of the victim. More attention is required regarding the industrial hazards of this substance.     

Nontraumatic clostridial myonecrosis.

We describe three cases of nontraumatic clostridial myonecrosis seen at the Victorian Institute of Forensic Medicine. Nontraumatic clostridial myonecrosis is an uncommon and often fatal condition that requires immediate institution of appropriate medical and surgical therapy. It is most commonly caused by Clostridium perfringens and Clostridium septicum and is associated with gastrointestinal and hematologic malignancies, diabetes mellitus, and peripheral vascular disease. The clinical features include a rapidly evolving acute illness with severe pain, marked tachycardia, and brawny discoloration of the skin with bullae formation and crepitus, followed by hypotension and acute renal failure. Features at autopsy include reddish brown skin discoloration with bullae formation and necrotic skeletal muscle. Radiographs may be of use prior to the postmortem in detecting gas within the soft tissues. Gram stain and microbiologic culture are important in establishing a definitive diagnosis; although the major factors in suggesting the diagnosis are the recognition of the typical clinical history and macroscopic autopsy findings.     

Glucose and lactate in vitreous humor compared with the determination of fructosamine for the postmortem diagnosis of diabetes mellitus

Diabetes mellitus is a chronic metabolic illness responsible for a great number of deaths. In postmortem diagnosis, because of the difficulty involved in interpreting blood glucose levels and relatively nonspecific pathologic features, biochemical markers in vitreous humor are useful. The aim of this study was to compare the results obtained for the combined determination of lactate and glucose with fructosamine levels recorded in the vitreous humor of two diagnostic groups (one diabetic and the other nondiabetic). The authors intended to ascertain the capacity of different markers measured in vitreous humor to diagnose diabetes mellitus. Fifty-one cadavers (mean age, 58.7 years; standard deviation, 17.09) were studied. The mean postmortem interval was 16.4 hours (standard deviation, 9.05). Cases were assigned to two diagnostic groups according to whether they were previously diagnosed as either diabetic or nondiabetic. Statistically significant differences for glucose, fructosamine, and the sum values of glucose and lactate were found between the two diagnostic groups. The highest levels were obtained in the group of cases with a previous diagnosis of diabetes mellitus. After the comparison of receiver operating characteristic curves, the sum values of glucose and lactate in vitreous humor is a better predictor of antemortem diabetes mellitus than the fructosamine.     

Degradation of biomolecules in artificially and naturally aged teeth: implications for age estimation based on aspartic acid racemization and DNA analysis

Postmortem teeth are the most stable structures, and can be used to gain different information (age estimation, genetic data). Over long postmortem intervals (PMI), degradation processes may alter the molecular integrity and thus affect the reliability of applied molecular methods. Whereas some knowledge on the degradation of biomolecules in bone during the PMI exists, data for teeth are lacking. In particular, the impact of degradation processes in dentine on age estimation based on aspartic acid racemization (AAR) cannot be estimated yet. Hence, the molecular stability of both collagen and DNA was analyzed systematically, and their impact on the reliability of age estimation based on AAR and genetic analyses was checked. Two hundred and ten human and 59 porcine teeth were heated (90 degrees C in water) to simulate collagen and DNA diagenesis; 14 naturally aged teeth (PMI: 3 days to 1700 years) were analyzed comparatively. Peptide patterns of cyanogen bromide (CNBr)-cleaved collagen were employed as a new approach to check the collagen integrity. In the same samples, collagen yields, amino acid compositions, AAR in different protein fractions, and DNA integrity were analyzed. In heated human and porcine teeth the collagen content declined during the heating experiment. The amino acid composition in human samples was collagen-like until 12 days of heating. In naturally aged teeth, the collagen yielded from 9.5 to 15%, and no discrepancy of amino acid composition to that of modern collagen was observed. Electrophoresis of CNBr-peptides showed an altered pattern in experimentally degraded samples from day 10 on; naturally aged collagen displayed the typical collagen pattern. AAR increased in all protein fractions with increasing duration of the heating experiment; naturally aged samples displayed a slow accumulation of AAR. DNA degraded progressively, and after 32 h of heat exposure no more DNA was detectable, whereas the amplification of nuclear and mitochondrial DNA was successful up to 48 h. STR typing was reliable up to 16 h, and sex determination up to 40 h of heat exposure. In naturally aged samples of DNA quality, yield and typing success did not correlate with PMI. The data highlight a remarkable stability of collagen dental proteins. Within relevant forensic periods a postmortem rise of AAR under normal conditions is negligible, and analyses of dental DNA has a high chance to be successful. However, after large PMI and/or extreme postmortem conditions age estimation based on AAR and genetic analyses lose their reliability.     

Age at death determinations for autopsied remains based on aspartic acid racemization in tooth dentin: importance of postmortem conditions

The extent of aspartic acid racemization in human dentin can be used to assess chronological age of the individual. This novel chemical method of aging human remains has potential applications in forensic sciences. Six autopsy cases have been analyzed for D/L aspartic acid contents, and their ages at death calculated. Independent age information came from known identity, tooth maturation, or tooth wear estimations. The six cases represented a range of postmortem fates: recent demise, burial, and ground surface exposure. Five samples, four recently deceased and one a burial, showed racemization ages in close agreement with other age data. One specimen, subjected to 51 days of open air exposure, exhibited a vastly inflated racemization age. Postmortem conditions are crucial to the accuracy of racemization age at death measurements.     

A Characterization of Sources of Isopropanol Detected on Postmortem Toxicologic Analysis

Abstract: Isopropanol is an important chemical to forensic pathologists in that intoxication can result in death yet presence does not necessarily indicate intoxication. Several reports have been published, which indicate that isopropanol can be created endogenously in certain situations including diabetes mellitus, starvation, dehydration, and chronic ethanol use; however, a large‐scale analysis addressing all of the possible causes of postmortem isopropanol detection has not been performed. A retrospective review of all cases examined at the Bexar County Medical Examiner’s Office between 1993 and 2008 in which isopropanol was detected in routine alcohol screening was undertaken. The cases were categorized by the source of the isopropanol, and the concentrations of isopropanol and acetone were analyzed. Analysis revealed isopropanol concentrations to be low (<100 mg/dL) in cases of antemortem and postmortem creation and in postmortem contamination and high (>100 mg/dL) in cases of antemortem exposure. These results are consistent with other published reports.     

Site-dependent production of gamma-hydroxybutyric acid in the early postmortem period

This study compared endogenous gamma-hydroxybutyric acid (GHB) concentrations in various postmortem fluid samples of 25 autopsy cases. All bodies were stored between 10-20 degrees C until autopsy, and the intervals between death and autopsy were less than 2 days (6-48 h). GHB concentrations were measured by headspace gas chromatography after GHB was converted to gamma-butyrolactone. Endogenous GHB concentrations were significantly higher in femoral venous blood (4.6+/-3.4 microg/ml, n=23) than in cerebrospinal fluid (1.8+/-1.5 microg/ml, n=9), vitreous humor (0.9+/-1.7 microg/ml, n=8), bile (1.0+/-1.1 microg/ml, n=9) and urine (0.6+/-1.2 microg/ml, n=12). GHB concentrations were similar in blood samples taken from different sites. Cut-off limits of 30 and 10 microg/ml are proposed for blood and urine, respectively, to discriminate between exogenous and endogenous GHB in decedents showing no or little putrefaction (postmortem intervals usually 48 h or less). The criterion established for endogenous GHB in postmortem urine may also be applicable to analytical results in cerebrospinal fluid, vitreous humor and bile from deceased persons.     

Fatal methanol intoxication with different survival times--morphological findings and postmortem methanol distribution

Three corresponding cases of fatal methanol intoxication with different survival times were investigated ante-mortem and postmortem. Ante-mortem serum methanol concentrations were determined during treatment in hospital for 4 days. Furthermore, postmortem distribution of methanol in various tissues and fluids was measured after autopsy. Morphological and toxicological findings are discussed based on the literature. The morphological findings correlated with the different survival times. The results of the toxicological analyses were partly in keeping with previously published data. Interestingly, very high methanol levels were determined in brain with very low concentrations in femoral venous blood. These results may have implications for postmortem toxicological analysis, brain death diagnosis and organ explanation for transplantation.     

Postmortem diffusion of drugs from the bladder into femoral venous blood.

We describe significantly elevated drug concentrations in the femoral venous blood due probably to postmortem diffusion from the bladder. A 16-year-old deceased male was found in a shallow ditch in winter. The estimated postmortem interval was 9 days and putrefaction was not advanced. The cardiac chambers contained fluid and coagulated blood and a small amount of buffy coat clots. Diffused hemorrhages were found in the gastric mucosa. The bladder contained approximately 600 ml of clear urine. Gas chromatographic-mass spectrometric analysis of the urine disclosed allylisopropylacetylurea (a fatty acid ureide sedative), diphenhydramine, chlorpheniramine and dihydrocodeine. The cause of death was considered to be drowning due to a drug overdose and cold exposure. The concentrations of diphenhydramine, free dihydrocodeine and total dihydrocodeine in the femoral venous blood (1.89, 3.27 and 3.30 microg/ml, respectively) were much higher than those in blood from the right cardiac chambers (0.294, 0.237 and 0.240 microg/ml, respectively). Urine concentrations of diphenhydramine, free dihydrocodeine and total dihydrocodeine were 22.6, 37.3 and 43.1 microg/ml, respectively. The stomach contained negligible amounts of diphenhydramine, free dihydrocodeine and total dihydrocodeine (0.029, 0.018 and 0.024 mg, respectively); concentrations of these drugs in the femoral muscle were 0.270, 0.246 and 0.314 microg/g, respectively. These results indicate that postmortem diffusion of diphenhydramine and dihydrocodeine from the bladder resulted in the elevated concentrations of these drugs in the femoral venous blood. Not only high urinary drug concentrations but also a large volume of urine in the bladder might accelerate the postmortem diffusion.     

Postmortem Changes of Female External Genitalia and Their Importance in Suspected Sexual Abuse

Examination of the female external genitalia to assess for sexual abuse is performed in living individuals, and the interpretation of the findings is based on evidence-based studies. However, in the deceased, no such studies are available, and postmortem changes could present as suspicious findings that can be mistaken for trauma. Patches of discoloration in the hymen were reported previously in one case as hypostasis (i.e., livor and lividity), and based on this finding, it was listed as a finding that is not associated with trauma. This was a retrospective study that was conducted in the Center of Forensic and Legal Medicine in Dammam, Saudi Arabia over a 4-year period. The study included 30 deceased women in whom photographic documentation of their external genitalia was assessed for postmortem changes. The postmortem interval ranged from less than 24 h to more than 100 days, and the ages of these deceased women were in the 20–40 year-old age group. In cases where the hymen, vagina, and/or fossa navicularis were clearly visible, none of these areas showed any hypostatic discoloration. A comparison between antemortem and postmortem appearance of the hymen in one case clearly showed the absence of hypostatic changes in the hymen. In conclusion, any discoloration of the external genitalia that is detected in a female decedent requires serious consideration.     

Interleukin-6 and C-reactive protein serum levels in sepsis-related fatalities during the early postmortem period

Postmortem interleukin-6 (IL-6) and C-reactive protein (CRP) serum levels were investigated prospectively in sepsis-related fatalities and non-septic fatalities by using a linear regression model. At least three blood samples were collected between 0.3 and 139 h postmortem from sepsis-related fatalities (n=8) and non-septic fatalities (n=16). In addition, one antemortem blood sample was collected shortly before death from the septic patients. Antemortem and postmortem IL-6 and CRP levels were highly elevated in all individuals included in the sepsis group. An excessive postmortem increase of IL-6 serum levels associated with progressive time after death was observed in five out of the eight septic patients. Both, IL-6 and CRP serum concentrations seem to be suitable biochemical postmortem markers of sepsis. The determination of IL-6 serum levels above 1500 pg/ml in peripheral venous blood obtained in the early postmortem interval can be considered as a diagnostic hint towards an underlying septic condition. A more precise postmortem discrimination between sepsis and non-septic underlying causes of death is provided by the postmortem measurement of serum CRP in peripheral venous blood: on condition that at least two postmortem CRP values have been determined at different time points postmortem, the CRP level of a deceased at the time of death can be calculated by using linear regression analysis. When assessing postmortem IL-6 and CRP concentrations as biochemical postmortem markers of sepsis, various clinical conditions, such as a preceding trauma or burn injury going along with elevated IL-6 and/or CRP levels prior to death as a result of the systemic inflammatory response syndrome (SIRS) should be taken into consideration, thus adding relevant information for the practical interpretation of the results.     

Serotonin, 5-hydroxyindolylacetic acid and cholesterol content in blood, cerebrospinal fluid and brain areas for differentiation of suicidal from non-suicidal cause of death

In the present study serotonin and its metabolite 5-hydroxyindoleacetic acid was investigated in the cerebrospinal fluid and in discrete brain areas of the left and right hemisphere collected from 34 bodies. Sixteen subjects were suicide victims, and 18 were matched as controls. Matching was done for gender, age, sex and cause of death. In suicide victims the concentration of 5-hydroxyindoleacetic acid in cerebrospinal fluid (occipital) was significantly decreased whereas there was no difference comparing the particular results established from the various brain areas. Nevertheless, there was a non-significant trend towards a higher concentration of serotonin in the thalamic area and towards a lower level in samples collected from the mesencephalon in suicide brains. In suicide subjects, the level of 5-hydroxyindoleacetic acid was often found to be increased in the hippocampus and to be decreased in the thalamus. A differentiation between suicide and homicide seems promising only on condition that the distribution of serotonin and metabolite concentrations in various brain areas is considered. The amount of total cholesterol in blood is suggested to be of limited value.