Medicolegal studies on alcohol detected in dead bodies — alcohol levels in skeletal muscle

An experiment was carried out on rats to determine whether or not a skeletal muscle sample was suitable for the determination of ethanol concentration in a carcass. Gas chromatography was used to estimate the ethanol and n-propanol concentrations in the femoral muscle and intracardial blood. The ethanol concentration of each sample was corrected according to the moisture ratio of circulating blood, viz., 78.5%.The ethanol concentration ratio of blood to muscle was 1.03 two hours after ethanol administration. When the carcasses of rats pre-treated with ethanol were stored at 15 °C and 25 °C, respectively, the ethanol concentrations in muscle and blood increased with time. At all times the concentration was higher in blood than in muscle, and also higher in samples collected from the carcass stored at 25 °C than at 15 °C.When the control carcass was stored in the same manner, the postmortem production of ethanol was noticed in both blood and muscle. As in the experimental rats, the control rats exhibited a higher blood ethanol than muscle ethanol level. Again, the ethanol concentration was higher in samples collected from the carcass stored at 25 °C than at 15 °C. The ratio of ethanol to n-propanol was less than 20:1 in blood and less than 10.1 in muscle.These results suggest that skeletal muscle may be a suitable tissue for the postmortem detection of ethanol.     

腐败血液中自生醇现象及法医学应用研究

目的检测分析腐败血液中乙醇、甲醇等物质的生成过程,为正确判断案发时人血液中醇类物质的实际浓度提供实验依据。方法以正常健康人血液制作腐败样本,分别模拟人死亡后正常人血液和糖尿病人高糖血液的腐败过程,借助顶空气相色谱仪测定两种血液腐败后醇/醛类物质的生成情况并对比含量差异。结果相同实验条件下,高糖血液较正常健康血液更早腐败产生乙醇,但前者乙醇浓度低于后者;血液腐败过程中还会产生乙醛、甲醇、异丙醇、正丙醇等物质,其中正丙醇含量与乙醇生成量存在一定相关性。结论血液腐败能够产生乙醇、甲醇等多种物质。研究还为糖尿病人血液腐败产生醇类物质的差异性评价提供了依据。     

Postmortem production of ethanol in different tissues under controlled experimental conditions

The aim of this study was to follow the postmortem ethanol production phenomenon under controlled experimental conditions (temperature, time interval) in different tissues. Specimens of blood, liver, skeletal muscle and kidney were taken from 30 corpses and no chemical preservatives were used in the specimens collected. Ethanol concentrations were detected by gas chromatography. All specimens stored at -20 degrees C and 4 degrees C did not show any change in ethanol concentration in an eight-day time interval. At 20 degrees C and 30 degrees C, all tissues, except blood, showed statistically significant ethanol production over the time interval tested. However, blood sample kept at 30 degrees C, showed statistically significant increase in ethanol production on the 2nd and 4th day comparing to the controls. Thus, we can state that postmortem ethanol production occurs in different tissues, and is increased at higher temperatures and, in general, it is in accordance with the course of time.     

PCR-based identification of postmortem microbial contaminants--a preliminary study.

Investigation of postmortem blood can reveal the presence of significant ethanol levels. However, in some instances it cannot easily be determined if the source of ethanol is from ingestion or from postmortem endogenous fermentation by contaminating microbes. Described here is a robust polymerase chain reaction (PCR)-based method for detecting the presence of common ethanol producing microbial contaminants in human blood. A set of DNA primers were designed for use in PCR to amplify and detect the genomic DNA from humans and three test microorganisms Escherichia coli, Proteus vulgaris, and Candida albicans. A rapid and reproducible protocol was developed for isolating genomic DNA from mixed human blood-microorganism samples that yields a suitable template for PCR. The organism-specific primer pairs can detect the presence of the target microorganisms in human blood at concentrations as low as 10 colony forming units/mL. The PCR products readily can be detected after agarose gel electrophoresis. This method provides an additional means of rapidly identifying microbial contaminants in postmortem blood samples.     

Ethanol formation in unadulterated postmortem tissues

During the investigation of aviation accidents, postmortem samples obtained from fatal accident victims are submitted to the FAA's Civil Aerospace Medical Institute (CAMI) for toxicological analysis. During toxicological evaluations, ethanol analysis is performed on all cases. Many species of bacteria, yeast, and fungi have the ability to produce ethanol and other volatile organic compounds in postmortem specimens. The potential for postmortem ethanol formation complicates the interpretation of ethanol-positive results from accident victims. Therefore, the prevention of ethanol formation at all steps following specimen collection is a priority. Sodium fluoride is the most commonly used preservative for postmortem specimens. Several studies have been published detailing the effectiveness of sodium fluoride for the prevention of ethanol formation in blood and urine specimens; however, our laboratory receives blood or urine in approximately 70% of cases. Thus, we frequently rely on tissue specimens for ethanol analysis. The postmortem tissue specimens received by our laboratory have generally been subjected to severe trauma and may have been exposed to numerous microbial species capable of ethanol production. With this in mind, we designed an experiment utilizing unadulterated tissue specimens obtained from aviation accident victims to determine the effectiveness of sodium fluoride at various storage temperatures for the prevention of microbial ethanol formation. We found that without preservative, specimens stored at 4 degrees C for 96 h showed an increase in ethanol concentration ranging from 22 to 75 mg/hg (average 42 +/- 15 mg/hg). At 25 degrees C, these same specimens showed an increase ranging from 19 to 84 mg/hg (average 45 +/- 22 mg/hg). With the addition of 1.00% sodium fluoride, there was no significant increase in ethanol concentration at either temperature.     

Differences between multisite postmortem ethanol concentrations as related to agonal events

In a study of postmortem ethanol concentrations, blood was withdrawn from the right atrium, ascending aorta, and inferior vena cava. These samples, vitreous humor, and gastric fluid were analyzed in 307 autopsies, where a minimum blood ethanol concentration of 0.05% weight/volume (w/v) was present. Premortem, agonal, and postmortem events were reviewed in an attempt to account for differences in blood ethanol concentrations between sites. The agonal aspiration of vomitus having at least 0.80% w/v ethanol appears to be associated with an increase in aortic ethanol concentrations. We conclude that valid interpretation of postmortem ethanol concentrations must take into consideration the possible entry of ethanol into the pulmonary venous circulation via the respiratory system.     

High urine ethanol and negative blood and vitreous ethanol in a diabetic woman: a case report, retrospective case survey, and review of the literature

Several studies have shown that ethanol can be produced in urine infected with yeast or bacteria in vitro. We present the unusual case of a diabetic woman in whom ethanol was produced in her urine in vivo. The decedent was a 19-year-old woman who was noncompliant with her diabetes treatment. She presented to a local hospital in severe diabetic ketoacidosis and died shortly thereafter. Upon arrival at the hospital, a blood glucose of 553 mg/dL was detected. A urinalysis was positive for ketones (> 80 mg/dL), glucose (> 1000 mg/dL), and large budding yeast forms. A drug screen performed on the urine was positive for ethanol. At the coroner/medical examiner office, an autopsy was negative for significant anatomic findings. Toxicology analysis revealed a urine ethanol level 0.32 g/dL, although no ethanol was detected in blood or vitreous samples. A urine gram stain and culture identified Candida glabrata. A retrospective case review of all deaths related to diabetes examined at the coroner/medical examiner office from 1986 to 2003 did not reveal other cases with similar findings. This case of a noncompliant, juvenile-diabetic woman illustrates a rare finding of apparent in vivo glucose fermentation by C. glabrata to form ethanol in the urine. This case also highlights a potential difficulty in toxicologic analysis and interpretation using urine only.     

Stability of drugs in stored postmortem femoral blood and vitreous humor

The stability of 46 drugs in postmortem femoral blood stored for one year at -20 degrees C was investigated. The drugs included benzodiazepines, antidepressants, analgetics and hypnotics. For seven drugs we found a significant change in the concentration between the first and second analysis. Five substances; ethanol, desmethylmianserin, 7-amino-nitrazepam, THC and zopiclone showed a decrease in the concentration whereas the concentrations of two substances; ketobemidone and thioridazine increased. However, the changes observed were not of such an order that it would affect the interpretation in normal forensic casework. We also investigated the possible influence of potassium fluoride on the concentrations of the 46 drugs in vitreous humor after storage for one year. For two substances, ethanol and zopiclone, there were significantly lower concentrations in the samples without potassium fluoride. Furthermore, we also studied the correlation between the concentrations in femoral blood and vitreous humor. For 23 substances there was a significant difference between the concentrations in the vitreous humor and femoral blood. Significant correlations between the concentrations in these two specimens were found for 23 substances, indicating that vitreous humor can be an alternative specimen when blood samples are not available, provided that such correlation exists for the particular substance. Statistical analysis also revealed a correlation between the degree of protein binding of the different drugs and percentage of vitreous/femoral blood concentrations.     

Postmortem production of ethanol and n-propanol in the brain of drowned persons

We examined endogenous ethanol and n-propanol levels in the brain in 29 drowning cases in which ethanol consumption was excluded. Based on the stage of putrefaction of the brain, our cases were classified into 4 groups: pulpified brain (PB, n = 11), softened brain (SB, n = 6), discolored brain (DB, n = 2), and normal brain (NB, n = 10). The endogenous ethanol and n-propanol levels (mg/g), respectively, in the brains from these groups were 1.06 +/- 0.401 and 0.076 +/- 0.032 in PB, 0.195 +/- 0.136 and 0.012 +/- 0.009 in SB, and 0.053 +/- 0.032 and 0.001 +/- 0.001 in DB. Ethanol and n-propanol were not detected in NB. The concentration ratios of ethanol to n-propanol were 16.2 +/- 7.1 in specimens with ethanol levels > or = 0.50 mg/g (n = 10), and 17.6 +/- 13.5 in specimens with ethanol levels of 0.10 to 0.49 mg/g (n = 9). Drinking may strongly be suspected when (1) ethanol concentration in the brain is > or = 0.50 mg/g and cerebral ethanol to n-propanol ratio is > or = 40; and (2) the concentration of ethanol is 0.10 to 0.49 mg/g and the ethanol to n-propanol ratio is > or = 60.     

Postmortem blood and vitreous humor ethanol concentrations in a victim of a fatal motor vehicle crash

A 20-year-old male was found on the passenger side of a small car after a collision with a semi-trailer truck. Postmortem blood, collected from the chest cavity, and vitreous humor samples were collected following harvesting of the heart and bones. Gas chromatographic analysis revealed a blood ethanol concentration of 0.32 g/dL and a vitreous humor ethanol concentration of 0.09 g/dL. The stomach was intact and full of fluid and food, but its contents were not collected. Possible explanations for the large difference between the two results include diffusion of ethanol from the stomach into the chest cavity, contamination of the blood sample prior to collection, and ingestion of a large quantity of ethanol shortly before death. This case demonstrates the importance of proper quality assurance procedures in collecting postmortem specimens and of collecting a vitreous humor sample for ethanol analysis in postmortem toxicology cases.     

Assessment of the Role Played by N‐propanol Found in Postmortem Blood in the Discrimination Between Antemortem Consumption and Postmortem Formation of Ethanol Using Rats

This study disproves the reliability of n‐propanol as a biomarker to establish whether the ethanol found in postmortem blood is derived from antemortem ingestion or postmortem putrefactive processes. Two groups of rats were given ethanol or normal saline solution, respectively, and sacrificed 1.5 h later. After putrefaction, blood and, in a few cases, urine samples from the rats were analyzed for ethanol and n‐propanol by head‐space gas chromatography equipped with flame ionization detection. Although the concentration ratios of ethanol/n‐propanol in the postmortem blood collected from the bodies without prior alcohol consumption were expected to be <20 (as per limited case reports and previous in vitro studies), in samples from several rats that were on saline solution, this ratio was found to exceed 20. In conclusion, the concentration ratio of ethanol/n‐propanol in postmortem blood does not allow for the discernment between antemortem ingestion and the postmortem synthesis of ethanol.     

Stability of diazepam in blood samples at different storage conditions and in the presence of alcohol

Diazepam is one of the mostly used benzodiazepines and it is frequently analyzed in different biological samples, especially blood samples. The diazepam stability in the sample matrices is an important factor regarding reliable data obtaining. The storage is the main factor determining the stability of diazepam in blood samples and it is the object of the study presented. Remaining diazepam amount in spiked whole blood and plasma samples were tested at different storage temperatures, in the absence or presence of sodium fluoride as stabilizer as well as the influence of ethanol on diazepam stability was evaluated. The results of the study indicated that the temperature is the main storage factor affecting diazepam stability. In the fluoride stabilized blood samples the amount of diazepam decreases up to 85% of initial level when stored at -20° C for the period of testing (12 weeks). The presence of low (0.5 g/L) or high (3g/L) ethanol concentrations influences the stability of diazepam at -20 °C. In whole blood samples, the combination of sodium fluoride and ethanol decreases additionally (15-25%) the concentration of the analyte. Freeze-thaw experiments of whole blood samples show about 5-9% decrease in diazepam concentration after the first cycle. The freeze-thaw experiments on plasma samples, containing ethanol and/or fluoride show insignificant decreases of analyte concentration. Further experiments on benzodiazepines stability at different storage conditions or in combination of different factors should be undertaken in forensic toxicology to ensure the data quality, their reliability and reproducibility.     

Origin of blood ethanol in decomposed bodies

Problems related to blood contamination by other postmortem fluids in decomposed bodies (DB) make the interpretation of medicolegal blood alcohol levels (B EtOH) a very difficult task. So the aim of this paper is to show the utilization of vitreous humor (VH) as the biological fluid for an unequivocal determination of ethanol origin in DB for forensic purposes. Alcohol was determined in VH, blood (chest fluid-CF) and urine (Ur) collected from 27 DB in different states of putrefaction. A simple head-space gas-chromatographic method was used. In fifteen cases alcohol was found to be of endogenous production due to its absence in VH. In the twelve remainders, alcohol was detected in VH and CF in an atypical distribution. Examining the reliable scene and historical information together with the analytical data, ethanol origin in these cases was classified: endogenous production (3 cases), ingested (2 cases), both (2 cases), contaminated plus endogenous production (3 cases) and unable to determine (2 cases). According to the results obtained it was possible to conclude that alcohol analysis in VH is fundamental for determining the origin of ethanol detected in CF of DB.     

Abnormally high alcohol concentration in the heart blood

A 46-year-old male alcoholic whose whereabouts had been unknown for about a month was found dead at the foot of a cliff 31 m deep. Fractures of the mandible, thorax and left patella were found at autopsy, but fatal injury to the brain or other organs was not observed. The alcohol distribution was 7.44 mg/g in the heart blood, 13.91 mg/g in the left thoracic cavity fluid and 1.88 mg/g in the urine. The high ethanol concentration in the heart blood was assumed to be mainly due to the diffusion of ethanol from the contents of the stomach and postmortem production of ethanol. It was decided that the cause of death was not acute alcohol intoxication but respiratory failure caused by fractures of the thorax.     

Headspace gas chromatographic determination of ethanol: the use of factorial design to study effects of blood storage and headspace conditions on ethanol stability and acetaldehyde formation in whole blood and plasma

Our headspace gas chromatographic flame ionization detection (HS-GC-FID) method for ethanol determination showed slightly, but consistently, low ethanol concentrations in whole blood (blood) in proficiency testing programs (QC-samples). Ethanol and acetaldehyde were determined using HS-GC-FID with capillary columns, headspace equilibration temperature (HS-T degrees ) of 70 degrees C and 20 min equilibration time (HS-EqT). Full factorial designs were used to study the variables HS-T degrees (50 degrees -70 degrees C), HS-EqT (15-25 min), ethanol concentration (0.20-1.20 g/kg) and storage at room temperature (0-6 days) with three sample-sets; plasma, hemolyzed blood and non-hemolyzed blood. A decrease in the ethanol concentration in blood was seen as a nearly equivalent increase in the acetaldehyde concentration. This effect was not observed in plasma, indicating chemical oxidation of ethanol to acetaldehyde in the presence of red blood cells. The variables showed different magnitude of effects in hemolyzed and non-hemolyzed blood. A decrease in ethanol concentration was seen even after a few days of storage and also when changing the HS-T degrees from 50 to 70 degrees C. The formation of acetaldehyde was dependent on all the variables and combinations of these (interactions) and HS-T degrees was involved in all the significant interaction effects. Favorable instrumental conditions were found to be HS-T degrees of 50 degrees C and HS-EqT of 15-25 min. The ethanol concentrations obtained for the range 0.04-2.5 g/kg after analyzing authentic forensic blood samples with a HS-T degrees of 50 degrees C were statistically significantly higher than at 70 degrees C (+0.0154 g/kg, p < 0.0001, n = 180). In conclusion, chemical oxidation of ethanol to acetaldehyde in the presence of red blood cells has been shown to contribute to lowered ethanol concentrations in blood samples. Storage conditions before analysis and the headspace equilibration temperature during analysis were important for the determination of blood ethanol concentrations.     

Site-dependent postmortem changes in blood cocaine concentrations

When a forensic toxicologist interprets postmortem blood cocaine findings he usually must make assumptions regarding perimortem drug concentrations. In-vitro studies have shown that cocaine rapidly hydrolyzes in unpreserved blood, particularly at elevated temperatures. However, other studies have demonstrated site-dependent postmortem release of some drugs from tissue stores accompanied by increases in drug concentrations in the blood. This study was undertaken to investigate whether blood cocaine concentrations change in the body during the postmortem interval and, if so, to measure the direction and magnitude of the changes. In medical examiner cases in which scene investigation suggested that the decreased was a cocaine user, blood samples were collected as soon after death as possible. At autopsy, a second set of samples was collected. Analysis of paired samples by gas chromatography/mass spectrometry (GC/MS) revealed dramatic differences in the cocaine concentration. The magnitude and direction of the change appears to be site dependent. Usually, but not invariably, cocaine concentration in subclavian vein blood decreases while that in heart, aorta, and femoral vein blood increases during the interval between death and autopsy. The findings emphasize the danger inherent in attempting to estimate the concentration of cocaine in blood at the time of death from postmortem data.     

尸检样本乙醇分析结果的解释策略

由于死后各种复杂的人为现象的存在,使得建立死后血液酒精含量(BAC)和死亡时醉酒状态之间的关系十分困难。在日常检案中必须考虑细菌污染、发酵是否引起死后乙醇再合成,死亡时胃中未被吸收的乙醇是否向周围组织以及血中扩散等问题。本文总结了关于死后乙醇分析及结果解释相关问题的研究文献,旨在为从事法医毒物学乙醇分析的同行在调查此类案件时提供良好的开端。     

Postmortem biochemical changes in canine blood

Antemortem and postmortem blood samples from 60 dogs were evaluated for sodium, chloride, potassium, urea nitrogen, glucose, creatinine, calcium, phosphorus, total protein, albumin, and carbon dioxide levels. Temperatures were 4, 20 and 37 degrees C. Postmortem intervals were 3, 6, 12, and 48 h. Blood urea nitrogen, calcium, and protein values remained stable after death, indicating diagnostic significance. Potassium, creatinine, and phosphorus levels increased with time and sodium, chloride, and total carbon dioxide levels decreased with time; therefore, determining these chemical values could be beneficial in estimating time of death. Glucose values were of limited value.     

Distribution of ethyl glucuronide in rib bone marrow, other tissues and body liquids as proof of alcohol consumption before death

Postmortem ethyl glucuronide (EtG) concentrations in rib bone marrow, liver, muscle, fat tissue, urine, blood and bile have been determined by LC-MS/MS. Samples have been taken from twelve corpses during autopsies. In nine corpses EtG could be detected, corresponding blood ethanol concentrations (BAC) were 0.04-0.37 g%. In three cases, no EtG was found; two of these cases showed postmortem BACs - possibly due to putrefaction - of 0.01 and 0.1g%. In rib bone marrow, which is easily accessible during autopsy, EtG concentrations (0.77-9.36 microg/g) have been lower than in blood (2.24-20.46 microg/mL) in eight of nine cases and comparable or higher than in muscle tissue. Therefore, rib bone marrow has been found suitable as matrix for EtG determination. The highest EtG concentrations have been found in urine in all but one case, where the resorption of ethanol had been incomplete. Second highest EtG concentrations have been detected in liver samples. In two cases with putrefaction, EtG could not be detected. In these cases, the detectable ethanol might have been produced partially or in total by postmortem fermentation. However, instability of EtG during putrefaction cannot be totally excluded which might result in a total loss of EtG.