Postmortem Ethanol Production Research Articles

B-294 Case report: Postmortem ethanol production following traumatic injury

Abstract Background Ethanol is the most commonly encountered psychoactive substance in forensic toxicology. Accurate interpretation of ethanol concentrations from postmortem samples is fraught with potential hurdles, including decomposition, redistribution, and postmortem production of ethanol. Through the fermentation of substrates like glucose, pyruvate, lactate, and amino acids, microorganisms present in the gut and/or environment can generate ethanol in contaminated human tissues, especially following traumatic injury. The degree of fermentation varies widely depending on the time between death and autopsy, temperature, humidity, tissue glucose concentrations, and the specimen type. Vitreous humor (VH) is the preferred specimen for postmortem toxicological analysis of ethanol because it accurately reflects the antemortem ethanol concentration in serum while being resistant to microbial contamination. This case highlights the utility of VH in an extreme instance of postmortem ethanol production. Methods Postmortem toxicological analysis of liver tissue and VH (NMS Labs, Horsham, PA) was performed by headspace-gas chromatography (HS-GC) Results Case presentation: A man in his sixties with a history of type 1 diabetes mellitus and hypertension was involved in a fatal mining accident while operating heavy machinery in which the equipment fell down a cliff, causing the man to be ejected onto the ground nearby. The decedent was discovered hours after the fall with extensive traumatic injuries to the left side of his body and head. Seven days later, an autopsy was performed by the medical examiner’s office. Postmortem toxicological analysis of liver tissue by HS-GC revealed ethanol at a concentration of 690 mg/100g. The liver tissue also tested positive for amlodipine, nicotine, cotinine, and caffeine. A vitreous humor (VH) sample tested negative (<10 mg/dL) for ethanol. Conclusions The toxicological findings reported herein are inconsistent with antemortem alcohol consumption and are instead an example of severe postmortem ethanol production by microbial contamination. Several factors likely led to the stark discrepancy between the concentrations of ethanol in VH and liver tissue. First, the decedent suffered traumatic injuries, creating conditions favorable for extensive microbial contamination. Second, the time between death and autopsy was seven days, allowing ample time for fermentation to occur, even under refrigerated conditions. Third, postmortem glycogenolysis in addition to the patient’s underlying T1DM likely provided a high concentration of glucose to fuel postmortem fermentation. Ultimately, this case underscores the importance of forensic specimen selection in accurately interpreting toxicology findings following traumatic injury.

Post-mortem formation of ethanol: Is 1-propanol a reliable marker? A proof-of-concept study using an invitro putrefactive environment setup.

Ethanol is the psychoactive substance identified most frequently in post-mortem specimens. Unfortunately, interpreting post-mortem ethanol concentrations can be difficult because of post-mortem alcohol redistribution and the possibility of post-mortem alcohol neogenesis. Indeed, in the time interval between death and sample collection, the decedent may be exposed to non-controlled environments for an extended period, promoting microbial colonization. Many authors report that in the presence of carbohydrates and other biomolecules, various species of bacteria, yeast, and fungi can synthesize ethanol and other volatile substances invitro and invivo. The aim of this study was to study the impact of several variables on microbial ethanol production as well as develop a mathematical model that could estimate the microbial-produced ethanol in correlation with the most significant consensual produced higher alcohol, 1-propanol. An experimental setup was developed using human blood samples and cadaveric fragments incubated under strictly anaerobic conditions to produce a novel substrate, "cadaveric putrefactive blood" mimicking post-mortem corpse conditions. The samples were analyzed daily for ethanol and 1-propanol using an HS-GC-FID validated method. The formation of ethanol was evaluated considering different parameters such as putrefactive stage, blood glucose concentration, storage temperature, and storage time. Statistical analysis was performed using the Mann-Whitney non-parametric test and simple linear regression. The results indicate that the early putrefactive stage, high blood glucose concentration, high temperature, and time of incubation increase microbial ethanol production. In addition, the developed mathematical equation confirms the feasibility of using 1-propanol as a marker of post-mortem ethanol production.

A primary study of ethanol production in postmortem liver and muscle tissue of rats

ObjectiveTo study the characteristics of postmortem ethanol production and its relation with alcohol congeners in postmortem rat liver and muscle tissues. MethodPostmortem liver and muscle tissues in Sprague–Dawley rats, from postmortem time interval (PMI) day 0–20, were analyzed via headspace gas chromatograph flame ionization detection to observe production of postmortem ethanol and 5 selected alcohol congeners. Result1. Putrid ethanol production increased gradually to a peak and then decreased with the prolongation of PMI; 2. Acetaldehyde, 1-propanol, and 3-methyl-butyraldehyde were produced along with postmortem ethanol; 1-butanol was only detected from day 11–20; 3. The concentrations of acetaldehyde, 1-propanol and 3-methyl-butyraldehyde was related with ethanol production. Fifteen mathematical models were constructed for putrid ethanol production based on acetaldehyde, 1-propanol, and 3-methyl-butyraldehyde. ConclusionA peak in postmortem ethanol production was identified. The production trends of acetaldehyde, 1-propanol, and 3-methyl-butyraldehyde in the liver, and of 1-propanol in muscle, were consistent with those of ethanol, and could potentially to be used as biomarkers of postmortem ethanol production. Further human samples and data analysis are needed to verify this.

Correlation between Postmortem Ethanol Production and Low Molecular Weight Volatiles (LMWVs) in a Thai Population

Objective: To determine the relationship between postmortem ethanol production and low molecular weight volatiles (LMWVs) in a Thai population.
 Materials and Methods: A retrospective study was conducted in 157 Thai cadavers. Various subject data were recorded, including degree of postmortem changes (transition period group vs. decomposition group classified by the total body score [TBS]) and blood profiles of ethanol, acetaldehyde, and 1-propanol concentrations from femoral blood samples that were diagnosed as postmortem production by vitreous humor analysis. Linear regression analysis was performed to determine the correlations between these three analytes and the TBS and between postmortem ethanol and the other two analytes.
 Results: This study comprised 44 females and 113 males with a mean age at death of 47.79 years old. Postmortem ethanol, acetaldehyde, and 1-propanol concentrations escalated along with the increased TBS (p<0.001). These three analytes were significantly correlated with the TBS (p<0.001) and the correlations of determination (R2) for postmortem ethanol and 1-propanol were better than for acetaldehyde (R2 = 0.488 and 0.414 vs. 0.269). Acetaldehyde and 1-propanol were positively correlated with postmortem ethanol (p<0.001). The correlations between postmortem ethanol and acetaldehyde, 1-propanol, and the combination of these two analytes produced R2 values of 0.413, 0.480, and 0.544, respectively.
 Conclusion: Acetaldehyde and 1-propanol concentrations were significantly correlated with postmortem ethanol concentrations in Thai cadavers, and the combination of these two markers produced a better correlation with the postmortem ethanol concentration.

Added value evidences of a simultaneous determination of two phosphatidylethanol isoforms and ethylglucuronide in dried blood spots in forensic contexts

In clinical or postmortem forensic medicine, acute or chronical alcohol abuse are of main concern and direct/indirect biomarkers are widely use in order to assess recent or retrospective alcohol abuse. We aim to illustrate that a simultaneous determination of 3 directs ethanol biomarkers [two phosphatidylethanol isoforms, 16:0/18:1 (PEth18:1) and 16:0/20:4 (PEth20:4), and ethylglucuronide (EtGB)] using dried blood spot (DBS) can be a useful additional tool in many situations. (Case#1) Sexual assault of a 17-year-old girl with negative urine and blood ethanol concentration (BAC) (but urine EtG concentration was 10 mg/L) sampled 19 hours after the facts. (Case#2) Only blood sampling (negative BAC) was performed 14 hours after a 36-year-old man (with heavy psychiatric disorders) tried to kill 3 persons. He was reported drunk at the time of the attack by witnesses. (Case#3) A 27-year-old man was implicated in a car-related fatality that occurred 11 days before sampling. Blood and hair sampling were performed in order to investigate his alcoholic status: hair EtG concentration was < 5 pg/mg. (Case#4) The alcoholic status of a 43-year-old woman killed in a car accident had to be established. BAC was 140 mg/L in the postmortem peripheral blood (only available biological sample; sampled less than one hour after death and kept refrigered until time of analysis, 2 days later). (Case#5) A low BAC (70 mg/L) was found in postmortem cardiac blood (only sample available) sampled on the corps of a 42-year-old man found submerged in the sea. (Case#6) Peripheral blood sampling (for BAC determination) and direct DBS sampling (MitraTM) were performed at the time of discovery of the 36-year-old man corpse, allegedly heavy drinker according to the testimonies (BAC was 2810 mg/L). Blood samples can alternatively consist in 10 μL from usual blood sampling tubes (e.g. fluoride preserved) transferred on DBS card, or direct DBS sampling using Volumetric Absorptive MicroSampling devices (VAMS; HemaxisTM, MitraTM). These DBS are analyzed using a LC-MS/MS (routinely used in clinical toxicology) method (LLOQ: 2 μg/L for the 3 analytes). Results (μg/L; PEth18:1, PEth20:4 and EtGB, respectively) were: – (Case#1) 22, 2 and 300, in coherence with both a recent alcohol intake hypothesis [EtGB above the 45 μg/L threshold supported an alcohol consumption [> 2 standard units (SU)] in the last 24 to 48 hours] and that this girl was not an excessive drinker [owing to the low PEths concentrations]; – (Case#2) 98, 103 and 71, consistent with an alcohol intake hypothesis arround the time of the attack by this man who had a significant consumption of alcohol in the last 10 days as PEth 20:4 concentration was similar to the PEth18:1 one; – (Case#3) 9, < 2 and < 2, and did not contradict the negative EtG hair result as these low values were, at most, compatible with an “occasional” or “social drinker” status (< 14 SU per week); – (Case#4) 817, 44 and 130. EtGB positive result supported a recent alcohol consumption and the very high PEth18:1 value strongly suggested chronic excessive alcohol consumption; – (Case#5) negative (< 2 μg/L) for the 3 analytes in postmortem cardiac blood, in agreement with both the hypothesis of a light postmortem ethanol production, and a probable “abstinent” or “occasional consumer” status of the victim; – (Case#6) 3470, 1030 and 13,400, supporting the massive alcohol intoxication hypothesis close to the death of a heavy drinker. This analytical tool is of high interest when only blood samples are available, although postmortem use (due to PEths neoformation issues) should be limited to cases with negative BAC, or when blood sampling is performed close to death using a VAMS or a refrigerated preserved (< 4 days) tube.

Effects of postmortem interval, putrefaction, diabetes, and location of death on the analysis of ethyl glucuronide and ethyl sulfate as ethanol biomarkers of antemortem alcohol consumption

This study evaluated the forensic value of ethanol biomarkers ethyl glucuronide (EtG) and ethyl sulfate (EtS) under different conditions, including diabetes mellitus, drug abuse, and advanced decomposition. In addition, we explored whether ethanol, EtG, or EtS formation occurred in patients who died as a result of diabetes mellitus. Fifty-two routine postmortem cases were divided into three groups. Group 1 included only the post-mortem cases in which at least blood samples were available (n=47). Group 2 included all cases with positive BAC (n=28). Group 3 included the cases with negative BAC while information surrounding the cases suspected antemortem alcohol consumption and cases that tested negative for ethanol but positive for EtG and EtS. We analyzed multiple bodily fluid specimens, including the vitreous humor, for ethanol biomarker analysis and accurately identified antemortem ethanol consumption or postmortem ethanol synthesis. We also determined the utility of urine samples for analyzing ethanol and its metabolites in putrefaction cases. If no urine sample was available at autopsy due to urination before death or diabetes-associated glucosuria, vitreous humor samples were an appropriate alternative for ethanol biomarker testing. We observed postmortem ethanol synthesis in diabetic individuals even with a short postmortem interval (PMI), however, glucose did not increase postmortem ethanol production in individuals with diabetes under appropriate preservation. The shorter the PMI, the better the ethanol source can be determined. Postmortem ethanol production occurred in all body fluid specimens analyzed herein, including the vitreous humor. EtG and EtS levels were stable and provided accurate insight into ethanol sources, even in cases of postmortem ethanol production. While the present study focused on the use of vitreous humor for the analysis, it is expected such samples may not be available in cases of advanced decay. In cases where no other bodily fluid specimens are available, solid tissue specimens are highly preferred.

Modeling Postmortem Ethanol Production/Insights into the Origin of Higher Alcohols.

The forensic toxicologist is challenged to provide scientific evidence to distinguish the source of ethanol (antemortem ingestion or microbial production) determined in the postmortem blood and to properly interpret the relevant blood alcohol concentration (BAC) results, in regard to ethanol levels at death and subsequent behavioral impairment of the person at the time of death. Higher alcohols (1-propanol, 1-butanol, isobutanol, 2-methyl-1-butanol (isoamyl-alcohol), and 3-methyl-2-butanol (amyl-alcohol)) are among the volatile compounds that are often detected in postmortem specimens and have been correlated with putrefaction and microbial activity. This brief review investigates the role of the higher alcohols as biomarkers of postmortem, microbial ethanol production, notably, regarding the modeling of postmortem ethanol production. Main conclusions of this contribution are, firstly, that the higher alcohols are qualitative and quantitative indicators of microbial ethanol production, and, secondly that the respective models of microbial ethanol production are tools offering additional data to interpret properly the origin of the ethanol concentrations measured in postmortem cases. More studies are needed to clarify current uncertainties about the origin of higher alcohols in postmortem specimens.

Modeling postmortem ethanol production by C. albicans: Experimental study and multivariate evaluation

In previous research, we modeled the ethanol production by certain bacteria under controlled experimental conditions in an attempt to quantify the production of microbial postmortem ethanol in cases where other alcohols were co-detected. This contribution on the modeling of postmortem ethanol production by Candida albicans is complementary to these previous studies. Τhis work aimed to study ethanol, higher alcohols (1-propanol, isobutanol, 2-methyl-1-butanol and 3-methyl-1-butanol), and 1-butanol production by Candida albicans: (i) in different culture media (Brain Heart Infusion, BHI and, Sabouraud Dextrose Broth, SDB), (ii) under mixed aerobic/anaerobic or strict anaerobic conditions, and (iii) at different temperatures (37 °C, 25 °C and, 4 °C), and develop simple mathematical models, resulted from fungal cultures at 25 °C, to predict the microbially produced ethanol in correlation with the other alcohols. The applicability of the models was tested in the C. albicans cultures in BHI and SDB media at 37 °C, in denatured human blood at 25 °C, acidic and neutral with different concentrations of additional glucose, in acidic denatured blood diluted with dextrose solution and in blood from autopsy cases. The received results indicated that the C. albicans models could apply in cases where yeasts have been activated in blood with elevated glucose levels. Overall, the in vitro ethanol production by C. albicans in blood depended on temperature, time, glucose (or carbohydrate) content, pH of the medium and endogenous changes in the medium composition through time. Our results showed that methyl-butanol is the most significant indicator of fungal ethanol production, followed by the equally important isobutanol and 1-propanol in qualitative and quantitative terms.

Estimating a reliable cutoff point of 1-propanol in postmortem blood as marker of microbial ethanol production

The interpretation of the ethanol analysis results in postmortem cases may be challenging when the origin of postmortem ethanol (antemortem ingestion or microbial production) is under dispute. In this study, we investigated the suitability of blood 1-propanol cutoff concentration as a reliable marker for the discrimination between “positive” and “negative” for postmortem ethanol production (PEP) autopsy blood samples by performing receiver operating characteristic (ROC) analysis. The results indicated that a threshold 1-propanol concentration of 0.104 mg/dL had an area under the curve of 0.90 (standard deviation = 0.03), sensitivity of 79%, and specificity of 91% for predicting PEP with 95% confidence interval. This means that the positive for PEP autopsy blood samples yield significantly differentiated PEP than approximately 90% of the controls. The estimated concentration of 1-propanol is an applicable threshold (cutoff) value for autopsy blood 1-propanol to discriminate between “positive” and “negative” samples for PEP. We named this threshold 1-propanol concentration (of 0.104 mg/dL) as “1-propanol criterion.” In an effort to test its applicability to postmortem cases, we evaluated blood ethanol and 1-propanol from 222 postmortem cases records. The results showed that 10% of the tested samples were positive for PEP, and only a few of them were from bodies with signs of putrefaction at autopsy. This finding indicates that PEP in a corpse could take place before the appearance of obvious putrefaction. We suggest the ROC-based calculation of the 1-propanol cutoff concentration, at 0.104 mg/dL, as an effective method for “flagging” blood samples as positive for PEP irrespectively of the presence or not of putrefaction.

Epidemiology of alcohol-related unintentional drowning: is post-mortem ethanol production a real challenge?

BackgroundPost-mortem (PM) ethanol production may hamper the interpretation of blood alcohol concentration (BAC) in victims of drowning. Different exclusion criteria (e.g. cases with low BAC or with protracted interval between death and toxicological analysis) have been proposed with no factual figures to reduce the potential bias due to PM ethanol production when examining the prevalence rates for alcohol-related drowning. The aim of this study is to verify the extent to which PM alcohol production may affect the accuracy of studies on drowning and alcohol.FindingsUnintentional fatal drowning cases (n = 967) for which a full medico-legal autopsy and toxicological analysis was performed, in Finland, from 2000 to 2013, and relevant variables (demographic data of the victims, month of incident, PM submersion time, blood alcohol concentration, urine alcohol concentration (UAC), vitreous humour alcohol concentration (VAC) were available. Overall, out of 967 unintentional drownings, 623 (64.4%) were positive for alcohol (BAC > 0 mg/dL), 595 (61.5%) had a BAC ≥ 50 mg/dL, and 567 (58.6%) a BAC ≥ 100 mg/dL. Simultaneous measurements, in each victim, of BAC, UAC, and VAC revealed PM ethanol production in only 4 victims (BAC: 25 mg/dL – 48 mg/dL). These false positive cases represented 0.4% of drownings with BAC > 0 mg/dL and 14.3% of drownings with BAC > 0 mg/dL and < 50 mg/dL.ConclusionsThe present study suggests that PM ethanol production has a limited impact on research addressing the prevalence rate for alcohol-related drowning and that the use of too rigorous exclusion criteria, such as those previously recommended, may led to a significant underestimation of actual alcohol-positive drowning cases.

Postmortem Ethanol Concentrations and Postmortem Ethanol Production

A review of the literature was conducted in reference to postmortem blood ethanol concentrations and the issue of postmortem production of ethanol. Ethanol in postmortem specimens may occur as a result of antemortem consumption (most common), postmortem production in the body or in vitro by microorganisms (less common), and endogenous production by microorganisms during life (rare). The portion of ethanol-positive postmortem cases in which postmortem production of ethanol is thought to have occurred is small, as is the portion of cases in which postmortem production of ethanol is a potentially important issue. It is unusual for postmortem production of ethanol to account for more than a 70 mg/dL concentration, but cases with higher concentrations occasionally occur. Cases in which blood ethanol is positive but vitreous ethanol concentration is much lower or negative, and/or in which the urine: blood ethanol concentration ratio is less than one are supportive of postmortem ethanol production. The presence of other alcohols such as n-propanol and/or n-butanol is suggestive of postmortem microbial production of ethanol. The ratio of serotonin metabolites in urine may be helpful in distinguishing postmortem production of ethanol from antemortem consumption, as may the concentration of ethyl glucuronide in blood. Candida and some other species may produce ethanol in urine antemortem and postmortem. In rare instances such as infections, endogenous ethanol production may occur in a living person. Recommendations are made concerning samples to collect and tests that may be helpful in distinguishing antemortem ethanol consumption from postmortem production of ethanol.

Postmortem endogenous ethanol production and diffusion from the lung due to aspiration of wood chip dust in the work place

We report an autopsy case of postmortem ethanol diffusion into the cardiac blood after aspiration of wood chips, although antemortem ethanol consumption was not evident. A man in his twenties, who was loading a truck with small wood chips in a hot, humid storehouse, was accidentally buried in a heap of chips. At the time the body was discovered, 20 h after the accident, rectal temperature was 36 °C. Autopsy showed the cause of death to be asphyxia due to obstruction of the airway by aspiration of wood chips. The ethanol and n-propanol levels were significantly higher in the lungs (left, 0.603 and 0.009 mg/g; right, 0.571 and 0.006 mg/g) than in other tissues. A significant difference in ethanol concentration was observed between the left cardiac blood (0.243 mg/g) and the right femoral blood (0.042 mg/g). Low levels of ethanol and n-propanol were detected in the stomach contents (0.105 and 0.001 mg/g, respectively). In order to determine whether aspiration of wood chips affects postmortem ethanol production in the lung, we measured the ethanol and n-propanol levels of homogenized rabbit lung tissue incubated with autoclaved or non-autoclaved wood chips. Levels of ethanol and n-propanol were significantly higher in the homogenates incubated with non-autoclaved chips for 24 h. The results of this animal experiment suggested that the ethanol detected in the lung was produced by putrefactive bacteria within the wood chips. After death, the ethanol produced endogenously in the lung appears to have diffused and affected the ethanol concentration of the left cardiac blood.

Clarification of Ethanol-Positive Case Using Urine Serotonin Metabolite Ratio

This paper intends to provide investigators with information useful in determining the presence of postmortem ethanol in fatal accidents and a case history of an accident that involved postmortem alcohol formation is presented. An ethanol-positive fatal case initially reported as being from ingestion was ultimately determined to be from postmortem ethanol production using the ratio of two serotonin metabolites found in urine. This case involved a transportation accident that could have resulted in additional hardships for the victim's family through loss of compensation and reputation.

Loss of Ethanol from Vitreous Humor in Drowning Deaths

Two separate cases of drowning with extended periods underwater (2 and 4 weeks) are reported. The postmortem ethanol concentrations were 260 and 280 in central blood, 50 and 80 in vitreous, and 330 and 320 in urine (mg/100 mL) for cases 1 and 2, respectively. Determination of the urine 5-hydroxytryptophol/5-hydroxyindole-3-acetic acid ratios produced results of 713 and 41 pmol/nmol, respectively. The serotonin metabolite ratios support the explanation of diffusion of ethanol from the vitreous fluid into the surrounding water, rather than postmortem production of ethanol in blood, as the primary reason for the blood-vitreous ethanol differences.

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.

Ethanol production by Candida albicans in postmortem human blood samples: Effects of blood glucose level and dilution

We present two cases in which the ethanol concentration in blood samples taken after death continued to increase in the absence of any remarkable increase in n-propanol concentration. Species of bacteria and yeasts, including Candida albicans were isolated from these samples. We then examined whether C. albicans, the most common yeast in the general environment, was able to produce ethanol in human blood stored at room temperature. Ethanol production increased as the glucose concentration increased, indicating that C. albicans produced ethanol from the glucose. Our results also suggested that C. albicans produced ethanol more easily in blood diluted by intravenous infusions that included glucose than in undiluted blood. These findings are useful for the evaluation of postmortem ethanol production in subjects whose blood has been diluted by infusions with glucose. Furthermore, there was no quantitative relationship between the amount of n-propanol detected and the amount of ethanol production: n-propanol appears to be an unreliable index of putrefaction and postmortem ethanol production by C. albicans. It is possible for the blood ethanol level to be high and n-propanol not to be detected, even if the subject has not been drinking alcohol. We reconfirmed the necessity of immediately adding sodium fluoride to samples for ethanol analysis to prevent postmortem ethanol production.

Elevated Postmortem Ethanol Concentrations in an Insulin-Dependent Diabetic

A 54-year-old woman (165 cm, 37 kg) was found dead in her home during a welfare check after not having been seen for at least three days. The body showed clear evidence of decomposition. Her head was in what appeared to be a pool of blood. The residence was clean, neat, and showed no evidence of violence. Insulin was found in the refrigerator, and syringes were in the kitchen cabinet. In agreement with these physical findings, her clinical history indicated that she suffered insulin-dependent diabetes mellitus. Autolytic changes were noticed at autopsy, and no obvious cause of death was demonstrated. The autopsy heart blood sample screened negative for cocaine and/or metabolite (benzoylecgonine), phenethylamines, opiates, and barbiturates by radioimmunoassay. The alcohol concentration was 0.51 g/dL in the blood, 0.04 g/dL in the brain, 0.08 g/dL in the liver, and 0.05 g/dL in the urine, and acetone levels were 42 mg/dL, 53 mg/dL, 14 mg/dL, and 19 mg/dL, respectively. Isopropanol was also present in all samples analyzed. The cause of death was ruled as metabolic acidosis due to diabetes mellitus. Possible bacterial postmortem production of ethanol is considered as an explanation for the increased concentration of ethanol found in the postmortem heart blood.

Comparative Alcohol Concentrations in Blood and Vitreous Fluid With Illustrative Case Studies

The Toxicology Bureau of the New Mexico Department of Health performs drug and alcohol testing on approximately 2800 medical examiner cases each year across the entire state. Although blood is usually the preferred specimen for alcohol analysis, the importance of multiple specimen analysis in alcohol-related death investigation is well understood. Quantitative alcohol determination in a variety of postmortem specimens may provide important interpretive information. In a total of 322 consecutive cases, blood and vitreous alcohol concentrations were compared. No alcohol was detected in either specimen in only 27 of the cases. In the remaining 295 investigations, alcohol was detected in the vitreous fluid, blood, or both. Analysis of the data and presentation of case studies reinforce the need for multiple specimen analysis in alcohol-related death investigation. Postmortem blood and vitreous alcohol concentrations were compared in a series of 295 alcohol-positive cases. The vitreous alcohol concentration (VAC) exceeded the blood alcohol concentration (BAC) in 209 cases (71%). Blood alcohol concentrations exceeded vitreous concentrations in 81 cases (27%), and the concentrations were equivalent in 5 cases (2%). For the purpose of this study, samples that were negative in both specimens were excluded. In casework where the VAC > BAC, linear regression analysis indicated an R2 value of 0.958 (n = 209) and a VAC approximately 16% higher than the BAC. The VAC/BAC ratio was more variable at lower BACs (< 0.1 g/100 mL). The source of blood for this data set was predominantly femoral (n = 203), followed by heart (n = 5) and pleural cavity (n = 1). Although VAC/BAC ratios were more consistent at concentrations of 0.1 g/100 mL and above, the overall ratio ranged from 1.01 to 2.20. Of the 81 cases where BAC > VAC, a total of 24 cases indicated no vitreous alcohol. The range of blood alcohol concentrations among these cases was widely variable (0.01 to 0.30 g/100 mL). Unlike the VAC/BAC data set which consisted of 97% femoral blood, the source of blood in the BAC > VAC data set was slightly more variable. Of the 81 cases where BAC > VAC the blood source consisted of femoral (n = 68), heart (n = 8), pleural cavity (n = 2), carotid (n = 1), jugular (n = 1), and chest blood (n = 1). All analyses were conducted using dual-column gas chromatography with flame-ionization detection (GC-FID) with a reporting limit of 0.01 g/100 mL ethanol in postmortem samples. A series of case studies are used to demonstrate postmortem interpretive issues and the benefits associated with multiple specimen analysis. Cases include postmortem production of ethanol, rapid or unexpected death during the absorptive phase, and site-dependent differences following traumatic injury. Actual case studies involving other volatile organic compounds are also presented including isopropanol and acetone from endogenous and exogenous sources. Many of these cases studies highlight the difficulty associated with postmortem alcohol interpretation in the absence of multiple specimens or adequate case history.

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.

Gas chromatographic procedures for determination of ethanol in postmortem blood using t-butanol and methyl ethyl ketone as internal standards

Three gas chromatographic procedures for the determination of ethanol in postmortem blood using alternative internal standards to n-propanol are presented: a direct injection procedure using t-butanol, and two headspace methods using t-butanol and methyl ethyl ketone. t-Butanol and methyl ethyl ketone were well resolved from ethanol, acetone, methanol and other commonly observed putrefactive volatiles using direct injection or headspace analysis. CVs for the direct injection method were below 5% for ethanol and below 10% for the other volatiles. The lower limits of detection (LOD) were 25–50 mg/L. The CVs for the headspace methods were below 5% for ethanol and below 6% for the other volatiles. The LODs were 10 mg/L using either t-butanol or methyl ethyl ketone as internal standards. The use of t-butanol or methyl ethyl ketone as alternatives to n-propanol avoids the possibility of error in the quantitation of ethanol due to the presence of n-propanol and allows for the identification of other volatiles that may aid in distinguishing antemortem ingestion from postmortem production of ethanol.