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.     

心肌梗死6项免疫组化指标的死后稳定性比较

目的　探讨用于早期心肌梗死死后诊断的维连接蛋白 (Fn)、纤维蛋白原 (Fg)、补体 (C5 )、肌红蛋白(Mb)、肌动蛋白 (HHF3 5 )、结蛋白 (Dm )等 6项免疫组化指标在死后不同时间的稳定性。方法　应用免疫组织化学法、图像分析和统计学处理系统 ,检测缺血心肌细胞内Mb、HHF3 5、Dm的缺失面积和Fn、Fg、C5的阳性反应面积 ,并对 6项免疫组化指标在死后不同时间的稳定性进行比较。结果　正常心肌组织 4℃放置 1～ 2d ,Dm、HHF3 5、Mb染色均匀 ,未见明显缺失 ;放置 3d以上 ,即可见Dm、HHF3 5、Mb明显缺失 ,且随放置时间延长 ,缺失面积逐步增大。缺血心肌组织随放置时间延长 ,Dm、HHF3 5、Mb的缺失面积逐步增大 ,Fg、C5、Fn阳性反应面积逐渐减少 ;放置 14d以上 ,Fg呈阴性反应 ;放置 2 1d以上 ,C5呈阴性反应 ;放置 2 8d ,Fn仍呈阳性反应。正常心肌组织放置不同时间 ,均未见Fg、C5、Fn阳性反应。图像分析结果显示阳性反应面积逐步减少。结论　Dm、HHF3 5、Mb的稳定性最差 ,易受死后自溶的影响 ,只适用于新鲜尸体 (死后 1～ 2d) ;Fg次之 ,可用于死后 4℃放置 7d的尸体 ;C5较好 ,可用于死后 4℃放置 14d的尸体 ;Fn稳定性最好 ,可用于死后 4℃放置 2 8d的尸体。     

Postmortem stability and interpretation of beta 2-agonist concentrations.

This paper describes a series of stability and redistribution studies aimed at understanding the presence and significance of beta 2-agonists in asthma deaths. Salbutamol and terbutaline were shown to be stable in postmortem blood at 23 degrees C for 1 week, 4 degrees C for 6 months and -20 degrees C for 1 to 2 years. However, fenoterol was shown to degrade at 23 degrees C (83% loss), 4 degrees C (93% loss) and -20 degrees C (66% loss) over the same time. Salbutamol concentrations detected in blood taken at the time of body admission to the mortuary were not significantly different from the concentrations detected in blood taken from the same cases at the time of autopsy (45 h later). This suggests that significant postmortem redistribution of salbutamol is unlikely to occur during this period. Postmortem blood concentrations of at least salbutamol are likely to reflect the concentration of these drugs in the body at the time of death.     

应用FTIR光谱技术推断死亡时间

目的 应用傅立叶变换红外(FTIR)光谱技术分析大鼠死后组织、人体离体组织随死亡时间推移的化学降解过程,为死亡时间推断的研究提供新的途径与方法.方法 大鼠断颈处死后置于4℃、20℃、30℃环境,在不同死亡时间点提取大鼠不同组织;收集有同样死亡时间的人尸体组织,离体取材,并运用FFIR光谱仪测定不同化学基团随死后和离体后...     

Blood, bone marrow and eye fluid ethanol concentrations in putrefied rabbits

The effect of putrefaction on postmortem blood, bone marrow and eye fluid ethanol levels was evaluated in rabbits. Control and dosed animals were sacrificed and stored at either room temperature (approx. 19 degrees C) or cold temperature (approx. 3.5 degrees C) for as long as 28 days. Control animals stored at room temperature showed ethanol levels in the bone marrow that peaked at 7 days after sacrifice, followed by decreases to a nondetectable level at 21 days. Overall decreases were demonstrated in bone marrow of dosed rabbits stored at room temperature for all postmortem intervals. The control animals stored at low temperature showed no ethanol in the bone marrow and blood until 21 days after sacrifice. Dosed rabbits stored at low temperature showed no significant changes in blood and marrow ethanol until 21 days after sacrifice.     

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.     

Postmortem interval estimation by creatinine levels in human psoas muscle.

A new method was tested for estimating time after death in the middle range of postmortem interval (weeks). Creatinine muscular concentration in human cadaver is positively correlated to postmortem estimation. Temperature should be mainly related to the creatinine transformation rate. A stronger correlation was found at 11 degrees C up to 30 days and at 20 degrees Celsius up to 15 days.     

Preliminary study of the effect of acute antemortem haemorrhage on postmortem abdominal impedance in rats

Studies over many years have revealed a consistent, inverse relationship between extracellular impedance of the rat abdomen and postmortem interval (PMI). Recent studies have shown that this relationship can be improved by correcting measured impedances to their theoretically predicted values at an arbitrarily chosen temperature of 40 degrees C, thus rendering them independent of the tissues' temperature at the time of impedance measurement. The present study, undertaken as a part of an ongoing effort to identify variables which might influence postmortem rate of change in abdominal impedance, was aimed at studying the possible effect of acute antemortem haemorrhage on abdominal impedance. Sudden loss of approximately 30% of the total blood volume, immediately prior to the death was without statistically significant effect on the pattern of postmortem change in abdominal impedance. Thus, in the control and experimental rats, respectively, impedance decreased progressively from 58.7 +/- 1.8 and 68.0 +/- 11.3 omega, 1 h postmortem, to 31.5 +/- 2.2 and 33.7 +/- 4.7 omega at a postmortem interval of 120 h (n = 6 in each group). In both groups, the relationship between impedance and postmortem interval was either linear or curvilinear. These findings are believed to mitigate in favour of continued effort to examine the potential usefulness of abdominal impedance, as an index of postmortem interval, under conditions encountered in routine forensic investigation.     

The forensic science implications of site and temporal influences on postmortem blood-drug concentrations

The dependence of postmortem blood-drug concentrations on the collection site and on the postmortem interval before specimen collection has been studied. These studies consisted of both sequential sampling from the same collection site at defined time intervals and a comparison of the drug concentrations of postmortem blood simultaneously collected from various sites. A site and time dependence was observed for postmortem blood-drug concentrations. The heart blood-drug concentrations were, in general, significantly higher than those of peripheral specimens. As a result of this phenomenon, the analysis of peripheral blood specimens and solid tissues is often necessary before a definitive interpretation of postmortem toxicological analyses is possible.     

Postmortem sera and cerebrospinal fluid enzymes

Antemortem and postmortem sera from 60 dogs were evaluated for lipase, amylase, alkaline phosphatase, gamma-glutamyltransferase, and alanine aminotransferase (AAT); cerebrospinal fluid was examined for AAT and alkaline phosphatase. The postmortem intervals were 3, 6, 12, 24, and 48 h at temperatures of 4, 20, and 37 degrees C. Amylase levels remained stable at 4 and 20 degrees C and may be beneficial for diagnosing pancreatitis. Lipase levels may be useful as an adjunct to amylase values. Serum alkaline phosphatase values increased with postmortem interval; values were higher at 37 degrees C than at 4 degrees C. Other enzymes were of little value for diagnosis.     

血液中乙醇保存稳定性研究

目的确定血液中乙醇最佳保存条件,探讨影响血液中乙醇含量稳定性的主要因素。方法对血液保存的温度(-20、4、20℃)、防腐剂(NaF、无防腐剂、Na2O2)、储存容器中空气所占比例(0%、25%、50%)和血醇质量浓度(0.2、0.8、2.0mg/mL)四个因素采用正交试验L9(34)方法分组,样本采用顶空气相色谱法进行测定,测定结果采用方差分析进行讨论。结果在20℃保存且不加入防腐剂的两组样本中血醇浓度变化明显,其余变化不明显。结论血液样本在4℃、储存容器中空气比例为50%和加防腐剂(NaF)的条件下保存,稳定性最佳;四个影响因素中温度为影响血液中乙醇含量稳定性的主要因素。     

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.     

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.     

肌红蛋白降解与人体死亡时间的相关性

目的检测人体死亡后骨骼肌中肌红蛋白的降解水平,探讨其与死亡时间的关系。方法分别于人体死后0、4、8、12、24、36、48、60、72h取骨骼肌,置于室温25℃,在不同时间点抽提蛋白质,用Western Blot方法检测肌红蛋白,随后利用生物电泳图像分析软件分析。结果肌红蛋白随死后时间逐步降解。结论肌红蛋白降解的检测可用于推断死亡时间。     

Peroxidase activity in traumatic skin lesions

Peroxidase activity was determined in experimental compression-excoriation lesions and incision wounds of rat skin after different periods of vital time. The peroxidase enzyme was extracted from the tissues by homogenization in 0.5% cetyltrimethylammoniumbromide, and the enzyme activity was measured from the supernatant by o-dianisidine-H2O2 assay. In the blood of the rats a mean activity of approx. 5.26 +/- 1.11 U/g dry weight was observed. In the control specimens of the skin the activity was very low and generally below the detection limit of the methods used. In 30-min-old compression-excoriation lesions the mean peroxidase activity was 0.38 +/- 0.21 U/g dry weight. In lesions older than 30 min the activity started to increase rapidly. In 4-h-old compression-excoriation lesions it was 10 times higher than the 30-min level and was 40 times higher in 12-h-old lesions and 70-100 times higher in 1-3-day-old compression-excoriation lesions, respectively. In 30-min-old incision wounds the mean peroxidase activity was 0.65 +/- 0.37 U/g dry weight. The increase of the activity compared with the 30-min level was even faster in the incision wounds: in 4-h-old wounds the mean activity was 50 times higher, in 12-h-old wounds 200 times higher and in those of 1-5 days it was several hundreds of times higher. Compression-excoriation lesions made after death showed activity similar to the control specimens. Postmortem autolysis at +22 degrees C resulted in a loss of the enzyme activity in 1-day-old compression-excoriation lesions so that after 3 days approx. 80% remained, and after 5 and 7 days approx. 40% was present. After 3 days of autolysis at +4 degrees C, nearly 100% of the activity remained and approx. 90% was present after 5 and 7 days of autolysis. Increased peroxidase activity was also detectable in human vital excoriations in the specimens which were taken in autopsies several days postmortem.     

死后大鼠脾脏组织FTIR测量结果的法医学分析

目的应用傅里叶变换红外（FTIR）光谱技术分析大鼠死后脾脏组织随死亡时间增加的化学变化过程,为死亡时间推断研究提供新的途径与研究数据。方法大鼠断颈处死后,在30℃、20℃及4℃环境中,不同死亡时间点提取大鼠脾脏组织,并运用FTIR光谱仪测定不同化学基团随死亡时间的变化。结果随着死亡时间的推移,大鼠脾脏组织FTIR光谱的主要吸收峰峰位没有明显变化,而其吸收峰强度有明显差异：（1）1080cm-1和1238cm-1被指认核酸谱带吸收峰的峰强呈下降趋势;（2）1541cm-1被指认酰胺Ⅱ吸收峰的峰强呈上升变化;（3）1396cm-1被指认脂肪酸吸收峰的峰强呈上升变化;（4）指认为C-H结构振动的2852、2871、2923、2958cm-1吸收峰的峰强呈现上升趋势。结论 FTIR光谱分析技术有望成为法医死亡时间推断的有效方法。     

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.     

心肌肌钙蛋白T在家兔缺血心肌中的表达及其死后稳定性

目的 探寻心肌肌钙蛋白T（cTnT）在家免缺血心肌中的表达规律及其死后稳定性．并对其在法医学实践中诊断早期心肌缺血的应用价值进行评价。方法用冠状动脉结扎法建立家兔急性心肌缺血模型．应用免疫组织化学法、图像分析和统计学处理系统,检测死后cTnT表达情况,并对实验组和对照组cTnT的表达进行比较。结果缺血区心肌组织可见明显不规则灶状、片状cTnT缺失区,间质呈阴性表达。在4℃放置．随放置时间延长,正常和缺血心肌组织cTnT阳性表达均有减弱趋势,至14d时均完全缺失。但仅在4℃放置1～7d内,缺血心肌和正常心肌标本之间cTnT阳性反应具有显著性差异。结论cTnT的免疫组化表达用于认定死后4℃放置的尸体是否有死前心肌缺血时．宜在7d以内进行。     

Interpreting results of ethanol analysis in postmortem specimens: a review of the literature

We searched the scientific literature for articles dealing with postmortem aspects of ethanol and problems associated with making a correct interpretation of the results. A person's blood-alcohol concentration (BAC) and state of inebriation at the time of death is not always easy to establish owing to various postmortem artifacts. The possibility of alcohol being produced in the body after death, e.g. via microbial contamination and fermentation is a recurring issue in routine casework. If ethanol remains unabsorbed in the stomach at the time of death, this raises the possibility of continued local diffusion into surrounding tissues and central blood after death. Skull trauma often renders a person unconscious for several hours before death, during which time the BAC continues to decrease owing to metabolism in the liver. Under these circumstances blood from an intracerebral or subdural clot is a useful specimen for determination of ethanol. Bodies recovered from water are particular problematic to deal with owing to possible dilution of body fluids, decomposition, and enhanced risk of microbial synthesis of ethanol. The relationship between blood and urine-ethanol concentrations has been extensively investigated in autopsy specimens and the urine/blood concentration ratio might give a clue about the stage of alcohol absorption and distribution at the time of death. Owing to extensive abdominal trauma in aviation disasters (e.g. rupture of the viscera), interpretation of BAC in autopsy specimens from the pilot and crew is highly contentious and great care is needed to reach valid conclusions. Vitreous humor is strongly recommended as a body fluid for determination of ethanol in postmortem toxicology to help establish whether the deceased had consumed ethanol before death. Less common autopsy specimens submitted for analysis include bile, bone marrow, brain, testicle, muscle tissue, liver, synovial and cerebrospinal fluids. Some investigators recommend measuring the water content of autopsy blood and if necessary correcting the concentration of ethanol to a mean value of 80% w/w, which corresponds to fresh whole blood. Alcoholics often die at home with zero or low BAC and nothing more remarkable at autopsy than a fatty liver. Increasing evidence suggests that such deaths might be caused by a pronounced ketoacidosis. Recent research has focused on developing various biochemical tests or markers of postmortem synthesis of ethanol. These include the urinary metabolites of serotonin and non-oxidative metabolites of ethanol, such as ethyl glucuronide, phosphatidylethanol and fatty acid ethyl esters. This literature review will hopefully be a good starting point for those who are contemplating a fresh investigation into some aspect of postmortem alcohol analysis and toxicology.     

Potential for error when assessing blood cyanide concentrations in fire victims.

The present study explores toxicologic significance of blood cyanide concentrations in fire victims. Headspace gas chromatography was used for cyanide detection. Analysis of blood samples from ten fire victims (postmortem interval = 8 h to 3 to 5 d) detected zero to 11.9 mg/L of cyanide and a large difference in cyanide concentrations among victims. Carboxyhemoglobin (COHb) saturation was in the range of 24.9 to 84.2%. To examine the effects of methemoglobinemia and postmortem interval on blood cyanide concentrations in fire victims, an experiment was carried out using rabbits as the animal model. The rabbits were sacrificed by intramuscular injection of 1 mL/kg 2% potassium cyanide 5 min after intravenous injection of 0.33 mL/kg of 3% sodium nitrite (Group A, n = 3) or physiological saline (Group B, n = 6). Average methemoglobin contents immediately before potassium cyanide administration were 6.9 and 0.8% in Groups A and B, respectively. Average cyanide concentrations in cardiac blood at the time of death were 47.4 and 3.56 mg/L, respectively. When blood-containing hearts of the rabbits (n = 3 for Group B) were left at 46 degrees C for the first 1 h, at 20 to 25 degrees C for the next 23 h and then at 4 degrees C for 48 h, approximately 85 and 46% of the original amounts of blood cyanide disappeared within 24 h in Groups A and B, respectively. After the 72-h storage period, 37 and 10%, respectively, of the original amounts of cyanide remained in the blood. When the other three hearts in Group B were left at 20 to 25 degrees C for the last 48 h without refrigeration, cyanide had disappeared almost completely by the end of the experiment. The present results and those published in the literature demonstrate that the toxic effects of cyanide on fire victims should not be evaluated based solely on the concentration in blood.