Forensic medical significance of enzymatic products of ethanol oxidation in cadaveric brain

The concentrations of ethanol, acetaldehyde, and the oxidizing enzymes alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AIDH) were measured in neuronal cytoplasm, limbic cortical capillaries, and cardiovascular center of the medulla oblongata. The measurements were carried out by histochemical methods, gas-liquid chromatography, etc. The results were processed with consideration for the degree and stage of ethanol intoxication in case of death from ethanol poisoning and asphyxia in hanging. Increase of ethanol concentration in the blood was associated with a decrease and then increase in the brain concentrations of ADH and with an increase of AIDH concentration. Enzymatic changes predominated in capillary walls; the minimum shifts were observed in the neuronal cytoplasm of the cerebral limbic cortex, which confirms the neurohumoral nature of detoxication regulation. Lethal ethanol poisoning could occur during any stage of ethanol intoxication. The detected changes in ethanol, acetaldehyde, and metabolizing oxidoreductases in brain tissue can be used for forensic medical diagnosis of ethanol poisoning.     

Histochemical changes in the hypothalamus, hypophysis, and adrenal glands observed in ethanol poisoning

A comprehensive morphological-and-histochemical study of neuroendocrinal internals in cases of ethanol poisonings was undertaken. Actual forensic medical materials were used (62 cadavers) to make morphometry examinations of the hypothesis and adrenal glands. Besides, the distribution of alcohol dehydrogenase and acetaldehyde dehydrogenase was investigated in the mediatory differential brain sections, i.e. cerebellum, locus coeruleus, dorsal raphe nucleus, hypothalamus and adrenal glands. A differential distribution of ethanol-oxidizing enzymes as well as their changes in ethanol lethal poisoning were established; additionally, a variety of morphological signs were defined, which enable the differential diagnosis of a death reason in acute alcoholic intoxication.     

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.     

Morphological signs of ethanol poisoning, alcohol abstinence and chronic alcoholic intoxication in the mesocorticolimbic dopaminergic system

Forensic medical diagnostics of ethanol poisoning, alcohol abstinence, and chronic alcoholic intoxication of the mesocorticolimbic dopaminergic system remains an unresolved problem and encounters difficulties. This situation is due not only to the marked vulnerability of the neurons of the dopaminergic system but also to the fact that its mechanisms are poorly understood. The objective of the present work was to substantiate and develop diagnostic criteria for ethanol poisoning, alcohol abstinence, and chronic alcoholic intoxication of the neurons both in the mesocorticolimbic dopaminergic system and in other brain regions. The object of the study was the brain of healthy adult subjects who died from alcohol intoxication (in the period of ethanol resorption) and under conditions of alcohol abstinence (completion of the abstinence course). The purpose of the study was to elucidate factors responsible for the different degree of damage to the neurons of various identification groups (intact, hypochromic, picnomorphic, shadow) and macrogliocytes. The cells of all these types were counted at an area of 0.25 sq. mm within 4 squares each having a side of 250 mcm in length. The absolute and relative number of neurons in each group as well as the number of polyneuronal satellite cells per one intact neuron was determined. It was shown that alcohol intoxication is associated with acute swelling of and severe damage to brain neurons caused by the combination of such factors as toxic effect of ethanol, excessive production of catecholamines, and functional overstrain of dopaminergic neurons. The severity of acute alcohol damage to the neurons decreased with the distance from the mid-brain dopaminergic nuclei. Restoration of neurons during alcohol abstinence was due to compensatory activation of interactions between neurons and glial cells. It decreased in the sequence from the paranigral nucleus of the ventral portion of mesencephalic tegumentum to the medial portion of the accumbence nucleus (field 24b, layer III of field la, layer V of field 1) depending on the initial severity of acute damage in the brain region being examined. The severity of damage to the neurons of the mesocorticolimbic dopaminergic system under conditions of chronic alcohol intoxication estimated from the number of shadow neurons was similar to the degree of acute swelling associated with ethanol poisoning and decreases from a maximum in the nuclei of the mesocorticolimbic dopaminergic system to a minimum in layer III of field 1.     

Automated headspace solid-phase microextraction and capillary gas chromatography analysis of ethanol in postmortem specimens

Solid-phase microextraction (SPME) is a relatively new solventless sample preparation technique that allows simultaneous sampling, extraction, pre-concentration, and introduction of analytes from a sample matrix in a single procedure. This methodology has been used for the analysis of several drugs of forensic toxicology interest including volatile compounds. This paper describes a methodology for analysis of ethanol and other volatile compounds using automatic headspace solid-phase microextraction (HS-SPME) and capillary gas chromatography in postmortem specimens. The methodology was initially developed using standard solutions of acetaldehyde, acetone, methanol, and ethanol. Isobutanol was used as internal standard. Postmortem samples of blood, urine, and vitreous humor were obtained during medico-legal autopsies. To date, there are no published paper regarding alcohol analysis in vitreous humor specimens using HS-SPME and limited literature analyzing blood and urine samples. HS-SPME analysis showed that, under optimized conditions, ethanol and isobutanol (internal standard) were well-separated from other volatile compounds such as acetaldehyde, acetone, and methanol considered to be potential interferents in ethanol analysis. The calibration curves for each volatile compound demonstrated good linearity throughout the concentration range from 0.001 to 1.0 g/dl and the detection limit of ethanol in the studied specimens was approximately 0.0001 g/dl.     

Acetaldehyde accumulation during headspace gas-chromatographic determination of ethanol

The acetaldehyde accumulation in blood during the equilibration time of samples processed by gas-liquid chromatographic headspace technique is reported. The quantitative loss of blood ethanol concentration is recorded at various temperatures and times of the preheating process.When the equilibration process is performed at 40 °C, minimal errors, due to ethanol oxidation, are observed.     

Acute ethanol poisoning: a two-year study of deaths in North Carolina

A 24-month study of fatalities in North Carolina with high blood ethanol levels (300 mg/100 ml or over) revealed 502 cases with either acute alcoholism or the effects of this range of blood ethanol concentration having caused or contributed to death. This investigation reassessed the criteria for ethanol poisoning, including its cause and manner of death, and revealed recurrent patterns common to this syndrome. This inquiry also contrasted the frequency of ethanol poisoning in different areas of the country.     

Effects of profound hypothermia on the blood-brain barrier permeability in acute and chronically ethanol treated rats

This study examines the effects of profound hypothermia on the blood-brain barrier (BBB) permeability in ethanol administrated rats. Vascular permeability to intravenously injected Evans blue (EB) was quantitatively examined in the brain regions of rats. Rats were treated with ethanol acute and chronically. Rectal temperature of rats was dropped into 20+/-1 degrees C during profound hypothermia. Mean arterial blood pressure in both acute and chronic ethanol treatments plus hypothermia significantly dropped into low levels as well as in hypothermia alone (P<0.01). Hypothermia led to a significant increase in the content of EB dye in the brain regions of rats (P<0.05). Both acute and chronic ethanol treatments plus hypothermia did not lead to a significant increase in the BBB permeability against intravenously injected EB dye. We conclude that ethanol intake protects the BBB against the effects of hypothermia.     

Experimental studies on the mechanism of ethanol formation in corpses

Various in vitro experiments were performed for the purpose of clarifying the mechanism of ethanol production in corpses. Whereas a negligible quantity of ethanol was produced in the blood alone, which was left at room temperature, the quantity of ethanol was slightly increased by addition of glucose to the blood. When saprogens were further added, the quantity was markedly increased. Various materials were added to blood-liver homogenates as specimens, and the mixtures were stored in an incubator at 37 degrees C. As a result of the addition of an antibiotic to the mixture every day, there was hardly any production of ethanol. When alcohol dehydrogenase (ADH) and reduced nicotinamide adenine dinucleotide (NADH) were added, ethanol production was slightly increased. When acetaldehyde was added first, ethanol production was inhibited the next day, but on and after day 2, the quantity of ethanol was more than that in the control material. When pyruvic acid was added first, the results were similar to the above. Pyrazole, cyanamide, and disulfiram completely inhibited the production of ethanol. Ethanol production in corpses is believed to take place through a pathway opposite to that of ethanol metabolism in the living body, under the influence of ADH, ALDH, etc., in saprogens using carbohydrates as substrates.     

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.     

Biochemical pathways generating post-mortem volatile compounds co-detected during forensic ethanol analyses

In this contribution are presented the fermentations of the main substrates present in a decaying corpse, namely carbohydrates, amino acids, glycerol and fatty acids, generating the post-mortem volatile compounds that could be detected along with ethanol during the forensic ethanol analysis. The available literature (preferably reviews) on microbial metabolic pathways (enzymes, substrates, conditions) that are implicated in the formation of these volatiles has been reviewed. The microbial formation of the following volatiles is supported by the presented biochemical data: ethanol, acetaldehyde, acetone, 2-propanol, 1-propanol, 1-butanol, isobutanol, isoamyl alcohol, d-amyl alcohol, acetate, propionate, butyrate, isobutyrate and ethyl esters (mainly ethyl acetate). The extracted information was correlated with the existing forensic literature on the post-mortem detected volatiles. The significance of the microbial produced volatiles on the selection of an appropriate internal standard for the ethanol analysis has been considered. Finally, the possible contribution of the presence of volatiles in the interpretation of ethanol analysis results in post-mortem cases is discussed.     

Urine/blood ratios of ethanol in deaths attributed to acute alcohol poisoning and chronic alcoholism

The concentrations of ethanol were determined in femoral venous blood (BAC) and urine (UAC) and the UAC/BAC ratios were evaluated for a large case series of forensic autopsies in which the primary cause of death was either acute alcohol poisoning (N=628) or chronic alcoholism (N=647). In alcohol poisoning deaths both UAC and BAC were higher by about 2g/l compared with chronic alcoholism deaths. In acute alcohol poisoning deaths the minimum BAC was 0.74 g/l and the distribution of UAC/BAC ratios agreed well with the shape of a Gaussian curve with mean+/-standard deviation (S.D.) and median (2.5th and 97.5th centiles) of 1.18+/-0.182 and 1.18 (0.87 and 1.53), respectively. In alcoholism deaths, when the BAC was above 0.74 g/l (N=457) the mean+/-S.D. and median (2.5th and 97.5th centiles) UAC/BAC ratios were 1.30+/-0.29 and 1.26 (0.87 and 2.1), respectively. When the BAC was below 0.74 g/l (N=190), the mean and median UAC/BAC ratios were considerably higher, being 2.24 and 1.58, respectively. BAC and UAC were highly correlated in acute alcohol poisoning deaths (r=0.84, residual S.D.=0.47 g/l) and in chronic alcoholism deaths (r=0.95, residual S.D.=0.41 g/l). For both causes of death (N=1275), the correlation between BAC and UAC was r=0.95 and the residual S.D. was 0.46 g/l. The lower UAC/BAC ratio observed in acute alcohol poisoning deaths (mean and median 1.18:1) suggests that these individuals died before absorption and distribution of ethanol in all body fluids were complete. The higher UAC/BAC ratio in chronic alcoholism (median 1.30:1) is closer to the value expected for complete absorption and distribution of ethanol in all body fluids.     

The significance of ethanolemia for the diagnosis of death from acute ethanol poisoning

Foci of myolysis of cardiac muscle fibers are suggested to be used for evaluation of thanatogenetic significance of ethanol concentration in cadaveric blood. This sign of acute ethanol poisoning is absent in case of other cause of death in a state of ethanol intoxication, even in the presence of high ethanolemia. Therefore, foci of myolysis are a sign of ethanol tolerance.     

Crystallization of cerebrospinal fluid in cases of death from ischemic heart disease and ethanol poisoning

Crystallographic analysis of the cerebrospinal fluid (CSF) was carried out in 18 cases of death from coronary disease and 19 cases of death from ethanol poisoning. The crystallograms were evaluated visually and by the stereoscopic picture. Specific features of the CSF crystal colonies growth in subjects dead from the above conditions are determined.     

慢性乙醇中毒小鼠脑神经细胞IP3R1表达的变化

目的研究慢性乙醇中毒引起小鼠脑神经细胞Ⅰ型1,4,5-三磷酸肌醇受体（IP3R1）表达的变化。方法 40只小鼠随机分为90d、180d组,各组再分为正常对照组、10%、20%、30%乙醇组,每组5只,乙醇组给予相应浓度乙醇饮用至相应时间;取各组小鼠脑组织,分别采用免疫组化和Western blot方法检测脑皮质神经细胞IP3R1表达的变化;SPSS 13.0软件对实验数据进行单因素方差分析。结果正常IP3R1免疫组化染色物分布于神经细胞胞浆内。90d组随乙醇浓度的增加,IP3R1免疫组化阳性细胞数和阳性细胞率逐步增加,组间比较,差异显著（P〈0.05）;180d组中10%、20%乙醇组阳性细胞数和阳性细胞率逐步增加,组间比较,差异显著（P〈0.05）,而30%乙醇组阳性细胞数和阳性细胞率反而减少,且低于90d组的相同浓度组（P〈0.05）。Western blot与免疫组化检测结果基本一致。结论慢性乙醇中毒可引起小鼠大脑皮质神经细胞IP3R1表达增加,而高浓度（30%）、长时间（180d）乙醇使IP3R1表达降低,可能与神经细胞变性、坏死、数目减少有关。     

The unreliability of using a urine ethanol concentration to predict a blood ethanol concentration

Of approximately 5,000 forensic cases with a positive ethanol result, over 1,000 were available in which both blood and urine were present for comparison of ethanol content. Data were examined for calculation of the urine to blood ethanol concentration ratio, with the intent of evaluating the validity of predicting a blood ethanol level given a urine ethanol level. The overall urine to blood ethanol concentration ratio was 1.57:1 with a range of 0.7 to 21.0:1. The extremely wide range of values implies that a large degree of error would be introduced if a mean ratio was used when predicting a blood ethanol level from a urine ethanol level.     

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...     

Simulation of parameters of alcohol-oxidizing enzyme systems and pathomorphological characteristics in situations of acute ethanol poisoning and ischemic heart disease

Morphometric characteristics and forensic chemical information used in diagnostics of acute ethanol intoxication and coronary heart disease in conjunction with macro- and microscopic pathomorphological signs of the changes in the heart, liver, and kidneys provide data that may suggest the presence of pathology but do not permit to reliably identify it. In this context, evaluation of activities of alcohol-oxidizing enzyme systems acquires clinical significance. The analysis of correlations between quantitative parameters supplemented by the construction of binary models allows to objectively interpret the conclusions about the cause of death in each concrete case of acute ethanol poisoning and coronary heart disease.     

气相色谱法同时测定血清中甲醇、乙醇、正丙醇

目的建立气相色谱同时测定血清中甲醇、乙醇、正丙醇含量的方法。方法改变气相色谱条件,以异戊醇为内标,采用气相色谱一氢火焰离子化检测器对血清直接进行检测。并通过待测组分与内标物的响应值比进行定量。结果GC／FID法检测血清中的甲醇、乙醇、正丙醇含量,得到了良好的线性关系。乙醇浓度从1～100mg／100ml。的线性关系式为Y=0．4145X＋0．0232（R2=0．9974）、浓度从100～1000mg／100mL的线性关系式为Y=0．4511X＋0．0746（R2=0．9911）,甲醇浓度从l-200mg／100mL的线性关系式为Y=0．2778X＋0．0493（R2=0．9983）。结论该方法操作简便快速,重现性好,通过检测正丙醇还可以推断腐败血样自身产生的乙醇量,是一种较为理想的血醇检测方法。