乙醇在犬体内的死后再分布及保存人血液中稳定性的研究

发布时间：2018-01-23 11:56

本文关键词： 乙醇顶空气相色谱稳定性死后再分布自生醇类　出处：《山西医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的: 1建立生物样品中乙醇和自生醇类的直接进样和顶空进样气相色谱检测方法; 2建立乙醇的死后再分布、保存检材中稳定性和自生醇类的研究模型和方法; 3研究犬体内乙醇的死后再分布规律、保存检材中乙醇的稳定性和自生醇产生规律,为涉及酒精案件检材的采集、保存、检测和结果分析提供科学依据。方法: 1乙醇及自生醇的气相色谱检测方法 1.1直接进样气相色谱法:体液、组织(匀浆)离心后取上清,加入内标液,柱头直接进样,TRACE2000气相色谱仪(FID检测器,PEG融硅石英毛细管柱)检测乙醇及自生醇,保留时间定性,内标法定量。 1.2顶空气相色谱法:取血样于顶空瓶中,加入内标液及蒸馏水,平衡后自动顶空进样器进样,Agilent6820气相色谱仪(FID检测器,DB-5毛细管柱)检测乙醇及自生醇,保留时间定性,内标法定量。 2死后再分布研究 2.1动物模型犬6只,分别于10min内经胃管灌入3.5ml/kg剂量的乙醇,1.5h后CO_2处死。 2.2样品采集与处理实验犬的心电、血压和呼吸全部消失后,置于室温下,分别于死后0、2、4、8、12、24、48、72、96、120h取左心室血、左心房血、右心室血、右心房血、下腔静脉血、玻璃体液、胆汁、肝脏、脾脏、肺脏、肾脏、大脑和肌肉,直接进样气相色谱法检测乙醇及正丙醇含量,同时观察是否有自生醇产生。 3保存检材中乙醇的稳定性 3.1模型死亡12h内人尸体血液,经检测无自生醇后,用乙醇溶液添加成初始浓度分别为20.12、51.73、77.9、142.29、314.05mg/100ml的样本。 3.2样品保存与处理样本置于不同器皿(塑料袋,试管,注射器)中,分别于室温、4℃和—20℃中保存,在2、4、7、15、30天经顶空气相色谱法检测其中乙醇浓度。 4保存检材中自生醇的研究 4.1保存人血液中自生醇的研究离体模型:空白人尸体样本分别置于不同器皿(塑料袋,试管,注射器)中,室温、4℃和—20℃条件中保存,在2、4、7、15、30天经顶空气相色谱法检测乙醇和正丙醇含量,并观察其它自生醇的产生情况。 4.2犬体内自生醇的研究在体模型:犬3只,经CO_2处死,心电、血压和呼吸全部消失后,置于室温下,分别于死后0、2、4、8、12、24、48、72、96、120h取左心室血、左心房血、右心室血、右心房血、下腔静脉血、玻璃体液、胆汁、肝脏、脾脏、肺脏、肾脏、大脑和肌肉,每次取材后将肌肉,皮肤逐层缝合,尽量保持机体的完整性。上述检材经直接进样气相色谱法检测乙醇及正丙醇含量,并观察其它自生醇的产生情况。 5统计学方法采用SPSS11.5统计软件处理数据,结果以均数±标准差((?)±s)表示结果: 1气相色谱法本研究建立的直接进样和顶空气相色谱检测生物样本中乙醇及自生醇(甲醇、丙酮、正丙醇、异丙醇、正丁醇和异丁醇)方法,使乙醇和自生醇分离良好,线性范围为2～400mg/100ml,最低检出浓度是0.15mg/100ml,回收率均在95%以上。 2死后再分布研究 2.1分布犬经灌胃后1.5h,体内乙醇含量顺序为玻璃体液(373.04±51.64)＞左心室血(266.52±39.47)、左心房血(275.67±52.29)、右心室血(255.06±35.28)、右心房血(277.65±28.91)、下腔静脉血(264.01±25.06)、尿(234.82±162.18)＞肝脏(162.01±5.45)、脾脏(177.07±43.35)、肺脏(199.70±29.28)、肾脏(203.27±34.20)、大脑(214.47±22.47)＞胆汁(169.27±52.74)＞肌肉(152.54±27.93)。玻璃体液中乙醇含量为心血和下腔静脉中乙醇含量的1.41倍;胆汁与心血和下腔静脉中乙醇含量之比为0.67。 2.2死后再分布与0h相比,2、4、8、12、24、48、72、96h左心室、左心房、右心室、右心房血中乙醇浓度变化无统计学意义,120h时左心室、左心房血乙醇浓度显著升高,且均高于右心室和右心房血中乙醇浓度(P＜0.05)。玻璃体液中乙醇浓度在0～2h明显下降(P＜0.05),2～72h变化无统计学意义。0～120h肌肉中乙醇浓度变化无统计学意义。 3保存检材中乙醇的稳定性研究保存温度、器皿类型和初始浓度均对血样中乙醇浓度有不同程度的影响。不同初始浓度的变化趋势大致相同,15天后,初始浓度为314.05mg/100ml的样本变化趋势趋于降低,其他组变化微小。随着保存温度的升高,样本中乙醇浓度的下降速度增加。4℃保存时样品中乙醇浓度在15天内无显著变化(P＞0.05),而-20℃保存的样品中乙醇浓度的变化均无显著性差异。塑料袋保存血中的乙醇浓度在第7天时就出现了明显的下降,而其他容器中乙醇浓度的变化趋势基本一致,在前15天变化平稳,之后有小幅度的下降,但均无显著性差异。 4保存检材中自尘醇的研究 4.1保存人血液中自生醇的研究室温保存大部分样本第7天时有少量乙醇、异丙醇、异丁醇和丙酮生成,30天也只有个别样本中检测出正丙醇;4℃保存样本在第30天时可检测到少量的乙醇;-20℃保存样本在30天时仍未检测到任何自生醇, 4.2犬体内自生醇的研究大部分组织如心血、下腔静脉血、尿、胆汁、肝脏、脾脏、肺脏和肾脏在死后12h就有不同程度乙醇和正丙醇产生,其中,120h尿中乙醇浓度可以达104.92mg/100ml。肌肉,玻璃体液,大脑分别在死后48、72、96h检出乙醇。犬体内乙醇和正丙醇的生成存在正相关。结论: 1本研究建立了生物样品中乙醇和自生醇类直接进样和顶空气相色谱检测方法,可使乙醇和自生醇达到良好分离,灵敏度高,可应用于酒精中毒的法医学研究和法医学鉴定。 2醉酒剂量灌胃后1.5h,犬体内乙醇含量顺序为玻璃体液＞左心室血、左心房血、右心室血、右心房血、下腔静脉血、尿＞肝脏、脾脏、肺脏、肾脏、大脑＞胆汁＞肌肉。玻璃体液中乙醇含量与心血和下腔静脉乙醇含量之比为1.41;胆汁与心血和下腔静脉中乙醇含量之比为0.63。 3乙醇在灌胃犬体内可发生死后再分布现象,左心血受到很大影响,比较而言,玻璃体液、肌肉和脑组织受到死后再分布影响较少。且死亡犬体内会产生乙醇、正丙醇、丙酮和异丙醇。在酒精中毒案件的法医学鉴定中,应综合考虑死后再分布对体内酒精含量的影响。 4保存温度,器皿类型和初始浓度均对血样中乙醇浓度有不同程度的影响,其中保存温度和器皿类型的影响较为显著。而且除了保存温度与保存时间不存在交互作用以外,其它因素两两之间都存在交互作用。鉴定人在保存和检测样本过程中只有加强各个环节的质量控制,才能提供客观准确的检测数据及保证复查结果的有效性。 5在体模型和离体模型均会产生自生醇,温度对其影响较大。大部分组织如心血、下腔静脉血、尿、胆汁、肝脏、脾脏、肺脏和肾脏在死后12h就有不同程度的乙醇和正丙醇产生,其中,120h尿中乙醇浓度可以达104.92mg/100ml。肌肉,玻璃体液,大脑分别在死后48,72,96h检出乙醇,犬体内乙醇和正丙醇的生成存在正相关;人空白血样在-20℃放置30天未检出任何自生醇。在涉及乙醇的法医学检案中,特别是腐败样品,应注意自生醇的检测,尿中检出乙醇不能判定生前饮酒,应采集不易腐败的组织如玻璃体液、肌肉和大脑,检测其乙醇含量,并观察与正丙醇比例关系来进行综合判定。
[Abstract]:Objective:
1 the determination of ethanol and autogenic alcohols in biological samples by direct injection and headspace injection gas chromatography (GC) was established.
2 the post - death redistribution of ethanol was established, and the research models and methods of the stability and autogenic alcohols in the materials were preserved.
3, we studied the rule of postmortem redistribution of ethanol in dogs, preserved the stability of ethanol and the rule of spontaneous alcohol production, and provided a scientific basis for the collection, preservation, detection and result analysis of samples collected from alcohol cases.
Method:
Determination of 1 ethanol and autogenic alcohol by gas chromatography
1.1, direct injection gas chromatography: body fluid, tissue (homogenate) centrifugation, take the supernatant, add the internal standard solution, column head directly into the sample, TRACE2000 gas chromatograph (FID detector, PEG fused silica capillary column) to detect ethanol and autogenic alcohol, the retention time is qualitative, internal standard method is quantitative.
1.2 headspace gas chromatography: blood samples were taken from headspace bottles, added the internal standard solution and distilled water. After balanced, the automatic headspace sampler was injected. The Agilent6820 gas chromatograph (FID detector and DB-5 capillary column) were used to detect ethanol and autogenic alcohol, the retention time was qualitative, and the internal standard method was used for quantitative analysis.
Postmortem redistribution study of 2
2.1 animal model
In 6 dogs, 3.5ml/kg was injected into the gastric tube in 10min, and CO_2 was executed after 1.5h.
2.2 sample collection and treatment
Dogs ECG, blood pressure and respiration all disappeared after being placed at room temperature, respectively, after the death of 0,2,4,8,12,24,48,72,96120h left ventricular blood, left atrial blood, right ventricular blood, right atrial blood, inferior vena cava blood, vitreous fluid, bile, liver, spleen, lung, kidney, brain and muscle, chromatography determination of ethanol and n-propanol content directly into the sample gas, and observe whether there is self generated alcohol.
3 the stability of ethanol in the stored material
3.1 model
After the autogenic alcohol was detected in the dead human body of 12h, a sample of 20.12,51.73,77.9142.29314.05mg/100ml was added to the initial concentration by the ethanol solution.
3.2 preservation and treatment of samples
Samples were placed in different containers (plastic bags, tubes, syringes), respectively at room temperature, save 4 DEG C and - 20 DEG C, in 2,4,7,15,30 days the top air chromatography of the ethanol concentration.
Study on the preservation of autogenic alcohol in 4 samples
4.1 study of autogenic alcohol in human blood
In vitro human cadaveric model: blank samples were placed in different containers (plastic bags, tubes, syringes), room temperature preservation, 4 C and 20 C and conditions, in 2,4,7,15,30: Headspace Gas Chromatography Determination of ethanol and n-propanol content, and observe the other authigenic alcohols produced conditions.
Study on autogenic alcohol in 4.2 dogs
In the model: the 3 dogs were sacrificed by CO_2, ECG, blood pressure and respiration all disappeared after being placed at room temperature, respectively, after the death of 0,2,4,8,12,24,48,72,96120h left ventricular blood, left atrial blood, right ventricular blood, right atrial blood, inferior vena cava blood, vitreous fluid, bile, liver, spleen, lung and kidney. The brain and muscles, each biopsy after muscle, skin sutured, try to keep the integrity of the body. The samples by direct injection gas chromatographic method for the determination of ethanol and n-propanol content, and observe the other self generated alcohol production.
5 statistical method
SPSS11.5 statistical software is used to deal with data, and the results are expressed in mean + + standard deviation ((?) + s)
Result:
1 gas chromatography
The detection of alcohol and self generated alcohol in biological sample directly into the sample and headspace gas chromatography was established in this study (methanol, acetone, isopropanol, n-propanol, n-butanol and isobutanol) method, ethanol and self generated alcohol were good. The linear range is 2 ~ 400mg/100ml, the minimum detectable concentration is 0.15mg/100ml, the recovery rate were more than 95%.
Postmortem redistribution study of 2
2.1 distribution
The dog after intragastric administration of 1.5h, in order for the ethanol content of vitreous humor (373.04 + 51.64), the left ventricular blood (266.52 + 39.47), left atrial blood (275.67 + 52.29), right ventricular blood (255.06 + 35.28), right atrial blood (277.65 + 28.91), inferior vena cava blood (264.01 + 25.06) urine, (234.82 + 162.18), liver (162.01 + 5.45), spleen (177.07 + 43.35), lung (199.70 + 29.28), kidney (203.27 + 34.20), brain (214.47 + 22.47), bile (169.27 + 52.74), muscle (152.54 + 27.93). The content of ethanol in the vitreous of 1.41 double heart and inferior vena cava blood alcohol content in blood and bile; ethanol content and ratio of 0.67. in inferior vena cava
2.2 postmortem redistribution
Compared with 0h, 2,4,8,12,24,48,72,96h, left ventricle, left atrium, right ventricle, right atrium blood alcohol concentration change was not statistically significant, left ventricular 120h, left atrial blood ethanol concentration was significantly increased, and the ethanol concentration was higher than that of the right ventricle and right atrium blood (P < 0.05). The concentration of ethanol in the vitreous fluid decreased significantly in 0 ~ 2H (P < 0.05), no significant changes in 2 ~ 72h ~.0 no significant change of ethanol concentration 120h muscle.
Study on the stability of ethanol in 3 stored samples
All types of vessels preservation temperature, and initial concentration have different effects on blood ethanol concentration. The change trend of different initial concentrations are roughly the same, 15 days after the initial concentration of sample 314.05mg/100ml change trend tends to decrease, the other group of small changes. With increasing storage temperature, ethanol concentration in the sample rate of decline increased.4 when the samples stored at the ethanol concentration in 15 days no significant change (P > 0.05), but there was no difference in ethanol concentration in the samples stored at -20. The concentration of ethanol in blood preservation bags appeared significantly decreased at day seventh, and the change trend of ethanol concentration in other container consistent changes smoothly in the first 15 days, after a small drop, but there was no significant difference.
Study on self dust alcohol in 4 preservation materials
4.1 study of autogenic alcohol in human blood
Most of the samples stored at room temperature for seventh days when there is a small amount of ethanol, isopropanol, isobutanol and acetone, 30 days only individual samples detected propanol; 4 C preservation sample can detect small amounts of ethanol on the thirtieth day; -20 samples stored at 30 days has not detected any self generated alcohol,
Study on autogenic alcohol in 4.2 dogs
Most organizations such as blood, urine, inferior vena cava blood, bile, liver, spleen, lung and kidney after the death of 12h have different degrees of ethanol and n-propanol, among them, 120h urine concentration of ethanol can reach 104.92mg/100ml. muscle, vitreous humor, brain 48,72,96h respectively after death. There was a positive correlation detection of ethanol formation in dogs ethanol and n-propanol.
Conclusion:
1, a direct injection and headspace gas chromatographic method for the determination of ethanol and autogenic alcohols in biological samples is established. The ethanol and self generated alcohols can be well separated, and the sensitivity is high. It can be applied to the forensic research and forensic identification of alcoholism.
2 drunken dose after intragastric administration of 1.5h dogs ethanol content order of vitreous, left ventricular blood, left atrial blood, right ventricular blood, right atrial blood, inferior vena cava blood, urine, liver, spleen, lung, kidney, brain, bile, muscle. In the vitreous fluid and blood alcohol content of ethanol and inferior vena cava the content ratio is 1.41; ethanol content of bile and blood and inferior vena cava in the ratio of 0.63.
3 ethanol in the postmortem redistribution phenomenon can be poured in the stomach of dog, left heart has been greatly affected, in comparison, vitreous fluid, muscle and brain tissue from postmortem redistribution is less affected. And death dogs will produce ethanol, propanol, acetone and isopropanol. In the case of alcoholism in forensic identification that should consider the postmortem redistribution effect on alcohol content.
4 types of vessels were temperature and initial concentration have different effects on blood alcohol concentration, the effect of temperature and the type of vessel is significant. In addition to storage temperature and storage time of interaction does not exist outside, there are other factors. The interaction between the 22 experts in the preservation and detection process only samples to strengthen the quality control of each link, in order to provide objective and accurate detection data and guarantee the validity of the review results.
5 in vivo and in vitro model model will produce self generated alcohol, temperature has more effect on it. Most tissues such as heart, inferior vena cava blood, urine, bile, liver, spleen, lung and kidney after the death of 12h have different levels of ethanol and n-propanol, the ethanol concentration can reach 120h in urine 104.92mg/100ml. muscle, vitreous humor, brain 48,72,96h detected ethanol after death respectively, are related to the generation of dogs ethanol and n-propanol; blank blood samples at -20 DEG C for 30 days were not detected in any spontaneous alcohol. Histologic examination involved in ethanol forensic case, especially corruption samples should be tested in situ alcohol could not determine his drinking, alcohol detection in urine should be collected is not easy to corrupt organizations such as vitreous fluid, muscle and brain, to detect the content of ethanol, and to observe the relationship with n-propanol in proportion to the comprehensive judgment.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：D919

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