Transferrin polymorphism detected in human urine using isoelectric focusing followed by immunoblotting

Genetic polymorphism of transferrin (TF) was revealed in human urine by isoelectric focusing and immunoblotting on thin-layer polyacrylamide gels. Using this technique more than 300 urine samples were examined, and correct TF typing from a small volume of urine (approx. 0.5 ml) was achieved, in comparison with the results of direct grouping for plasma. Three common phenotypes, TF C1, C2-1 and C2, were differentiated. In addition, the rare types TF C1D, C2D, and C1B were observed. The frequencies of the TF alleles in our samples were found to be: TF*C1 = 0.7265, TF*C2 = 0.2624, TF*D = 0.0083 and TF*B = 0.0028.     

用超薄层聚丙烯酰胺凝胶等电聚焦法调查西藏藏族群体GC亚型频率报道

作者采用超薄层聚丙烯酰胺凝胶等电聚焦法检测了142例西藏地区无关藏族青年男女GC亚型,发现6例变异型,其中2例1A变异;1例1c变异;3例1F1S-2变异,这种新的变异型,迄今国内外尚未见报道。GC亚型的基因频率分别为:GC*1F=0.3921、GC*1S=0.3705、GC*2=0.2266、GC*1A=0.0072、GC*1C=0.0036。个人识别机率为0.8079;非父排除率为0.3430。     

尿中氯胺酮及其代谢物检测的研究

目的建立氯胺酮滥用者尿中氯胺酮及其代谢物检测方法。方法尿液用有机溶剂液-液萃取,气相色谱/氮磷检测器、电子捕获检测器、氢火焰检测器和气-质联用仪测定。结果确认了尿液中氯胺酮的主要代谢物,尿液中氯胺酮及去甲氯胺酮的最小检测限均为2ng/mL,脱氢去甲氯胺酮的最小检测限为5ng/mL。结论所建方法快速、灵敏、准确,能够满足氯胺酮滥用者尿液检测的需要。     

Benzodiazepine findings in blood and urine by gas chromatography and immunoassay

Gas chromatography (GC) and immunoassay techniques applied to blood and urine specimens were compared for the screening of benzodiazepines in postmortem forensic toxicology. Five hundred and six such successive postmortem cases in which both urine and peripheral blood was sent for toxicological analysis by the medical examiners were selected. The urine specimens were tested by the Emit((R)) d.a.u. Benzodiazepine Assay, and in parallel, the blood and urine specimens were screened for benzodiazepine drugs and their metabolites by an established automated dual-column GC method. The lowest number of positives (153) was obtained when immunoassay was performed without enzyme hydrolysis. When urine samples were hydrolysed before immunoassay, the number of positives increased to 175. The highest number of positives (200) was obtained in urine by GC, and the screening of blood by GC yielded 185 quantitative results. Despite the urine GC screening produced the most positives, the quantitative screening of the blood by GC appears to be the most efficient approach in postmortem forensic toxicology, considering the fact that although urine findings confirm the presence of the drug, quantitative results in urine are irrelevant to acute toxicity.     

Genetic polymorphisms of orosomucoid ORM1 and ORM2 in a Japanese population: occurrence of new ORM1 alleles

Genetic polymorphisms of orosomucoid ORM1 and ORM2 in a Japanese population from northern Japan were investigated using isoelectric focusing (IEF) in ultrathin layer polyacrylamide gels containing Triton X-100 and immunofixation. Nine ORM1 phenotypes which are determined by four common and one rare alleles were observed. Two of the identified alleles at this locus were considered to be new. The ORM2 pattern was classified into 14 phenotypes as products of one common and two variant alleles. The estimated allele frequencies were ORM1*1 = 0.668, ORM1*2 = 0.170, ORM1*2.1 = 0.136, ORM1*5.2 = 0.022 and ORM1*7 = 0.004; ORM2*1 = 0.972, ORM2*3 = 0.006 and ORM2*6 = 0.022.     

大鼠血液、尿液中阿米替林的气相色谱快速分析

目的建专：m液及尿液中阿米替林（AMTL）的气相色谱分析方法,、方法以正常大鼠m液及尿液为空F1样奉,分别添加AMTI-标准品和内标SKF525A。实验大鼠以AMTL2倍LD50灌胃,致大鼠急性中毒后提取血液及尿液。用乙醚提取样本中AMTL,采用GC／FID法进行定量分析,并考察实验条件,结果采用该方法,血液及尿液中AMTL线性池用分别为5～150μg／mL（r=0．993）和5～150μg／mL（r=0．998）;最低检测限（S／N／〉3）均为1．0陆g／mL;口内、口间精密度均小于6％,同收率存95.5％～105．6％之间。结论该方法方操作便捷、捧确度高,适用=fAMTL临床治疗中血药浓度快速监测和法医毒物分析鉴定。     

尿中MDMA及其代谢物的GC和GC／MS分析

考察MDMA在人体内的代谢以及建立尿中MDMA和体内主要代谢物MDA的分析方法。尿样水解后经液-液提取处理,用GC／MS（EI、PCI）和GC／FID法分析。人摄入MDMA后尿中MDA和原体MDMA比约为0．10～0．14。GC／MS／SIM和GC／FID法的最低检出限为2ng／ml和50ng／ml,回收率大于85％,变异系数小于10％。该法简便快速、灵敏度高、结果可靠,可用于MDMA滥用者的尿样鉴定。MDA／MDMA浓度比可作为评判毒分结果的参考指标。     

Routine phenotyping of native and desialyzed alpha 2HS-glycoprotein from blood stains

The application of a polyacrylamide gel isoelectric focusing (PAGIEF) and immunoblotting procedure for the identification of native alpha 2HS-glycoprotein (AHSG) in routine casework blood stains has produced reportable results on 57.2% of samples. This reporting rate is lower than that for group specific component (GC) (83.8%) and phosphoglucomutase (PGM 1) (72.8%) phenotyping of the same samples. Blood stain samples were desialyzed with 1 U/ml neuraminidase, overnight at room temperature prior to PAGIEF in gels containing pharmalyte pH 5-6 and 2.5 M urea. Simple AHSG patterns were developed by immunoblotting. This procedure was five times as sensitive as the native AHSG method and desialyzation was reproducible over a range of incubation times and neuraminidase concentrations. The application of the desialyzed AHSG analysis to routine casework samples has resulted in a significant increase in the number of reportable results (762 reported out of 1027 samples). This reporting rate (74.2%) compares favourably with that for GC (79.1%) and PGH 1 (69.6%) phenotyping of the same samples. The three AHSG alleles (AHSG*1, 2 and 3) are clearly resolved after sample desialyzation and separation in gels containing pharmalyte pH 5-6 and 2.5 M urea. The sensitivity of desialyzed AHSG phenotyping approaches that of GC and this technique is worthy of inclusion in blood stain screening protocols of forensic laboratories in regions where the population has a limited range of rare AHSG alleles.     

Plasminogen (PLG): a useful genetic marker for paternity examinations

The genetically determined polymorphism of plasminogen (PLG) was analyzed by isoelectric focusing on polyacrylamide gels. For analysis neuraminidase-pretreated sera were used. PLG was developed functionally by activation with urokinase and subsequent lysis of casein in an agar overlay. In a random sample of 957 unrelated healthy individuals from Southern Germany, three common phenotypes, PLG1, 2-1, and 2, and five rare variants were found. The allele frequencies were: PLG*1 = 0.7174, PLG*2 = 0.2780, and PLG*Var = 0.0046. The theoretical exclusion rate in cases of disputed paternity is 16.5%.     

气相色谱法快速分析人血、尿中地芬尼多

目的建立人血、尿中地芬尼多的气相色谱快速分析方法。方法用氯仿提取血、尿中的地芬尼多,采用气相色谱法进行定性、定量分析。以正常人血浆、尿液为空白样本,分别添加标准地芬尼多及SKF525（内标物）对方法进行考察和优化,并对1例大剂量误服地芬尼多中毒者体液浓度进行快速测定和检测。结果所建方法分析血、尿中地芬尼多的线性范围均为5.0～200.0μg/mL;最低检测限均为1.0μg/mL（S/N≥3）;日内、日间精密度RSD≤5.6%（n=5）;回收率：血≤（106.23±2.05）%;尿≤（104.19±5.51）%。结论该分析方法操作便捷、实用,适用于地芬尼多的临床血药浓度快速监测和法医毒物鉴定。     

Detection of bis(2-chlorethyl)-sulfide (Yperite) in urine by high resolution gas chromatography-mass spectrometry

Bis(2-chlorethyl)-sulfide (Yperite) could be detected by GC/MS in urine samples, concentration ranging from 1 to 30 ppb. These results were supported by high resolution mass spectral data.     

中毒致死者体内芬氟拉明的GC/NPD,GC/MS法测定

建立了生物检材中芬氟拉明的定性定量分析方法。体液及脏器组织经有机溶剂提取后 ,用GC/MS法进行药物筛选、定性 ,生物检材中的芬氟拉明浓度用4 -苯丁胺作内标、GC/NPD法测定。测得芬氟拉明中毒致死者的血液、尿液、肝等组织中浓度分别为7.8μg/ml、64.2μg/ml、31.3μg/g。并对尸体解剖所见及方法可行性进行讨论     

中国人群补体C_1R的遗传多态性

采用聚丙烯酰胺等电聚焦电泳，结合免疫印迹技术，对中国辽宁地区360名无关个体的补体C1R遗传多态性进行了研究。共检出6种常见表现型和4种变异型。基因频率C1R*1=0．5181，C1R*2=0．3291，C1R*3=0．1472，CIR*R=0．0056，分布符合Hardy-Weinberg法则。C1R的血型鉴别机率（DP值）为0．7694，是一种具有高度鉴别能力的血清多态性遗传标记。     

C6 phenotyping in sera and bloodstains by isoelectric focusing followed by electroimmunoblotting

The genetic polymorphism of C6 was investigated in 329 unrelated Japanese individuals using isoelectric focusing in polyacrylamide gels followed by an electroimmunoblotting technique. Besides six common phenotypes C6 A, AB, B, AB2, BB2 and B2, six rare variants were observed. The allele frequencies were: C6*A = 0.4422, C6*B = 0.4757, C6*B2 = 0.0714, C6*A3 = 0.0015, C6*M1 = 0.0046 and C6*B3 = 0.0046. The population data confirmed that the C6*B2 allele is the third common allele characterizing Japanese. The present electroimmunoblotting technique was applied to demonstrate C6 types in dried bloodstains. The C6 types were determined from bloodstains stored at 4 degrees C for up to 10 weeks, at room temperature for up to 2 weeks and at 37 degrees C for up to 4 days. The results show that this component system offers a new powerful means for the medico-legal grouping of bloodstains.     

Polymorphism of EsD by isoelectric focusing: description of the new allele EsD*Kofu and phenotyping in bloodstains

The polymorphism of EsD was investigated in 1115 unrelated Japanese individuals by isoelectric focusing. Besides the three common phenotypes two heterozygotes EsD 7-1 and EsD 7-2 were observed. The gene frequencies were: EsD*1 = 0.6234, EsD*2 = 0.3663, and EsD*7 = 0.0103. In addition, a rare variant was detected in a probandus living in the city of Kofu. The family analysis suggested the hereditary occurrence of a new allele EsD*Kofu. The isoelectric focusing method was successfully applied to phenotyping EsD in bloodstains; each phenotype was demonstrated at 37 degrees C for up to 2 weeks, at room temperature for up to 9 weeks, and at 4 degrees C for over 20 weeks after stain formation.     

Simultaneous analyses of cocaine, cocaethylene, and their possible metabolic and pyrolytic products

A method was developed for simultaneously analyzing cocaine (COC), benzoylecgonine (BZE), norbenzoylecgonine (BNE), norcocaine (NCOC), ecgonine (ECG), ecgonine methyl ester (EME), m-hydroxybenzoylecgonine (HBZE), anhydroecgonine methyl ester (AEME), cocaethylene (CE), norcocaethylene (NCE), and ecgonine ethyl ester (EEE) in blood, urine, and muscle. Available deuterated analogs of these analytes were used as internal standards. Proteins from blood and muscle homogenate were precipitated with cold acetonitrile. After the removal of acetonitrile by evaporation, the supernatants and urine were subjected to solid-phase extraction. The eluted analytes were converted to their hydrochloride salts and derivatized with pentafluoropropionic anhydride and 2,2,3,3,3-pentafluoro-1-propanol. The derivatized products were analyzed by a gas chromatograph (GC)/mass spectrometer by selected ion monitoring. The limit of detection (LOD) for COC, BZE, NCOC, EME, CE, NCE, and EEE was 2ng/ml, while the LODs for BNE, ECG, HBZE, and AEME were 25, 640, 50, and 13 ng/ml, respectively. This method was successfully applied in analyzing 13 case samples from aviation accident pilot fatalities and motor vehicle operators. AEME concentrations found in the 13 samples were consistent with those produced solely by the GC inlet pyrolysis of COC controls in blood. Anhydroecgonine cannot be used as a marker for the abuse of COC by smoking because it is also pyrolytically produced from COC metabolites on the GC inlet. The developed method can be effectively adopted for analyzing COC and related compounds in urine, blood, and muscle by a single extraction with increased sensitivity through formation of hydrochloride salts and using a one-step derivatization.     

Isomeric amphetamines--a problem for urinalysis?

Alkyl amphetamine isomers (amphetamine, 1-phenyl-2-butylamine (PBA), methamphetamine, N-methyl-PBA, N,N-dimethylamphetamine, N-ethylamphetamine, N-ethyl-PBA and N,N-diethylamphetamine) were purchased or synthesized and tested by immunoassay and GC/MS for their detectability in urine. Some cross reactivity was observed with PBA, N-methyl-PBA N-ethylamphetamine, and N-ethyl-PBA when analyzed using a series of commercial amphetamine and methamphetamine immunoassays. Chromatographic co-elution problems were observed for the underivatized isomeric group N,N-dimethylamphetamine, N-ethylamphetamine, and N-methyl-PBA under GC/MS conditions used; and their GC/MS spectra were quite similar. Of the potential derivatives, pentafluoropropionyl (PFP) anhydride and heptafluorobutyryl (HFB) anhydride provided adequate separation and easily distinguishable spectra using the electron-impact GC/MS conditions specified.     

北京地区人群血清型α2HS频率调查与血痕中α2HS的检测

本文采用聚丙烯酰胺凝胶等电聚焦和免疫固定技术,调查北京地区随机人群的α2HS糖蛋白(α2HS)的频率分布。在185名无亲缘关系的健康人中,发现3种常见表型,即α2HS1-1型(99人)、2-1型(74人)、2-2型12人。未发现稀有型。基因频率为:α2HS~1=0.7351,α2HS~2=0.6490。室温中保存6个月的血痕,可检出其α2HS表型。     

Plasminogen (PLG) polymorphism in northern Japanese: confirmation of PLG*M6 allele

Plasminogen (PLG) phenotyping has been performed on 450 unrelated individuals from northern Japan, using wide-scale ultrathin layer polyacrylamide gel isoelectric focusing combined with immunoblotting. One common phenotype and six rare ones were observed. The rare phenotypes included the recently detected allele PLG*M6 in a new combination with PLG*M5 allele. The estimated allele frequencies for PLG*A, PLG*A3, PLG*M2, PLG*M5, PLG*M6, PLG*B, and PLG*B2 were 0.961, 0.009, 0.001, 0.016, 0.001, 0.003, and 0.009, respectively.