A sandwich enzyme immunoassay for pulmonary surfactant protein D and measurement of its blood levels in drowning victims

A sensitive sandwich enzyme immunoassay for human pulmonary surfactant protein D (SP-D) was developed and used to examine the blood SP-D levels of drowning victims. Human SP-D was purified from amniotic fluid by chromatographic methods, and an antibody against human SP-D was prepared. A polystyrene ball coated with anti-SP-D IgG was incubated with purified human SP-D, and then with anti-SP-D Fab'-peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorometry using 3-(4-hydroxyphenyl)propionic acid as the hydrogen donor. The detection limit of human SP-D was 5.2 pg per assay tube. Examination of cross-reactions of this sandwich enzyme immunoassay with proteins from other human organs showed it to be highly specific for lung, and Northern blot analysis detected specific SP-D mRNA expression only in lung. The SP-D concentration of normal human serum was 6.4+/-2.7 (mean+/-S.D.) ng ml(-1) (n=20). The recovery rates of 0.52 ng and 5.2 ng SP-D added to 5 microl normal human serum were 93.6+/-2.7% and 93.6+/-6.1%, respectively. Blood SP-D levels of victims from the saltwater drowning group (n=14) revealed higher concentrations (105.8+/-53.7 ng ml(-1)), while freshwater drowning victims (n=12) were estimated to be 74.1+/-43.9 ng ml(-1). The SP-D levels of 15 subjects who died of hemorrhage (n=5), heart failure (n=8), traumatic shock (n=1), and electrocution (n=1) were lower (22.0+/-8.5 ng ml(-1)), and those of asphyxia victims (n=10) were slightly higher (36.2+/-17.1 ng ml(-1)) than those of other causes of death, except for drowning. These results suggest that in drowning victims, SP-D flowed into the systemic circulation by physiological and physical mechanisms, and the differences of blood SP-D levels between saltwater drowning and freshwater drowning victims are presumed to be influenced by the type of agony and/or the length of survival time in water.     

The effects of sarin-like and soman-like organophosphorus agents on MAPK and JNK in rat brains

One sarin-like and one soman-like organophosphorus agent [bis(isopropyl methyl)phosphonate, BIMP and bis(pinacolyl methyl)phosphonate, BPMP] were injected intravenously (iv) in rats. An increase in the tyrosine phosphorylation of several proteins in the cytosol fraction of the brain was observed. Activation of c-Jun N-terminal kinase (JNK) and slight activation of mitogen-activated protein kinase (MAPK) in the cytosol were also observed. The activation of these enzymes may be related to the high toxicity of these nerve agents.     

Lectin-histochemical detection of degenerative glycoconjugate deposits in human brain

Several lectins were used to study the localization of glycoconjugates in brain of elderly people and patients with Alzheimer type dementia (ATD) and Down's syndrome (DS). Five kinds of degenerated or deposited materials stained clearly by lectins specific to GalNAC, Gal, Fuc, and/or Man were recognized much in ATD and DS, less in elderly peoples, in addition to the binding of the lectins to neurons. (i) Round shape deposits called corpora amylacea (CA) which consisted of various sizes of round material, existed mainly on the surface of cerebral cortex and some in white matter of the brain. They were colored by Alcian blue (AB), Aldehyde fucsin (AF) and periodic acid shiff (PAS) and weakly by Hematoxylin (H), but not by Eosin (B). They showed clear reactivity with lectins specific to GalNAC, Gal, Fuc and Gal-GalNAC. (ii) Amorphous and variform amyloid deposits existed around blood vessels in the white matter were stained by thioflavin and lectins specific to GalNAC, Gal and Fuc, but not with Man specific lectins and PAS, AB, AF and HE. (iii) Another kind of amyloid deposits which showed a similar characteristic to the previous one and were recognized mainly in white matter and independent blood vessels. These deposits were stained by thioflavin but not by PAS, AB, AF and HE and showed good reactivity with lectins specific to GalNAC, Gal, Fuc, Gal-GalNAC, Gal-GIcNAc and Man. The reactivity with lectins specific to Gal, Fuc, and Man was seen in senile plaques (iv) and neurofibrillary tangles (v). Although at present we are unable to explain the origin of these deposits, it is clear from this study that the glycoconjugates form an integral part of the degeneration in the brain. The lectin staining with GS-I is useful in the forensic pathology to diagnose brain disorders at postmortem examination, since these lectin were able to detect five types of degeneration changes and/or deposits.     

Screening and determination of benzodiazepines in whole blood using solid-phase extraction and gas chromatography/mass spectrometry

Benzodiazepines are one of the most widely prescribed drugs for the treatment of a wide spectrum of clinical disorders. They are used as anticonvulsants, anxiolytics, hypnotics or muscle relaxants with different duration of action. In this paper, a simple and sensitive method for the determination of benzodiazepines in whole blood using solid-phase extraction and gas chromatography/mass spectrometry (GC/MS) is described. The drugs spiked in whole blood were extracted with an Oasis HLB solid-phase extraction cartridge (Waters), which contains a copolymer designed to have a hydrophilic-lipophilic balance. GC/MS analysis was performed using a Shimadzu QP-5000 equipped with a BPX5 capillary column (15 mx0.32 mm I.D., film thickness 0.25 microm, SGE). Nineteen benzodiazepines and two thienodiazepines were well separated from each other on their SIM chromatograms and also on the TIC with the exception of oxazolam to cloxazolam separation. The blank extract from whole blood gave no peaks that interfered with all benzodiazepines and thienodiazepines on the chromatogram. The calibration curves for selected benzodiazepines with fludiazepam as an internal standard showed excellent linearity over the concentration range 5-500 ng/ml blood with a correlation coefficients of >0.995. The detection limits ranged from 0.2 to 20 ng/ml blood. The method is simple and sensitive for the determination of benzodiazepines in whole blood and seems to be useful in the practice of forensic science.     

A sandwich enzyme immunoassay for human pancreatic elastase III

To develop a method for the determination of pancreas injuries using a pancreas-specific antigen as a marker, human elastase III was purified from the pancreas by chromatographic methods. A rabbit anti-human elastase III antibody was prepared, and this antibody was confirmed using immunoblotting to react only with elastase III among proteins from the pancreas. A sensitive sandwich enzyme immunoassay for human elastase III was developed. The detection limit for human elastase III was 0.3 pg (10 amol) per assay. Proteins extracted from the pancreas showed the strongest response, whereas reactions of the other organs were less than the detection limit. These results suggest that a sandwich enzyme immunoassay for human elastase III is useful for the determination of pancreas injury.     

体外培养FBs损伤模型的建立及细胞免疫组化研究

首次应用培养人胎肺成纤维细胞 (FBs)损伤模型 ,体外研究创缘FBs合成细胞型纤维连接蛋白 (cellu larfibronectins,cFn)的变化与损伤时间的关系。结果表明 ,损伤使处于静息状态的融合FBs成为具有运动能力和增殖能力的活跃细胞。应用免疫组化ABC法结合图像分析技术 ,观察伤后不同时间创缘FBs内cFn的含量变化。伤后 1h ,可以检测到cFn有变化 ,且在伤后 6h内 ,cFn逐渐增多 ,其变化与损伤时间呈正相关。这对今后应用cFn与损伤时间推断的研究提供了科学依据 ,同时为法医学损伤时间研究开辟了新的途径。     

大鼠肝细胞DNA含量与早期死亡时间关系的图像分析研究

实验选择 15只大鼠 ,处死后 ,在 2 4h内 ,每隔 1h取肝组织块进行细胞学涂片、福尔马林液固定、Feul gen染色、自动图像分析仪测量、统计学处理数据。结果表明 ,肝细胞DNA含量在死后 2 4h内 ,随死亡时间的延长而较有规律的下降。其中积分光密度、平均光密度、异形指数是用来研究死亡 2 4h内肝细胞DNA降解规律 ,并准确推断死亡时间的较好指标。     

应用40Hz听觉相关电位与听性脑干反应评估听阈

对 42例 74耳（正常听力组 32耳,检案中主诉听力下降 42耳）分别进行纯音测定,声导抗测定,听性脑干反应 (Auditory Brainstem Response,ABR)及 0.5～ 2kHz范围的 40Hz听觉相关电位 (40Hertz Auditory Event－ related Potential,40Hz AERP)测定,对其中 20耳进行睡眠及清醒两种状态的 40Hz AERP测定。将纯音听阈、 40Hz AERP反应阈、 ABR反应阈三者之间进行比较分析。结果表明,正常听力耳 40Hz AERP反应阈值较纯音测听阈值高,在不同频率的校正值 (差值 )不同, 0.5 kHz为 12.7± 6.4(dBnHL); 1kHz为 14.7± 6.3(dBnHL); 2 kHz为 15± 5.6(dBnHL)。 ABR阈值比行为听阈高,校正值为 8.9± 5.3(dBnHL)。睡眠状态时 40Hz AERP阈值较清醒状态阈值高,校正值为 9.7± 2.45(dBnHL)。检案中主诉听力下降者主、客观语音频率均值之间明显不相符 ,误差率为 61％。本研究表明单纯使用 ABR、纯音测听中任何一种方法估计语音频率听阈均有一定的误差。