胆碱酯酶的基础与临床研究进展

简述红细胞乙酰胆碱酯酶（ＡＣｈＥ）和血浆丁酰胆碱酯酶（ＢｕＣｈＥ）的基础和临床应用研究的若干进展。红细胞ＡＣｈＥ活性测定已广泛应用于有机磷酸酯（ＯＰ）和氨基甲酸酯杀虫剂中毒的诊断以及职业接触的健康监护。血浆ＢｕＣｈＥ由肝细胞合成,是评价肝细胞合成功能的灵敏指标。急性重型肝炎、慢性活动性肝炎和肝硬化时血浆ＢｕＣｈＥ活性明显降低,可作为这些肝病的诊断标准之一。氨基甲酰化ＢｕＣｈＥ和有机磷酰化ＢｕＣｈＥ之间动力学特征的差别可用于这两类杀虫剂中毒的鉴别诊断：前者在酶抑制率≥４０％时,经时反应曲线呈非线性,而后者呈线性。     

微量羟胺比色法测量胆碱酯酶活性

微量羟胺比色法是根据Hestrin常量法改良的测量胆碱酯酶活性的微量法,操作简化,节省试剂及酶样品90%,适用于各种来源的胆碱酯酶活性测定、抑制实验、重活化及老化等实验。     

红细胞乙酰胆碱酯酶动力学法自动化测定

目的：建立适用于测定实验动物红细胞乙酰胆碱酯酶（ＡＣｈＥ）活性的自动化动力学法。方法：根据Ｗｈｉｔａｋｅｒ法稍加修改，编制适用于自动生化仪的ＡＣｈＥ动力学法分析程序，并作方法学性能评价。结果：在本法条件下，酶反应吸光度和时间之间的线性可达≥５ｍｉｎ；ＡＣｈＥ活性在２１０Ｕ／Ｌ范围内，呈线性关系，最低检测限为≤１．５Ｕ／Ｌ；批内和批间ＣＶ分别为１．１％～１．７％和３．０％～４．５％；正常参考值（ｘ±ｓ，ｋＵ／ＬＲＢＣ）：恒河猴１１．９０±１．７４（ｎ＝１９），比格狗１．８１±０．４７（ｎ＝４５），ＳＤ大鼠０．９７±０．２０（ｎ＝３０）。结论：自动化动力学法测定红细胞ＡＣｈＥ简便，精密度高，尤其适用于大鼠和狗等动物红细胞低活性ＡＣｈＥ测定。     

微量乙酰靛酚比色法测量胆碱酯酶活性

微量乙酰靛酚比色法是采用脂溶性底物乙酰靛酚测量胆碱酯酶活性的方法,适用于膜结合胆碱酶、抗原抗体复合物中的胆碱酯酶等必需用脂溶性底物测定胆碱酯酶活性的实验.     

颅脑损伤后肌酸磷酸激酶活性和循环血内皮细胞变化

目的：寻找有利于颅脑外伤早期诊断的指标．方法：制作大鼠颅脑撞击损伤模型．伤后测定循环血内皮细胞（ＣＥＣ）数和ＢＢ型肌酸磷酸激酶（ＣＫ－ＢＢ）活性，２４小时活杀后测脑含水量．结果：伤后损伤组血浆ＣＫ总活性明显高于对照组，尤以伤后０．５、２、２４小时最为显著（Ｐ＜０．０１）．脑脊液（ＣＳＦ）中ＣＫ－ＢＢ活性于伤后０．５小时即有显著升高，但血浆ＣＫ－ＢＢ此时未见升高．伤后２小时无论ＣＳＦ和血浆ＣＫ－ＢＢ均达峰值，ＣＳＦ－ＣＫ－ＢＢ较血浆ＣＫ－ＢＢ升高更为明显（Ｐ＜０．０１）．伤后８小时和２４小时ＣＳＦ－ＣＫ－ＢＢ虽稍有回落，但仍高于正常值（Ｐ＜０．０１），而血浆ＣＫ－ＢＢ已回至正常水平．ＣＥＣ计数在伤后各时相点均显著高于正常（Ｐ＜０．０１）．脑水含量伤后显著增加（Ｐ＜０．０５）．结论：ＣＥＣ计数和Ｃ－ＢＢ活性测定在脑损伤早期诊断中具有重要的作用，其中ＣＳＦ－ＣＫ－ＢＢ活性变化敏感性更高．     

脂多糖刺激肺血管内巨噬细胞环氧合酶-2表达的意义

探讨脂多糖（ＬＰＳ）对肺血管内巨噬细胞（ＰＩＭ）环氧合酶－２（ＣＯＸ－２）的作用及意义。方法改良法分离、培养猪ＰＩＭ，以ＬＰＳ（１０μｇ／ｍｌ）刺激，反转录滞麦链反应（ＲＴ－ＰＣＲ）检测ＣＯＸ－２ｍＲＮＡ的变化；经染色法观察ＣＯＸ－２酶蛋白的改变；放射免疫分析法（ＲＩＡ）测定前列腺素Ｅ２）（ＰＧＥ２）变化，以间接显示ＣＯＸ活性的改变。结果ＬＰＳ刺激后，ＰＩＭ ＣＯＸ－２ｍＲＮＡ和酶蛋白表达ＰＧＥ２     

L-NAME对小肠上皮细胞辐射防护作用机理的探讨

目的探讨硝基左旋精氨酸甲基酯（Ｌ－ＮＡＭＥ）对小肠上皮细胞辐射防护的机理。方法实验用大鼠ＩＥＣ－６细胞，６０Ｃｏγ射线照射；荧光分光光度法测定培养液上清中一氧化氮（ＮＯ）含量；ＮＡＤＰＨ黄递酶组化法（ＮＤ组化法）和免疫细胞化学法观察一氧化氮合酶（ＮＯＳ）活性变化；ＭＴＴ法检测细胞的生存力。结果ＩＥＣ－６细胞经射线照射后，其培养液上清中ＮＯ含量明显增加，细胞内结构型一氧化氮合酶（ｃＮＯＳ）活性升高，诱导型一氧化氮合酶（ｉＮＯＳ）活性降低，Ｌ－ＮＡＭＥ能明显提高细胞的生存力，ＮＯ底物－左旋精氨酸（Ｌ－Ａｒｇ）部分逆转其作用。结论ＮＯ参与了ＩＥＣ－６细胞的辐射损伤，Ｌ－ＮＡＭＥ对ＩＥＣ－６细胞具有辐射防护作用。     

烟雾吸入伤后肺表面活性物质蛋白A含量的动态变化及意义

目的探讨烟雾吸入伤后肺表面活性物质蛋白Ａ（ＳＰ－Ａ）含量的变化、可能机理及其与肺表面活性物质（ＰＳ）活性抑制和肺功能损害的关系。方法采用大鼠烟雾吸入伤模型，分别检测正常对照及致伤２ｈ、６ｈ、１２ｈ和２４ｈ动物的动脉血气，肺水量，静态肺顺应性（Ｃｓｔ），支气管肺泡灌洗液（ＢＡＬＦ）表面张力特性，ＢＡＬＦ中ＳＰ－Ａ、丙二醛（ＭＤＡ）、总蛋白（ＴＰ）含量及弹性蛋白酶（Ｅｌａ）活性。结果动物伤后出现急性呼吸衰竭和肺水肿，Ｃｓｔ显著降低；ＢＡＬＦ的最小表面张力（ＳＴｍｉｎ）升高，滞后环面积下降；ＢＡＬＦ中ＳＰ－Ａ、ＭＤＡ、ＴＰ含量及Ｅｌａ活性明显升高，但ＳＰＡ－Ａ的相对含量（％ＴＰ）降低，且与Ｅｌａ、Ｃｓｔ和ＳＴｍｉｎ的变化相关显著。结论烟雾吸入伤早期ＳＰ－Ａ相对含量减少，可能是导致肺表面活性物质活性抑制及肺功能障碍的主要原因之一。氧化及Ｅｌａ可能是引起ＳＰ－Ａ含量和（或）功能异常的重要因素。     

60Coγ射线对BEP2D细胞氧化损伤的研究

目的：研究＾６０Ｃｏγ射线照射人乳头状病毒永生化的人支气管上皮细胞（ＢＥＰ２Ｄ）的产生的活性氧（ＲＯＳ）及期 对ＤＮＡ的氧化损伤,以及纳米硒的保护作用。方法细胞上清中过氧化氢（Ｈ２Ｏ２）、超氧阴离子（ＮＯ＾－２）以及羟自由基（．ＯＨ）含量分别用辣根过氧化物酶介导的酚红氧化法、细胞色素Ｃ还原法和番红花红退色法进行测定;细胞内Ｈ２Ｏ２和Ｈ＾－２分别用２’,７’－二氯荧光黄双乙酸盐（ＤＣＦＨ－ＤＡ）和氢     

欧芹素乙和已酮可可碱对白细胞介素-1_β诱导脑血管内皮细胞与单核细胞粘附和迁移的抑制作用

目的：研究白细胞介素－１β（ＩＬ－ １β） 对培养的牛脑微血管内皮细胞（ＢＣＭＥＣ） 与单核细胞（ＭＮＣ） 粘附、迁移的影响及药物的保护作用。方法：通过测定粘附于内皮细胞单层上的ＭＮＣ及胶原层中的ＭＮＣ,计算ＭＮＣ的粘附率及迁移率。结果：ＩＬ－ １β（５００ｋＵ·Ｌ－ １） 可促进单核细胞与脑微血管内皮细胞的粘附与迁移,其促粘附与迁移作用具明显的时效关系。ＩＬ－ １β促粘附达最大效应时间为２ｈ,较对照组提高４６．１％ ;促迁移作用２ｈ 达最大值,较对照提高１１８ ．１ ％ 。欧芹素乙、己酮可可碱在浓度１ ～１００μｍｏｌ·Ｌ－１ 范围时,可剂量依赖性地拮抗ＩＬ－ １β诱导的粘附及迁移,药物浓度最高（１００μｍｏｌ·Ｌ－１） 时,抑制率达最大。结论：欧芹素乙与己酮可可碱呈剂量依赖性地抑制ＭＮＣ与ＩＬ－１β诱导的ＢＣＭＥＣ的粘附及迁移     

Facile synthesis of new carbon-11 labeled conformationally restricted rivastigmine analogues as potential PET agents for imaging AChE and BChE enzymes.

Rivastigmine is a newer-generation inhibitor with a dual inhibitory action on both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes, and is used for the treatment of AChE- and BChE-related diseases such as brain Alzheimer's disease and cardiovascular disease. New carbon-11 labeled conformationally restricted rivastigmine analogues radiolabeled quaternary ammonium triflate salts, (3aR,9bS)-1-[(11)C]methyl-1-methyl-6-(methylcarbamoyloxy)-2,3,3a,4,5,9b-hexahydro-1H-benzo[g]indolium triflate ([(11)C]8) and (3aR,9bS)-1-[(11)C]methyl-1-methyl-6-(heptylcarbamoyloxy)-2,3,3a,4,5,9b-hexahydro-1H-benzo[g]indolium triflate ([(11)C]9), were designed and synthesized as potential positron emission tomography (PET) agents for imaging AChE and BChE enzymes. The appropriate precursors were labeled with [(11)C]CH(3)OTf through N-[(11)C]methylation, and the target tracers were isolated by solid-phase extraction (SPE) using a cation-exchange CM Sep-Pak cartridge in 40-50% radiochemical yields decay corrected to end of bombardment (EOB), 15-20 min overall synthesis time, and 148-222 GBq/micromol specific activity at EOB.     

四唑蓝比色法测定肝素酶活性

目的：建立一种检测肝素酶活性的简便快捷的方法。方法：肝素酶作用于肝素／硫酸肝素特定位点的β-1，4糖苷键生成还原性醛基末端，还原性醛基末端可将生色底物四唑蓝还原成蓝色产物。颜色变化反映还原性醛基基团数量的改变的特性，被用于肝素酶活性的检测。结果：四唑蓝比色法的结果表明，颜色的改变程度与酶活性戍正比；凝胶和SDS-PAGE电泳法验证了四唑蓝比色法的结果。结论：四唑蓝比色法可较准确地反映肝素酶活性，可以成为测定肝素酶活性的简便、经济、有效的方法。     

Dosimetric characterization of an encapsulated interstitial brachytherapy source of 125I on a tungsten substrate

PURPOSE: Recently, a new design of an encapsulated 125I source using a tungsten substrate has been introduced by Best Medical International and named as Best Model 2301 source. In contrast to model 6711 source that uses silver as substrate, the model 2301 source does not yield fluorescent x rays (22.1 keV and 25.5 keV) in the energy range of dosimetric interest. This changes the dosimetric characteristics of the source and experimental determination of these characteristics is needed. METHODS AND MATERIALS: In this work, the dosimetric characteristics of the tungstenbased 125I source were measured using LiF TLDs in a Solid Water phantom. The dose rate constant as well as the radial dose function and anisotropy function were measured. RESULTS: The dose rate constant for the tungsten-based source was determined to be 1.02 +/- 0.07 cGy h(-1) U(-1) in contrast to the previously reported value of 0.98 for the silver-based model 6711 source. The radial dose function for the tungsten-based model 2301 source decreases slightly less rapidly with distance than that for the silver-based model 6711 source. Considerable differences in the anisotropy functions between the two sources were observed. CONCLUSIONS: Dosimetric parameters of the Model 2301 source, based on AAPM TG-43 formalism, have been experimentally determined.     

二胺氧化酶活性的测定方法

介绍了用测量~3H标记的底物被分解的量反映肠匀浆和血浆中二胺氧化酶活性的方法。条件实验结果表明,血浆和肠匀浆分别保温1～5h和0.25～5h,分解的~3H标记底物的dpm随保温时间延长而增高,不同稀释浓度的肠匀浆分解~3H标记的底物,也随稀释倍数增加而降低。取样后保存时间对酶活性影响较大,取样后,1d内可测得满意的结果。     

Validity and reliability of combining three methods to determine ventilatory threshold.

PURPOSE: This research was undertaken to validate a combination of methodologies to determine ventilatory threshold (VT). METHODS: Three methods were used individually and then combined to determine VT as follows: 1) ventilatory equivalencies, 2) excess CO2 production, and 3) a modified V-slope method. Three groups of participants-endurance athletes (N = 132), healthy, aerobically active adults (N = 31), and healthy, sedentary/low-active adults (N = 22)-were independently evaluated for VT and compared with the criterion standard lactate threshold (LT) defined as the first rise in blood lactate with increasing intensity of exercise. RESULTS: VT and LT were significantly correlated using the combined VT method within each study group (r = 0.98, 0.97, and 0.95, respectively; P < 0.001). Mean VO2 values at VT and LT were not significantly different between the three groups (P > 0.20). The combined method improved the determination rate of VT and reduced the standard deviation of the LT - VT difference by 80-170% over the individual methods. During test-retest procedures VO2lt and VO2vt determined by the combined method met criteria demonstrating further reliability. CONCLUSION: The combined method to determine VT is valid and reliable across a wide fitness range in healthy individuals and improves the determination rate and accuracy of VT determination over the use of single methods.     

Influence of errors in sampling time and in activity measurement on the single sample clearance determination

INTRODUCTION: Plasma clearance rate of 51Cr-EDTA estimated by using one blood sample is commonly used for the calculation of glomerular filtration rate. AIM: To estimate the error on single-sample clearance determination induced by errors in sampling time and activity measurement, and to compare it with the error observed on the clearance determination obtained using the slope-intercept method. METHODS: Forty-five adult patients were chosen from a data base of 51Cr-EDTA plasma clearance values determined by using two blood samples taken around 2 and 4 h. Patients were selected in such a way as to include clearances from 30 ml.min-1 to 155 ml.min-1, with steps of 3 ml.min-1. Based on the slope and the intercept of the slope with the y-axis, the plasma concentration at exactly 2 and 4 h was determined. Normally distributed random errors were then introduced in the sampling time (SD of 0, 1 and 2 min) as well as in the activity measurement (SD of 0, 1, 2 and 5%). Then, clearance was calculated using two single-sample methods (i.e. the algorithms of Groth and Tauxe), and the slope-intercept method, which requires two blood samples. For each setting, the simulation was repeated 200 times. The effects on clearance of a random error on the time sampling and/or the activity measurement were then evaluated. RESULTS: The error on single-sample clearance induced by a 2 min error in sampling time associated with a 5% error in activity measurement was negligible. For all clearance levels, the SD of the error on the calculated clearance was less than 3.8 ml.min-1. Whatever algorithm was chosen, the errors on the single-sample clearance were systematically lower than those observed with the slope-intercept method, for the whole clearance range. CONCLUSION: Errors in sampling time and in activity measurement induced only a very small error on the single-sample EDTA clearance, which is systematically lower compared to that observed on the slope-intercept method using two blood samples.     

Thin film thickness determination with neutron activation analysis.

Thickness determination of Ta2O5 thin films, deposited on the glass substrates and metallic indium and gold thin films on both glass and aluminum substrates, were performed by neutron activation analysis. Thickness determination of these thin films were made by comparing gamma-rays emitted from the radio-isotopes in the thin film with the substrate material followed by the neutron irradiations. The method led to determination of the film thicknesses without using any standard sample. A complementary optical transmission measurement was also applied on multi-layered Ta2O5 thin films for determining the individual layer densities.     

Influence of measurement errors on temperature-based death time determination

Temperature-based methods represent essential tools in forensic death time determination. Empirical double exponential models have gained wide acceptance because they are highly flexible and simple to handle. The most established model commonly used in forensic practice was developed by Henssge. It contains three independent variables: the body mass, the environmental temperature, and the initial body core temperature. The present study investigates the influence of variations in the input data (environmental temperature, initial body core temperature, core temperature, time) on the standard deviation of the model-based estimates of the time since death. Two different approaches were used for calculating the standard deviation: the law of error propagation and the Monte Carlo method. Errors in environmental temperature measurements as well as deviations of the initial rectal temperature were identified as major sources of inaccuracies in model based death time estimation.     

The determination of calcium in faecal samples by instrumental fast neutron activation analysis

A method is described for the determination of calcium in faecal material by a purely instrumental technique, namely fast neutron activation analysis. The major advantage of the technique over existing methods is that it requires minimal sample preparation. In addition, only a single standard is used and the method is also fairly rapid and simple. The method is well suited to the routine analysis of large numbers of samples and the results are shown to be comparable with atomic absorption spectrophotometry in accuracy. Finally, the technique can be extended to include the determination of several other elements providing a standard for each element is included.     

Vitamin B2 content determination in liver paste by using acid and acid-enzyme hydrolysis

BACKGROUND/AIM: Vitamin B2 is available in foodstuff in the form of coenzyme and in free form. For its content determination a few procedures should be performed (deliberation from a complex, extraction of free and deliberated form) and detection, identification and quantification. There is a particular problem in determination of vitamin B2 in the meat products. For a determination of total vitamin B2 content in liver paste two preparation procedures are compared: acid and acid-enzymatic hydrolysis. The aim of this study thus, was to compare the effectivenes of these two different procedures for vitamin B2 content determination in liver paste. METHODS: High pressure liquid chromatography (HPLC) method with fluorescence detector, as specific and adequately sensitive for the foodstuff of a complex composition with a natural vitamin content, was used for determination of vitamin B2 in liver paste. Acid hydrolysis was performed with the application 0.1 M hydrochloric acid in a pressure cooker, and enzymatic hydrolysis was performed with the 10% takadiastase on 45 degrees C within four hours. Ten samples of liver paste from the supply of the Serbian Army were examined. Separation was performed on the analytical column Nucleosil 50-5 C18 with mobile phase 450 ml CH3OH + 20 ml 5 mM CH3COONH4, and detection on the fluorescent detector with the variable wave length. Both methods were validated: examining a detection limit, quantification limit, specificity (because of a possible B2 vitamin interference with reagents), linearity of a peak area and standard concentration of B2 vitamin ratio in the range from 0.05 microg/ml to 2 microg/ml, precision for the 0.05 microg/ml concentration and recovery. RESULTS: All the previously examined parameters validated both methods as specific, precise and reproductive, with a high recovery (98.5% for acid and 98.2% for acid-enzymatic hydrolysis), as well as linearity in a range that significantly superseded the expected content in the samples (r = 0.9994, and r = 0.99987). Hydrolysis procedures make a sample suitable for vitamin B2 determination. In the liver paste samples a high content of vitamin B2 was determined: 0.83 mg/100 g after acid hydrolysis, and 0.909 mg/100 g after acid-enzyme hydrolysis. There were statistically significantly higher values determined after the acid-enzyme hydrolysis (p < 0.05). CONCLUSION: Using acid-enzyme hydrolysis and separation instrument technique (liquid chromatography) with a fluorescent detector as detection system, statistically significantly greater vitamin B2 quantities were determined than after using acid hydrolysis procedure. Vitamin B2 content determined in ten liver paste samples was high (0.881-0.936 mg/100 g) indicating that this meat product is a good vitamin B2 source.