难治性肾病综合征患者谷胱甘肽硫转移酶活性和基因多态性研究

目的:研究谷胱甘肽硫转移酶(GSTs)活性与基因多态性在难治性肾病综合征(RNS)患者中的分布特征。方法:运用等位基因特异性PCR(ASPCR)和PCR-限制性片段长度多态性(PCR-RFLP)的方法分析健康人和难治性肾病综合征患者谷胱甘肽硫转移酶基因型;用紫外分光光度法分别测定两者红细胞GSTs活性。结果:RNS患者的GSTs基因型分布与健康者差异无显著性;难治性肾病综合征患者GSTs活性与健康人存在差异,难治性肾病综合征患者平均GSTs活性(5.9±2.0)U/gHb高于健康人(4.3±2.6)U/gHb,杂合子I/V基因型组的平均GSTs活性比野生I/I基因型的GST活性低,比突变v/v基因型的GSTs活性高。在RNS患者中,GSTM1基因型之间GSTs活性差异不大。结论:借鉴健康人的基因型研究结果,综合分析RNS患者GST的活性和基因多态性特征,为疾病个体化治疗提供研究基础。     

离子对高效液相色谱法测定人体红细胞内核苷二磷酸激酶活性

目的:建立测定人体红细胞(RBC)内核苷二磷酸激酶(NDPK)活性的反相离子对高效液相色谱法。方法:稀释RBC中加入反应底物(dADP和dGTP)及其他反应混合液后,37℃孵育5 min,100℃灭活10 min,离心取上清液进样分析。以反应产物dATP定量分析NDPK活性。色谱柱为SymmetryShield TMRP18(150 mm×3.9 mm,5μm),检测波长为260 nm,柱温为25℃;采用梯度洗脱,流动相A为20 mmol.L-1 KH2PO4-K2HPO4缓冲液、加入离子对试剂5 mmol.L-1四丁基硫酸氢铵(TBAHS)溶液,流动相B为乙腈。结果:NDPK催化反应dADP+dGTPdATP+dGDP反应特异性良好,RBC内dATP的线性范围为4.33～43.34μmol.L-1(r2=1.000),定量下限为4.33μmol.L-1。日内及日间RSD分别小于5.53%和6.69%,准确度为98.4%～104.2%,平均绝对回收率大于83%;dATP样品在室温或4℃、-80℃保存30 d及反复冻融3次稳定性良好。结论:该法操作简便快速、灵敏、专属性强,可应用于人体RBC内NDPK活性测定,为研究NDPK活性与嘌呤类药物反应相关性提供基础。     

中国健康汉族和瑶族人群巯基嘌呤甲基转移酶活性的分布

目的研究巯基嘌呤甲基转移酶(TPMT)活性在中国汉族和瑶族人群中的分布。方法建立改良的反相高效液相色谱法,测定人红细胞(RBC)中TPMT活性。结果测定了273名中国健康汉族成年人、88名汉族儿童和148名健康瑶族儿童红细胞中TPMT活性,未发现酶缺陷者,其平均活性分别为(11.96±3.27),(13.02±2.78)和(13.27±3.92)nmol·(h·mL)-1packed红细胞。瑶族儿童TPMT活性较同龄汉族儿童差异无显著性;TPMT活性在中国汉族和瑶族人群中都呈正态分布;在中国汉族和瑶族人群中均未发现有与性别有关的差异;汉族人群TPMT活性与年龄无关;瑶族儿童TPMT活性与年龄呈正相关,但相关关系较弱。结论中国汉族和瑶族人TPMT活性分布均呈正态分布。     

LC-MS/MS法测定全血中西罗莫司的药物浓度

目的建立测定人全血中西罗莫司(免疫抑制剂)浓度的LC-MS/MS的方法。方法全血样品经硫酸锌乙腈混合液沉淀后,用Zobox XDB-CN色谱柱,0.1 mmol.L-1乙酸水-0.1 mmol.L-1乙酸乙腈(60∶40)为流动相,流速0.45 mL.min-1,通过电喷雾离子化电离源,经多反应监测模式检测。结果全血中西罗莫司的线性范围为1.12～33.47μg.L-1,日内、日间RSD均小于10%,提取回收率为81.4%～92.4%。结论本法灵敏、准确、快速,可用于人全血中西罗莫司浓度监测和药代动力学研究。     

三氧化二砷对人肺腺癌耐药细胞A549/R的逆转作用

目的探讨三氧化二砷(As2O3)对肺腺癌耐药细胞株(A549/R)耐药性的逆转作用.方法采用四甲基偶氮唑蓝(MTT)法及荧光分光光度计分别检测As203的细胞毒性、A549/R细胞的药敏性及As203作用前后细胞内药浓的变化;采用生化方法检测A549/R细胞谷胱甘肽-S-转移酶(GSTs)活性,观察非细胞毒性剂量(0.15μmol·L-1)和低毒剂量(0.375 μmol·L-1)的As203对肺腺癌A549/R耐药细胞内GSTs活性的影响.结果As203在非细胞毒剂量下可显著降低耐药细胞对ADM的IC50值;As203作用后A549/R细胞内ADM积聚增加,其逆转倍数为2.3倍;A549/R细胞中GSTs活性增高,不同浓度的As203可降低GSTs活性;结论As203可通过降低GSTs活性而部分逆转A549/R细胞对ADM耐药.     

利福平抑制对乙酰氨基酚代谢物耗竭谷胱甘肽的作用

目的研究小鼠肝微粒体温孵体系中利福平(RFP)抑制对乙酰氨基酚(APAP)毒性代谢物耗竭谷胱甘肽(GSH)的保护作用.方法取健康空白小鼠制备肝微粒体,体外温孵,通过高效液相色谱法(HPLC)测定GSH浓度.结果在体外P450体系中,50 mg·L-1 GSH温孵1 h后10.0%代谢,加入5 mmol·L-1 APAP温孵1 h后43.3%GSH耗竭,而加入100 mg·L-1 RFP和5mmol·L-1 APAP温孵1 h后仅19.7%GSH耗竭.结论利福平抑制对乙酰氨基酚的毒性代谢物导致的谷胱甘肽耗竭.     

离子色谱法测定天麻中二氧化硫残留量

目的建立离子色谱法测定天麻中二氧化硫残留量的检测方法。方法样品经30mL.L-1盐酸水溶液蒸煮,氮气作载气,导出后用30mL.L-1双氧水氧化吸收,再煮沸除去剩余的双氧水,用去离子水转移至25mL量瓶中,并稀释至刻度作为供试溶液,使用离子色谱仪分析。用A-SUPP5-150阴离子分析柱,3.2mmol.L-1碳酸钠溶液-1.0mmol.L-1碳酸氢钠溶液为淋洗液,流速为0.7mL.min-1;进样量为20μL。结果线性范围(以SO2计)为1.667～133.38μg.mL-1,平均回收率为98.0%。结论测定方法简便,精密度、重复性良好,准确度较高。     

RP-HPLC法同时测定复方盐酸二甲双胍片中盐酸二甲双胍和格列美脲的含量

目的建立同时测定复方盐酸二甲双胍片中格列美脲和盐酸二甲双胍含量的方法。方法采用RP-HPLC法。色谱柱为Diamonsil C18柱(150 mm×4.6 mm,5μm),流动相为乙腈-20 mmol.L-1磷酸氢二钠溶液(含8 mmol.L-1十二烷基硫酸钠,磷酸调pH值7.5)(体积比为33∶67),流速为1 mL.min-1,检测波长为228 nm。结果格列美脲和盐酸二甲双胍的线性范围分别为0.1～0.6 mg.L-1(r=0.999 1,n=6)和25.0～150.0 mg.L-1(r=0.999 6,n=6);平均回收率分别为99.4%(RSD=1.0%,n=9)和99.8%(RSD=0.39%,n=9)。结论本方法简便、准确,可用于复方盐酸二甲双胍片的质量控制。     

高效液相色谱法测定血中胺碘酮及其活性代谢物浓度

目的:建立反相高效液相色谱测定胺碘酮及其活性代谢物去乙基胺碘酮血药浓度的方法.方法:依利特分析柱HypersilODS2(4.6 mm×250mm,5μm),流动相为乙腈-100 mmol·L-1醋酸(内含15 mmol·L-1二乙胺)(55:45),检测波长242 nm,流速为1.0mL·min,柱温为室温.结果:线性范围0.1～4.8 mg·L,方法回收率90.3%～101.9%,日内RSD小于7%,日间RSD小于11%.结论:适于胺碘酮及其活性代谢物去乙基胺碘酮的临床监测.     

川芎嗪对大鼠背根神经节细胞P2X嘌呤受体介导反应的作用

目的探讨川芎嗪(tetram ethy1pyrazine,TMP)对嘌呤2X(P2X)受体介导反应的作用。方法在大鼠新鲜分离的背根神经节(dorsal root ganglion,DRG)神经元标本上应用全细胞膜片钳技术记录川芎嗪对P2X受体激动剂激活电流的影响。结果外加ATP(1~1 000μmol.L-1)可引起DRG神经元产生激活电流(n=102),ATP-激活电流(IATP)显示快失敏和慢失敏两种形式的内向电流。预加川芎嗪(0.1~10mmol.L-1)后,大部分(89.2%,91/102)受检细胞可观察到ATP(100μmol.L-1)-激活电流出现明显的抑制作用。川芎嗪(1 mmol.L-1)使α,-βm eATP(10μmol.L-1)-激活电流减小。预加川芎嗪(1 mmol.L-1)后ATP(1~1 000μmol.L-1)激活电流的剂量-效应曲线明显下移。预加川芎嗪(1 mmol.L-1)前后ATP(100μmol.L-1)的I-V曲线反转电位值不变,均接近0 mV。川芎嗪(1 mmol.L-1)可明显抑制被前列腺素E2(100μmol.L-1)或P物质(0.1μmol.L-1)增大的ATP激活电流。通过微电极胞内透析注入PKA抑制剂H89(10μmol.L-1)至胞内,使川芎嗪(1 mmol.L-1)抑制ATP(100μmol.L-1)激活电流的作用减小。结论川芎嗪可能是通过PKA系统以及P2X受体离子通道复合体细胞外环的变构调制点影响P2X受体激动剂在大鼠DRG神经元的激活电流。     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

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Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

Aquatic Insects and Trace Metals: Bioavailability,Bioaccumulation, and Toxicity

Abstract

The uptake of metals from food and water sources by insects is thought to be additive. For a given metal, the proportions taken up from water and food will depend both on the bioavailable concentration of the metal associated with each source and the mechanism and rate by which the metal enters the insect. Attempts to correlate insect trace metal concentrations with the trophic level of insects should be made with a knowledge of the feeding relationships of the individual taxa concerned. Pathways for the uptake of essential metals, such as copper and zinc, exist at the cellular level, and other nonessential metals, such as cadmium, also appear to enter via these routes. Within cells, trace metals can be bound to proteins or stored in granules. The internal distribution of metals among body tissues is very heterogeneous, and distribution patterns tend to be both metal and taxon specific. Trace metals associated with insects can be both bound on the surface of their chitinous exoskeleton and incorporated into body tissues. The quantities of trace meals accumulated by an individual reflect the net balance between the rate of metal influx from both dissolved and particulate sources and the rate of metal efflux from the organism. The toxicity of metals has been demonstrated at all levels of biological organization: cell, tissue, individual, population, and community. Much of the literature pertaining to the toxic effects of metals on aquatic insects is based on laboratory observations and, as such, it is difficult to extrapolate the data to insects in nature. The few experimental studies in nature suggest that trace metal contaminants can affect both the distribution and the abundance of aquatic insects. Insects have a largely unexploited potential as biomonitors of metal contamination in nature. A better understanding of the physico-chemical and biological mechanisms mediating trace metal bioavailability and exchange will facilitate the development of general predictive models relating trace metal concentrations in insects to those in their environment. Such models will facilitate the use of insects as contaminant biomonitors.     

Steroidal sapogenin contents in some domestic plants

In order to find out the values of the steroid resources for the future use. the compositions and contents of steroidal sapogenins from 13 domestic plants have been investigated. As a result,Dioscorea nipponica, D. quinqueloba andSmilax china were found to have large amount of diosgenin. And pennogenin inTrillium kamtschaticum andParis verticillata, yuccagenin inAllium fistulosum, hecogenin inAgave americana and neochlorogenin inSolanum nigum were appeared to be major steroidal sapogenins.     

Alzheimer’s disease – current trends in basic and clinical research. Highlights from the 16th ECNP

Advances in the molecular biological knowledge of neuronal nicotinic acetylcholine receptors (nAChRs) have led to a growing interest by the pharmaceutical industry in the development of novel compounds that selectively modulate nAChR function. The ability of (-)-nicotine, an activator of nAChRs, to enhance attentional aspects of cognition in animals and humans, to exert neuroprotective and anxiolytic-like effects, and presumably to mediate the negative correlation between smoking and Alzheimer's (and Parkinson's) Disease, has focused interest on the potential therapeutic utility of modulators of nAChR function for treatment of some of the deficits associated with these progressive, neurodegenerative conditions. Numerous compounds are known which activate nAChRs and which might serve as lead compounds toward the development of such agents. The pharmacologic diversity of neuronal nAChR subtypes suggests the possibility of developing selective compounds which would have more favourable side-effect profiles than existing agents. This broader class of agents, collectively called cholinergic channel modulators (ChCMs), is anticipated to encompass compounds which would have more favourable side-effect profiles than existing agents, which generally exhibit low selectivity. This selectivity may be achieved by preferentially activating some subtypes of nAChRs (i.e., Cholinergic Channel Activators, ChCAs) or inhibiting the function of other subtypes (Cholinergic Channel Inhibitors, ChCIs). An overview of the biology of nAChRs and the rationale for the use of ChCMs for the treatment of dementia related to neurodegenerative diseases are presented, followed by a discussion of lead compounds and compounds under consideration for clinical evaluation.