LC-MS/MS法研究1-[1-(6-甲氧基-2-萘基)乙基]-2-(4-硝基苄基)-6,7-二甲氧基-1,2,3,4-四氢异喹啉氢溴酸盐在大鼠体内的代谢产物

采用液相色谱-串联质谱法对大鼠灌胃1-［1-(6-甲氧基-2-萘基)乙基］-2-(4-硝基苄基)-6,7-二甲氧基-1,2,3,4-四氢异喹啉氢溴酸盐(编号P91024)后粪便、尿液、胆汁和血浆中的主要代谢产物进行研究。通过比较给药样品和空白样品的全扫描总离子流色谱和选择离子扫描色谱图差别寻找I相代谢产物；根据其一级和二级质谱图，确定I相代谢产物的分子结构。完全提取I相代谢产物后的样品溶液，再用葡糖醛酸酶酶解，得II相结合物的苷元部分，采用与I相代谢产物鉴定同样方法寻找和鉴定II相代谢产物苷元的结构，进而确证II相代谢产物的分子结构。从大鼠粪便中鉴定出P91024的2个I相代谢物，从胆汁中鉴定出1个I相和5个II相代谢产物，从尿液中鉴定出1个I相和3个II相代谢产物，从血浆中鉴定出4个I相和1个II相代谢产物；并分别分析推测出它们的结构。P91024在大鼠体内被代谢转化为多种产物，利用LC-MS/MS可以快速寻找和鉴定。     

槲皮素代谢产物定性检出条件研究

目的:研究给予大鼠槲皮素后血浆中代谢产物的定性检出条件。方法:给予大鼠槲皮素单体(100mg.kg-1)后腹腔静脉取血,血浆样品经2mol.L-1盐酸(甲醇-水溶液)水解,采用高效液相色谱法对血浆样品中代谢产物进行定性分析。结果:大鼠给药后3h内取血、血浆样品经盐酸水解处理4h,可稳定检测槲皮素和异鼠李素,样品在—20℃条件下贮存稳定。结论:血浆样品采集时间、盐酸水解时间及贮存方式等不同均可影响代谢产物的有效检出。     

液相色谱-串联质谱法分析抗肿瘤新药乙烷硒啉在大鼠体内的代谢产物

本研究对抗肿瘤新药1,2-[二(1,2-苯并异硒唑-3(2H)-酮)]乙烷(乙烷硒啉,BBSKE)在大鼠体内的代谢产物进行鉴定。在灌胃给予大鼠单剂量乙烷硒啉200mg·kg-1后,采用液相色谱-串联质谱法(LC-MSn)对大鼠尿液、粪样、胆汁和血浆中的代谢产物进行检测,通过全扫描和选择离子扫描,以及根据多级质谱裂解规律对代谢物的结构进行分析。研究发现在大鼠尿样、粪样、胆汁和血浆中检测到3种Ⅰ相代谢产物和1种Ⅱ相代谢产物,其代谢途径分别为氧化、甲基化、硫甲基化和葡萄糖醛酸化反应,提示乙烷硒啉在大鼠体内的代谢方式可能是通过氧化、甲基化及葡萄糖醛酸化反应形成代谢产物。     

桂枝茯苓胶囊在原发性痛经模型大鼠血浆、胆汁、尿液和粪便中的代谢产物研究

目的:研究桂枝茯苓胶囊在原发性痛经模型大鼠体内的代谢产物。方法:基于超高效液相色谱-线性离子阱-静电轨道阱串联质谱联用(UHPLC-LTQ-Orbitrap-MS)方法,采用Waters ACQUITY UPLC BEH C₁₈色谱柱(100 mm×2.1 mm,1.7μm),以乙腈-0.1%甲酸水为流动相,梯度洗脱,流速0.4 mL·min^-1,质谱采用电喷雾(ESI)离子源,以正负离子模式采集多级质谱碎片信息,对大鼠灌服桂枝茯苓胶囊混悬液后的血浆、胆汁、尿液和粪便进行分析,利用质量亏损过滤(MDF)技术进行代谢产物鉴定分析。结果:共鉴定出8个原形及50个代谢产物,在体内主要发生Ⅰ相代谢反应(脱羧、水解、脱羟基和脱水等)及Ⅱ相代谢反应(甲基化、乙酰化、硫酸化及葡萄糖醛酸化等)。结论:采用LC-MS方法鉴定了原发性痛经模型大鼠体内桂枝茯苓胶囊代谢产物,为该复方制剂的药物代谢动力学研究提供依据。     

硝克柳胺在大鼠和人肝脏的体外代谢研究

研究硝克柳胺在大鼠肝微粒体和胞浆中的代谢动力学，鉴定硝克柳胺在大鼠和人肝微粒体中的主要代谢产物及参与代谢的药物代谢酶。采用高效液相色谱-紫外检测(HPLC-UV)方法测定大鼠肝微粒体和胞浆中硝克柳胺浓度，应用选择性抑制剂鉴定参与硝克柳胺代谢的药物代谢酶类型，采用液相色谱-串联质谱联用(LC-MS/MS)法分离鉴定硝克柳胺在大鼠和人肝微粒体中的主要代谢产物。硝克柳胺在大鼠和人肝微粒体的主要代谢产物(M₁)为硝基还原产物［3-(3′-羧基-4′-羟基苯胺羰基)-6-氨基-7-羟基-8-甲基香豆素］，大鼠体内(血浆、尿液、胆汁及肝组织)主要代谢产物与M₁一致。硝克柳胺的体外代谢是依赖多个药物代谢酶参与的酶促反应，包括微粒体CYP450还原酶、细胞色素b₅还原酶和CYP2C6以及胞浆NAD(P)H脱氢酶和黄嘌呤氧化酶。     

白及有效成分Militarine在肝微粒体中的体外代谢途径及其酶促动力学特征

目的:研究白及有效成分Militarine在肝微粒体中的体外代谢途径及其酶促反应动力学特征。方法:建立大鼠和人肝微粒体体外孵育体系,对Militarine进行体外孵育反应;采用超高效液相色谱-四极杆-飞行时间质谱联用技术,结合UNIFI数据库并参考文献对其代谢产物进行结构鉴定。同时,以葛根素为内标,采用超高效液相色谱-三重四极杆串联质谱联用技术对Militarine在大鼠肝微粒体中的代谢转化量进行定量分析,通过GraphPad Prism 5.0软件拟合代谢转化曲线,并计算在有/无还原性辅酶Ⅱ(NADPH)参与的反应条件下的Militarine酶促动力学参数[最大清除速率(v_(max))、米氏常数(k_m)、固有清除率(CL_(int))]。结果:Militarine在大鼠和人肝微粒体中孵育后,生成了1个化学式为C_(21)H_(29)O_(11)的化合物,推测其为Militarine的酯键水解代谢产物。酶促动力学研究显示,有/无NADPH参与的Militarine酶促反应的v_(max)分别为1.955、2.129 nmol/(h·mg),k_m分别为8.601、9.854 nmol/mL,CL_(int)分别为0.227 3、0.216 1 mL/(h·mg),两者无明显差异。结论:Militarine在肝微粒体中的主要代谢途径为C1或C4位酯键的水解;其代谢不依赖于由NADPH启动反应的细胞色素P_(450)酶代谢途径。     

HPLC-ECD法测定大鼠血浆中银杏叶黄酮成分及药代动力学研究

目的:建立大鼠灌胃银杏叶提取物后血浆中黄酮类成分的灵敏分析方法,并进行药代动力学研究。方法:血浆样品经葡萄糖醛酸苷水解酶水解后,采用高效液相色谱分离及电化学检测,同时分析大鼠血浆样品中槲皮素、异鼠李素和山柰酚。色谱柱为Agilent Zorbax Ec lipse XDB-C8(150 mm×4.6 mm,5μm),流动相为甲醇-异丙醇-磷酸盐缓冲液(磷酸二氢钠-磷酸,pH=2)(15?15?70),流速0.8 mL.m in-1,检测电压为200 mV,柱温30℃。结果:槲皮素、异鼠李素和山柰酚与其他内源性和药源性成分达到良好分离;最低检测浓度达到0.5 ng.mL-1;平均提取回收率分别为88.7%,86.2%,87.0%;准确度在92.7%~105.3%之间,日内精密度RSD小于6.6%,日间精密度RSD小于14.1%,样品分析时间为20 m in。大鼠灌胃给予银杏叶提取物10 mg.kg-1后,酶水解血浆中槲皮素、异鼠李素和山柰酚的半衰期分别为(3.53±1.88)h,(6.94±4.05)h,(3.97±2.30)h;3个黄酮类成分在大鼠血浆中都显示明显的二次达峰现象。结论:建立的检测方法能灵敏、特...     

基于UHPLC-Q-Orbitrap HRMS技术的白术内酯I在大鼠体内代谢产物的鉴定及代谢途径分析

目的 采用超高效液相色谱-四级杆/静电场轨道阱高分辨质谱技术(UHPLC-Q-Orbitrap HRMS)鉴定白术内酯I在大鼠体内的代谢产物，并探讨其可能的代谢途径。方法 SD大鼠单次灌胃给予白术内酯I后，收集大鼠血浆、尿液、粪便样品。以体积分数0.1%甲酸水溶液(A)-乙腈(B)为流动相，梯度洗脱。在电喷雾电离源(ESI源)正、负离子模式下分析大鼠生物样本。根据质谱提供的准分子离子峰、碎片离子及准确相对分子质量，鉴定白术内酯I体内代谢产物。结果 共鉴定出大鼠体内代谢产物30个，其中血浆样品中5个，尿液样品中17个，粪便样品中16个。氧化、水合、脱饱和、硫酸化、葡萄糖醛酸化、甘氨酸结合、半胱氨酸结合等是白术内酯I体内主要的代谢途径。结论 首次阐明了白术内酯I在大鼠体内的代谢产物及代谢途径，为其进一步的药效学评价和开发利用提供参考，此外也为单体药物代谢产物鉴定提供了一种综合研究方法。     

采用UHPLC-MS和质量亏损过滤技术分析二苯乙烯苷在大鼠体内的代谢产物和代谢途径

目的:分析二苯乙烯苷在大鼠体内的代谢产物并推测代谢途径。方法:将雄性SD大鼠随机分为血浆组(n=3)、尿液组(n=3)、胆汁组(n=3)和组织组(n=9),各组大鼠均单次灌胃二苯乙烯苷200 mg/kg,分别收集给药后10、30 min和1、1.5、2、4 h的血浆,给药后0~6 h的尿液,给药后0~4 h的胆汁以及给药后30 min和1、2 h(每个时间点3只)的心、肝、脾、肺、肾、胃组织样品,经甲醇沉淀蛋白后,采用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱联用技术和质量亏损过滤技术联合分析、鉴定各样本中的代谢产物,推测代谢途径。结果:从血浆、尿液、胆汁、心、肝、脾、肺、肾、胃样品中分别检出6、7、11、1、5、1、3、4、4个代谢产物,包括Ⅰ相代谢(如水解、加氢、羟化)产物2个、Ⅱ相代谢(如葡萄糖醛酸结合和硫酸化)产物18个,其中葡萄糖醛酸结合产物有12个。结论:二苯乙烯苷在胆汁中的代谢产物种类居多,以Ⅱ相代谢产物二苯乙烯苷的葡萄糖醛酸结合产物为主;代谢途径主要涉及葡萄糖水解、加氢、羟化、葡萄糖醛酸结合、硫酸化反应等。     

HPLC-ESI/MS分析5-羟基黄酮在大鼠体内的代谢产物

目的研究5-羟基黄酮在大鼠体内的代谢产物。方法应用高效液相色谱-电喷雾质谱(HPLC-ESI/MS)检测大鼠灌胃5-羟基黄酮后血浆、尿液、胆汁和粪便中的代谢产物。实验采用Zorbax C18色谱柱,0.05%甲酸乙腈-0.05%甲酸水二元线性梯度洗脱进行色谱分离,并与电喷雾质谱联用,根据正离子模式的分子离子峰获得化合物分子量信息,推测化合物的可能结构。结果仅在大鼠粪便中检测到原形成分,在大鼠尿液、粪便、血浆、胆汁中检测到5-羟基黄酮葡糖醛酸结合物。结论 5-羟基黄酮吸收差,在大鼠体内主要以II相代谢产物葡萄糖醛酸结合物的形式存在。     

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.     

Pharmacological treatment of COVID-19: an opinion paper

The precocity and efficacy of the vaccines developed so far against COVID-19 has been the most significant and saving advance against the pandemic. The development of vaccines has not prevented, during the whole period of the pandemic, the constant search for therapeutic medicines, both among existing drugs with different indications and in the development of new drugs. The Scientific Committee of the COVID-19 of the Illustrious College of Physicians of Madrid wanted to offer an early, simplified and critical approach to these new drugs, to new developments in immunotherapy and to what has been learned from the immune response modulators already known and which have proven effective against the virus, in order to help understand the current situation.     

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.     

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.