基于脂类代谢组学研究对乙酰氨基酚对小鼠药物性肝损伤的早期毒性

目的:基于脂类代谢组学研究对乙酰氨基酚(APAP)对小鼠的早期肝损伤毒性,为寻找潜在生物标志物提供参考。方法:将20只小鼠随机分为正常组和APAP肝损伤组,每组10只。APAP肝损伤组小鼠腹腔注射APAP 300 mg/kg建立急性肝损伤模型,正常组小鼠腹腔注射等体积生理盐水。1 h后取血并分离血浆,采用超高效液相色谱串联三重四级杆飞行时间质谱(UPLC-Triple-TOF-MS)法检测小鼠血浆代谢产物进行代谢组学分析,采用主成分分析(PCA)、偏最小二乘法分析(PLS-DA)以及正交偏二乘法分析(OPLS-DA)区分组间代谢轮廓的整体差异,并根据HMDB、Metlin、LIPID MAPS数据库对脂类代谢物进行筛选和鉴定,同时检测小鼠血浆中APAP水平变化。认定OPLS-DA分析中变量权重值(VIP)大于1且P<0.05的脂类代谢物为差异代谢物,并将脂类差异代谢物与血浆中APAP水平进行相关性分析。结果:PCA、PLS-DA以及OPLS-DA结果显示,正常组和APAP肝损伤组样品点位于图形不同的区域,具有良好的区分度。APAP肝损伤组与正常组比较,血浆中5个脂肪酰类代谢产物水平出现显著的升高或降低,8个甘油磷脂类代谢产物水平均显著降低,1个鞘脂类代谢产物水平显著上升;9-硫杂硬脂酸、十四烷二酸、9-过氧化氢-10,12-十八碳二烯酸、三-肉豆蔻酰基肉碱(脂肪酰类)和Scyphostatin A(鞘脂类)水平与血浆中APAP水平显著相关。结论:APAP染毒1 h后血浆脂类代谢组学显示异常,共发现14个相关脂类差异代谢物,其中5个与血浆中APAP水平显著相关。     

基于UPLC-Q-TOF/MS平台的结晶肾损伤小鼠的尿液代谢组学研究

目的 通过研究草酸钙结石小鼠尿液中代谢物的变化,探究草酸钙结晶导致肾损伤的内在机制。方法 以乙醛酸盐诱导的小鼠草酸钙结晶模型为对象,采用基于超高效液相-四极杆飞行时间串联质谱（UPLC-Q-TOF/MS）的代谢组学方法测定尿液中内源性代谢物的变化,并采用SIMCA-P进行多元统计分析,Metabo Analyst软件进行代谢物通路分析。结果 与正常组相比,模型组小鼠的肾组织出现明显的钙盐沉积且血清中的肌酐和尿素氮含量异常升高,肾脏出现损伤;从尿液中筛选出尿酸、牛磺酸、苯丙氨酸等21个差异代谢物。结论 通过代谢物通路分析,差异代谢物主要涉及氨基酸代谢、能量代谢、牛磺酸代谢、嘌呤代谢和VB₆代谢,为进行结石疾病机制研究以及早期标志物的筛选提供了重要参考。     

基于HPLC-三重四极杆线性离子阱杂交质谱技术的代谢组学分析方法的建立

目的:建立基于高效液相色谱-三重四极杆线性离子阱杂交质谱(HPLC-QTRAP)技术的代谢组学分析方法。方法:采用HPLC-QTRAP技术对尿液中28种生理浓度的代谢物进行检测,计算检出率并比较正负离子同时检测与正负离子独立检测两种方法的优劣;以外加的阿托伐他汀和苯烯莫德为内标,采用混合标准品溶液考察仪器的日内与日间精密度,采用加入混合标准品的健康人尿液样品考察分析方法的日内与日间精密度。结果:在选定的色谱及质谱条件下,28种代谢物的检出率为53.57%(15/28),正负离子独立检测的方法优于正负离子同时检测的方法,混合标准品溶液与尿样模拟样品的日内RSD均<20%,但日间RSD较大。与外标法比较,内标校正法未显示出明显优势。结论:HPLC-QTRAP技术应用于代谢组学生物标志物的筛选还有很大的改进空间。     

双氢青蒿素对肝癌细胞氨基酸代谢的影响及机制研究

目的:考察双氢青蒿素(DHA)对人肝癌细胞Huh7、BEL-7402中氨基酸类代谢产物的影响,为阐明DHA参与肝癌细胞代谢的调控机制提供理论基础。方法:采用CCK-8法,测定不同浓度DHA(12.5、25、50、100μmol/L)作用24、48、72 h后对两种细胞活性的影响。将两种细胞分别分为对照组和给药组(DHA,25μmol/L),加入不含药或含药培养基后培养24 h,均平行操作3份。对各组细胞样品进行衍生化处理后,采用气相色谱联用质谱(GC-MS)法检测细胞中氨基酸代谢物含量变化,结合SIMCA-P软件分析和化合物谱库的比对,筛选出两种细胞中的差异代谢物;采用Metaboanalyst 4.0软件对差异代谢物进行通路富集分析。结果:与对照组比较,给药组Huh7细胞或BEL-7402细胞中谷氨酰胺、谷胱甘肽、苯丙氨酸、富马酸、牛磺酸等含量均呈下降趋势。上述两种细胞中分别筛选获得28、29个差异代谢物,两者的共同差异代谢物有10个,包括谷氨酰胺、谷胱甘肽、牛磺酸、富马酸、苯丙氨酸等;两者的差异代谢物分别富集到8、6条通路上,其共同富集通路为氨基酸-tRNA生物合成、天冬氨酸-丙氨酸-谷氨酸代谢、氮代谢、苯丙氨酸代谢、戊糖磷酸途径。结论:DHA能明显降低Huh7细胞和BEL-7402细胞的活性,并能显著降低细胞中谷氨酰胺、谷氨酸、谷胱甘肽、苯丙氨酸等的含量;其可能通过影响天冬氨酸-丙氨酸-谷氨酸代谢通路等机制来调控两种细胞的生长。     

UPLC-Q-Exactive Plus Orbitrap-MS鉴定桑色素在大鼠体内的代谢产物

目的 利用UPLC-Q-Exactive PlusSOrbitrap-MS技术对大鼠灌胃给予桑色素后体内的的主要代谢产物进行研究。方法 大鼠灌胃给予桑色素20 mg/kg后,分别收集血浆、尿液和粪便样品,采用超高压液相色谱串联高分辨质谱技术测定桑色素的体内代谢物。结果 根据一级质谱分子离子信息和二级质谱碎裂离子信息,在大鼠血浆和尿液中均发现2个葡萄糖醛酸代谢产物,在粪便中发现脱氢产物。结论 桑色素在大鼠体内的主要代谢途径为葡萄糖醛酸化反应。本研究初步阐明了桑色素在大鼠体内的代谢情况,为进一步药理作用机制研究提供依据。     

基于脂质代谢组学评价五味子素B诱导的急性高甘油三酯血症小鼠模型

目的利用脂质代谢组学技术对五味子素B(schisandrin B,Sch B)诱导的小鼠高甘油三酯血症模型进行评价和提供新的实验依据。方法♂ICR小鼠分为4组:正常饮食(ND)组; ND+Sch B组;高脂高糖饮食(HFFD)组; HFFD+Sch B组。生化法检测血清甘油三酯(TG)和总胆固醇水平;利用超高效液相色谱-四极杆飞行时间质谱联用仪(UPLCQ-TOF/MS)的代谢组学技术方法测定各组小鼠血清中脂类代谢物的变化。结果 ND+Sch B组与ND组比,筛选出27个差异代谢物,分别为TG类18个、磷脂酰胆碱(PC) 7个、磷脂酰乙醇胺(PE) 2个; HFFD组与ND组比,筛选出27个差异代谢物,分别为神经鞘磷脂6个、PC 13个、胆甾醇酯(CE) 2个、TG类5个、磷脂酰肌醇1个; HFFD+Sch B组与HFFD组比,筛选出25个差异代谢物,分别为TG类14个、CE 1个、PC 6个、PE 4个。结论 Sch B诱导的高甘油三酯血症动物模型涉及血清脂质代谢组学的改变。     

基于代谢组学探讨快松饮防治便秘的作用机制

目的 探讨快松饮防治便秘的作用机制。方法 采用复方地芬诺酯片建立小鼠和大鼠的慢传输型便秘模型。取2批小鼠，分别分为空白组，模型组，阳性对照组（麻仁软胶囊，0.64 g/kg），快松饮低、中、高剂量组（3.2、6.4、12.8 g/kg），每组10只；通过小鼠肠推进实验、小鼠排便实验考察快松饮防治便秘的效果。取大鼠分为空白组，模型组，阳性对照组（麻仁软胶囊，0.36 g/kg），快松饮低、高剂量组（2.4、4.8 g/kg），每组7或8只；每天给药1次，连续给药1周后，利用超高效液相色谱-飞行时间质谱技术对大鼠血清、尿液进行代谢组学分析。结果 与模型组比较，快松饮高剂量组小鼠墨汁推进率显著升高、5 h内排便数量显著增加（P<0.05），快松饮中、高剂量组小鼠的首次排便时间显著缩短、5 h内排便质量显著增加（P<0.05或P<0.01）。血清代谢组学筛选得到16个差异代谢物（如脯氨酸、丙酰基肉碱、硬脂酰溶血性磷脂酰胆碱等）和6条代谢通路（如鞘磷脂代谢、精氨酸和脯氨酸代谢、鞘糖脂生物合成-乳糖和新内酯系列等）；尿液代谢组学筛选得到20个差异代谢物（如前列腺素A2、L-缬氨酸...     

右美托咪啶预处理脂多糖诱导的巨噬细胞代谢物研究

摘要：目的:应用代谢组学方法,研究右美托咪啶(DEX)预处理LPS(脂多糖)诱导的巨噬细胞炎症模型代谢物的变化,探讨其抗炎机制可能性代谢途径。方法:DEX预处理LPS诱导的巨噬细胞炎症模型,利用超高效液相色谱-三重四级杆-线性离子阱质谱(UPLC-Q-TRAP-MS/MS)采集细胞代谢物信息,并采用多元变量统计方法:主成分分析法、正交偏最小二乘法判别分析法筛选细胞差异代谢物。结果:与对照组相比,DEX干预后筛选出甜菜碱、胆碱、乳酸、谷氨酰胺、蛋氨酸、S-腺苷L-甲硫氨酸、磷脂酰甘油、磷脂酰胆碱、瓜氨酸、脯氨酸、二磷酸腺苷(ADP)、环磷酸腺苷(c AMP)、丙酮酸等22种差异代谢物。结论:代谢组学方法显示DEX预处理炎症细胞特征性代谢物分子,为DEX抗炎生物机制奠定基础。     

糖尿病周围神经病变患者尿代谢组学研究

目的：采用气相色谱-质谱联用技术（GC/MS）技术,分析糖尿病周围神经病变患者尿液中小分子代谢物,为糖尿病周围神经病变的发生机制和早期诊断提供代谢水平上的依据。方法：采用气相色谱-质谱联用技术（GC/MS）检测40例正常人（正常组）、20例单纯糖尿病患者（单纯组）以及40例糖尿病周围神经病变患者（合并组）的尿液样本中小分子代谢物。采用主成分分析法（PCA）和偏最小二乘法判别分析方法（PLS-DA）观察正常组、单纯组以及合并组的代谢谱的变化。结果：正常组和单纯组的代谢谱区分良好,正常组和合并组的代谢谱也能良好的区分。利用Wiley和NIST等数据库筛选和及本实验室建立的标准品代谢物谱库,鉴定了17个与糖尿病周围神经病变密切相关的潜在生物标志物。经过代谢通路分析,糖尿病周围神经病变患者体内存在牛磺酸和亚牛磺酸代谢、甘氨酸、丝氨酸和苏氨酸代谢等代谢异常。结论：糖尿病周围神经病变发生与牛磺酸和亚牛磺酸代谢、甘氨酸,丝氨酸和苏氨酸代谢等代谢异常有关,尿液中鉴定的代谢物有望成为糖尿病周围神经病变诊断的潜在生物标志物。     

基于广泛靶向代谢组学研究甘草不同年限差异代谢物

目的:通过对不同栽培年限甘草中的代谢成分进行定性定量分析,寻找其差异代谢物,探究甘草体内代谢物的累积规律。方法:采用Agilent SB-C₁₈(100 mm×2.1 mm, 1.8μm)色谱柱,以0.1%甲酸水溶液为流动相A,0.1%甲酸乙腈溶液为流动相B,梯度洗脱,流速0.35 mL·min^-1,柱温40℃,进样量4μL;质谱采用正负离子扫描,多反应监测模式,进行样品质谱信号采集,基于自建二级质谱数据库对不同年限甘草体内的代谢物进行定性与定量分析,结合主成分分析、正交偏最小二乘法判别分析、聚类热图分析等手段对不同年限甘草的代谢物进行多元统计分析。结果:(1)从不同年限甘草样品共检测到1 038个代谢物,其中一年生与二年生甘草间存在201个差异代谢物,125个上调,76个下调;二年生与三年生之间存在223个差异代谢物,64个上调,159个下调;一年生与三年生之间存在185个差异代谢物,59个上调,126个下调;发现一年生甘草特有代谢物4个,二年生6个,三年生1个。(2)对差异代谢物进行K-均值聚类分析,按照积累趋势不同将差异代谢物进行分类,...     

终末期肾衰竭维持血液透析患者的血清代谢组学研究

目的：采用气相色谱-质谱联用（GC/MS）为基础的代谢组学技术分析终末期肾病（end stage renal disease,ESRD）维持血液透析患者血清中代谢物的变化,筛选出可用于ESRD早期临床诊断的潜在生物标志物,探讨ESRD血液透析患者代谢紊乱的发生机制。方法：采用GC/MS检测63例ESRD患者（病例组）和相匹配的20例健康人（对照组）血清中代谢物,采用偏最小二乘判别分析法（partial least squares discrimination analysis,PLS-DA）分析两组样本血清代谢谱,寻找两组之间的差异代谢物。结果：对照组和病例组的代谢谱区分良好,利用数据库NIST、Wiley Registry代谢组数据库和标准品数据库鉴定出34个差异代谢物,绘制34个差异代谢物ROC曲线,计算曲线下面积,羟胺、尿素、富马酸等28个代谢物面积大于0.5。结论：ESRD患者存在能量代谢、氨基酸代谢、脂质代谢、尿素循环、嘌呤代谢等代谢异常,血清中鉴定的羟胺、尿素、富马酸等28个代谢物有望成为诊断ESRD的潜在生物标志物。     

Novel metabolites of trichloroethylene through dechlorination reactions in rats, mice and humans

The excretion and biotransformation of [14C]trichloroethylene (Tri) has been studied in female rats and mice. Seventy-two hours after a single oral dose of 200 mg/kg, rats exhaled 52% and mice 11% of the recovered radioactivity as unchanged Tri, and 1.9% and 6%, respectively, as 14CO2. Rats excreted 41.2% of the recovered radioactivity in the urine, in contrast to mice where urinary activity amounted to 76%. The isolation of urinary metabolites was accomplished by reversed-phase HPLC, using a water-methanol gradient. After chemical derivatization, a combination of radio-GC and GC/MS was used for identification. The metabolites identified in rat urine were: trichloroacetic acid (15.3%); trichloroethanol, free (11.7%) and as the glucuronide (61.9%); dichloroacetic acid (2.0%); oxalic acid (1.3%) and N-(hydroxyacetyl)-aminoethanol (HAAE) (7.2%). In mice, trichloroethanol (free and in several conjugated forms) is the main metabolite of Tri (94.3%), but small amounts of HAAE (4.1%) and oxalic acid (0.7%) are also excreted. Only traces of dichloro- and trichloroacetic acids were found in this species. In human male subjects, HAAE was also identified as a urinary metabolite of Tri after exposure of two volunteers to 200 ppm Tri for 6 hr. The identification of HAAE and oxalic acid as metabolites indicates hydrolytic dechlorination reactions in the metabolism of Tri.     

Metabolism of the antineoplastic and immunosuppressive drug 2-CdA (Leustatin) in animals and humans

1. The in vivo metabolism of the antineoplastic and immunosuppressive drug 2-CdA (Leustatin) was investigated in mice, monkeys and humans after a single subcutaneous dose of cladribine 60 mg kg(-1) to eight male and eight female mice and 10 mg kg(-1) to one male and one female monkey, and an intravenous infusion dose of cladribine 22-45 mg(-1) per subject to 12 male patients. 2. Plasma (1 h), red blood cells (1 h) and faecal samples (0-24 h) were obtained from mice and monkeys, and urine samples (0-24 h) were obtained from these species and humans. 3. Unchanged cladribine (urine: 47% of the sample in human; 60% of the sample in mouse; 73% of the sample in monkey) and 10 metabolites, consisting of four phase I metabolites (M1-3, M7) and six phase II metabolites -- five glucuronides (M4, M6, M8-10) and one sulfate (M5) -- were profiled, characterized and tentatively identified in plasma, red blood cells, and faecal and urine samples on the basis of API ionspray-mass spectrometry (MS) and MS/MS data. 4. Metabolites were formed via the following three metabolic pathways: oxidative cleavage at the adenosine and deoxyribose linkage (A); oxidation at adenosine/deoxyribose (B); and conjugation (C). 5. Pathways A and B appear to be major steps, forming four oxidative/cleavage metabolites (M1-3, M7) (each 3-20% of the sample). 6. Pathway C along or in conjunction with pathways A and B produced cladribine glucuronide, cladribine sulfate and four glucuronides of oxidative/cleavage metabolites in minor/trace quantities (each < or = 5% of the sample). 7. In addition, the in vitro metabolism of cladribine was conducted using rat and human liver microsomal fractions in the presence of an beta-nicotinamide adenine dinucleotide phosphate-generating system. Unchanged cladribine (> or = 90% of the sample) plus three minor metabolites, M1-3 (each < 8% of the sample), were profiled and tentatively identified by thin-layer chromatography and MS data. 8. Cladribine is not extensively metabolized in vitro and in vivo in all species. However, humans appear to metabolize cladribine to a greater extent than other animals.     

基于GC-TOF/MS技术分析肾性高血压大鼠血清代谢组学变化

目的 基于气相色谱-质谱（GC-TOF/MS）技术分析两肾一夹（2K1C）肾性高血压大鼠血清代谢物的变化 情况。方法 20只健康雄性SD大鼠按照随机数字表法分为假手术（Sham）组和2K1C组,每组10只。比较建模前及 建模4周后2组大鼠体质量及尾动脉收缩压变化。采用GC-TOF/MS检测血清代谢产物的表达水平,正交偏最小二乘 法-判别分析（OPLS-DA）筛选出差异表达代谢物,并对筛选出的差异表达代谢物进行聚类分析及KEGG通路分析。 结果 4周后2K1C组共7只大鼠建模成功。与Sham组相比,2K1C组共筛选到14种差异表达代谢物,其中,花生四 烯酸（arachidonic acid）、马尿酸（hippuric acid）、七烷酸（heptadecanoic acid）、吲哚乳酸酯（indolelactate）、木糖（xylose）、 顺-巨头鲸鱼酸（cis-gondoic acid）、富马酸（fumaric acid）、胞苷-磷酸（cytidine-monophosphate）、乳酰胺（lactamide）、2- 羟基-3-异丙基丁二酸（2-hydroxy-3-isopropylbutanedioic acid）、双缩脲（biuret）这 11 种代谢物表达上调,酪氨酸 （tyrosine）、胆酸（cholic acid）、豆甾醇（stigmasterol）表达下调。以上差异表达代谢物与能量代谢、氨基酸代谢、脂质代 谢、初级胆汁酸生物合成等生物过程密切相关。结论 2K1C肾性高血压大鼠血清代谢物发生变化,并涉及能量代谢 等多种通路,进而影响肾性高血压的病理生理过程。     

姜黄素对非酒精性脂肪肝小鼠代谢组学的调控研究

目的 探讨姜黄素（CUR）对非酒精性脂肪肝病（NAFLD）的代谢调节作用。方法 小鼠适应性饲养3 d后,随机分为3组：对照（C）组、高脂饮食模型（HFD）组、CUR （50 mg/kg）组。C组饲喂普通饲料,HFD和CUR组饲喂高脂饲料,CUR同时ig给药10周,C组和HFD组ig给予0.5% CMC-Na溶液,给药体积均为10 mL/kg。给药结束后,取血和肝脏,肝组织切片后进行HE染色和油红O染色,光学显微镜观察病理变化;采用基于气相色谱-质谱联用（GC/MS）的代谢组学方法检测血清和肝脏组织样品,数据通过Simca-P13.0及Metaboanalysis网络工具进行分析,找出代谢组变化趋势和相关差异化合物。此外,制备小鼠肝原代细胞,棕榈酸（PA）100 mmol/L诱导细胞NAFLD体外模型,观察CUR （10 μmol/L）对代谢组变化的影响。结果 油红O染色结果显示,HFD小鼠肝脏组织存在大量红染油滴,CUR组小鼠肝脏组织中的脂质油滴明显减少。HE染色分析显示,与C组比较,HFD组小鼠肝脏组织有空泡状,CUR组小鼠肝脏组织与C组比较没有明显区别。对血清样和肝组织样品的GC-MS数据进行PLS-DA分析发现C组与HFD组偏离较远,而CUR组与HFD组相比出现向C组偏移的趋势,进一步S-plot分析发现影响较大的代谢为胆固醇的合成、酮体的生成以及氨基酸代谢,细胞实验得到了类似的结果;经通路MetPA分析和代谢产物富集,血清和细胞中各组间差异化合物代谢主要集中在氨基酸代谢,肝脏中代谢差异主要集中在氨基酸代谢和部分脂质代谢通路;进一步进行差异代谢物的筛选,HFD组生糖氨基酸和生酮氨基酸较对照组均有不同程度的升高,CUR能够降低这些氨基酸的水平。结论 高脂饮食会造成小鼠三大代谢的异常,姜黄素能够一定程度上改善小鼠代谢紊乱。     

Metabolism of the antineoplastic and immunosuppressive drug 2-CdA (Leustatin®) in animals and humans

1. The in vivo metabolism of the antineoplastic and immunosuppressive drug 2-CdA (Leustatin®) was investigated in mice, monkeys and humans after a single subcutaneous dose of cladribine 60?mg?kg^?1 to eight male and eight female mice and 10?mg?kg^?1 to one male and one female monkey, and an intravenous infusion dose of cladribine 22–45 mg^?1 per subject to 12 male patients.

2. Plasma (1?h), red blood cells (1?h) and faecal samples (0–24?h) were obtained from mice and monkeys, and urine samples (0–24?h) were obtained from these species and humans.

3. Unchanged cladribine (urine: 47%?of the sample in human; 60%?of the sample in mouse; 73%?of the sample in monkey) and 10 metabolites, consisting of four phase I metabolites (M1–3, M7) and six phase II metabolites—five glucuronides (M4, M6, M8–10) and one sulfate (M5)?—?were profiled, characterized and tentatively identified in plasma, red blood cells, and faecal and urine samples on the basis of API ionspray-mass spectrometry (MS) and MS/MS data.

4. Metabolites were formed via the following three metabolic pathways: oxidative cleavage at the adenosine and deoxyribose linkage (A); oxidation at adenosine/deoxyribose (B); and conjugation (C).

5. Pathways A and B appear to be major steps, forming four oxidative/cleavage metabolites (M1–3, M7) (each 3–20%?of the sample).

6. Pathway C along or in conjunction with pathways A and B produced cladribine glucuronide, cladribine sulfate and four glucuronides of oxidative/cleavage metabolites in minor/trace quantities (each?≤?5%?of the sample).

7. In addition, the in vitro metabolism of cladribine was conducted using rat and human liver microsomal fractions in the presence of an?β-nicotinamide adenine dinucleotide phosphate-generating system. Unchanged cladribine (≥?90%?of the sample) plus three minor metabolites, M1–3 (each?8. Cladribine is not extensively metabolized in vitro and in vivo in all species. However, humans appear to metabolize cladribine to a greater extent than other animals.     

Metabolic study of new psychoactive substance methoxpropamine in mice by UHPLC-QTOF-HRMS

Methoxpropamine (MXPr) is an arylcyclohexylamine dissociative drug structurally similar to 3-methoxyeticyclidine, ketamine, and deschloroketamine, recently appeared in the European illegal market, and was classified within the new psychoactive substances (NPS). Our study investigated the metabolism of MXPr to elucidate the distribution of the parent drug and its metabolites in body fluids and fur of 16 mice. After the intraperitoneal administration of MXPr (1, 3, and 10 mg/kg), urine samples from eight male and eight female mice were collected every hour for six consecutive hours and then at 12- to 24-h intervals. Additionally, plasma samples were collected 24 h after MXPr (1 and 3 mg/kg) administration. Urine and plasma were diluted 1:3 with acetonitrile/methanol (95:5) and directly injected into the UHPLC-QTOF-HRMS system. The phase-I and phase-II metabolites were preliminarily identified by means of the fragmentation patterns and the exact masses of both their precursor and fragment ions. Lastly, the mice fur was analyzed following an extraction procedure specific for the keratin matrix. Desmethyl-MXPr-glucoronide was identified in urine as the main metabolite, detected up to 24 h after administration. The presence of norMXPr in urine, plasma, and fur was also relevant, following a N-dealkylation process of the parent drug. Other metabolites that were identified in fur and plasma included desmethyl-MXPr and dihydro-MXPr. Knowledge of the MXPr metabolites evolution is likely to support their introduction as target compounds in NPS toxicological screening analysis on real samples, both to confirm intake and extend the detection window of the dissociative drug MXPr in the biological matrices.     

基于非靶向代谢组学的促乳腺癌曲妥珠单抗耐药 代谢产物分析

摘 要 目的：探究人类表皮生长因子受体2（HER2）阳性乳腺癌曲妥珠单抗（trastuzumab）耐药细胞（HCC1954）和敏感细胞（SK-BR-3）之间的代谢物差异。方法：利用超高效液相色谱-串联质谱（UHPLC-MS/MS）非靶向代谢组学方法进行代谢物的鉴定与定量,结合多元统计方法主成分分析（PCA）和正交偏最小二乘-判别分析（OPLS-DA）以筛选差异代谢物,并对差异代谢物进行KEGG通路富集分析。结果：在正负离子模式下鉴定筛选出15种显著性变化差异代谢物,在曲妥珠单抗耐药细胞中丙酰肉碱和谷胱甘肽等7种代谢物上调,肉豆蔻酸和D-葡萄糖酸等8种代谢物下调。KEGG通路富集分析发现,9条代谢通路具有统计学意义（P<0.05）,其中氮代谢和谷氨酰胺与谷氨酸代谢最为显著,P值为0.001 3,还发现谷氨酸和谷氨酰胺均在这两条通路中发挥作用。结论：本研究初步揭示了HER2阳性乳腺癌曲妥珠单抗耐药和敏感细胞之间的代谢物差异,为HER2阳性乳腺癌细胞曲妥珠单抗诊断标志物进一步研究提供了代谢组学的数据支持。     

Metabolite profiling of [14C]hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in Yucatan miniature pigs

The study reported herein examined the metabolism of 14C-labeled hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) resulting from a single oral gavage of 5 ml/kg to male and female Yucatan miniature pigs (43 mg/kg, 56 microCi/kg in 0.5% carboxymethylcellulose in water). Blood, urine, and feces were collected at selected times of 1, 6, 12, and 24 h postdose. At 24 h postdose, liver samples were collected. Blood, plasma, liver, and excreta were analyzed for total RDX-derived radioactivity and metabolites were identified. Urine was the major route of elimination of 14C-RDX-derived radioactivity in both males and females. Relatively low levels of radioactivity were found in gastrointestinal contents and in feces, suggesting nearly complete absorption of 14C-RDX following an oral dose. Analysis of urine by liquid chromatography-mass spectrometry (LC/MS) identified quantifiable levels of two ring-cleavage metabolites, 4-nitro-2,4-diazabutanal and 4-nitro-2,4-diaza-butanamide, as well as parent RDX. The 4-nitro-2,4-diazabutanal, was seen in earlier studies of aerobic metabolism of RDX. The 4-nitro-2,4-diaza-butanamide, an amide, was not previously reported but was tentatively identified in this study. Analysis by a more sensitive method (LC/MS/MS) also showed trace amounts of the RDX metabolites 1-nitroso-3,5-dinitro-1,3,5-triazacyclohexane (MNX) (in both male and female urine) and 1-nitro-3,5-dinitroso-1,3,5-triazacyclohexane (DNX) (in male urine). Analysis of plasma by LC/MS/MS also revealed quantifiable levels of RDX and trace levels of MNX, DNX, and 1,3,5-trinitroso-1,3,5-triazacyclohexane (TNX). None of the liver extracts showed quantifiable levels of RDX or any identifiable metabolites. Most of the radioactivity was in the form of water-soluble high-molecular-weight compounds. RDX when given orally to pigs was rapidly metabolized by loss of two nitro groups followed by ring cleavage.     

Sulfate metabolites as alternative markers for the detection of 4‐chlorometandienone misuse in doping control

Sulfate metabolites have been described as long‐term metabolites for some anabolic androgenic steroids (AAS). 4‐chlorometandienone (4Cl‐MTD) is one of the most frequently detected AAS in sports drug testing and it is commonly detected by monitoring metabolites excreted free or conjugated with glucuronic acid. Sulfation reactions of 4Cl‐MTD have not been studied. The aim of this work was to evaluate the sulfate fraction of 4Cl‐MTD metabolism by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) to establish potential long‐term metabolites valuable for doping control purposes. 4Cl‐MTD was administered to two healthy male volunteers and urine samples were collected up to 8 days after administration. A theoretical selected reaction monitoring (SRM) method working in negative mode was developed. Ion transitions were based on ionization and fragmentation behaviour of sulfate metabolites as well as specific neutral losses (NL of 15 Da and NL of 36 Da) of compounds with related chemical structure. Six sulfate metabolites were detected after the analysis of excretion study samples. Three of the identified metabolites were characterized by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) and gas chromatography‐tandem mass spectrometry (GC‐MS/MS). Results showed that five out of the six identified sulfate metabolites were detected in urine up to the last collected samples from both excretion studies. Copyright © 2016 John Wiley & Sons, Ltd.