基于惩罚岭型线性判别对乙酰氨基酚肝毒性代谢组学的研究

目的:研究基于惩罚岭型线性判别分析(PRLDA)对乙酰氨基酚(APAP)为肝毒性模型药物,与传统的主成分分析(PCA)比较建立的代谢组学模型的方法学。方法:应用APAP对大鼠肝毒性造模,利用UPLC-MS检测尿液样本,以总离子流色谱为毒性表达变量,运用PRLDA和PCA分别建立APAP肝毒性代谢组学模型。结果:在PRLDA代谢组学模型中,APAP组明显偏离正常组,可以直观看出正常组与模型组的类间差异,具有较强的分类能力;而PCA的分类能力较差。结论:PRLDA代谢组学模型的建立能准确、灵敏地表达药物毒性,并且该方法优于PCA模型。     

石榴皮鞣质对慢性肾小球肾炎大鼠内源性代谢物的影响

目的: 运用代谢组学方法考察石榴皮鞣质对慢性肾小球肾炎大鼠尿液内源性物质代谢的影响,寻找潜在的生物标志物并分析代谢途径,为慢性肾小球肾炎的治疗和石榴皮鞣质的利用提供相关理论依据。方法: SD大鼠随机分为5组,正常对照组、模型组、阳性对照组、石榴皮鞣质高剂量组、低剂量组。除正常对照组,其余各组均采取大鼠尾静脉注射阳离子化牛血清白蛋白(C-BSA)构建大鼠慢性肾小球肾炎(CGN)模型。收集各组大鼠术后3周尿样,经预处理后,进行液相色谱-高分辨质谱分析,获得总离子流图。对LC-MS所得数据进行预处理,导入SIMCA-P软件进行主成分分析(PCA)和偏最小二乘法判别分析(PLS-DA),根据获得的得分图、载荷图以及VIP值的分析结果,寻找潜在的生物标志物。结果: 代谢组学分析发现造模后第3周各组大鼠尿液有明显的聚类现象,通过对重要变量的分析鉴定,筛选出12个潜在的生物标志物。结论: 石榴皮鞣质干预下的慢性肾小球肾炎大鼠内源性代谢物聚类性趋近正常水平,代谢组学分析法寻找潜在生物标志物可行且有效。     

高血压病肝阳上亢证患者血清样品的核磁共振谱代谢组学研究

目的采用1HNMR结合主成分分析（PCA）方法研究高血压病肝阳上亢证患者和健康志愿者的血液成分谱差异,寻找可能的生物标记物,揭示高血压病肝阳上亢的证候本质。方法根据临床高血压病肝阳上亢证的诊断标准,选择典型病例,以1HNMR分析高血压病肝阳上亢证患者和健康志愿者的血液成分,使用PCA方法进行模式识别,寻找标记物并做出相关代谢途径的可能解释。结果PCA方法处理高血压病肝阳上亢证患者和健康志愿者的血清1HNMR谱数据显示,两组1HNMR谱数据可以在得分图实现分类,与健康志愿者作比较,患者机体相关代谢发生显著变化,体内部分氨基酸代谢和葡萄糖代谢明显异于健康志愿者。结论1HNMR结合PCA模式识别的代谢组学方法具有研究复杂条件下机体病理生理变化的优势,为理解高血压病肝阳上亢证的证侯本质和诊断该类病证提供了科学依据。     

基于细胞代谢组学的商陆皂苷甲诱导HKC细胞损伤的机制研究

目的 基于细胞代谢组学技术,考察商陆皂苷甲（EsA）诱导人肾小管上皮HKC细胞损伤前后对细胞代谢产物的影响,寻找潜在的生物标志物。方法 MTT法检测EsA 0（对照组）、0.125、0.250、0.500、0.750、1.000 mmol/L对HKC细胞活力的影响;试剂盒法检测EsA 0（对照组）、0.25、0.75 mmol/L对HKC细胞上清LDH含量的影响,明确EsA肾细胞毒性;利用UPLC-Q/TOF-MS分析技术,将EsA 0（对照组）、0.25、0.75 mmol/L作用24 h的HKC细胞进行代谢组学分析,采用Progenesis QI软件进行数据处理,将得到的数据导入SIMCA-P12.0 software进行多元统计分析,利用主成分分析（PCA）剔除离群值,进行正交偏最小二乘分析（OPLS-DA）得到变量权重值（VIP）,选取VIP＞1的差异代谢物,进行检验,获得具有统计学意义的生物标志物。通过HMDB、KEGG、METLIN等代谢物数据库对生物标记物进行鉴定,利用MetPA平台对进行代谢通路分析。结果 与对照组比较,EsA在大于0.25 mmol/L时对HKC细胞活力有显著的抑制作用（P<0.05、0.01）;细胞上清液中LDH含量显著上升（P<0.05）。通过代谢组学技术和相关数据库分析筛选出了15个生物标志物,主要涉及甘油磷脂代谢、嘧啶代谢、鞘脂代谢、氨基酸代谢和能量代谢途径。结论 EsA可诱导肾细胞损伤,可能与氧化应激损伤,氨基酸代谢失调,能量代谢异常以及脂质代谢紊乱相关。     

慢性温和不可预知应激抑郁模型大鼠脑组织1H-NMR代谢组学研究

目的 采用高分辨核磁共振¹H谱（¹H-NMR）代谢组学技术研究慢性温和不可预知应激（CUMS）抑郁大鼠脑组织中代谢物及代谢通路的变化，探讨抑郁症的发病机制。方法 12只雄性SD大鼠随机分为模型组和对照组，采用CUMS对模型组大鼠进行为期4周的造模，进行称体质量、旷场实验和糖水偏爱实验验证模型是否成功，造模结束后收集大鼠脑组织。采用两相提取法（甲醇/氯仿/水）对脑组织进行提取，得到水溶性和脂溶性代谢物。应用¹H-NMR技术结合多元统计和代谢通路分析筛选出与抑郁相关的脑内差异代谢物，并构建其代谢通路。结果 行为学数据显示，与对照组比较，模型组大鼠的体质量、糖水偏爱率、旷场的穿格数和直立次数均显著降低（P<0.05），显示了抑郁状态。在大鼠脑组织的¹H-NMR图谱中共指认出35种内源性代谢产物。脑组织中水溶性和脂溶性代谢物主成分分析（PCA）均显示模型组与对照组分开，与行为学结果一致，表明造模成功；OPLS-DA分析找到9个水溶性差异代谢物和6个脂溶性差异代谢物。就水溶性代谢物而言，与对照组比较，模型组中肌酐、谷氨酰胺、牛磺酸和γ-氨基丁酸含量显著增加（P<0.05、0.01、0.001），丙二醇、谷氨酸、亮氨酸、缬氨酸和赖氨酸含量显著降低（P<0.05、0.01、0.001）。就脂溶性代谢物而言，与对照组比较，模型组-（CH₂） n和-N （Me₃）₃含量显著升高（P<0.05、0.001），胆固醇的C18/19甲基、R-CH₃、CH₂OPO₂-、-CH=CH-含量显著降低（P<0.05、0.01、0.001）。与对照组比较，CUMS造模后5条代谢通路发生显著变化：缬氨酸、亮氨酸和异亮氨酸生物合成，牛磺酸和亚硫磺酸代谢，丙氨酸、天冬氨酸和谷氨酸代谢，糖降解和糖异生通路和丙酮酸代谢。结论 运用¹H-NMR代谢组学技术结合多元统计分析和代谢通路分析，阐明抑郁症的发病机制与能量代谢、氨基酸代谢和神经递质合成等相关。     

基于代谢组学和网络药理学分析福建金线莲叶和台湾银线兰叶治疗肝纤维化作用差异

目的 初步分析福建金线莲(Anoectochilus roxburghii)和台湾银线兰(Anoectochilus formosanus)的叶片在治疗肝纤维化疾病的作用机制差异，为分析两者的临床疗效差异及其资源的精准利用提供参考信息。方法 利用代谢组学技术分析福建金线莲叶(the leaves of A.roxburghii，Arl)和台湾银线兰叶(the leaves of A.formosanus，Afl)的差异代谢物；并结合网络药理学方法分析其差异代谢物治疗肝纤维化疾病的作用差异。结果 利用代谢组学的方法分析初步得到Arl和Afl的46个差异代谢物，主要为黄酮类、糖苷类和甘油磷脂类等，其中有23种差异化合物具有潜在药用活性。基于网络药理学的方法，预测了23种差异化合物的759个相关靶点，与肝纤维化疾病的相关靶点进行映射，得到交集靶点249个，其中AKT1、STAT3、VEGFA等48个关键靶点被预测为治疗肝纤维化的主要差异靶点；作用靶点较多的关键差异化合物为Arl相比Afl含量较高的化合物，预示Arl中富含更多可以用于治疗肝纤维化的有效化合物。GO富集功能和KEGG富集分析表明，福建金线莲与台湾银线兰的差异成分参与治疗肝纤维化疾病的信号通路有所不同，VEGF信号通路为Arl相比Afl含量较高的差异化合物的主要差异信号通路，磷脂酰肌酶3-激酶-蛋白激酶B(PI3K-Akt)信号通路为Arl相比Afl含量较低的差异化合物治疗肝纤维化疾病的主要差异信号通路。分子对接结果表明各差异成分与其对应的关键靶点均有较好的结合活性。结论 本研究表明福建金线莲与台湾银线兰叶片中存在一些差异代谢物，预测了其治疗肝纤维化疾病作用机制差异，并初步验证了其作用靶点，为金线莲资源的精准利用具有指导性意义。     

基于液质联用技术的急性淋巴细胞白血病血清代谢组学分析

目的:对急性淋巴细胞白血病(ALL)患者血清进行代谢组学分析,寻找与ALL相关的潜在代谢生物标志物,以探索ALL患者血清代谢组学一般规律。方法:运用代谢组学方法,采用超高效液相色谱-质谱联用技术(UPLC-MS)对ALL患者和正常人的血清样本进行代谢组学检测,原始数据经过Compound Discoverer 2.0软件处理,进一步应用SIMCA-P+软件对代谢组学数据进行主成分分析(PCA)及正交-偏最小二乘判别分析(OPLS-DA),寻找与ALL相关的差异代谢物。结果:多变量统计结果显示,与健康对照组相比,ALL患者存在异常的糖类、脂肪酸和氨基酸代谢,并初步鉴定出8种差异代谢产物,分别为癸酰肉碱、花生四烯酸、鞘氨醇-1-磷酸盐、乳酸、哌啶酸、柠檬酸、肌酸和棕榈酰肉碱。结论:ALL患者与对照组血清的代谢谱存在明显的差异,鉴定出的差异代谢物对ALL的诊断和治疗具有重要意义,可为研究ALL及临床用药提供新的思路和方法。     

基于毛细管电泳法人血清的代谢组学研究

目的 本文以毛细管电泳(CE)技术对肿瘤患者血清代谢组学规律的研究.方法 非涂层石英毛细管柱(57cm×75μm);分离电压30kv,毛细管柱温20℃,检测波长为200nm,毛细管电泳背景电解质:40mmol/L硼砂+5mmol/LMSDS,pH10.0;进样量为5s×0.5psi;图谱中数据用32Karat软件导出,导出数据经SIMCA-P 12.0的PLS-DA法进行模式识别分析.结果 血清中各代谢组分被完全分离,日内和日间精密度(RSD)分别为0.18％～4.25％和1.14％～4.65％;健康组血清和肿瘤组血清全组分分析能较好聚类区分;提取出11个高贡献率的代谢物并能对健康组和肿瘤组聚类区分;采用该方法获得的代谢物变化与同时检测出CA125值的变化相对一致.结论 代谢组学的CE法检测和研究可能成为肿瘤早期诊断潜在标志物研究的又一种方法,所体现的关联代谢组分可为进一步肿瘤的早期诊断生物标志物的研究提供一定的基础依据.     

代谢组学数据处理方法——主成分分析

代谢组学在生命科学领域得到了越来越广泛的应用并展现出良好的前景。代谢组学分析产生的含有大量变量的数据难以用常规方法进行分析,如何正确分析和解释代谢组学的数据是研究的关键。本文主要介绍了在代谢组学数据分析中占主导地位的主成分分析基本方法,旨在加强代谢组学数据分析的基础知识并规范数据分析的方法。     

人体肠道菌群体外生物转化大黄过程优化及成分变化研究

目的 探究人体肠道菌群体外生物转化大黄过程条件及成分变化。方法 采集3名健康儿童（1～3岁）新鲜粪便制备菌群悬液;以活菌数保持最多为依据进行初始培养基的选择;按照初始培养基中原有的0.5%碳源和0.5%氮源的添加量,进行不同碳源/氮源组合,依据活菌计数结果判断最优组合;通过指纹图谱技术筛选菌群体外培养条件,包括大黄添加方式（大黄粉或大黄水浸液）、转化时间（24或48 h）、氧需求（有氧或无氧）;基于LC-MS代谢组学进行转化前后成分分析,采用SIMCA软件（V16.0.2）进行主成分分析（PCA）,差异代谢物分析采用MetaboAnalyst 5.0。结果 菌落计数和指纹图谱结果显示,蔗糖/大豆胨为改良YCFA培养基最适组合,粉状大黄为添加方式,培养基与大黄粉最适添加体积质量比为32∶1,有氧培养24 h为最优生物转化条件。代谢组学结果表明,主要代谢产物共计763种,数目较多的代谢物类别分别为脂质和类脂质分子（244种）、苯丙烷和聚酮化合物（138种）、有机杂环化合物（101种）、有机酸及其衍生物（92种）和苯类（59种）;转化前后代谢物PCA图能良好区分,确定18种关键差异代谢物,山柰酚-3-O-刺槐二糖苷和薯蓣皂苷上调最明显。结论 采用指纹图谱技术联合代谢组学方法揭示了大黄在菌群生物转化前后存在的成分差异,通过菌群生物转化可提高大黄活性成分的溶出和转变。     

Progress toward automated metabolic profiling of human serum: comparison of CPMG and gradient-filtered NMR analytical methods

The investigation of drug delivery and metabolism requires the analysis of molecules in complicated biological matrices such as human serum. In NMR-based metabonomic analysis, T(2) relaxation editing with a CPMG filter is commonly used to suppress background signals from proteins and other endogenous components. Radio frequency pulse imperfections and incomplete irradiation across the spectral bandwidth can cause phase and baseline distortions in CPMG spectra. These distortions are exacerbated by water suppression techniques. Baseline correction methods included in commercially available data processing software packages may be incapable of producing artifact-free spectra. To increase the analytical precision of metabolic profiling, one NMR spectroscopist may be responsible for manually phasing and baseline correcting hundreds of spectra individually to remove operator-dependent variations, significantly reducing throughput. For metabonomic analysis of human serum, it was observed that the application of a pulsed field gradient filter produced (1)H NMR spectra well suited to automatic phasing routines. Superior baseline characteristics, an increased tolerance to radio frequency pulse imperfections, and improved water suppression were achieved. A concomitant reduction in signal intensity compared with the CPMG method was easily recovered by increasing the number of scans. Principal component analysis (PCA) of spectra, acquired under a variety of experimental conditions, revealed the improved reproducibility and robustness of (1)H NMR pulsed field gradient-filtered metabonomic analyses of serum compared to the CPMG method.     

多中心临床试验中实验室数据的定性分析方法

目的：探讨多中心临床试验中实验室数据的定性分析方法.方法：通过计算疗前正常者疗后异常的发生率,判断药物对实验室指标的影响;计算疗后实验室检查结果高于疗前者的比例,检验试验结果的随机性.结果：多中心临床试验中实验室定性资料的分析可以抵消年龄、性别、中心间差异的影响,为判定药物的安全性提供重要线索.结论：多中心临床试验中实验室资料既要进行定性分析,又要进行定量分析.     

气阴两虚证心肌缺血模型方证对应的代谢组学表征

为了探索方证对应的科学内涵,本研究以气阴两虚证心肌缺血为模型,以基于气相色谱/飞行时间质谱(GC-TOF/MS)检测技术的代谢组学方法为技术手段,对比分析了生脉注射液、丹参注射液和普萘洛尔的保护作用。数据处理和模式识别后,无论从PLS-DA散点图直观观察还是药物纠正的异常的内源性化合物个数来看,生脉注射液都显示优于其他两种药物的效果,进一步用与心血管和睡眠密切相关的指标内皮素-1验证了代谢组学的结果。最后分析了受到调节的化合物及相关生化代谢通路,推测生脉注射液的作用与调节能量代谢、脂代谢、氨基酸代谢等多条代谢途径有关,这为生脉方对应用于气阴两虚证心肌缺血提供了科学的证据。此发现说明代谢组学在中医药研究中具有巨大的潜力,这为传统中医理论的现代化研究提供了新的研究思路与方法。     

Development and validation of urinary nucleosides and creatinine assay by capillary electrophoresis with solid phase extraction

For the analysis of metabolite nucleoside profiles, capillary electrophoretic (CE) methods preceded by appropriate solid phase extraction procedures have been developed. The approach has been proposed for the determination of 13 nucleosides and creatinine in human urine. A background solution composed of 100 mM borate–72 mM phosphate–160 mM SDS and a fused silica capillary of 70 cm length to detector and 50 μm i.d. were used. The methods developed were statistically validated for their linearity, trueness, precision and selectivity. Stability of the analyzed nucleoside profiles in urine during storage was checked. Validation parameters of solid phase extraction procedures for urinary nucleosides were evaluated. The developed analytical methods were employed for the analysis of 22 urine samples from healthy patients and cancer patients from the urological ward. Nucleoside profiles were compared among the subjects. It was proved that the methods proposed were suitable for a fast and reliable determination of urinary creatinine and modified nucleoside profiles, which can be further submitted for the metabonomic analysis of cancer patients.     

Application of adsorption method to the chromatographic analysis of free drug concentration

The development of analytical techniques for estimation of free drug concentration is crucial for the needs of modern pharmacology. Up to now, many different methods of free drug assay have been used. The methods involving isolation of free drug from a sample are most frequently applied because they can be easily adopted for the processing of real samples. Among the methods that do not require free drug isolation only the adsorption method could be promising in application to samples of this kind. The successful use of the adsorption method is possible only if two requirements are fulfilled: (I) the chosen adsorbent does not bind proteins from the sample and (II) the processes of adsorption of the assayed drug on the adsorbent used and on the protein should be independent. This paper discusses the possibilities of application of the adsorption method for determination of free drug concentration using propofol as the model drug. The presented results show that the fulfillment of the above conditions may be very difficult if not impossible not only for propofol, but for other drugs as well.     

Confidence interval of the difference between two proportions with overdispersion

In confidence interval estimation of the difference between two proportions with overdispersion due to positive correlations, the usual asymptotic normality-based method generally has lower coverage rates than desired, especially when sample size is moderate. Applying the concept of effective sample size to existing methods for independent data, we propose three new asymptotic normality-based methods. It is demonstrated through an extensive Monte Carlo study that the proposed methods generally perform better than the usual method. The proposed methods are illustrated in the application to a motivating data example.     

NMR-based metabonomic analysis of the hepatotoxicity induced by combined exposure to PCBs and TCDD in rats

A metabonomic approach using ¹H NMR spectroscopy was adopted to investigate the metabonomic pattern of rat urine after oral administration of environmental endocrine disruptors (EDs) polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) alone or in combination and to explore the possible hepatotoxic mechanisms of combined exposure to PCBs and TCDD. ¹H NMR spectra of urines collected 24 h before and after exposure were analyzed via pattern recognition by using principal component analysis (PCA). Serum biochemistry and liver histopathology indicated significant hepatotoxicity in the rats of the combined group. The PCA scores plots of urinary ¹H NMR data showed that all the treatment groups could be easily distinguished from the control group, so could the PCBs or TCDD group and the combined group. The loadings plots of the PCA revealed remarkable increases in the levels of lactate, glucose, taurine, creatine, and 2-hydroxy-isovaleric acid and reductions in the levels of 2-oxoglutarate, citrate, succinate, hippurate, and trimethylamine-N-oxide in rat urine after exposure. These changes were more striking in the combined group. The changed metabolites may be considered possible biomarker for the hepatotoxicity. The present study demonstrates that combined exposure to PCBs and TCDD induced significant hepatotoxicity in rats, and mitochondrial dysfunction and fatty acid metabolism perturbations might contribute to the hepatotoxicity. There was good conformity between changes in the urine metabonomic pattern and those in serum biochemistry and liver histopathology. These results showed that the NMR-based metabonomic approach may provide a promising technique for the evaluation of the combined toxicity of EDs.     

Cross-Validation for Linear Model with Unequal Variances in Genomic Analysis

Abstract

In recent years, genomic studies are usually conducted to identify genes that may have an impact on clinical outcomes. The identified genes are then used to establish a predictive model for identifying subjects who are most likely to respond to the test treatment in clinical trials. This information is useful in early and later phases of clinical development. The United States Food and Drug Administration (FDA) requires that such a predictive model be validated before it can be used in clinical development. Shao [Shao, J. (1993). Linear model selection by cross-validation. J. Amer. Statist. Assoc. 88(422):486–494] proposed a cross-validation method for linear model with equal variances, which is found useful in genomic studies. In practice, however, genomic data may be obtained from different sources with unequal variances. As a result, Shao’s method may not be applied directly. In this paper, we extend Shao’s method for cross-validation of a linear model with unequal variances. Along this line, two re-sampling methods were proposed to account for the heterogeneity in variance. Several simulations were performed to evaluate the finite samples performances of the proposed methods. An example concerning a breast cancer research is present to illustrate the use of the proposed methods.     

Applying biofluid 1H NMR-based metabonomic techniques to distinguish between HIV-1 positive/AIDS patients on antiretroviral treatment and HIV-1 negative individuals

Metabonomics, the study of metabolites and their role in various physiological states, is a novel methodology arising from the post-genomics era and has extensive biomedical application. This technology has exhibited vast success in the identification and study of human diseases and may find further application in the study of HIV/AIDS. Specifically, the wide range of clinical and metabolic abnormalities associated with the use of antiretroviral (ARV) treatment may be investigated. To this end, this preliminary study evaluated whether metabonomic techniques could distinguish between HIV-1 positive/AIDS patients utilizing antiretroviral therapy and HIV-1 negative individuals. Serum metabolic profiles determined by 1H nuclear magnetic resonance (NMR) spectroscopy combined with pattern recognition analysis of the data showed that this distinction was attainable; suggesting that ARV-associated side-effects could be monitored utilizing NMR metabonomic techniques.     

基于化学计量学分析的桃仁炮制前后指纹图谱研究

目的 建立桃仁炮制前、后HPLC指纹图谱，比较桃仁炮制前后的质量差异以及桃仁与种皮的差异。方法 采用高效液相色谱法测定桃仁饮片（生桃仁、燀桃仁、炒桃仁）各10批的指纹图谱，使用"中药色谱指纹图谱相似度评价系统（2012.130723版）"进行相似度评价，并采用SIMCA统计软件对结果进行偏最小二乘法判别分析。结果 生桃仁、燀桃仁、炒桃仁HPLC指纹图谱均含8个共有峰、种皮HPLC指纹图谱含有6个共有峰，相对应的指纹图谱相似度均>0.90。统计结果显示桃仁饮片炮制前、后的指纹图谱可明显区分，并揭示了对此区分贡献最大的4个潜在标志性色谱峰。结论 该方法准确、稳定，反映了桃仁饮片炮制前后以及桃仁与种皮的差异性，为桃仁饮片临床用药、质量控制提供一定的参考。