Urinary signature of anabolic steroids and glucocorticoids in humans by LC-MS

A metabonomic strategy based on LC-MS was employed to investigate the metabolic profile of urine samples from 20 athletes who had been tested positive for corticoids and anabolic steroids and 29 controls. In this aim, different sample preparations and chromatographic conditions were compared. The acquired LC-MS data of doped athletes and controls were subjected to analysis of variance (ANOVA) and principal component analysis (PCA). Using this approach, molecular signature of human urine was obtained showing that metabonomics could be a complementary tool to discriminate different urinary profiles and to track down metabolic changes in humans.     

Human Plasma Metabolic Profiles of Coronary Heart Disease by Gas Chromatography-Mass Spectrometry with Monte Carlo Tree Approach

Metabolomics is a useful approach to explore systemic metabolic variation and to elucidate disease mechanisms. In this study, human plasma metabolic profiles of coronary heart disease (CHD) patients and healthy controls were obtained by gas chromatography-mass spectrometry (GC-MS). A relatively new pattern recognition method, the Monte Carlo tree (MCTree) approach, was used to explore metabolic differences between CHD patients and healthy controls. In this way, CHD patients with different severity of coronary atherosclerosis were classified by the corresponding metabolic profiles. Furthermore, important metabolites contributing to the classification were screened and identified by their mass spectra. Several potential biomarkers were discussed in some detail. The results demonstrated that the proposed method might be a useful tool for discovering metabolic abnormalities and potential biomarkers for diseases.     

基于气相色谱-质谱联用和图模型分析的脂代谢异常患者血浆代谢组学研究

利用气相色谱-质谱联用技术(GC-MS)和图模型分析方法,寻找脂代谢异常患者可能的血浆代谢标志物群。采用GC-MS技术对37例脂代谢异常患者和10例健康人的血浆样品进行分析,得到血浆代谢物的表达谱。偏最小二乘-判别分析(Partial least squares-discriminant analysis,PLS-DA)得分图可区分脂代谢异常患者与健康人,运用PLS-DA载荷图及t检验发现有9个代谢物在两组间存在显著性差异。经NIST谱库检索,它们分别为缬氨酸、甘氨酸、丙氨酸、焦谷氨酸、葡萄糖醛酸、半乳糖、甘露糖、亚油酸和甘油。在脂代谢异常患者血浆中,除甘油浓度显著高于健康人外,其余8种代谢物浓度均明显低于健康人。图模型分析结果发现这些代谢物与脂代谢异常临床常用诊断指标之间具有很好的相关性。它们可能是脂代谢异常疾病早期诊断和预后新的特异性代谢标志物群。     

Serum fatty acid profiles and potential biomarkers of ankylosing spondylitis determined by gas chromatography–mass spectrometry and multivariate statistical analysis

Ankylosing spondylitis (AS) is a common chronic inflammatory rheumatic disease. Early and accurate detection is essential for effective disease treatment. Recently, research has focused on genomics and proteomics. However, the associated metabolic variations, especially fatty acid profiles, have been poorly discussed. In this study, the gas chromatography–mass spectrometry (GC‐MS) approach and multivariate statistical analysis were used to investigate the metabolic profiles of serum free fatty acids (FFAs) and esterified fatty acids (EFAs) in AS patients. The results showed that significant differences in most of the FFA (C12:0, C16:0, C16:1, C18:3, C20:4, C20:5, C22:5 and C22:6) and EFA (C12:0, C16:1, C18:0, C18:1, C18:2, C18:3, C20:4 and C22:6) concentrations were found between the AS patients and healthy controls (p < 0.05). Principal component analysis and partial least squares discriminant analysis were performed to classify the AS patients and controls. Additionally, FFAs C20:4, C12:0, C18:3 and EFAs C22:6, C12:0 were confirmed as potential biomarkers to identify AS patients and healthy controls. The present study highlights that differences in the serum FFA and EFA profiles of AS patients reflect the metabolic disorder. Moreover, FFA and EFA biomarkers appear to have clinical applications for the screening and diagnosis of AS. Copyright © 2014 John Wiley & Sons, Ltd.     

基于液相色谱-质谱联用技术的代谢组学方法研究薄荷烟对大鼠代谢的影响

将基于液相色谱-质谱联用(LC-MS)技术的代谢组学分析平台用于薄荷烟对大鼠代谢影响的研究。分析了3组大鼠的尿样,包括对照大鼠、吸食普通烟大鼠和吸食薄荷烟大鼠,并采用主成分分析(PCA)方法对数据进行模式识别。PCA得分图表明吸食薄荷烟大鼠与对照组大鼠尿样的代谢差异要小于吸食普通烟大鼠。从PCA载荷图中找到并鉴定了犬尿喹啉酸等8种重要代谢物。通过考察代谢物在对照大鼠、吸食薄荷烟大鼠和吸食普通烟大鼠尿样中的相对含量变化,进一步说明了烟草中添加薄荷醇可减少烟草对大鼠代谢的影响。     

Metabonomics study on the effect of Siwu Decoction for blood deficiency syndrome in rats using UPLC–Q/TOF–MS analysis

Siwu decoction (SWD), a traditional Chinese medicinal formula with over 1000 years of clinical history, is widely used for gynecological disease, especially blood deficiency syndrome, which is similar to anemia in modern medicine. In view of metabonomics being useful approach to investigate the potential mechanisms of action from the point of view of systems biology, in this study an ultra‐performance liquid chromatography–quadrupole‐time of flight mass spectrometry method was employed for a holistic evaluation of SWD on a blood‐deficiency rat model induced by N‐acetylphenylhydrazine and cyclophosphamide via plasma metabonomics study. Routine blood examination results showed that SWD could significantly improve the declining hemogram indices. Meanwhile, the plasma metabonomics profiles in different groups were analyzed and differentiating metabolites were primarily visualized through chemometric analysis. Seven biomarkers were identified in plasma samples of blood‐deficiency rat model compared with the normal group. Five main metabolism pathways were suggested using the Kyoto Encyclopedia of Genes and Genomes Pathway Analysis and Pathway Activity Profiling algorithm analysis. This indicated that SWD played a therapeu role in blood deficiency by regulating the aberrant endogenous metabolites. To sum up, this study provides clear evidence that a metabonomics study could serve as a useful tool to elucidate the systematic therapeutic profiles and mechanisms for blood deficiency syndrome of Chinese herbal medicines.     

Elucidation of urinary metabolites of fluoxymesterone by liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry

The suitability of liquid chromatography tandem mass spectrometry (LC-MS/MS) and gas chromatography mass spectrometry (GC-MS) for the elucidation of fluoxymesterone metabolism has been evaluated. Electrospray ionization (ESI) and collision induced dissociation (CID) fragmentation in LC-MS/MS and electron impact spectra (EI) in GC-MS have been studied for fluoxymesterone and two commercially available metabolites. MS(n) experiments and accurate mass measurements performed by an ion-trap analyser and a QTOF instrument respectively have been used for the elucidation of the fragmentation pathway. The neutral loss scan of 20 Da (loss of HF) in LC-MS/MS has been applied for the selective detection of fluoxymesterone metabolites. In a positive fluoxymesterone doping control sample, 9 different analytes have been detected including the parent compound. Seven of these metabolites were also confirmed by GC-MS including 5 previously unreported metabolites. On the basis of the ionization, the CID fragmentation, the accurate mass of the product ions and the EI spectra of these analytes, a tentative elucidation as well as a proposal for the metabolic pathway of fluoxymesterone has been suggested. The presence of these compounds has also been confirmed by the analysis of five other positive fluoxymesterone urine samples.     

Postprandial metabolomics: A pilot mass spectrometry and NMR study of the human plasma metabolome in response to a challenge meal

The study of postprandial metabolism is relevant for understanding metabolic diseases and characterizing personal responses to diet. We combined three analytical platforms – gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) – to validate a multi-platform approach for characterizing individual variation in the postprandial state. We analyzed the postprandial plasma metabolome by introducing, at three occasions, meal challenges on a usual diet, and 1.5 years later, on a modified background diet. The postprandial response was stable over time and largely independent of the background diet as revealed by all three analytical platforms. Coverage of the metabolome between NMR and GC-MS included more polar metabolites detectable only by NMR and more hydrophobic compounds detected by GC-MS. The variability across three separate testing occasions among the identified metabolites was in the range of 1.1–86% for GC-MS and 0.9–42% for NMR in the fasting state at baseline. For the LC-MS analysis, the coefficients of variation of the detected compounds in the fasting state at baseline were in the range of 2–97% for the positive and 4–69% for the negative mode. Multivariate analysis (MVA) of metabolites detected with GC-MS revealed that for both background diets, levels of postprandial amino acids and sugars increased whereas those of fatty acids decreased at 0.5 h after the meal was consumed, reflecting the expected response to the challenge meal. MVA of NMR data revealed increasing postprandial levels of amino acids and other organic acids together with decreasing levels of acetoacetate and 3-hydroxybutanoic acid, also independent of the background diet. Together these data show that the postprandial response to the same challenge meal was stable even though it was tested 1.5 years apart, and that it was largely independent of background diet. This work demonstrates the efficacy of a multi-platform metabolomics approach followed by multivariate and univariate data analysis for a broad-scale screen of the individual metabolome, particularly for studies using repeated measures to determine dietary response phenotype.     

Metabonomics Study of Heart Homogenates from Myocardial Infarction Rats Using Liquid Chromatography/Time of Flight Mass Spectrometry

In this study, a metabonomics analysis of heart homogenates from myocardial ischemic rats was performed by LC–TOF–MS. Hydrophilic interaction chromatography (HILIC) was used to separate the endogenous metabolites in heart homogenates. Partial least squares to latent structure-discriminant analysis (PLS-DA) was used for data analysis. Good separations were observed between the normal and model groups and 15 potential biomarkers were identified. The major disturbed metabolic pathways were purine metabolism, pyrimidine metabolism, urea cycle, and energy metabolism. The results demonstrated that a metabonomics approach based on HILIC-MS was useful for studying metabolic mechanism on target tissue of the myocardial infarction rat.

     

Classification and differential metabolite discovery of liver diseases based on plasma metabolic profiling and support vector machines

Discovery of differential metabolites is the focus of metabonomics study. It has very important applications in pathogenesis and disease classification. The aim of this work is to identify differential metabolites for classifying the patients with hepatocellular carcinoma, cirrhosis and hepatitis based on metabolic profiling data analyzed by gas chromatography-time of flight mass spectrometry. A two-stage feature selection algorithm, F-SVM, combining F-score in analysis of variance and support vector machine (SVM), was applied in discovering discriminative metabolites for three different types of liver diseases. The results show that the accuracy rate of the double cross-validation was 73.68±2.98%. 22 important differential metabolites selected by F-SVM were identified and related pathophysiological process of liver diseases was set forth. We conclude that F-SVM is quite feasible to be applied in the selection of biologically relevant features in metabonomics.     

Metabolomic characterization of semen from asthenozoospermic patients using ultra-high-performance liquid chromatography–tandem quadrupole time-of-flight mass spectrometry

Asthenozoospermia (AS) is a common factor of male infertility, and its pathogenesis remains unclear. The purpose of this study was to investigate the differential seminal plasma metabolic pattern in asthenozoospermic men and to identify potential biomarkers in relation to spermatogenic dysfunction using sensitive ultra-high-performance liquid chromatography–tandem quadruple time-of-flight MS (UHPLC–Q-TOF/MS). The samples of seminal plasma from patients with AS (n = 20) and healthy controls (n = 20) were checked and differentiated by UHPLC–Q-TOF/MS. Compared with the control group, the AS group showed a total of nine significantly different metabolites, including increases in creatinine, uric acid, N⁶-methyladenosine (m⁶A), uridine, and taurine and decreases in carnitine, nicotinamide, N-acetylputrescine and l -palmitoylcarnitine. By analyzing the correlation among these metabolites and clinical computer-assisted semen analysis reports, we found that m⁶A is significantly correlated with not only the four decreased metabolites but also with sperm count, motility, and curvilinear velocity. Furthermore, nicotinamide was shown to correlate with other identified metabolites, indicating its important role in the metabolic pathway of AS. Current results implied that sensitive untargeted seminal plasma metabolomics could identify distinct metabolic patterns of AS and would help clinicians by offering novel cues for discovering the pathogenesis of male infertility.     

A new metabonomics method for simultaneous determination of EFAs and NEFAs in plasma using GC-MS and its application

A new metabonomics method was developed for simultaneous qualitative and quantitative analysis on esterified and nonesterified fatty acids(EFAs and NEFAs) in plasma.Merely 10μL of plasma was required.The pretreatment of the sample was simple without disposing the protein.After simple extraction and derivation,15 FAs in plasma were precisely quantified.Gas chromatography tandem mass spectrometry(GC-MS) was used in the study and the quantities of the analytes,which varied in abundance over three orders of ...     

细胞代谢组学用于羽扇豆醇干预人乳腺癌细胞MCF-7的机理探究

应用基于气相色谱-质谱联用（GC-MS）的代谢组学方法结合细胞周期实验,研究羽扇豆醇体外抑制人乳腺癌细胞MCF-7增殖的作用机理。代谢组学的研究结果表明：通过正交偏最小方差判别分析（OPLS-DA）可以很好地区分羽扇豆醇作用的MCF-7细胞代谢谱与对照组细胞代谢谱,模型参数为：R²Ycum=0.988,Q²Ycum=0.964。VIP（variable importance in the projection）值大于1的差异代谢物进一步用t检验进行单位分析,选择t<0.05（VIP>1）的代谢物作为羽扇豆醇作用组的生物标志物,得到琥珀酸、磷酸、亮氨酸、异亮氨酸等11种代谢差异物。结合羽扇豆醇将细胞周期抑制在G1期这一现象,推测羽扇豆醇可能是主要抑制了三羧酸循环中的琥珀酰辅酶A的生成和底物磷酸化生成ATP的反应来抑制MCF-7细胞的增殖。本实验从代谢组学角度为乳腺癌抗肿瘤机制提供新的线索。     

In vivo toxicity screening programs using metabonomics

Metabonomics is an emerging technology that enables rapid in vivo screening for toxicity, disease state, or drug efficacy. The technology combines the power of high-resolution nuclear magnetic resonance (NMR) techniques with statistical data analysis methods to rapidly evaluate the metabolic "status" of an animal. Complimentary to other profiling technologies like proteomics and genomics, metabonomics provides a fingerprint of the small-molecules contained in a given biofluid through the time course of a study. This article reviews the steps in implementing a metabonomics-based screening program from study design through data analysis. While metabonomics is still a relatively new technology in comparison to the other "omics", published results from metabonomics studies demonstrate its potential impact in the drug discovery process by enabling the incorporation of safety endpoints much earlier in the drug discovery process, reducing the likelihood (and cost) of later stage attrition.     

一种基于液相色谱-质谱技术进行血清代谢组学研究的方法：从代谢指纹到潜在标志物

本文描述了一种基于液相色谱-质谱技术（LC-MS）的代谢组学发现疾病潜在标志物的方法.该方法利用LC-MS获得代谢指纹图谱,并通过多种统计分析方法对产生的海量数据进行分析,最终筛选出潜在标志物.数据分析过程包括：通过归一化、修正80%规则、数据集分割和数据缩放等方法对数据集进行预处理 通过正交校正的偏最小二乘（OPLS）模式识别方法对样品进行分型 根据模型的变量重要性因子（VIP值）、非参数检验结果和z值筛选潜在标志物.以宫颈癌血清样本为例,应用上述方法,15个变量被确认为潜在标志物,操作者接受曲线（ROC）下的面积为0.667～0.956.经过相关性分析和结构鉴定,发现这15个变量来自9个化合物.其中7个化合物被鉴定为色氨酸、硬脂酸、花生四烯酸、溶血磷脂酰胆碱（0：0/16：0,16：0/0：0,18：1/0：0和18：0/0：0）,说明在宫颈癌中花生四烯酸和溶血磷脂酰胆碱的代谢发生异常.     

Metabonomics Study of the Hematopoietic Effect of Medicinal Wine Maoji Jiu on a Blood Deficiency Rat Model by Ultra-High-Performance Liquid Chromatography Coupled to Quadrupole Time-of-Flight Mass Spectrometry and a Pattern Recognition Approach

Maoji Jiu (MJ) is a kind of medicinal wine that has been widely used by Chinese people for many years to nourish and promote blood circulation. The purpose of this study was to investigate the hematopoietic effect of MJ on the metabolism of blood deficient rats and to explore the underlying hematopoietic regulation mechanisms. Blood deficiency model rats were induced by subcutaneous injection of N-acetylphenylhydrazine (APH) and intraperitoneal injection of cyclophosphamide (CTX). The plasma metabolic fingerprints of blood deficiency model rats with and without MJ treatment were obtained by using metabonomics based on ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC–QTOF/MS). Orthogonal partial least squares-discriminant analysis (OPLS–DA) was used to evaluate the hematopoietic effect of MJ and identify potential biomarkers in the plasma of blood deficiency model rats. The levels of white blood cells (WBC), red blood cells (RBC) and hemoglobin (HGB) and the activity of antioxidant capacity showed a recovery trend to the control group after MJ treatment, while the dose of 10 mL/kg showed the best effect. In this study, thirteen potential biomarkers were identified, which were mainly related to seven metabolic pathways, including linoleic acid metabolism, d-glutamine and d-glutamate metabolism, alanine, aspartate and glutamate metabolism, tryptophan metabolism, pyrimidine metabolism, porphyrin and chlorophyll metabolism and arginine biosynthesis. Metabolomics was applied frequently to reflect the physiological and metabolic state of organisms comprehensively, indicating that the rapid plasma metabonomics may be a potentially powerful tool to reveal the efficacy and enriching blood mechanism of MJ.     

代谢组学的整合化发展及其新进展

代谢组学迅猛发展,自身特点及科研使命决定了其整合化的必然趋势.近年来,代谢组学经历了内部整合(包括:检测技术整合、多样本整合分析、数据处理方法整合)以及外部整合(代谢组学与其它组学的整合分析).整合化发展增强了代谢组学在其应用领域的科学创新性与说服力.本文综述了代谢组学的整合化及其最新研究进展,并展望了其应用前景.     

Plasma fingerprinting with GC-MS in acute coronary syndrome

New biomarkers of cardiovascular disease are needed to augment the information obtained from traditional indicators and to illuminate disease mechanisms. One of the approaches used in metabolomics/metabonomics for that purpose is metabolic fingerprinting aiming to profile large numbers of chemically diverse metabolites in an essentially nonselective way. In this study, gas chromatography-mass spectrometry was employed to evaluate the major metabolic changes in low molecular weight plasma metabolites of patients with acute coronary syndrome (n = 9) and with stable atherosclerosis (n = 10) vs healthy subjects without significant differences in age and sex (n = 10). Reproducible differences between cases and controls were obtained with pattern recognition techniques, and metabolites accounting for higher weight in the classification have been identified through their mass spectra. On this basis, it seems inherently plausible that even a simple metabolite profile might be able to offer improved clinical diagnosis and prognosis, but in addition, specific markers are being identified.     

Lipidomics reveals the dysregulated ceramide metabolism in oxidized low-density lipoprotein-induced macrophage-derived foam cell

Atherosclerosis (AS) is associated with increasing lipid peroxidation. Oxidative modification of low-density lipoproteins (ox-LDL) is one most important factors contributing to the pathogenesis and clinical features of AS. The lipid composition influenced by ox-LDL is not known clearly. In this work, a UHPLC/Orbitrap MS-based lipidomics approach integrated pathway analysis was performed to advance understanding of the lipid composition and feature pathway in an ox-LDL-induced foamy macrophage cell. In the lipid metabolic profiling, 196 lipid species from 15 (sub)classes were identified. Lipid profiling indicated that increasing ox-LDL caused lipid metabolic alternations, manifesting as phospholipids being down-regulated and sphingolipids being up-regulated. Pathway analysis explored glycerophospholipid and sphingolipid metabolism, which was involved in atherogenic changes. Notably, dysregulated ceramide metabolism was a typical feature of foamy cell formation. qRT-PCR analysis was conducted to explore the differentially expressed genes. It indicated that ceramide metabolic balance might be disordered, performing higher synthesis and lower hydrolysis, with the ratio of SMPD1/SGMS2 being significantly up-regulated (p < 0.05) in the ox-LDL induced group. Our work offers a comprehensive understanding of macrophage-derived foam cells and screen feature pathways associated with foamy cell formation, which provides a reference for the clinic diagnosis of AS and drug interventions.