慢性乙型肝炎肝气郁结证患者血液代谢指纹图谱研究初探

目的:借助代谢组学技术和现代分析技术,找出慢性乙型肝炎(CHB)肝气郁结证患者内源性标志物,绘制肝气郁结证患者血液代谢指纹图谱,揭示肝气郁结证的生物学本质。方法:通过中医半定量辨证收集31例CHB肝气郁结证患者,对照组肝血瘀证16例以及健康人组30例,通过GC-MS(气相色谱和质谱)技术检测各组人员生物学标志物及内源性代谢谱。结果:与健康者组相比,肝气郁结证患者有明显差异的代谢物质,包括丙酸、丙二酸、嘧啶、吲哚胺、5-恶唑胺、萘啶、吲哚乙酸、呋喃半乳糖、吡喃葡萄糖、肌醇、乙醛胺、十七酸、雄甾烷;但与对照组肝血瘀证患者代谢物质的差异性分析提示,PCA(主成分分析)得分图上有一定分离趋势,但并不能完全分开。结论:本研究利用代谢组学的方法,找到了肝气郁结证与健康者之间的差异性代谢物质,提示中医证候是有其生物学物质基础的,并且代谢组学是对其研究的很好的工具之一。     

口腔鳞状细胞癌患者唾液的代谢组学分析

目的利用基于液相-质谱联用的代谢组学研究方法寻找口腔鳞状细胞癌(OSCC)患者唾液中潜在的生物标志物。方法采用高分离度快速液相色谱-四极杆-飞行时间质谱(RRLC/Q-TOF/MS)技术对OSCC患者和健康人的唾液样品进行分析,分别获得两组样品的代谢轮廓,同时运用主成分分析法(PCA)技术对两组代谢物进行分类,并寻找潜在的生物标志物。结果 RRLC/Q-TOF/MS检测结果显示,OSCC组和健康组人群唾液代谢轮廓存在明显的差异,发现并鉴定得到了7个OSCC相关的生物标志物,分别为胆碱、葡萄糖、色氨酸、苯丙氨酸、谷氨酸、谷氨酰胺、肉毒碱,与正常健康人相比,胆碱的代谢趋势上调,其余代谢物的趋势均下调。结论与健康人相比,OSCC患者主要存在着氨基酸代谢和能量代谢失衡的现象,同时三羧酸循环代谢异常,为将来的临床诊断和治疗提供了重要的信息。     

高血压病肝火亢盛证型代谢组学的初步研究

目的初步探讨高血压病肝火亢盛证型患者的代谢组学变化,并确定其相关生物标志物群,初步阐明高血压病肝火亢盛证型的代谢组学内涵。方法选取2008年8月—2008年12月在江苏省中医院就诊的原发性高血压中医辨证为肝火亢盛证型患者24例,同时选取同期进行体检的健康人群21名,抽取受试者空腹12h静脉血、留取受试者晨尿。经样品前处理后,采用气相色谱/飞行时间质谱(GC/TOF-MS)技术对高血压病肝火亢盛证型患者与健康人群的血清、尿液进行全谱代谢组学分析,并进一步采用主成分分析(PCA)进行主成分因子负荷矩阵分析,并作变量散点图,寻找高血压病肝火亢盛证型相关的生物标志物群。结果高血压病肝火亢盛证型组与健康人群组血清和尿液的代谢谱图均有改变,两组血清代谢谱图差异有统计学意义(P<0.05)。高血压病肝火亢盛证型组与健康人群组血清和尿液的代谢产物中均存在发生显著变化的化合物,并且两组血清中的3-羟基丁酸和葡萄糖,以及尿液中的甘氨酸和磷酸差异均有统计学意义(P<0.05)。结论高血压病肝火亢盛证型患者与健康人群代谢谱之间存在差异,而且可从代谢组学分析中找出特异的标志性代谢物,阐释中医证候的生物学本质。代谢组学是一种有良好发展前景的中医证候学研究方法。     

基于非靶向代谢组学技术的胆石症血浆代谢标志物的初步探讨

背景:胆石症是胆道系统最常见的疾病之一,有关胆石症代谢组学的研究目前尚无报道。目的:应用非靶向代谢组学技术筛选胆石症患者的血浆代谢标志物。方法:基于上海市男、女性健康队列开展胆石症血浆代谢谱预初实验。应用超高效液相色谱-四级杆飞行时间质谱(UPLC-QTOFMS)和气相色谱-飞行时间质谱(GC-TOFMS)技术检测43例基线期胆石症患者和43名年龄配对健康对照者的血浆代谢物,采用主成分分析(PCA)、正交偏最小二乘判别分析(OPLS-DA)以及t检验筛选胆石症患者的特异性代谢物。通过Logistic回归分析构建胆石症预测模型,以ROC曲线对模型进行初步评价。结果:UPLC-QTOFMS和GC-TOFMS共检测出血浆代谢物618个,根据OPLSDA VIP 1. 0且t检验P 0. 05的筛选条件,筛选出差异代谢物30个,由其中的2-甲基-4-戊烯酸、磷脂酰乙醇胺PE(p-16∶0e/0∶0)、顺式-4,7,10,13,16,19-二十二碳六烯酸、2-氨基丁酸、尿酸、瓜氨酸、S-异戊烯基-L-半胱氨酸、羟环己脲和2,3-环氧甲基萘醌九个代谢物构建的胆石症预测模型ROC曲线下面积(AUC)为0. 97,敏感性和特异性分别为95. 4%和88. 4%。结论:胆石症患者的血浆代谢谱发生明显变化,由其中9个差异代谢物构建的预测模型能较好地区分胆石症患者与健康对照者。     

轻度认知功能障碍患者尿液生物标志物

目的探讨轻度认知功能障碍(MCI)患者尿液中潜在的生物标志物。方法通过超高效液相色谱-质谱联用和代谢组学的方法,考查MCI患者和正常老年人尿液中代谢产物的扰动。对于超高效液相色谱-质谱联用所产生的色谱图,进行主成分分析显示出疾病组和正常组之间的代谢物的变化。结果 MCI患者的鞘氨醇类相对于正常组变化明显,5个潜在的与MCI相关的生物标志物被发现。结论 MCI患者的代谢物谱发生了明显改变,这可以为MCI的诊断提供依据。     

阿尔茨海默病患者血浆生物标记物代谢组学分析

目的探寻阿尔茨海默病(AD)患者血浆中潜在的生物标记物,为AD的早期诊断提供依据。方法通过超高效液相色谱-质谱联用和代谢组学的方法,考查AD患者和正常老年人血浆中,代谢产物的扰动。对于超高效液相色谱-质谱联用所产生的色谱图,进行主成分分析显示出疾病组和正常组之间的代谢物的变化。结果 AD患者的溶血卵磷脂类,鞘氨醇类,色氨酸相对于正常组变化明显。9个潜在的与AD相关的生物标记物被发现。结论 AD患者的代谢物谱发生了明显改变,这可以为AD的诊断提供依据。     

老年脑卒中后认知障碍伴失眠患者的血清代谢组学

目的 基于血清代谢组学分析老年脑卒中后认知障碍伴失眠患者差异代谢标志物。方法 对比老年缺血性脑卒中后认识障碍伴失眠患者和单纯脑卒中患者各9例,通过对这18例受试者进行血清液相色谱-质谱联用(LC-MS)非靶向代谢组学分析,筛选差异代谢物,探讨脑卒中后继发认知和失眠障碍的发生机制。结果 基于非靶向代谢组研究,发现老年脑卒中后认知障碍伴失眠患者的差异代谢中脂类占绝大多数,其最多的是甘油磷脂类,多数呈含量下调趋势,4种不饱和脂肪酸(二十二碳二烯酸、十八碳二烯酸、类花生酸、6-酮-前列腺素F1α)和两种饱和脂肪酸(二十二烷酸、棕榈酸)在观察组含量均上调,鞘磷脂代谢产物神经酰胺和神经鞘氨醇磷酸胆碱含量下调,而二氢神经鞘氨醇含量上调;氨基酸代谢中两种差异代谢物(半胱氨酸硫酸酯和左旋精氨酸)含量在观察组中上调;剩余的5种差异代谢物上调的次黄嘌呤、左旋肉碱、睾酮和异丙肾上腺素,下调的有尿苷二磷酸葡萄糖。结论 缺血性脑卒中后认知障碍伴失眠患者的血清代谢谱与单纯脑卒中者存在明显差别,多种代谢物及代谢通路可能参与老年脑卒中患者继发认知、失眠障碍的发病过程。     

大鼠急性坏死性胰腺炎和慢性胰腺炎代谢特征分析

目的 用代谢组学方法研究大鼠胰腺组织代谢特征,以期发现胰腺炎症的标记性代谢物.方法 Wistar大鼠22只,按数字表法随机分成急性坏死性胰腺炎组(ANP,7只)、慢性胰腺炎组(CP,6只)和对照组(9只).ANP组经腹腔注射20％L-精氨酸溶液制模;CP组经尾静脉注射二丁基二氯基锡(DBTC)溶液制模;对照组注射等量生理盐水.检测血清淀粉酶含量,胰腺组织行病理学检查.利用高分辨魔角旋转核磁共振波谱对离体胰腺组织进行代谢成分分析,并对实验数据进行主成分分析,比较ANP与CP的代谢特征.结果 与对照组相比,ANP组小分子代谢物亮氨酸、异亮氨酸、缬氨酸含量增加,而CP组这些代谢物含量减少.ANP组和CP组的磷酸胆碱、甘油磷酸胆碱、胆碱含量均升高,脂肪酸、乳酸、甜菜碱、甘氨酸含量均下降,CP组中脂类代谢产物含量明显高于ANP组,且仅在CP组中观察到牛磺酸含量升高.结论 胰腺炎症疾病造成胰腺组织内代谢异常,升高的牛磺酸水平可能是区分CP和ANP的标记物.     

基于气相色谱质谱联用对冠心病“痰”“瘀”证候血清代谢组学的研究

目的研究冠心病痰证、瘀证病人血清代谢组学特征。方法选择冠心病稳定期病人102例,其中痰证52例、瘀证50例,31名健康者,利用核磁共振谱(GCMS)和色谱/质谱(LC/MS)联用、模式识别技术等现代分析技术,比较痰证、瘀证冠心病病人和健康人的代谢物组谱差异,对同病异证病人间代谢物组谱差异进行分析。结果在多种氨基酸、有机酸、长链脂肪酸类、溶血磷脂类、磷脂类、鞘磷脂等代谢物方面,冠心病组相对于健康对照组表现出明显的差异,其中大部分的差异代谢物也是痰浊组和血瘀组的共性代谢物。冠心病组有机酸、麦芽糖、多种氨基酸较健康对照组明显升高,而多种不饱和脂肪酸及衍生物、维生素E、胆固醇等显著降低。痰浊组和血瘀组的特性差异代谢物主要有苹果酸和琥珀酸,果糖和葡萄糖,甘氨酸和丙氨酸以及棕榈烯酸等。其中7,10-顺十六碳二烯酸和DPA在痰浊组中降低,其余则在血瘀组中浓度升高。结论冠心病有特异性的代谢组特征,痰浊和血瘀既有特性差异性代谢物,也有共性代谢物,二者异中有同,同中有异。痰浊和血瘀有共同的物质基础,这与中医理论的"痰瘀同源"是一致的,作为致病因子,二者在体内可以相互转化,共同致病,亦即"痰瘀同病",因而在治疗时可以考虑痰瘀同治,提高疗效。     

2型糖尿病胃轻瘫大鼠的血液代谢组学改变

[目的]检测2型糖尿病胃轻瘫大鼠的血液代谢谱的变化,探讨代谢组学在糖尿病胃轻瘫研究中的应用.[方法]Wistar大鼠腹腔注射链脲菌素(Streptozotoein,STZ) 80 mg/kg 1周后,高糖高脂饲料喂养8周后,建立2型糖尿病胃轻瘫模型.动态检测空腹血糖(FBG)变化,三酰甘油(TG)、总胆固醇(TC)、游离脂肪酸(FFA)及胰岛素(INS)水平,采用气相色谱质谱联用(GC/MS)的方法检测造模前,造模1、2、4、6、8周后大鼠血液代谢谱的变化;并与正常对照组大鼠作比较.[结果]与正常对照组相比,模型组大鼠FBG持续升高,FFA、TG、TC明显升高,血清INS含量明显降低;模型组大鼠代谢谱与正常对照组完全区分,并随时间变化逐渐偏离;PLS-DA分析显示,2组的血液代谢物能很好的区别开.[结论]2型糖尿病胃轻瘫大鼠血液代谢组学发生明显变化,血液代谢组学在一定程度上反映其病理变化.     

A metabonomic investigation on the biochemical perturbation in post-stroke patients with depressive disorder (PSD)

A metabonomics study based on GC/MS and multivariate statistical analysis was performed involving 28 post stroke depressed (PSD) patients, 27 post-stroke non-depressed (PSND) patients and 33 healthy subjects to investigate the biochemical perturbation in their plasma samples. The outcome of this study showed that there was distinctive metabolic profile for PSD patients. Seven sentinel metabolites showed marked perturbations in PSD patients' blood. The introduction of metabonomics approach may provide a novel metabonomic insight about PSD and the sentinel metabolites for classifying PSD.     

磁共振对代谢综合征患者血浆代谢组学分析的作用

目的从代谢组学角度研究代谢综合征(MS)的特征代谢物,并探讨其在疾病发生、发展演变过程中的整体作用机制。方法采集36例受试者的血浆,包括正常人(n=11)、MS高危患者(n=13)和MS患者(n=12),采用1 H NMR技术检测血浆的代谢谱,并对谱图的积分数据进行主成分分析(PCA)和偏最小二乘法判别分析(PLS-DA),以辨识血浆代谢产物的变化,识别并分析出特征代谢物的组分。结果代谢组学可以成功区分正常组、MS高危组、MS患者组,通过对各组特征代谢物的比较和分析,可知脂代谢异常在代谢综合征演变过程中起着重要的桥梁作用,代谢综合征的发生是机体多个系统功能改变共同造成的结果,体内碳水化合物、糖类、脂类、蛋白质等代谢紊乱是代谢综合征发病的关键环节。结论代谢组学能较清晰地反映代谢综合征血浆样本间的代谢差异和变化,作为一种新的技术手段研究代谢综合征具有重要的应用价值。     

Urinary metabolic insights into host-gut microbial interactions in healthy and IBD children

AIMTo identify metabolic signatures in urine samples from healthy and inflammatory bowel disease(IBD)children.METHODSWe applied liquid chromatography and gas chromatography coupled to targeted mass spectrometry(MS)-based metabolite profiling to identify and quantify bile acids and host-gut microbial metabolites in urine samples collected from 21 pediatric IBD patients monitored three times over one year(baseline,6 and 12 mo),and 27age-and gender-matched healthy children.RESULTSurinary metabolic profiles of IBD children differ significantly from healthy controls.Such metabolic differences encompass central energy metabolism,amino acids,bile acids and gut microbial metabolites.In particular,levels of pyroglutamic acid,glutamic acid,glycine and cysteine,were significantly higher in IBD children in the course of the study.This suggests that glutathione cannot be optimally synthesized and replenished.Whilst alterations of the enterohepatic circulation of bile acids in pediatric IBD patients is known,we show here that non-invasive urinary bile acid profiling can assess those altered hepatic and intestinal barrier dysfunctions.CONCLUSIONThe present study shows how non-invasive sampling of urine followed by targeted MS-based metabonomic analysis can elucidate and monitor the metabolic status of children with different GI health/disease status.     

Study of the metabolomics characteristics of patients with metabolic syndrome based on liquid chromatography quadrupole time-of-flight mass spectrometry

BackgroundMetabolic syndrome (MS) is a disease with complex pathophysiology and pathogenesis involving multiple systems of the human body. This study aimed to identify serum metabolites that are relevant to MS.Material and methodsThis study involved 40 patients with MS and 28 healthy adults, and the following data were statistically analyzed: basic clinical data, blood lipids, fasting blood glucose, blood pressure, waist circumference, and visceral fat coefficient. Serum samples from both groups were collected and analyzed by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF/MS); multivariate and univariate statistical methods were used to identify potential MS biomarkers and MS-related metabolic pathways. In addition, leucine and valine levels in serum from MS patients and normal subjects were measured using enzyme-linked immunosorbent assays (ELISAs).ResultsIn this study, 23 potential biomarkers were identified in the plasma of MS patients. These biomarkers were mainly related to metabolism; the tricarboxylic acid cycle; galactose metabolism; arachidonic acid metabolism; valine, leucine, and isoleucine degradation; and valine, leucine, and isoleucine biosynthesis. ELISAs were utilized to verify serum leucine and valine levels, and the results supported the experimental metabolomics results.ConclusionsIn total, 23 MS-related metabolites were identified in the serum; these differential metabolites were mainly associated with lipid metabolism, amino acid metabolism, glucose metabolism, purine metabolism, and other related metabolic pathways. This study shows that LC/MS-based metabolomics methods can be used to investigate the pathological changes in MS patients and identify biomarkers for the early diagnosis of MS.     

LC-MS-based serum metabolomics reveals a distinctive signature in patients with rheumatoid arthritis

Metabolomics has been applied to explore altered metabolite profiles in disease and identify unique metabolic signatures in recent years. We aim to characterize the metabolic profile of rheumatoid arthritis patients and explore its underlying pathological processes using metabolomics approach. Serum samples from 30 rheumatoid arthritis (RA) patients, 30 primary Sjogren’s syndrome (pSS) patients, and 32 healthy controls (HC) were collected. The sample was analyzed by ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-HRMS). Potential biomarkers were screened from orthogonal projection to latent structure discriminate analysis (OPLS-DA) and further evaluated by receiver operating characteristic analysis (ROC). Compared with HC and pSS patients, the RA patients had increased serum levels of 4-methoxyphenylacetic acid, glutamic acid, L-leucine, L-phenylalanine, L-tryptophan, L-proline, glyceraldehyde, fumaric acid, and cholesterol as well as decreased capric acid, argininosuccinic acid, and billirubin. A total of eight potential biomarkers were screened and tentatively identified for RA. A panel of three metabolites (4-methoxyphenylacetic acid, L-phenylalanine, and L-leucine) was identified as specific biomarkers of RA. ROC analysis showed that the panel had a sensitivity of 93.30% with a specificity of 95.20% in discrimination RA from other groups. UPLC-HRMS-based quantification of circulating metabolites was a useful tool for identifying RA patients from pSS patients and healthy controls. The potential biomarkers indicated that the RA metabolic disturbance might be associated with inflammation injury, amino acid metabolism, oxidative stress, and phospholipid metabolism.     

Urine metabonomic profiling of a female adolescent with PIT-1 mutation before and during growth hormone therapy: Insights into the metabolic effects of growth hormone

ObjectiveGrowth hormone (GH) is a protein hormone with important roles in growth and metabolism. The objective of this study was to investigate the metabolism of a human subject with severe GH deficiency (GHD) due to a PIT-1 gene mutation and the metabolic effects of GH therapy using Nuclear Magnetic Resonance (NMR)-based metabonomics. NMR-based metabonomics is a platform that allows the metabolic profile of biological fluids such as urine to be recorded, and any alterations in the profile modulated by GH can potentially be detected.DesignUrine samples were collected from a female subject with severe GHD before, during and after GH therapy, and from healthy age- and sex-matched controls and analysed with NMR-based metabonomics.SettingThe samples were collected at a hospital and the study was performed at a research facility.ParticipantsWe studied a 17 year old female adolescent with severe GHD secondary to PIT-1 gene mutation who had reached final adult height and who had ceased GH therapy for over 3 years. The subject was subsequently followed for 5 years with and without GH therapy. Twelve healthy age-matched female subjects acted as control subjects.InterventionThe GH-deficient subject re-commenced GH therapy at a dose of 1 mg/day to normalise serum IGF-1 levels.Main outcome measuresUrine metabolic profiles were recorded using NMR spectroscopy and analysed with multivariate statistics to distinguish the profiles at different time points and identify significant metabolites affected by GH therapy.ResultsNMR-based metabonomics revealed that the metabolic profile of the GH-deficient subject altered with GH therapy and that her profile was different from healthy controls before, and during withdrawal of GH therapy.ConclusionThis study illustrates the potential use of NMR-based metabonomics for monitoring the effects of GH therapy on metabolism by profiling the urine of GH-deficient subjects. Further controlled studies in larger numbers of GH-deficient subjects are required to determine the clinical benefits of NMR-based metabonomics in subjects receiving GH therapy.     

代谢组学的研究进展及在糖尿病中的应用

代谢组学是继基因组学和蛋白质组学之后新发展的一门系统科学,主要研究代谢产物的变化规律,揭示机体生命活动的代谢本质.本文主要介绍了代谢组学的研究方法如核磁共振波谱技术(NMR)、质谱技术(MS)等,同时回顾了近年来代谢组学技术在临床诊断、药物开发、毒理学等研究领域的应用及进展.着重综述了代谢组学在糖尿病研究中的应用,并对其发展前景进行展望.     

Fast and accurate quantitative organic acid analysis with LC-QTOF/MS facilitates screening of patients for inborn errors of metabolism

Since organic acid analysis in urine with gaschromatography-mass spectrometry (GC-MS) is a time-consuming technique, we developed a new liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) method to replace the classical analysis for diagnosis of inborn errors of metabolism (IEM). Sample preparation is simple and experimental time short. Targeted mass extraction and automatic calculation of z-scores generated profiles characteristic for the IEMs in our panel consisting of 71 biomarkers for defects in amino acids, neurotransmitters, fatty acids, purine, and pyrimidine metabolism as well as other disorders. In addition, four medication-related metabolites were included in the panel. The method was validated to meet Dutch NEN-EN-ISO 15189 standards. Cross validation of 24 organic acids from 28 urine samples of the ERNDIM scheme showed superiority of the UPLC-QTOF/MS method over the GC-MS method. We applied our method to 99 patient urine samples with 32 different IEMs, and 88 control samples. All IEMs were unambiguously established/diagnosed using this new QTOF method by evaluation of the panel of 71 biomarkers. In conclusion, we present a LC-QTOF/MS method for fast and accurate quantitative organic acid analysis which facilitates screening of patients for IEMs. Extension of the panel of metabolites is easy which makes this application a promising technique in metabolic diagnostics/laboratories.     

Hepatitis C virus infection diagnosis using metabonomics

Summary. Metabonomics based on nuclear magnetic resonance (NMR) can reveal the profile of endogenous metabolites of low molecular weight in biofluids related to disease. The profile is identified a ‘metabolic fingerprint’ like from the pathological process, why this metabonomics has been used as a diagnostic method. The aim of the present study was to apply metabonomics to identify patients infected with the hepatitis C virus (HCV) through an analysis of ¹H NMR spectra of urine samples associated with multivariate statistical methods. A pilot study was carried out for the diagnostic test evaluation, involving two groups: (i) 34 patients positive for anti‐HCV and HCV‐RNA and negative for anti‐HBc (disease group); and (ii) 32 individuals positive for anti‐HBc and negative for HBsAg and anti‐HCV. The urine samples were analyzed through ¹H NMR, applying principal component analysis and discriminant analysis for classification. The metabonomics model was capable of identifying 32 of the 34 patients in the disease group as positive and 31 of the 32 individuals in the control group as negative, demonstrating 94% sensitivity and specificity of 97% as well as positive and negative predictive values of 97% and 94%, respectively, and 95% accuracy (P < 0.001). In conclusion, the metabonomics model based on ¹H NMR spectra of urine samples in this preliminary study discriminated patients with HCV infection with high sensitivity and specificity, thereby demonstrating this model to be a potential tool for use in medical practice in the near future.