基于血浆中脂肪酸代谢谱及非线性判别分析方法的糖尿病中医证候分型

糖尿病是严重威胁人类健康的代谢综合征之一,中医因在治疗糖尿病方面有着自身的优势和特色而广泛受到重视。该文以血浆中脂肪酸代谢谱及血脂代谢指标为研究对象,结合化学计量学方法,对5种糖尿病中医证候(气虚、阴虚、气阴两虚、热盛和血瘀)进行关联分析。通过正交信号校正的偏最小二乘(OSC-PLS)方法将5种证候与健康组较明显地区分开。同时,采用非线性判别分析(ULDA)对健康组、中医虚证(气虚、阴虚、气阴两虚)、中医实证(热盛、血瘀)进行分析,3组样本体现明显的聚类效果,正判率达到95.7%。其中对分类贡献较大的标志物为二十碳二烯酸(C20:2)、二十碳五烯酸(C20:5)、甘油三酯(TG)和高密度脂蛋白(HDL),这一结果为辅助糖尿病中医临床诊断提供了重要的信息。     

血浆氨基酸代谢谱与糖尿病相关性研究

为了利用代谢组学手段研究血浆氨基酸代谢谱与糖尿病相关性, 采用邻苯二甲醛(OPA)柱前在线衍生反相高效液相色谱法,建立了血浆中21种氨基酸代谢谱的相对定量分析方法.衍生由自动进样器在线自动完成,紫外检测器检测,流动相A为10 mmol/L Na2HPO4-Na2B4O7缓冲液(pH 7.95),B为乙腈-甲醇-水 (45: 45∶ 10,V/V), 线性梯度洗脱,流动相B在30 min内由5%增加至46%,27 min内21种氨基酸全部得到良好分离.用此方法测定了51位临床糖尿病人血浆氨基酸代谢谱.以相对峰面积作为原始数据,按血糖值高低预分组,通过Spss和Matlab软件平台,用线性判别分析、逐步判别分析和得分图进行关联研究,结果表明:血浆中氨基酸代谢谱与血糖值的高低存在相关性,对血糖值低于6.2的志愿者和血糖值高于9.5的志愿者,判别分析的总正判率达到了94.1%.逐步判别分析结果表明,有7种氨基酸(Arg, Cit, Asp, Asn, Thr, Leu, Trp)承载了上述分组的重要信息.其对上述分组的判别分析总正判率为90.2%,可作为反映血糖值高低变化的标志物组,对于糖尿病的早期诊断及深入研究具有潜在的科研及临床价值.     

基于气相色谱的血清中游离脂肪酸代谢轮廓分析区分糖尿病与正常人

建立了以硫酸/甲醇为甲酯化试剂的气相色谱法,对人血清中7种游离脂肪酸(FFA)进行定量代谢轮廓分析。该法以十七酸为内标,血清样品直接甲酯化处理,采用Rtx-Wax色谱柱分离,氢火焰离子化检测器检测。研究结果表明,7种脂肪酸在2～500μg/mL范围内线性关系良好(r>0.999),相对标准偏差(RSD)为1.15%～5.39%,回收率为76.6%～121.9%。用建立的FFA定量轮廓分析方法结合主成分分析区分了31例糖尿病患者和30例正常人,结果显示糖尿病病人与正常人血清中的脂肪酸代谢存在差异。所建立的方法操作简单,结果准确可靠,可用于FFA血清浓度的监测。而肉豆蔻酸(C14∶0)和油酸(C18∶1)承载了两组人群的重要信息,作为可能的标志物,对糖尿病的临床诊断具有重要意义。     

基于1H-NMR的模式识别方法应用于异常黑胆质糖尿病患者的尿液代谢组学研究

探讨核磁共振氢谱结合模式识别方法应用于异常黑胆质糖尿病患者的尿液代谢组研究可行性。对32 例异常黑胆质糖尿病患者和29 例健康人尿液进行核磁共振氢谱检测,采用主成分分析(principal component analysis, PCA)、偏最小二乘法判别分析(partial least squares dis-criminant analysis, PLS-DA)、正交偏最小二乘法判别分析(orthogonal to partial least squares discriminant analysis,OPLS-DA)进行模式识别分析,比较3种模式识别方法的判别能力。运用3种模式识别均可以对2组数据进行有效的区分,但OPLS-DA较PCA、P[1]LS-DA更加有效,不仅提高了模式识别方法的判断能力,可以清楚的判断两组中有差异的代谢物。基于核磁共振氢谱结合模式识别分析方法可以为异常黑胆质糖尿病代谢标志物的寻找提供理论依据。OPLS-DA的模式识别方法较其它2种方法更具优势,在揭示维医理论本质上有着广阔的应用前景。     

基于超高效液相色谱-高分辨质谱联用技术研究冠心病及冠心病合并2型糖尿病患者代谢特征

采用超高效液相色谱-四极杆-轨道阱质谱联用技术开展冠心病及冠心病合并2型糖尿病患者的血浆代谢组学研究,以探寻冠心病及冠心病合并2型糖尿病患者的代谢特征,为基于代谢组学的临床治疗方案优化奠定基础。本研究鉴定出78种血浆中的内源性代谢物。在代谢物定性与定量分析基础上,结合偏最小二乘-判别分析和变量重要性投影等方法,筛选出20种区分健康人和冠心病患者的潜在特征代谢物、35种区分冠心病及冠心病合并2型糖尿病患者的潜在特征代谢物、37种区分健康人和冠心病合并2型糖尿病的潜在特征代谢物。代谢通路分析结果表明,氨基酸代谢在这两类患者的生理代谢过程中发挥重要作用,而脂肪酸代谢很可能与冠心病合并2型糖尿病相关。     

基于UPLC-MS/MS的2型糖尿病患者血清胆汁酸代谢产物分析

应用基于超高效液相色谱-串联质谱(UPLC-MS/MS)技术的代谢组学方法检测2型糖尿病患者血清胆汁酸代谢谱,获得代谢标志物,发掘可预测糖尿病的潜在预防治疗靶点。采用Waters ACQUITY UPLC BEH C18色谱柱(150 mm×2.1 mm,1.7 μm)分离,0.05%乙酸水溶液-乙腈为流动相,以0.40 mL/min流速进行梯度洗脱,用此方法测定了30例糖尿病患者和 50例正常人血清胆汁酸代谢谱。41种胆汁酸标准品在30 min内均得到良好分离,各成分在一定浓度范围内呈良好线性关系,检出限(LOD)为0.24~7.81 nmol/L,定量下限(LOQ)为0.49~15.63 nmol/L,相关系数(r2)为 0.986 7~0.999 7。血清中胆汁酸代谢谱与两组间存在相关性,通过正交偏最小二乘法-判别分析(OPLS-DA)发现,有12种胆汁酸(包括甘氨熊脱氧胆酸、甘氨胆酸、牛磺鹅脱氧胆酸、胆酸、熊去氧胆酸、甘氨鹅脱氧胆酸、甘氨脱氧胆酸、12 酮基石胆酸、鹅脱氧胆酸、脱氧胆酸、异石胆酸、石胆酸)承载了分组的重要信息。结合受试者工作特征(ROC)分析,最终获得牛磺鹅脱氧胆酸(AUC=0.68)、甘氨鹅脱氧胆酸(AUC=0.66)、甘氨熊脱氧胆酸(AUC=0.63)、胆酸(AUC=0.63)、脱氧胆酸(AUC=0.63)、甘氨脱氧胆酸(AUC=0.58) 6种胆汁酸代谢标志物,对于糖尿病的早期诊断及深入研究具有潜在的科研及临床价值。     

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。     

糖肾方对自发性Ⅱ型糖尿病鼠磷脂代谢的影响

利用正相液相色谱飞行时间质谱建立了大鼠血浆磷脂的轮廓谱。通过对磷脂轮廓谱的PLS-DA判别分析及14种化合物的定量,考察了中药糖肾方对自发性II型糖尿病鼠磷脂代谢的影响,并对比了糖肾方与西药蒙诺在糖尿病发展过程中的不同作用。结果表明：与正常对照组相比,糖尿病鼠的血浆磷脂代谢发生异常。糖肾方能够调节糖尿病鼠的磷脂代谢紊乱,蒙诺则加剧了病鼠的磷脂代谢异常。此外,8种磷脂化合物可能成为糖尿病发展过程中的潜在生物标志物。     

代谢组学应用于糖肾方治疗糖尿病肾病的疗效评价

建立了基于超高效液相色谱-飞行时间质谱(UPLC/TOF MS)分析技术的血浆代谢指纹谱,应用多元统计分析方法评价糖尿病肾病患者血浆代谢物变化差异及糖肾方的干预效果。通过研究糖肾方干预糖尿病肾病血浆内源性代谢物的变化,探索与该疾病密切相关的代谢途径,评价糖肾方的治疗效果。结果发现: 经糖肾方治疗后,血浆内源性代谢物发生了明显变化,磷脂代谢、脂肪酸代谢、氨基酸代谢、嘌呤嘧啶代谢、固醇类代谢等多个代谢途径得到纠正。本研究基于UPLC/TOF MS的代谢组学方法,能够从整体水平反映疾病治疗过程中代谢网络的变化趋势,证实糖肾方具有治疗糖尿病肾病的临床疗效并有助于阐释药物作用机理。     

非靶向尿液代谢组学方法研究五味子治疗精尿病并发症作用机制

采用超高效液相色谱-串联四级杆飞行时间质谱联用技术(Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry,UPLC/Q-TOF-MS),结合主成分分析技术(Principal component analysis,PCA),进行了五味子治疗糖尿病并发症大鼠尿液代谢组学研究,对五味子治疗糖尿病并发症大鼠后尿液生物标志物变化进行了考察.采用UPLC/Q-TOF-MS方法分析了健康组、糖尿病并发症模型组和五味子给药组的大鼠尿液,采用偏最小二乘法-判别分析(Partial least squares discriminant analysis,PLS-DA)和正交偏最小二乘法-判别分析(Orthogonal partial least squares discriminant analysis,OPLS-DA)载荷图筛选,通过分析对各组分离贡献较大(VIP>1,P°0.05)化合物的串联质谱数据,再经Human Metabolome Data-base(HMDB)等数据库检索,进行质谱信息匹配,尿液中共鉴定出28种潜在生物标记物,其中正谱13种,负谱15种.这些生物标记物主要影响戊糖和葡糖醛酸相互转化通路、核黄素代谢、泛酸和CoA合成、精氨酸和脯氨酸代谢、肠内菌代谢、嘌吟代谢、Vc代谢胆酸合成、色氨酸代谢等通路,五味子从能量代谢、氨基酸代谢、肠内菌代谢、脂类代谢等各个角度发挥治疗糖尿病并发症的作用.从各个通路的相关生物功能分析,五味子治疗糖尿病肾病的作用较强,此外还具有降脂、抗氧化的功效.     

Analysis of plasma free fatty acid cyanomethyl derivatives by GC-NPD for the diagnosis of mitochondrial fatty acid oxidation disorders

Summary Early diagnosis of fatty acid oxidation (FAO) disorders is important to reduce severe morbidity and mortality. Although analysis of plasma free fatty acids (FFAs) is frequently performed using stable isotope-dilution gas chromatography-mass-spectrometry (GC-MS), there are institutions where the required instrumentation is not available to support a rapid work-up of acutely ill patients. For this reason, we have developed a novel cyanomethyl derivatization method for FFAs which is followed by GC analysis of the resulting esters using nitrogen-phosphorus detector (NPD) for the rapid diagnosis of mitochondrial fatty acid oxidation disorders. FFAs were extracted from plasma and derivatized to the cyanomethyl ester by heating with bromoacetonitrile at 60°C for 30 min GC-NPD analysis was then performed. The mean recoveries of C6:0-C18:0FFAs were between 87% abd 96%. The method detection limits (S/N=3) were 0.1–0.5 ng for C6:0-C14:0 FFAs, and 0.001–0.01 ng for C16:0-C18:0 FFAs. We succesfully performed differential diagnosis of representative FAO disorders from the confimed patient's plasmas. This simple method offers cost-effective and time-saving alternative to GC-MS for the biochemical diagnosis of selected FAO disorders.     

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.     

Identifying potential serum biomarkers of breast cancer through targeted free fatty acid profiles screening based on a GC–MS platform

Recent advances suggest that abnormal fatty acid metabolism highly correlates with breast cancer, which provide clues to discover potential biomarkers of breast cancer. This study aims to identify serum free fatty acid (FFA) metabolic profiles and screen potential biomarkers for breast cancer diagnosis. Gas chromatography–mass spectrometry and our in-house fatty acid methyl ester standard substances library were combined to accurately identify FFA profiles in serum samples of breast cancer patients and breast adenosis patients (as controls). Potential biomarkers were screened by applying statistical analysis. A total of 18 FFAs were accurately identified in serum sample. Two groups of patients were correctly discriminated by the orthogonal partial least squares–discriminant analysis model based on FFA profiles. Seven FFA levels were significantly higher in serum from breast cancer patients than that in controls, and exhibited positive correlation with malignant degrees of disease. Furthermore, five candidates (palmitic acid, oleic acid, cis-8,11,14-eicosatrienoic acid, docosanoic acid and the ratio of oleic acid to stearic acid) were selected as potential serum biomarkers for differential diagnosis of breast cancer. Our study will help to reveal the metabolic signature of FFAs in breast cancer patients, and provides valuable information for facilitating clinical noninvasive diagnosis.     

Free fatty acid determination in plasma by GC-MS after conversion to Weinreb amides

Circulating free fatty acids (FFAs) play important physiological roles as contributing components in cellular structure as well as energy utilization. Elevated levels of circulating FFAs are associated with metabolic aberrations in humans. FFAs differ in chain length and saturation and may be altered in metabolically dysregulated conditions, such as type 2 diabetes mellitus. Potentially, alterations in circulating levels of specific FFAs could also be important in terms of prognostic value. Various methods have been used to analyze FFAs. In this study, a straightforward and accurate method for the determination of FFAs in plasma has been established and evaluated, through conversion of plasma FFAs into acid fluorides followed by conversion to Weinreb amides (dimethylamide). The method is mild, efficient, selective, and quantitative for FFAs, when analyzed with capillary gas chromatography tandem mass spectrometry. Standard curves were linear over the range of 1,000–20,000 ng/mL with a correlation coefficient (r ²) of 0.998, and coefficient of variation of triplicate analysis was <10 %. The gas chromatography–mass spectrometry (GC-MS) technique was reproducible and repeatable, and recoveries were above 90 %. From the generated MS spectra, five specific FFAs were identified. An explicit interest was the quantification of palmitate (C16:0) and palmitoleate (C16:1), which have been connected with detrimental and positive effects on the insulin-producing beta cells, respectively. The results demonstrate the suitability of Weinreb amides for efficient and rapid isolation of FFAs in plasma, prior to quantitative GC-MS analysis. We suggest that the method can be used as a routine standardized way of quantifying FFAs.     

NMR metabolic profiling of organic and aqueous sea bass extracts: implications in the discrimination of wild and cultured sea bass

The nuclear magnetic resonance (NMR) technique was used as analytical tool to determine the complete metabolic profiling of sea bass extracts: water-soluble metabolites belonging to different classes such as sugars, amino acids, dipeptides and organic acids as well as metabolites soluble in organic solvent such as lipids, sterols and fatty acids were identified. The metabolite profiling together with a suitable statistical analysis were used to discriminate between wild and cultured sea bass samples. Preliminary results show that discrimination between wild and cultured sea bass was obtained not only using fatty acid composition but also cholesterol and phosphatidylethanolamine and some water-soluble metabolites such as choline, trimethylamine oxide, glutamine, fumaric and malic acids.     

Analysis of free fatty acids and fatty acid phenacyl esters in vegetable oils and margarine by capillary electrochromatography

Capillary electrochromatography (CEC) has been utilized to analyze free fatty acids (FFAs) and fatty acid phenacyl esters (FAPEs) originating from vegetable oils and margarine. The analyses were performed on capillaries 25 and 40 cm long, 100 microm ID, and packed with 3 microm Hypersil ODS. Isocratic elution was achieved with the mobile phase acetonitrile/50 mM 2-(N-morpholino)ethane sulfonic acid (MES), pH 6, at a ratio of 9:1. For quantitative analysis, the formation of FAPE derivatives is preferred. Moreover, the number of double bonds in the FAPEs can be elucidated by measuring the UV absorbance ratio of 240:210 nm. For the determination of the oleate/elaidate ratio in margarines, the FFAs are analyzed because of overlap of elaidate/palmitate in FAPE analysis. Data obtained with CEC and micro liquid chromatography (LC) were compared and CEC was found to be far superior in terms of efficiency and speed of analysis. Important selectivity differences were noted between micro LC on highly endcapped ODS and CEC on the silanol-rich Hypersil ODS.     

Lipid Biomarkers of Lens Aging

Lipids are important structural components of cell membranes and have profound effect on membrane fluidity. Lipid profiling and lipidomics have captured increased attention due to the well-recognized roles of lipids in numerous human diseases. Investigating lipid profiles not only provides insights into the specific roles of lipid molecular species in health and diseases, but can also help in identifying potential preventive or therapeutic biomarkers. Cataract, the loss of transparency of eye lens, is a disease of protein aggregation. There are several factors contributing to the stability in protein conformation. Age-related changes in lipid composition could be a contributing factor for altered protein–lipid interaction leading to protein aggregation and cataract. Keeping this in view, in the present study, fatty acid profiling from different age groups of lenses was carried out, using a freshwater catfish as the model. Total lipids were extracted from lenses of three different age groups of fishes (young, adult, and aged) and fatty acid methyl esters (FAME) were prepared and FAME analysis was carried out using gas chromatography–mass spectrometry. The results showed that three fatty acids viz. heneicosylic acid (C21), docosahexaenoic acid (C22:6), nervonic acid (C24:1) which were not present in the adult lens, appeared in the aged lens. On the other hand, eicosenoic acid (C20:1) present in the adult lens was found to be absent in the aged lens. The appearance or disappearance of these fatty acids can possibly serve as biomarkers of aging lens which is the most vulnerable stage for cataract development.     

Simultaneous determination of six free fatty acids in alcohol extract of <Emphasis Type="Italic">P. japonicus</Emphasis> C. A. Mey. van <Emphasis Type="Italic">major</Emphasis> (Burk.) C. Y. Wu et K. M. Feng by gas chromatography-mass spectrometry

P. japonicus C. A. Mey. var. major (Burk.) C. Y. Wu et K. M. Feng is an important medicinal plant and has special beneficial effects on human health. Six types of free fatty acids (FFAs) were identified and quantified in the alcohol extract of P. japonicus C. A. Mey. var. major with a GC-MS method, including two fatty acids essential to the human body. All the six FFAs identified in alcohol extract were quantified using nonadecanoic acid as an internal standard. The results showed that the alcohol extract was abundant in four types of FFAs, unsaturated FFAs amounted to 47.04% of the total FFA content, and there were no trans FFAs. Palmitic acid (38.82%, 4.74 ± 0.09 mg/g) was the predominant fatty acid, followed by linoleic acid (27.92%, 2.31 ± 0.07 mg/g), stearic acid (14.14%, 1.54 ± 0.04 mg/g) and oleic acid (11.04%, 1.02 ± 0.03 mg/g).