A metabolomic study of urinary and serum changes in response to therapy in early rheumatoid arthritis in humans

Venous blood from DMARD naive Rheumatoid arthritis patientswas taken into plain tubes, allowed to clot centrifuged at 800xg 4oC and the serum removed for storage at -80oC. Urine from the same patients was collected, centrifuged at 800xg 4oC then filtered through 0.2uM filter before storage at -80oC.Serum samples were thawed at 4°C centrifuged at 15,000g at 4°C for 5 minutes. To remove proteins, 200μl from the middle of the sample was placed into a Nanosep® Omega 3000 Da (Pall Lifesciences, UK) molecular weight cut-off (MWCO) and centrifuged at 10,000g at 4°C for 15 minutes. Immediately prior to use, to remove the preservative glycerol, the filters were had been washed 6 times in distilled water at 37°C by centrifugation at 3000g for 15 minutes (6). The resulting serum filtrate was diluted in a 1+3 ratio with NMR buffer containing 1.6mM Difluorotrimethylsilylmethylphosphonic acid (DFTMP, Manchester Organics, Manchester, UK), 400mM phosphate, 40% D2O, 0.4% azide and 2mM 3-(Trimethylsilyl)-1-propanesulfonic acid-d6 sodium salt (DSS-d6, all from Merck, Southampton, UK). An aliquot (60ul) was removed to a glass champagne vials (Cole-Parmer, Saint Neots, UK) and stored at -80°C until analysis.<br>After thawing, urine samples (1ml) at 4¬oC were centrifuged at 15,000g for 5 minutes. A cleared sample (0.5ml) was mixed at 1:3 ratio with the 4x NMR buffer as for the serum above. The pH was adjusted (twice over a period of 30minutes) to pH 7.0. The samples were centrifuged at 15000g for 5 minutes and a sample (60ul) was removed to a glass champagne vial and frozen at -80oC prior to NMR spectroscopy." "Samples were defrosted and transferred to 1.7mm NMR tubes (Bruker Biospin, Coventry, UK) using an Anachem Autosampler. After capping the tubes and wiping with dust-free paper, one-dimensional 1H spectra were acquired in a Bruker AVANCE II 600 MHz NMR spectrometer (Bruker Corp., USA) equipped with a 1.7 mm cryoprobe<br>One-dimensional 1H spectra were acquired at 300K using a standard 1D-1H-Nuclear Overhauser Effect spectroscopy (NOESY) pulse sequence with water saturation using pre-sat in a Bruker AVANCE II 600 MHz NMR spectrometer (Bruker Corp., USA) equipped with a 1.7 mm cryoprobe. Spectral width was set to 12 ppm and the scans were repeated 128 times. Samples series were loaded into 96-tube racks and held at 6oC in the SampleJet sample handing device until processed.<br>Automated data processing, Fourier transformation, and phasing were carried out in Metabolab. The spectra were then normalised to the median and bucketed per peak into a matrix of metabolite peak intensities using MetabolabChenomx v8.2 software used for initial metabolite identification using an initial automated fit to the Chenomx library, followed by manual final annotation.The prepared buckted table of NMR peak intesnties and the table of identified metabolies was subjected to univariate and multivariate analysis. Unsupervised multivariate analysis of the spectra was carried after out using Principal Component Analysis (PCA). The data was scaled by mean centering of each variable followed by division of the square root of the standard deviation (Pareto scaling) before analysis. <br>

未接受改善病情抗风湿药（DMARD, Disease-Modifying Antirheumatic Drug）的类风湿关节炎患者的静脉血采集于普通采血管，静置凝血后于4℃、800×g条件下离心，分离血清并于-80℃保存。同批次患者的尿液样本采集后，于4℃、800×g离心，经0.2μm滤器过滤后，于-80℃保存。
血清样本于4℃解冻后，在4℃、15000×g条件下离心5分钟。为去除蛋白，取样本中部200μL移至Nanosep® Omega 3000 Da（分子量截留值（MWCO），英国Pall Lifesciences公司）超滤管中，于4℃、10000×g离心15分钟。临用前，为去除防腐剂甘油，将超滤管于37℃、3000×g条件下离心15分钟，用蒸馏水洗涤6次以完成去污。所得血清超滤滤液以1:3的比例与核磁共振（NMR）缓冲液混合稀释，该缓冲液含1.6mM二氟三甲基硅基甲基膦酸（DFTMP，英国曼彻斯特Manchester Organics公司）、400mM磷酸盐、40%氘代水（D₂O）、0.4%叠氮化物以及2mM 3-(三甲基硅基)-1-丙磺酸-d6钠盐（DSS-d6，均购自英国南安普敦Merck公司）。取60μL等分试样置于玻璃香槟型进样瓶（英国Saint Neots Cole-Parmer公司）中，于-80℃保存直至检测。
解冻后，将1mL尿液样本置于4℃环境下，以15000×g离心5分钟。取澄清后的0.5mL尿液样本，按照与血清样本相同的比例，以1:3混合于4×NMR缓冲液中。随后将样本pH值调整至7.0（30分钟内调整两次）。再次以15000×g离心5分钟后，取60μL样本置于玻璃香槟型进样瓶中，于-80℃冷冻保存，待进行核磁共振光谱检测。
样本解冻后，使用Anachem自动进样器将其转移至1.7mm核磁共振管（英国考文垂Bruker Biospin公司）。封管并用无尘纸擦拭后，在配备1.7mm低温探头的布鲁克AVANCE II 600MHz核磁共振波谱仪（美国布鲁克公司）上采集一维氢谱。采用配备1.7mm低温探头的布鲁克AVANCE II 600MHz核磁共振波谱仪，以300K温度采集一维氢谱，使用标准1D-¹H-核Overhauser效应光谱（NOESY）脉冲序列，并通过预饱和法实现水峰抑制。谱宽设置为12ppm，扫描次数为128次。将样本组装入96管架，置于SampleJet样本处理装置中6℃保存，直至完成上机检测。
采用Metabolab软件完成自动化数据处理、傅里叶变换以及相位校正。随后将谱图以中位数进行归一化处理，并通过MetabolabChenomx v8.2软件将各峰按积分值归集为代谢物峰强度矩阵；该软件首先通过自动化拟合Chenomx数据库完成初始代谢物鉴定，随后由人工进行最终注释。
将整理好的核磁共振峰强度表格与已鉴定代谢物表格进行单变量与多变量统计分析。对谱图采用主成分分析（PCA）进行无监督多变量统计分析。分析前，对数据进行帕累托（Pareto）标准化处理：先对每个变量进行均值中心化，再除以标准差的平方根。