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>