Metabolic profiling detects biomarkers of protein degradation in COPD patients On‐line data supplement Baljit K. Ubhi, John H. Riley, Paul A. Shaw, David A. Lomas, Ruth Tal-Singer, William MacNee, Jules L Griffin, Susan C Connor Materials and Methods: ECLIPSE is a 3 year longitudinal study with samples collected at baseline, 3 and 6 months and then every 6 months for 3 years. Control subjects consisting of current or former smokers and individuals who have never smoked (n=566), were recruited as comparators with the COPD subjects (2756). The COPD patients were selected according to the following criteria; an forced expiratory volume in the first second (FEV1) of less than 80% of predicted normal and a baseline FEV1/ forced vital capacity(FVC) ratio of less than 0.7, aged 40-75 and either current or ex-smokers with a greater than 10 pack-year history. Patients were categorised according to the criteria from the Global Initiative for Chronic Obstructive Lung Disease (GOLD status; (E1)) i.e. COPD patients had an FEV1/FVC ratio of less than 70% and were subdivided into GOLD stage II, stage III and stage IV based on an 50%<FEV1<80%, 30%<FEV1<50% and <30% FEV1 predicted respectively. The control group consists of a cohort of 343 current and ex-smokers with greater than 10 pack-year smoking history and a second cohort of 223 who have never smoked (less than 1 pack-year smoking history). Both cohorts that make up the control groups have a baseline FEV1 greater than 85% predicted and a FEV1/FVC ratio of greater than 0.7. The metabolomics cohort was selected as a subset of these patients, excluding current smokers. Patients were in a stable state, free of exacerbations, for one month prior to sampling. Initial pair-matched selection of metabolomics patients excluded current smokers so that power for detecting COPD-related effects was not reduced by the confounding effects of smoking. However a few individuals (n=5) were found by cotinine assay to be current smokers and these were excluded from further analysis. The ECLIPSE study was ethically approved in each of the participating centres and all participants provided written informed consent. (Clinicaltrials.gov identifier NCT00292552; GSK Study Identifier SCO104960). Study Assessments: All subjects performed standardized spirometry according to international guidelines (E2) before and after salbutamol (180 ug) with reversible airflow obstruction being defined as an increase in FEV1 of 15% and at least 200ml post- bronchdilator. Study assessments also included pulmonary function measurements, computed chest tomography, biomarkers (in blood, sputum, urine and exhaled breath condensate), health outcome questionnaires and exercise testing (6 minute walk test, measured according to international guidelines (E3)). Several known and potential biomarkers of pulmonary, vascular, cardiac, and systemic disease including cachexia were also measured. Body mass index (BMI) was calculated and the subjects split into two groups <21 cachectic and >40 extremely obese. The BMI and fat free mass (measured by bioelectrical impedence) were also used as continous variables for PLS and Pearson correlation calculations. Body composition was determined by whole body plethmysography as described in the clinical protocol. These variables were treated independently as continuous variables. Exercise testing was carried out according to the protocol and utilized the 6 minute walking test. Subjects were divided into 2 groups based on the distance walked < 350m or >= 350 m. CT Scan All subjects underwent a low-dose computed tomography (CT) scan of the chest All of the CT scans were evaluated at the central imaging unit at the University of British Columbia, Vancouver, Canada. All ECLIPSE subjects underwent a low-dose CT scan of the chest to quantitfy the degree of emphysema using multi-detector-row CT scanners (GE Healthcare or Siemens Healthcare) with a minimum of 4 rows, obtained in supine position at suspended full inspiration without administration of intravenous contrast. Exposure settings were 120 kVp and 40 mAs and images were reconstructed using 1.0 mm (Siemens) or 1.25 mm (GE) contiguous slices and a low spatial frequency reconstruction algorithm (GE: Standard; Siemens: b35f). CT scanners were calibrated regularly using industry and institutional standards. The CT scans were evaluated at the central imaging unit at the University of British Columbia, Vancouver. The percentage of lung CT voxels below a threshold of -950 Hounsfield Units (%LAA) as a representative of the presence of emphysema, was performed using the software Pulmonary Workstation 2.0 (VIDA Diagnostics, Iowa City, IA, U.S.A.). A cut off of <10% low attenuation areas (LAA) was utilised for minimal emphysema and 20% for emphysema [18]. The %LAA was also used as a continuous variable . Open Profiling Metabolomics Experiments Sampling and Data acquisition 2 and 4 ml aliquots of blood were collected into 4ml BD Serum Vacutainer with Silica Act Clot Activator, incubated at room temperature for 30 minutes and centrifuged at 1500 x g for 10 minutes. 5 x 500ul of serum were then aliquoted into cryotubes and stored at –80C until analysis. Serum samples were thawed once at the time of NMR sample preparation and data acquisition. NMR samples were partly prepared using a Tecan robot; 150ul of serum were added to 450ul of cold isotonic saline containing D2O ((10% (v/v) D2O; 90% (v/v) deionised H2O; 0.9% (w/v) NaCl (BDH Lab. Supplies, UK)) and 4,4-dimethyl-4-silapentane-1- ammonium trifluoroacetate (DSA; Onyx Scientific Limited, Silverbriar, Sunderland Enterprise Park East, Sunderland, UK; Alum et al 2008), mixed and then centrifuged at 2400g for 20 minutes at 4 degrees. 550ul of each supernatant was transferred into 5mm Wilmad NMR tubes (507PP). Proton NMR spectra were acquired for each sample using a TXI probe on a Bruker Avance III 700MHz NMR spectrometer operating at 699.72 MHz.. A Carr- Purcell-Meiboom-Gill (CPMG) pulse sequence was used to reduce interference from background proteins with a total spin-spin relaxation delay of 320ms. The free induction decays were collected into 64k datapoints with a spectral width of 20 ppm. The spectra were zero-filled to double size and an exponential line broadening factor of 1 Hz applied prior to Fourier transformation. The water resonance was suppressed by presaturation during the relaxation delay prior to the 90 degree pulse. The experimental parameters used have been previously described (E4). Amino acid analysis A targeted LC-MS/MS method was developed to provide analytical validation of the COPD- related amino acid changes observed by open profiling metabolomics and subsequently to assist in the biological evaluation of their biomarker potential. Analytical validation of one dimensional NMR findings is usually required due to the high degree of signal overlap and this can only be partly achieved for some metabolites (eg proline) by additional metabolite spiking and two dimensional NMR experiments. There are also other limitations to open profiling metabolomics including semi rather than absolute quantitative measurement and, in the case of NMR, lower sensitivity compared to other techniques. A targeted amino acid assay using an LC-MS/MS platform was chosen because it could provide quantitative amino acid information on metabolites present in low concentration. A smaller sample size (male smoker control (n=30) versus GOLD IV (n=30) was selected from the original NMR dataset (n=244) for the purposes of the analytical validation. In this case the most severely affected COPD patients were compared with controls with the main purpose of confirming the NMR findings. Chemicals HPLC grade methanol was purchased from Fisher (Fisher Scientific GmbH, Ulm, Germany) and ammonium formate was purchased from Sigma/Aldrich (MO, USA). The Phenomenex EZ:Faast LC-MS kit (Phenomenex Inc, Torrence, CA, USA) was used for the preparation of samples for amino acid analysis. Sample Preparation Samples were first prepared according to the EZ:Faast amino acid analysis procedure ((www.phenomenex.com) and the dried down organic extracts were reconstituted in 100 μl of a 1:2 (v/v) 10 mM ammonium formate in water: 10 mM ammonium formate in methanol solution. Liquid Chromatography The LC-MS/MS assay was developed using a Waters Acquity UPLC system attached to a Waters Quattro Premier XE Triple Quadrupole mass spectrometer to detect chloroformate amino acid derivatives prepared by the EZ: Faast amino acid derivatisation procedure (www.phenomenex.com). Sample aliquots of 5 μl were injected onto a Phenomenex EZ:Faast AAA-MS column (250 mm × 2.0 mm; room temperature). A linear gradient of 68-83% buffer B in buffer A (10mM ammonium formate buffer A: 10mM ammonium formate in methanol buffer B) was applied over 11 minutes and equibrated at 68% buffer B for 2 minutes between injections at constant eluent flow of 0.25ml/min. Carry-over was eliminated by using a strong and weak needle wash in between each sample injection (83:17 methanol:water and 10:90 methanol:water were strong and weak washes respectively). A 5-point calibration curve at the beginning and end of each batch was acquired for a standard amino acid mix and a QC sample was run every ten injections to test for system reproducibility. The QC sample consisted of the three internal standards, homoarginine (HARG), methionine-d3 (MET-d3) and homophenylalanine (HPHE), diluted 1:100. A derivatised, pooled sample of 10 μl of each sample was injected every ten samples. Mass Spectrometry Mass spectrometric data was collected using a Waters Quattro Premier XE Triple Quadrupole mass spectrometer equipped with an electrospray source in positive ionisation mode. The source temperature was set to 120˚C with a cone gas flow of 50 l/hr, a desolvation temperature of 350˚C and a desolvation gas flow of 700 l/hr. A capillary voltage of 1000 V was applied.