#METABOLOMICS WORKBENCH martiroca_20210121_031601 DATATRACK_ID:2413 STUDY_ID:ST001662 ANALYSIS_ID:AN002713 PROJECT_ID:PR001067 VERSION 1 CREATED_ON January 26, 2021, 11:32 am #PROJECT PR:PROJECT_TITLE Urinary LCMS metabolimc study in bladder cancer PR:PROJECT_SUMMARY Bladder cancer (BC) is among the most frequent malignancies worldwide. Novel PR:PROJECT_SUMMARY non-invasive markers are needed to diagnose and stage BC with more accuracy than PR:PROJECT_SUMMARY invasive procedures such as cystoscopy. Our aim was to discover novel urine PR:PROJECT_SUMMARY metabolomic profiles to diagnose and stage non-muscle invasive (NMIBC) and PR:PROJECT_SUMMARY muscle-invasive (MIBC) patients using ultra-performance liquid chromatography PR:PROJECT_SUMMARY analysis (UPLC)-based metabolomics. We prospectively recruited 64 BC patients PR:PROJECT_SUMMARY (19 TaG1, 11 TaG3, 20 T1G3, 12 T2G3, 1 T2G2, 1 T3G3) and 20 age- and sex-matched PR:PROJECT_SUMMARY healthy volunteers without evidence of renal or bladder condition confirmed by PR:PROJECT_SUMMARY ultrasound, from whom we collected a first morning urine sample (before surgery PR:PROJECT_SUMMARY in patients). We conducted a UPLC-quadrupole-time-of-flight mass spectrometry PR:PROJECT_SUMMARY (UPLC-Q-ToF MS) untargeted metabolomic analysis in all urine samples. We PR:PROJECT_SUMMARY selected the discriminant variables between groups with a supervised PR:PROJECT_SUMMARY orthogonal-least-squares discriminant analysis (OPLS-DA) analysis and we PR:PROJECT_SUMMARY identified them by querying their exact mass against those presented in online PR:PROJECT_SUMMARY databases through a mediator platform. Subsequently, we confirmed the PR:PROJECT_SUMMARY dysregulated metabolites when chemical standards were commercially available. We PR:PROJECT_SUMMARY compared all clinical groups of patients with controls and we identified PR:PROJECT_SUMMARY dysregulated metabolites in every comparison. Of these, we confirmed p-cresol PR:PROJECT_SUMMARY glucuronide as potential diagnostic biomarker, and potential staging tool for PR:PROJECT_SUMMARY NMIBC patients. Among NMIBC patients, we identified p-coumaric acid as a PR:PROJECT_SUMMARY potential staging biomarker for milder NMIBC stages (TaG1). Additionally, we PR:PROJECT_SUMMARY confirmed spermine and adenosine as potential staging biomarkers for MIBC. This PR:PROJECT_SUMMARY is the first study conducted in urine samples of most stages of NMIBC and MIBC PR:PROJECT_SUMMARY and healthy controls to identify non-invasive biomarkers. Once confirmed, these PR:PROJECT_SUMMARY may improve BC management thus reducing the use of current harmful diagnostic PR:PROJECT_SUMMARY techniques. PR:INSTITUTE Health Research Institute Hospital La Fe PR:LABORATORY Analytical Unit PR:LAST_NAME Roca Marugán PR:FIRST_NAME Marta PR:ADDRESS Avenida Fernando Abril Martorell 106, Torre A, Valencia, Valencia, 46026, Spain PR:EMAIL marta_roca@iislafe.es PR:PHONE 680888576 #STUDY ST:STUDY_TITLE LC-MS Metabolomics of Urine Reveals Distinct Profiles for Low- and High-Grade ST:STUDY_TITLE Bladder Cancer ST:STUDY_SUMMARY Bladder cancer (BC) is among the most frequent malignancies worldwide. Novel ST:STUDY_SUMMARY non-invasive markers are needed to diagnose and stage BC with more accuracy than ST:STUDY_SUMMARY invasive procedures such as cystoscopy. Our aim was to discover novel urine ST:STUDY_SUMMARY metabolomic profiles to diagnose and stage non-muscle invasive (NMIBC) and ST:STUDY_SUMMARY muscle-invasive (MIBC) patients using ultra-performance liquid chromatography ST:STUDY_SUMMARY analysis (UPLC)-based metabolomics. We prospectively recruited 64 BC patients ST:STUDY_SUMMARY (19 TaG1, 11 TaG3, 20 T1G3, 12 T2G3, 1 T2G2, 1 T3G3) and 20 age- and sex-matched ST:STUDY_SUMMARY healthy volunteers without evidence of renal or bladder condition confirmed by ST:STUDY_SUMMARY ultrasound, from whom we collected a first morning urine sample (before surgery ST:STUDY_SUMMARY in patients). We conducted a UPLC-quadrupole-time-of-flight mass spectrometry ST:STUDY_SUMMARY (UPLC-Q-ToF MS) untargeted metabolomic analysis in all urine samples. We ST:STUDY_SUMMARY selected the discriminant variables between groups with a supervised ST:STUDY_SUMMARY orthogonal-least-squares discriminant analysis (OPLS-DA) analysis and we ST:STUDY_SUMMARY identified them by querying their exact mass against those presented in online ST:STUDY_SUMMARY databases through a mediator platform. Subsequently, we confirmed the ST:STUDY_SUMMARY dysregulated metabolites when chemical standards were commercially available. We ST:STUDY_SUMMARY compared all clinical groups of patients with controls and we identified ST:STUDY_SUMMARY dysregulated metabolites in every comparison. Of these, we confirmed p-cresol ST:STUDY_SUMMARY glucuronide as potential diagnostic biomarker, and potential staging tool for ST:STUDY_SUMMARY NMIBC patients. Among NMIBC patients, we identified p-coumaric acid as a ST:STUDY_SUMMARY potential staging biomarker for milder NMIBC stages (TaG1). Additionally, we ST:STUDY_SUMMARY confirmed spermine and adenosine as potential staging biomarkers for MIBC. This ST:STUDY_SUMMARY is the first study conducted in urine samples of most stages of NMIBC and MIBC ST:STUDY_SUMMARY and healthy controls to identify non-invasive biomarkers. Once confirmed, these ST:STUDY_SUMMARY may improve BC management thus reducing the use of current harmful diagnostic ST:STUDY_SUMMARY techniques. ST:INSTITUTE Health Research Institute Hospital La Fe ST:LABORATORY Analytical Unit ST:LAST_NAME Roca Marugán ST:FIRST_NAME Marta ST:ADDRESS Avenida Fernando Abril Martorell 106, Torre A, Valencia, Valencia, 46026, Spain ST:EMAIL marta_roca@iislafe.es ST:PHONE 680888576 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Pooled #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - mv08618_05.mzXML Disease:QC | Type of tumor:QC RAW_FILE_NAME=mv08618_05.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_08.mzXML Disease:TAG1 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_08.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_09.mzXML Disease:TAG1 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_09.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_11.mzXML Disease:T1G3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_11.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_12.mzXML Disease:Control | Type of tumor:Control RAW_FILE_NAME=mv08618_12.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_13.mzXML Disease:Control | Type of tumor:Control RAW_FILE_NAME=mv08618_13.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_14.mzXML Disease:T2G3 | Type of tumor:MIBC RAW_FILE_NAME=mv08618_14.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_15.mzXML Disease:T2G3 | Type of tumor:MIBC RAW_FILE_NAME=mv08618_15.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_16.mzXML Disease:TAG1 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_16.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_17.mzXML Disease:Control | Type of tumor:Control RAW_FILE_NAME=mv08618_17.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_18.mzXML Disease:QC | Type of tumor:QC RAW_FILE_NAME=mv08618_18.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_19.mzXML Disease:T1G3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_19.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_20.mzXML Disease:T1G3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_20.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_21.mzXML Disease:T2G3 | Type of tumor:MIBC RAW_FILE_NAME=mv08618_21.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_22.mzXML Disease:T2G3 | Type of tumor:MIBC RAW_FILE_NAME=mv08618_22.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_23.mzXML Disease:TAG1 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_23.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_24.mzXML Disease:T1G3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_24.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_25.mzXML Disease:T2G3 | Type of tumor:MIBC RAW_FILE_NAME=mv08618_25.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_26.mzXML Disease:TAG3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_26.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_27.mzXML Disease:TAG3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_27.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_28.mzXML Disease:T2G3 | Type of tumor:MIBC RAW_FILE_NAME=mv08618_28.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_29.mzXML Disease:QC | Type of tumor:QC RAW_FILE_NAME=mv08618_29.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_31.mzXML Disease:TAG1 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_31.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_32.mzXML Disease:Control | Type of tumor:Control RAW_FILE_NAME=mv08618_32.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_33.mzXML Disease:T1G3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_33.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_34.mzXML Disease:TAG3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_34.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_35.mzXML Disease:T1G3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_35.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_36.mzXML Disease:T2G3 | Type of tumor:MIBC RAW_FILE_NAME=mv08618_36.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_37.mzXML Disease:Control | Type of tumor:Control RAW_FILE_NAME=mv08618_37.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_38.mzXML Disease:TAG1 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_38.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_39.mzXML Disease:T1G3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_39.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_40.mzXML Disease:QC | Type of tumor:QC RAW_FILE_NAME=mv08618_40.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_41.mzXML Disease:T1G3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_41.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_42.mzXML Disease:T1G3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_42.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_43.mzXML Disease:Control | Type of tumor:Control RAW_FILE_NAME=mv08618_43.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_44.mzXML Disease:TAG1 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_44.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_45.mzXML Disease:Control | Type of tumor:Control RAW_FILE_NAME=mv08618_45.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_46.mzXML Disease:T1G3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_46.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_47.mzXML Disease:TAG1 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_47.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_48.mzXML Disease:Control | Type of tumor:Control RAW_FILE_NAME=mv08618_48.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_49.mzXML Disease:TAG3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_49.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_50.mzXML Disease:QC | Type of tumor:QC RAW_FILE_NAME=mv08618_50.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_51.mzXML Disease:T1G3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_51.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_52.mzXML Disease:TAG1 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_52.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_53.mzXML Disease:TAG1 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_53.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_54.mzXML Disease:T2G3 | Type of tumor:MIBC RAW_FILE_NAME=mv08618_54.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_55.mzXML Disease:Control | Type of tumor:Control RAW_FILE_NAME=mv08618_55.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_56.mzXML Disease:T1G3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_56.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_57.mzXML Disease:Control | Type of tumor:Control RAW_FILE_NAME=mv08618_57.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_58.mzXML Disease:T1G3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_58.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_59.mzXML Disease:TAG3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_59.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_60.mzXML Disease:T1G3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_60.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_61.mzXML Disease:QC | Type of tumor:QC RAW_FILE_NAME=mv08618_61.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_62.mzXML Disease:T1G3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_62.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_63.mzXML Disease:T1G3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_63.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_64.mzXML Disease:Control | Type of tumor:Control RAW_FILE_NAME=mv08618_64.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_65.mzXML Disease:Control | Type of tumor:Control RAW_FILE_NAME=mv08618_65.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_66.mzXML Disease:TAG3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_66.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_67.mzXML Disease:TAG3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_67.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_68.mzXML Disease:T1G3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_68.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_69.mzXML Disease:TAG3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_69.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_70.mzXML Disease:T1G3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_70.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_71.mzXML Disease:TAG1 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_71.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_72.mzXML Disease:QC | Type of tumor:QC RAW_FILE_NAME=mv08618_72.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_73.mzXML Disease:TAG3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_73.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_74.mzXML Disease:T2G3 | Type of tumor:MIBC RAW_FILE_NAME=mv08618_74.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_75.mzXML Disease:Control | Type of tumor:Control RAW_FILE_NAME=mv08618_75.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_76.mzXML Disease:T1G3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_76.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_77.mzXML Disease:TAG1 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_77.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_78.mzXML Disease:Control | Type of tumor:Control RAW_FILE_NAME=mv08618_78.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_79.mzXML Disease:TAG1 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_79.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_80.mzXML Disease:Control | Type of tumor:Control RAW_FILE_NAME=mv08618_80.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_81.mzXML Disease:Control | Type of tumor:Control RAW_FILE_NAME=mv08618_81.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_82.mzXML Disease:TAG1 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_82.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_83.mzXML Disease:QC | Type of tumor:QC RAW_FILE_NAME=mv08618_83.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_84.mzXML Disease:TAG3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_84.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_85.mzXML Disease:TAG1 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_85.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_86.mzXML Disease:Control | Type of tumor:Control RAW_FILE_NAME=mv08618_86.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_87.mzXML Disease:Control | Type of tumor:Control RAW_FILE_NAME=mv08618_87.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_88.mzXML Disease:T2G3 | Type of tumor:MIBC RAW_FILE_NAME=mv08618_88.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_89.mzXML Disease:T2G3 | Type of tumor:MIBC RAW_FILE_NAME=mv08618_89.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_90.mzXML Disease:TAG1 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_90.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_91.mzXML Disease:TAG1 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_91.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_93.mzXML Disease:Control | Type of tumor:Control RAW_FILE_NAME=mv08618_93.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_94.mzXML Disease:QC | Type of tumor:QC RAW_FILE_NAME=mv08618_94.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_95.mzXML Disease:TAG3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_95.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_96.mzXML Disease:TAG1 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_96.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_97.mzXML Disease:TAG1 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_97.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_98.mzXML Disease:Control | Type of tumor:Control RAW_FILE_NAME=mv08618_98.mzXML SUBJECT_SAMPLE_FACTORS - mv08618_99.mzXML Disease:T1G3 | Type of tumor:NMIBC RAW_FILE_NAME=mv08618_99.mzXML #COLLECTION CO:COLLECTION_SUMMARY Study subjects Sixty-four BC patients were recruited between May 2016 and April CO:COLLECTION_SUMMARY 2018 at La Fe University and Polytechnic Hospital (Valencia, Spain). Twenty age- CO:COLLECTION_SUMMARY and sex-matched healthy volunteers (control group) who underwent an ultrasound CO:COLLECTION_SUMMARY scan to rule out the presence of urological malignancies or other alterations CO:COLLECTION_SUMMARY were also recruited. Patients and controls were clinically followed-up until May CO:COLLECTION_SUMMARY 2020. Pre-operative clinical staging was performed through physical examination, CO:COLLECTION_SUMMARY urine cytology and CT scans of the chest, abdomen and pelvis (in case of CO:COLLECTION_SUMMARY invasive bladder cancer). The tumor histological classification was done CO:COLLECTION_SUMMARY according to grade in the WHO 1973 and 2004 classifications. Demographic and CO:COLLECTION_SUMMARY clinical data were collected. The exclusion criteria were lack of informed CO:COLLECTION_SUMMARY consent, absence of histological confirmation and presence of other CO:COLLECTION_SUMMARY malignancies. Informed consent was obtained from all participants according to CO:COLLECTION_SUMMARY protocols approved by the ethics review board at La Fe University and CO:COLLECTION_SUMMARY Polytechnic Hospital. The study was performed according to the declaration of CO:COLLECTION_SUMMARY Helsinki, as amended in Edinburgh in 2000. Urine collection A first morning CO:COLLECTION_SUMMARY urine sample of 25-50 ml was collected in sterile containers from all CO:COLLECTION_SUMMARY participants. Urine was kept at 4 ºC until processing and centrifuged at 805 x CO:COLLECTION_SUMMARY g for 5 min at 4 ºC to remove cellular debris. Supernatant was aliquoted and CO:COLLECTION_SUMMARY frozen at -80 ºC until analyzed. The concentration of creatinine in each urine CO:COLLECTION_SUMMARY sample was measured by clinical laboratory standardized methods. CO:SAMPLE_TYPE Urine #TREATMENT TR:TREATMENT_SUMMARY A first morning urine sample of 25-50 ml was collected in sterile containers TR:TREATMENT_SUMMARY from all participants. Urine was kept at 4 ºC until processing and centrifuged TR:TREATMENT_SUMMARY at 805 x g for 5 min at 4 ºC to remove cellular debris. Supernatant was TR:TREATMENT_SUMMARY aliquoted and frozen at -80 ºC until analyzed. The concentration of creatinine TR:TREATMENT_SUMMARY in each urine sample was measured by clinical laboratory standardized methods #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Sample treatment was performed according to a previous study 22. Briefly, after SP:SAMPLEPREP_SUMMARY centrifugation, 40 µl of urine cleaned supernatant were transferred to a 96 SP:SAMPLEPREP_SUMMARY well-plate for LC-MS (liquid chromatography-mass spectrometry) analysis and SP:SAMPLEPREP_SUMMARY diluted by adding 50 µl of H2O (0.1 % v/v HCOOH). Each sample was spiked with SP:SAMPLEPREP_SUMMARY 10 µl of 20 µM IS solution containing phenylalanine-d5, caffeine-d9, leukine SP:SAMPLEPREP_SUMMARY enkephaline and reserpine in H2O:CH3OH (1:1, 0.1% v/v HCOOH). Blank samples were SP:SAMPLEPREP_SUMMARY prepared by replacing urine by ultrapure H2O. A quality control (QC) sample was SP:SAMPLEPREP_SUMMARY prepared by mixing 10 µl from each urine sample. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The metabolomic analysis was carried out using an Ultra-Performance Liquid CH:CHROMATOGRAPHY_SUMMARY Chromatography (UPLC) system coupled to an iFunnel Q-ToF CH:CHROMATOGRAPHY_SUMMARY (quadrupole-time-of-flight) Agilent 6550 mass spectrometer (Agilent CH:CHROMATOGRAPHY_SUMMARY Technologies, CA, USA) using an UPLC BEH C18 (100 x 2.1 mm, 1.7 µm, Waters, CH:CHROMATOGRAPHY_SUMMARY Wexford, Ireland) column from Waters (Wexford, Ireland). Autosampler and column CH:CHROMATOGRAPHY_SUMMARY temperatures were set to 4 °C and 40 °C, respectively, and the injection CH:CHROMATOGRAPHY_SUMMARY volume was 5 µl. Mobile phase A and mobile phase B consisted of H2O and CH:CHROMATOGRAPHY_SUMMARY acetonitrile, both containing 0.1% of formic acid. A gradient elution was CH:CHROMATOGRAPHY_SUMMARY performed at a flow rate of 400 µl min-1 along 14 min as follows: initial CH:CHROMATOGRAPHY_SUMMARY conditions of 98% of mobile phase A was maintained for 1 min, and then decreased CH:CHROMATOGRAPHY_SUMMARY until 75% in 2 min, 50% in 3 min and 5% in 3 more min. 95% of mobile phase B was CH:CHROMATOGRAPHY_SUMMARY held for 3 min and then, a 0.55 min gradient was used to return to the initial CH:CHROMATOGRAPHY_SUMMARY conditions, which were held for 2.5 min to totally column recovery. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 6550 CH:COLUMN_NAME Waters Acquity BEH C18 (100 x 2mm, 1.7um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6550 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS Full scan MS data from 50 to 1700 m/z with a scan frequency of 6 Hz was MS:MS_COMMENTS collected both in positive (ESI+) and negative (ESI-) electrospray ionization MS:MS_COMMENTS modes. The following electrospray ionization parameters were used: gas MS:MS_COMMENTS temperature, 200 °C; drying gas, 14 l min-1; nebulizer, 60 psi; sheath gas MS:MS_COMMENTS temperature, 350 °C; sheath gas flow, 11 l min-1. Urine samples were randomly MS:MS_COMMENTS injected in the chromatographic system in order to avoid intra-batch MS:MS_COMMENTS variability, as well as to enhance quality and reproducibility. QC sample was MS:MS_COMMENTS analyzed every 7 injections to monitor and correct changes in the instrument MS:MS_COMMENTS response. QC sample was also repeatedly analysed under auto MS/MS and All-ion MS:MS_COMMENTS (MSE) fragmentation modes to provide useful information of fragment ions for MS:MS_COMMENTS identification purposes. Sample stability and analytical drift were investigated MS:MS_COMMENTS through IS intensities. Blank analysis was performed at the end of the sequence MS:MS_COMMENTS and used to identify artefacts from sampling, preparation of samples and MS:MS_COMMENTS analysis. MS:MS_RESULTS_FILE ST001662_AN002713_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Seconds #END