#METABOLOMICS WORKBENCH Davidobe12_20200327_124023 DATATRACK_ID:1961 STUDY_ID:ST001369 ANALYSIS_ID:AN002285 PROJECT_ID:000000 VERSION 1 CREATED_ON April 29, 2020, 10:46 am #PROJECT PR:PROJECT_TITLE Metabolomics analysis of serum samples from patients during 2 years of IAT PR:PROJECT_TITLE trial, double blind placebo controled study PR:PROJECT_TYPE Study the changes produced by the use of AIT treatment or placebo PR:PROJECT_SUMMARY 47 patients were enrolled in a double-blind, placebo-controlled, multicenter PR:PROJECT_SUMMARY trial using with GRAZAX® (Phleum pretense) during 2 years of therapy (T2). PR:PROJECT_SUMMARY Immunological assays such as sIgE, sIgG4 and ISAC were carried out to the 31 PR:PROJECT_SUMMARY patients who finished the trial. Additionally, serum and PBMCs samples from PR:PROJECT_SUMMARY these samples were analyzed by metabolomics and transcriptomics, respectively. PR:PROJECT_SUMMARY Based on their sensitization level, 22 patients were grouped in Mono and Poli PR:PROJECT_SUMMARY groups, excluding epithelial allergic patients. Individuals were studied based PR:PROJECT_SUMMARY on their treatment in Active and Placebo and their sensitization level. PR:INSTITUTE Institute of Applied Molecular Medicine, CEU; The Centre of Metabolomics and PR:INSTITUTE Bioanalysis, CEU PR:LAST_NAME Barber Hernández PR:FIRST_NAME Domingo PR:ADDRESS Avda. Monteprincipe s/n 28668 Boadilla del Monte, Madrid, España PR:EMAIL domingo.barberhernandez@ceu.es PR:PHONE Tlf: 91 372 47 00 ext. 4662 #STUDY ST:STUDY_TITLE Grass pollen sublingual immunotherapy treatment induces transcriptomic and ST:STUDY_TITLE metabolic changes due to AIT treatment ST:STUDY_SUMMARY 47 patients were enrolled in a double-blind, placebo-controlled, multicenter ST:STUDY_SUMMARY trial using with GRAZAX® (Phleum pretense) during 2 years of therapy (T2). ST:STUDY_SUMMARY Immunological assays such as sIgE, sIgG4 and ISAC were carried out to the 31 ST:STUDY_SUMMARY patients who finished the trial. Additionally, serum and PBMCs samples from ST:STUDY_SUMMARY these samples were analyzed by metabolomics and transcriptomics, respectively. ST:STUDY_SUMMARY Based on their sensitization level, 22 patients were grouped in Mono and Poli ST:STUDY_SUMMARY groups, excluding epithelial allergic patients. Individuals were studied based ST:STUDY_SUMMARY on their treatment in Active and Placebo and their sensitization level. For ST:STUDY_SUMMARY metabolomics, samples were analyzed by Liquid and Gas Chromatography coupled to ST:STUDY_SUMMARY Mass Spectrometry (LC-MS and GC-MS, respectively). ST:INSTITUTE Institute of Applied Molecular Medicine, CEU; The Centre of Metabolomics and ST:INSTITUTE Bioanalysis, CEU ST:LAST_NAME Obeso Montero ST:FIRST_NAME David ST:ADDRESS Avda. Monteprincipe s/n 28668 Boadilla del Monte, Madrid, España ST:EMAIL david.obesomontero@beca.ceu.es ST:PHONE Tlf: 91 372 47 00 ext. 4662 ST:NUM_GROUPS 2 main groups: Active and Placebo, and 2 subgroups: Monosensitized and ST:NUM_GROUPS Polisensitized patients. ST:TOTAL_SUBJECTS 22 ST:STUDY_COMMENTS https://www.ceu.es;http://www.metabolomica.uspceu.es #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 Subject 1 P1_t0 Treatment:Active | Sensitization:Mono RAW_FILE_NAME=P1_t0.d SUBJECT_SAMPLE_FACTORS Subject 2 P3_t0 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P3_t0.d SUBJECT_SAMPLE_FACTORS Subject 3 P4_t0 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P4_t0.d SUBJECT_SAMPLE_FACTORS Subject 4 P8_t0 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P8_t0.d SUBJECT_SAMPLE_FACTORS Subject 5 P9_t0 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P9_t0.d SUBJECT_SAMPLE_FACTORS Subject 6 P11_t0 Treatment:Active | Sensitization:Poli RAW_FILE_NAME=P11_t0.d SUBJECT_SAMPLE_FACTORS Subject 7 P12_t0 Treatment:Active | Sensitization:Poli RAW_FILE_NAME=P12_t0.d SUBJECT_SAMPLE_FACTORS Subject 8 P13_t0 Treatment:Active | Sensitization:Poli RAW_FILE_NAME=P13_t0.d SUBJECT_SAMPLE_FACTORS Subject 9 P14_t0 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P14_t0.d SUBJECT_SAMPLE_FACTORS Subject 10 P15_t0 Treatment:Active | Sensitization:Poli RAW_FILE_NAME=P15_t0.d SUBJECT_SAMPLE_FACTORS Subject 11 P16_t0 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P16_t0.d SUBJECT_SAMPLE_FACTORS Subject 12 P17_t0 Treatment:Active | Sensitization:Mono RAW_FILE_NAME=P17_t0.d SUBJECT_SAMPLE_FACTORS Subject 13 P20_t0 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P20_t0.d SUBJECT_SAMPLE_FACTORS Subject 14 P21_t0 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P21_t0.d SUBJECT_SAMPLE_FACTORS Subject 15 P22_t0 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P22_t0.d SUBJECT_SAMPLE_FACTORS Subject 16 P23_t0 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P23_t0.d SUBJECT_SAMPLE_FACTORS Subject 17 P24_t0 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P24_t0.d SUBJECT_SAMPLE_FACTORS Subject 18 P25_t0 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P25_t0.d SUBJECT_SAMPLE_FACTORS Subject 19 P26_t0 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P26_t0.d SUBJECT_SAMPLE_FACTORS Subject 20 P27_t0 Treatment:Active | Sensitization:Mono RAW_FILE_NAME=P27_t0.d SUBJECT_SAMPLE_FACTORS Subject 21 P28_t0 Treatment:Active | Sensitization:Mono RAW_FILE_NAME=P28_t0.d SUBJECT_SAMPLE_FACTORS Subject 22 P30_t0 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P30_t0.d SUBJECT_SAMPLE_FACTORS Subject 1 P1_t2 Treatment:Active | Sensitization:Mono RAW_FILE_NAME=P1_t2.d SUBJECT_SAMPLE_FACTORS Subject 2 P3_t2 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P3_t2.d SUBJECT_SAMPLE_FACTORS Subject 3 P4_t2 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P4_t2.d SUBJECT_SAMPLE_FACTORS Subject 4 P8_t2 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P8_t2.d SUBJECT_SAMPLE_FACTORS Subject 5 P9_t2 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P9_t2.d SUBJECT_SAMPLE_FACTORS Subject 6 P11_t2 Treatment:Active | Sensitization:Poli RAW_FILE_NAME=P11_t2.d SUBJECT_SAMPLE_FACTORS Subject 7 P12_t2 Treatment:Active | Sensitization:Poli RAW_FILE_NAME=P12_t2.d SUBJECT_SAMPLE_FACTORS Subject 8 P13_t2 Treatment:Active | Sensitization:Poli RAW_FILE_NAME=P13_t2.d SUBJECT_SAMPLE_FACTORS Subject 9 P14_t2 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P14_t2.d SUBJECT_SAMPLE_FACTORS Subject 10 P15_t2 Treatment:Active | Sensitization:Poli RAW_FILE_NAME=P15_t2.d SUBJECT_SAMPLE_FACTORS Subject 11 P16_t2 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P16_t2.d SUBJECT_SAMPLE_FACTORS Subject 12 P17_t2 Treatment:Active | Sensitization:Mono RAW_FILE_NAME=P17_t2.d SUBJECT_SAMPLE_FACTORS Subject 13 P20_t2 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P20_t2.d SUBJECT_SAMPLE_FACTORS Subject 14 P21_t2 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P21_t2.d SUBJECT_SAMPLE_FACTORS Subject 15 P22_t2 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P22_t2.d SUBJECT_SAMPLE_FACTORS Subject 16 P23_t2 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P23_t2.d SUBJECT_SAMPLE_FACTORS Subject 17 P24_t2 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P24_t2.d SUBJECT_SAMPLE_FACTORS Subject 18 P25_T2 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P25_t2.d SUBJECT_SAMPLE_FACTORS Subject 19 P26_t2 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P26_t2.d SUBJECT_SAMPLE_FACTORS Subject 20 P27_T2 Treatment:Active | Sensitization:Mono RAW_FILE_NAME=P27_t2.d SUBJECT_SAMPLE_FACTORS Subject 21 P28_t2 Treatment:Active | Sensitization:Mono RAW_FILE_NAME=P28_t2.d SUBJECT_SAMPLE_FACTORS Subject 22 P30_t2 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P30_t2.d #COLLECTION CO:COLLECTION_SUMMARY For immunological analyses and metabolomics, serum samples were obtained and CO:COLLECTION_SUMMARY stored at -20°C until analysis. In the case of transcriptomics, Peripheral CO:COLLECTION_SUMMARY blood mononuclear cells (PBMCs) were isolated from whole blood and stored at CO:COLLECTION_SUMMARY -20°C in Buffer RLT until analysis. CO:SAMPLE_TYPE Blood (serum) CO:STORAGE_CONDITIONS -20℃ #TREATMENT TR:TREATMENT_SUMMARY Subjects were randomized (1:1) during autumn 2013 to receive either active TR:TREATMENT_SUMMARY treatment with GRAZAX® (Phleum pratense, 75,000SQ‐T tablets ALK, Hørsholm, TR:TREATMENT_SUMMARY Denmark) or placebo as Investigational Medical Products (IMP) during 2 years. TR:TREATMENT_SUMMARY Twenty-five patients were assigned to daily sublingual administration of TR:TREATMENT_SUMMARY GRAZAX®, while the rest received placebo tablets that were similar to the TR:TREATMENT_SUMMARY active IMP with regard to appearance, smell and taste. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For LC-MS, proteins were removed adding 300 µL of cold (-20 °C) SP:SAMPLEPREP_SUMMARY methanol:ethanol (1:1) to 100 µL of sample. Samples were then vortex-mixed and SP:SAMPLEPREP_SUMMARY stored on ice for 5 min. Supernatant containing the metabolites was separated SP:SAMPLEPREP_SUMMARY from the pellet by centrifugation (16,000× g for 20 min at 4 °C), and put into SP:SAMPLEPREP_SUMMARY a LC vial for analysis. For GC-MS, serum samples were first deproteinized using SP:SAMPLEPREP_SUMMARY cold acetonitrile (ACN) in a 3:1 proportion (120 μL of ACN were added to 40 μL SP:SAMPLEPREP_SUMMARY of serum). Samples were kept on ice for 5 minutes. Afterwards, the metabolites SP:SAMPLEPREP_SUMMARY were separated by centrifugation (10 min at 15,400 x g and 4°C). 100 µL of the SP:SAMPLEPREP_SUMMARY resulting supernatant were transferred to a GC vial with insert and were SP:SAMPLEPREP_SUMMARY evaporated to dryness for 2 hours at 30°C (SpeedVac Concentrator, Thermo Fisher SP:SAMPLEPREP_SUMMARY Scientific, Waltham, MA, USA). Afterwards, a methoximation was performed by SP:SAMPLEPREP_SUMMARY addition of 10 µL of O-methoxyamine hydrochloride 15mg/mL in pyridine, with the SP:SAMPLEPREP_SUMMARY purpose of protecting the reactive oxygen groups in the metabolites. The mixture SP:SAMPLEPREP_SUMMARY was vigorously vortex-mixed (1 minute each vial), ultrasonicated (3 times, 20 SP:SAMPLEPREP_SUMMARY seconds each) and further vortexed another 2 minutes. Then, the samples were SP:SAMPLEPREP_SUMMARY left in darkness at room temperature for 16 h for the completion of the SP:SAMPLEPREP_SUMMARY methoximation reaction. Finally, the samples were derivatized by the addition of SP:SAMPLEPREP_SUMMARY 10 µL of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% SP:SAMPLEPREP_SUMMARY trimethylchlorosilane (TMCS). SP:PROCESSING_METHOD Protein precipitation and metabolite extraction SP:PROCESSING_STORAGE_CONDITIONS On ice #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY GC-MS analysis was performed by a GC system (Agilent Technologies 7890A) CH:CHROMATOGRAPHY_SUMMARY equipped with an autosampler (Agilent 7693) coupled to a mass spectrometer with CH:CHROMATOGRAPHY_SUMMARY triple-Axis detector (5975C, Agilent). Two microliters (2 μL) of the CH:CHROMATOGRAPHY_SUMMARY derivatized sample were injected through a GC-Column DB5-MS (30 m length, 0.25 CH:CHROMATOGRAPHY_SUMMARY mm i.d., 0.25 μm film 95% dimethyl/5% diphenylpolysiloxane) with an integrated CH:CHROMATOGRAPHY_SUMMARY precolumn (10 m J&W, Agilent). Carrier gas (He) flow rate was set at 1 mL/min CH:CHROMATOGRAPHY_SUMMARY and injector temperature at 250 °C. Split ratio was fixed from 1:5 to 1:10 with CH:CHROMATOGRAPHY_SUMMARY 3 to 10 mL/min. He split flow into a Restek 20782 (Bellefonte, PA, USA) CH:CHROMATOGRAPHY_SUMMARY deactivated glass-wool split liner. The temperature gradient was programmed as CH:CHROMATOGRAPHY_SUMMARY follows: the initial oven temperature was set at 60 ºC (held for 1 min), CH:CHROMATOGRAPHY_SUMMARY increased to 325 ºC at 10 ºC/min rate (within 26.5 min) and hold 325 ºC for CH:CHROMATOGRAPHY_SUMMARY 10 min. The total run time was 37.5 min. A cool-down period was applied for 10 CH:CHROMATOGRAPHY_SUMMARY min before the next injection. Detector transfer line, filament source and the CH:CHROMATOGRAPHY_SUMMARY quadrupole temperature were set at 280 ºC, 230 ºC and 150 ºC, respectively. CH:CHROMATOGRAPHY_SUMMARY MS detection was performed with electron ionization (EI) mode at -70 eV. The CH:CHROMATOGRAPHY_SUMMARY mass spectrometer was operated in scan mode over a mass range of m/z 50-600 at a CH:CHROMATOGRAPHY_SUMMARY rate of 2.7 scan/s. Several IS injections, a standard mix of fatty acid methyl CH:CHROMATOGRAPHY_SUMMARY esters (FAME C8-C30), extraction blanks and 3 QCs samples were injected at the CH:CHROMATOGRAPHY_SUMMARY beginning of analysis, following QCs injections every 5 experimental samples and CH:CHROMATOGRAPHY_SUMMARY 1 QC injection at the end of worklist. CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890A CH:COLUMN_NAME GC-Column DB5-MS (30 m length, 0.25 mm i.d., 0.25 µm film 95% dimethyl/5% diphenylpolysiloxane) with an integrated precolumn (10 m J&W, Agilent) CH:COLUMN_NAME diphenylpolysiloxane) with an integrated precolumn (10 m J&W, Agilent) CH:COLUMN_TEMPERATURE As Oven CH:INJECTION_TEMPERATURE 250 CH:INTERNAL_STANDARD Standard mix of fatty acid methyl esters (FAME C8-C30) CH:SAMPLE_INJECTION 2 μL CH:ANALYTICAL_TIME 37.5 min CH:OVEN_TEMPERATURE The initial oven temperature was set at 60 ºC (held for 1 min), increased to CH:OVEN_TEMPERATURE 325 ºC at 10 ºC/min rate (within 26.5 min) and hold 325 ºC for 10 min. CH:TRANSFERLINE_TEMPERATURE 280 #ANALYSIS AN:ANALYSIS_TYPE MS AN:DATA_FORMAT .D #MS MS:INSTRUMENT_NAME Agilent 5975C MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS GC-MS analysis was performed by a GC system (Agilent Technologies 7890A) MS:MS_COMMENTS equipped with an autosampler (Agilent 7693) coupled to a mass spectrometer with MS:MS_COMMENTS triple-Axis detector (5975C, Agilent). MS:HELIUM_FLOW 1 mL/min MS:IONIZATION_ENERGY -70 eV MS:MS_RESULTS_FILE ST001369_AN002285_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END