#METABOLOMICS WORKBENCH Davidobe12_20210118_021528_mwtab.txt DATATRACK_ID:2401 STUDY_ID:ST001655 ANALYSIS_ID:AN002702 PROJECT_ID:PR001061 VERSION 1 CREATED_ON January 20, 2021, 9:51 am #PROJECT PR:PROJECT_TITLE Characterization of anaphylaxis reveals different metabolic changes depending on PR:PROJECT_TITLE severity and triggers. PR:PROJECT_SUMMARY Despite its increasing incidence, the underlying molecular processes of PR:PROJECT_SUMMARY anaphylaxis remain unclear and there are not known biomarkers for appropriate PR:PROJECT_SUMMARY diagnosis. The mechanism associated to the reactions still needs to be clarified PR:PROJECT_SUMMARY in humans. The rapid onset and potentially fatal outcome in the absence of PR:PROJECT_SUMMARY managed treatment, prevent its study and prompt obvious technical and ethical PR:PROJECT_SUMMARY implications. Twenty episodes of anaphylaxis were analyzed. Sera was collected PR:PROJECT_SUMMARY at different times: during the acute phase (T1), the recovery phase (T2) and PR:PROJECT_SUMMARY around 2-3 months after the anaphylactic reaction (T0). The analysis included PR:PROJECT_SUMMARY untargeted metabolomics combining liquid chromatography coupled to mass PR:PROJECT_SUMMARY spectrometry (LC-MS) and proton-nuclear magnetic resonance (1H-NMR). Reactions PR:PROJECT_SUMMARY were classified according to the trigger (food and/or drug) and severity PR:PROJECT_SUMMARY (moderate and severe). “Food T1 vs T2” and “moderate T1 vs T2” PR:PROJECT_SUMMARY anaphylaxis comparisons showed clear metabolic patterns during the onset of an PR:PROJECT_SUMMARY anaphylactic reaction, which differed from those induced by drugs, food+drug or PR:PROJECT_SUMMARY severe anaphylaxis “T1 vs T2”. Moreover, the model of food anaphylaxis was PR:PROJECT_SUMMARY able to distinguish the well-characterized IgE (beta-lactam) from non-IgE- PR:PROJECT_SUMMARY mediated anaphylaxis (NSAIDs), suggesting a differential metabolic pathway PR:PROJECT_SUMMARY associated with the mechanism of action. Moreover, metabolic differences between PR:PROJECT_SUMMARY “moderate vs severe” at T1 and T0 were studied. Among the metabolites, PR:PROJECT_SUMMARY glucose, lipids, cortisol, betaine and oleamide were observed altered. The PR:PROJECT_SUMMARY results of the study provide the first evidence that different anaphylactic PR:PROJECT_SUMMARY triggers, induce differential metabolic changes. Besides, the basal status might PR:PROJECT_SUMMARY identify high risk patients, thus opening new ways to understand, diagnose and PR:PROJECT_SUMMARY treat anaphylaxis. PR:INSTITUTE Hospital Universitari i Politècnic La Fe PR:DEPARTMENT Subdirección Médica, Departament de Salut València La Fe. PR:LAST_NAME Hernández Fernández de Rojas PR:FIRST_NAME Dolores PR:ADDRESS Avinguda de Fernando Abril Martorell, 106, 46026 València, Valencia, España. PR:EMAIL hernandez_dol@gva.es PR:PHONE 91 372 47 00 ext. 4662 #STUDY ST:STUDY_TITLE Characterization of anaphylaxis reveals different metabolic changes depending on ST:STUDY_TITLE severity and triggers. ST:STUDY_SUMMARY Background: Despite its increasing incidence, the underlying molecular processes ST:STUDY_SUMMARY of anaphylaxis remain unclear and there are not known biomarkers for appropriate ST:STUDY_SUMMARY diagnosis. The mechanism associated to the reactions still needs to be clarified ST:STUDY_SUMMARY in humans. The rapid onset and potentially fatal outcome in the absence of ST:STUDY_SUMMARY managed treatment, prevent its study and prompt obvious technical and ethical ST:STUDY_SUMMARY implications. Methods: Twenty episodes of anaphylaxis were analyzed. Sera was ST:STUDY_SUMMARY collected at different times: during the acute phase (T1), the recovery phase ST:STUDY_SUMMARY (T2) and around 2-3 months after the anaphylactic reaction (T0). The analysis ST:STUDY_SUMMARY included untargeted metabolomics combining liquid chromatography coupled to mass ST:STUDY_SUMMARY spectrometry (LC-MS) and proton-nuclear magnetic resonance (1H-NMR). Reactions ST:STUDY_SUMMARY were classified according to the trigger (food and/or drug) and severity ST:STUDY_SUMMARY (moderate and severe). Results: “Food T1 vs T2” and “moderate T1 vs T2” ST:STUDY_SUMMARY anaphylaxis comparisons showed clear metabolic patterns during the onset of an ST:STUDY_SUMMARY anaphylactic reaction, which differed from those induced by drugs, food+drug or ST:STUDY_SUMMARY severe anaphylaxis “T1 vs T2”. Moreover, the model of food anaphylaxis was ST:STUDY_SUMMARY able to distinguish the well-characterized IgE (beta-lactam) from non-IgE- ST:STUDY_SUMMARY mediated anaphylaxis (NSAIDs), suggesting a differential metabolic pathway ST:STUDY_SUMMARY associated with the mechanism of action. Moreover, metabolic differences between ST:STUDY_SUMMARY “moderate vs severe” at T1 and T0 were studied. Among the metabolites, ST:STUDY_SUMMARY glucose, lipids, cortisol, betaine and oleamide were observed altered. ST:STUDY_SUMMARY Conclusions: The results of the study provide the first evidence that different ST:STUDY_SUMMARY anaphylactic triggers, induce differential metabolic changes. Besides, the basal ST:STUDY_SUMMARY status might identify high risk patients, thus opening new ways to understand, ST:STUDY_SUMMARY diagnose and treat anaphylaxis. ST:INSTITUTE The Centre of Metabolomics and Bioanalysis ST:DEPARTMENT Analytical chemistry ST:LAST_NAME Obeso Montero ST:FIRST_NAME David ST:ADDRESS Av. de Montepríncipe, s/n ST:EMAIL david.obesomontero@beca.ceu.es ST:PHONE 607535650 ST:NUM_GROUPS 2 groups ST:TOTAL_SUBJECTS 20 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Male and female #FACTORS #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 - P1_t0 Trigger:Drug | Severity:Moderate | Time:Time 0 RAW_FILE_NAME=P1_t0.d SUBJECT_SAMPLE_FACTORS - P1_t1 Trigger:Drug | Severity:Moderate | Time:Time 1 RAW_FILE_NAME=P1_t1.d SUBJECT_SAMPLE_FACTORS - P1_t2 Trigger:Drug | Severity:Moderate | Time:Time 2 RAW_FILE_NAME=P1_t2.d SUBJECT_SAMPLE_FACTORS - P10_t1 Trigger:Food | Severity:Moderate | Time:Time 1 RAW_FILE_NAME=P10_t1.d SUBJECT_SAMPLE_FACTORS - P10_t2 Trigger:Food | Severity:Moderate | Time:Time 2 RAW_FILE_NAME=P10_t2.d SUBJECT_SAMPLE_FACTORS - P11_t0 Trigger:Food | Severity:Severe | Time:Time 0 RAW_FILE_NAME=P11_t0.d SUBJECT_SAMPLE_FACTORS - P11_t1 Trigger:Food | Severity:Severe | Time:Time 1 RAW_FILE_NAME=P11_t1.d SUBJECT_SAMPLE_FACTORS - P11_t2 Trigger:Food | Severity:Severe | Time:Time 2 RAW_FILE_NAME=P11_t2.d SUBJECT_SAMPLE_FACTORS - P12_t0 Trigger:Idiopatic | Severity:Severe | Time:Time 0 RAW_FILE_NAME=P12_t0.d SUBJECT_SAMPLE_FACTORS - P12_t1 Trigger:Idiopatic | Severity:Severe | Time:Time 1 RAW_FILE_NAME=P12_t1.d SUBJECT_SAMPLE_FACTORS - P12_t2 Trigger:Idiopatic | Severity:Severe | Time:Time 2 RAW_FILE_NAME=P12_t2.d SUBJECT_SAMPLE_FACTORS - P13_t0 Trigger:Drug | Severity:Severe | Time:Time 0 RAW_FILE_NAME=P13_t0.d SUBJECT_SAMPLE_FACTORS - P13_t1 Trigger:Drug | Severity:Severe | Time:Time 1 RAW_FILE_NAME=P13_t1.d SUBJECT_SAMPLE_FACTORS - P13_t2 Trigger:Drug | Severity:Severe | Time:Time 2 RAW_FILE_NAME=P13_t2.d SUBJECT_SAMPLE_FACTORS - P14_t0 Trigger:Drug | Severity:Severe | Time:Time 0 RAW_FILE_NAME=P14_t0.d SUBJECT_SAMPLE_FACTORS - P14_t1 Trigger:Drug | Severity:Severe | Time:Time 1 RAW_FILE_NAME=P14_t1.d SUBJECT_SAMPLE_FACTORS - P14_t2 Trigger:Drug | Severity:Severe | Time:Time 2 RAW_FILE_NAME=P14_t2.d SUBJECT_SAMPLE_FACTORS - P15_t0 Trigger:Drug | Severity:Mild | Time:Time 0 RAW_FILE_NAME=P15_t0.d SUBJECT_SAMPLE_FACTORS - P15_t1 Trigger:Drug | Severity:Mild | Time:Time 1 RAW_FILE_NAME=P15_t1.d SUBJECT_SAMPLE_FACTORS - P16_t0 Trigger:Food | Severity:Moderate | Time:Time 0 RAW_FILE_NAME=P16_t0.d SUBJECT_SAMPLE_FACTORS - P16_t1 Trigger:Food | Severity:Moderate | Time:Time 1 RAW_FILE_NAME=P16_t1.d SUBJECT_SAMPLE_FACTORS - P16_t2 Trigger:Food | Severity:Moderate | Time:Time 2 RAW_FILE_NAME=P16_t2.d SUBJECT_SAMPLE_FACTORS - P17_t0 Trigger:Drug | Severity:Moderate | Time:Time 0 RAW_FILE_NAME=P17_t0.d SUBJECT_SAMPLE_FACTORS - P17_t1 Trigger:Drug | Severity:Moderate | Time:Time 1 RAW_FILE_NAME=P17_t1.d SUBJECT_SAMPLE_FACTORS - P17_t2 Trigger:Drug | Severity:Moderate | Time:Time 2 RAW_FILE_NAME=P17_t2.d SUBJECT_SAMPLE_FACTORS - P18_t0 Trigger:Food | Severity:Moderate | Time:Time 0 RAW_FILE_NAME=P18_t0.d SUBJECT_SAMPLE_FACTORS - P18_t1 Trigger:Food | Severity:Moderate | Time:Time 1 RAW_FILE_NAME=P18_t1.d SUBJECT_SAMPLE_FACTORS - P18_t2 Trigger:Food | Severity:Moderate | Time:Time 2 RAW_FILE_NAME=P18_t2.d SUBJECT_SAMPLE_FACTORS - P19_t0 Trigger:Drug | Severity:Severe | Time:Time 0 RAW_FILE_NAME=P19_t0.d SUBJECT_SAMPLE_FACTORS - P19_t1 Trigger:Drug | Severity:Severe | Time:Time 1 RAW_FILE_NAME=P19_t1.d SUBJECT_SAMPLE_FACTORS - P19_t2 Trigger:Drug | Severity:Severe | Time:Time 2 RAW_FILE_NAME=P19_t2.d SUBJECT_SAMPLE_FACTORS - P2_t0 Trigger:Food | Severity:Moderate | Time:Time 0 RAW_FILE_NAME=P2_t0.d SUBJECT_SAMPLE_FACTORS - P2_t1 Trigger:Food | Severity:Moderate | Time:Time 1 RAW_FILE_NAME=P2_t1.d SUBJECT_SAMPLE_FACTORS - P2_t2 Trigger:Food | Severity:Moderate | Time:Time 2 RAW_FILE_NAME=P2_t2.d SUBJECT_SAMPLE_FACTORS - P20_t0 Trigger:Other | Severity:Moderate | Time:Time 0 RAW_FILE_NAME=P20_t0.d SUBJECT_SAMPLE_FACTORS - P20_t1 Trigger:Other | Severity:Moderate | Time:Time 1 RAW_FILE_NAME=P20_t1.d SUBJECT_SAMPLE_FACTORS - P3_t0 Trigger:Drug | Severity:Moderate | Time:Time 0 RAW_FILE_NAME=P3_t0.d SUBJECT_SAMPLE_FACTORS - P3_t1 Trigger:Drug | Severity:Moderate | Time:Time 1 RAW_FILE_NAME=P3_t1.d SUBJECT_SAMPLE_FACTORS - P4_t0 Trigger:Drug | Severity:Severe | Time:Time 0 RAW_FILE_NAME=P4_t0.d SUBJECT_SAMPLE_FACTORS - P4_t1 Trigger:Drug | Severity:Severe | Time:Time 1 RAW_FILE_NAME=P4_t1.d SUBJECT_SAMPLE_FACTORS - P4_t2 Trigger:Drug | Severity:Severe | Time:Time 2 RAW_FILE_NAME=P4_t2.d SUBJECT_SAMPLE_FACTORS - P5_t1 Trigger:Idiopatic | Severity:Moderate | Time:Time 1 RAW_FILE_NAME=P5_t1.d SUBJECT_SAMPLE_FACTORS - P5_t2 Trigger:Idiopatic | Severity:Moderate | Time:Time 2 RAW_FILE_NAME=P5_t2.d SUBJECT_SAMPLE_FACTORS - P6_t0 Trigger:Idiopatic | Severity:Severe | Time:Time 0 RAW_FILE_NAME=P6_t0.d SUBJECT_SAMPLE_FACTORS - P6_t1 Trigger:Idiopatic | Severity:Severe | Time:Time 1 RAW_FILE_NAME=P6_t1.d SUBJECT_SAMPLE_FACTORS - P6_t2 Trigger:Idiopatic | Severity:Severe | Time:Time 2 RAW_FILE_NAME=P6_t2.d SUBJECT_SAMPLE_FACTORS - P7_t0 Trigger:Drug | Severity:Severe | Time:Time 0 RAW_FILE_NAME=P7_t0.d SUBJECT_SAMPLE_FACTORS - P7_t1 Trigger:Drug | Severity:Severe | Time:Time 1 RAW_FILE_NAME=P7_t1.d SUBJECT_SAMPLE_FACTORS - P7_t2 Trigger:Drug | Severity:Severe | Time:Time 2 RAW_FILE_NAME=P7_t2.d SUBJECT_SAMPLE_FACTORS - P8_t0 Trigger:Other | Severity:Moderate | Time:Time 0 RAW_FILE_NAME=P8_t0.d SUBJECT_SAMPLE_FACTORS - P8_t1 Trigger:Other | Severity:Moderate | Time:Time 1 RAW_FILE_NAME=P8_t1.d SUBJECT_SAMPLE_FACTORS - P8_t2 Trigger:Other | Severity:Moderate | Time:Time 2 RAW_FILE_NAME=P8_t2.d SUBJECT_SAMPLE_FACTORS - P9_t0 Trigger:Food | Severity:Moderate | Time:Time 0 RAW_FILE_NAME=P9_t0.d SUBJECT_SAMPLE_FACTORS - P9_t1 Trigger:Food | Severity:Moderate | Time:Time 1 RAW_FILE_NAME=P9_t1.d SUBJECT_SAMPLE_FACTORS - P9_t2 Trigger:Food | Severity:Moderate | Time:Time 2 RAW_FILE_NAME=P9_t2.d #COLLECTION CO:COLLECTION_SUMMARY A prospective clinical and observational study of patients with anaphylactic CO:COLLECTION_SUMMARY reactions was performed. Patients of all ages and both sexes were recruited at CO:COLLECTION_SUMMARY outpatient clinics and the departments of Emergency, and other services at CO:COLLECTION_SUMMARY Hospital La Fe. All fulfilled clinical criteria of anaphylaxis and severity was CO:COLLECTION_SUMMARY graded following the classification by Brown, et al3. Patients were classified CO:COLLECTION_SUMMARY as food, drug or idiopathic origin, as well as in mild, moderate or severe CO:COLLECTION_SUMMARY according to the number of organs affected and clinical symptoms. The allergy CO:COLLECTION_SUMMARY evaluation was conducted by the Allergology Service of Hospital La Fe. The CO:COLLECTION_SUMMARY ethical committee approved the study protocol and all subjects were informed and CO:COLLECTION_SUMMARY provided written consent. Serum samples were taken during the acute moment of CO:COLLECTION_SUMMARY the reaction at the first moment of medical attention (< 2h, hereafter referred CO:COLLECTION_SUMMARY as ‘T1’), and after clinical recovery (approximately 2-4h later, referred to CO:COLLECTION_SUMMARY as ‘T2’). Patients were treated according to the Galaxy 2016 practical CO:COLLECTION_SUMMARY guide, using all necessary drugs to rescue them. Samples were collected, CO:COLLECTION_SUMMARY following specific standard operating procedures (SOPs)31-33. Briefly, these CO:COLLECTION_SUMMARY were collected in a vacutainer tube (Ref. 368965) and processed within the first CO:COLLECTION_SUMMARY 30 to 60 min after blood extraction. Sample aliquots were stored at -80ºC until CO:COLLECTION_SUMMARY further analyses. Subsequently, between 2-3 months after the anaphylaxis, a CO:COLLECTION_SUMMARY serum sample was taken when the allergy evaluation was performed (basal state, CO:COLLECTION_SUMMARY called ‘T0’). CO:SAMPLE_TYPE Blood (serum) CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Patients were treated according to the Galaxy 2016 practical guide, using all TR:TREATMENT_SUMMARY necessary drugs to rescue them. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY After thawing the samples, 150 µL of cold acetonitrile (0.1% formic acid, v/v) SP:SAMPLEPREP_SUMMARY was added to 50 µL of serum, vortex and kept at -20ºC for 30 min for protein SP:SAMPLEPREP_SUMMARY precipitation. Then, 25 µl of cleaned supernatant were transferred to a 96 SP:SAMPLEPREP_SUMMARY well-plate for UPLC-MS analysis. Finally, 125 µL of H2O (0.1% formic acid, SP:SAMPLEPREP_SUMMARY v/v), and 2 µL of internal standard (IS) mix solution, containing reserpine, SP:SAMPLEPREP_SUMMARY leucine enkephaline, caffeine-d9 and phenylalanine-d5 in H20:CH3OH (1:1, 0.1% SP:SAMPLEPREP_SUMMARY v/v formic acid) at 20 µM were added to each sample. Blank samples were SP:SAMPLEPREP_SUMMARY prepared by replacing serum by ultrapure H20 in order to identify potential SP:SAMPLEPREP_SUMMARY artefacts from the tube, reagents and other materials. A quality control (QC) SP:SAMPLEPREP_SUMMARY was prepared by mixing 10 µL from each prepared sample. Samples were randomly SP:SAMPLEPREP_SUMMARY injected in the chromatographic system (UPLC-ToF-MS) injection a QC every 6 SP:SAMPLEPREP_SUMMARY serum samples in order to avoid intra-batch variability, as well as to enhance SP:SAMPLEPREP_SUMMARY quality and reproducibility. Blank analysis was performed at the beginning and SP:SAMPLEPREP_SUMMARY at end of the sequence. Sample stability and analytical drift were investigated SP:SAMPLEPREP_SUMMARY through the internal standard intensities. #CHROMATOGRAPHY _1 CH:CHROMATOGRAPHY_SUMMARY The chromatographic separation was performed by using an UPLC BEH C18 (100 x 2.1 _1 CH:CHROMATOGRAPHY_SUMMARY mm, 1.7 µm, Waters, Wexford, Ireland) column from Waters (Wexford, Ireland). _1 CH:CHROMATOGRAPHY_SUMMARY Autosampler and column temperatures were set to 4°C and 40°C, respectively, _1 CH:CHROMATOGRAPHY_SUMMARY and the injection volume was 5 µL. Mobile phase A and B consisted of H20 and _1 CH:CHROMATOGRAPHY_SUMMARY acetonitrile, respectively, both containing 0.1% formic acid. The gradient _1 CH:CHROMATOGRAPHY_SUMMARY elution lasted 14 minutes at a flow rate of 400 µl min-1. The mobile phase A _1 CH:CHROMATOGRAPHY_SUMMARY was maintained at 98% for 1 min, and then decreased until 75% in 2 minutes, 50% _1 CH:CHROMATOGRAPHY_SUMMARY in 3 minutes and 5% in 3 more minutes. 95% of mobile phase B was held for 3 min _1 CH:CHROMATOGRAPHY_SUMMARY and then, a 0.55 min gradient was used to return to the initial conditions, _1 CH:CHROMATOGRAPHY_SUMMARY which were held for 2.5 min to full column recovery. _1 CH:CHROMATOGRAPHY_TYPE None (Direct infusion) _1 CH:INSTRUMENT_NAME none _1 CH:COLUMN_NAME none #ANALYSIS AN:ANALYSIS_TYPE NMR #NMR NM:INSTRUMENT_NAME Bruker 500MHz spectrometer NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:SPECTROMETER_FREQUENCY 500MHz NM:NMR_RESULTS_FILE ST001655_AN002702_Results.txt UNITS:ppm #END