#METABOLOMICS WORKBENCH Codreags00_20240321_134237 DATATRACK_ID:4734 STUDY_ID:ST003139 ANALYSIS_ID:AN005152 PROJECT_ID:PR001951 VERSION 1 CREATED_ON March 21, 2024, 2:15 pm #PROJECT PR:PROJECT_TITLE Endothelial-Dependent Vascular Reactivity After Cardiopulmonary Bypass is PR:PROJECT_TITLE Associated with Unique Metabolomic Signatures PR:PROJECT_TYPE Untargeted Metabolomics analysis PR:PROJECT_SUMMARY Cardiopulmonary bypass (CPB), an extracorporeal method necessary for the PR:PROJECT_SUMMARY surgical correction of complex congenital heart defects, incites significant PR:PROJECT_SUMMARY inflammatory and vascular changes. Along with these changes are alterations in PR:PROJECT_SUMMARY cellular metabolism that promote energy production to deal with this stress. PR:PROJECT_SUMMARY Utilizing laser-doppler perfusion monitoring coupled with iontophoresis (LDPMI) PR:PROJECT_SUMMARY in patients undergoing corrective heart surgery, we hypothesized that temporal, PR:PROJECT_SUMMARY untargeted metabolomics could be performed to assess the link between metabolism PR:PROJECT_SUMMARY and vascular function. The data give insight into the metabolic landscape of PR:PROJECT_SUMMARY children undergoing CPB for corrective heart surgery and provide detail into how PR:PROJECT_SUMMARY these metabolites relate to physiological aberrations in the vasculature. PR:INSTITUTE Vanderbilt University PR:DEPARTMENT Chemistry PR:LABORATORY Center for Innovative Technology PR:LAST_NAME CODREANU PR:FIRST_NAME SIMONA PR:ADDRESS 1234 STEVENSON CENTER LANE PR:EMAIL SIMONA.CODREANU@VANDERBILT.EDU PR:PHONE 6158758422 #STUDY ST:STUDY_TITLE Endothelial-Dependent Vascular Reactivity After Cardiopulmonary Bypass is ST:STUDY_TITLE Associated with Unique Metabolomic Signatures ST:STUDY_TYPE untargeted metabolomics analysis ST:STUDY_SUMMARY Cardiopulmonary bypass (CPB), an extracorporeal method necessary for the ST:STUDY_SUMMARY surgical correction of complex congenital heart defects, incites significant ST:STUDY_SUMMARY inflammatory and vascular changes. Along with these changes are alterations in ST:STUDY_SUMMARY cellular metabolism that promote energy production to deal with this stress. ST:STUDY_SUMMARY Utilizing laser-doppler perfusion monitoring coupled with iontophoresis (LDPMI) ST:STUDY_SUMMARY in patients undergoing corrective heart surgery, we hypothesized that temporal, ST:STUDY_SUMMARY untargeted metabolomics could be performed to assess the link between metabolism ST:STUDY_SUMMARY and vascular function. Globally, we found 2404 unique metabolites in the plasma ST:STUDY_SUMMARY of patients undergoing CPB. Metabolites related to arginine biosynthesis were ST:STUDY_SUMMARY the most altered in the CPB period. When examining metabolic profiles in ST:STUDY_SUMMARY correlation with endothelial-dependent (acetylcholine, ACh) or ST:STUDY_SUMMARY endothelial-independent (sodium nitroprusside, SNP) vascular reactivity, purine ST:STUDY_SUMMARY metabolism was most consistently associated with either vascular response. With ST:STUDY_SUMMARY ACh-mediated responses, L-acetylcarnitine levels were most strongly associated, ST:STUDY_SUMMARY while L-glutamine levels were associated with both ACh and SNP responsiveness. ST:STUDY_SUMMARY These data give insight into the metabolic landscape of children undergoing CPB ST:STUDY_SUMMARY for corrective heart surgery and provide detail into how these metabolites ST:STUDY_SUMMARY relate to physiological aberrations in the vasculature. ST:INSTITUTE Vanderbilt University ST:DEPARTMENT Chemistry ST:LABORATORY Center for Innovative Technology ST:LAST_NAME CODREANU ST:FIRST_NAME SIMONA ST:ADDRESS 1234 STEVENSON CENTER LANE ST:EMAIL SIMONA.CODREANU@VANDERBILT.EDU ST:PHONE 6158758422 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENOTYPE_STRAIN Congenital heart defects (CHD) undergoing cardiopulmonary bypass (CPB) SU:AGE_OR_AGE_RANGE less than 1 year of age #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 Sample source:Plasma | time of collection:1-pre RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_pre_P1 SUBJECT_SAMPLE_FACTORS - P2 Sample source:Plasma | time of collection:2-pre RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_pre_P2 SUBJECT_SAMPLE_FACTORS - P3 Sample source:Plasma | time of collection:3-pre RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_pre_P3 SUBJECT_SAMPLE_FACTORS - P4 Sample source:Plasma | time of collection:4-pre RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_pre_P4 SUBJECT_SAMPLE_FACTORS - P5 Sample source:Plasma | time of collection:6-pre RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_pre_P5 SUBJECT_SAMPLE_FACTORS - P6 Sample source:Plasma | time of collection:7-pre RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_pre_P6 SUBJECT_SAMPLE_FACTORS - P7 Sample source:Plasma | time of collection:9-pre RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_pre_P7 SUBJECT_SAMPLE_FACTORS - P8 Sample source:Plasma | time of collection:12-pre RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_pre_P8 SUBJECT_SAMPLE_FACTORS - P9 Sample source:Plasma | time of collection:14-pre RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_pre_P9 SUBJECT_SAMPLE_FACTORS - P10 Sample source:Plasma | time of collection:15-pre RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_pre_P10 SUBJECT_SAMPLE_FACTORS - P11 Sample source:Plasma | time of collection:16-pre RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_pre_P11 SUBJECT_SAMPLE_FACTORS - P12 Sample source:Plasma | time of collection:20-pre RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_pre_P12 SUBJECT_SAMPLE_FACTORS - P13 Sample source:Plasma | time of collection:21-pre RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_pre_P13 SUBJECT_SAMPLE_FACTORS - P14 Sample source:Plasma | time of collection:1-post1 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post1_P14 SUBJECT_SAMPLE_FACTORS - P15 Sample source:Plasma | time of collection:2-post1 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post1_P15 SUBJECT_SAMPLE_FACTORS - P16 Sample source:Plasma | time of collection:3-post1 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post1_P16 SUBJECT_SAMPLE_FACTORS - P17 Sample source:Plasma | time of collection:4-post1 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post1_P17 SUBJECT_SAMPLE_FACTORS - P18 Sample source:Plasma | time of collection:6-post1 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post1_P18 SUBJECT_SAMPLE_FACTORS - P19 Sample source:Plasma | time of collection:7-post1 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post1_P19 SUBJECT_SAMPLE_FACTORS - P20 Sample source:Plasma | time of collection:9-post1 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post1_P20 SUBJECT_SAMPLE_FACTORS - P21 Sample source:Plasma | time of collection:12-post1 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post1_P21 SUBJECT_SAMPLE_FACTORS - P22 Sample source:Plasma | time of collection:14-post1 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post1_P22 SUBJECT_SAMPLE_FACTORS - P23 Sample source:Plasma | time of collection:15-post1 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post1_P23 SUBJECT_SAMPLE_FACTORS - P24 Sample source:Plasma | time of collection:20-post1 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post1_P24 SUBJECT_SAMPLE_FACTORS - P25 Sample source:Plasma | time of collection:21-post1 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post1_P25 SUBJECT_SAMPLE_FACTORS - P26 Sample source:Plasma | time of collection:1-post2 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post2_P26 SUBJECT_SAMPLE_FACTORS - P27 Sample source:Plasma | time of collection:2-post2 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post2_P27 SUBJECT_SAMPLE_FACTORS - P28 Sample source:Plasma | time of collection:3-post2 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post2_P28 SUBJECT_SAMPLE_FACTORS - P29 Sample source:Plasma | time of collection:6-post2 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post2_P29 SUBJECT_SAMPLE_FACTORS - P30 Sample source:Plasma | time of collection:7-post2 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post2_P30 SUBJECT_SAMPLE_FACTORS - P31 Sample source:Plasma | time of collection:9-post2 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post2_P31 SUBJECT_SAMPLE_FACTORS - P32 Sample source:Plasma | time of collection:12-post2 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post2_P32 SUBJECT_SAMPLE_FACTORS - P33 Sample source:Plasma | time of collection:14-post2 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post2_P33 SUBJECT_SAMPLE_FACTORS - P34 Sample source:Plasma | time of collection:15-post2 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post2_P34 SUBJECT_SAMPLE_FACTORS - P35 Sample source:Plasma | time of collection:21-post2 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post2_P35 SUBJECT_SAMPLE_FACTORS - P36 Sample source:Plasma | time of collection:22-post2 RAW_FILE_NAME(MS_Raw_File)=SC_20230131_HILICn_FMS_Stark_post2_P36 #COLLECTION CO:COLLECTION_SUMMARY Patients underwent laser Doppler perfusion monitoring with iontophoresis (LDPMI) CO:COLLECTION_SUMMARY as well as blood collection at the following time points: preoperatively (within CO:COLLECTION_SUMMARY 7 days of surgical date), 2 to 4 hours after CPB, and 24 hours after CPB. LDPMI CO:COLLECTION_SUMMARY measurements were performed using a Periflux 5010 coupled to a Perilont 382b CO:COLLECTION_SUMMARY (Perimed, Stockholm, Sweden) as previously described 6. Briefly, 180 μL of 2% CO:COLLECTION_SUMMARY acetylcholine (ACh, Sigma-Aldrich, St. Louis, MO) was pulsed with a 0.1 mA CO:COLLECTION_SUMMARY anodal current for 20 seconds for a total of five doses separated by 120 seconds CO:COLLECTION_SUMMARY over 10 minutes. After a 10-minute rest and at a separate site, 180 μL of 1% CO:COLLECTION_SUMMARY sodium nitroprusside (SNP, Sigma) was pulsed with a 0.2 mA cathodal current CO:COLLECTION_SUMMARY using identical dosing intervals and duration. Blood was collected at the end of CO:COLLECTION_SUMMARY the LDPMI measurements into EDTA-containing vacutainers. Blood was then CO:COLLECTION_SUMMARY centrifuged at 2200 RPM for 5 minutes and the plasma was removed and stored at CO:COLLECTION_SUMMARY −80°C. CO:SAMPLE_TYPE Blood (plasma) CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Patients underwent laser Doppler perfusion monitoring with iontophoresis (LDPMI) TR:TREATMENT_SUMMARY as well as blood collection at the following time points: preoperatively (within TR:TREATMENT_SUMMARY 7 days of surgical date), 2 to 4 hours after CPB, and 24 hours after CPB. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Briefly, plasma samples collected at three different time points (pre, post1 and SP:SAMPLEPREP_SUMMARY post2) were normalized by total volume (20µL/sample). Metabolites were SP:SAMPLEPREP_SUMMARY extracted with methanol/water 80:20. Heavy labeled phenylalanine-D8 and SP:SAMPLEPREP_SUMMARY biotin-D2 were added to individual samples prior to protein precipitation. SP:SAMPLEPREP_SUMMARY Following overnight incubation at -80°C, precipitated proteins were pelleted by SP:SAMPLEPREP_SUMMARY centrifugation at 10,000 rpm for 10 min and metabolite extracts were dried down SP:SAMPLEPREP_SUMMARY in vacuo. Metabolite extracts were further cleaned up of the high lipid content SP:SAMPLEPREP_SUMMARY using Captiva EMR lipid cartridges (Agilent Technologies, Santa Clara, CA) under SP:SAMPLEPREP_SUMMARY controlled positive pressure (3-4psi). Briefly, dry samples of metabolite SP:SAMPLEPREP_SUMMARY extracts were reconstituted in 100µL of methanol:water (4:1, v:v) and directly SP:SAMPLEPREP_SUMMARY applied to individual pre-equilibrated cartridges. Metabolite elution of the SP:SAMPLEPREP_SUMMARY cartridges was completed using 500µL crash solvent of acetonitrile:water (5:1, SP:SAMPLEPREP_SUMMARY v:v) with 1% formic acid and dried down in vacuo. Individual clean extracts were SP:SAMPLEPREP_SUMMARY reconstituted in 100 µl of acetonitrile/water (80:20, v/v) containing SP:SAMPLEPREP_SUMMARY heavy-labeled carnitine-D9, tryptophan-D3, valine-D8, and inosine-4N15, and SP:SAMPLEPREP_SUMMARY centrifuged for 5 min at 10,000 rpm to remove insoluble material. A pooled SP:SAMPLEPREP_SUMMARY quality control sample (QC) was prepared by pooling equal volumes of individual SP:SAMPLEPREP_SUMMARY samples. The pooled QC sample was used for column conditioning (8 injections SP:SAMPLEPREP_SUMMARY prior to sample analysis), retention time alignment and to assess mass SP:SAMPLEPREP_SUMMARY spectrometry instrument reproducibility throughout the sample set. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Hydrophylic compounds separation CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) CH:SOLVENT_A 90% water, 10% acetonitrile, 5mM Ammonium Formate, 0.1%FA CH:SOLVENT_B 10% water, 90% acetonitrile, 5mM Ammonium Formate, 0.1%FA CH:FLOW_GRADIENT 30 min; 95%A, 5%B CH:FLOW_RATE 0.20mL/min CH:COLUMN_TEMPERATURE 30 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Full mass scan was acquired at 120,000 resolution with a scan rate of 3.5 Hz, MS:MS_COMMENTS automatic gain control (AGC) target of 1x106, and maximum ion injection time of MS:MS_COMMENTS 100 ms, and MS/MS spectra were collected at 15,000 resolution, AGC target of MS:MS_COMMENTS 2x105 ions, with a maximum ion injection time of 100 ms. Mass spectrometry raw MS:MS_COMMENTS data was imported, processed, normalized and reviewed using Progenesis QI v.3.0 MS:MS_COMMENTS (Non-linear Dynamics, Newcastle, UK). All MS and MS/MS sample runs were aligned MS:MS_COMMENTS against a pooled QC reference run. MS:MS_RESULTS_FILE ST003139_AN005152_Results.txt UNITS:peak intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END