#METABOLOMICS WORKBENCH mchughce_20191213_075454 DATATRACK_ID:1867 STUDY_ID:ST001294 ANALYSIS_ID:AN002155 PROJECT_ID:PR000876 VERSION 1 CREATED_ON December 22, 2019, 5:17 pm #PROJECT PR:PROJECT_TITLE Estimating Platelet Mitochondrial Function in Patients with Sepsis PR:PROJECT_TYPE Quantitative NMR Metabolomics PR:PROJECT_SUMMARY Sepsis patients and ill controls admitted to the ED had matched whole blood (WB) PR:PROJECT_SUMMARY and isolated platelet samples collected. The control group was chosen to PR:PROJECT_SUMMARY represent an acutely ill 127cohort of the same racial, sex, and age as the PR:PROJECT_SUMMARY sepsis cohort. NMR was run on extracted WB and platelets to determine how the PR:PROJECT_SUMMARY metabolome reflects mitochondrial function PR:INSTITUTE University of Michigan, University of Mississippi, University of Minnesota PR:DEPARTMENT Clinical Pharmacy (UMich); Emergency Medicine (UMiss) PR:LABORATORY Stringer NMR Metabolomics Laboratory PR:LAST_NAME McHugh PR:FIRST_NAME Cora PR:ADDRESS 428 Church St, Ann Arbor, MI, 48103, USA PR:EMAIL mchughce@med.umich.edu PR:PHONE 7343530164 PR:FUNDING_SOURCE GM111400, K23GM113041 PR:CONTRIBUTORS Kathleen Stringer, Mike Puskarich, Marc McCann #STUDY ST:STUDY_TITLE Estimating Platelet Mitochondrial Function in Patients with Sepsis - Platelet ST:STUDY_TITLE NMRs (part-I) ST:STUDY_TYPE single timepoint ST:STUDY_SUMMARY Relationships between platelet mitochondrial oxygen consumption rates (mOCR) and ST:STUDY_SUMMARY metabolites in platelets as measured by quantitative 1H-NMR metabolomics. ST:STUDY_SUMMARY Samples collected in ED at a single timepoint. WB and platelets isolated from ST:STUDY_SUMMARY the same blood samples. Comparison of mitochondrial function and metabolomics in ST:STUDY_SUMMARY patients with sepsis and non-sepsis ED patients ST:INSTITUTE University of Michigan, University of Mississippi, University of Minnesota ST:DEPARTMENT Clinical Pharmacy (UMich); Emergency Medicine (UMiss) ST:LABORATORY Stringer NMR Metabolomics Laboratory ST:LAST_NAME McHugh ST:FIRST_NAME Cora ST:ADDRESS 428 Church St, Ann Arbor, MI, 48103, USA ST:EMAIL mchughce@med.umich.edu ST:PHONE 7343530164 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 23 ST:NUM_MALES 12 ST:NUM_FEMALES 11 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:AGE_OR_AGE_RANGE 32-67 SU:GENDER Male and female SU:HUMAN_RACE Caucasian (C) and African American (AA) SU:HUMAN_ETHNICITY non-hispanic SU:HUMAN_INCLUSION_CRITERIA Sepsis: 1) Suspected or confirmed infection; 2) Any two of four criteria of SU:HUMAN_INCLUSION_CRITERIA systemic inflammatory response in ED(18); 3) Age ≥ 18; 4) Lactate ≥ 2.0 SU:HUMAN_INCLUSION_CRITERIA mmol/L; 5) Enrollment within 2 hours of initiation of quantitative resuscitation SU:HUMAN_INCLUSION_CRITERIA protocol. Controls: admitted to the emergency department and had no medical SU:HUMAN_INCLUSION_CRITERIA conditions that required chronic administration of medication expected to affect SU:HUMAN_INCLUSION_CRITERIA platelet function (aspirin, PGY12 inhibitors, etc) SU:HUMAN_EXCLUSION_CRITERIA Exclusion criteria were:1) any primary diagnosis other than sepsis; 2) SU:HUMAN_EXCLUSION_CRITERIA established Do Not Resuscitate status; 3) transferred from another hospital with SU:HUMAN_EXCLUSION_CRITERIA sepsis therapy already initiated; 4) cardiopulmonary resuscitation (chest SU:HUMAN_EXCLUSION_CRITERIA compression or defibrillation) prior to enrollment; 5) patient or legal SU:HUMAN_EXCLUSION_CRITERIA representative unable to understand and sign informed consent. #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 C01H08 233_33 Condition:Control Age=29; Gender=F; Race=Caucasian; RAW_FILE_NAME=233.33 SUBJECT_SAMPLE_FACTORS C02H03 233_11 Condition:Control Age=53; Gender=M; Race=Caucasian; RAW_FILE_NAME=233.11 SUBJECT_SAMPLE_FACTORS C03H02 233_34 Condition:Control Age=56; Gender=M; Race=African-American; RAW_FILE_NAME=233.34 SUBJECT_SAMPLE_FACTORS C06H14 233_30 Condition:Control Age=35; Gender=F; Race=African-American; RAW_FILE_NAME=233.30 SUBJECT_SAMPLE_FACTORS C07H06 233_37 Condition:Control Age=53; Gender=M; Race=African-American; RAW_FILE_NAME=233.37 SUBJECT_SAMPLE_FACTORS C08H11 233_39 Condition:Control Age=55; Gender=M; Race=African-American; RAW_FILE_NAME=233.39 SUBJECT_SAMPLE_FACTORS C09H13 233_35 Condition:Control Age=30; Gender=F; Race=African-American; RAW_FILE_NAME=233.35 SUBJECT_SAMPLE_FACTORS C10H09 233_01 Condition:Control Age=56; Gender=M; Race=African-American; RAW_FILE_NAME=233.01 SUBJECT_SAMPLE_FACTORS C11H21 233_07 Condition:Control Age=33; Gender=F; Race=African-American; RAW_FILE_NAME=233.07 SUBJECT_SAMPLE_FACTORS C12H05 233_02 Condition:Control Age=64; Gender=F; Race=African-American; RAW_FILE_NAME=233.02 SUBJECT_SAMPLE_FACTORS C13H15 233_17 Condition:Control Age=61; Gender=M; Race=Caucasian; RAW_FILE_NAME=233.17 SUBJECT_SAMPLE_FACTORS C14H27 233_06 Condition:Control Age=24; Gender=F; Race=African-American; RAW_FILE_NAME=233.06 SUBJECT_SAMPLE_FACTORS C15H30 233_05 Condition:Control Age=36; Gender=M; Race=African-American; RAW_FILE_NAME=233.05 SUBJECT_SAMPLE_FACTORS C16H19 233_18 Condition:Control Age=37; Gender=M; Race=Caucasian; RAW_FILE_NAME=233.18 SUBJECT_SAMPLE_FACTORS HAPS01 233_19 Condition:Sepsis Age=89; Gender=M; Race=Caucasian; RAW_FILE_NAME=233.19 SUBJECT_SAMPLE_FACTORS HAPS02 233_38 Condition:Sepsis Age=55; Gender=M; Race=African-American; RAW_FILE_NAME=233.38 SUBJECT_SAMPLE_FACTORS HAPS03 233_10 Condition:Sepsis Age=59; Gender=M; Race=Caucasian; RAW_FILE_NAME=233.10 SUBJECT_SAMPLE_FACTORS HAPS04 233_22 Condition:Sepsis Age=78; Gender=M; Race=African-American; RAW_FILE_NAME=233.22 SUBJECT_SAMPLE_FACTORS HAPS05 233_28 Condition:Sepsis Age=73; Gender=F; Race=African-American; RAW_FILE_NAME=233.28 SUBJECT_SAMPLE_FACTORS HAPS07 233_20 Condition:Sepsis Age=48; Gender=F; Race=Caucasian; RAW_FILE_NAME=233.20 SUBJECT_SAMPLE_FACTORS HAPS08 233_31 Condition:Sepsis Age=33; Gender=F; Race=Caucasian; RAW_FILE_NAME=233.31 SUBJECT_SAMPLE_FACTORS HAPS09 233_32 Condition:Sepsis Age=66; Gender=M; Race=African-American; RAW_FILE_NAME=233.32 SUBJECT_SAMPLE_FACTORS HAPS10 233_04 Condition:Sepsis Age=55; Gender=M; Race=African-American; RAW_FILE_NAME=233.04 SUBJECT_SAMPLE_FACTORS HAPS12 233_24 Condition:Sepsis Age=67; Gender=M; Race=African-American; RAW_FILE_NAME=233.24 SUBJECT_SAMPLE_FACTORS HAPS14 233_21 Condition:Sepsis Age=39; Gender=F; Race=African-American; RAW_FILE_NAME=233.21 SUBJECT_SAMPLE_FACTORS HAPS15 233_25 Condition:Sepsis Age=68; Gender=M; Race=Caucasian; RAW_FILE_NAME=233.25 SUBJECT_SAMPLE_FACTORS HAPS16 233_29 Condition:Sepsis Age=43; Gender=M; Race=Caucasian; RAW_FILE_NAME=233.29 SUBJECT_SAMPLE_FACTORS HAPS18 233_08 Condition:Sepsis Age=67; Gender=F; Race=Caucasian; RAW_FILE_NAME=233.08 SUBJECT_SAMPLE_FACTORS HAPS19 233_12 Condition:Sepsis Age=38; Gender=M; Race=Caucasian; RAW_FILE_NAME=233.12 SUBJECT_SAMPLE_FACTORS HAPS21 233_16 Condition:Sepsis Age=28; Gender=F; Race=African-American; RAW_FILE_NAME=233.16 SUBJECT_SAMPLE_FACTORS HAPS22 233_13 Condition:Sepsis Age=52; Gender=M; Race=Caucasian; RAW_FILE_NAME=233.13 SUBJECT_SAMPLE_FACTORS HAPS24 233_09 Condition:Sepsis Age=69; Gender=F; Race=African-American; RAW_FILE_NAME=233.09 SUBJECT_SAMPLE_FACTORS HAPS25 233_27 Condition:Sepsis Age=70; Gender=M; Race=African-American; RAW_FILE_NAME=233.27 SUBJECT_SAMPLE_FACTORS HAPS26 233_03 Condition:Sepsis Age=49; Gender=F; Race=Caucasian; RAW_FILE_NAME=233.03 SUBJECT_SAMPLE_FACTORS HAPS27 233_15 Condition:Sepsis Age=24; Gender=F; Race=African-American; RAW_FILE_NAME=233.15 SUBJECT_SAMPLE_FACTORS HAPS28 233_14 Condition:Sepsis Age=54; Gender=M; Race=Caucasian; RAW_FILE_NAME=233.14 SUBJECT_SAMPLE_FACTORS HAPS29 233_26 Condition:Sepsis Age=62; Gender=F; Race=Caucasian; RAW_FILE_NAME=233.26 SUBJECT_SAMPLE_FACTORS HAPS30 233_23 Condition:Sepsis Age=32; Gender=M; Race=African-American; RAW_FILE_NAME=233.23 SUBJECT_SAMPLE_FACTORS HAPS31 233_40 Condition:Sepsis Age=49; Gender=M; Race=Caucasian; RAW_FILE_NAME=233.40 #COLLECTION CO:COLLECTION_SUMMARY WB samples (12 mL each) were collected by direct venipuncture or from an CO:COLLECTION_SUMMARY indwelling line into K2 EDTA-containing tubes. Platelets were then isolated. CO:COLLECTION_SUMMARY centrifuged (200x gfor 6 min at room temperature).The platelet-rich plasma layer CO:COLLECTION_SUMMARY was transferred to another tube and centrifuged (4500x gfor 5 minat 4C) to CO:COLLECTION_SUMMARY pellet the platelets.The nearly cell-free plasma layer was transferred to a new CO:COLLECTION_SUMMARY tube, leaving approximately 0.25 mL of plasma in the original tube. Remaining CO:COLLECTION_SUMMARY plasma was used to resuspend the platelet pellet to produce ultra-rich plasma. CO:SAMPLE_TYPE Platelets CO:COLLECTION_METHOD direct venipuncture or indwelling line CO:COLLECTION_FREQUENCY once CO:VOLUMEORAMOUNT_COLLECTED 12mL CO:STORAGE_CONDITIONS Described in summary CO:COLLECTION_VIALS K2 EDTA-containing Vacutainer tubes (BD 134367863; Becton-Dickinson, Franklin CO:COLLECTION_VIALS Lakes, NJ USA) CO:ADDITIVES K2 EDTA #TREATMENT TR:TREATMENT_SUMMARY No treatment. Sepsis and non-sepsis controls had platelets isolated. TR:TREATMENT_SUMMARY Mitochondrial function was measured via mitochondrial oxygen consumption (mOCR) #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Methanol chlorform extraction of platelet pellet after 2 freeze-thaw cycles in SP:SAMPLEPREP_SUMMARY the presence of methanol. SP:SAMPLEPREP_PROTOCOL_ID Platelet Pellet MeOHCHCl3 Extraction SP:SAMPLEPREP_PROTOCOL_FILENAME 233.2.1_-_HAPS_Platelet_Pellet_MeOHCHCl3_Extraction SP:PROCESSING_STORAGE_CONDITIONS On ice SP:EXTRACTION_METHOD Methanol chloroform extraction SP:EXTRACT_ENRICHMENT Lyophilization SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION 500uL 50mM sodium phosphate buffer in D2O SP:SAMPLE_SPIKING 4.99 mM DSS internal standard #ANALYSIS AN:DATA_FORMAT .fid #NMR NM:INSTRUMENT_NAME Bruker 18.8 Tesla (800 MHz) NMR spectrometer ascend NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:NMR_COMMENTS Arrayed for water saturation frequency and 90deg. pulse width for each sample NM:STANDARD_CONCENTRATION 10% (~0.5mM) NM:SPECTROMETER_FREQUENCY 800 MHz NM:NMR_PROBE Triple resonance inverse detection TCI cryoprobe NM:NMR_SOLVENT D20 NM:NMR_TUBE_SIZE 5mm NM:SHIMMING_METHOD Auto shim (gradient shimming) NM:PULSE_SEQUENCE noesygppr1d NM:WATER_SUPPRESSION saturation at 80 Hz induced field strength NM:PULSE_WIDTH 5.5ms NM:OFFSET_FREQUENCY around -178Hz NM:PRESATURATION_POWER_LEVEL 80db NM:CHEMICAL_SHIFT_REF_CPD DSS-d6 NM:TEMPERATURE 25 NM:NUMBER_OF_SCANS 128 #NMR_METABOLITE_DATA NMR_METABOLITE_DATA:UNITS μM NMR_METABOLITE_DATA_START Samples 233_01 233_02 233_03 233_04 233_05 233_06 233_07 233_08 233_09 233_10 233_11 233_12 233_13 233_14 233_15 233_16 233_17 233_18 233_19 233_20 233_21 233_22 233_23 233_24 233_25 233_26 233_27 233_28 233_29 233_30 233_31 233_32 233_33 233_34 233_35 233_37 233_38 233_39 233_40 Factors Condition:Control Condition:Control Condition:Sepsis Condition:Sepsis Condition:Control Condition:Control Condition:Control Condition:Sepsis Condition:Sepsis Condition:Sepsis Condition:Control Condition:Sepsis Condition:Sepsis Condition:Sepsis Condition:Sepsis Condition:Sepsis Condition:Control Condition:Control Condition:Sepsis Condition:Sepsis Condition:Sepsis Condition:Sepsis Condition:Sepsis Condition:Sepsis Condition:Sepsis Condition:Sepsis Condition:Sepsis Condition:Sepsis Condition:Sepsis Condition:Control Condition:Sepsis Condition:Sepsis Condition:Control Condition:Control Condition:Control Condition:Control Condition:Sepsis Condition:Control Condition:Sepsis ADP 37.2 42.1 16.7 46.6 33.8 32 43.7 37 27.8 15.4 42.6 42.7 48.5 32.6 20.7 16.9 29.8 51.9 26.2 48.8 41 24.8 35.9 14.2 15.9 ND 49.8 20.8 17.4 41.8 82.8 10 62.9 18.7 51.2 56.5 11.5 19.5 31 AMP 9.5 10.1 8.1 18.6 8.3 6.2 9.1 7 4.3 5.8 8.7 9 7.6 7 5.6 6 6.1 11.9 6.4 17.1 5.6 6.7 8.3 5 5 ND 9.4 4.9 4.9 5.5 14.2 4.6 12.6 4.1 8.8 11.2 6.1 10.5 ATP 62.6 63.9 40.7 91.6 53.7 51.1 60.3 69 36.5 31.7 35.8 75.8 62.8 48.8 27.5 47.7 44.2 79.2 32.8 69.2 63 24.9 63 26.3 36.1 ND 87.8 27.8 25.6 54.1 161.1 14.3 120.8 30.1 78.8 88.3 16.8 39.6 63.8 Alanine 9.3 12.1 6.4 13.3 14.5 11.5 12.5 17 6.7 15.9 9.6 13 13 11.6 7.4 7.5 18.2 11.7 12.2 34.5 14.7 9.4 9.1 ND 14.1 ND 15.2 10.1 4.4 9.2 27.3 4.1 67 5.5 15 15.5 9 6.8 12 Carnitine 3 3.5 1.7 9 1.8 4.2 4 3.2 ND 1.8 3.6 3.3 1.9 1.6 1.2 6.3 2.9 4.1 1.9 3.1 5.1 1.3 2.6 1.2 1.9 0.6 5.6 2.8 1 3.1 8 0.7 4.6 ND 3 5.3 1.3 2.7 4.1 Choline 6.1 7.1 3.3 10.6 3.9 3.6 4.9 7 4.3 2.3 4.7 5.9 3.7 2.2 2.6 8.3 3.4 4.9 2.6 3.8 6.3 1.4 4.5 3.6 4.1 ND 6.2 4.1 1.2 3.6 15.1 1.2 5.8 ND 6.6 4.5 1.9 1.9 8.8 Creatine 2.8 2.8 2.4 42.2 3.7 3 4.8 5 5.4 4.6 3.5 2.6 3.9 ND 2.2 2.7 2.7 6.2 2.1 9.1 7.7 ND 6.2 3.9 2.2 5.3 4.1 10 2.9 2.2 16.3 2.1 5 1.6 8.7 5.1 1.7 1.3 2.6 Formate 12.9 24.1 11.8 13.5 13.3 13 17.7 12.2 11.8 14 17.8 14.8 18.2 17.7 14.3 19.6 20.2 14.5 15.5 28.4 23.4 20 26.3 19.9 29.6 34.5 12.9 18 15.6 17.8 26.2 18.2 25.7 21.6 20.8 23.2 19.8 22 14.6 GTP 14.3 21.8 11.9 23.5 15.8 15.9 14.2 18.9 7.3 7.1 16.2 18.4 15.4 11.3 ND 13.1 12.2 16.9 12.2 21.7 16.3 ND 16.6 ND 11.8 ND 21.2 ND ND 15.4 47.7 ND 26.7 ND 19.4 16.2 ND 8 20.7 Glucose 36.1 64.9 ND 58.9 61.8 42.7 72.3 76.5 37.9 62 67.9 74.4 39.4 58.8 107.2 45.6 29.8 37.1 22 51.9 38.8 78.7 37.4 58.7 86.5 64.9 49.3 65.2 ND ND 61.6 ND ND ND 46.3 59.6 ND ND ND Glutamate 75.8 95.5 56.5 183.9 74.1 76.5 90.1 105 49.9 65.4 90.9 125.5 85.7 76 41 112.7 67.8 117 79.6 212.4 109.4 45.3 89.3 44 89.1 ND 164.5 64.9 47.5 84.4 284.4 27.3 212.6 34.1 82 94.7 51.1 50.9 147.9 Glutamine ND ND 26.3 46.1 27.9 23.6 28.9 41.1 10.7 17.4 36.8 28.7 59.7 29.1 16.9 32.3 16.2 40.5 21.6 110.7 45.7 ND 26.8 ND 26.9 ND 48.4 28.6 26.9 29.4 94.7 ND 60.5 ND 33.1 26.1 ND ND 43.5 Glycine 7.9 10.5 8.9 18.2 8 9.7 11.9 12.3 4.8 8 12.5 14.8 11.3 8.1 4.9 7.9 12.1 14.6 8.6 29.5 13 5.5 12 4.9 7.1 5.6 10.9 7.8 4.2 8.4 67.9 ND 22.4 ND 17.1 16.4 5.3 6.7 23.1 Lactate 53.3 67.6 30.6 219.6 61.9 67.1 93.9 149.7 32.3 101.2 61.2 86.6 97.1 41.1 69.8 39.1 37.3 67.4 44.9 87.5 66.1 43.6 71.8 19.3 80.3 14.5 164 61.6 23.5 47.2 215.5 32.1 119.7 27.4 68.9 116.9 51.4 24.3 65.6 O-Acetylcholine 5.2 7.2 2.9 3.5 4.1 2.8 4.4 3.3 3.8 3.1 2.9 4 2.9 3.3 1.8 1.6 5.9 6.5 1.9 4.3 4.5 1.7 3.5 2.9 3.4 0.4 6 2 2.1 2.1 12.9 1 7.1 ND 3.8 3.6 1.6 2.1 4.3 O-Phosphocholine 10.2 13.3 5.2 16.7 9.9 6.3 9.5 9 11.3 6.4 7.5 13.9 8 6.6 4.3 5.9 9.6 15.7 5.3 18.8 12.1 3.8 9.7 10.5 6.6 1.1 10.4 5.2 4 6.7 27.7 2.2 16.4 ND 8.2 13.5 4.1 4.4 15 O-Phosphoethanolamine 54.5 55.8 37 102.6 48 56.1 52.4 58.8 32 28.5 52 68.4 44.7 39.3 28 35.5 42.3 76.2 38.2 121 60.3 26.4 52 20 41.3 12.1 118.8 36.7 21.3 47.8 162.9 13.6 118.9 ND 72.9 70.5 22.8 28.9 67.9 Taurine 578.1 709.7 449.9 965.4 578.5 563.6 718 717.5 398.9 380.1 548.6 643.3 443.2 476.3 242 510.8 411.8 853.3 389.8 950.3 596.9 241.2 564.6 207.3 432.3 47.2 981.2 310 298.8 460.2 1755 142.2 883.3 189 794.1 722.7 279.4 318.7 674.6 myo-Inositol 22.8 27.8 15 60.2 22.2 23 25.8 45.7 16.2 18.2 16.1 42.3 17 14.3 16.3 50.5 20.2 24.2 36.6 57.9 38.6 25 33.8 10 27.7 ND 58.9 42.5 14 17.6 107.8 9.7 52.6 14.9 40.2 51.9 28.3 15.7 62.5 NMR_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID ADP C00008 AMP C00020 ATP C00002 Alanine C00041 Carnitine C00318 Choline C00114 Creatine C00300 Formate C00058 GTP C00044 Glucose C00221 Glutamate C00025 Glutamine C00064 Glycine C00037 Lactate C00186 O-Acetylcholine C01996 O-Phosphocholine C00588 O-Phosphoethanolamine C00346 Taurine C00245 myo-Inositol C00137 METABOLITES_END #END