#METABOLOMICS WORKBENCH srjhogan@gatech.edu_20180119_070355 DATATRACK_ID:1301 STUDY_ID:ST000920 ANALYSIS_ID:AN001508 PROJECT_ID:PR000636 VERSION 1 CREATED_ON January 19, 2018, 11:56 am #PROJECT PR:PROJECT_TITLE Discovery of Lipidome Alterations Following Traumatic Brain Injury via PR:PROJECT_TITLE High-Resolution Metabolomics PR:PROJECT_TYPE Untargeted Lipidomics PR:PROJECT_SUMMARY Traumatic brain injury (TBI) can occur across wide segments of the population, PR:PROJECT_SUMMARY presenting in a heterogeneous manner that makes diagnosis inconsistent and PR:PROJECT_SUMMARY management challenging. Biomarkers offer the potential to objectively identify PR:PROJECT_SUMMARY injury status, severity, and phenotype by measuring the relative concentrations PR:PROJECT_SUMMARY of endogenous molecules in readily accessible biofluids. Through a data-driven, PR:PROJECT_SUMMARY discovery approach, novel biomarker candidates for TBI were identified in the PR:PROJECT_SUMMARY serum lipidome of adult male Sprague-Dawley rats in the first week following PR:PROJECT_SUMMARY moderate controlled cortical impact (CCI). Serum samples were analyzed in PR:PROJECT_SUMMARY positive and negative ion modes by Ultra Performance Liquid Chromatography Mass PR:PROJECT_SUMMARY Spectrometry (UPLC-MS). A predictive panel for the classification of injured and PR:PROJECT_SUMMARY uninjured sera samples, consisting of 26 dysregulated species belonging to a PR:PROJECT_SUMMARY variety of lipid classes, was developed with a cross-validated accuracy of 85.3% PR:PROJECT_SUMMARY using omniClassifier software to optimize feature selection,. Polyunsaturated PR:PROJECT_SUMMARY fatty acids (PUFAs) and PUFA-containing diacylglycerols were found to be PR:PROJECT_SUMMARY upregulated in sera from injured rats, while changes in sphingolipids and other PR:PROJECT_SUMMARY membrane phospholipids were also observed, many of which map to known secondary PR:PROJECT_SUMMARY injury pathways. Overall, the identified biomarker panel offers viable molecular PR:PROJECT_SUMMARY candidates representing lipids that may readily cross the blood brain barrier PR:PROJECT_SUMMARY (BBB) and aid in the understanding of TBI pathophysiology. PR:INSTITUTE Georgia Institute of Technology PR:DEPARTMENT Chemistry PR:LABORATORY Fernández PR:LAST_NAME Hogan PR:FIRST_NAME Scott PR:ADDRESS 901 Atlantic Drive, Atlanta, GA, 30332, USA PR:EMAIL srjhogan@gatech.edu PR:PHONE 2156924657 #STUDY ST:STUDY_TITLE Discovery of Lipidome Alterations Following Traumatic Brain Injury via ST:STUDY_TITLE High-Resolution Metabolomics ST:STUDY_TYPE Untargeted lipidomics ST:STUDY_SUMMARY Traumatic brain injury (TBI) can occur across wide segments of the population, ST:STUDY_SUMMARY presenting in a heterogeneous manner that makes diagnosis inconsistent and ST:STUDY_SUMMARY management challenging. Biomarkers offer the potential to objectively identify ST:STUDY_SUMMARY injury status, severity, and phenotype by measuring the relative concentrations ST:STUDY_SUMMARY of endogenous molecules in readily accessible biofluids. Through a data-driven, ST:STUDY_SUMMARY discovery approach, novel biomarker candidates for TBI were identified in the ST:STUDY_SUMMARY serum lipidome of adult male Sprague-Dawley rats in the first week following ST:STUDY_SUMMARY moderate controlled cortical impact (CCI). Serum samples were analyzed in ST:STUDY_SUMMARY positive and negative ion modes by Ultra Performance Liquid Chromatography Mass ST:STUDY_SUMMARY Spectrometry (UPLC-MS). A predictive panel for the classification of injured and ST:STUDY_SUMMARY uninjured sera samples, consisting of 26 dysregulated species belonging to a ST:STUDY_SUMMARY variety of lipid classes, was developed with a cross-validated accuracy of 85.3% ST:STUDY_SUMMARY using omniClassifier software to optimize feature selection,. Polyunsaturated ST:STUDY_SUMMARY fatty acids (PUFAs) and PUFA-containing diacylglycerols were found to be ST:STUDY_SUMMARY upregulated in sera from injured rats, while changes in sphingolipids and other ST:STUDY_SUMMARY membrane phospholipids were also observed, many of which map to known secondary ST:STUDY_SUMMARY injury pathways. Overall, the identified biomarker panel offers viable molecular ST:STUDY_SUMMARY candidates representing lipids that may readily cross the blood brain barrier ST:STUDY_SUMMARY (BBB) and aid in the understanding of TBI pathophysiology. ST:INSTITUTE Georgia Institute of Technology ST:DEPARTMENT Chemistry ST:LABORATORY Fernández ST:LAST_NAME Hogan ST:FIRST_NAME Scott ST:ADDRESS 311 Ferst Dr ST:EMAIL srjhogan@gatech.edu ST:PHONE 2156924657 ST:NUM_GROUPS 5 ST:TOTAL_SUBJECTS 34 ST:NUM_MALES 34 #SUBJECT SU:SUBJECT_TYPE Rat SU:SUBJECT_SPECIES Rattus norvegicus SU:TAXONOMY_ID 10116 SU:GENOTYPE_STRAIN Sprague-dawley SU:AGE_OR_AGE_RANGE 8 weeks SU:WEIGHT_OR_WEIGHT_RANGE 300-400 g SU:GENDER male #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - TC7 Class:TBI SUBJECT_SAMPLE_FACTORS - TL3 Class:TBI SUBJECT_SAMPLE_FACTORS - TN3 Class:TBI SUBJECT_SAMPLE_FACTORS - TI7 Class:TBI SUBJECT_SAMPLE_FACTORS - TK3 Class:TBI SUBJECT_SAMPLE_FACTORS - TE7 Class:TBI SUBJECT_SAMPLE_FACTORS - TF7 Class:TBI SUBJECT_SAMPLE_FACTORS - TG7 Class:TBI SUBJECT_SAMPLE_FACTORS - TH7 Class:TBI SUBJECT_SAMPLE_FACTORS - TO3 Class:TBI SUBJECT_SAMPLE_FACTORS - TA7 Class:TBI SUBJECT_SAMPLE_FACTORS - TB7 Class:TBI SUBJECT_SAMPLE_FACTORS - TD7 Class:TBI SUBJECT_SAMPLE_FACTORS - TJ3 Class:TBI SUBJECT_SAMPLE_FACTORS - TP3 Class:TBI SUBJECT_SAMPLE_FACTORS - TM3 Class:TBI SUBJECT_SAMPLE_FACTORS - SA3 Class:SHAM SUBJECT_SAMPLE_FACTORS - NA8 Class:naïve SUBJECT_SAMPLE_FACTORS - SC7 Class:SHAM SUBJECT_SAMPLE_FACTORS - SA7 Class:SHAM SUBJECT_SAMPLE_FACTORS - NA6 Class:naïve SUBJECT_SAMPLE_FACTORS - SB3 Class:SHAM SUBJECT_SAMPLE_FACTORS - NA2 Class:naïve SUBJECT_SAMPLE_FACTORS - NA4 Class:naïve SUBJECT_SAMPLE_FACTORS - SC3 Class:SHAM SUBJECT_SAMPLE_FACTORS - NA3 Class:naïve SUBJECT_SAMPLE_FACTORS - NA0 Class:naïve SUBJECT_SAMPLE_FACTORS - SD7 Class:SHAM SUBJECT_SAMPLE_FACTORS - NA9 Class:naïve SUBJECT_SAMPLE_FACTORS - NA7 Class:naïve SUBJECT_SAMPLE_FACTORS - NA5 Class:naïve SUBJECT_SAMPLE_FACTORS - SD3 Class:SHAM SUBJECT_SAMPLE_FACTORS - SB7 Class:SHAM SUBJECT_SAMPLE_FACTORS - NA1 Class:naïve #COLLECTION CO:COLLECTION_SUMMARY During the afternoon, approximately 200 µL of whole blood was collected from a CO:COLLECTION_SUMMARY tail vein punctured by 20-gauge Precision Glide needles (Beckton Dickinson) and CO:COLLECTION_SUMMARY stored on ice. Whole blood samples were allowed to coagulate at room temperature CO:COLLECTION_SUMMARY for 45 minutes, and all sample collection followed literature guidelines for CO:COLLECTION_SUMMARY limiting the potential for hemolysis.37-39 Samples were then centrifuged at 4 CO:COLLECTION_SUMMARY °C for 15 min at 2500 x g, and serum was collected in 50 μL aliquots and CO:COLLECTION_SUMMARY stored at -80 °C. CO:SAMPLE_TYPE Serum #TREATMENT TR:TREATMENT_SUMMARY Unilateral contusions of the lateral frontoparietal cortex were created using a TR:TREATMENT_SUMMARY controlled cortical impact (CCI) device (Pittsburgh Precision Instruments, TR:TREATMENT_SUMMARY Pittsburgh, PA) following published procedures. Rats were anesthetized TR:TREATMENT_SUMMARY (induction, 5% isoflurane; maintenance 2-3% isoflurane), mounted in a TR:TREATMENT_SUMMARY stereotaxic frame and a 6 mm craniectomy was made over the left frontoparietal TR:TREATMENT_SUMMARY cortex (center: -3.0 mm AP, +2.0 mm ML from bregma). A pneumatic piston (tip TR:TREATMENT_SUMMARY diameter=5 mm; positioned 15 degrees from vertical in the coronal plane) TR:TREATMENT_SUMMARY impacted the cortical tissue to a depth of 2 mm (velocity=4 m/s, duration=200 TR:TREATMENT_SUMMARY ms), values consistent with a moderate TBI insult #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Prior to analysis, lipids and small non-polar metabolites were separated from SP:SAMPLEPREP_SUMMARY proteins in blood using isopropyl alcohol (IPA) for protein precipitation, shown SP:SAMPLEPREP_SUMMARY to be advantageous over the Folch or Bligh and Dyer methods for a variety of SP:SAMPLEPREP_SUMMARY experimental considerations.40-42 For protein precipitation purposes, serum SP:SAMPLEPREP_SUMMARY samples were thawed on ice for 1 h and serum was mixed 1:3 v/v with IPA. Samples SP:SAMPLEPREP_SUMMARY were centrifuged at 16,000 x g for 7 min, and the supernatant was collected. SP:SAMPLEPREP_SUMMARY Samples were stored at -80 °C until analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity H-Class CH:COLUMN_NAME Acquity BEH C18 CH:FLOW_RATE .3 mL/min CH:COLUMN_TEMPERATURE 60° C CH:SOLVENT_A water: acetonitrile (40:60) + 10 mM ammonium formate + 0.1% formic acid CH:SOLVENT_B 10% acetonitrile in IPA + 10 mM ammonium formate + 0.1% formic acid #ANALYSIS AN:ANALYSIS_TYPE MS AN:SOFTWARE_VERSION MassLyxnx V4.1 #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Waters Synapt G2 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:CAPILLARY_VOLTAGE 3.0 kV MS:SOURCE_TEMPERATURE 80° C MS:DESOLVATION_GAS_FLOW 600 L/h MS:DESOLVATION_TEMPERATURE 150° C MS:MS_RESULTS_FILE ST000920_AN001508_Results.txt UNITS:normalized abundance #END