#METABOLOMICS WORKBENCH Gpacheco_20240702_174547 DATATRACK_ID:4970 STUDY_ID:ST003303 ANALYSIS_ID:AN005413 PROJECT_ID:PR002053 VERSION 1 CREATED_ON July 2, 2024, 9:16 pm #PROJECT PR:PROJECT_TITLE Metabolic Alterations in Aneurysmal Subarachnoid Hemorrhage PR:PROJECT_SUMMARY Aneurysmal subarachnoid hemorrhage (aSAH) is a severe type of stroke that is PR:PROJECT_SUMMARY associated with poor outcome. A subset of patients with aSAH will develop PR:PROJECT_SUMMARY secondary complications, most notably delayed cerebral ischemia (DCI), which PR:PROJECT_SUMMARY potentiates neurological injury. In this study, we investigate the relationship PR:PROJECT_SUMMARY between cerebrospinal fluid (CSF) iron accumulation, brain metabolism, and PR:PROJECT_SUMMARY neuronal injury in aSAH patients with or without DCI. We collected longitudinal PR:PROJECT_SUMMARY CSF samples of patients immediately after hospitalization and 5-8 days after PR:PROJECT_SUMMARY onset of ictus. CSF was analyzed with metabolomics to determine metabolic PR:PROJECT_SUMMARY alterations associated with aSAH and DCI. Metabolomic profiling of the CSF PR:PROJECT_SUMMARY samples uncovered significant dysregulation of metabolic pathways associated PR:PROJECT_SUMMARY with energy generation and amino acid utilization, consistent with mitochondrial PR:PROJECT_SUMMARY dysfunction. Using machine learning, we identified a set of metabolites that PR:PROJECT_SUMMARY predicted ICU length of stay (LOS). aSAH alters the CSF metabolome involved in PR:PROJECT_SUMMARY mitochondrial function and a subset of these metabolites are predictive of ICU PR:PROJECT_SUMMARY stay. These results identify potential biomarkers for mitochondrial pathology PR:PROJECT_SUMMARY and provide insight into alterations in brain iron metabolism triggered by aSAH. PR:INSTITUTE University of Akron PR:DEPARTMENT Chemistry PR:LAST_NAME Pacheco PR:FIRST_NAME Gardenia PR:ADDRESS 190 E. Buchtel Common, Akron, OH 44325 PR:EMAIL gardenia.pacheco2@gmail.com PR:PHONE 815-299-2731 #STUDY ST:STUDY_TITLE Metabolic Alterations in Aneurysmal Subarachnoid Hemorrhage ST:STUDY_SUMMARY Aneurysmal subarachnoid hemorrhage (aSAH) is a severe type of stroke that is ST:STUDY_SUMMARY associated with poor outcome. A subset of patients with aSAH will develop ST:STUDY_SUMMARY secondary complications, most notably delayed cerebral ischemia (DCI), which ST:STUDY_SUMMARY potentiates neurological injury. In this study, we investigate the relationship ST:STUDY_SUMMARY between cerebrospinal fluid (CSF) iron accumulation, brain metabolism, and ST:STUDY_SUMMARY neuronal injury in aSAH patients with or without DCI. We collected longitudinal ST:STUDY_SUMMARY CSF samples of patients immediately after hospitalization and 5-8 days after ST:STUDY_SUMMARY onset of ictus. CSF was analyzed with metabolomics to determine metabolic ST:STUDY_SUMMARY alterations associated with aSAH and DCI. Metabolomic profiling of the CSF ST:STUDY_SUMMARY samples uncovered significant dysregulation of metabolic pathways associated ST:STUDY_SUMMARY with energy generation and amino acid utilization, consistent with mitochondrial ST:STUDY_SUMMARY dysfunction. Using machine learning, we identified a set of metabolites that ST:STUDY_SUMMARY predicted ICU length of stay (LOS). aSAH alters the CSF metabolome involved in ST:STUDY_SUMMARY mitochondrial function and a subset of these metabolites are predictive of ICU ST:STUDY_SUMMARY stay. These results identify potential biomarkers for mitochondrial pathology ST:STUDY_SUMMARY and provide insight into alterations in brain iron metabolism triggered by aSAH. ST:INSTITUTE University of Akron ST:DEPARTMENT Chemistry ST:LAST_NAME Pacheco ST:FIRST_NAME Gardenia ST:ADDRESS 190 E. Buchtel Common, Akron, OH 44325 ST:EMAIL gardenia.pacheco2@gmail.com ST:PHONE 815-299-2731 #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 - 1C Sample_Group:Control | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_1C_56.mzML SUBJECT_SAMPLE_FACTORS - 3C Sample_Group:Control | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_3C_35.mzML SUBJECT_SAMPLE_FACTORS - 4C Sample_Group:Control | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_4C_45.mzML SUBJECT_SAMPLE_FACTORS - 5C Sample_Group:Control | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_5C_29.mzML SUBJECT_SAMPLE_FACTORS - 6C Sample_Group:Control | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_6C_46.mzML SUBJECT_SAMPLE_FACTORS - 7C Sample_Group:Control | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_7C_59.mzML SUBJECT_SAMPLE_FACTORS - 9C Sample_Group:Control | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_9C_51.mzML SUBJECT_SAMPLE_FACTORS - 10C Sample_Group:Control | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_10C_36.mzML SUBJECT_SAMPLE_FACTORS - 11C Sample_Group:Control | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_11C_28.mzML SUBJECT_SAMPLE_FACTORS - 12C Sample_Group:Control | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_12C_38.mzML SUBJECT_SAMPLE_FACTORS - 5P1 Sample_Group:SAH_Early | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_5P1_30.mzML SUBJECT_SAMPLE_FACTORS - 6P1 Sample_Group:SAH_Early | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_6P1_57.mzML SUBJECT_SAMPLE_FACTORS - 7P1 Sample_Group:SAH_Early | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_7P1_41.mzML SUBJECT_SAMPLE_FACTORS - 11P1 Sample_Group:SAH_Early | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_11P1_24.mzML SUBJECT_SAMPLE_FACTORS - 12P1 Sample_Group:SAH_Early | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_12P1_66.mzML SUBJECT_SAMPLE_FACTORS - 13P1 Sample_Group:SAH_Early | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_13P1_54.mzML SUBJECT_SAMPLE_FACTORS - 14P1 Sample_Group:SAH_Early | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_14P1_61.mzML SUBJECT_SAMPLE_FACTORS - 15P1 Sample_Group:SAH_Early | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_15P1_26.mzML SUBJECT_SAMPLE_FACTORS - 17P1 Sample_Group:SAH_Early | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_17P1_60.mzML SUBJECT_SAMPLE_FACTORS - 19P1 Sample_Group:SAH_Early | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_19P1_43.mzML SUBJECT_SAMPLE_FACTORS - 20P1 Sample_Group:SAH_Early | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_20P1_37.mzML SUBJECT_SAMPLE_FACTORS - 1P2 Sample_Group:SAH_Late | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_1P2_62.mzML SUBJECT_SAMPLE_FACTORS - 2P2 Sample_Group:SAH_Late | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_2P2_50.mzML SUBJECT_SAMPLE_FACTORS - 3P2 Sample_Group:SAH_Late | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_3P2_64.mzML SUBJECT_SAMPLE_FACTORS - 4P2 Sample_Group:SAH_Late | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_4P2_23.mzML SUBJECT_SAMPLE_FACTORS - 5P2 Sample_Group:SAH_Late | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_5P2_34.mzML SUBJECT_SAMPLE_FACTORS - 6P2 Sample_Group:SAH_Late | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_6P2_25.mzML SUBJECT_SAMPLE_FACTORS - 7P2 Sample_Group:SAH_Late | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_7P2_65.mzML SUBJECT_SAMPLE_FACTORS - 8P2 Sample_Group:SAH_Late | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_8P2_31.mzML SUBJECT_SAMPLE_FACTORS - 9P2 Sample_Group:SAH_Late | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_9P2_52.mzML SUBJECT_SAMPLE_FACTORS - 10P2 Sample_Group:SAH_Late | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_10P2_53.mzML SUBJECT_SAMPLE_FACTORS - 11P2 Sample_Group:SAH_Late | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_11P2_27.mzML SUBJECT_SAMPLE_FACTORS - 12P2 Sample_Group:SAH_Late | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_12P2_32.mzML SUBJECT_SAMPLE_FACTORS - 13P2 Sample_Group:SAH_Late | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_13P2_39.mzML SUBJECT_SAMPLE_FACTORS - 14P2 Sample_Group:SAH_Late | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_14P2_42.mzML SUBJECT_SAMPLE_FACTORS - 15P2 Sample_Group:SAH_Late | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_15P2_40.mzML SUBJECT_SAMPLE_FACTORS - 16P2 Sample_Group:SAH_Late | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_16P2_58.mzML SUBJECT_SAMPLE_FACTORS - 17P2 Sample_Group:SAH_Late | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_17P2_63.mzML SUBJECT_SAMPLE_FACTORS - 18P2 Sample_Group:SAH_Late | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_18P2_48.mzML SUBJECT_SAMPLE_FACTORS - 19P2 Sample_Group:SAH_Late | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_19P2_49.mzML SUBJECT_SAMPLE_FACTORS - 20P2 Sample_Group:SAH_Late | Sample source:CSF RAW_FILE_NAME(Raw_File_Name)=LS_iHILIC_NEG_CSF_20P2_47.mzML #COLLECTION CO:COLLECTION_SUMMARY Samples of cerebrospinal fluid (CSF) were obtained from the EVD within the first CO:COLLECTION_SUMMARY 24 hours (early sample), and once between days 5 and 8 (late sample) following CO:COLLECTION_SUMMARY the onset of ictus. Fluid was obtained from the burette attached to the EVD CO:COLLECTION_SUMMARY system, allowing only sampling of fresh CSF. A total of 6 mL were collected at CO:COLLECTION_SUMMARY each time point and immediately centrifuged at 2,000 g for 10 minutes in the CO:COLLECTION_SUMMARY Cleveland Clinic Genetics core laboratory. The (non-cellular) supernatant was CO:COLLECTION_SUMMARY aliquoted in small polypropylene cryovials and stored in liquid nitrogen to CO:COLLECTION_SUMMARY avoid auto-oxidation of samples (9). The time elapsed from sample collection to CO:COLLECTION_SUMMARY storage in liquid nitrogen was kept under 30 minutes. Control CSF was obtained CO:COLLECTION_SUMMARY from patients with suspected neurological disease and seen at the “lumbar CO:COLLECTION_SUMMARY puncture clinic” that resulted normal after testing analysis and imaging CO:COLLECTION_SUMMARY studies. This study was conducted in accordance with all local IRB guidelines, CO:COLLECTION_SUMMARY and informed consent was obtained from all individual participants or their next CO:COLLECTION_SUMMARY of kin/legally authorized representatives. Additional CSF samples from aSAH CO:COLLECTION_SUMMARY patients and controls were obtained as diagnostic remnants from Accio Biobank CO:COLLECTION_SUMMARY Online. CO:SAMPLE_TYPE Cerebrospinal fluid CO:VOLUMEORAMOUNT_COLLECTED 6 mL CO:STORAGE_CONDITIONS Described in summary CO:STORAGE_VIALS Polypropylene cryovials #TREATMENT TR:TREATMENT_SUMMARY Samples of cerebrospinal fluid (CSF) were obtained from the EVD within the first TR:TREATMENT_SUMMARY 24 hours (early sample), and once between days 5 and 8 (late sample) following TR:TREATMENT_SUMMARY the onset of ictus. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Small molecules were extracted from patient CSF samples using a modified method SP:SAMPLEPREP_SUMMARY prior to metabolomic analysis (11). A 100 μL CSF aliquot was selected and SP:SAMPLEPREP_SUMMARY thawed on ice for all patients analyzed. Each sample was transferred to a 1.5 mL SP:SAMPLEPREP_SUMMARY microcentrifuge tube. For protein precipitation, 400 μL of cold methanol (4X SP:SAMPLEPREP_SUMMARY sample volume) was added to each sample, vortexed, and incubated at -20°C for 2 SP:SAMPLEPREP_SUMMARY h. Following this incubation period, samples were centrifuged at 13,200 rpm for SP:SAMPLEPREP_SUMMARY 20 min at 4°C. The supernatant was transferred to a new 1.5 mL microcentrifuge SP:SAMPLEPREP_SUMMARY tube and then dried down in a CentriVap Concentrator (LABCONCO, Kansas, MO, SP:SAMPLEPREP_SUMMARY USA). The dry samples were maintained at -80°C until analysis was performed. SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Ultra-high performance LC (UHPLC)/MS was performed with an Agilent 1290 Infinity CH:CHROMATOGRAPHY_SUMMARY II LC System interfaced with an Agilent QTOF 6545 Mass Spectrometer. Hydrophilic CH:CHROMATOGRAPHY_SUMMARY interaction liquid chromatography (HILIC) was conducted with a HILICON CH:CHROMATOGRAPHY_SUMMARY iHILIC-(P) Classic HILIC column (100 mm x 2.1 mm, 5 µm). Mobile-phase solvents CH:CHROMATOGRAPHY_SUMMARY were composed of A = 20 mM ammonium bicarbonate, 0.1 % ammonium hydroxide and CH:CHROMATOGRAPHY_SUMMARY 2.5 µM medronic acid in water:acetonitrile (95:5) and B = 2.5 µM medronic acid CH:CHROMATOGRAPHY_SUMMARY in acetonitrile:water (95:5). The column compartment was maintained at 45 ºC CH:CHROMATOGRAPHY_SUMMARY for all experiments. The following linear gradient was applied at a flow rate of CH:CHROMATOGRAPHY_SUMMARY 250 µL min-1: 0-1 min: 90 % B, 1-12 min: 90-35 % B, 12-12.5 min: 35-25 % B, CH:CHROMATOGRAPHY_SUMMARY 12.5-14.5 min: 25 % B. The column was re-equilibrated with 20 column volumes of CH:CHROMATOGRAPHY_SUMMARY 90% B. The injection volume was 4 µL for all samples. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME HILICON iHILIC-(P) Classic HILIC column (100 mm x 2.1 mm, 5 µm) CH:SOLVENT_A Water:acetonitrile (95:5); 20 mM ammonium bicarbonate; 0.1% ammonium hydroxide; CH:SOLVENT_A 2.5 µM medronic acid CH:SOLVENT_B Acetonitrile:water (95:5); 2.5 µM medronic acid CH:FLOW_GRADIENT 0-1 min: 90 % B, 1-12 min: 90-35 % B, 12-12.5 min: 35-25 % B, 12.5-14.5 min: 25 CH:FLOW_GRADIENT % B CH:FLOW_RATE 250 µL min CH:COLUMN_TEMPERATURE 45 CH:SAMPLE_INJECTION 4 µL CH:CAPILLARY_VOLTAGE -3 kV #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6545 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Data were collected with the following settings: capillary voltage, -3 kV; gas, MS:MS_COMMENTS 200ºC at 10 L/min; nebulizer, 44 psi; sheath gas, 300ºC at 12 L/min; MS:MS_COMMENTS fragmentor voltage, 100 V; scan rate, one scan per second; mass range, 67-1500 MS:MS_COMMENTS Da; polarity, negative. LC/MS data were processed and analyzed with the MS:MS_COMMENTS open-source Skyline software (12) and XCMS (13). Metabolomics data analysis and MS:MS_COMMENTS visualization was completed in MetaboAnalyst 5.0 (14). Features were putatively MS:MS_COMMENTS identified via DecoID by matching MS/MS fragmentation to library standards15 and MS:MS_COMMENTS identifications confirmed with level 1 or 2 confidence according to the MS:MS_COMMENTS Metabolomics Standards Initiative (16). Heatmaps of row Z-score values for the MS:MS_COMMENTS 49 metabolites identified were generated with the open-source Morpheus (Broad MS:MS_COMMENTS Institute, https://software.broadinstitute.org/morpheus) software. Hierarchical MS:MS_COMMENTS clustering of the patients (columns) was completed using the one minus Pearson MS:MS_COMMENTS correlation metric with an average linkage. Graphs were made using Prism 9 MS:MS_COMMENTS software (GraphPad Software, San Diego, CA). Machine learning was performed as MS:MS_COMMENTS previously described (17). Five different machine learning models: logistic MS:MS_COMMENTS regression, ElasticNet linear regression, partial least squares discriminant MS:MS_COMMENTS analysis (PLSDA), support vector machine (SVM), and random forest were tested on MS:MS_COMMENTS CSF samples collected at 24 hours and 5-8 days after aSAH ictus using a MS:MS_COMMENTS leave-one-out cross validation. The significance of the model fit was evaluated MS:MS_COMMENTS with a permutation test. MS:MS_RESULTS_FILE ST003303_AN005413_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END