#METABOLOMICS WORKBENCH epannkuk_20240711_062357 DATATRACK_ID:4998 STUDY_ID:ST003334 ANALYSIS_ID:AN005463 PROJECT_ID:PR002073 VERSION 1 CREATED_ON July 17, 2024, 5:09 am #PROJECT PR:PROJECT_TITLE Impact of partial body shielding from very high dose rates on untargeted PR:PROJECT_TITLE metabolomics in biodosimetry PR:PROJECT_SUMMARY A realistic exposure to ionizing radiation (IR) from an improvised nuclear PR:PROJECT_SUMMARY device will likely include individuals that are partially shielded from the PR:PROJECT_SUMMARY initial blast delivered at a very high-dose rate (VHDR). As different tissues PR:PROJECT_SUMMARY have varying levels of radiosensitivity, e.g. hematopoietic vs. gastrointestinal PR:PROJECT_SUMMARY tissues, the effects of shielding on radiation biomarkers needs to be addressed. PR:PROJECT_SUMMARY Here, we explore how biofluid (urine and serum) metabolite signatures from male PR:PROJECT_SUMMARY and female C57BL/6 mice exposed to VHDR (5 – 10 Gy/sec) total body irradiation PR:PROJECT_SUMMARY (TBI, 0, 4, and 8 Gy) compare to individuals exposed to partial body irradiation PR:PROJECT_SUMMARY (PBI) (lower body irradiated [LBI] or upper body irradiated [UBI] at an 8 Gy PR:PROJECT_SUMMARY dose) using a data-independent acquisition untargeted metabolomics approach. PR:PROJECT_SUMMARY Although sex differences were observed in the spatial groupings of urine PR:PROJECT_SUMMARY signatures from TBI and PBI mice, a metabolite signature PR:PROJECT_SUMMARY (N6,N6,N6-trimethyllysine, carnitine, propionylcarnitine, PR:PROJECT_SUMMARY hexosamine-valine-isoleucine, taurine, and creatine) previously developed from PR:PROJECT_SUMMARY variable dose rate experiments was able to identify individuals with high PR:PROJECT_SUMMARY sensitivity and specificity irrespective of radiation shielding. A panel of PR:PROJECT_SUMMARY serum metabolites composed from previous untargeted studies on nonhuman primates PR:PROJECT_SUMMARY had excellent performance for separating irradiated cohorts; however, a PR:PROJECT_SUMMARY multi-omic approach to complement the metabolome could increase dose estimation PR:PROJECT_SUMMARY confidence intervals. Overall, these results support the inclusion of small PR:PROJECT_SUMMARY molecule markers in biodosimetry assays without substantial interference from PR:PROJECT_SUMMARY upper or lower body shielding. PR:INSTITUTE Georgetown University PR:LAST_NAME Pannkuk PR:FIRST_NAME Evan PR:ADDRESS 3970 Reservoir Rd, NW New Research Build, washington dc, District of Columbia, PR:ADDRESS 20057, USA PR:EMAIL elp44@georgetown.edu PR:PHONE 2026875650 #STUDY ST:STUDY_TITLE Impact of partial body shielding from very high dose rates on untargeted ST:STUDY_TITLE metabolomics in biodosimetry ST:STUDY_SUMMARY A realistic exposure to ionizing radiation (IR) from an improvised nuclear ST:STUDY_SUMMARY device will likely include individuals that are partially shielded from the ST:STUDY_SUMMARY initial blast delivered at a very high-dose rate (VHDR). As different tissues ST:STUDY_SUMMARY have varying levels of radiosensitivity, e.g. hematopoietic vs. gastrointestinal ST:STUDY_SUMMARY tissues, the effects of shielding on radiation biomarkers needs to be addressed. ST:STUDY_SUMMARY Here, we explore how biofluid (urine and serum) metabolite signatures from male ST:STUDY_SUMMARY and female C57BL/6 mice exposed to VHDR (5 – 10 Gy/sec) total body irradiation ST:STUDY_SUMMARY (TBI, 0, 4, and 8 Gy) compare to individuals exposed to partial body irradiation ST:STUDY_SUMMARY (PBI) (lower body irradiated [LBI] or upper body irradiated [UBI] at an 8 Gy ST:STUDY_SUMMARY dose) using a data-independent acquisition untargeted metabolomics approach. ST:STUDY_SUMMARY Although sex differences were observed in the spatial groupings of urine ST:STUDY_SUMMARY signatures from TBI and PBI mice, a metabolite signature ST:STUDY_SUMMARY (N6,N6,N6-trimethyllysine, carnitine, propionylcarnitine, ST:STUDY_SUMMARY hexosamine-valine-isoleucine, taurine, and creatine) previously developed from ST:STUDY_SUMMARY variable dose rate experiments was able to identify individuals with high ST:STUDY_SUMMARY sensitivity and specificity irrespective of radiation shielding. A panel of ST:STUDY_SUMMARY serum metabolites composed from previous untargeted studies on nonhuman primates ST:STUDY_SUMMARY had excellent performance for separating irradiated cohorts; however, a ST:STUDY_SUMMARY multi-omic approach to complement the metabolome could increase dose estimation ST:STUDY_SUMMARY confidence intervals. Overall, these results support the inclusion of small ST:STUDY_SUMMARY molecule markers in biodosimetry assays without substantial interference from ST:STUDY_SUMMARY upper or lower body shielding. ST:INSTITUTE Georgetown University ST:LAST_NAME Pannkuk ST:FIRST_NAME Evan ST:ADDRESS 3970 Reservoir Rd, NW New Research Build, washington dc, District of Columbia, ST:ADDRESS 20057, USA ST:EMAIL elp44@georgetown.edu ST:PHONE 2026875650 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENDER Male and female #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 8Gy24 POS_009 Sample source:Serum | Sex:F | Exposure:LB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy27 POS_010 Sample source:Serum | Sex:F | Exposure:LB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS CTL5 POS_011 Sample source:Serum | Sex:F | Exposure:No exposure | Irradiation:0Gy SUBJECT_SAMPLE_FACTORS 4Gy7 POS_012 Sample source:Serum | Sex:F | Exposure:TB | Irradiation:4Gy SUBJECT_SAMPLE_FACTORS CTL2 POS_013 Sample source:Serum | Sex:F | Exposure:No exposure | Irradiation:0Gy SUBJECT_SAMPLE_FACTORS 8Gy12 POS_014 Sample source:Serum | Sex:M | Exposure:TB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy23 POS_015 Sample source:Serum | Sex:F | Exposure:UB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy17 POS_016 Sample source:Serum | Sex:M | Exposure:UB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 4Gy6 POS_017 Sample source:Serum | Sex:F | Exposure:TB | Irradiation:4Gy SUBJECT_SAMPLE_FACTORS 8Gy18 POS_018 Sample source:Serum | Sex:M | Exposure:UB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS CTL1 POS_022 Sample source:Serum | Sex:F | Exposure:No exposure | Irradiation:0Gy SUBJECT_SAMPLE_FACTORS 4Gy6 POS_023 Sample source:Serum | Sex:F | Exposure:TB | Irradiation:4Gy SUBJECT_SAMPLE_FACTORS CTL5 POS_024 Sample source:Serum | Sex:M | Exposure:No exposure | Irradiation:0Gy SUBJECT_SAMPLE_FACTORS 8Gy19 POS_025 Sample source:Serum | Sex:M | Exposure:UB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy23 POS_026 Sample source:Serum | Sex:M | Exposure:LB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS CTL4 POS_027 Sample source:Serum | Sex:F | Exposure:No exposure | Irradiation:0Gy SUBJECT_SAMPLE_FACTORS 8Gy30 POS_028 Sample source:Serum | Sex:F | Exposure:LB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy15 POS_029 Sample source:Serum | Sex:F | Exposure:TB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy20 POS_030 Sample source:Serum | Sex:M | Exposure:UB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS CTL1 POS_031 Sample source:Serum | Sex:F | Exposure:No exposure | Irradiation:0Gy SUBJECT_SAMPLE_FACTORS 8Gy11 POS_036 Sample source:Serum | Sex:F | Exposure:TB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 4Gy9 POS_037 Sample source:Serum | Sex:F | Exposure:TB | Irradiation:4Gy SUBJECT_SAMPLE_FACTORS 4Gy8 POS_038 Sample source:Serum | Sex:F | Exposure:TB | Irradiation:4Gy SUBJECT_SAMPLE_FACTORS CTL1 POS_039 Sample source:Serum | Sex:M | Exposure:No exposure | Irradiation:0Gy SUBJECT_SAMPLE_FACTORS CTL4 POS_040 Sample source:Serum | Sex:F | Exposure:No exposure | Irradiation:0Gy SUBJECT_SAMPLE_FACTORS 4Gy6 POS_041 Sample source:Serum | Sex:M | Exposure:TB | Irradiation:4Gy SUBJECT_SAMPLE_FACTORS 8Gy21 POS_042 Sample source:Serum | Sex:F | Exposure:UB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS CTL2 POS_043 Sample source:Serum | Sex:F | Exposure:No exposure | Irradiation:0Gy SUBJECT_SAMPLE_FACTORS 4Gy8 POS_044 Sample source:Serum | Sex:M | Exposure:TB | Irradiation:4Gy SUBJECT_SAMPLE_FACTORS 8Gy22 POS_045 Sample source:Serum | Sex:M | Exposure:LB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 4Gy7 POS_049 Sample source:Serum | Sex:F | Exposure:TB | Irradiation:4Gy SUBJECT_SAMPLE_FACTORS 8Gy12 POS_050 Sample source:Serum | Sex:F | Exposure:TB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy22 POS_051 Sample source:Serum | Sex:F | Exposure:UB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy13 POS_052 Sample source:Serum | Sex:F | Exposure:TB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy11 POS_053 Sample source:Serum | Sex:F | Exposure:TB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 4Gy10 POS_054 Sample source:Serum | Sex:M | Exposure:TB | Irradiation:4Gy SUBJECT_SAMPLE_FACTORS 8Gy18 POS_055 Sample source:Serum | Sex:F | Exposure:UB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy16 POS_056 Sample source:Serum | Sex:F | Exposure:UB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy14 POS_057 Sample source:Serum | Sex:F | Exposure:TB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 4Gy9 POS_058 Sample source:Serum | Sex:F | Exposure:TB | Irradiation:4Gy SUBJECT_SAMPLE_FACTORS 8Gy21 POS_063 Sample source:Serum | Sex:F | Exposure:LB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy25 POS_064 Sample source:Serum | Sex:M | Exposure:LB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy25 POS_065 Sample source:Serum | Sex:F | Exposure:LB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS CTL4 POS_066 Sample source:Serum | Sex:M | Exposure:No exposure | Irradiation:0Gy SUBJECT_SAMPLE_FACTORS 8Gy15 POS_067 Sample source:Serum | Sex:F | Exposure:TB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy29 POS_068 Sample source:Serum | Sex:F | Exposure:LB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy13 POS_069 Sample source:Serum | Sex:M | Exposure:TB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy23 POS_070 Sample source:Serum | Sex:F | Exposure:LB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy26 POS_071 Sample source:Serum | Sex:F | Exposure:LB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 4Gy10 POS_072 Sample source:Serum | Sex:F | Exposure:TB | Irradiation:4Gy SUBJECT_SAMPLE_FACTORS 8Gy28 POS_076 Sample source:Serum | Sex:F | Exposure:LB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS CTL3 POS_077 Sample source:Serum | Sex:M | Exposure:No exposure | Irradiation:0Gy SUBJECT_SAMPLE_FACTORS 4Gy10 POS_078 Sample source:Serum | Sex:F | Exposure:TB | Irradiation:4Gy SUBJECT_SAMPLE_FACTORS 8Gy12 POS_079 Sample source:Serum | Sex:F | Exposure:TB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS CTL3 POS_080 Sample source:Serum | Sex:F | Exposure:No exposure | Irradiation:0Gy SUBJECT_SAMPLE_FACTORS 8Gy17 POS_081 Sample source:Serum | Sex:F | Exposure:UB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy25 POS_082 Sample source:Serum | Sex:F | Exposure:UB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy22 POS_083 Sample source:Serum | Sex:F | Exposure:LB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 4Gy9 POS_084 Sample source:Serum | Sex:M | Exposure:TB | Irradiation:4Gy SUBJECT_SAMPLE_FACTORS CTL2 POS_085 Sample source:Serum | Sex:M | Exposure:No exposure | Irradiation:0Gy SUBJECT_SAMPLE_FACTORS 8Gy24 POS_090 Sample source:Serum | Sex:M | Exposure:LB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy14 POS_091 Sample source:Serum | Sex:F | Exposure:TB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 4Gy7 POS_092 Sample source:Serum | Sex:M | Exposure:TB | Irradiation:4Gy SUBJECT_SAMPLE_FACTORS 8Gy14 POS_093 Sample source:Serum | Sex:M | Exposure:TB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy13 POS_094 Sample source:Serum | Sex:F | Exposure:TB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy16 POS_095 Sample source:Serum | Sex:M | Exposure:UB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy21 POS_096 Sample source:Serum | Sex:M | Exposure:LB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 4Gy8 POS_097 Sample source:Serum | Sex:F | Exposure:TB | Irradiation:4Gy SUBJECT_SAMPLE_FACTORS CTL3 POS_098 Sample source:Serum | Sex:F | Exposure:No exposure | Irradiation:0Gy SUBJECT_SAMPLE_FACTORS 8Gy20 POS_099 Sample source:Serum | Sex:F | Exposure:UB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy24 POS_103 Sample source:Serum | Sex:F | Exposure:UB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy19 POS_104 Sample source:Serum | Sex:F | Exposure:UB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS 8Gy15 POS_105 Sample source:Serum | Sex:M | Exposure:TB | Irradiation:8Gy SUBJECT_SAMPLE_FACTORS CTL5 POS_106 Sample source:Serum | Sex:F | Exposure:No exposure | Irradiation:0Gy #COLLECTION CO:COLLECTION_SUMMARY Serum samples were prepared using BD Microtainer Tube (REF 365967) with ~100 µL CO:COLLECTION_SUMMARY of whole blood added to each tube, kept at room temperature for 30 min, then CO:COLLECTION_SUMMARY spun at 1300× g at 4 °C for 10 min. Serum was stored at −80 °C and then CO:COLLECTION_SUMMARY shipped on dry ice to Georgetown University Medical Center CO:SAMPLE_TYPE Blood (serum) CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY All animal experiments were approved by the Columbia University Institutional TR:TREATMENT_SUMMARY Animal Care and Use Committee (IACUC, protocol #AABA9603) and were conducted TR:TREATMENT_SUMMARY under all relevant federal and state guidelines. Male (n=5) and female (n=10) TR:TREATMENT_SUMMARY C57BL/6 mice (ages 12 – 14 weeks) were purchased from Charles River TR:TREATMENT_SUMMARY Laboratories (Frederick, MD) and randomly assigned to the zero-dose sham (0 Gy) TR:TREATMENT_SUMMARY and irradiated (4 and 8 Gy, total and partial body exposure) cohorts. Samples TR:TREATMENT_SUMMARY were collected at 1 day. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY A 5 μl aliquot of serum was mixed with 195 μl of cold 66% acetonitrile SP:SAMPLEPREP_SUMMARY containing internal standards (2 μM debrisoquine [M+H]+ = 176.1188; 5 μM SP:SAMPLEPREP_SUMMARY chlorpropamide [M+H]+ = 277.0414, [M-H]- = 275.0257; 30 μM 4-nitrobenzoic acid SP:SAMPLEPREP_SUMMARY [M-H]- = 166.0141). The samples were vortexed and then incubated on ice for 10 SP:SAMPLEPREP_SUMMARY min. Residual solids were pelleted to the bottom by centrifugation for 10 min SP:SAMPLEPREP_SUMMARY (10,000 x g, 4°C), and then an aliquot was placed in a liquid chromatography SP:SAMPLEPREP_SUMMARY (LC) vial. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The LC and MS conditions for serum was as follows: LC solvent A (water/0.1% CH:CHROMATOGRAPHY_SUMMARY formic acid [FA]), solvent B (acetonitrile/0.1% FA), and solvent C CH:CHROMATOGRAPHY_SUMMARY (isopropanol/0.1% FA). Operating conditions for ESI were, capillary voltage 3 CH:CHROMATOGRAPHY_SUMMARY kV, cone voltage 30 V, desolvation temperature 500°C, desolvation gas flow 600 CH:CHROMATOGRAPHY_SUMMARY L/Hr. The gradient for serum was: 4 min 98% A 2% B, 4 min 40% A 60% B, 1.5 min CH:CHROMATOGRAPHY_SUMMARY 2% A 98% B, 2 min 11.8% B 88.2% C, 0.5 min 50% A 50% B, and 1 min 98% A 2% B at CH:CHROMATOGRAPHY_SUMMARY a flow rate of 0.5 ml/min, column temp 60 °C (S3 Fig). CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters ACQUITY UPLC BEH C18 (50 x 2.1mm,1.7um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:SOLVENT_C 100% isopropanol; 0.1% formic acid CH:FLOW_GRADIENT 4 min 98% A 2% B, 4 min 40% A 60% B, 1.5 min 2% A 98% B, 2 min 11.8% B 88.2% C, CH:FLOW_GRADIENT 0.5 min 50% A 50% B, and 1 min 98% A 2% B CH:FLOW_RATE 0.5 ml/min CH:COLUMN_TEMPERATURE 60 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Xevo-G2-S MS:INSTRUMENT_TYPE QTRAP MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Operating conditions for ESI were, capillary voltage 3 kV, cone voltage 30 V, MS:MS_COMMENTS desolvation temperature 500°C, desolvation gas flow 600 L/Hr. MS:MS_RESULTS_FILE ST003334_AN005463_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END