#METABOLOMICS WORKBENCH epannkuk_20220414_115412 DATATRACK_ID:3202 STUDY_ID:ST002175 ANALYSIS_ID:AN003563 VERSION 1 CREATED_ON 10-26-2022 #PROJECT PR:PROJECT_TITLE Effect of external high-dose rate radiation on mouse biofluid metabolomics PR:PROJECT_SUMMARY In the event of an improvised nuclear device (IND), a complex IR exposure will PR:PROJECT_SUMMARY occur consisting of both low (LDR) and high-dose rates (HDR). We have previously PR:PROJECT_SUMMARY addressed LDR exposures from internal emitters or externally deposited PR:PROJECT_SUMMARY radionuclides on biofluid small molecule signatures, but further research on the PR:PROJECT_SUMMARY HDR component is required. Here, we exposed 8 − 10 week old male C57BL/6 mice PR:PROJECT_SUMMARY to a cumulative dose of 3 Gy using a reference dose rate of 0.7 Gy/min or a HDR PR:PROJECT_SUMMARY of 7 Gy/sec, collected urine and serum at 1 and 7 d, then compared the PR:PROJECT_SUMMARY metabolite signatures using a untargeted (urine) approache with liquid PR:PROJECT_SUMMARY chromatography mass spectrometry platforms. PR:INSTITUTE Georgetown University PR:LAST_NAME Pannkuk PR:FIRST_NAME Evan PR:ADDRESS 3970 Reservoir Rd, NW New Research Building E504 PR:EMAIL elp44@georgetown.edu PR:PHONE 2026875650 PR:PUBLICATIONS https://www.mdpi.com/2218-1989/12/6/520 PR:DOI http://dx.doi.org/10.21228/M8TH8J #STUDY ST:STUDY_TITLE Effect of external high-dose rate radiation on mouse biofluid metabolomic ST:STUDY_TITLE signatures ST:STUDY_SUMMARY In the event of an improvised nuclear device (IND), a complex IR exposure will ST:STUDY_SUMMARY occur consisting of both low (LDR) and high-dose rates (HDR). We have previously ST:STUDY_SUMMARY addressed LDR exposures from internal emitters or externally deposited ST:STUDY_SUMMARY radionuclides on biofluid small molecule signatures, but further research on the ST:STUDY_SUMMARY HDR component is required. Here, we exposed 8 − 10 week old male C57BL/6 mice ST:STUDY_SUMMARY to a cumulative dose of 3 Gy using a reference dose rate of 0.7 Gy/min or a HDR ST:STUDY_SUMMARY of 7 Gy/sec, collected urine and serum at 1 and 7 d, then compared the ST:STUDY_SUMMARY metabolite signatures using either untargeted (urine) or targeted (serum) ST:STUDY_SUMMARY approaches with liquid chromatography mass spectrometry platforms. ST:INSTITUTE Georgetown University ST:LAST_NAME Pannkuk ST:FIRST_NAME Evan ST:ADDRESS 3970 Reservoir Rd, NW New Research Building E504 ST:EMAIL elp44@georgetown.edu ST:PHONE 2026875650 ST:SUBMIT_DATE 2022-04-14 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:AGE_OR_AGE_RANGE 8 - 10 weeks SU:GENDER Male #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS 18-2 113 Factor:CTL | Factor:D1 | Factor:post RAW_FILE_NAME=POS_146 SUBJECT_SAMPLE_FACTORS 16-2 13 Factor:CTL | Factor:D1 | Factor:post RAW_FILE_NAME=POS_023 SUBJECT_SAMPLE_FACTORS 13-2 14 Factor:CTL | Factor:D1 | Factor:post RAW_FILE_NAME=POS_024 SUBJECT_SAMPLE_FACTORS 15-2 16 Factor:CTL | Factor:D1 | Factor:post RAW_FILE_NAME=POS_026 SUBJECT_SAMPLE_FACTORS 14-2 62 Factor:CTL | Factor:D1 | Factor:post RAW_FILE_NAME=POS_082 SUBJECT_SAMPLE_FACTORS 17-2 84 Factor:CTL | Factor:D1 | Factor:post RAW_FILE_NAME=POS_112 SUBJECT_SAMPLE_FACTORS 15-2 115 Factor:CTL | Factor:D1 | Factor:pre RAW_FILE_NAME=POS_151 SUBJECT_SAMPLE_FACTORS 13-2 127 Factor:CTL | Factor:D1 | Factor:pre RAW_FILE_NAME=POS_166 SUBJECT_SAMPLE_FACTORS 18-2 132 Factor:CTL | Factor:D1 | Factor:pre RAW_FILE_NAME=POS_170 SUBJECT_SAMPLE_FACTORS 14-2 5 Factor:CTL | Factor:D1 | Factor:pre RAW_FILE_NAME=POS_012 SUBJECT_SAMPLE_FACTORS 16-2 70 Factor:CTL | Factor:D1 | Factor:pre RAW_FILE_NAME=POS_093 SUBJECT_SAMPLE_FACTORS 17-2 85 Factor:CTL | Factor:D1 | Factor:pre RAW_FILE_NAME=POS_113 SUBJECT_SAMPLE_FACTORS 33-2 120 Factor:CTL | Factor:D7 | Factor:post RAW_FILE_NAME=POS_156 SUBJECT_SAMPLE_FACTORS 32-2 24 Factor:CTL | Factor:D7 | Factor:post RAW_FILE_NAME=POS_037 SUBJECT_SAMPLE_FACTORS 34-2 51 Factor:CTL | Factor:D7 | Factor:post RAW_FILE_NAME=POS_069 SUBJECT_SAMPLE_FACTORS 36-2 78 Factor:CTL | Factor:D7 | Factor:post RAW_FILE_NAME=POS_104 SUBJECT_SAMPLE_FACTORS 33-2 116 Factor:CTL | Factor:D7 | Factor:pre RAW_FILE_NAME=POS_152 SUBJECT_SAMPLE_FACTORS 32-2 133 Factor:CTL | Factor:D7 | Factor:pre RAW_FILE_NAME=POS_171 SUBJECT_SAMPLE_FACTORS 36-2 142 Factor:CTL | Factor:D7 | Factor:pre RAW_FILE_NAME=POS_182 SUBJECT_SAMPLE_FACTORS 31-2 146 Factor:CTL | Factor:D7 | Factor:pre RAW_FILE_NAME=POS_186 SUBJECT_SAMPLE_FACTORS 34-2 18 Factor:CTL | Factor:D7 | Factor:pre RAW_FILE_NAME=POS_028 SUBJECT_SAMPLE_FACTORS 35-2 73 Factor:CTL | Factor:D7 | Factor:pre RAW_FILE_NAME=POS_099 SUBJECT_SAMPLE_FACTORS 1-2 108 Factor:HDR | Factor:D1 | Factor:post RAW_FILE_NAME=POS_142 SUBJECT_SAMPLE_FACTORS 6-2 111 Factor:HDR | Factor:D1 | Factor:post RAW_FILE_NAME=POS_144 SUBJECT_SAMPLE_FACTORS 5-2 114 Factor:HDR | Factor:D1 | Factor:post RAW_FILE_NAME=POS_147 SUBJECT_SAMPLE_FACTORS 4-2 117 Factor:HDR | Factor:D1 | Factor:post RAW_FILE_NAME=POS_153 SUBJECT_SAMPLE_FACTORS 3-2 139 Factor:HDR | Factor:D1 | Factor:post RAW_FILE_NAME=POS_179 SUBJECT_SAMPLE_FACTORS 2-2 48 Factor:HDR | Factor:D1 | Factor:post RAW_FILE_NAME=POS_066 SUBJECT_SAMPLE_FACTORS 1-2 122 Factor:HDR | Factor:D1 | Factor:pre RAW_FILE_NAME=POS_158 SUBJECT_SAMPLE_FACTORS 4-2 128 Factor:HDR | Factor:D1 | Factor:pre RAW_FILE_NAME=POS_167 SUBJECT_SAMPLE_FACTORS 3-2 45 Factor:HDR | Factor:D1 | Factor:pre RAW_FILE_NAME=POS_064 SUBJECT_SAMPLE_FACTORS 2-2 57 Factor:HDR | Factor:D1 | Factor:pre RAW_FILE_NAME=POS_077 SUBJECT_SAMPLE_FACTORS 6-2 86 Factor:HDR | Factor:D1 | Factor:pre RAW_FILE_NAME=POS_114 SUBJECT_SAMPLE_FACTORS 5-2 88 Factor:HDR | Factor:D1 | Factor:pre RAW_FILE_NAME=POS_116 SUBJECT_SAMPLE_FACTORS 19-2 103 Factor:HDR | Factor:D7 | Factor:post RAW_FILE_NAME=POS_134 SUBJECT_SAMPLE_FACTORS 22-2 11 Factor:HDR | Factor:D7 | Factor:post RAW_FILE_NAME=POS_018 SUBJECT_SAMPLE_FACTORS 21-2 129 Factor:HDR | Factor:D7 | Factor:post RAW_FILE_NAME=POS_168 SUBJECT_SAMPLE_FACTORS 24-2 145 Factor:HDR | Factor:D7 | Factor:post RAW_FILE_NAME=POS_185 SUBJECT_SAMPLE_FACTORS 23-2 54 Factor:HDR | Factor:D7 | Factor:post RAW_FILE_NAME=POS_075 SUBJECT_SAMPLE_FACTORS 20-2 92 Factor:HDR | Factor:D7 | Factor:post RAW_FILE_NAME=POS_120 SUBJECT_SAMPLE_FACTORS 24-2 137 Factor:HDR | Factor:D7 | Factor:pre RAW_FILE_NAME=POS_177 SUBJECT_SAMPLE_FACTORS 20-2 141 Factor:HDR | Factor:D7 | Factor:pre RAW_FILE_NAME=POS_181 SUBJECT_SAMPLE_FACTORS 22-2 41 Factor:HDR | Factor:D7 | Factor:pre RAW_FILE_NAME=POS_057 SUBJECT_SAMPLE_FACTORS 21-2 60 Factor:HDR | Factor:D7 | Factor:pre RAW_FILE_NAME=POS_080 SUBJECT_SAMPLE_FACTORS 23-2 64 Factor:HDR | Factor:D7 | Factor:pre RAW_FILE_NAME=POS_087 SUBJECT_SAMPLE_FACTORS 9-2 106 Factor:REF | Factor:D1 | Factor:post RAW_FILE_NAME=POS_140 SUBJECT_SAMPLE_FACTORS 8-2 33 Factor:REF | Factor:D1 | Factor:post RAW_FILE_NAME=POS_049 SUBJECT_SAMPLE_FACTORS 11-2 38 Factor:REF | Factor:D1 | Factor:post RAW_FILE_NAME=POS_054 SUBJECT_SAMPLE_FACTORS 10-2 65 Factor:REF | Factor:D1 | Factor:post RAW_FILE_NAME=POS_088 SUBJECT_SAMPLE_FACTORS 12-2 68 Factor:REF | Factor:D1 | Factor:post RAW_FILE_NAME=POS_091 SUBJECT_SAMPLE_FACTORS 9-2 121 Factor:REF | Factor:D1 | Factor:pre RAW_FILE_NAME=POS_157 SUBJECT_SAMPLE_FACTORS 10-2 151 Factor:REF | Factor:D1 | Factor:pre RAW_FILE_NAME=POS_190 SUBJECT_SAMPLE_FACTORS 12-2 22 Factor:REF | Factor:D1 | Factor:pre RAW_FILE_NAME=POS_035 SUBJECT_SAMPLE_FACTORS 8-2 34 Factor:REF | Factor:D1 | Factor:pre RAW_FILE_NAME=POS_050 SUBJECT_SAMPLE_FACTORS 11-2 61 Factor:REF | Factor:D1 | Factor:pre RAW_FILE_NAME=POS_081 SUBJECT_SAMPLE_FACTORS 25-2 131 Factor:REF | Factor:D7 | Factor:post RAW_FILE_NAME=POS_169 SUBJECT_SAMPLE_FACTORS 30-2 150 Factor:REF | Factor:D7 | Factor:post RAW_FILE_NAME=POS_189 SUBJECT_SAMPLE_FACTORS 27-2 26 Factor:REF | Factor:D7 | Factor:post RAW_FILE_NAME=POS_039 SUBJECT_SAMPLE_FACTORS 28-2 30 Factor:REF | Factor:D7 | Factor:post RAW_FILE_NAME=POS_043 SUBJECT_SAMPLE_FACTORS 26-2 40 Factor:REF | Factor:D7 | Factor:post RAW_FILE_NAME=POS_056 SUBJECT_SAMPLE_FACTORS 29-2 96 Factor:REF | Factor:D7 | Factor:post RAW_FILE_NAME=POS_127 SUBJECT_SAMPLE_FACTORS 25-2 125 Factor:REF | Factor:D7 | Factor:pre RAW_FILE_NAME=POS_164 SUBJECT_SAMPLE_FACTORS 27-2 136 Factor:REF | Factor:D7 | Factor:pre RAW_FILE_NAME=POS_173 SUBJECT_SAMPLE_FACTORS 26-2 17 Factor:REF | Factor:D7 | Factor:pre RAW_FILE_NAME=POS_027 SUBJECT_SAMPLE_FACTORS 29-2 23 Factor:REF | Factor:D7 | Factor:pre RAW_FILE_NAME=POS_036 SUBJECT_SAMPLE_FACTORS 30-2 77 Factor:REF | Factor:D7 | Factor:pre RAW_FILE_NAME=POS_103 SUBJECT_SAMPLE_FACTORS 28-2 94 Factor:REF | Factor:D7 | Factor:pre RAW_FILE_NAME=POS_125 #COLLECTION CO:COLLECTION_SUMMARY Urine was collected after irradiation CO:SAMPLE_TYPE Urine CO:COLLECTION_METHOD Spot Urine CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Male 8 – 10 week old C57BL/6 mice were obtained from Charles River TR:TREATMENT_SUMMARY Laboratories (Frederick, MD, USA) and were irradiated using the FLASH irradiator TR:TREATMENT_SUMMARY at the Radiological Research Accelerator Facility [16] (Figure S1). This novel TR:TREATMENT_SUMMARY irradiator is based on a Clinac 2100C (Varian Medical Systems, Corona, CA, USA) TR:TREATMENT_SUMMARY where the pulse delivery is controlled using in house software. All irradiations TR:TREATMENT_SUMMARY were performed using 9 MeV electrons with no scatterer or flattening filter. For TR:TREATMENT_SUMMARY these experiments, mice were placed in a 72 mm x 41mm x41mm acrylic box in which TR:TREATMENT_SUMMARY air holes had been drilled (The Container Store, Coppell, TX, USA). For 0.7 TR:TREATMENT_SUMMARY Gy/min irradiations, mice (n=6) were individually placed at 120 cm above the TR:TREATMENT_SUMMARY clinac head and irradiation delivered at 3.25 pulses per second. In this TR:TREATMENT_SUMMARY configuration, 3 Gy was delivered in 580 pulses. For 7 Gy/sec mice (n=6) were TR:TREATMENT_SUMMARY individually placed 20cm above the clinac head (Figure S1) and dose delivered at TR:TREATMENT_SUMMARY 180 pulses/sec after allowing 20 sec where the acceleration and electron source TR:TREATMENT_SUMMARY were both on but operated asynchronously so that no beam is delivered. In this TR:TREATMENT_SUMMARY configuration, 3 Gy was delivered in 78 pulses. Dosimetry was performed prior to TR:TREATMENT_SUMMARY irradiation using a NIST-traceable Advanced Marcus Ion Chamber (AMIC) and Unidos TR:TREATMENT_SUMMARY E electrometer (PTW, Freiburg, Germany). Verification of dosimetry was performed TR:TREATMENT_SUMMARY using OBT3 radiochromic film (Ashland Specialty Chemicals, Wayne, NJ, USA). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY urine (20 μl) was deproteinized (80 μl 50% cold acetonitrile) with internal SP:SAMPLEPREP_SUMMARY standards (2 μM debrisoquine [M+H]+ = 176.1188; 30 μM 4-nitrobenzoic acid SP:SAMPLEPREP_SUMMARY [M-H]- = 166.0141), vortexed, incubated on ice (10 min), and centrifuged for 10 SP:SAMPLEPREP_SUMMARY min (max speed, 4 °C). A 1 μl aliquot of each sample was combined for a SP:SAMPLEPREP_SUMMARY quality control (QC) sample. SP:PROCESSING_STORAGE_CONDITIONS -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Mobile phases consisted of the following: solvent A (water/0.1% formic acid CH:CHROMATOGRAPHY_SUMMARY [FA]) and solvent B (acetonitrile/0.1% FA). The gradient for urine was (solvent CH:CHROMATOGRAPHY_SUMMARY A and B) 4.0 min 5% B, 4.0 min 20% B, 5.1 min 95% B, and 1.9 min 5% B at a flow CH:CHROMATOGRAPHY_SUMMARY rate of 0.5 ml/min, column temp 40 °C. CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters Acquity BEH C18 (50 x 2.1mm, 1.7 um) CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Synapt G2 S QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:MS_COMMENTS Samples were injected (2 μl) into a Waters Acquity Ultra Performance Liquid MS:MS_COMMENTS Chromatography (UPLC) with a BEH C18 1.7 μm, 2.1 x 50 mm column and coupled to MS:MS_COMMENTS a Xevo® G2-S quadrupole time-of-flight (QTOF) MS (Waters, Milford, MA, USA). MS:MS_COMMENTS Positive and negative electrospray ionization (ESI) data-independent modes were MS:MS_COMMENTS used for data acquisition with leucine enkephalin ([M+H]+ = 556.2771, [M-H]- = MS:MS_COMMENTS 554.2615) as Lock-Spray®. Operating conditions for ESI were: capillary voltage MS:MS_COMMENTS 2.75 kV, cone voltage 30 V, desolvation temperature 500°C, desolvation gas flow MS:MS_COMMENTS 1000 L/Hr. MS:ION_MODE POSITIVE MS:MS_RESULTS_FILE ST002175_AN003563_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END