#METABOLOMICS WORKBENCH Jianzhi.Hu@pnnl.gov_20161102_155557 DATATRACK_ID:775 STUDY_ID:ST000501 ANALYSIS_ID:AN000770 PROJECT_ID:PR000378 VERSION 1 CREATED_ON November 2, 2016, 4:02 pm #PROJECT PR:PROJECT_TITLE NMR based Metabolomics Analysis of Mouse Exposed to Gamma Radiation PR:PROJECT_TYPE Biofluids Extracts Comparison PR:PROJECT_SUMMARY Biofluid extracts from gamma readiation exposed mouse plasma and controls were PR:PROJECT_SUMMARY compared via NMR based metabolomic analysis PR:INSTITUTE Pacific Northwest National Laboratory PR:LAST_NAME Hu PR:FIRST_NAME Jian Zhi PR:ADDRESS 3335 Innovation Blvd, Richland, Washington, 99354, USA PR:EMAIL Jianzhi.Hu@pnnl.gov PR:PHONE (509)371-6544 #STUDY ST:STUDY_TITLE NMR based Metabolomics Analysis of Mouse Exposed to Gamma Radiation ST:STUDY_TYPE Biofluids Extracts Comparison ST:STUDY_SUMMARY Biofluid extracts from gamma readiation exposed mouse plasma and controls were ST:STUDY_SUMMARY compared via NMR based metabolomic analysis ST:INSTITUTE Pacific Northwest National Laboratory ST:LAST_NAME Hu ST:FIRST_NAME Jian Zhi ST:ADDRESS 3335 Innovation Blvd, Richland, Washington, 99354, USA ST:EMAIL Jianzhi.Hu@pnnl.gov ST:PHONE (509)371-6544 #SUBJECT SU:SUBJECT_TYPE Animal SU:SUBJECT_SPECIES Mus musculus #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - G041 radiation dose:- SUBJECT_SAMPLE_FACTORS - G042 radiation dose:- SUBJECT_SAMPLE_FACTORS - G043 radiation dose:- SUBJECT_SAMPLE_FACTORS - G044 radiation dose:- SUBJECT_SAMPLE_FACTORS - G341 radiation dose:3.0 SUBJECT_SAMPLE_FACTORS - G342 radiation dose:3.0 SUBJECT_SAMPLE_FACTORS - G343 radiation dose:3.0 SUBJECT_SAMPLE_FACTORS - G344 radiation dose:3.0 SUBJECT_SAMPLE_FACTORS - G345 radiation dose:3.0 SUBJECT_SAMPLE_FACTORS - G741 radiation dose:7.8 SUBJECT_SAMPLE_FACTORS - G742 radiation dose:7.8 SUBJECT_SAMPLE_FACTORS - G743 radiation dose:7.8 SUBJECT_SAMPLE_FACTORS - G744 radiation dose:7.8 #COLLECTION CO:COLLECTION_SUMMARY Plasma was prepared and snap-frozen at -80°C before NMR spectroscopy and CO:COLLECTION_SUMMARY morphology analysis. #TREATMENT TR:TREATMENT_SUMMARY A total of 13 seven-week-old C57BL/6 female mice randomly into 3 groups, and TR:TREATMENT_SUMMARY then whole body being exposed to gamma radiation. The individually housed TR:TREATMENT_SUMMARY animals were fed a standard diet and maintained in an environment controlled TR:TREATMENT_SUMMARY facility with an ambient temperature of 22-25°C and 45% relative humidity on 12 TR:TREATMENT_SUMMARY h light-dark cycle. Mice were exposed to whole body gamma radiation using a TR:TREATMENT_SUMMARY high-activity source (1250 keV 60Co) with the linear energy transfer (LET) TR:TREATMENT_SUMMARY associated with these fields in the range of 0.2-2 keV/μm. The mice were TR:TREATMENT_SUMMARY isolated in the corner of their polymer cages and placed a minimum of 100 cm TR:TREATMENT_SUMMARY from the collimated 6000 Ci (222 TBq) 60Co source, and then irradiation to the TR:TREATMENT_SUMMARY proposed dosage, respectively. The result of absorbed dose rate at a depth of TR:TREATMENT_SUMMARY approximately 600 mg/cm2 were ~0.83 Gy/min relative to tissue. After irradiation TR:TREATMENT_SUMMARY the isolation barrier was removed and mice were transferred to Animal Facility. TR:TREATMENT_SUMMARY All procedures (i.e., placement in aerated boxes and subsequent tissue TR:TREATMENT_SUMMARY harvesting) for the control mice were identical to those of irradiated animals, TR:TREATMENT_SUMMARY except that they received no radiation. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Step 1: After randomization of the samples, every preweighted intact frozen SP:SAMPLEPREP_SUMMARY plasma was added into MeOH and then vortex 2 mins. After centrifuge at 7200rpm SP:SAMPLEPREP_SUMMARY for 5 minutes at 4C, the liqiuds layer in translted into a glass vial. Add 4 ml SP:SAMPLEPREP_SUMMARY MeOH and 0.85 ml deionized H2O per gram of liver tissue, followed by vortexing SP:SAMPLEPREP_SUMMARY the mixture and then 2 ml chloroform per gram of tissue was added and vortexed SP:SAMPLEPREP_SUMMARY again. This process took 6 minutes and kept exactly the same for each sample. SP:SAMPLEPREP_SUMMARY Step 2: 2 ml chloroform and 2 ml deionized H2O per gram of tissue were added in SP:SAMPLEPREP_SUMMARY the mixture then vortexed again, followed by transferring the different layers SP:SAMPLEPREP_SUMMARY into glass vials separately with syringes after the mixture (in ice bath) SP:SAMPLEPREP_SUMMARY centrifuged. Finally, the solvents of hydrophilic metabolites were removed by SP:SAMPLEPREP_SUMMARY employing lyophilizer and then stored at -80oC freezer before NMR spectral SP:SAMPLEPREP_SUMMARY measurements. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE - CH:INSTRUMENT_NAME - CH:COLUMN_NAME - #ANALYSIS AN:ANALYSIS_TYPE NMR #NMR NM:INSTRUMENT_NAME Varian INOVA 800 NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:SPECTROMETER_FREQUENCY 800MHz #NMR_METABOLITE_DATA NMR_METABOLITE_DATA:UNITS µM/mg NMR_METABOLITE_DATA_START metabolite_name G041 G042 G043 G044 G341 G342 G343 G344 G345 G741 G742 G743 G744 1,3-Dimethylurate 0.487 0.549 0.492 0.504 0.603 0.578 0.559 0.560 0.552 0.482 0.510 0.503 0.470 2-Aminobutyrate 0.122 0.111 0.111 0.101 0.144 0.132 0.153 0.117 0.131 0.119 0.111 0.106 0.110 2-Hydroxyvalerate 0.171 0.192 0.173 0.172 0.227 0.207 0.213 0.211 0.211 0.104 0.113 0.110 0.102 2-Oxobutyrate 0.160 0.174 0.142 0.126 0.099 0.092 0.087 0.087 0.092 0.083 0.102 0.097 0.085 3-Hydroxybutyrate 1.875 1.642 1.530 1.717 1.444 1.697 1.853 1.382 1.464 0.285 0.158 0.162 0.136 3-Hydroxyisobutyrate 0.057 0.075 0.067 0.070 0.083 0.077 0.088 0.070 0.065 0.050 0.063 0.056 0.051 3-Methylxanthine 0.028 0.043 0.053 0.036 0.036 0.036 0.038 0.036 0.029 0.047 0.064 0.043 0.051 4-Hydroxyphenylacetate 0.061 0.075 0.082 0.070 0.096 0.088 0.107 0.089 0.102 0.144 0.129 0.120 0.117 ADP 0.006 0.004 0.007 0.005 0.005 0.004 0.002 0.003 0.003 0.009 0.008 0.005 0.005 ATP 0.004 0.006 0.007 0.004 0.018 0.020 0.010 0.011 0.008 0.015 0.013 0.006 0.007 Acetoacetate 0.087 0.069 0.073 0.070 0.057 0.083 0.087 0.073 0.047 0.024 0.022 0.024 0.031 Alanine 0.367 0.441 0.328 0.374 0.350 0.304 0.352 0.354 0.402 0.855 0.904 0.765 0.774 Alloisoleucine 0.130 0.136 0.140 0.113 0.151 0.110 0.141 0.111 0.114 0.083 0.081 0.094 0.079 Citrate 0.528 0.484 0.417 0.417 0.526 0.553 0.528 0.501 0.500 0.376 0.379 0.345 0.352 Creatine 0.028 0.028 0.042 0.035 0.064 0.059 0.068 0.060 0.059 0.039 0.040 0.038 0.040 Creatine phosphate 0.200 0.253 0.193 0.273 0.307 0.280 0.326 0.286 0.290 0.210 0.211 0.209 0.191 Dimethyl sulfone 1.360 1.350 1.472 1.208 1.557 1.316 1.305 1.502 1.312 0.998 1.133 0.989 1.185 Dimethylamine 0.897 0.810 0.871 0.795 0.835 0.830 0.784 0.791 0.837 0.798 0.934 0.767 0.874 Fucose 0.144 0.132 0.169 0.185 0.232 0.211 0.312 0.305 0.218 0.180 0.214 0.215 0.192 Fumarate 0.004 0.006 0.007 0.007 0.004 0.004 0.004 0.004 0.007 0.010 0.012 0.005 0.007 Glucose 10.773 8.883 7.854 9.619 8.677 12.760 13.068 10.340 12.113 9.786 11.775 10.054 13.039 Glutamine 0.905 0.856 0.747 0.747 0.716 0.714 0.696 0.699 0.786 0.938 1.037 1.071 0.947 Glycolate 0.587 0.899 0.902 0.767 0.469 0.500 0.498 0.468 0.516 0.902 1.119 0.933 1.113 Histidine 0.006 0.006 0.009 0.005 0.006 0.007 0.010 0.007 0.007 0.008 0.017 0.008 0.012 Hydroxyacetone 0.035 0.040 0.058 0.040 0.036 0.039 0.051 0.045 0.051 0.047 0.053 0.050 0.051 IMP 0.003 0.004 0.004 0.003 0.002 0.005 0.002 0.004 0.004 0.014 0.014 0.010 0.008 Isobutyrate 0.099 0.079 0.098 0.083 0.086 0.075 0.090 0.077 0.080 0.076 0.083 0.081 0.082 Lactate 3.061 2.939 2.271 3.042 2.828 2.720 2.781 2.814 2.939 6.053 5.863 5.517 5.520 Leucine 0.222 0.225 0.213 0.201 0.186 0.189 0.222 0.179 0.215 0.310 0.297 0.301 0.288 Lysine 0.264 0.291 0.237 0.267 0.450 0.433 0.463 0.439 0.430 0.401 0.408 0.432 0.424 Malonate 6.692 6.498 6.690 6.314 6.082 6.008 5.889 6.261 6.338 5.934 6.045 5.921 5.873 O-Acetylcarnitine 0.045 0.093 0.069 0.047 0.042 0.039 0.037 0.029 0.039 0.038 0.045 0.038 0.036 Phenylalanine 0.079 0.069 0.084 0.063 0.075 0.077 0.094 0.069 0.079 0.110 0.112 0.096 0.107 Pyruvate 0.063 0.057 0.069 0.070 0.075 0.074 0.094 0.078 0.074 0.105 0.089 0.097 0.090 Sarcosine 0.076 0.115 0.089 0.102 0.098 0.096 0.113 0.112 0.097 0.145 0.160 0.141 0.154 Succinate 0.289 0.302 0.224 0.237 0.288 0.243 0.241 0.276 0.318 0.440 0.450 0.344 0.376 Threonine 0.224 0.259 0.142 0.166 0.311 0.289 0.293 0.301 0.272 0.329 0.398 0.337 0.367 Tryptophan 0.085 0.091 0.111 0.111 0.101 0.120 0.122 0.108 0.134 0.141 0.129 0.133 0.145 Uridine 0.017 0.018 0.022 0.017 0.014 0.011 0.011 0.011 0.017 0.014 0.021 0.015 0.021 Valine 0.324 0.294 0.310 0.271 0.312 0.281 0.316 0.246 0.243 0.351 0.370 0.366 0.409 cis-Aconitate 0.048 0.042 0.042 0.032 0.067 0.080 0.097 0.100 0.096 0.005 0.009 0.006 0.010 τ-Methylhistidine 0.031 0.038 0.044 0.023 0.049 0.039 0.055 0.053 0.043 0.079 0.067 0.062 0.052 NMR_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name PubChem ID KEGG ID 1,3-Dimethylurate 428 C00986 2-Aminobutyrate 5493 C02356 2-Hydroxyvalerate 98009 2-Oxobutyrate 51 C00026 3-Hydroxybutyrate 441 C01089 3-Hydroxyisobutyrate 4414 C01188 3-Methylxanthine 70639 4-Hydroxyphenylacetate 127 C00642 ADP 6022 C00008 ATP 5957 C00002 Acetoacetate 96 C00164 Alanine 5950 C00041 Alloisoleucine 99288 Citrate 311 C00158 Creatine 586 C00300 Creatine phosphate 587 C02305 Dimethyl sulfone 6213 C11142 Dimethylamine 3824 C00543 Fucose 17106 C01019 Fumarate 284 C00058 Glucose 5793 C00031 Glutamine 5961 C00064 Glycolate 757 C00160 Histidine 6274 C00135 Hydroxyacetone 7630 C05235 IMP 8582 C00130 Isobutyrate 5612 C02632 Lactate 107689 C00186 Leucine 6106 C00123 Lysine 5962 C00047 Malonate 3673 C00383 O-Acetylcarnitine 18230 C02571 Phenylalanine 6140 C00079 Pyruvate 1060 C00022 Sarcosine 3513 C00213 Succinate 1110 C00042 Threonine 6288 C00188 Tryptophan 6305 C00078 Uridine 6029 C00299 Valine 6287 C00183 cis-Aconitate 309 C00417 τ-Methylhistidine 92105 C01152 METABOLITES_END #END