#METABOLOMICS WORKBENCH Jianzhi.Hu@pnnl.gov_20160815_215942 DATATRACK_ID:702 STUDY_ID:ST000479 ANALYSIS_ID:AN000745 PROJECT_ID:PR000365 VERSION 1 CREATED_ON August 23, 2016, 10:55 am #PROJECT PR:PROJECT_TITLE NMR based quantitative metabolomics studies of heart and lung from mouse exposed PR:PROJECT_TITLE to whole body high dose gamma and proton radiation PR:PROJECT_SUMMARY Tissue extracts from ionizing readiation exposed mouse heart and controls were PR:PROJECT_SUMMARY compared via NMR based metabolomic analysis PR:INSTITUTE Pacific Northwest National Laboratory PR:DEPARTMENT Fundamental & Computational Sciences PR:LAST_NAME Hu PR:FIRST_NAME Jianzhi PR:ADDRESS Pacific Northwest National Laboratory, Richland, WA 99352 PR:EMAIL Jianzhi.Hu@pnnl.gov PR:PHONE (+1)5093716544 #STUDY ST:STUDY_TITLE NMR based quantitative metabolomics studies of heart and lung from mouse exposed ST:STUDY_TITLE to whole body high dose gamma and proton radiation ST:STUDY_SUMMARY Tissue extracts from ionizing readiation exposed mouse heart 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 Jianzhi ST:ADDRESS Pacific Northwest National Laboratory, Richland, WA 99352 ST:EMAIL Jianzhi.Hu@pnnl.gov ST:PHONE (+1)5093716544 #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 days post to radiation:4 | radiation dose:- | radiation source:sham SUBJECT_SAMPLE_FACTORS - G042 days post to radiation:4 | radiation dose:- | radiation source:sham SUBJECT_SAMPLE_FACTORS - G043 days post to radiation:4 | radiation dose:- | radiation source:sham SUBJECT_SAMPLE_FACTORS - G044 days post to radiation:4 | radiation dose:- | radiation source:sham SUBJECT_SAMPLE_FACTORS - G341 days post to radiation:4 | radiation dose:3.0 | radiation source:gamma SUBJECT_SAMPLE_FACTORS - G342 days post to radiation:4 | radiation dose:3.0 | radiation source:gamma SUBJECT_SAMPLE_FACTORS - G343 days post to radiation:4 | radiation dose:3.0 | radiation source:gamma SUBJECT_SAMPLE_FACTORS - G344 days post to radiation:4 | radiation dose:3.0 | radiation source:gamma SUBJECT_SAMPLE_FACTORS - G345 days post to radiation:4 | radiation dose:3.0 | radiation source:gamma SUBJECT_SAMPLE_FACTORS - G741 days post to radiation:4 | radiation dose:7.8 | radiation source:gamma SUBJECT_SAMPLE_FACTORS - G742 days post to radiation:4 | radiation dose:7.8 | radiation source:gamma SUBJECT_SAMPLE_FACTORS - G743 days post to radiation:4 | radiation dose:7.8 | radiation source:gamma SUBJECT_SAMPLE_FACTORS - G744 days post to radiation:4 | radiation dose:7.8 | radiation source:gamma SUBJECT_SAMPLE_FACTORS - G011 days post to radiation:11 | radiation dose:- | radiation source:sham SUBJECT_SAMPLE_FACTORS - G012 days post to radiation:11 | radiation dose:- | radiation source:sham SUBJECT_SAMPLE_FACTORS - G013 days post to radiation:11 | radiation dose:- | radiation source:sham SUBJECT_SAMPLE_FACTORS - G014 days post to radiation:11 | radiation dose:- | radiation source:sham SUBJECT_SAMPLE_FACTORS - G311 days post to radiation:11 | radiation dose:3.0 | radiation source:gamma SUBJECT_SAMPLE_FACTORS - G312 days post to radiation:11 | radiation dose:3.0 | radiation source:gamma SUBJECT_SAMPLE_FACTORS - G313 days post to radiation:11 | radiation dose:3.0 | radiation source:gamma SUBJECT_SAMPLE_FACTORS - G314 days post to radiation:11 | radiation dose:3.0 | radiation source:gamma SUBJECT_SAMPLE_FACTORS - G315 days post to radiation:11 | radiation dose:3.0 | radiation source:gamma SUBJECT_SAMPLE_FACTORS - P311 days post to radiation:11 | radiation dose:3.0 | radiation source:proton SUBJECT_SAMPLE_FACTORS - P312 days post to radiation:11 | radiation dose:3.0 | radiation source:proton SUBJECT_SAMPLE_FACTORS - P313 days post to radiation:11 | radiation dose:3.0 | radiation source:proton SUBJECT_SAMPLE_FACTORS - P314 days post to radiation:11 | radiation dose:3.0 | radiation source:proton SUBJECT_SAMPLE_FACTORS - P315 days post to radiation:11 | radiation dose:3.0 | radiation source:proton #COLLECTION CO:COLLECTION_SUMMARY Hearts were dissected and snap-frozen at -80°C before NMR spectroscopy and CO:COLLECTION_SUMMARY morphology analysis. #TREATMENT TR:TREATMENT_SUMMARY A total of 27 seven-week-old C57BL/6 female mice randomly into 6 groups, and TR:TREATMENT_SUMMARY then whole body being exposed to either gamma or proton radiation. The TR:TREATMENT_SUMMARY individually housed animals were fed a standard diet and maintained in an TR:TREATMENT_SUMMARY environment controlled facility with an ambient temperature of 22-25°C and 45% TR:TREATMENT_SUMMARY relative humidity on 12 h light-dark cycle. Mice were exposed to whole body TR:TREATMENT_SUMMARY gamma radiation using a high-activity source (1250 keV 60Co) with the linear TR:TREATMENT_SUMMARY energy transfer (LET) associated with these fields in the range of 0.2-2 TR:TREATMENT_SUMMARY keV/μm. The mice were isolated in the corner of their polymer cages and placed TR:TREATMENT_SUMMARY a minimum of 100 cm from the collimated 6000 Ci (222 TBq) 60Co source, and then TR:TREATMENT_SUMMARY irradiation to the proposed dosage, respectively. The result of absorbed dose TR:TREATMENT_SUMMARY rate at a depth of approximately 600 mg/cm2 were ~0.83 Gy/min relative to TR:TREATMENT_SUMMARY tissue. After irradiation the isolation barrier was removed and mice were TR:TREATMENT_SUMMARY transferred to Animal Facility.Mice placed into aerated polystyrene boxes TR:TREATMENT_SUMMARY immediately before exposure to the entrance region of proton at the BNL TR:TREATMENT_SUMMARY Alternating Gradient Synchrotron (AGS) (1055 MeV/nucleon at the target, with a TR:TREATMENT_SUMMARY track-averaged LET =148.2 keV/μm at target). Doses of 0 and 3.0 Gy were TR:TREATMENT_SUMMARY delivered to a maximum of four mice at ~0.83 Gy/min in a single fraction. All TR:TREATMENT_SUMMARY procedures (i.e., placement in aerated boxes and subsequent tissue harvesting) TR:TREATMENT_SUMMARY for the control mice were identical to those of irradiated animals, except that TR:TREATMENT_SUMMARY they received no radiation. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY "Step 1: After randomization of the samples, every preweighted intact frozen SP:SAMPLEPREP_SUMMARY heart tissue was homogenized by a Tissue Tearsor (Model 985-370, BioSpec SP:SAMPLEPREP_SUMMARY Product, Inc.) in ice-cold after adding 4 ml MeOH and 0.85 ml deionized H2O per SP:SAMPLEPREP_SUMMARY gram of liver tissue, followed by vortexing the mixture and then 2 ml chloroform SP:SAMPLEPREP_SUMMARY per gram of tissue was added and vortexed again. This process took 6 minutes and SP:SAMPLEPREP_SUMMARY kept exactly the same for each sample. Step 2: 2 ml chloroform and 2 ml SP:SAMPLEPREP_SUMMARY deionized H2O per gram of tissue were added in the mixture then vortexed again, SP:SAMPLEPREP_SUMMARY followed by transferring the different layers into glass vials separately with SP:SAMPLEPREP_SUMMARY syringes after the mixture (in ice bath) centrifuged. Finally, the solvents of SP:SAMPLEPREP_SUMMARY hydrophilic metabolites were removed by employing lyophilizer and then stored at SP:SAMPLEPREP_SUMMARY -80oC freezer before NMR spectral measurements." #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE - CH:INSTRUMENT_NAME - CH:COLUMN_NAME - #ANALYSIS AN:ANALYSIS_TYPE NMR #NMR NM:INSTRUMENT_NAME Varian INOVA 600 NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:SPECTROMETER_FREQUENCY 600MHz #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 G011 G012 G013 G014 G311 G312 G313 G314 G315 P311 P312 P313 P314 P315 2-Aminobutyrate 0.443 0.411 0.437 0.407 0.354 0.291 0.299 0.334 0.333 0.405 0.412 0.393 0.400 0.367 0.355 0.345 0.373 0.341 0.335 0.350 0.357 0.343 0.359 0.390 0.353 0.329 0.344 3-Hydroxybutyrate 2.265 2.375 2.462 2.306 2.198 2.214 2.305 2.113 2.272 0.505 0.481 0.494 0.511 2.204 2.099 2.061 2.111 1.618 1.529 1.699 1.716 1.518 0.521 0.547 0.479 0.478 0.468 ADP 1.106 1.013 0.988 1.020 0.842 0.918 1.258 1.085 1.182 1.115 1.262 1.197 1.203 1.173 1.023 0.981 1.158 0.884 0.832 1.076 1.048 0.707 1.141 1.249 1.074 1.043 1.153 AMP 0.296 0.296 0.351 0.347 0.404 0.418 0.275 0.258 0.377 0.479 0.427 0.451 0.435 0.316 0.290 0.313 0.314 0.440 0.605 0.635 0.686 0.517 0.299 0.317 0.296 0.272 0.296 ATP 1.587 1.449 1.289 1.435 1.072 0.916 1.349 1.452 1.453 1.886 2.128 2.183 1.956 1.282 1.308 1.054 1.346 1.405 1.571 1.331 1.328 1.293 1.380 1.657 1.614 1.439 1.300 Adenosine 0.875 0.929 1.117 1.088 1.201 1.249 1.227 1.217 1.210 0.890 0.892 0.890 0.911 0.999 1.339 1.177 1.387 0.997 0.768 0.799 1.036 0.912 1.411 1.317 1.385 1.232 1.212 Alanine 6.425 6.537 5.973 6.594 6.959 6.382 6.819 7.009 6.401 7.534 8.390 7.389 6.546 6.782 6.544 6.365 6.763 7.174 7.621 6.940 7.423 6.881 7.216 6.637 6.374 6.300 7.182 Aspartate 5.187 5.042 6.037 5.630 6.947 5.736 5.309 5.542 7.151 3.560 3.946 3.872 4.098 5.436 5.606 5.757 5.482 4.041 4.147 4.318 3.918 3.966 3.856 3.884 3.790 3.285 3.256 Cholate 0.188 0.163 0.181 0.159 0.275 0.272 0.274 0.276 0.298 0.214 0.233 0.216 0.227 0.187 0.176 0.210 0.207 0.234 0.272 0.280 0.231 0.242 0.250 0.248 0.252 0.233 0.243 Choline 0.823 0.721 0.842 0.691 0.784 0.853 0.864 0.794 0.874 0.832 0.895 0.855 0.837 0.925 0.906 0.850 0.846 0.838 0.792 0.824 0.909 0.897 0.963 1.008 0.991 0.932 0.887 Creatine 27.212 26.233 27.834 27.074 26.532 25.555 25.599 26.468 26.722 27.274 27.309 28.300 27.426 28.949 28.434 29.058 28.807 28.032 28.751 28.077 30.669 29.942 31.095 31.121 26.734 26.365 28.066 Fumarate 0.100 0.106 0.147 0.140 0.094 0.074 0.101 0.072 0.085 0.110 0.137 0.125 0.113 0.159 0.122 0.130 0.159 0.112 0.158 0.146 0.121 0.111 0.116 0.083 0.093 0.102 0.086 Glucose-6-phosphate 1.722 1.691 1.503 1.686 0.941 1.192 1.406 1.389 1.422 2.288 2.644 2.424 2.341 1.185 1.135 1.161 1.170 1.303 1.163 1.007 1.245 1.003 1.434 1.519 1.315 1.471 1.278 Glutamate 8.436 7.188 8.446 7.764 8.071 9.041 8.273 9.068 6.473 7.500 8.693 8.253 7.559 8.011 8.774 8.751 8.465 7.826 8.844 8.995 8.592 8.191 10.275 11.366 10.376 11.002 10.134 Glutamine 16.244 15.520 15.276 16.018 13.131 14.062 14.289 12.919 14.125 14.576 16.516 15.265 15.273 14.238 13.932 15.026 13.974 15.007 14.005 13.395 13.615 14.715 13.064 13.287 11.942 12.642 12.447 IMP 0.229 0.197 0.228 0.194 0.251 0.203 0.232 0.251 0.209 0.246 0.304 0.314 0.269 0.228 0.219 0.227 0.243 0.262 0.219 0.201 0.204 0.263 0.275 0.276 0.270 0.245 0.240 Inosine 0.782 0.696 0.821 0.738 1.145 1.060 0.808 0.800 0.917 0.713 0.606 0.624 0.762 1.001 0.908 0.995 0.956 0.577 0.513 0.609 0.538 0.525 0.870 0.861 1.043 0.879 0.990 Isoleucine 0.313 0.264 0.305 0.264 0.311 0.262 0.320 0.268 0.259 0.192 0.199 0.177 0.184 0.207 0.212 0.177 0.189 0.225 0.218 0.215 0.210 0.239 0.153 0.153 0.137 0.131 0.134 Lactate 22.256 20.793 19.722 20.460 15.233 16.252 18.891 15.953 18.854 29.960 32.543 33.349 30.486 24.469 24.431 20.084 22.720 20.154 22.627 17.921 18.608 21.588 19.092 18.501 19.506 22.621 21.329 NAD+ 1.306 1.189 1.197 1.321 1.243 1.454 1.213 1.382 1.503 1.538 1.538 1.481 1.511 1.218 1.130 1.270 1.239 1.360 1.386 1.136 1.158 1.259 1.151 0.987 1.051 1.097 1.186 NADH 0.240 0.247 0.238 0.224 0.174 0.176 0.154 0.159 0.179 0.232 0.230 0.279 0.262 0.160 0.206 0.197 0.159 0.177 0.162 0.164 0.176 0.161 0.191 0.171 0.195 0.196 0.170 NADP+ 0.099 0.099 0.101 0.104 0.126 0.111 0.121 0.118 0.109 0.112 0.115 0.110 0.108 0.128 0.111 0.117 0.145 0.137 0.140 0.135 0.132 0.141 0.132 0.113 0.132 0.116 0.111 Nicotinurate 0.158 0.129 0.146 0.135 0.097 0.089 0.098 0.082 0.095 0.216 0.193 0.209 0.203 0.189 0.203 0.196 0.195 0.187 0.179 0.185 0.155 0.191 0.161 0.155 0.144 0.173 0.164 O-Acetylcarnitine 0.777 0.654 0.730 0.634 0.831 0.844 0.829 0.913 0.928 0.741 0.861 0.770 0.797 0.840 0.852 0.810 0.799 0.848 0.772 0.772 0.859 0.767 0.864 0.927 0.837 0.901 0.853 Phenylalanine 0.162 0.152 0.168 0.142 0.175 0.160 0.175 0.159 0.152 0.197 0.201 0.185 0.188 0.154 0.138 0.147 0.141 0.187 0.185 0.172 0.161 0.161 0.145 0.161 0.142 0.159 0.160 Pyridoxine 0.058 0.054 0.066 0.063 0.034 0.033 0.026 0.025 0.029 0.045 0.050 0.038 0.035 0.062 0.060 0.058 0.064 0.037 0.039 0.040 0.042 0.033 0.073 0.073 0.066 0.065 0.072 Succinate 4.902 5.429 4.917 5.327 5.019 5.192 5.365 4.502 4.465 4.932 5.100 4.743 3.596 4.776 4.593 4.500 4.762 4.918 5.254 4.820 5.248 5.461 3.816 3.835 3.497 3.447 3.880 Taurine 74.085 70.890 73.878 72.899 75.342 72.811 75.421 72.382 74.259 74.970 74.499 72.338 75.207 79.439 76.103 77.213 78.171 80.330 80.310 81.256 82.785 82.109 78.698 72.502 72.422 76.452 78.558 Threonine 1.589 1.468 1.696 1.444 2.547 2.209 2.267 2.593 2.417 2.550 2.770 2.631 2.528 1.899 1.800 1.888 1.802 2.409 2.559 2.439 2.395 2.459 2.122 2.022 2.019 2.062 2.016 Thymidine 0.044 0.042 0.042 0.041 0.031 0.030 0.050 0.044 0.050 0.046 0.054 0.041 0.050 0.048 0.054 0.049 0.049 0.049 0.050 0.050 0.049 0.043 0.052 0.046 0.043 0.046 0.044 Tryptophan 0.090 0.089 0.103 0.100 0.108 0.101 0.124 0.124 0.111 0.117 0.105 0.113 0.120 0.091 0.107 0.091 0.091 0.118 0.118 0.089 0.083 0.103 0.085 0.091 0.095 0.086 0.091 Tyrosine 0.177 0.186 0.189 0.180 0.228 0.220 0.241 0.224 0.207 0.293 0.287 0.243 0.253 0.172 0.161 0.169 0.162 0.225 0.219 0.226 0.221 0.225 0.268 0.294 0.260 0.303 0.240 UDP-N-Acetylglucosamine 0.081 0.083 0.080 0.080 0.076 0.086 0.090 0.076 0.079 0.084 0.089 0.072 0.070 0.113 0.112 0.109 0.114 0.087 0.072 0.069 0.070 0.065 0.086 0.090 0.071 0.082 0.089 UDP-glucose 0.216 0.191 0.205 0.206 0.212 0.204 0.194 0.227 0.219 0.248 0.257 0.253 0.249 0.212 0.208 0.208 0.216 0.247 0.238 0.243 0.247 0.233 0.253 0.259 0.224 0.248 0.220 Uracil 0.053 0.044 0.051 0.043 0.058 0.053 0.058 0.043 0.043 0.039 0.041 0.031 0.033 0.044 0.036 0.041 0.039 0.043 0.040 0.046 0.044 0.045 0.039 0.031 0.028 0.038 0.031 Uridine 0.197 0.184 0.194 0.191 0.206 0.175 0.155 0.150 0.206 0.174 0.176 0.189 0.178 0.212 0.215 0.222 0.226 0.193 0.186 0.201 0.208 0.202 0.212 0.219 0.207 0.192 0.216 Valine 0.599 0.495 0.594 0.497 0.609 0.491 0.614 0.537 0.494 0.454 0.496 0.466 0.451 0.389 0.381 0.347 0.368 0.434 0.397 0.431 0.397 0.431 0.408 0.403 0.309 0.290 0.304 τ-Methylhistidine 0.295 0.278 0.298 0.268 0.261 0.317 0.304 0.269 0.263 0.299 0.310 0.253 0.286 0.274 0.237 0.273 0.246 0.324 0.321 0.262 0.255 0.271 0.349 0.343 0.285 0.307 0.284 NMR_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name PubChem ID KEGG ID 2-Aminobutyrate 80283 C02356 3-Hydroxybutyrate 441 C01089 ADP 6022 C00008 AMP 6083 C00020 ATP 5957 C00002 Adenosine 60961 C00212 Alanine 5950 C00041 Aspartate 5960 C00049 Cholate 221493 C00695 Choline 305 C00114 Creatine 586 C00300 Fumarate 284 C00058 Glucose-6-phosphate 5958 C00092 Glutamate 33032 C00025 Glutamine 5961 C00064 IMP 8582 C00130 Inosine 6021 C00294 Isoleucine 6306 C00407 Lactate 107689 C00186 NAD+ 5893 C00003 NADH 439153 C00004 NADP+ 5886 C00006 Nicotinurate 68499 C05380 O-Acetylcarnitine 18230 C02571 Phenylalanine 6140 C00079 Pyridoxine 1054 C00314 Succinate 1110 C00042 Taurine 1123 C00245 Threonine 6288 C00188 Thymidine 5789 C00214 Tryptophan 6305 C00078 Tyrosine 6057 C00082 UDP-N-Acetylglucosamine 445675 C00043 UDP-glucose 8629 C00029 Uracil 1174 C00106 Uridine 6029 C00299 Valine 6287 C00183 τ-Methylhistidine 92105 C01152 METABOLITES_END #END