#METABOLOMICS WORKBENCH jburgess_20160305_135129 STUDY_ID:ST000361 ANALYSIS_ID:AN000593 PROJECT_ID:PR000287 VERSION 1 CREATED_ON March 11, 2016, 12:19 pm #PROJECT PR:PROJECT_TITLE Characterization and Plasticity of the Metabolome in Peripheral Cells in Bipolar PR:PROJECT_TITLE I Disorder PR:PROJECT_TYPE Broad Spectrum LCMS PR:PROJECT_SUMMARY Bipolar disorder is a serious condition characterized by severe and debilitating PR:PROJECT_SUMMARY elevations and depressions in mood and energy (1). Bipolar I disorder (BPI) is a PR:PROJECT_SUMMARY particularly severe phenotype which is generally defined as having periods of PR:PROJECT_SUMMARY abnormal moods ranging from “highs,” (mania or hypomania), to “lows” PR:PROJECT_SUMMARY (depression), alternating with periods of relatively normal moods. Key problems PR:PROJECT_SUMMARY in treating bipolar disorder relate to the fact that the pathophysiology of the PR:PROJECT_SUMMARY disorder remains elusive; as a result, there are currently no laboratory tests PR:PROJECT_SUMMARY for diagnosis, to aid treatment decisions, or to predict treatment responses. PR:PROJECT_SUMMARY Recent advances in the study of bipolar disorder increasingly implicate PR:PROJECT_SUMMARY mitochondrial and bioenergetic dysfunction as a common feature of the PR:PROJECT_SUMMARY pathophysiology of the disorder (2). Shifts in metabolism from mitochondrial PR:PROJECT_SUMMARY oxidative phosphorylation toward glycolysis have been observed in the brains of PR:PROJECT_SUMMARY bipolar patients experiencing depressive mood states (3). Increased oxidative PR:PROJECT_SUMMARY stress has also been shown in postmortem brain from bipolar patients (4), and in PR:PROJECT_SUMMARY cultured peripheral fibroblasts from patients with major depression (5). PR:PROJECT_SUMMARY However, little is known about the inherent metabolic state of peripheral cells PR:PROJECT_SUMMARY during bipolar disorder, which may reflect the overall metabolic profile of the PR:PROJECT_SUMMARY individual and may also impact the supply of nutrients to the brain. We propose PR:PROJECT_SUMMARY to characterize the baseline metabolomic profiles of peripheral cells in PR:PROJECT_SUMMARY cultured fibroblasts from patients with BPI and matched non-affected controls. PR:PROJECT_SUMMARY Patients with Bipolar I Disorder (BPI) and matched non-affected controls were PR:PROJECT_SUMMARY recruited. Males and females from all races and ethnicities between the ages of PR:PROJECT_SUMMARY 19-65 participated in the research. Skin biopsies were obtained and fibroblasts PR:PROJECT_SUMMARY were isolated from each of these biopsies. A total of 1 x 〖10〗^7 fibroblasts PR:PROJECT_SUMMARY cells were used for metabolomics. Cell pellets were flash frozen in liquid PR:PROJECT_SUMMARY nitrogen and shipped on dry ice to the NIH Eastern Regional Comprehensive PR:PROJECT_SUMMARY Metabolomic Resource Core at RTI International in North Carolina. Metabolomics PR:PROJECT_SUMMARY will be determined using a broad spectrum metabolomics protocol by RTI. PR:INSTITUTE University of Alabama, Birmingham PR:DEPARTMENT School of Medicine PR:LABORATORY Department of Pathology PR:LAST_NAME Landar PR:FIRST_NAME Aimee PR:ADDRESS BMR2 506 901 9th St South, Birmingham AL 35205 PR:EMAIL landar@uab.edu PR:PHONE 205-975-9507 PR:FUNDING_SOURCE NIH, Common Fund, Pilot and Feasibility #STUDY ST:STUDY_TITLE Characterization and Plasticity of the Metabolome in Peripheral Cells in Bipolar ST:STUDY_TITLE I Disorder ST:STUDY_TYPE Broad Spectrum LCMS ST:STUDY_SUMMARY Patients with Bipolar I Disorder (BPI) and matched non-affected controls were ST:STUDY_SUMMARY recruited. Males and females from all races and ethnicities between the ages of ST:STUDY_SUMMARY 19-65 participated in the research. Skin biopsies were obtained and fibroblasts ST:STUDY_SUMMARY were isolated from each of these biopsies. A total of 1 x 〖10〗^7 fibroblasts ST:STUDY_SUMMARY cells were used for metabolomics. Cell pellets were flash frozen in liquid ST:STUDY_SUMMARY nitrogen and shipped on dry ice to the NIH Eastern Regional Comprehensive ST:STUDY_SUMMARY Metabolomic Resource Core at RTI International in North Carolina. Metabolomics ST:STUDY_SUMMARY will be determined using a broad spectrum metabolomics protocol by RTI ST:INSTITUTE RTI ST:DEPARTMENT Discovery Sciences ST:LABORATORY System and Translational Sciences ST:LAST_NAME Sumner ST:FIRST_NAME Susan ST:ADDRESS 3040 Cornwallis Rd, RTP,NC ST:EMAIL ssumner@rti.org ST:PHONE 919-541-7479 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 18 ST:NUM_MALES 7 ST:NUM_FEMALES 11 #SUBJECT SU:SUBJECT_TYPE human SU:SUBJECT_SPECIES Homo sapiens SU:AGE_OR_AGE_RANGE 27-61 years SU:GENDER male/female SU:HUMAN_RACE caucasian, hispanic, african american #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS 131 A_0131 Group:Bipolar Age=51; Sex=M; Race=Hispanic SUBJECT_SAMPLE_FACTORS 318 B_0318 Group:Control Age=61; Sex=F; Race=Caucasian SUBJECT_SAMPLE_FACTORS 255 B_0255 Group:Control Age=55; Sex=M; Race=African American SUBJECT_SAMPLE_FACTORS 110 A_0110 Group:Bipolar Age=42; Sex=F; Race=African-American SUBJECT_SAMPLE_FACTORS 191 A_0191 Group:Bipolar Age=38; Sex=M; Race=White SUBJECT_SAMPLE_FACTORS 61 B_0061 Group:Control Age=41; Sex=F; Race=African American SUBJECT_SAMPLE_FACTORS 245 A_0245 Group:Bipolar Age=38; Sex=F; Race=Hispanic SUBJECT_SAMPLE_FACTORS 183 B_0183 Group:Control Age=27; Sex=F; Race=White SUBJECT_SAMPLE_FACTORS 302 A_0302 Group:Bipolar Age=50; Sex=M; Race=Caucasian SUBJECT_SAMPLE_FACTORS 203 B_0203 Group:Control Age=28; Sex=M; Race=Caucasian SUBJECT_SAMPLE_FACTORS 182 B_0182 Group:Control Age=50; Sex=F; Race=White SUBJECT_SAMPLE_FACTORS 247 B_0247 Group:Control Age=49; Sex=M; Race=Caucasian SUBJECT_SAMPLE_FACTORS 293 A_0293 Group:Bipolar Age=39; Sex=F; Race=African American SUBJECT_SAMPLE_FACTORS 276 A_0276 Group:Bipolar Age=40; Sex=F; Race=African-American SUBJECT_SAMPLE_FACTORS 107 B_0107 Group:Control Age=39; Sex=M; Race=Caucasian SUBJECT_SAMPLE_FACTORS 151 B_0151 Group:Control Age=55; Sex=F; Race=White SUBJECT_SAMPLE_FACTORS 128 B_0128 Group:Control Age=55; Sex=F; Race=African-American SUBJECT_SAMPLE_FACTORS 312 B_0312 Group:Control Age=43; Sex=F; Race=Hispanic SUBJECT_SAMPLE_FACTORS - Total_Pool_01 Group:- Age=-; Sex=-; Race=- SUBJECT_SAMPLE_FACTORS - Total_Pool_02 Group:- Age=-; Sex=-; Race=- SUBJECT_SAMPLE_FACTORS - Total_Pool_03 Group:- Age=-; Sex=-; Race=- SUBJECT_SAMPLE_FACTORS - Total_Pool_04 Group:- Age=-; Sex=-; Race=- SUBJECT_SAMPLE_FACTORS - Total_Pool_05 Group:- Age=-; Sex=-; Race=- #COLLECTION CO:COLLECTION_SUMMARY For extraction, 500 µL of an ice-cold solution of 0.005 mg/mL Tryptohan-d5 in CO:COLLECTION_SUMMARY 90:10 Methanol:Chloroform (v/v) was added to the tubes containing the thawed CO:COLLECTION_SUMMARY human dermal fibroblasts cell pellets. MagNA Lyser ceramic beads were added to CO:COLLECTION_SUMMARY the tubes, and a MagNA Lyser was used to beat the samples for two thirty seconds CO:COLLECTION_SUMMARY pulses at 2,000 rpm, placing the samples on cold block for 5 minutes in between CO:COLLECTION_SUMMARY pulses. Sonication for five minutes was performed to the beaten cell samples, CO:COLLECTION_SUMMARY followed by centrifugation at room temperature and at 16,000 rcf for 4 minutes. CO:COLLECTION_SUMMARY A 300 µL aliquot of each experimental cell sample homogenized supernatant was CO:COLLECTION_SUMMARY transferred to new pre-labeled 2.0 mL LoBind Eppendorf tubes and stored at -80 CO:COLLECTION_SUMMARY °C for one hour. CO:SAMPLE_TYPE fibroblasts #TREATMENT TR:TREATMENT_SUMMARY NA #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Whole study pooled QC samples were created by combining 175 µL aliquot from SP:SAMPLEPREP_SUMMARY each of the study samples into a 20 mL scintillation vial. The QC pooled sample SP:SAMPLEPREP_SUMMARY was then vortexed for 30 seconds on an analog vortex mixer. Aliquots, 300 µL, SP:SAMPLEPREP_SUMMARY were transferred to five new pre-labeled 2.0 mL LoBind Eppendorf tubes and SP:SAMPLEPREP_SUMMARY stored at -80 °C for one hour. Next, the supernatants of the study and whole SP:SAMPLEPREP_SUMMARY study pooled QC samples, generated above, were dried overnight using a SP:SAMPLEPREP_SUMMARY lyophilizer. The residue was reconstituted in 75 µL of reconstitution buffer ( SP:SAMPLEPREP_SUMMARY 95:5 Water:Methanol, v/v) and mixed on a multiple tube vortexer for five minutes SP:SAMPLEPREP_SUMMARY at 5,000 rpm. Then, the samples were centrifuged at room temperature at 16,000 SP:SAMPLEPREP_SUMMARY rcf for four minutes, and the supernatants were transferred to clean pre-labeled SP:SAMPLEPREP_SUMMARY autosampler vials for data acquisition. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY UPLC-MS spectra were collected for all samples. UPLC was performed on a Waters CH:CHROMATOGRAPHY_SUMMARY Acquity UPLC with an Acquity HSS T3 column (2.1x 100mm x 1.8 µm) at 50 °C CH:CHROMATOGRAPHY_SUMMARY using the reversed-phase separation. Water with 0.1% formic acid (mobile phase CH:CHROMATOGRAPHY_SUMMARY A) and methanol with 0.1% formic acid (mobile phase B) were used as mobile CH:CHROMATOGRAPHY_SUMMARY phases and the metabolites were chromatographically separated using a gradient CH:CHROMATOGRAPHY_SUMMARY separation: (see the 3. Sheridan-Mice-Microglia Cells_RP-Metadata and Analytical CH:CHROMATOGRAPHY_SUMMARY Metadata.xlsx file for the flow gradient). Mass spectroscopy analysis was CH:CHROMATOGRAPHY_SUMMARY performed using a Synapt G2 Si ESI-Q-TOF using a 10 µL injection volume. CH:CHROMATOGRAPHY_SUMMARY UPLC-MS data were collected over 70-1000 m/z in both positive and negative CH:CHROMATOGRAPHY_SUMMARY modes. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Acquity HSS T3 CH:FLOW_RATE .4ml/min CH:METHODS_FILENAME RTI-RCMRC-RP CH:SOLVENT_A Water with 0.1% formic acid CH:SOLVENT_B Methanol with 0.1% formic acid CH:INJECTION_TEMPERATURE 8 C #ANALYSIS AN:ANALYSIS_TYPE MS AN:OPERATOR_NAME Suraj Dhungana AN:DETECTOR_TYPE TOF AN:SOFTWARE_VERSION Mass Lynx 4.1 AN:ACQUISITION_DATE 8/05/15 AN:ANALYSIS_PROTOCOL_FILE RTI-RCMRC-RP-NEG AN:DATA_FORMAT Continuum #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Waters Synapt G2 Si QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS intensity MS_METABOLITE_DATA_START Samples Total_Pool_01 Total_Pool_02 Total_Pool_03 Total_Pool_04 Total_Pool_05 A_0110 A_0131 A_0191 A_0245 A_0276 A_0293 A_0302 B_0061 B_0107 B_0128 B_0151 B_0182 B_0183 B_0203 B_0247 B_0255 B_0312 B_0318 Factors Group:- Group:- Group:- Group:- Group:- Group:Bipolar Group:Bipolar Group:Bipolar Group:Bipolar 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4101.566929 1601.973009 3390.409978 3779.741371 5037.362869 4608.649745 3962.79459 3084.03457 2545.349659 2617.360904 2517.5345 2894.657684 4402.495822 1308.799257 6173.964707 1988.212384 2990.289745 3248.288282 3250.788761 4423.477192 4910.091754 5676.970849 15.52_963.5084m/z 468.6451708 2033.356815 3415.59938 2885.990628 4555.466965 1215.54559 2469.063986 4207.809577 1042.3338 1604.56415 2157.31501 4405.095979 59.86703928 1410.40719 4912.396274 2731.722303 2574.635462 4255.759798 3742.280934 880.252134 3408.397588 6448.046819 2717.58745 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name m/z Retention time (min) 14.02_649.1216m/z 649.1216199 14.01731667 15.70_761.1578m/z 761.1578085 15.70231667 0.74_817.7076m/z 817.7076184 0.736633333 12.37_377.0592m/z 377.0592229 12.3734 2.93_641.1535m/z 641.1534544 2.926066667 15.35_300.1561m/z 300.1561003 15.3542 13.63_416.3689m/z 416.3689296 13.62825 15.92_569.1619m/z 569.1618736 15.9202 0.74_396.1128m/z 396.1128228 0.736633333 2.56_958.9936m/z 958.9935907 2.557466667 0.72_572.1416m/z 572.1416201 0.716166667 3.83_624.7055m/z 624.705478 3.8332 1.30_92.0254m/z 92.02536524 1.302633333 0.68_253.0448m/z 253.044839 0.675216667 0.65_351.9708m/z 351.9707948 0.654733333 4.26_796.3433m/z 796.3433237 4.263216667 10.35_143.0492m/z 143.0491658 10.3478 0.72_818.2124m/z 818.2124462 0.716166667 13.94_241.1789m/z 241.1789385 13.93541667 0.70_368.0525m/z 368.0525452 0.695683333 0.68_424.0026m/z 424.0025903 0.675216667 13.79_176.0706m/z 176.0705606 13.79206667 3.08_325.1069m/z 325.1068619 3.082666667 12.94_375.0190m/z 375.0190274 12.9394 1.81_110.0245m/z 110.0244758 1.807183333 0.74_806.7200m/z 806.720039 0.736633333 3.25_878.3269m/z 878.3268743 3.246483333 0.68_277.0489m/z 277.0488932 0.675216667 4.35_624.2417m/z 624.2416639 4.345133333 0.65_500.0043n 499.031261 0.654733333 2.64_968.1329m/z 968.1329388 2.639383333 0.79_166.0174m/z 166.0173768 0.7907 7.49_93.0343m/z 93.03432779 7.490016667 1.95_891.6261m/z 891.6260929 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3.87_432.0165m/z 432.0164947 3.87415 1.60_196.0609m/z 196.0609366 1.602416667 10.31_320.0601m/z 320.0600535 10.30685 2.64_840.2269m/z 840.2268908 2.639383333 1.43_93.0456m/z 93.04562762 1.4255 1.32_286.0552m/z 286.0552301 1.3231 1.75_611.2419m/z 611.2418894 1.74575 15.92_847.1286m/z 847.1285648 15.9202 1.32_545.9747m/z 545.9747179 1.3231 15.74_421.2241m/z 421.2241354 15.74326667 0.63_576.9564m/z 576.9563639 0.63425 14.02_833.3355m/z 833.3355011 14.01731667 1.77_487.1302m/z 487.130248 1.766233333 0.72_628.5572m/z 628.557171 0.716166667 1.77_577.0975m/z 577.0975373 1.766233333 11.48_811.3384m/z 811.3383926 11.47978333 0.70_366.0574m/z 366.0574375 0.695683333 15.92_371.2184m/z 371.2183913 15.9202 9.50_673.0249m/z 673.024855 9.495133333 0.72_575.6322m/z 575.6322224 0.716166667 1.60_342.0450m/z 342.0450177 1.602416667 1.60_172.0606m/z 172.0606407 1.602416667 2.64_909.1709m/z 909.1708516 2.639383333 1.79_140.0348m/z 140.0348291 1.786716667 15.92_779.1412m/z 779.1411726 15.9202 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8.62_333.1197m/z 333.1197352 8.622 13.14_227.1973m/z 227.1973465 13.14416667 7.99_411.1476m/z 411.147647 7.994566667 8.26_486.2515m/z 486.2515019 8.260783333 7.99_368.1476n 367.136555 7.994566667 8.62_413.1523m/z 413.1522639 8.622 12.54_855.3945m/z 855.3945458 12.53721667 2.64_498.0005m/z 498.0005387 2.639383333 13.25_315.1369m/z 315.1368571 13.24655 1.02_259.0064m/z 259.0064274 1.01595 0.70_486.0172m/z 486.0172259 0.695683333 2.66_793.0296m/z 793.0295919 2.65985 15.07_149.0063m/z 149.0062874 15.06751667 15.19_802.5493m/z 802.5493219 15.19038333 15.52_963.5084m/z 963.5084244 15.51801667Compound m/z Retention time (min) 15.52_963.5084m/z 963.5084244 15.51801667 METABOLITES_END #END