{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002721","ANALYSIS_ID":"AN004411","VERSION":"1","CREATED_ON":"07-31-2023"},

"PROJECT":{"PROJECT_TITLE":"Repeated Exposure to Wood Smoke Alters Pulmonary Gene and Metabolic Profiles","PROJECT_SUMMARY":"This project aims to evaluate the association of eucalyptus wood smoke exposure with perturbations to lung metabolism in a pre-clinical rat model. This cross-sectional study of male Long-Evans rats were exposed whole body to eucalyptus smoke for 4 consecutive days on week 1, followed by a 3-day break, and 3 subsequent days of smoke exposure on week 2. The smoke exposures were set to a target CO of 10ppm for the low smoke group and 20ppm for the high smoke group. Animal were sacrificed and lungs were extracted for metabolomics analysis. Exposures to eucalyptus smoke and biological perturbations to lung metabolism will be associated with known metabolic dysregulation associated with smoke exposures.","INSTITUTE":"Emory University","DEPARTMENT":"School of Medicine","LABORATORY":"Clinical Biomarkers Laboratory","LAST_NAME":"Tran","FIRST_NAME":"ViLinh","ADDRESS":"615 Michael St, Suite 225, Atlanta, GA 30322, USA","EMAIL":"vtran6@emory.edu","PHONE":"404-727-5091","DOI":"http://dx.doi.org/10.21228/M8NB07","CONTRIBUTORS":"Matthew R. Smith, PdD, Collette M. Miller, PhD, Kymberly M. Gowdy, PhD, Dean P. Jones, PhD"},

"STUDY":{"STUDY_TITLE":"Repeated Exposure to Wood Smoke Alters Pulmonary Gene and Metabolic Profiles","STUDY_SUMMARY":"This project aims to evaluate the association of eucalyptus wood smoke exposure with perturbations to lung metabolism in a pre-clinical rat model. This cross-sectional study of male Long-Evans rats were exposed whole body to eucalyptus smoke for 4 consecutive days on week 1, followed by a 3-day break, and 3 subsequent days of smoke exposure on week 2. The smoke exposures were set to a target CO of 10ppm for the low smoke group and 20ppm for the high smoke group. Animal were sacrificed and lungs were extracted for metabolomics analysis. Exposures to eucalyptus smoke and biological perturbations to lung metabolism will be associated with known metabolic dysregulation associated with smoke exposures.","INSTITUTE":"Emory University","DEPARTMENT":"School of Medicine","LABORATORY":"Clinical Biomarkers Laboratory","LAST_NAME":"Tran","FIRST_NAME":"ViLinh","ADDRESS":"615 Michael St, Suite 225, Atlanta, GA 30322, USA","EMAIL":"vtran6@emory.edu","PHONE":"4047275091","SUBMIT_DATE":"2023-05-12"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Rattus norvegicus","TAXONOMY_ID":"10116","AGE_OR_AGE_RANGE":"12 weeks","GENDER":"Male"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"41_1",
"Factors":{"Class":"15"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_049 VT_210113_M63_051 VT_210113_M63_053"}
},
{
"Subject ID":"-",
"Sample ID":"42_1",
"Factors":{"Class":"15"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_031 VT_210113_M63_033 VT_210113_M63_035"}
},
{
"Subject ID":"-",
"Sample ID":"43_1",
"Factors":{"Class":"15"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_079 VT_210113_M63_081 VT_210113_M63_083"}
},
{
"Subject ID":"-",
"Sample ID":"44_1",
"Factors":{"Class":"15"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_115 VT_210113_M63_117 VT_210113_M63_119"}
},
{
"Subject ID":"-",
"Sample ID":"45_1",
"Factors":{"Class":"15"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_067 VT_210113_M63_069 VT_210113_M63_071"}
},
{
"Subject ID":"-",
"Sample ID":"46_1",
"Factors":{"Class":"15"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_085 VT_210113_M63_087 VT_210113_M63_089"}
},
{
"Subject ID":"-",
"Sample ID":"47_1",
"Factors":{"Class":"15"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_025 VT_210113_M63_027 VT_210113_M63_029"}
},
{
"Subject ID":"-",
"Sample ID":"48_1",
"Factors":{"Class":"15"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_127 VT_210113_M63_129 VT_210113_M63_131"}
},
{
"Subject ID":"-",
"Sample ID":"25_1",
"Factors":{"Class":"7.5"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_091 VT_210113_M63_093 VT_210113_M63_095"}
},
{
"Subject ID":"-",
"Sample ID":"26_1",
"Factors":{"Class":"7.5"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_097 VT_210113_M63_099 VT_210113_M63_101"}
},
{
"Subject ID":"-",
"Sample ID":"27_1",
"Factors":{"Class":"7.5"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_163 VT_210113_M63_165 VT_210113_M63_167"}
},
{
"Subject ID":"-",
"Sample ID":"28_1",
"Factors":{"Class":"7.5"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_133 VT_210113_M63_135 VT_210113_M63_137"}
},
{
"Subject ID":"-",
"Sample ID":"29_1",
"Factors":{"Class":"7.5"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_037 VT_210113_M63_039 VT_210113_M63_041"}
},
{
"Subject ID":"-",
"Sample ID":"30_1",
"Factors":{"Class":"7.5"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_043 VT_210113_M63_045 VT_210113_M63_047"}
},
{
"Subject ID":"-",
"Sample ID":"31_1",
"Factors":{"Class":"7.5"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_019 VT_210113_M63_021 VT_210113_M63_023"}
},
{
"Subject ID":"-",
"Sample ID":"32_1",
"Factors":{"Class":"7.5"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_103 VT_210113_M63_105 VT_210113_M63_107"}
},
{
"Subject ID":"-",
"Sample ID":"10_1",
"Factors":{"Class":"Air"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_109 VT_210113_M63_111 VT_210113_M63_113"}
},
{
"Subject ID":"-",
"Sample ID":"11_1",
"Factors":{"Class":"Air"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_169 VT_210113_M63_171 VT_210113_M63_173"}
},
{
"Subject ID":"-",
"Sample ID":"12_1",
"Factors":{"Class":"Air"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_061 VT_210113_M63_063 VT_210113_M63_065"}
},
{
"Subject ID":"-",
"Sample ID":"13_1",
"Factors":{"Class":"Air"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_073 VT_210113_M63_075 VT_210113_M63_077"}
},
{
"Subject ID":"-",
"Sample ID":"14_1",
"Factors":{"Class":"Air"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_151 VT_210113_M63_153 VT_210113_M63_155"}
},
{
"Subject ID":"-",
"Sample ID":"15_1",
"Factors":{"Class":"Air"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_055 VT_210113_M63_057 VT_210113_M63_059"}
},
{
"Subject ID":"-",
"Sample ID":"16_1",
"Factors":{"Class":"Air"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_121 VT_210113_M63_123 VT_210113_M63_125"}
},
{
"Subject ID":"-",
"Sample ID":"9_1",
"Factors":{"Class":"Air"},
"Additional sample data":{"Sample_Data:Sample type":"Lung Tissue","Sample matrix":"Lung Tissue","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_157 VT_210113_M63_159 VT_210113_M63_161"}
},
{
"Subject ID":"-",
"Sample ID":"nist1_1",
"Factors":{"Class":"NA"},
"Additional sample data":{"Sample_Data:Sample type":"Quality Control","Sample matrix":"plasma","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_001 VT_210113_M63_003 VT_210113_M63_005"}
},
{
"Subject ID":"-",
"Sample ID":"nist2_1",
"Factors":{"Class":"NA"},
"Additional sample data":{"Sample_Data:Sample type":"Quality Control","Sample matrix":"plasma","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_283 VT_210113_M63_285 VT_210113_M63_287"}
},
{
"Subject ID":"-",
"Sample ID":"q3June2014_1a_1",
"Factors":{"Class":"NA"},
"Additional sample data":{"Sample_Data:Sample type":"Quality Control","Sample matrix":"plasma","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_007 VT_210113_M63_009 VT_210113_M63_011"}
},
{
"Subject ID":"-",
"Sample ID":"q3June2014_1b_1",
"Factors":{"Class":"NA"},
"Additional sample data":{"Sample_Data:Sample type":"Quality Control","Sample matrix":"plasma","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_013 VT_210113_M63_015 VT_210113_M63_017"}
},
{
"Subject ID":"-",
"Sample ID":"q3June2014_1c_1",
"Factors":{"Class":"NA"},
"Additional sample data":{"Sample_Data:Sample type":"Quality Control","Sample matrix":"plasma","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_139 VT_210113_M63_141 VT_210113_M63_143"}
},
{
"Subject ID":"-",
"Sample ID":"q3June2014_1d_1",
"Factors":{"Class":"NA"},
"Additional sample data":{"Sample_Data:Sample type":"Quality Control","Sample matrix":"plasma","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_145 VT_210113_M63_147 VT_210113_M63_149"}
},
{
"Subject ID":"-",
"Sample ID":"q3June2014_1e_1",
"Factors":{"Class":"NA"},
"Additional sample data":{"Sample_Data:Sample type":"Quality Control","Sample matrix":"plasma","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_271 VT_210113_M63_273 VT_210113_M63_275"}
},
{
"Subject ID":"-",
"Sample ID":"q3June2014_1f_1",
"Factors":{"Class":"NA"},
"Additional sample data":{"Sample_Data:Sample type":"Quality Control","Sample matrix":"plasma","Date analyzed":"1/14/21","RAW_FILE_NAME":"VT_210113_M63_277 VT_210113_M63_279 VT_210113_M63_281"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"The intact left lung was removed, and a longitudinal section was made to produce tissue samples for downstream metabolomic assessments. Lung section were frozen in liquid nitrogen and stored at -80°C until use. The samples were shipped on dry ice from the clinical centers to the Clinical Biomarkers Laboratory at Emory University.","SAMPLE_TYPE":"Lung","STORAGE_CONDITIONS":"-80℃"},

"TREATMENT":{"TREATMENT_SUMMARY":"Samples were received frozen in aliquots of lung tissue. Prior to analysis, samples were thawed and prepared for HRM analysis using the standard protocols described in the Sample Preparation section. Eucalyptus was selected as the biomass of interest due to its prevalence and likeness of chaparral ecosystems present in Southern California and Australia, areas that have experienced growing severity of wildland fires in recent years. To mimic the deployment of common fire line shifts, rats were exposed to whole-body eucalyptus smoke over the course of two weeks. Exposure occurred for 4 consecutive days on week 1, followed by a 3-day break, and 3 subsequent days of smoke exposure on week 2. An n = 24 rats were randomized by weight into three groups based on body weight (n = 8 / group): filtered air, low smoke, and high smoke concentrations. The smoke exposures were set to a target CO of 10 ppm for the low smoke group and 20 ppm for the high smoke group. These concentrations bracket the National Wildfire Coordinating Group’s recommended exposure limit of 15 ppm, which is lower than the 35 ppm limit set by the National Institute for Occupational Safety and Health. Further, these concentrations were selected to avoid respiratory suppression and the potential for anti-inflammatory signaling from high concentrations of CO in the rodent. Eucalyptus smoke was generated in a quartz-tube furnace system set to a smoldering condition (~ 500°C) with a primary air flow at ~ 2 L/min. Additional details on the design of the tube furnace system can be found in Kim, et al. Generated smoke was diluted using a secondary air flow to maintain our target CO concentrations and was subsequently delivered to a whole-body inhalation chamber. CO and CO2 were continuously monitored using a nondispersive infrared analyzer (602 CO/CO2; CAI, Inc.) and nitrogen oxides were monitored using a chemiluminescent analyzer (42i NO/NO2/NOx; ThermoScientific). Environmental conditions in the chambers were measured in real-time using Dasylab Software (v.13.0; National Instruments). Particle size distribution within the exposure chamber was measured for 5 minutes at the mid-point of the exposure using an electrical low-pressure impactor (ELPI model 97-2E; Dekati Ltd.) and analyzed for surface area-weighted distributions (ELPIvi v.3.0; Dekati Ltd.). Final PM concentrations following each exposure was determined by gravimetrically weighing a glass-fiber filtered placed in the exhaust line of the quartz-tube furnace system before and after the combustion. Due to staffing limitations, VOCs were not able to be characterized for the specific exposures within this study. However, the chemicals found in eucalyptus smoke and concentration ranges generated in our tube furnace system can be found in Kim, et al. (36) and Kim, et al. Attributable to the length of the quartz tube used in our system, each daily exposure was limited to 1 hour."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Samples were prepared for metabolomics analysis using established methods(Johnson et al. (2010). Analyst; Go et al. (2015). Tox Sci). Prior to analysis, lung samples were removed from storage at -80 degrees C and thawed on ice. The 20-30mg of each lung tissue samples was then mixed with ice cold LC-MS grade acetonitrile: water mixture (Sigma-Aldrich; 2:1 ration; 15uL per mg tissue) which contains 2.5uL per 100uL of internal standard solution with eight stable isotopic chemicals selected to cover a range of chemical properties. Samples are then sonicated on ice to ensure metabolite extraction as previously described (Chandler et al (2016) AJ Physiology; Hu et al. (2019) AJ Pathology). Following sonication, tissue lysates are then equilibrated for 30 mins on ice, upon which precipitated proteins are removed by centrifuge (16.1xg at 4C for 10 mins). The resulting supernatant containing metabolite extract (100uL) is removed, added to a low volume autosampler vial and maintained at 4C until analysis (<22h).","SAMPLEPREP_PROTOCOL_FILENAME":"EmoryUniversity_HRM_SamplePreparation_tissue_062016_v1.pdf","PROCESSING_STORAGE_CONDITIONS":"-80℃","EXTRACT_STORAGE":"-80℃"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"The HILIC column is operated parallel to reverse phase column for simultaneous analytical separation and column flushing through the use of a dual head HPLC pump equipped with 10-port and 6-port switching valves. During operation of HILIC separation method, the MS is operated in positive ion mode and 10 microliters of sample is injected onto the HILIC column while the reverse phase column is flushing with wash solution. Flow rate is maintained at 0.35 mL/min until 1.5 min, increased to 0.4 mL/min at 4 min and held for 1 min. Solvent A is 100% LC-MS grade water, solvent B is 100% LC-MS grade acetonitrile and solvent C is 2% formic acid (v/v) in LC-MS grade water. Initial mobile phase conditions are 22.5% A, 75% B, 2.5% C hold for 1.5 min, with linear gradient to 77.5% A, 20% B, 2.5% C at 4 min, hold for 1 min, resulting in a total analytical run time of 5 min. During the flushing phase (reverse phase analytical separation), the HILIC column is equilibrated with a wash solution of 77.5% A, 20% B, 2.5% C.","METHODS_FILENAME":"EmoryUniversity_HRM-QEHF_chromatography_5min_092017_v1.pdf","CHROMATOGRAPHY_COMMENTS":"Triplicate injections for each chromatography mode","INSTRUMENT_NAME":"Thermo Dionex Ultimate 3000","COLUMN_NAME":"Waters XBridge BEH Amide (50 x 2.1mm,2.5um)","COLUMN_TEMPERATURE":"60","FLOW_GRADIENT":"A= water, B= acetonitrile, C= 2% formic acid in water; 22.5% A, 75% B, 2.5% C hold for 1.5 min, linear gradient to 77.5% A, 20% B, 2.5% C at 4 min, hold for 1 min","FLOW_RATE":"0.35 mL/min for 1.5 min; linear increase to 0.4 mL/min at 4 min, hold for 1 min","SOLVENT_A":"100% water","SOLVENT_B":"100% acetonitrile","CHROMATOGRAPHY_TYPE":"HILIC","SOLVENT_C":"100% water; 2% formic acid"},

"ANALYSIS":{"LABORATORY_NAME":"Clinical Biomarkers Laboratory","ANALYSIS_TYPE":"MS","ANALYSIS_PROTOCOL_FILE":"EmoryUniversity_HRM_DataAnalysis-MS_092017_v1.pdf","ACQUISITION_DATE":"1/14/21","SOFTWARE_VERSION":"Xcalibur 3.0.63","OPERATOR_NAME":"ViLinh Tran","DETECTOR_TYPE":"Orbitrap","DATA_FORMAT":"Profile"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive HF hybrid Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","MS_COMMENTS":"Xcalibur 3.0.63","ION_MODE":"POSITIVE","CAPILLARY_TEMPERATURE":"250C","COLLISION_GAS":"N2","DRY_GAS_FLOW":"45","DRY_GAS_TEMP":"150C","MASS_ACCURACY":"< 3ppm","SPRAY_VOLTAGE":"3500","ACTIVATION_PARAMETER":"5.00E+05","ACTIVATION_TIME":"118ms","INTERFACE_VOLTAGE":"S-Lens RF level= 55","MS_RESULTS_FILE":"ST002721_AN004411_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Seconds"},

"MS_METABOLITE_DATA":{
"Units":"peak area",

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