{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001526","ANALYSIS_ID":"AN002547","VERSION":"1","CREATED_ON":"November 12, 2020, 11:15 am"},

"PROJECT":{"PROJECT_TITLE":"Mitochondrial health is enhanced in rats with higher vs. lower intrinsic exercise capacity and extended lifespan","PROJECT_TYPE":"Untargeted analysis of primary metabolism by GCTOF","PROJECT_SUMMARY":"The intrinsic aerobic capacity of an organism is thought to play a role in aging and longevity. Maximal respiratory rate capacity, a metabolic performance measure, is one of the best predictors of cardiovascular- and all-cause mortality. Rats selectively bred for high-(HCR) vs. low-(LCR) intrinsic running-endurance capacity have up to 31% longer lifespan. We found that positive changes in indices of mitochondrial health in cardiomyocytes (respiratory reserve, maximal respiratory capacity, resistance to mitochondrial permeability transition, autophagy/mitophagy, higher lipids-over-glucose utilization) are uniformly associated with the extended longevity in HCR vs. LCR female rats. Cross-sectional heart metabolomics revealed pathways from lipid metabolism in the heart which were significantly enriched by a select group of strain dependent metabolites, consistent with enhanced lipids utilization by HCR cardiomyocytes. Heart-liver-serum metabolomics further revealed shunting of lipidic substrates between liver and heart via serum during aging. Thus, mitochondrial health in cardiomyocytes is associated with extended longevity in rats with higher intrinsic exercise capacity, and likely these findings can be translated to other populations as predictors of outcomes of health and survival.","INSTITUTE":"National Institute on Aging","DEPARTMENT":"Cardioprotection Section","LABORATORY":"Laboratory of Cardiovascular Science","LAST_NAME":"Sollott","FIRST_NAME":"Steven","ADDRESS":"Laboratory of Cardiovascular Science, National Institute on Aging, NIH, Baltimore, MD 21224, USA","EMAIL":"sollotts@grc.nia.nih.gov","PHONE":"410-558-8657","FUNDING_SOURCE":"Intramural Research Program of the National Institutes of Health, National Institute on Aging."},

"STUDY":{"STUDY_TITLE":"Mitochondrial health is enhanced in rats with higher vs. lower intrinsic exercise capacity and extended lifespan","STUDY_SUMMARY":"The intrinsic aerobic capacity of an organism is thought to play a role in aging and longevity. Maximal respiratory rate capacity, a metabolic performance measure, is one of the best predictors of cardiovascular- and all-cause mortality. Rats selectively bred for high-(HCR) vs. low-(LCR) intrinsic running-endurance capacity have up to 31% longer lifespan. We found that positive changes in indices of mitochondrial health in cardiomyocytes (respiratory reserve, maximal respiratory capacity, resistance to mitochondrial permeability transition, autophagy/mitophagy, higher lipids-over-glucose utilization) are uniformly associated with the extended longevity in HCR vs. LCR female rats. Cross-sectional heart metabolomics revealed pathways from lipid metabolism in the heart which were significantly enriched by a select group of strain dependent metabolites, consistent with enhanced lipids utilization by HCR cardiomyocytes. Heart-liver-serum metabolomics further revealed shunting of lipidic substrates between liver and heart via serum during aging. Thus, mitochondrial health in cardiomyocytes is associated with extended longevity in rats with higher intrinsic exercise capacity, and likely these findings can be translated to other populations as predictors of outcomes of health and survival.","INSTITUTE":"National Institute on Aging","DEPARTMENT":"Cardioprotection Section","LABORATORY":"Laboratory of Cardiovascular Science","LAST_NAME":"Sollott","FIRST_NAME":"Steven","ADDRESS":"Laboratory of Cardiovascular Science, National Institute on Aging, NIH, Baltimore, MD 21224, USA","EMAIL":"sollotts@grc.nia.nih.gov","PHONE":"410-558-8657"},

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},
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},
{
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"Sample ID":"LIVER_LCR29203_052",
"Factors":{"treatment":"LCR","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"190409cadsa04_1"}
},
{
"Subject ID":"-",
"Sample ID":"Serum HCR18729_042",
"Factors":{"treatment":"HCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190409cadsa23_1"}
},
{
"Subject ID":"-",
"Sample ID":"Serum LCR28069_041",
"Factors":{"treatment":"LCR","Tissue":"serum"},
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},
{
"Subject ID":"-",
"Sample ID":"SERUM_HCR18866_025",
"Factors":{"treatment":"HCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190409cadsa42_1"}
},
{
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"Sample ID":"SERUM_HCR18871_027",
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},
{
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},
{
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"Sample ID":"SERUM_HCR18895_028",
"Factors":{"treatment":"HCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190409cadsa28_1"}
},
{
"Subject ID":"-",
"Sample ID":"SERUM_HCR18927_029",
"Factors":{"treatment":"HCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190410cadsa06_1"}
},
{
"Subject ID":"-",
"Sample ID":"SERUM_HCR18947_030",
"Factors":{"treatment":"HCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190410cadsa04_1"}
},
{
"Subject ID":"-",
"Sample ID":"SERUM_HCR19139_073",
"Factors":{"treatment":"HCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190410cadsa01_1"}
},
{
"Subject ID":"-",
"Sample ID":"SERUM_HCR19140_074",
"Factors":{"treatment":"HCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190409cadsa13_1"}
},
{
"Subject ID":"-",
"Sample ID":"SERUM_HCR19143_075",
"Factors":{"treatment":"HCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190410cadsa11_1"}
},
{
"Subject ID":"-",
"Sample ID":"SERUM_HCR19153_072",
"Factors":{"treatment":"HCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190409cadsa47_1"}
},
{
"Subject ID":"-",
"Sample ID":"SERUM_HCR19157_070",
"Factors":{"treatment":"HCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190410cadsa29_1"}
},
{
"Subject ID":"-",
"Sample ID":"SERUM_HCR19158_071",
"Factors":{"treatment":"HCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190409cadsa22_1"}
},
{
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"Sample ID":"SERUM_LCR28344_033",
"Factors":{"treatment":"LCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190409cadsa30_1"}
},
{
"Subject ID":"-",
"Sample ID":"SERUM_LCR28369_036",
"Factors":{"treatment":"LCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190409cadsa48_1"}
},
{
"Subject ID":"-",
"Sample ID":"SERUM_LCR28397_035",
"Factors":{"treatment":"LCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190409cadsa21_1"}
},
{
"Subject ID":"-",
"Sample ID":"SERUM_LCR28404_031",
"Factors":{"treatment":"LCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190410cadsa05_1"}
},
{
"Subject ID":"-",
"Sample ID":"SERUM_LCR28415_034",
"Factors":{"treatment":"LCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190409cadsa20_1"}
},
{
"Subject ID":"-",
"Sample ID":"SERUM_LCR28438_032",
"Factors":{"treatment":"LCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190409cadsa25_1"}
},
{
"Subject ID":"-",
"Sample ID":"SERUM_LCR29029_081",
"Factors":{"treatment":"LCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190409cadsa09_1"}
},
{
"Subject ID":"-",
"Sample ID":"SERUM_LCR29097_078",
"Factors":{"treatment":"LCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190410cadsa24_1"}
},
{
"Subject ID":"-",
"Sample ID":"SERUM_LCR29098_077",
"Factors":{"treatment":"LCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190410cadsa03_1"}
},
{
"Subject ID":"-",
"Sample ID":"SERUM_LCR29099_079",
"Factors":{"treatment":"LCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190410cadsa26_1"}
},
{
"Subject ID":"-",
"Sample ID":"SERUM_LCR29200_080",
"Factors":{"treatment":"LCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190410cadsa31_1"}
},
{
"Subject ID":"-",
"Sample ID":"SERUM_LCR29203_076",
"Factors":{"treatment":"LCR","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"190409cadsa43_1"}
},
{
"Subject ID":"-",
"Sample ID":"Heart Pool 1",
"Factors":{"treatment":"control","Tissue":"Heart"},
"Additional sample data":{"RAW_FILE_NAME":"190409cadsa34_1"}
},
{
"Subject ID":"-",
"Sample ID":"Heart Pool 2",
"Factors":{"treatment":"control","Tissue":"Heart"},
"Additional sample data":{"RAW_FILE_NAME":"190410cadsa16_1"}
},
{
"Subject ID":"-",
"Sample ID":"Liver Pool 1",
"Factors":{"treatment":"control","Tissue":"Liver"},
"Additional sample data":{"RAW_FILE_NAME":"190409cadsa38_1"}
},
{
"Subject ID":"-",
"Sample ID":"Liver Pool 2",
"Factors":{"treatment":"control","Tissue":"Liver"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa20_1"}
},
{
"Subject ID":"-",
"Sample ID":"Serum Pool 4",
"Factors":{"treatment":"control","Tissue":"Serum"},
"Additional sample data":{"RAW_FILE_NAME":"170808dngsa06_1"}
},
{
"Subject ID":"-",
"Sample ID":"Serum Pool 1",
"Factors":{"treatment":"control","Tissue":"Serum"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa47_1"}
},
{
"Subject ID":"-",
"Sample ID":"Serum Pool 2",
"Factors":{"treatment":"control","Tissue":"Serum"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa19_1"}
},
{
"Subject ID":"-",
"Sample ID":"Serum Pool 3",
"Factors":{"treatment":"control","Tissue":"Serum"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa15_1"}
},
{
"Subject ID":"-",
"Sample ID":"13-H_031",
"Factors":{"treatment":"control","Tissue":"heart"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa25_1"}
},
{
"Subject ID":"-",
"Sample ID":"14-H_032",
"Factors":{"treatment":"control","Tissue":"heart"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa38_1"}
},
{
"Subject ID":"-",
"Sample ID":"15-H_033",
"Factors":{"treatment":"control","Tissue":"heart"},
"Additional sample data":{"RAW_FILE_NAME":"170808dngsa03_1"}
},
{
"Subject ID":"-",
"Sample ID":"21-H_034",
"Factors":{"treatment":"control","Tissue":"heart"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa24_1"}
},
{
"Subject ID":"-",
"Sample ID":"22-H_035",
"Factors":{"treatment":"control","Tissue":"heart"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa03_1"}
},
{
"Subject ID":"-",
"Sample ID":"23-H_036",
"Factors":{"treatment":"control","Tissue":"heart"},
"Additional sample data":{"RAW_FILE_NAME":"170808dngsa02_1"}
},
{
"Subject ID":"-",
"Sample ID":"24-H_037",
"Factors":{"treatment":"control","Tissue":"heart"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa01_1"}
},
{
"Subject ID":"-",
"Sample ID":"25-H_038",
"Factors":{"treatment":"control","Tissue":"heart"},
"Additional sample data":{"RAW_FILE_NAME":"170808dngsa01_1"}
},
{
"Subject ID":"-",
"Sample ID":"26-H_039",
"Factors":{"treatment":"control","Tissue":"heart"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa40_3"}
},
{
"Subject ID":"-",
"Sample ID":"1-H_027",
"Factors":{"treatment":"control","Tissue":"heart"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa50_1"}
},
{
"Subject ID":"-",
"Sample ID":"2-H_028",
"Factors":{"treatment":"control","Tissue":"heart"},
"Additional sample data":{"RAW_FILE_NAME":"170808dngsa08_1"}
},
{
"Subject ID":"-",
"Sample ID":"5-H_029",
"Factors":{"treatment":"control","Tissue":"heart"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa34_1"}
},
{
"Subject ID":"-",
"Sample ID":"6-H_030",
"Factors":{"treatment":"control","Tissue":"heart"},
"Additional sample data":{"RAW_FILE_NAME":"170808dngsa15_1"}
},
{
"Subject ID":"-",
"Sample ID":"10-L_008",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa30_1"}
},
{
"Subject ID":"-",
"Sample ID":"11-L_009",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa29_1"}
},
{
"Subject ID":"-",
"Sample ID":"12-L_010",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa13_1"}
},
{
"Subject ID":"-",
"Sample ID":"13-L_011",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170808dngsa07_2"}
},
{
"Subject ID":"-",
"Sample ID":"14-L_012",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa28_1"}
},
{
"Subject ID":"-",
"Sample ID":"15-L_013",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa35_1"}
},
{
"Subject ID":"-",
"Sample ID":"16-L_014",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa10_2"}
},
{
"Subject ID":"-",
"Sample ID":"9-L_007",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa22_1"}
},
{
"Subject ID":"-",
"Sample ID":"17-L_015",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa37_3"}
},
{
"Subject ID":"-",
"Sample ID":"18-L_016",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa42_1"}
},
{
"Subject ID":"-",
"Sample ID":"19-L_017",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa17_1"}
},
{
"Subject ID":"-",
"Sample ID":"20-L_018",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa11_1"}
},
{
"Subject ID":"-",
"Sample ID":"21-L_019",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170808dngsa04_1"}
},
{
"Subject ID":"-",
"Sample ID":"22-L_020",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa16_1"}
},
{
"Subject ID":"-",
"Sample ID":"23-L_021",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa33_1"}
},
{
"Subject ID":"-",
"Sample ID":"24-L_022",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170808dngsa13_1"}
},
{
"Subject ID":"-",
"Sample ID":"25-L_023",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa36_1"}
},
{
"Subject ID":"-",
"Sample ID":"26-L_024",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170808dngsa12_2"}
},
{
"Subject ID":"-",
"Sample ID":"27-L_025",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170808dngsa05_1"}
},
{
"Subject ID":"-",
"Sample ID":"28-L_026",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa08_1"}
},
{
"Subject ID":"-",
"Sample ID":"1-L_001",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa14_1"}
},
{
"Subject ID":"-",
"Sample ID":"2-L_002",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa26_1"}
},
{
"Subject ID":"-",
"Sample ID":"3-L_003",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa48_2"}
},
{
"Subject ID":"-",
"Sample ID":"4-L_004",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170808dngsa11_3"}
},
{
"Subject ID":"-",
"Sample ID":"5-L_005",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa45_3"}
},
{
"Subject ID":"-",
"Sample ID":"6-L_006",
"Factors":{"treatment":"control","Tissue":"liver"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa12_1"}
},
{
"Subject ID":"-",
"Sample ID":"C2-HC-1_046",
"Factors":{"treatment":"control","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa49_1"}
},
{
"Subject ID":"-",
"Sample ID":"C2-HC-2_047",
"Factors":{"treatment":"control","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa44_1"}
},
{
"Subject ID":"-",
"Sample ID":"HC-4_049",
"Factors":{"treatment":"control","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa31_1"}
},
{
"Subject ID":"-",
"Sample ID":"HC-5_050",
"Factors":{"treatment":"control","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa05_1"}
},
{
"Subject ID":"-",
"Sample ID":"HC-6_051",
"Factors":{"treatment":"control","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa39_1"}
},
{
"Subject ID":"-",
"Sample ID":"HC-7_052",
"Factors":{"treatment":"control","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"170808dngsa10_1"}
},
{
"Subject ID":"-",
"Sample ID":"HC-8_053",
"Factors":{"treatment":"control","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa43_1"}
},
{
"Subject ID":"-",
"Sample ID":"MA-10_063",
"Factors":{"treatment":"control","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"170808dngsa09_1"}
},
{
"Subject ID":"-",
"Sample ID":"MA-11_064",
"Factors":{"treatment":"control","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa46_1"}
},
{
"Subject ID":"-",
"Sample ID":"MA-12_065",
"Factors":{"treatment":"control","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa41_1"}
},
{
"Subject ID":"-",
"Sample ID":"MA-5_058",
"Factors":{"treatment":"control","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa27_1"}
},
{
"Subject ID":"-",
"Sample ID":"MA-7_060",
"Factors":{"treatment":"control","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa32_1"}
},
{
"Subject ID":"-",
"Sample ID":"MA-8_061",
"Factors":{"treatment":"control","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa18_2"}
},
{
"Subject ID":"-",
"Sample ID":"MA-9_062",
"Factors":{"treatment":"control","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"170808dngsa14_1"}
},
{
"Subject ID":"-",
"Sample ID":"Rat 1_040",
"Factors":{"treatment":"control","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa09_1"}
},
{
"Subject ID":"-",
"Sample ID":"Rat 2_041",
"Factors":{"treatment":"control","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa07_1"}
},
{
"Subject ID":"-",
"Sample ID":"Rat 3_042",
"Factors":{"treatment":"control","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa23_1"}
},
{
"Subject ID":"-",
"Sample ID":"Rat 4_043",
"Factors":{"treatment":"control","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa06_1"}
},
{
"Subject ID":"-",
"Sample ID":"Rat 5_044",
"Factors":{"treatment":"control","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa02_1"}
},
{
"Subject ID":"-",
"Sample ID":"Rat 6_045",
"Factors":{"treatment":"control","Tissue":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"170807dngsa04_1"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Samples were flash-frozen.","SAMPLE_TYPE":"Blood;Liver;Heart"},

"TREATMENT":{"TREATMENT_SUMMARY":"Information not provided."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Extraction of Mammalian Tissue Samples: Lungs/Muscle/Heart 1. References: Fiehn O, Kind T (2006) Metabolite profiling in blood plasma. In: Metabolomics: Methods and Protocols. Weckwerth W (ed.), Humana Press, Totowa NJ (in press) 2.Starting material: Mammalian tissue: Lung/Muscle/Heart: Whole tissue sample is prepared OR stein mill whole tissue sample and weigh 50mL aliquot. 3. Equipment: Centrifuge (Eppendorf 5415 D) Calibrated pipettes 1-200μl and 100-1000μl Eppendorf tubes 2ml, uncoloured (Cat. No. 022363204) Centrifuge tubes, various sizes, polypropylene Eppendorff Tabletop Centrifuge (Proteomics core Lab.) ThermoElectron Neslab RTE 740 cooling bath at –20°C MiniVortexer (VWR) Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments) Speed vacuum concentration system (Labconco Centrivap cold trap) Turex mini homogenizer 4. Chemicals Acetonitrile, LCMS grade (JT Baker; Cat. No.9829-02) Isopropanol, HPLC grade (JT Baker; Cat. No. 9095-02) Crushed ice pH paper 5-10 (EMD Chem. Inc.) Nitrogen line with pipette tip 18 MΩ pure water (Millipore) 5. Procedure Preparation of extraction mix and material before experiment: Switch on bath to pre-cool at –20°C (±2°C validity temperature range) Check pH of acetonitrile and isopropanol (pH7) using wetted pH paper Make the extraction solution by missing acetonitrile, isopropanol and water in proportions 3 : 3 : 2 Rinse the extraction solution for 5 min with nitrogen. Make sure that the nitrogen line was flushed out of air before using it for degassing the extraction solvent solution Sample Preparation Weigh 50 mg tissue sample in to a 25 ml conical polypropylene centrifuge tube. Add 2.5mL extraction solvent to the tissue sample and homogenize for 45 seconds ensuring that sample resembles a powder. In between samples, clean the homogenizer in solutions of methanol, acetone, water, and the extraction solvent. Centrifuge the samples at 2500 rpm. for 5 minutes. Aliquot 2 X 500µl supernatant, one for analysis and one for a backup sample. Store backup aliquot in the -20°C freezer. Evaporate one 500µl aliquot of the sample in the Labconco Centrivap cold trap concentrator to complete dryness The dried aliquot is then re-suspended with 500μl 50% acetonitrile (degassed as given) Centrifuge for 2 min at 14000 rcf using the centrifuge Eppendorf 5415. Remove supernatant to a new Eppendorff tube. Evaporate the supernatant to dryness in the the Labconco Centrivap cold trap concentrator. Submit to derivatization. The residue should contain membrane lipids because these are supposedly not soluble enough to be found in the 50% acetonitrile solution. Therefore, this ‘membrane residue’ is now taken for membrane lipidomic fingerprinting using the nanomate LTQ ion trap mass spectrometer. Likely, a good solvent to redissolve the membrane lipids is e.g. 75% isopropanol (degassed as given above). If the ‘analysis’ aliquot is to be used for semi lipophilic compounds such as tyrosine pathway intermediates (incl. dopamine, serotonine etc, i.e. polar aromatic compounds), then these are supposedly to be found together with the ‘GCTOF’ aliquot. We can assume that this mixture is still too complex for Agilent chipLCMS. Therefore, in order to develop and validate target analysis for such aromatic compounds, we should use some sort of Solid Phase purification. We re-suspend the dried ‘GCTOF’ aliquot in 300 l water (degassed as before) to take out sugars, aliphatic amino acids, hydroxyl acids and similar logP compounds. The residue should contain our target aromatics .We could also try to adjust pH by using low concentration acetate or phosphate buffer. The residue could then be taken up in 50% acetonitrile and used for GCTOF and Agilent chipMS experiments. The other aliquot should be checked how much of our target compounds would actually be found in the ‘sugar’ fraction. 6. Problems To prevent contamination disposable material is used. Control pH from extraction mix. 7. Quality assurance For each sequence of sample extractions, perform one blank negative control extraction by applying the total procedure (i.e. all materials and plastic ware) without biological sample. 8. Disposal of waste Collect all chemicals in appropriate bottles and follow the disposal rules. Sample preparation of blood plasma or serum samples for GCTOF analysis Purpose: This SOP describes sample extraction and sample preparation for primary metabolism profiling by gas chromatography/time-of-flight mass spectrometry (GCTOF). References: Fiehn O, Kind T (2006) Metabolite profiling in blood plasma. In: Metabolomics: Methods and Protocols. Weckwerth W (ed.), Humana Press, Totowa NJ. Fiehn, O. Metabolomics by gas chromatography - mass spectrometry: combined targeted and untargeted profiling. 2016. Curr. Protoc. Mol. Biol. 114:30.4.1-30.4.32. doi: 10.1002/0471142727.mb3004s114. Starting material: Plasma/serum: 30 µL sample volume or aliquot Equipment: Centrifuge Eppendorf 5415 D Calibrated pipettes 1-200µL and 100-1000µL Multi-Tube Vortexer (VWR VX-2500) Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments) Speed vacuum concentration system (Labconco Centrivap cold trap) Nitrogen line with Pasteur pipette Chemicals and consumables: Product Manufacturer & Part Number Eppendorf tubes 1.5 mL, uncolored Eppendorf 022363204 Crushed ice UC Davis Water, LC/MS Grade Fisher Optima W6-4 Acetonitrile, LC/MS Grade Fisher Optima A955-4 Isopropanol, LC/MS Grade Fisher A461-4 pH paper 5-10 Millipore Sigma 1095330001 Bioreclamation human plasma (disodium EDTA) Bioreclamation HMPLEDTA Sample Preparation: Preparation of extraction solvent For 1 L of extraction solvent, combine 375 mL of acetonitrile, 375 mL of isopropanol, and 250 mL water in a 1 L bottle conditioned with the aforementioned chemicals. If a different total volume of extraction solvent is needed, simply mix acetonitrile, isopropanol, and water in volumes in proportion 3:3:2. Purge the extraction solution mix for 5 min with nitrogen with small bubbles. Make sure that the nitrogen line is flushed out of air before using it for degassing the extraction solvent solution. Store at -20°C until use. Note: if solvent freezes, sonicate until thawed and mix before use. Extraction Thaw raw samples at room temperature (or in the refrigerator at 4˚C) and vortex 10 sec at low speed to homogenize. Aliquot 30 μL of plasma sample into a 1.5 mL Eppendorf tube. Keep all samples on ice. Add 1 mL 3:3:2 (v/v/v) ACN:IPA:H2O extraction solvent (prechilled in a -20°C freezer). Vortex the sample for 10 sec. Shake for 5 min at 4°C using the Orbital Mixing Chilling/Heating Plate. Continue to keep all extracted samples on ice. Centrifuge samples for 2 min at 14000 rcf. Aliquot two 450 μL portions of the supernatant into 1.5 mL Eppendorf tubes (one for analysis and one as a backup sample). Transfer 100 μL of the remaining supernatant from each sample to a 2, 15, or 50 mL tube for pools, depending on number of samples in the study. Evaporate one 450 μL aliquot of the sample in the Labconco Centrivap cold trap concentrator to complete dryness. Proceed with cleanup or store tubes at -20°C until cleanup. Pooling Transfer multiple 475 µL aliquots of pooled samples to 1.5 mL Eppendorf tubes, one aliquot for every 10 samples in the study. If there is still pool remaining, prepare additional aliquots for backup. Centrifuge pool samples for 2 min at 14000 rcf. Remove 450 µL supernatant to new 1.5 mL Eppendorf tube. Evaporate to complete dryness in the Labconco Centrivap cold trap concentrator. Proceed with cleanup or store tubes at -20°C until cleanup. Cleanup Resuspend the dried aliquot with 500 μL 50:50 (v/v) ACN:H2O (degassed as given above) and vortex for about 10 sec. Centrifuge for 2 min at 14000 rcf. Remove 475 μL supernatant to a new 1.5 mL Eppendorf tube. Evaporate the transferred supernatant to complete dryness in the Labconco Centrivap cold trap concentrator. Submit to derivatization (see SOP “Derivatization of GC Samples & Standards”) or store at -20°C until ready for analysis. Quality assurance For every 50 samples, perform one method blank negative control extraction by applying the total procedure (i.e. all materials and plastic ware) without biological sample. If no combined pool was made from the extracted samples, use one commercial plasma/serum pool sample per 10 authentic subject samples as control instead. Disposal of waste Collect all chemicals in appropriate bottles and follow the disposal rules. Collect residual plasma/serum samples in specifically designed red ‘biohazard’ waste bags."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"A 30 m long, 0.25 mm i.d. Rtx-5Sil MS column (0.25 μm 95% dimethyl 5% diphenyl polysiloxane film) with additional 10 m integrated guard column is used (Restek, Bellefonte PA). 99.9999% pure Helium with built-in purifier (Airgas, Radnor PA) is set at constant flow of 1 ml/min. The oven temperature is held constant at 50°C for 1 min and then ramped at 20°C/min to 330°C at which it is held constant for 5 min.","CHROMATOGRAPHY_TYPE":"GC","INSTRUMENT_NAME":"Leco","COLUMN_NAME":"Rtx-5Sil MS column"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Leco Pegasus IV TOF","INSTRUMENT_TYPE":"GC-TOF","MS_TYPE":"EI","ION_MODE":"POSITIVE","MS_COMMENTS":"GC-TOF Method: Instruments: Gerstel CIS4 –with dual MPS Injector/ Agilent 6890 GC- Pegasus III TOF MS Injector conditions: Agilent 6890 GC is equipped with a Gerstel automatic liner exchange system (ALEX) that includes a multipurpose sample (MPS2) dual rail, and a Gerstel CIS cold injection system (Gerstel, Muehlheim, Germany) with temperature program as follows: 50°C to 275°C final temperature at a rate of 12 °C/s and hold for 3 minutes. Injection volume is 0.5 μl with 10 μl/s injection speed on a splitless injector with purge time of 25 seconds. Liner (Gerstel #011711-010-00) is changed after every 10 samples, (using the Maestro1 Gerstel software vs. 1.1.4.18). Before and after each injection, the 10 μl injection syringe is washed three times with 10 μl ethyl acetate. Gas Chromatography conditions: A 30 m long, 0.25 mm i.d. Rtx-5Sil MS column (0.25 μm 95% dimethyl 5% diphenyl polysiloxane film) with additional 10 m integrated guard column is used (Restek, Bellefonte PA). 99.9999% pure Helium with built-in purifier (Airgas, Radnor PA) is set at constant flow of 1 ml/min. The oven temperature is held constant at 50°C for 1 min and then ramped at 20°C/min to 330°C at which it is held constant for 5 min. Mass spectrometer settings: A Leco Pegasus IV time of flight mass spectrometer is controlled by the Leco ChromaTOF software vs. 2.32 (St. Joseph, MI). The transfer line temperature between gas chromatograph and mass spectrometer is set to 280°C. Electron impact ionization at 70V is employed with an ion source temperature of 250°C. Acquisition rate is 17 spectra/second, with a scan mass range of 85-500 Da."},

"MS_METABOLITE_DATA":{
"Units":"normalized peak height",

"Data":[{"Metabolite":"xylulose NIST","Heart HCR18659_018":"23173","Heart HCR18698_017":"34728","Heart HCR18729_016":"12941","Heart HCR18730_015":"24145","Heart HCR18731_014":"9581","Heart LCR28026_013":"28052","Heart LCR28069_012":"25558","Heart LCR28135_010":"8191","Heart LCR28208_011":"30182","Liver HCR18659_009":"13288","Liver HCR18698_008":"9577","Liver HCR18729_007":"15097","Liver HCR18730_006":"9739","Liver HCR18731_005":"3877","_001":"19175","_002":"8566","_003":"7490","Liver LCR28026_004":"6968","Liver LCR28069_003":"11063","Liver LCR28135_001":"1932","Liver LCR28208_002":"8333","_004":"9259","_005":"8360","MA5-HCR_030":"116630","MA7-HCR_028":"10461","MA8-HCR_029":"84972","Serum HCR18659_027":"11996","Serum HCR18729_025":"42333","Serum HCR18730_024":"25542","Serum HCR18731_023":"27581","SerumHCR18698_026":"27588","_006":"173632","_007":"12007","_008":"17964","Serum LCR28026_022":"10633","Serum LCR28069_021":"22035","Serum LCR28135_019":"29747","Serum 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}

}