{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002783","ANALYSIS_ID":"AN004530","VERSION":"1","CREATED_ON":"July 18, 2023, 9:42 am"},

"PROJECT":{"PROJECT_TITLE":"Metabolic changes during normothermic isolated kidney perfusion","PROJECT_TYPE":"Experimental","PROJECT_SUMMARY":"Normothermic isolated kidney perfusion is being developed as a method to preserve donor kidneys and to assess their future function before they are transplanted. Donor kidneys can be exposed to different types and degrees of ischemic injury before they are preserved. This project studies how the metabolome changes in relation to different types and degrees of ischemia.","INSTITUTE":"KU Leuven","DEPARTMENT":"Microbiology, Immunology and Transplantation","LABORATORY":"Lab of Abdominal Transplantation","LAST_NAME":"Jochmans","FIRST_NAME":"Ina","ADDRESS":"Herestraat 49, Leuven, 3000, Belgium","EMAIL":"ina.jochmans@kuleuven.be","PHONE":"None","FUNDING_SOURCE":"FTBO, KOOR, FWO","CONTRIBUTORS":"Julie De Beule, Bart Ghesquière, Ina Jochmans"},

"STUDY":{"STUDY_TITLE":"Metabolic changes of the pig kidney during isolated normothermic perfusion with autologous whole blood - perfusate","STUDY_SUMMARY":"This study investigates how glucose, lactate and 20 amino acids in the perfusate of normothermically perfused pig kidneys changes over time. It also studies how type and severity of ischemia before perfusion (cold or warm ischemia) change this profile.","INSTITUTE":"KU Leuven","DEPARTMENT":"Microbiology, Immunology and Transplantation","LABORATORY":"Lab of Abdominal Transplantation","LAST_NAME":"Jochmans","FIRST_NAME":"Ina","ADDRESS":"Herestraat 49, Leuven, 3000, Belgium","EMAIL":"ina.jochmans@kuleuven.be","NUM_GROUPS":"3","TOTAL_SUBJECTS":"18 kidneys","NUM_MALES":"9 male pigs","STUDY_TYPE":"Experimental","PHONE":"None"},

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"Sample ID":"33_T3",
"Factors":{"Group":"CI","Timepoint":"180"},
"Additional sample data":{"RAW_FILE_NAME":"33_T3.mzML"}
},
{
"Subject ID":"33",
"Sample ID":"33_T4",
"Factors":{"Group":"CI","Timepoint":"240"},
"Additional sample data":{"RAW_FILE_NAME":"33_T4.mzML"}
},
{
"Subject ID":"71",
"Sample ID":"71_T0",
"Factors":{"Group":"CI","Timepoint":"0"},
"Additional sample data":{"RAW_FILE_NAME":"71_T0.mzML"}
},
{
"Subject ID":"71",
"Sample ID":"71_T0.25",
"Factors":{"Group":"CI","Timepoint":"15"},
"Additional sample data":{"RAW_FILE_NAME":"71_T0.25.mzML"}
},
{
"Subject ID":"71",
"Sample ID":"71_T0.50",
"Factors":{"Group":"CI","Timepoint":"30"},
"Additional sample data":{"RAW_FILE_NAME":"71_T0.5.mzML"}
},
{
"Subject ID":"71",
"Sample ID":"71_T0.75",
"Factors":{"Group":"CI","Timepoint":"45"},
"Additional sample data":{"RAW_FILE_NAME":"71_T0.75.mzML"}
},
{
"Subject ID":"71",
"Sample ID":"71_T1",
"Factors":{"Group":"CI","Timepoint":"60"},
"Additional sample data":{"RAW_FILE_NAME":"71_T1.mzML"}
},
{
"Subject ID":"71",
"Sample ID":"71_T2",
"Factors":{"Group":"CI","Timepoint":"120"},
"Additional sample data":{"RAW_FILE_NAME":"71_T2.mzML"}
},
{
"Subject ID":"71",
"Sample ID":"71_T3",
"Factors":{"Group":"CI","Timepoint":"180"},
"Additional sample data":{"RAW_FILE_NAME":"71_T3.mzML"}
},
{
"Subject ID":"71",
"Sample ID":"71_T4",
"Factors":{"Group":"CI","Timepoint":"240"},
"Additional sample data":{"RAW_FILE_NAME":"71_T4.mzML"}
},
{
"Subject ID":"72",
"Sample ID":"72_T0",
"Factors":{"Group":"CI","Timepoint":"0"},
"Additional sample data":{"RAW_FILE_NAME":"72_T0.mzML"}
},
{
"Subject ID":"72",
"Sample ID":"72_T0.25",
"Factors":{"Group":"CI","Timepoint":"15"},
"Additional sample data":{"RAW_FILE_NAME":"72_T0.25.mzML"}
},
{
"Subject ID":"72",
"Sample ID":"72_T0.50",
"Factors":{"Group":"CI","Timepoint":"30"},
"Additional sample data":{"RAW_FILE_NAME":"72_T0.5.mzML"}
},
{
"Subject ID":"72",
"Sample ID":"72_T0.75",
"Factors":{"Group":"CI","Timepoint":"45"},
"Additional sample data":{"RAW_FILE_NAME":"72_T0.75.mzML"}
},
{
"Subject ID":"72",
"Sample ID":"72_T1",
"Factors":{"Group":"CI","Timepoint":"60"},
"Additional sample data":{"RAW_FILE_NAME":"72_T1.mzML"}
},
{
"Subject ID":"72",
"Sample ID":"72_T2",
"Factors":{"Group":"CI","Timepoint":"120"},
"Additional sample data":{"RAW_FILE_NAME":"72_T2.mzML"}
},
{
"Subject ID":"72",
"Sample ID":"72_T3",
"Factors":{"Group":"CI","Timepoint":"180"},
"Additional sample data":{"RAW_FILE_NAME":"72_T3.mzML"}
},
{
"Subject ID":"72",
"Sample ID":"72_T4",
"Factors":{"Group":"CI","Timepoint":"240"},
"Additional sample data":{"RAW_FILE_NAME":"72_T4.mzML"}
},
{
"Subject ID":"75",
"Sample ID":"75_T0",
"Factors":{"Group":"CI","Timepoint":"0"},
"Additional sample data":{"RAW_FILE_NAME":"75_T0.mzML"}
},
{
"Subject ID":"75",
"Sample ID":"75_T0.25",
"Factors":{"Group":"CI","Timepoint":"15"},
"Additional sample data":{"RAW_FILE_NAME":"75_T0.25.mzML"}
},
{
"Subject ID":"75",
"Sample ID":"75_T0.50",
"Factors":{"Group":"CI","Timepoint":"30"},
"Additional sample data":{"RAW_FILE_NAME":"75_T0.5.mzML"}
},
{
"Subject ID":"75",
"Sample ID":"75_T0.75",
"Factors":{"Group":"CI","Timepoint":"45"},
"Additional sample data":{"RAW_FILE_NAME":"75_T0.75.mzML"}
},
{
"Subject ID":"75",
"Sample ID":"75_T1",
"Factors":{"Group":"CI","Timepoint":"60"},
"Additional sample data":{"RAW_FILE_NAME":"75_T1.mzML"}
},
{
"Subject ID":"75",
"Sample ID":"75_T2",
"Factors":{"Group":"CI","Timepoint":"120"},
"Additional sample data":{"RAW_FILE_NAME":"75_T2.mzML"}
},
{
"Subject ID":"75",
"Sample ID":"75_T3",
"Factors":{"Group":"CI","Timepoint":"180"},
"Additional sample data":{"RAW_FILE_NAME":"75_T3.mzML"}
},
{
"Subject ID":"75",
"Sample ID":"75_T4",
"Factors":{"Group":"CI","Timepoint":"240"},
"Additional sample data":{"RAW_FILE_NAME":"75_T4.mzML"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Perfusate samples were collected in ethylenediaminetetraacetic acid (EDTA) tubes, at baseline (before the kidney was mounted on the normothermic perfusion device), every 15 minutes during the first hour and hourly thereafter. One milliliter perfusate aliquots (centrifuged 1000g, 10 minutes, 4°C) were snap frozen in liquid nitrogen and stored at -80°C until analysis.","SAMPLE_TYPE":"Perfusate","COLLECTION_FREQUENCY":"Baseline (T0) and during perfusion at 15 min (T0.25), 30 min (T0.50), 45 min (T0.75), 1 hour (T1), 2 hours (T2), 3 hours (T3), 4 hours (T4)","STORAGE_CONDITIONS":"-80℃","ADDITIVES":"EDTA"},

"TREATMENT":{"TREATMENT_SUMMARY":"Three experimental conditions were investigated (n=6/group): (a) Controls; (b) warm ischemia (WI) simulating hypoxic acute kidney injury; (c) cold ischemia (CI) replicating clinical cold storage. Control kidneys were retrieved, flushed with cold IGL-1, and immediately reperfused. CI group, kidneys were exposed to 22h of CI by submerging them in IGL- 1 (a clinical preservation solution for cold storage) and storing them on ice. In WI, the renal artery and vein were clamped for 60 min before retrieval. In the All kidneys were flushed with 200 ml of Ringer’s solution before mounting them on the ex situ circuit to wash out IGL-1.","ANIMAL_ANESTHESIA":"Pigs were sedated by an intramuscular injection of Tiletamine/Zolazepam (8 mg/kg, Zoletil®, Virbac, Belgium) and Xylazine (2 mg/kg, Xylazine®, VMD pharma, Belgium) to allow orotracheal intubation. Anesthesia was maintained by inhalation of isoflurane (1% Isovet®, Piramal Critical Care B.V., Belgium) and continuous infusion of fentanyl (8 µg/kg, Fentanyl®, Janssen Pharmaceutica, Belgium).","ANIMAL_ACCLIMATION_DURATION":"Minimum of 2 days","ANIMAL_FASTING":"Overnight","ANIMAL_ENDP_EUTHANASIA":"Non-recovery study"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Samples were extracted in an 80% methanol (80:20 methanol:water) (Methanol ≥99.9%, HiPerSolv CHROMANORM®, ULTRA for LC-MS, suitable for UPLC/UHPLC-MS instruments, VWR, Belgium) extraction buffer containing 1 µM of deuterated D27 myristic acid, 5 µM D12 glucose, 3 µM 13C5-D5-15N Glutamic acid and 3 µM D7-15N4-Arginine as internal standards. 10 µl of sample was added to 990 µl of the extraction buffer and stored overnight at -80 °C. Insolubilities and precipitated proteins were removed by centrifugation at 20.000 g, for 15 min at 4 °C. 200 µL of the supernatant was transferred to an appropriate mass-spectrometry vial. 10 µl of sample was added to 990 µl of the extraction buffer and stored overnight at -80 °C.","EXTRACT_STORAGE":"-80℃"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"10 µl was loaded onto an Ultra Performance Liquid Chromatograph (UPLC) equipped with a Hydrophilic Interaction Liquid Chromatography (HILIC) column (InfinityLab Poroshell 120 HILIC-Z PEEK-lined 2.1 x 150 mm, 2.7 µm column; Agilent, Santa Clara, USA)) and connected in-line to a Q-exactive Orbitrap Focus (Thermo Fisher Scientific, Massachusetts, USA) mass spectrometer. A linear gradient was built up starting with 90% solvent A (LC-MS grade acetonitrile, Acetonitrile hypergrade for LC-MS LiChrosolv, Supelco (Merck), Germany) and 10% solvent B (10 mM ammonium acetate (LiChropur™, eluent additive for LC-MS, (Merck), Germany), pH 9.3). At 2 min the gradient increased to 60% of solvent B and maintained at 60% until 15 min. The gradient returned to 10% solvent B at 16 min and remained until 25 min. The flow rate was 250 µl/min and the column was kept at 25°C throughout the analysis. The MS operated in negative ion mode, with a spray voltage of 2.9 kV and a temperature of the capillary of 325 °C. Gas settings were as follows: sheath gas 40 and auxiliary gas 15. The vaporizer temperature was set at 300 °C. A full scan (resolution of 70.000 and scan range of m/z 70-1050) was applied. XCalibur version to operate the LC-MS was 4.2.47.","CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Agilent 1290 Infinity","COLUMN_NAME":"Agilent InfinityLab Poroshell 120 (2.1 x 150 mm, 2.7 um)","SOLVENT_A":"100% acetonitrile","SOLVENT_B":"100% water; 10 mM ammonium acetate, pH 9.3","FLOW_GRADIENT":"A linear gradient was built up starting with 90% solvent A and 10% solvent B. At 2 min the gradient increased to 60% of solvent B and maintained at 60% until 15 min.","FLOW_RATE":"250 µl/min","COLUMN_TEMPERATURE":"25°C"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","LABORATORY_NAME":"Laboratory of Applied Mass Spectrometry, KU Leuven"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Focus","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"Raw MS data were converted into mzML using the MSConvert tool of Proteowizard (version 3.0.20247). Peak picking was performed with El_Maven (El_Maven, Elucidata, Massachusetts, USA, version 0.12.0). Metabolites were identified using an in-house library containing exact mass and retention time. The mass accuracy during data processing in El Maven was set at 10 ppm. Calculation of abundances was done in the LC-MS Workflow of El_Maven. Raw abundances (peak area values) for each metabolite were corrected for internal standard (Myristic acid d27)."},

"MS_METABOLITE_DATA":{
"Units":"Peak area",

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}

}