{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST003137","ANALYSIS_ID":"AN005150","VERSION":"1","CREATED_ON":"March 24, 2024, 2:33 am"},

"PROJECT":{"PROJECT_TITLE":"Lipid class-specific kinetics of plasma fatty acids, oxylipins, endocannabinoids, lysophospholipids and bile acids upon a lipopolysaccharide challenge of healthy humans and their modulation by anti-oxidative supplements","PROJECT_SUMMARY":"While molecular mechanisms of inflammatory processes are well characterized, the systemic responses of humans exposed to pathogen-associated molecular pattern with regard to fatty acid derivatives and other lipids have hardly been determined. Here, we present a dual stage controlled clinical intervention study with healthy individuals challenged with lipopolysaccharide. While in a first stage, plasma proteomics and lipidomics was applied to observe the kinetics of inflammatory modulators within eight hours, the effects of a placebocontrolled anti-oxidative intervention were determined in the second stage. Plasma proteome profiling demonstrated the early involvement of platelets detectable within two hours after lipopolysaccharide challenge, followed by the characteristic induction of liver-derived acute phase proteins and innate immune cell-derived alarmins. Untargeted lipidomics demonstrated the early release of fatty acids and taurocholic acid within two hours, followed by complex time courses of various oxylipins and the downregulation of numerous lysophospholipids and deoxycholic acid. Groups of molecules with similar kinetics during the time course analysis upon lipopolysaccharide challenge were observed to have common precursors or synthesizing enzymes. Dietary supplementation with antioxidants did not affect the kinetics of detectable proteins, but significantly downregulated the pro-inflammatory sphingosine-1-phosphate and increased the levels of oxylipins described to facilitate the resolution of inflammation, 20-HEPE and 22-HDoHE. The present study identified a complex network of oxylipins, bile acids, lysophospholipids and endocannabinoids deregulated in plasma upon lipopolysaccharide challenge, introduces platelets as powerful inflammatory modulators and suggests that dietary antioxidant supplementation hardly interferes with the induction of inflammatory processes, but may rather support the resolution of inflammation.","INSTITUTE":"University of Vienna","DEPARTMENT":"Department of Analytical Chemistry","LABORATORY":"Gerner lab","LAST_NAME":"Hagn","FIRST_NAME":"Gerhard","ADDRESS":"Währingerstraße 38, 1090 Vienna, Austria","EMAIL":"gerhard.hagn@univie.ac.at","PHONE":"+43 1 4277 52375"},

"STUDY":{"STUDY_TITLE":"Lipid class-specific kinetics of plasma fatty acids, oxylipins, endocannabinoids, lysophospholipids and bile acids upon a lipopolysaccharide challenge of healthy humans and their modulation by anti-oxidative supplements (Part 1/2 - negative mode)","STUDY_SUMMARY":"While molecular mechanisms of inflammatory processes are well characterized, the systemic responses of humans exposed to pathogen-associated molecular pattern with regard to fatty acid derivatives and other lipids have hardly been determined. Here, we present a dual stage controlled clinical intervention study with healthy individuals challenged with lipopolysaccharide. While in a first stage, plasma proteomics and lipidomics was applied to observe the kinetics of inflammatory modulators within eight hours, the effects of a placebocontrolled anti-oxidative intervention were determined in the second stage. Plasma proteome profiling demonstrated the early involvement of platelets detectable within two hours after lipopolysaccharide challenge, followed by the characteristic induction of liver-derived acute phase proteins and innate immune cell-derived alarmins. Untargeted lipidomics demonstrated the early release of fatty acids and taurocholic acid within two hours, followed by complex time courses of various oxylipins and the downregulation of numerous lysophospholipids and deoxycholic acid. Groups of molecules with similar kinetics during the time course analysis upon lipopolysaccharide challenge were observed to have common precursors or synthesizing enzymes. Dietary supplementation with antioxidants did not affect the kinetics of detectable proteins, but significantly downregulated the pro-inflammatory sphingosine-1-phosphate and increased the levels of oxylipins described to facilitate the resolution of inflammation, 20-HEPE and 22-HDoHE. The present study identified a complex network of oxylipins, bile acids, lysophospholipids and endocannabinoids deregulated in plasma upon lipopolysaccharide challenge, introduces platelets as powerful inflammatory modulators and suggests that dietary antioxidant supplementation hardly interferes with the induction of inflammatory processes, but may rather support the resolution of inflammation.","INSTITUTE":"University of Vienna","DEPARTMENT":"Department of Analytical Chemistry","LABORATORY":"Gerner lab","LAST_NAME":"Hagn","FIRST_NAME":"Gerhard","ADDRESS":"Währingerstraße 38, 1090 Vienna, Austria","EMAIL":"gerhard.hagn@univie.ac.at","PHONE":"+43 1 4277 52375"},

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{
"Subject ID":"-",
"Sample ID":"Donor33_stage2_480_min_Placebo_1",
"Factors":{"Sample source":"Plasma","Treatment":"Placebo"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"22_Eicos_767"}
},
{
"Subject ID":"-",
"Sample ID":"Donor33_stage2_480_min_Placebo_2",
"Factors":{"Sample source":"Plasma","Treatment":"Placebo"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"22_Eicos_767_230206231832"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Full blood was collected using EDTA-anticoagulated collection tubes at baseline (BL), 60 min, 120 min, 240 min and 480 min after LPS infusion and plasma was obtained immediately by centrifugation at 4 °C at 2000 g for 10 min. After centrifugation, all samples were immediately frozen in pre-labelled Eppendorf safe-lock tubes at -80 °C until analysis.","SAMPLE_TYPE":"Blood (plasma)"},

"TREATMENT":{"TREATMENT_SUMMARY":"Subjects were recruited by the Department of Clinical Pharmacology at the Medical University of Vienna. The study protocol was approved by the Ethics Committee of the Medical University of Vienna (EC No.: 64/2009) and was conducted in accordance with the guidelines of the Helsinki Declaration. For this randomized, double-masked, placebo-controlled parallel group study, 30 healthy male human individuals were included upon passing a screening examination and signed written informed consent prior to study entry. Subjects with any clinically relevant illness, intake of medication, including vitamin or mineral supplements, or blood donation within 3 weeks prior to the study were excluded and subjects who did not complete the study were replaced. In addition, participants had to abstain from alcohol or caffeine containing beverages within 12 hours prior to each study day of the dual stage controlled clinical intervention study. To induce systemic inflammation and oxidative stress, an intravenous infusion of a bolus containing 2 ng/kg bodyweight Escherichia coli endotoxin known as lipopolysaccharide (LPS; NIH-CC, Bethesda, MD, USA) was used. Full blood was collected using EDTA-anticoagulated collection tubes at baseline (BL), 60 min, 120 min, 240 min and 480 min after LPS infusion and plasma was obtained immediately by centrifugation at 4 °C at 2000 g for 10 min. After centrifugation, all samples were immediately frozen in pre-labelled Eppendorf safe-lock tubes at -80 °C until analysis. After this first stage, participants were randomly assigned to take either the omega-3 fatty acid containing food supplement Vitamac (n = 17; Croma Pharma GmbH, Korneuburg, Austria) or matching lactose and wheat starch containing placebo capsules (n = 13) for 14 days. After the 14 days intervention, all participants were treated again with LPS in the second stage and plasma was collected at baseline (BL), 60 min, 120 min, 240 min and 480 min after LPS infusion applying the same protocol as described for the first stage."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Frozen EDTA-anticoagulated plasma was freshly thawed on ice. For precipitation of proteins, plasma (400 µL) was mixed with cold EtOH (1.6 mL, abs. 99%, -20°C; AustrAlco) including an internal standard mixture of 12S-HETE-d8, 15S-HETE-d8, 5-Oxo-ETE-d7, 11,12-DiHETrE-d11, PGE2-d4 and 20-HETE-d6 (concentrations can be found below). The samples were stored over-night at -20°C. After centrifugation (30 min, 4536 g, 4°C), the supernatant was transferred into a new 15 mL FalconTM tube. EtOH was evaporated via vacuum centrifugation at 37°C until the original sample volume (400 µL) was restored. For solid phase extraction (SPE) samples were loaded onto preconditioned StrataX SPE columns (30 mg mL-1; Phenomenex, Torrance, CA, USA) using Pasteur pipettes. After sample loading, the SPE columns were washed with 5 mL of MS grade water and eluted with ice-cold MeOH (500 µL; MeOH abs.; VWR International, Vienna, Austria) containing 2% formic acid (FA; Sigma-Aldrich). MeOH was evaporated using a gentle nitrogen stream at room temperature and the dried samples were reconstituted in 150 µL reconstitution solvent (H2O:ACN:MeOH + 0.2% FA–vol% 65:31.5:3.5). The samples were then transferred into an autosampler held at stored at 4°C and subsequently measured via LC-MS/MS. 12S-HETE-d8: 6.67 pg/µL 15S-HETE-d8: 6.67 pg/µL 5-Oxo-ETE-d7: 20 pg/µL 11,12-DiHETrE-d11: 6.67 pg/µL PGE2-d4: 13.33 pg/µL 20-HETE-d6: 6.67 pg/µL"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"For LC-MS analyses, analytes were separated using a Thermo Scientific Vanquish (UHPLC) system equipped with a Kinetex C18-column (2.6 µm, XB-C18, 100 A° , LC Column 150 * 2.1 mm; Phenomenex) applying a gradient flow profile (mobile phase A: H2O + 0.2% FA, mobile phase B: ACN:MeOH (vol% 90:10) + 0.2% FA) starting at 35% B and increasing to 90% B (1–10 min), further increasing to 99% B within 0.5 min and held for 5 min. Solvent B was then decreased to the initial level of 35% within 0.5 min and the column was equilibrated for 4 min, resulting in a total run time of 20 min. The flow rate was kept at 200 µL min-1 and the column oven temperature at 40°C. The injection volume was 20 µL and all samples were analysed in technical duplicates.","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"Phenomenex Kinetex XB-C18 (150 x 2.1mm, 2.6um)","SOLVENT_A":"100% water; 0.2% formic acid","SOLVENT_B":"90% acetonitrile/10% methanol; 0.2% formic acid","FLOW_GRADIENT":"0min with 35% B to 90% B (1–10 min), further increasing to 99% B within 0.5 min and held for 5 min. Solvent B was then decreased to the initial level of 35% within 0.5 min and the column was equilibrated for 4 min, resulting in a total run time of 20 min.","FLOW_RATE":"200 µL/min","COLUMN_TEMPERATURE":"40"},

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

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive HF hybrid Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"The Vanquish UHPLC system was coupled to a Q ExactiveTM HF Quadrupole-OrbitrapTM high-resolution mass spectrometer (Thermo Fisher Scientific, Austria), equipped with a HESI source for negative ionization to perform the mass spectrometric analysis. The MS scan range was 250-700 m/z with a resolution of 60,000 (at m/z 200) on the MS1 level. A Top 2 method was applied for fragmentation (HCD 24 normalized collision energy), preferable 33 m/z values specific for well-known eicosanoids and precursor molecules from an inclusion list. The resulting fragments were analysed on the MS2 level at a resolution of 15,000 (at m/z 200). Operating in negative ionization mode, a spray voltage of 3.5 kV and a capillary temperature of 253°C were applied. Sheath gas was set to 46 and the auxiliary gas to 10 (arbitrary units)."},

"MS_METABOLITE_DATA":{
"Units":"normalized AUC",

"Data":[{"Metabolite":"11,12-DiHETrE","Donor1_stage1_0_min_con_1":"18.19","Donor1_stage1_0_min_con_2":"18.11","Donor1_stage1_60_min_con_1":"17.84","Donor1_stage1_60_min_con_2":"17.88","Donor1_stage1_120_min_con_1":"18.04","Donor1_stage1_120_min_con_2":"18.18","Donor1_stage1_240_min_con_1":"18.66","Donor1_stage1_240_min_con_2":"18.68","Donor1_stage1_480_min_con_1":"18.38","Donor1_stage1_480_min_con_2":"18.24","Donor1_stage2_0_min_Placebo_1":"18.31","Donor1_stage2_0_min_Placebo_2":"18.38","Donor1_stage2_60_min_Placebo_1":"18.07","Donor1_stage2_60_min_Placebo_2":"18.06","Donor1_stage2_120_min_Placebo_1":"18.23","Donor1_stage2_120_min_Placebo_2":"18.11","Donor1_stage2_240_min_Placebo_1":"18.12","Donor1_stage2_240_min_Placebo_2":"18.19","Donor1_stage2_480_min_Placebo_1":"17.75","Donor1_stage2_480_min_Placebo_2":"17.7","Donor2_stage1_0_min_con_1":"17.39","Donor2_stage1_0_min_con_2":"17.42","Donor2_stage1_60_min_con_1":"17.61","Donor2_stage1_60_min_con_2":"17.5","Donor2_stage1_120_min_con_1":"17.7","Donor2_stage1_120_min_con_2":"17.78","Donor2_stage1_240_min_con_1":"17.84","Donor2_stage1_240_min_con_2":"17.8","Donor2_stage1_480_min_con_1":"17.27","Donor2_stage1_480_min_con_2":"17.14","Donor2_stage2_0_min_Verum_1":"18.02","Donor2_stage2_0_min_Verum_2":"18.11","Donor2_stage2_60_min_Verum_1":"17.94","Donor2_stage2_60_min_Verum_2":"17.99","Donor2_stage2_120_min_Verum_1":"17.94","Donor2_stage2_120_min_Verum_2":"17.92","Donor2_stage2_240_min_Verum_1":"18.74","Donor2_stage2_240_min_Verum_2":"18.78","Donor2_stage2_480_min_Verum_1":"18.85","Donor2_stage2_480_min_Verum_2":"18.8","Donor3_stage1_0_min_con_1":"17.93","Donor3_stage1_0_min_con_2":"17.95","Donor3_stage1_60_min_con_1":"18.02","Donor3_stage1_60_min_con_2":"17.98","Donor3_stage1_120_min_con_1":"18.07","Donor3_stage1_120_min_con_2":"18.1","Donor3_stage1_240_min_con_1":"18.31","Donor3_stage1_240_min_con_2":"18.25","Donor3_stage1_480_min_con_1":"17.58","Donor3_stage1_480_min_con_2":"17.69","Donor3_stage2_0_min_Verum_1":"17.87","Donor3_stage2_0_min_Verum_2":"17.81","Donor3_stage2_60_min_Verum_1":"17.86","Donor3_stage2_60_min_Verum_2":"17.8","Donor3_stage2_120_min_Verum_1":"17.79","Donor3_stage2_120_min_Verum_2":"17.93","Donor3_stage2_240_min_Verum_1":"18.43","Donor3_stage2_240_min_Verum_2":"18.49","Donor3_stage2_480_min_Verum_1":"17.39","Donor3_stage2_480_min_Verum_2":"17.44","Donor5_stage1_0_min_con_1":"17.65","Donor5_stage1_0_min_con_2":"17.56","Donor5_stage1_60_min_con_1":"17.09","Donor5_stage1_60_min_con_2":"17.19","Donor5_stage1_120_min_con_1":"17.47","Donor5_stage1_120_min_con_2":"17.69","Donor5_stage1_240_min_con_1":"18.23","Donor5_stage1_240_min_con_2":"18.36","Donor5_stage1_480_min_con_1":"17.91","Donor5_stage1_480_min_con_2":"17.99","Donor5_stage2_0_min_Verum_1":"17.46","Donor5_stage2_0_min_Verum_2":"17.55","Donor5_stage2_60_min_Verum_1":"17.48","Donor5_stage2_60_min_Verum_2":"17.69","Donor5_stage2_120_min_Verum_1":"17.28","Donor5_stage2_120_min_Verum_2":"17.53","Donor5_stage2_240_min_Verum_1":"18.34","Donor5_stage2_240_min_Verum_2":"18.5","Donor5_stage2_480_min_Verum_1":"17.12","Donor5_stage2_480_min_Verum_2":"16.96","Donor6_stage1_0_min_con_1":"18.37","Donor6_stage1_0_min_con_2":"18.48","Donor6_stage1_60_min_con_1":"18.83","Donor6_stage1_60_min_con_2":"18.72","Donor6_stage1_120_min_con_1":"18.62","Donor6_stage1_120_min_con_2":"18.5","Donor6_stage1_240_min_con_1":"18.9","Donor6_stage1_240_min_con_2":"19.12","Donor6_stage1_480_min_con_1":"18.49","Donor6_stage1_480_min_con_2":"18.3","Donor6_stage2_0_min_Verum_1":"18.04","Donor6_stage2_0_min_Verum_2":"18.05","Donor6_stage2_60_min_Verum_1":"18.26","Donor6_stage2_60_min_Verum_2":"18.02","Donor6_stage2_120_min_Verum_1":"17.56","Donor6_stage2_120_min_Verum_2":"17.68","Donor6_stage2_240_min_Verum_1":"18.54","Donor6_stage2_240_min_Verum_2":"18.6","Donor6_stage2_480_min_Verum_1":"17.52","Donor6_stage2_480_min_Verum_2":"17.44","Donor7_stage1_0_min_con_1":"17.84","Donor7_stage1_0_min_con_2":"18.02","Donor7_stage1_60_min_con_1":"18.09","Donor7_stage1_60_min_con_2":"18.15","Donor7_stage1_120_min_con_1":"18.16","Donor7_stage1_120_min_con_2":"17.94","Donor7_stage1_240_min_con_1":"18.42","Donor7_stage1_240_min_con_2":"18.5","Donor7_stage1_480_min_con_1":"18.08","Donor7_stage1_480_min_con_2":"17.99","Donor7_stage2_0_min_Placebo_1":"17.91","Donor7_stage2_0_min_Placebo_2":"17.67","Donor7_stage2_60_min_Placebo_1":"18.07","Donor7_stage2_60_min_Placebo_2":"17.87","Donor7_stage2_120_min_Placebo_1":"17.91","Donor7_stage2_120_min_Placebo_2":"17.96","Donor7_stage2_240_min_Placebo_1":"18.47","Donor7_stage2_240_min_Placebo_2":"18.59","Donor7_stage2_480_min_Placebo_1":"18.05","Donor7_stage2_480_min_Placebo_2":"17.93","Donor8_stage1_0_min_con_1":"18.37","Donor8_stage1_0_min_con_2":"18.45","Donor8_stage1_60_min_con_1":"18","Donor8_stage1_60_min_con_2":"18.06","Donor8_stage1_120_min_con_1":"18.25","Donor8_stage1_120_min_con_2":"18.17","Donor8_stage1_240_min_con_1":"18.72","Donor8_stage1_240_min_con_2":"18.7","Donor8_stage1_480_min_con_1":"18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}