Summary of Study ST003137

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001950. The data can be accessed directly via it's Project DOI: 10.21228/M8NX50 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST003137
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
Submit Date	2024-03-18
Raw Data Available	Yes
Raw Data File Type(s)	mzML, raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2025-06-02
Release Version	1

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Project:

Project ID:	PR001950
Project DOI:	doi: 10.21228/M8NX50
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

Subject:

Subject ID:	SU003254
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male
Species Group:	Mammals

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment
SA339888	Donor33_stage1_480_min_con_1	con
SA339889	Donor25_stage1_480_min_con_2	con
SA339890	Donor25_stage1_480_min_con_1	con
SA339891	Donor25_stage1_240_min_con_2	con
SA339892	Donor25_stage1_240_min_con_1	con
SA339893	Donor33_stage1_120_min_con_2	con
SA339894	Donor33_stage1_240_min_con_1	con
SA339895	Donor25_stage1_120_min_con_2	con
SA339896	Donor24_stage1_60_min_con_1	con
SA339897	Donor33_stage1_0_min_con_1	con
SA339898	Donor33_stage1_480_min_con_2	con
SA339899	Donor33_stage1_240_min_con_2	con
SA339900	Donor33_stage1_0_min_con_2	con
SA339901	Donor32_stage1_60_min_con_1	con
SA339902	Donor24_stage1_240_min_con_1	con
SA339903	Donor24_stage1_120_min_con_2	con
SA339904	Donor24_stage1_120_min_con_1	con
SA339905	Donor24_stage1_60_min_con_2	con
SA339906	Donor24_stage1_240_min_con_2	con
SA339907	Donor24_stage1_480_min_con_1	con
SA339908	Donor24_stage1_480_min_con_2	con
SA339909	Donor25_stage1_0_min_con_1	con
SA339910	Donor25_stage1_0_min_con_2	con
SA339911	Donor25_stage1_60_min_con_1	con
SA339912	Donor32_stage1_480_min_con_2	con
SA339913	Donor33_stage1_60_min_con_1	con
SA339914	Donor32_stage1_60_min_con_2	con
SA339915	Donor33_stage1_60_min_con_2	con
SA339916	Donor33_stage1_120_min_con_1	con
SA339917	Donor25_stage1_60_min_con_2	con
SA339918	Donor32_stage1_120_min_con_1	con
SA339919	Donor32_stage1_120_min_con_2	con
SA339920	Donor32_stage1_480_min_con_1	con
SA339921	Donor32_stage1_240_min_con_2	con
SA339922	Donor32_stage1_240_min_con_1	con
SA339923	Donor25_stage1_120_min_con_1	con
SA339924	Donor26_stage1_480_min_con_1	con
SA339925	Donor28_stage1_0_min_con_1	con
SA339926	Donor27_stage1_480_min_con_2	con
SA339927	Donor28_stage1_0_min_con_2	con
SA339928	Donor29_stage1_480_min_con_2	con
SA339929	Donor29_stage1_240_min_con_2	con
SA339930	Donor29_stage1_480_min_con_1	con
SA339931	Donor27_stage1_480_min_con_1	con
SA339932	Donor27_stage1_240_min_con_2	con
SA339933	Donor30_stage1_0_min_con_1	con
SA339934	Donor30_stage1_0_min_con_2	con
SA339935	Donor27_stage1_60_min_con_2	con
SA339936	Donor27_stage1_120_min_con_1	con
SA339937	Donor27_stage1_240_min_con_1	con
SA339938	Donor27_stage1_120_min_con_2	con
SA339939	Donor29_stage1_240_min_con_1	con
SA339940	Donor29_stage1_120_min_con_2	con
SA339941	Donor28_stage1_480_min_con_1	con
SA339942	Donor28_stage1_240_min_con_2	con
SA339943	Donor28_stage1_480_min_con_2	con
SA339944	Donor24_stage1_0_min_con_2	con
SA339945	Donor29_stage1_0_min_con_1	con
SA339946	Donor29_stage1_0_min_con_2	con
SA339947	Donor28_stage1_240_min_con_1	con
SA339948	Donor28_stage1_120_min_con_2	con
SA339949	Donor29_stage1_60_min_con_2	con
SA339950	Donor29_stage1_120_min_con_1	con
SA339951	Donor29_stage1_60_min_con_1	con
SA339952	Donor28_stage1_60_min_con_1	con
SA339953	Donor28_stage1_120_min_con_1	con
SA339954	Donor28_stage1_60_min_con_2	con
SA339955	Donor27_stage1_60_min_con_1	con
SA339956	Donor27_stage1_0_min_con_2	con
SA339957	Donor31_stage1_120_min_con_1	con
SA339958	Donor31_stage1_120_min_con_2	con
SA339959	Donor31_stage1_60_min_con_2	con
SA339960	Donor31_stage1_60_min_con_1	con
SA339961	Donor31_stage1_0_min_con_1	con
SA339962	Donor31_stage1_0_min_con_2	con
SA339963	Donor31_stage1_240_min_con_1	con
SA339964	Donor31_stage1_240_min_con_2	con
SA339965	Donor26_stage1_0_min_con_1	con
SA339966	Donor32_stage1_0_min_con_1	con
SA339967	Donor26_stage1_0_min_con_2	con
SA339968	Donor26_stage1_60_min_con_1	con
SA339969	Donor31_stage1_480_min_con_1	con
SA339970	Donor31_stage1_480_min_con_2	con
SA339971	Donor26_stage1_60_min_con_2	con
SA339972	Donor26_stage1_120_min_con_1	con
SA339973	Donor30_stage1_120_min_con_2	con
SA339974	Donor30_stage1_240_min_con_1	con
SA339975	Donor30_stage1_120_min_con_1	con
SA339976	Donor30_stage1_60_min_con_2	con
SA339977	Donor27_stage1_0_min_con_1	con
SA339978	Donor30_stage1_60_min_con_1	con
SA339979	Donor30_stage1_240_min_con_2	con
SA339980	Donor30_stage1_480_min_con_1	con
SA339981	Donor26_stage1_240_min_con_1	con
SA339982	Donor26_stage1_120_min_con_2	con
SA339983	Donor26_stage1_240_min_con_2	con
SA339984	Donor26_stage1_480_min_con_2	con
SA339985	Donor30_stage1_480_min_con_2	con
SA339986	Donor32_stage1_0_min_con_2	con
SA339987	Donor19_stage1_0_min_con_1	con

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Collection:

Collection ID:	CO003247
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 ID:	TR003263
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.

Treatment ID:

TR003263

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.

Sample Preparation:

Sampleprep ID:	SP003261
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

Sampleprep ID:

SP003261

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

Combined analysis:

Analysis ID	AN005150
Chromatography ID	CH003898
MS ID	MS004886
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Vanquish
Column	Phenomenex Kinetex XB-C18 (150 x 2.1mm, 2.6um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive HF hybrid Orbitrap
Ion Mode	NEGATIVE
Units	normalized AUC

Chromatography:

Chromatography ID:	CH003898
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.
Instrument Name:	Thermo Vanquish
Column Name:	Phenomenex Kinetex XB-C18 (150 x 2.1mm, 2.6um)
Column Temperature:	40
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
Solvent A:	100% water; 0.2% formic acid
Solvent B:	90% acetonitrile/10% methanol; 0.2% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004886
Analysis ID:	AN005150
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
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).
Ion Mode:	NEGATIVE