Summary of Study ST003050

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 PR001899. The data can be accessed directly via it's Project DOI: 10.21228/M88147 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	ST003050
Study Title	Plasma instead of serum avoids critical confounding of clinical metabolomics studies by platelets (Part 1/3 - Plasma and serum eicosadomics)
Study Summary	Metabolomics is an emerging and powerful molecular profiling method supporting clinical investigations. Serum and plasma are commonly used without rational prioritization. Serum is collected after blood coagulation, a complex biochemical process involving active platelet metabolism. This may affect the metabolome and increase the variance as platelet counts and function may vary substantially in individuals. A multi-omics approach systematically investigating the suitability of serum and plasma for clinical studies demonstrated that metabolites correlated well (n=461, R2=0.991), whereas lipid mediators (n=104, R2=0.906) and proteins (n=322, R2=0.860) differed substantially between specimen. Independently, analysis of platelet releasates identified most biomolecules significantly enriched in serum when compared to plasma. A prospective, randomized, controlled parallel group metabolomics trial with acetylsalicylic acid administered for 7 days demonstrated that the apparent drug effects significantly differ depending on analyzed specimen. Only serum analyses of healthy individuals suggested a significant downregulation of TXB2 and 12-HETE, which were specifically formed during coagulation in vitro. Plasma analyses reliably identified acetylsalicylic acid effects on metabolites and lipids occurring in vivo such as a decrease in polyunsaturated fatty acids. The present data suggests that plasma should be preferred above serum for clinical metabolomics studies as the serum metabolome may be substantially confounded by platelets.
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	2024-01-17
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2024-04-12
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001899
Project DOI:	doi: 10.21228/M88147
Project Title:	Plasma instead of serum avoids critical confounding of clinical metabolomics studies by platelets
Project Summary:	Metabolomics is an emerging and powerful molecular profiling method supporting clinical investigations. Serum and plasma are commonly used without rational prioritization. Serum is collected after blood coagulation, a complex biochemical process involving active platelet metabolism. This may affect the metabolome and increase the variance as platelet counts and function may vary substantially in individuals. A multi-omics approach systematically investigating the suitability of serum and plasma for clinical studies demonstrated that metabolites correlated well (n=461, R2=0.991), whereas lipid mediators (n=104, R2=0.906) and proteins (n=322, R2=0.860) differed substantially between specimen. Independently, analysis of platelet releasates identified most biomolecules significantly enriched in serum when compared to plasma. A prospective, randomized, controlled parallel group metabolomics trial with acetylsalicylic acid administered for 7 days demonstrated that the apparent drug effects significantly differ depending on analyzed specimen. Only serum analyses of healthy individuals suggested a significant downregulation of TXB2 and 12-HETE, which were specifically formed during coagulation in vitro. Plasma analyses reliably identified acetylsalicylic acid effects on metabolites and lipids occurring in vivo such as a decrease in polyunsaturated fatty acids. The present data suggests that plasma should be preferred above serum for clinical metabolomics studies as the serum metabolome may be substantially confounded by platelets.
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
Publications:	https://doi.org/10.1021/acs.jproteome.3c00761

Subject:

Subject ID:	SU003165
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

Factors:

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

mb_sample_id	local_sample_id	Sample material	Treatment
SA331296	Donor_1_Plasma_after_Aspirin_1	Plasma	after_Aspirin
SA331297	Donor_5_Plasma_after_Aspirin_1	Plasma	after_Aspirin
SA331298	Donor_6_Plasma_after_Aspirin_1	Plasma	after_Aspirin
SA331299	Donor_6_Plasma_after_Aspirin_2	Plasma	after_Aspirin
SA331300	Donor_1_Plasma_after_Aspirin_2	Plasma	after_Aspirin
SA331301	Donor_4_Plasma_after_Aspirin_2	Plasma	after_Aspirin
SA331302	Donor_5_Plasma_after_Aspirin_2	Plasma	after_Aspirin
SA331303	Donor_2_Plasma_after_Aspirin_1	Plasma	after_Aspirin
SA331304	Donor_4_Plasma_after_Aspirin_1	Plasma	after_Aspirin
SA331305	Donor_2_Plasma_after_Aspirin_2	Plasma	after_Aspirin
SA331306	Donor_3_Plasma_after_Aspirin_2	Plasma	after_Aspirin
SA331307	Donor_3_Plasma_after_Aspirin_1	Plasma	after_Aspirin
SA331308	Donor_9_Plasma_after_Omega3_2	Plasma	after_Omega3
SA331309	Donor_9_Plasma_after_Omega3_1	Plasma	after_Omega3
SA331310	Donor_7_Plasma_after_Omega3_1	Plasma	after_Omega3
SA331311	Donor_8_Plasma_after_Omega3_1	Plasma	after_Omega3
SA331312	Donor_8_Plasma_after_Omega3_2	Plasma	after_Omega3
SA331313	Donor_12_Plasma_after_Omega3_2	Plasma	after_Omega3
SA331314	Donor_12_Plasma_after_Omega3_1	Plasma	after_Omega3
SA331315	Donor_7_Plasma_after_Omega3_2	Plasma	after_Omega3
SA331316	Donor_11_Plasma_after_Omega3_2	Plasma	after_Omega3
SA331317	Donor_11_Plasma_after_Omega3_1	Plasma	after_Omega3
SA331318	Donor_10_Plasma_after_Omega3_2	Plasma	after_Omega3
SA331319	Donor_10_Plasma_after_Omega3_1	Plasma	after_Omega3
SA331320	Donor_4_Plasma_before_Aspirin_1	Plasma	before_Aspirin
SA331321	Donor_2_Plasma_before_Aspirin_2	Plasma	before_Aspirin
SA331322	Donor_2_Plasma_before_Aspirin_1	Plasma	before_Aspirin
SA331323	Donor_1_Plasma_before_Aspirin_1	Plasma	before_Aspirin
SA331324	Donor_1_Plasma_before_Aspirin_2	Plasma	before_Aspirin
SA331325	Donor_3_Plasma_before_Aspirin_1	Plasma	before_Aspirin
SA331326	Donor_3_Plasma_before_Aspirin_2	Plasma	before_Aspirin
SA331327	Donor_6_Plasma_before_Aspirin_1	Plasma	before_Aspirin
SA331328	Donor_5_Plasma_before_Aspirin_2	Plasma	before_Aspirin
SA331329	Donor_5_Plasma_before_Aspirin_1	Plasma	before_Aspirin
SA331330	Donor_6_Plasma_before_Aspirin_2	Plasma	before_Aspirin
SA331331	Donor_4_Plasma_before_Aspirin_2	Plasma	before_Aspirin
SA331332	Donor_8_Plasma_before_Omega3_2	Plasma	before_Omega3
SA331333	Donor_9_Plasma_before_Omega3_2	Plasma	before_Omega3
SA331334	Donor_8_Plasma_before_Omega3_1	Plasma	before_Omega3
SA331335	Donor_7_Plasma_before_Omega3_2	Plasma	before_Omega3
SA331336	Donor_7_Plasma_before_Omega3_1	Plasma	before_Omega3
SA331337	Donor_10_Plasma_before_Omega3_1	Plasma	before_Omega3
SA331338	Donor_9_Plasma_before_Omega3_1	Plasma	before_Omega3
SA331339	Donor_12_Plasma_before_Omega3_1	Plasma	before_Omega3
SA331340	Donor_10_Plasma_before_Omega3_2	Plasma	before_Omega3
SA331341	Donor_11_Plasma_before_Omega3_2	Plasma	before_Omega3
SA331342	Donor_12_Plasma_before_Omega3_2	Plasma	before_Omega3
SA331343	Donor_11_Plasma_before_Omega3_1	Plasma	before_Omega3
SA331284	Donor_3_Plasma_2	Plasma	Plasma_Serum_comparison
SA331285	Donor_3_Plasma_1	Plasma	Plasma_Serum_comparison
SA331286	Donor_2_Plasma_1	Plasma	Plasma_Serum_comparison
SA331287	Donor_4_Plasma_1	Plasma	Plasma_Serum_comparison
SA331288	Donor_2_Plasma_2	Plasma	Plasma_Serum_comparison
SA331289	Donor_5_Plasma_1	Plasma	Plasma_Serum_comparison
SA331290	Donor_6_Plasma_2	Plasma	Plasma_Serum_comparison
SA331291	Donor_6_Plasma_1	Plasma	Plasma_Serum_comparison
SA331292	Donor_5_Plasma_2	Plasma	Plasma_Serum_comparison
SA331293	Donor_1_Plasma_2	Plasma	Plasma_Serum_comparison
SA331294	Donor_4_Plasma_2	Plasma	Plasma_Serum_comparison
SA331295	Donor_1_Plasma_1	Plasma	Plasma_Serum_comparison
SA331356	Donor_5_Serum_after_Aspirin_1	Serum	after_Aspirin
SA331357	Donor_5_Serum_after_Aspirin_2	Serum	after_Aspirin
SA331358	Donor_4_Serum_after_Aspirin_2	Serum	after_Aspirin
SA331359	Donor_6_Serum_after_Aspirin_2	Serum	after_Aspirin
SA331360	Donor_6_Serum_after_Aspirin_1	Serum	after_Aspirin
SA331361	Donor_1_Serum_after_Aspirin_1	Serum	after_Aspirin
SA331362	Donor_2_Serum_after_Aspirin_1	Serum	after_Aspirin
SA331363	Donor_4_Serum_after_Aspirin_1	Serum	after_Aspirin
SA331364	Donor_2_Serum_after_Aspirin_2	Serum	after_Aspirin
SA331365	Donor_1_Serum_after_Aspirin_2	Serum	after_Aspirin
SA331366	Donor_3_Serum_after_Aspirin_2	Serum	after_Aspirin
SA331367	Donor_3_Serum_after_Aspirin_1	Serum	after_Aspirin
SA331368	Donor_9_Serum_after_Omega3_1	Serum	after_Omega3
SA331369	Donor_9_Serum_after_Omega3_2	Serum	after_Omega3
SA331370	Donor_8_Serum_after_Omega3_2	Serum	after_Omega3
SA331371	Donor_8_Serum_after_Omega3_1	Serum	after_Omega3
SA331372	Donor_10_Serum_after_Omega3_1	Serum	after_Omega3
SA331373	Donor_7_Serum_after_Omega3_1	Serum	after_Omega3
SA331374	Donor_12_Serum_after_Omega3_1	Serum	after_Omega3
SA331375	Donor_7_Serum_after_Omega3_2	Serum	after_Omega3
SA331376	Donor_12_Serum_after_Omega3_2	Serum	after_Omega3
SA331377	Donor_11_Serum_after_Omega3_2	Serum	after_Omega3
SA331378	Donor_11_Serum_after_Omega3_1	Serum	after_Omega3
SA331379	Donor_10_Serum_after_Omega3_2	Serum	after_Omega3
SA331380	Donor_1_Serum_before_Aspirin_1	Serum	before_Aspirin
SA331381	Donor_4_Serum_before_Aspirin_2	Serum	before_Aspirin
SA331382	Donor_5_Serum_before_Aspirin_1	Serum	before_Aspirin
SA331383	Donor_5_Serum_before_Aspirin_2	Serum	before_Aspirin
SA331384	Donor_6_Serum_before_Aspirin_1	Serum	before_Aspirin
SA331385	Donor_4_Serum_before_Aspirin_1	Serum	before_Aspirin
SA331386	Donor_3_Serum_before_Aspirin_2	Serum	before_Aspirin
SA331387	Donor_1_Serum_before_Aspirin_2	Serum	before_Aspirin
SA331388	Donor_2_Serum_before_Aspirin_1	Serum	before_Aspirin
SA331389	Donor_2_Serum_before_Aspirin_2	Serum	before_Aspirin
SA331390	Donor_3_Serum_before_Aspirin_1	Serum	before_Aspirin
SA331391	Donor_6_Serum_before_Aspirin_2	Serum	before_Aspirin
SA331392	Donor_7_Serum_before_Omega3_1	Serum	before_Omega3
SA331393	Donor_10_Serum_before_Omega3_2	Serum	before_Omega3
SA331394	Donor_11_Serum_before_Omega3_1	Serum	before_Omega3
SA331395	Donor_11_Serum_before_Omega3_2	Serum	before_Omega3

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Collection:

Collection ID:	CO003158
Collection Summary:	SERUM/PLASMA: Blood samples were obtained at baseline and after 7 days intake of the study medication. On both study days two blood samples using 6 mL K3EDTA and serum collection tubes (both Vacuette, Greiner Bio-One GmbH, Kremsmünster, Austria) were obtained from each subject. EDTA-anticoagulated tubes were carefully inverted two times after blood draw and centrifuged immediately at room temperature at 2000 g for 10 min. In contrast, filled serum tubes were carefully inverted after blood draw and placed to sit upright for 15 to 30 minutes to allow clot formation. Then, tubes were centrifuged at room temperature at 2000 g for 10 min. Directly after centrifugation, 500 µL of plasma or serum, respectively, were transferred into pre-labelled Eppendorf safe-lock tubes and stored at -80°C until analysis.
Sample Type:	Blood (serum) and blood (plasma)

Treatment:

Treatment ID:	TR003174
Treatment Summary:	Subjects were randomized to receive either acetylsalicylic acid or Omega-3 capsules for 7 days. One study cohort was instructed to take 500 mg acetylsalicylic acid (Aspirin® 500 mg acetylsalicylic acid, Cellulose powder, maize starche) per day in the evening whereas the second study cohort was instructed to take two Omega-3 complex 870 mg capsules (Br. Böhm Omega-3 capsules, 1017 mg cold water fish oil equivalent to 870 mg Omega-3, consisting of 420 mg EPA, 330 mg DHA, 5μg Vitamin D equivalent to 200 IU, 6 mg Vitamin E, 30 mg Co-enzyme Q10) per day in the evening.

Treatment ID:

TR003174

Treatment Summary:

Subjects were randomized to receive either acetylsalicylic acid or Omega-3 capsules for 7 days. One study cohort was instructed to take 500 mg acetylsalicylic acid (Aspirin® 500 mg acetylsalicylic acid, Cellulose powder, maize starche) per day in the evening whereas the second study cohort was instructed to take two Omega-3 complex 870 mg capsules (Br. Böhm Omega-3 capsules, 1017 mg cold water fish oil equivalent to 870 mg Omega-3, consisting of 420 mg EPA, 330 mg DHA, 5μg Vitamin D equivalent to 200 IU, 6 mg Vitamin E, 30 mg Co-enzyme Q10) per day in the evening.

Sample Preparation:

Sampleprep ID:	SP003171
Sampleprep Summary:	SERUM/PLASMA: Frozen EDTA-anticoagulated plasma or serum was freshly thawed on ice. For precipitation of proteins, plasma or serum (400 µL) was mixed with cold EtOH (1.6 mL, abs. 99%, -20°C; AustroAlco) 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 buffer (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:

SP003171

Sampleprep Summary:

SERUM/PLASMA: Frozen EDTA-anticoagulated plasma or serum was freshly thawed on ice. For precipitation of proteins, plasma or serum (400 µL) was mixed with cold EtOH (1.6 mL, abs. 99%, -20°C; AustroAlco) 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 buffer (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	AN005001
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:	CH003779
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:	MS004741
Analysis ID:	AN005001
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