Summary of Study ST002461

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 PR001588. The data can be accessed directly via it's Project DOI: 10.21228/M8F71Q This work is supported by NIH grant, U2C- DK119886.

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Study IDST002461
Study TitleUntargeted metabolomics of HUVECs subjected to hypoxia-reoxygenation
Study SummaryAcute hemorrhage commonly leads to coagulopathy and organ dysfunction or failure. Recent evidence suggests that damage to the endothelial glycocalyx contributes to these adverse outcomes. The physiological events mediating acute glycocalyx shedding are undefined, however. Here, we show that succinate accumulation within endothelial cells drives glycocalyx degradation through a membrane reorganization-mediated mechanism. We investigated this mechanism in a cultured endothelial cell hypoxia-reoxygenation model, in a rat model of hemorrhage, and in trauma patient plasma samples. We found that succinate metabolism by succinate dehydrogenase mediates glycocalyx damage through lipid oxidation and phospholipase A2-mediated membrane reorganization (increasing lysophospholipids), promoting the interaction of MMP24 and MMP25 with glycocalyx constituents. In trauma patients, we found that succinate levels were associated with glycocalyx damage and the development of coagulopathy, and that interaction of MMP24 and syndecan-1 were elevated compared to healthy controls. This establishes a novel metabolic cascade mediating the endotheliopathy of traumatic hemorrhage.
Institute
Tulane University School of Medicine
DepartmentSurgery
LaboratoryTulane Trauma and Critical Care Research Lab
Last NameJackson-Weaver
First NameOlan
Address1430 Tulane Ave, Department of Surgery, School of Medicine
Emailojacksonweaver@tulane.edu
Phone5049885111
Submit Date2023-01-30
Num Groups2
Total Subjects12
Analysis Type DetailLC-MS
Release Date2023-03-31
Release Version1
Olan Jackson-Weaver Olan Jackson-Weaver
https://dx.doi.org/10.21228/M8F71Q
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001588
Project DOI:doi: 10.21228/M8F71Q
Project Title:Untargeted metabolomics of HUVECs subject to hypoxia/reoxygenation
Project Summary:Metabolomics analysis was performed on HUVECs subjected to hypoxia-reoxygenation to investigate metabolic alterations caused by this perturbation.
Institute:Tulane University
Department:Surgery
Laboratory:Tulane Trauma and Critical Care Research Lab
Last Name:Jackson-Weaver
First Name:Olan
Address:1430 Tulane Ave, Department of Surgery, School of Medicine, New Orleans, LA, 70112, USA
Email:ojacksonweaver@tulane.edu
Phone:5049885111

Subject:

Subject ID:SU002551
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

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

mb_sample_id local_sample_id Treatment
SA246367HR3hypoxia/reoxygenation
SA246368HR4hypoxia/reoxygenation
SA246369HR6hypoxia/reoxygenation
SA246370HR2hypoxia/reoxygenation
SA246371HR5hypoxia/reoxygenation
SA246372HR1hypoxia/reoxygenation
SA246373N3normoxia
SA246374N2normoxia
SA246375N4normoxia
SA246376N5normoxia
SA246377N6normoxia
SA246378N1normoxia
Showing results 1 to 12 of 12

Collection:

Collection ID:CO002544
Collection Summary:HUVECs were grown to confluence and subjected to normoxia or hypoxia (2% oxygen for 30 minutes) followed by reoxygenation for 30 minutes. Cells were washed with PBS and scraped from the plates. They were then flash frozen and saved for analysis.
Collection Protocol Filename:HUVECmetabolomics.docx
Sample Type:Endothelial cells
Storage Conditions:-80℃

Treatment:

Treatment ID:TR002563
Treatment Summary:Cells were subjected to either normoxia or 30 minutes of hypoxia (2%) followed by 30 minutes of reoxygenation.

Sample Preparation:

Sampleprep ID:SP002557
Sampleprep Summary:Samples were thawed and add with 800μL of 80% methanol, vortexed for 30 s. Then all samples were extracted at 4°C with ultrasound for 30 mins, kept at -40°C for 1 h, and centrifuged at 12000 rpm and 4°C for 15 mins. Finally, supernatant dried down under a nitrogen gas, the residue was resuspended with 100μL of 80% methanol and 5μL of DL-o-Chlorophenylalanine (140μg/mL) for LC-MS analysis.

Combined analysis:

Analysis ID AN004015 AN004016
Analysis type MS MS
Chromatography type Normal phase Normal phase
Chromatography system Ultimate 3000LC (Thermo) Ultimate 3000LC (Thermo)
Column Waters ACQUITY UPLC HSS CN (100 x 2.1mm,1.8um) Waters ACQUITY UPLC HSS CN (100 x 2.1mm,1.8um)
MS Type ESI ESI
MS instrument type Single quadrupole Single quadrupole
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE NEGATIVE
Units Peak area Peak area

Chromatography:

Chromatography ID:CH002966
Chromatography Summary:Separation is performed by Ultimate 3000LC combined with Q Exactive MS (Thermo) and screened with ESI-MS. The LC system is comprised of an ACQUITY UPLC HSS T3 (100×2.1mm 1.8 μm) with Ultimate 3000LC. The mobile phase is composed of solvent A (0.05% formic acid-water) and solvent B (acetonitrile) with a gradient elution (0-1 min, 5% B; 1-12 min, 5-95% B; 12-13.5 min, 95% B; 13.5-13.6 min, 95-5% B; 13.6-16.0 min, 5% B). The flow rate of the mobile phase is 0.3 mL·min-1 . The column temperature is maintained at 40°C, and the sample manager temperature is set at 4°C. Mass spectrometry parameters in ESI+ and ESI- mode are listed as follows: ESI+: Heater Temp 300 °C; Sheath Gas Flow rate, 45 arb; Aux Gas Flow Rate, 15 arb; Sweep Gas Flow Rate, 1 arb; spray voltage, 3.0 kV; Capillary Temp, 350 °C; S-Lens RF Level, 30%. ESI-: Heater Temp 300 °C, Sheath Gas Flow rate, 45 arb; Aux Gas Flow Rate, 15arb; Sweep Gas Flow Rate, 1 arb; spray voltage, 3.2 kV; Capillary Temp,350 °C; S-Lens RF Level, 60%.
Instrument Name:Ultimate 3000LC (Thermo)
Column Name:Waters ACQUITY UPLC HSS CN (100 x 2.1mm,1.8um)
Column Temperature:40°C
Flow Gradient:0-1 min, 5% B; 1-12 min, 5-95% B; 12-13.5 min, 95% B; 13.5-13.6 min, 95-5% B; 13.6-16.0 min, 5% B
Flow Rate:0.3 mL·min-1
Solvent A:100% water; 0.05% formic acid
Solvent B:100% acetonitrile
Chromatography Type:Normal phase

MS:

MS ID:MS003762
Analysis ID:AN004015
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Single quadrupole
MS Type:ESI
MS Comments:ESI+: Heater Temp 300 °C; Sheath Gas Flow rate, 45 arb; Aux Gas Flow Rate, 15 arb; Sweep Gas Flow Rate, 1 arb; spray voltage, 3.0 kV; Capillary Temp, 350 °C; S-Lens RF Level, 30%.
Ion Mode:POSITIVE
  
MS ID:MS003763
Analysis ID:AN004016
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Single quadrupole
MS Type:ESI
MS Comments:ESI-: Heater Temp 300 °C, Sheath Gas Flow rate, 45 arb; Aux Gas Flow Rate, 15arb; Sweep Gas Flow Rate, 1 arb; spray voltage, 3.2 kV; Capillary Temp,350 °C; S-Lens RF Level, 60%.
Ion Mode:NEGATIVE
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