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.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST002461 |
| Study Title | Untargeted metabolomics of HUVECs subjected to hypoxia-reoxygenation |
| Study Summary | Acute 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 |
| 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 |
| ojacksonweaver@tulane.edu | |
| Phone | 5049885111 |
| Submit Date | 2023-01-30 |
| Num Groups | 2 |
| Total Subjects | 12 |
| Analysis Type Detail | LC-MS |
| Release Date | 2023-03-31 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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 |
|---|---|---|
| SA246367 | HR3 | hypoxia/reoxygenation |
| SA246368 | HR4 | hypoxia/reoxygenation |
| SA246369 | HR6 | hypoxia/reoxygenation |
| SA246370 | HR2 | hypoxia/reoxygenation |
| SA246371 | HR5 | hypoxia/reoxygenation |
| SA246372 | HR1 | hypoxia/reoxygenation |
| SA246373 | N3 | normoxia |
| SA246374 | N2 | normoxia |
| SA246375 | N4 | normoxia |
| SA246376 | N5 | normoxia |
| SA246377 | N6 | normoxia |
| SA246378 | N1 | normoxia |
| 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 |
