Summary of Study ST004453
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 PR002813. The data can be accessed directly via it's Project DOI: 10.21228/M82554 This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST004453 |
| Study Title | The eicosanoid cascade promotes inflammaging in C. elegans |
| Study Summary | Chronic inflammation is a hallmark of aging, but the underlying mechanisms of how inflammatory signaling drives aging remain unclear. Inflammation is in part controlled by the lipid-based eicosanoid signaling cascade consisting of three branches defined by their lipid-metabolizing enzymes, namely cyclooxygenases (COXs), lipoxygenases (LOXs), and cytochrome P450 (CYPs) enzymes. In an unbiased small molecule screen for lifespan extension in C. elegans, we identify the sulfonyl fluoride SF-3 and show it to extend lifespan by inhibiting soluble epoxide hydrolase 1 (CEEH-1) of C. elegans, a key enzyme in the CYP branch of the eicosanoid cascade. In C. elegans, the hydrolysis of epoxy fatty acids by CEEH-1 generates toxic dihydroxyeicosanoids (DiHETs) that shorten lifespan by the chronic activation of the ATF-6 arm of the UPRER. Our results causally link the CYP branch of the eicosanoid cascade to aging via the hydrolysis of epoxides into DiHETs, some of which are toxic and thus provide a mechanism for how inflammatory signaling drives aging. |
| Institute | Scripps Research Institute |
| Department | Department of Molecular and Cellular Biology |
| Last Name | Clay |
| First Name | Khalyd |
| Address | 10550 N. Torrey Pines Rd., La Jolla, Ca, 92037, USA |
| khalydc@scripps.edu | |
| Phone | 9283698108 |
| Submit Date | 2025-07-21 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML, raw(Waters) |
| Analysis Type Detail | LC-MS |
| Release Date | 2026-01-12 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002813 |
| Project DOI: | doi: 10.21228/M82554 |
| Project Title: | The eicosanoid cascade promotes inflammaging in C. elegans: Quantification of oxylipins in C. elegans following knockout of CEEH-1 |
| Project Summary: | Chronic inflammation is a hallmark of aging, but the underlying mechanisms of how inflammatory signaling drives aging remain unclear. Inflammation is in part controlled by the lipid-based eicosanoid signaling cascade consisting of three branches defined by their lipid-metabolizing enzymes, namely cyclooxygenases (COXs), lipoxygenases (LOXs), and cytochrome P450 (CYPs) enzymes. In an unbiased small molecule screen for lifespan extension in C. elegans, we identify the sulfonyl fluoride SF-3 and show it to extend lifespan by inhibiting soluble epoxide hydrolase 1 (CEEH-1) of C. elegans, a key enzyme in the CYP branch of the eicosanoid cascade. In C. elegans, the hydrolysis of epoxy fatty acids by CEEH-1 generates toxic dihydroxyeicosanoids (DiHETs) that shorten lifespan by the chronic activation of the ATF-6 arm of the UPRER. Our results causally link the CYP branch of the eicosanoid cascade to aging via the hydrolysis of epoxides into DiHETs, some of which are toxic and thus provide a mechanism for how inflammatory signaling drives aging. |
| Institute: | Scripps Research Institute |
| Department: | Molecular and Cellular Biology |
| Laboratory: | Petrascheck |
| Last Name: | Clay |
| First Name: | Khalyd |
| Address: | 10550 N. Torrey Pines Rd., La Jolla, Ca, 92037, USA |
| Email: | khalydc@scripps.edu |
| Phone: | 9283698108 |
Subject:
| Subject ID: | SU004615 |
| Subject Type: | Invertebrate |
| Subject Species: | Caenorhabditis elegans |
| Taxonomy ID: | 6239 |
Factors:
Subject type: Invertebrate; Subject species: Caenorhabditis elegans (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Genotype | Treatment |
|---|---|---|---|---|
| SA526986 | TQXS_04292022_019 | Blank | - | Ethanol |
| SA526987 | TQXS_05202021_003 | Blank | - | Ethanol |
| SA526988 | TQXS_02102021_008 | Blank | - | Ethanol |
| SA526992 | TQXS_05202021_026 | C.elegans lysate | ceeh-1 | - |
| SA526993 | TQXS_05202021_025 | C.elegans lysate | ceeh-1 | - |
| SA526994 | TQXS_05202021_024 | C.elegans lysate | ceeh-1 | - |
| SA526995 | TQXS_04292022_038 | C.elegans lysate | ceeh-2 | - |
| SA526996 | TQXS_04292022_039 | C.elegans lysate | ceeh-2 | - |
| SA526997 | TQXS_04292022_040 | C.elegans lysate | ceeh-2 | - |
| SA526989 | TQXS_02102021_023 | C.elegans lysate | N2 | - |
| SA526990 | TQXS_02102021_024 | C.elegans lysate | N2 | - |
| SA526991 | TQXS_02102021_025 | C.elegans lysate | N2 | - |
| SA526998 | TQXS_02102021_018 | Standard | - | Oxy STD 3 |
| SA526999 | TQXS_05202021_014 | Standard | - | Oxy STD 3 |
| SA527000 | TQXS_04292022_025 | Standard | - | Oxy STD 3 |
| SA527001 | TQXS_05202021_013 | Standard | - | Oxy STD 4 |
| SA527002 | TQXS_04292022_024 | Standard | - | Oxy STD 4 |
| SA527003 | TQXS_02102021_017 | Standard | - | Oxy STD 4 |
| SA527004 | TQXS_05202021_012 | Standard | - | Oxy STD 5 |
| SA527005 | TQXS_04292022_023 | Standard | - | Oxy STD 5 |
| SA527006 | TQXS_02102021_016 | Standard | - | Oxy STD 5 |
| SA527007 | TQXS_05202021_011 | Standard | - | Oxy STD 6 |
| SA527008 | TQXS_04292022_022 | Standard | - | Oxy STD 6 |
| SA527009 | TQXS_02102021_015 | Standard | - | Oxy STD 6 |
| SA527010 | TQXS_02102021_014 | Standard | - | Oxy STD 7 |
| SA527011 | TQXS_05202021_010 | Standard | - | Oxy STD 7 |
| SA527012 | TQXS_04292022_021 | Standard | - | Oxy STD 7 |
| SA527013 | TQXS_02102021_013 | Standard | - | Oxy STD 8 |
| SA527014 | TQXS_05202021_009 | Standard | - | Oxy STD 8 |
| SA527015 | TQXS_04292022_020 | Standard | - | Oxy STD 8 |
| Showing results 1 to 30 of 30 |
Collection:
| Collection ID: | CO004608 |
| Collection Summary: | Worms were grown on nematode growth media (NGM) + E. Coli OP50 petri dishes. At the first day of adulthood, worms were collected in a 10mL falcon tun using S-basel, and then washed five times with S-basal to remove any bacteria from the cuticle of the worms, and placed into 1.5 mL Eppendorf tubes. From here, samples were centrifuged at 4000 rpm for one minute, and the S-basal from above the worm pellet was removed. Following this, the samples were then centrifuged at 10,000 rpm at 4 °C for 10 minutes (Eppendorf Centrifuge 5340R), all liquid from above the pellet was again removed, and then a long-stemmed 20 µL pipet tip was used to remove any liquid from the bottom of the pellet. Filter paper cut into triangles was then placed in the tube, touching the samples to remove any remaining liquid from the sample for at least five minutes. Once the filter paper was removed and samples were as dry as possible, the Eppendorf tubes were shut and placed in liquid nitrogen before being placed in a -80 °C freezer until sample preparation. |
| Sample Type: | Worms |
Treatment:
| Treatment ID: | TR004624 |
| Treatment Summary: | "Blank" refers to ethanol only. "Standards" (i.e Oxy Std X) are standards prepared according to Table 1 in SamplePrep. "C. elegans lysate" - no treatment was administered to animals, but deuterated standards prepared in Table 2 were spiked in as described in Sampleprep. |
Sample Preparation:
| Sampleprep ID: | SP004621 |
| Sampleprep Summary: | Low-binding Eppendorf tubes were weighed, then frozen worm pellet was dislodged from its previous Eppendorf tube using a hypodermic needle and transferred to the low-binding tube to weigh again for worm weight. 10 µL of antioxidant (mix of 0.2 mg/mL triphenylphosphine (TPP), 0.2 mg/mL butylated hydroxytoluene (BHT), 1 mg/mL ethylenediaminetetraacetic acid (EDTA)), 10 µL of Type I internal standard mix of deuterated fatty acid standards (, 5-HETE-d8, 8,9-EET-d11, 15(S)-HETE-d8, PGB2-d4, 8,9-DiHETrE-d11, 9-HODE-d4, LTB4-d4), 1.2 µL of 10 mmol AUDA, 100 µL of PBS, and two to three stainless steel beads were added to the worm sample before being homogenized three times consecutively at 5 M/s for 30 seconds with five second pauses between on an Omni Bead Ruptor (Kennesaw, GA). After homogenization, 900 µL of PBS was added, then these samples were then set aside while the Oasis HLB 3 cc (60 mg) solid phase extraction (SPE) cartridges (Waters Co.) were being prepared. The SPE columns were prepared by sequentially adding 2 mL ethyl acetate, 4 mL methanol, and 2 mL 95:5 v/v DI water:methanol. The samples were then loaded onto the SPE column and washed with 1.5 mL 95:5 v/v DI water:methanol. The collection tube below the SPE column was then replaced and collection of samples was performed using 0.5 mL methanol, followed by 1 mL ethyl acetate. These samples were then transferred to 2 mL low-binding Eppendorf tubes, 6 µL of 30% glycerol in methanol was added, and samples were concentrated in SpeedVac Concentrator (Savant, Hyannis, MA) until all solvent was evaporated. Once all solvent was evaporated, samples were resuspended in 100 µL of 75:25 methanol:water containing 10 nM of CUDA (12-[[(cyclohexylamino)carbonyl]amino]-dodecanoic acid), a Type II internal standard, and vortexed for 10 minutes. Samples were then transferred to amber vials (Wheaton, Millville, NJ) with 350 µL glass inserts (Wheaton), purged with argon, and placed in a -80 °C freezer until mass spectrometry analysis. A standard curve was prepared using a mixture of the Type I deuterated internal standards mentioned above along with all the PUFAs, hydroxy-, epoxy-, and dihydroxy-PUFAs, prostaglandins, thromboxanes, leukotrienes, lipoxins, and resolvins listed in chemicals. Five calibration curve standards were prepared via serial dilution from the stock concentration of the non-deuterated compounds (Table 1), based on biological levels while the Type I deuterated standard mixture were added at the same concentration in all calibration standards (Table 2). |
| Sampleprep Protocol Filename: | Sampleprep_updated.pdf |
| Sampleprep Protocol Comments: | Tables are located in document. |
Combined analysis:
| Analysis ID | AN007457 |
|---|---|
| Chromatography ID | CH005647 |
| MS ID | MS007149 |
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Waters Acquity |
| Column | Waters ACQUITY UPLC CSH C18 (150 x 2.1mm,1.7um) |
| MS Type | ESI |
| MS instrument type | Triple quadrupole |
| MS instrument name | Waters Xevo TQ-XS |
| Ion Mode | NEGATIVE |
| Units | nM (STDs) pmol/g (analyte) |
Chromatography:
| Chromatography ID: | CH005647 |
| Methods Filename: | Elution_Gradient.pdf |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters ACQUITY UPLC CSH C18 (150 x 2.1mm,1.7um) |
| Column Temperature: | 50 |
| Flow Gradient: | 0min, 35% B; 1min, 40% B; 3min, 55% B; 8.5min, 65% B; 12.5min, 72% B; 15min, 82% B; 16min, 100% B; 18.1min, 35% B |
| Flow Rate: | 250 uL/min |
| Solvent A: | 100% water; 0.1% acetic acid |
| Solvent B: | 84% acetonitrile/16% methanol; 0.1% acetic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS007149 |
| Analysis ID: | AN007457 |
| Instrument Name: | Waters Xevo TQ-XS |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Targeted oxylipin analysis was performed using negative mode electrospray ionization (ESI-) triple quadrupole extra sensitive tandem mass spectrometer (TQ-XS LC-MS/MS) from Waters Co. (Milford, MA), equipped with a Waters ACQUITY SDS pump and Waters ACQUITY CM detector. A Waters ACQUITY FTN autosampler was kept at 10 °C. These studies are performed utilizing a Waters ACQUITY UPLC CSH C18 column (2.1 x 150 mm, 1.7 µm, serial no. 01853301115122), equipped with a Waters ACQUITY ULPC CSH C18 (2.1 x 5 mm, 1.7 µm) VanGuard Pre-Column. An aqueous phase of 99.9:0.1 water:acetic acid and an organic phase of 84:16:0.1 acetonitrile:methanol:acetic acid were used for LC separation, beginning at 35% organic (65% aqueous) ramping to 100% organic over 22 minutes. The column temperature was maintained at 50 °C. The gradient elution was performed at a 250 µL/min flow rate. As over 100 oxylipins are analyzed with this method, ten batches are utilized each with their own deuterium standard with similar properties and retention times to the metabolites in that batch, and CUDA is used an internal standard overall. All optimized parameters (CV, CE, LLOD, ULOD, RT, and MRM for the analysis of the hydroxy-, epoxy-, and dihydroxy-PUFAs in this study by mass spectrometry is shown in Table 3. Samples consisted of at least five mg of worms per replicate. Raw mass spectrometry data was analyzed using MassLynx V4.2, and processed data was plotted via GraphPad Prism. Calibration standards were excluded for signal-to-noise (S/N) <10 and deviation between predicted standard concentration and measured concentration >20%. Measured analyte concentrations within the samples were excluded from further analysis if below the lower limit of detection (LLOD), above the upper LOD (ULOD), or S/N<10. |
| Ion Mode: | NEGATIVE |
| Acquisition Parameters File: | MS_aquisition_updated.pdf |
