#METABOLOMICS WORKBENCH patchthr33_20190922_071733 DATATRACK_ID:1818 STUDY_ID:ST001252 ANALYSIS_ID:AN002078 PROJECT_ID:PR000839 VERSION 1 CREATED_ON September 24, 2019, 3:24 pm #PROJECT PR:PROJECT_TITLE Eicosanoid Expression in Dermal Fibroblasts PR:PROJECT_SUMMARY Quantitative lipidomics studies examining eicosanoid levels in primary dermal PR:PROJECT_SUMMARY fibroblasts PR:INSTITUTE University of South Florida, Tampa PR:LAST_NAME Chalfant PR:FIRST_NAME Charles PR:ADDRESS 4202 E Fowler Ave, Tampa, FL 33620 PR:EMAIL cechalfant@usf.edu PR:PHONE 8139747103 #STUDY ST:STUDY_TITLE Eicosanoid profiles of dermal fibroblasts ST:STUDY_SUMMARY The sphingolipid, ceramide-1-phosphate (C1P), directly binds and activates Group ST:STUDY_SUMMARY IVA cytosolic phospholipase A2 (cPLA2) to generate eicosanoids. Due to the role ST:STUDY_SUMMARY of eicosanoids in wound healing, we choose to use our novel genetic mouse model ST:STUDY_SUMMARY expressing cPLA2 with an ablated C1P interaction site (KI) to examine the ST:STUDY_SUMMARY cPLA2/C1P interaction in wound healing. Wound closure rate was not affected, but ST:STUDY_SUMMARY wound maturation was enhanced by loss of the C1P/cPLA2α interaction based on ST:STUDY_SUMMARY the following findings. Wounds in KI mice displayed: i) increased infiltration ST:STUDY_SUMMARY of dermal fibroblasts into the wound environment; ii) increased wound tensile ST:STUDY_SUMMARY strength; and iii) higher Type I/Type III collagen ratios. These findings were ST:STUDY_SUMMARY recapitulated in vitro as primary dermal fibroblasts (pDFs) from KI mice showed ST:STUDY_SUMMARY significantly increased collagen deposition and migration velocity compared to ST:STUDY_SUMMARY WT and KO pDFs. Additionally, the KI showed an altered eicosanoid profile of ST:STUDY_SUMMARY reduced pro-inflammatory prostaglandins (PGE2) and increased levels of specific ST:STUDY_SUMMARY HETE species (5-HETE). Elevated 5-HETE levels promoted increased dermal ST:STUDY_SUMMARY fibroblast migration and collagen deposition. This “gain of function” role ST:STUDY_SUMMARY for the mutant cPLA2 was also linked to differential cellular localization of ST:STUDY_SUMMARY cPLA2α and 5-HETE biosynthetic factors. These studies demonstrate regulation of ST:STUDY_SUMMARY key biological mechanisms by a defined protein:lipid interaction in vivo and ST:STUDY_SUMMARY provide new insights into cPLA2 function. ST:INSTITUTE University of South Florida, Tampa ST:LAST_NAME Chalfant ST:FIRST_NAME Charles ST:ADDRESS 4202 E Fowler Ave, Tampa, FL 33620 ST:EMAIL cechalfant@usf.edu ST:PHONE 8139747103 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - Patrick Media 1 Genotype:Wild Type | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 2 Genotype:Wild Type | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 3 Genotype:Wild Type | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 4 Genotype:Wild Type | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 5 Genotype:Wild Type | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 6 Genotype:Wild Type | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 7 Genotype:Wild Type | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 8 Genotype:Wild Type | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 9 Genotype:cPLA2α KO | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 10 Genotype:cPLA2α KO | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 11 Genotype:cPLA2α KO | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 12 Genotype:cPLA2α KO | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 13 Genotype:cPLA2α KO | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 14 Genotype:cPLA2α KO | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 15 Genotype:cPLA2α KO | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 16 Genotype:cPLA2α KO | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 17 Genotype:cPLA2α KI | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 18 Genotype:cPLA2α KI | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 19 Genotype:cPLA2α KI | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 20 Genotype:cPLA2α KI | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 21 Genotype:cPLA2α KI | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 22 Genotype:cPLA2α KI | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 23 Genotype:cPLA2α KI | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 24 Genotype:cPLA2α KI | Treatment:Unscratched SUBJECT_SAMPLE_FACTORS - Patrick Media 25 Genotype:Wild Type | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 26 Genotype:cPLA2α KI | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 27 Genotype:cPLA2α KI | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 28 Genotype:Wild Type | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 29 Genotype:cPLA2α KI | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 30 Genotype:cPLA2α KI | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 31 Genotype:cPLA2α KO | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 32 Genotype:cPLA2α KO | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 33 Genotype:Wild Type | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 34 Genotype:Wild Type | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 35 Genotype:cPLA2α KI | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 36 Genotype:cPLA2α KI | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 37 Genotype:Wild Type | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 38 Genotype:Wild Type | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 39 Genotype:cPLA2α KI | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 40 Genotype:cPLA2α KI | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 41 Genotype:cPLA2α KO | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 42 Genotype:cPLA2α KO | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 43 Genotype:cPLA2α KO | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 44 Genotype:Wild Type | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 45 Genotype:Wild Type | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 46 Genotype:cPLA2α KO | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 47 Genotype:cPLA2α KO | Treatment:Scratched SUBJECT_SAMPLE_FACTORS - Patrick Media 48 Genotype:cPLA2α KO | Treatment:Scratched #COLLECTION CO:COLLECTION_SUMMARY pDFs obtained from WT, KI, and KO mice were plated at a density of 2 x 106 on 10 CO:COLLECTION_SUMMARY cm tissue culture plates in high glucose DMEM supplemented with 20% FBS (Gibco) CO:COLLECTION_SUMMARY and 2% penicillin/streptomycin. Following the overnight incubation cells were CO:COLLECTION_SUMMARY rested in 2% FBS (Gibco) 2% penicillin/streptomycin (Bio Whittaker) high glucose CO:COLLECTION_SUMMARY DMEM (Gibco) for 2 hours, then the media was exchanged with 0% FBS 2% CO:COLLECTION_SUMMARY penicillin/ high glucose DMEM, and mechanical trauma was induced on the CO:COLLECTION_SUMMARY monolayer by performing scratched across the diameter of the plate in an CO:COLLECTION_SUMMARY asterisk pattern using 4 x 20 ul pipette tips on a multichannel micro pipettor. CO:COLLECTION_SUMMARY Media was taken for lipidomic analysis at 0 h and 2 h. CO:SAMPLE_TYPE Fibroblasts #TREATMENT TR:TREATMENT_SUMMARY Primary Dermal Fibroblasts were plated at a density of 2 x 106 on 10 cm tissue TR:TREATMENT_SUMMARY culture plates in high glucose DMEM supplemented with 20% FBS (Gibco) and 2% TR:TREATMENT_SUMMARY penicillin/streptomycin. Following the overnight incubation cells were rested in TR:TREATMENT_SUMMARY 2% FBS (Gibco) 2% penicillin/streptomycin (Bio Whittaker) high glucose DMEM TR:TREATMENT_SUMMARY (Gibco) for 2 hours, then the media was exchanged with 0% FBS 2% penicillin/ TR:TREATMENT_SUMMARY high glucose DMEM, and mechanical trauma was induced on the monolayer by TR:TREATMENT_SUMMARY performing scratched across the diameter of the plate in an asterisk pattern TR:TREATMENT_SUMMARY using 4 x 20 l pipette tips on a multichannel micro pipettor. Media was taken TR:TREATMENT_SUMMARY for lipidomic analysis at 0 h and 2 h. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Samples were stored at − 80 °C until the time of analysis. Lipids were SP:SAMPLEPREP_SUMMARY extracted for eicosanoids. Eicosanoid Preparation Eicosanoids were extracted SP:SAMPLEPREP_SUMMARY using a modified extraction process from previously described. (1, 2). 4 mL of SP:SAMPLEPREP_SUMMARY media was combined with an IS mixture comprised of comprised of 10% methanol SP:SAMPLEPREP_SUMMARY (400 μl), glacial acetic acid (20 μl), and internal standard (20 μl) SP:SAMPLEPREP_SUMMARY containing the following deuterated eicosanoids (1.5 pmol/μl, 30 pmol total) SP:SAMPLEPREP_SUMMARY (All standards purchased from Cayman Chemicals): (d4) 6keto-Prostaglandin F1α, SP:SAMPLEPREP_SUMMARY (d4) Prostaglandin F2α, (d4) Prostaglandin E2, (d4) Prostaglandin D2, (d8) SP:SAMPLEPREP_SUMMARY 5-Hydroxyeicosa-tetranoic acid (5-HETE), (d8) 12-Hydroxyeicosa-tetranoic acid SP:SAMPLEPREP_SUMMARY (12-HETE) (d8) 15-Hydroxyeicosa-tetranoic acid (15-HETE), (d6) SP:SAMPLEPREP_SUMMARY 20-Hydroxyeicosa-tetranoic acid (20-HETE), (d11) 8,9 Epoxyeicosa-trienoic acid, SP:SAMPLEPREP_SUMMARY (d8) 14,15 Epoxyeicosa-trienoic acid, (d8) Arachidonic acid, (d5) SP:SAMPLEPREP_SUMMARY Eicosapentaenoic acid, (d5) Docosahexaenoic acid, (d4) Prostaglandin A2, (d4) SP:SAMPLEPREP_SUMMARY Leukotriene B4, (d4) Leukotriene C4, (d4) Leukotriene D4, (d4) Leukotriene E4, SP:SAMPLEPREP_SUMMARY (d5) 5(S),6(R)-Lipoxin A4, (d11) 5-iPF2α-VI, (d4) 8-iso Prostaglandin F2α, SP:SAMPLEPREP_SUMMARY (d11) (±)14,15-DHET, (d11) (±)8,9-DHET, (d11) (±)11,12-DHET, (d4) SP:SAMPLEPREP_SUMMARY Prostaglandin E1, (d4) Thromboxane B2, (d6) dihmo gamma linoleic acid, (d5) SP:SAMPLEPREP_SUMMARY Resolvin D2, (d5) Resolvin D1, (d5) Maresin 2, and (d5) Resolvin D3. Samples and SP:SAMPLEPREP_SUMMARY vial rinses (5% MeOH; 2 ml) were applied to Strata-X SPE columns (Phenomenex), SP:SAMPLEPREP_SUMMARY previously washed with methanol (2 ml) and then dH2O (2 ml). Eicosanoids eluted SP:SAMPLEPREP_SUMMARY with isopropanol (2 ml), were dried in vacuuo and reconstituted in EtOH:dH2O SP:SAMPLEPREP_SUMMARY (50:50;100 μl) prior to UPLC ESI-MS/MS analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Shimadzu Nexera X2 CH:COLUMN_NAME Acentis Express C18 column #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 5500 QTrap MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE MALDI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Sciex Analyst software was used for analysis #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS pmol of lipid MS_METABOLITE_DATA_START Samples Patrick Media 1 Patrick Media 2 Patrick Media 3 Patrick Media 4 Patrick Media 5 Patrick Media 6 Patrick Media 7 Patrick Media 8 Patrick Media 9 Patrick Media 10 Patrick Media 11 Patrick Media 12 Patrick Media 13 Patrick Media 14 Patrick Media 15 Patrick Media 16 Patrick Media 17 Patrick Media 18 Patrick Media 19 Patrick Media 20 Patrick Media 21 Patrick Media 22 Patrick Media 23 Patrick Media 24 Patrick Media 25 Patrick Media 26 Patrick Media 27 Patrick Media 28 Patrick Media 29 Patrick Media 30 Patrick Media 31 Patrick Media 32 Patrick Media 33 Patrick Media 34 Patrick Media 35 Patrick Media 36 Patrick Media 37 Patrick Media 38 Patrick Media 39 Patrick Media 40 Patrick Media 41 Patrick Media 42 Patrick Media 43 Patrick Media 44 Patrick Media 45 Patrick Media 46 Patrick Media 47 Patrick Media 48 Factors Genotype:Wild Type | Treatment:Unscratched Genotype:Wild Type | Treatment:Unscratched Genotype:Wild Type | Treatment:Unscratched Genotype:Wild Type | Treatment:Unscratched Genotype:Wild Type | Treatment:Unscratched Genotype:Wild Type | Treatment:Unscratched Genotype:Wild Type | Treatment:Unscratched Genotype:Wild Type | Treatment:Unscratched Genotype:cPLA2α KO | Treatment:Unscratched Genotype:cPLA2α KO | Treatment:Unscratched Genotype:cPLA2α KO | Treatment:Unscratched Genotype:cPLA2α KO | Treatment:Unscratched Genotype:cPLA2α KO | Treatment:Unscratched Genotype:cPLA2α KO | Treatment:Unscratched Genotype:cPLA2α KO | Treatment:Unscratched Genotype:cPLA2α KO | Treatment:Unscratched Genotype:cPLA2α KI | Treatment:Unscratched Genotype:cPLA2α KI | Treatment:Unscratched Genotype:cPLA2α KI | Treatment:Unscratched Genotype:cPLA2α KI | Treatment:Unscratched Genotype:cPLA2α KI | Treatment:Unscratched Genotype:cPLA2α KI | Treatment:Unscratched Genotype:cPLA2α KI | Treatment:Unscratched Genotype:cPLA2α KI | Treatment:Unscratched Genotype:Wild Type | Treatment:Scratched Genotype:cPLA2α KI | Treatment:Scratched Genotype:cPLA2α KI | Treatment:Scratched Genotype:Wild Type | Treatment:Scratched Genotype:cPLA2α KI | Treatment:Scratched Genotype:cPLA2α KI | Treatment:Scratched Genotype:cPLA2α KO | Treatment:Scratched Genotype:cPLA2α KO | Treatment:Scratched Genotype:Wild Type | Treatment:Scratched Genotype:Wild Type | Treatment:Scratched Genotype:cPLA2α KI | Treatment:Scratched Genotype:cPLA2α KI | Treatment:Scratched Genotype:Wild Type | Treatment:Scratched Genotype:Wild Type | Treatment:Scratched Genotype:cPLA2α KI | Treatment:Scratched Genotype:cPLA2α KI | Treatment:Scratched Genotype:cPLA2α KO | Treatment:Scratched Genotype:cPLA2α KO | Treatment:Scratched Genotype:cPLA2α KO | Treatment:Scratched Genotype:Wild Type | Treatment:Scratched Genotype:Wild Type | Treatment:Scratched Genotype:cPLA2α KO | Treatment:Scratched Genotype:cPLA2α KO | Treatment:Scratched Genotype:cPLA2α KO | Treatment:Scratched 6-keto PGF1α 16.49 11.97 17.57 15.61 15.95 14.03 17.54 13.99 8.27 7.64 11.62 9.03 9.64 11.05 12.61 10.52 168.60 134.90 152.63 187.27 180.00 214.10 200.92 218.25 204.81 140.28 173.43 161.94 141.67 139.49 50.95 46.87 605.81 220.29 122.39 136.79 229.85 628.05 133.77 174.69 44.19 55.05 55.57 184.25 281.08 64.91 58.74 51.56 8-iso PGF2α 0.08 0.08 0.11 0.09 0.08 0.10 0.09 0.07 0.08 0.07 0.10 0.08 0.09 0.10 0.10 0.11 0.38 0.23 0.30 0.35 0.33 0.42 0.45 0.38 0.43 0.25 0.24 0.49 0.20 0.21 0.13 0.09 1.31 0.40 0.19 0.20 0.45 1.10 0.23 0.22 0.11 0.10 0.09 0.35 0.52 0.11 0.14 0.11 TXB2 2.34 1.82 2.25 2.67 2.31 2.09 2.52 2.35 2.45 2.32 3.22 2.70 3.20 2.94 3.26 3.14 2.16 1.69 2.04 2.59 2.33 2.94 2.96 2.95 0.31 0.35 0.38 0.27 0.29 0.38 0.40 0.35 0.46 0.30 0.34 0.38 0.33 0.48 0.31 0.41 0.25 0.25 0.27 0.29 0.31 0.32 0.26 0.30 5-iPF2α-VI 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.03 0.01 0.01 0.01 0.00 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 PGE2 2.49 1.86 2.81 2.14 2.54 2.08 2.87 2.05 1.90 1.79 2.74 2.07 2.04 2.56 3.03 2.18 54.42 46.71 50.76 58.15 59.83 69.54 65.14 76.98 19.02 39.50 41.97 23.80 36.17 42.52 5.22 4.86 54.33 18.40 37.65 37.54 21.30 40.87 32.38 38.52 4.89 5.20 5.58 16.46 22.18 5.54 5.79 5.29 PGF2α 0.42 0.45 0.54 0.54 0.54 0.47 0.60 0.45 0.42 0.44 0.61 0.51 0.51 0.58 0.63 0.54 3.01 2.25 2.70 3.17 3.12 3.87 3.54 3.58 5.80 2.06 2.37 7.91 1.96 2.00 0.83 0.72 22.08 5.45 1.79 1.90 6.82 17.22 1.72 2.22 0.83 0.78 0.81 4.37 8.17 0.89 0.90 0.76 PGD2 0.25 0.11 0.25 0.27 0.17 0.12 0.27 0.28 0.24 0.26 0.38 0.18 0.31 0.30 0.40 0.38 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 5.39 1.80 3.02 10.77 2.25 2.32 1.26 0.94 18.23 5.25 2.20 2.65 7.26 14.03 2.82 3.83 0.86 1.13 1.10 5.96 7.90 1.25 1.20 1.09 PGE1 0.37 0.26 0.38 0.28 0.34 0.29 0.38 0.32 0.31 0.30 0.48 0.31 0.34 0.45 0.49 0.38 3.32 2.58 2.94 3.39 3.29 3.82 3.67 3.87 1.54 2.02 2.02 1.61 1.85 2.16 0.56 0.50 4.15 1.53 1.95 1.96 1.87 3.77 1.78 2.09 0.51 0.58 0.58 1.52 1.87 0.58 0.61 0.53 Resolvin D1 0.34 0.35 0.35 0.35 0.35 0.35 0.34 0.34 0.35 0.34 0.33 0.35 0.33 0.34 0.35 0.34 0.34 0.35 0.35 0.34 0.34 0.35 0.33 0.35 0.35 0.33 0.34 0.34 0.33 0.35 0.34 0.33 0.33 0.34 0.34 0.34 0.33 0.34 0.35 0.33 0.35 0.33 0.34 0.34 0.33 0.34 0.33 0.34 PGA2 0.61 0.58 0.65 0.60 0.62 0.63 0.63 0.61 0.59 0.62 0.65 0.61 0.64 0.65 0.66 0.63 1.70 1.42 1.57 1.93 1.64 1.91 1.99 1.88 0.87 1.32 1.31 0.97 1.18 1.28 0.70 0.66 1.70 0.92 1.20 1.23 0.98 1.41 1.18 1.41 0.67 0.67 0.70 0.87 1.05 0.69 0.68 0.65 (±)14,15-DHET 0.26 0.22 0.24 0.25 0.26 0.23 0.26 0.24 0.24 0.25 0.30 0.26 0.30 0.28 0.33 0.33 0.23 0.18 0.21 0.25 0.24 0.30 0.29 0.28 0.15 0.12 0.13 0.15 0.13 0.13 0.15 0.13 0.24 0.17 0.12 0.14 0.17 0.23 0.13 0.15 0.11 0.12 0.13 0.15 0.16 0.12 0.13 0.13 (±)11,12-DHET 0.14 0.11 0.15 0.16 0.13 0.13 0.14 0.13 0.14 0.12 0.17 0.14 0.16 0.14 0.15 0.15 0.10 0.08 0.11 0.12 0.11 0.14 0.14 0.14 0.07 0.04 0.03 0.09 0.04 0.04 0.04 0.04 0.18 0.07 0.04 0.04 0.08 0.17 0.04 0.05 0.04 0.04 0.03 0.06 0.08 0.04 0.04 0.04 (±)8,9-DHET 0.07 0.05 0.06 0.06 0.05 0.05 0.06 0.06 0.05 0.05 0.07 0.06 0.07 0.05 0.07 0.09 0.05 0.04 0.04 0.06 0.05 0.07 0.05 0.07 0.03 0.01 0.02 0.04 0.02 0.02 0.02 0.01 0.10 0.04 0.02 0.01 0.04 0.12 0.02 0.02 0.01 0.02 0.01 0.03 0.05 0.02 0.02 0.02 20-HETE 15.30 15.86 15.29 15.19 15.01 14.58 15.68 15.95 15.90 15.84 15.84 16.70 16.47 15.56 16.43 16.81 16.45 15.87 16.56 17.72 16.49 17.53 17.31 16.98 22.61 20.56 20.74 22.50 21.43 21.88 20.56 21.00 22.39 19.91 21.87 22.29 21.52 21.58 21.87 20.51 21.01 21.62 21.43 22.67 22.70 21.79 22.41 23.08 15 HETE 0.22 0.19 0.22 0.19 0.24 0.19 0.25 0.18 0.20 0.19 0.24 0.23 0.24 0.24 0.29 0.25 0.98 0.72 0.83 1.06 0.93 1.24 1.20 1.12 2.37 0.40 0.38 2.66 0.29 0.38 0.16 0.17 15.14 2.53 0.31 0.27 2.61 11.03 0.34 0.40 0.17 0.15 0.15 2.26 2.93 0.19 0.17 0.16 12 HETE 2.34 1.59 2.47 2.47 2.17 1.89 2.89 2.22 2.02 1.93 2.91 2.35 2.67 2.63 3.11 2.65 1.68 0.98 1.37 2.02 1.56 2.17 2.51 1.99 0.26 0.08 0.09 0.22 0.06 0.08 0.10 0.09 0.79 0.24 0.08 0.09 0.25 0.71 0.07 0.09 0.07 0.07 0.07 0.24 0.25 0.09 0.07 0.08 (±)14(15)-EET 0.04 0.04 0.05 0.05 0.03 0.03 0.05 0.04 0.03 0.03 0.06 0.05 0.05 0.05 0.04 0.05 0.04 0.03 0.04 0.04 0.05 0.04 0.05 0.05 0.09 0.03 0.02 0.08 0.02 0.03 0.03 0.03 0.15 0.08 0.03 0.02 0.06 0.14 0.02 0.02 0.02 0.02 0.02 0.06 0.06 0.02 0.02 0.02 5 HETE 0.03 0.03 0.03 0.03 0.04 0.03 0.06 0.04 0.04 0.04 0.05 0.05 0.05 0.05 0.05 0.05 0.04 0.04 0.04 0.07 0.04 0.05 0.06 0.05 0.21 0.02 0.03 0.28 0.03 0.03 0.04 0.03 0.94 0.22 0.02 0.02 0.20 0.79 0.03 0.03 0.02 0.03 0.03 0.16 0.25 0.03 0.03 0.05 EPA 27.77 23.67 27.85 31.51 27.43 24.62 29.91 0.00 30.65 30.27 39.27 33.83 40.78 36.36 40.54 38.84 27.58 20.80 25.02 31.02 29.33 34.84 37.13 35.18 38.73 9.13 8.81 38.50 7.45 10.56 9.60 8.87 147.71 43.42 7.85 8.05 46.63 169.80 8.12 10.06 7.42 7.17 7.21 37.20 54.77 8.42 7.53 8.08 DHA 220.99 192.31 205.66 237.42 216.38 198.25 240.49 210.24 229.40 237.74 318.93 276.92 316.84 294.79 318.64 303.51 190.86 164.29 176.73 213.77 208.42 244.40 251.95 248.08 193.94 52.11 48.52 178.03 43.11 65.98 66.94 64.38 526.72 205.06 45.30 47.80 230.88 647.62 48.67 57.02 48.16 47.41 45.79 188.02 259.72 57.37 50.70 55.81 AA 205.66 178.21 202.05 229.95 204.91 187.12 219.22 193.60 190.53 190.51 281.84 217.59 263.10 242.86 274.75 248.92 200.00 141.62 176.67 224.79 206.11 243.48 253.81 269.86 807.69 145.66 160.39 857.14 124.66 162.83 139.01 119.20 2501.75 947.62 118.88 125.38 1015.35 2984.49 126.86 168.97 138.83 128.04 113.33 760.00 1123.50 138.85 126.25 122.89 DHGLA 26.13 22.92 24.52 28.08 25.87 23.76 27.63 23.86 23.25 23.83 32.91 27.95 32.90 31.03 33.56 31.13 24.76 19.96 21.89 26.31 25.70 29.59 30.60 32.39 67.23 15.06 15.63 64.00 13.29 20.67 13.88 13.08 278.44 69.75 11.96 12.63 77.91 363.77 14.83 16.85 13.26 13.10 13.28 63.21 92.34 14.08 14.13 12.30 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention Time m/z PubChem ID KEGG ID 6-keto PGF1α 4.33 369.2 5280888 C05961 8-iso PGF2α 5.09 353.4 5282263 C14794 TXB2 5.09 369.2 5283137 C05963 5-iPF2α-VI 5.41 353.2 53394506 C05959 PGE2 4.56 351.2 5280360 C00584 PGF2α 5.5 353.2 5280636 C00639 PGD2 5.59 351.201 448457 C00696 PGE1 5.63 353.2 5280723 C04741 Resolvin D1 6.09 375.201 44251266 C18178 PGA2 6.4 333.2 5280880 C05953 (±)14,15-DHET 7.52 337.2 5283147 C14775 (±)11,12-DHET 7.61 337.2 129590 C14774 (±)8,9-DHET 7.73 337.2 5283144 C14773 20-HETE 7.75 319.2 5283157 C14748 15 HETE 7.99 319.2 9966861 C04742 12 HETE 8.15 319.2 13786989 C14777 (±)14(15)-EET 8.33 319.2 5283205 C14771 5 HETE 8.41 319.2 5280733 C04805 EPA 9.54 301.2 446284 C06428 DHA 10.04 327.2 15608515 C06429 AA 10.21 303.2 444899 C00219 DHGLA 10.72 305.2 3014613 C03242 METABOLITES_END #END