#METABOLOMICS WORKBENCH shancock_20250220_141052 DATATRACK_ID:5659 STUDY_ID:ST003761 ANALYSIS_ID:AN006176 PROJECT_ID:PR002344 VERSION 1 CREATED_ON 03-03-2025 #PROJECT PR:PROJECT_TITLE Fatty acids profiling of gemcitabine based chemotherapy resistant PDAC cells PR:PROJECT_SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with few PR:PROJECT_SUMMARY treatment options and poor survivability. In this work we sought to characterise PR:PROJECT_SUMMARY metabolic adaptations to gemcitabine (GEMC)-based chemotherapy exposure to PR:PROJECT_SUMMARY discover new therapeutic targets for improving treatment efficacy. We show that PR:PROJECT_SUMMARY gemcitabine-based chemotherapy resistance (GEMR) upregulates de novo lipogenesis PR:PROJECT_SUMMARY in Panc1 and MiaPaCa2 cells. PR:INSTITUTE Victor Chang Cardiac Research Institute PR:LAST_NAME Hancock PR:FIRST_NAME Sarah PR:ADDRESS Level 7 Lowy Packer Building, 405 Liverpool Street, Darlinghurst, NSW 2010, PR:ADDRESS Australia PR:EMAIL s.hancock@victorchang.edu.au PR:PHONE +61414537526 PR:DOI http://dx.doi.org/10.21228/M8NC1Z #STUDY ST:STUDY_TITLE Fatty acids profiling of gemcitabine based chemotherapy resistant PDAC cells ST:STUDY_SUMMARY In this work produced combination gemcitabine/paclitaxel attenuated (CombAT) ST:STUDY_SUMMARY Panc1 and MiaPaCa2 cells. We profiled AMP-derivatised fatty acids from both cell ST:STUDY_SUMMARY lines and compared them to control (CON) cells. Also included are a reference ST:STUDY_SUMMARY standard produced from AMP-derivatised fatty acid methyl esters (FAMEs), ST:STUDY_SUMMARY internal quality controls (iQC) made from pooled samples, and a blank sample for ST:STUDY_SUMMARY background subtraction. This study found increased 16:1n-7 and decreased ST:STUDY_SUMMARY 16:1n-10 in Panc1 CombAT cells, with no change in MiaPaCa2 CombAT cells. Also ST:STUDY_SUMMARY apparent were changes in polyunsaturated fatty acids in Panc1 CombAT cells ST:STUDY_SUMMARY consistent with a decrease in fatty acid desaturase 2 (FADS2) activity. Data ST:STUDY_SUMMARY were analysed using Agilent Profinder 10.0 with fatty acids identified using a ST:STUDY_SUMMARY Personal Compound Databases and Libraries (PCDL) developed from the FAMEs ST:STUDY_SUMMARY reference standard. ST:INSTITUTE Victor Chang Cardiac Research Institute ST:LAST_NAME Hancock ST:FIRST_NAME Sarah ST:ADDRESS Level 7 Lowy Packer Building, 405 Liverpool Street, Darlinghurst, NSW 2010, ST:ADDRESS Australia ST:EMAIL s.hancock@victorchang.edu.au ST:PHONE +61414537526 ST:SUBMIT_DATE 2025-02-20 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - Blank Sample source:- | group:Blank | group:Blank RAW_FILE_NAME(Raw file name)=Blank.mzML; protein amount (ug)=- SUBJECT_SAMPLE_FACTORS - FAMEs-mix_01 Sample source:- | group:FAMEs | group:FAMEs RAW_FILE_NAME(Raw file name)=FAMEs-mix_01.mzML; protein amount (ug)=- SUBJECT_SAMPLE_FACTORS - FAMEs-mix_02 Sample source:- | group:FAMEs | group:FAMEs RAW_FILE_NAME(Raw file name)=FAMEs-mix_02.mzML; protein amount (ug)=- SUBJECT_SAMPLE_FACTORS - iQC_01 Sample source:- | group:pool | group:iQC RAW_FILE_NAME(Raw file name)=iQC_01.mzML; protein amount (ug)=- SUBJECT_SAMPLE_FACTORS - iQC_02 Sample source:- | group:pool | group:iQC RAW_FILE_NAME(Raw file name)=iQC_02.mzML; protein amount (ug)=- SUBJECT_SAMPLE_FACTORS - MiaPaCa2_COMBR_p1 Sample source:Pancreas | group:MiaPaCa2 cells | group:CombAT RAW_FILE_NAME(Raw file name)=MiaPaCa2_COMBR_p1.mzML; protein amount (ug)=81.83384 SUBJECT_SAMPLE_FACTORS - MiaPaCa2_COMBR_p2 Sample source:Pancreas | group:MiaPaCa2 cells | group:CombAT RAW_FILE_NAME(Raw file name)=MiaPaCa2_COMBR_p2.mzML; protein amount (ug)=62.98929 SUBJECT_SAMPLE_FACTORS - MiaPaCa2_COMBR_p3 Sample source:Pancreas | group:MiaPaCa2 cells | group:CombAT RAW_FILE_NAME(Raw file name)=MiaPaCa2_COMBR_p3.mzML; protein amount (ug)=101.68031 SUBJECT_SAMPLE_FACTORS - MiaPaCa2_CON_p1 Sample source:Pancreas | group:MiaPaCa2 cells | group:CON RAW_FILE_NAME(Raw file name)=MiaPaCa2_CON_p1.mzML; protein amount (ug)=79.05266 SUBJECT_SAMPLE_FACTORS - MiaPaCa2_CON_p2 Sample source:Pancreas | group:MiaPaCa2 cells | group:CON RAW_FILE_NAME(Raw file name)=MiaPaCa2_CON_p2.mzML; protein amount (ug)=61.26514 SUBJECT_SAMPLE_FACTORS - MiaPaCa2_CON_p3 Sample source:Pancreas | group:MiaPaCa2 cells | group:CON RAW_FILE_NAME(Raw file name)=MiaPaCa2_CON_p3.mzML; protein amount (ug)=75.58103 SUBJECT_SAMPLE_FACTORS - Panc1_COMBR_p1 Sample source:Pancreas | group:Panc1 cells | group:CombAT RAW_FILE_NAME(Raw file name)=Panc1_COMBR_p1.mzML; protein amount (ug)=66.71797 SUBJECT_SAMPLE_FACTORS - Panc1_COMBR_p2 Sample source:Pancreas | group:Panc1 cells | group:CombAT RAW_FILE_NAME(Raw file name)=Panc1_COMBR_p2.mzML; protein amount (ug)=57.54543 SUBJECT_SAMPLE_FACTORS - Panc1_COMBR_p3 Sample source:Pancreas | group:Panc1 cells | group:CombAT RAW_FILE_NAME(Raw file name)=Panc1_COMBR_p3.mzML; protein amount (ug)=92.50384 SUBJECT_SAMPLE_FACTORS - Panc1_CON_p1 Sample source:Pancreas | group:Panc1 cells | group:CON RAW_FILE_NAME(Raw file name)=Panc1_CON_p1.mzML; protein amount (ug)=102.66325 SUBJECT_SAMPLE_FACTORS - Panc1_CON_p2 Sample source:Pancreas | group:Panc1 cells | group:CON RAW_FILE_NAME(Raw file name)=Panc1_CON_p2.mzML; protein amount (ug)=77.17731 SUBJECT_SAMPLE_FACTORS - Panc1_CON_p3 Sample source:Pancreas | group:Panc1 cells | group:CON RAW_FILE_NAME(Raw file name)=Panc1_CON_p3.mzML; protein amount (ug)=57.68309 #COLLECTION CO:COLLECTION_SUMMARY Samples were obtained from established immortalised adherent human pancreatic CO:COLLECTION_SUMMARY ductal adenocarcinoma cells (Panc1 & MiaPaCa2). Cells were cultured in high CO:COLLECTION_SUMMARY glucose DMEM containing 4.5 g/L glucose and 4 mM glutamine and were supplemented CO:COLLECTION_SUMMARY with 10% FCS. Cells were cultured under 5% CO2 at 37ºC and routinely passaged CO:COLLECTION_SUMMARY every 2-3 days (when ~80-90% confluent). Cell lines were regularly screened for CO:COLLECTION_SUMMARY mycoplasma infection. CO:SAMPLE_TYPE Pancreas #TREATMENT TR:TREATMENT_SUMMARY Combination gemcitabine/paclitaxel attenuated (CombAT) cells were produced by TR:TREATMENT_SUMMARY serial treatment with escalating doses of gemcitabine/paclitaxel (10:1) for TR:TREATMENT_SUMMARY approximately 12 weeks. The final concentration of gemcitabine for Panc1 cells TR:TREATMENT_SUMMARY was 28 nM (2.8 nM paclitaxel), and 8 nM (0.8 nM paclitaxel) for MiaPaCa2 cells. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Lipids were extracted from cells grown to 80-90% confluency in 6 well plates SP:SAMPLEPREP_SUMMARY using a modified methyl tert-butyl ether (MTBE) method. Adherent cells were SP:SAMPLEPREP_SUMMARY washed with ice-cold PBS and scraped into methanol containing 0.01% butylated SP:SAMPLEPREP_SUMMARY hydroxytoluene and 100 pmol each of internal standards (phosphatidylcholine SP:SAMPLEPREP_SUMMARY 19:0/19:0, phosphatidylethanolamine 17:0/17:0, phosphatidylserine 17:0/17:0, SP:SAMPLEPREP_SUMMARY lysophosphatidylcholine 17:0, lysophosphatidylethanolamine 14:0, SP:SAMPLEPREP_SUMMARY phosphatidylglycerol 17:0/17:0, phosphatidic acid 17:0/17:0, ceramide SP:SAMPLEPREP_SUMMARY d18:1/17:0, dihydrosphingomyelin 12:0, cholesteryl ester 22:1, diacylglycerol SP:SAMPLEPREP_SUMMARY 17:0/17:0, D5-triacylglycerol 16:0/16:0/16:0 and cardiolipin SP:SAMPLEPREP_SUMMARY 14:0/14:0/14:0/14:0, and 20 nmol of methyl nonadecanoate). An empty well was SP:SAMPLEPREP_SUMMARY also scraped for use in background subtraction. To this, MTBE was added at a SP:SAMPLEPREP_SUMMARY ratio of 3:1 v/v, and extracts were rotated overnight at 4ºC. Following SP:SAMPLEPREP_SUMMARY overnight rotation, 1 part of ice-cold 150 mM ammonium acetate was added, SP:SAMPLEPREP_SUMMARY samples were vortexed vigorously and centrifuged at 2000 x g for 5 min. The SP:SAMPLEPREP_SUMMARY upper organic phase containing lipids was transferred into a 1.5 mL autosampler SP:SAMPLEPREP_SUMMARY vial and dried under N2 at 37ºC Dried lipids were reconstituted in SP:SAMPLEPREP_SUMMARY chloroform:methanol:water (60:30:4.5 v/v/v), transferred to a sleeved vial, and SP:SAMPLEPREP_SUMMARY stored at -20ºC until analysis. The aqueous phase containing the protein pellet SP:SAMPLEPREP_SUMMARY was dried under N2 and digested with 1M NaOH overnight at 4ºC. Digested protein SP:SAMPLEPREP_SUMMARY was diluted 1:2 v/v with water and protein concentration was determined using a SP:SAMPLEPREP_SUMMARY Pierce BCA assay kit (ThermoFisher Scientific) Following MTBE extraction of SP:SAMPLEPREP_SUMMARY lipids, an aliquot was taken and hydrolysed in 0.6 M KOH in 75% v/v methanol at SP:SAMPLEPREP_SUMMARY 60ºC for 30 min. Samples were then cooled to room temperature and neutralised SP:SAMPLEPREP_SUMMARY with 25% v/v acetic acid. Water and n-hexane (3:2 v/v) were added separately and SP:SAMPLEPREP_SUMMARY samples were vortexed vigorously before being centrifuged at 2000 x g for 5 min. SP:SAMPLEPREP_SUMMARY The upper phase containing hydrolysed fatty acids was removed, and the aqueous SP:SAMPLEPREP_SUMMARY phase was washed with a second volume of n-hexane and combined. The combined SP:SAMPLEPREP_SUMMARY organic phase was then dried under N2 at 37°C. Dried hydrolysed fatty acids SP:SAMPLEPREP_SUMMARY were derivatised with an AMP+ MaxSpec kit (Cayman Chemical, Ann Arbor, MI, USA) SP:SAMPLEPREP_SUMMARY as per manufacturer instructions. Following this, AMP-derivatised fatty acids SP:SAMPLEPREP_SUMMARY were re-extracted using MTBE:water (1:1 v/v), and the upper MTBE phase was dried SP:SAMPLEPREP_SUMMARY under N2 before being resuspended in methanol. Samples were stored at -20°C SP:SAMPLEPREP_SUMMARY until analysis. A 37-component fatty acid methyl ester standard (Merck Life SP:SAMPLEPREP_SUMMARY Sciences) containing an additional 3.184 nmol of methyl nonadecanoate was SP:SAMPLEPREP_SUMMARY concurrently subjected to the same derivatisation procedure and used as an SP:SAMPLEPREP_SUMMARY external quality control for fatty acid identification by LC-MS. #CHROMATOGRAPHY CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Thermo Accucore C30 (150 x 2.1 mm, 2.6 μm) CH:COLUMN_TEMPERATURE 30°C CH:FLOW_GRADIENT 0-3.25 min increase from 50% B to 56% B, 3.25-6.5 min increase to 58% B, 6.5-7.5 CH:FLOW_GRADIENT min increase to 80% B, 7.5-9.5 min increase to 100% B, hold at 100% B for 1.5 CH:FLOW_GRADIENT mins, re-equilibrated at 50% B for 4.5 mins. CH:FLOW_RATE 0.4 mL/min CH:SOLVENT_A 100% Water; 0.1% Formic acid CH:SOLVENT_B 100% Acetonitrile; 0.1% Formic acid CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6560 Ion Mobility MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:MS_COMMENTS AMP-derivatised fatty acids were detected as positive ions in QTOF-only mode on MS:MS_COMMENTS an Agilent 6560 IM-MS QTOF mass spectrometer (Agilent Technologies). Source MS:MS_COMMENTS conditions were as follows: capillary voltage of 3.5 kV, nozzle voltage of 1 kV MS:MS_COMMENTS and fragmentor at 360 V. Nitrogen was used as the sheath and collision gas, with MS:MS_COMMENTS drying gas set at 5 L/min and sheath gas at 12 L/min (both gases heated to MS:MS_COMMENTS 300°C) and the nebuliser pressure set at 20 psi. Ions were acquired in Auto MS:MS_COMMENTS MS/MS mode at an MS1 mass range of m/z 100-1000 and MS2 mass range of m/z MS:MS_COMMENTS 50-600. Spectra were acquired in MS1 mode at 5 spectra/s and 200 ms/spectrum, MS:MS_COMMENTS and in MS2 mode at 10 spectra/s and 100 ms/spectrum. Precursor ions were MS:MS_COMMENTS isolated using a window of 1.3 Th, acquisition time of 50 ms/spectra, and MS:MS_COMMENTS collision energy of 45 eV. Calibration mix A was infused throughout and masses MS:MS_COMMENTS were automatically detected and corrected using a 100 ppm window and minimum MS:MS_COMMENTS height of 1x10^3 counts to ensure 5 ppm mass accuracy, with m/z 121.050873 and MS:MS_COMMENTS 922.009798 used as reference ions. AMP-derivatised fatty acids as [M+H]+ were MS:MS_COMMENTS aligned using Mass Profinder v10.0 (Agilent Technologies) with an RT tolerance MS:MS_COMMENTS of 0.5 min and MS1 tolerance of 10 ppm. Lipids were identified using a custom MS:MS_COMMENTS personal compound database and library (PCDL) generated from the RT of external MS:MS_COMMENTS quality control samples (see attached RT list) and MS/MS spectra were manually MS:MS_COMMENTS inspected to ensure correct MUFA isomer identification. Using this method, FA MS:MS_COMMENTS 18:1 isomers were only partially separated and so these data were not analysed MS:MS_COMMENTS further. Data were exported, blank subtracted and quantified from internal MS:MS_COMMENTS standards before being normalised to total protein. MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS nmol/mg protein MS_METABOLITE_DATA_START Samples MiaPaCa2_COMBR_p1 MiaPaCa2_COMBR_p2 MiaPaCa2_COMBR_p3 MiaPaCa2_CON_p1 MiaPaCa2_CON_p2 MiaPaCa2_CON_p3 Panc1_COMBR_p1 Panc1_COMBR_p2 Panc1_COMBR_p3 Panc1_CON_p1 Panc1_CON_p2 Panc1_CON_p3 Factors Sample source:Pancreas | group:MiaPaCa2 cells | group:CombAT Sample source:Pancreas | group:MiaPaCa2 cells | group:CombAT Sample source:Pancreas | group:MiaPaCa2 cells | group:CombAT Sample source:Pancreas | group:MiaPaCa2 cells | group:CON Sample source:Pancreas | group:MiaPaCa2 cells | group:CON Sample source:Pancreas | group:MiaPaCa2 cells | group:CON Sample source:Pancreas | group:Panc1 cells | group:CombAT Sample source:Pancreas | group:Panc1 cells | group:CombAT Sample source:Pancreas | group:Panc1 cells | group:CombAT Sample source:Pancreas | group:Panc1 cells | group:CON Sample source:Pancreas | group:Panc1 cells | group:CON Sample source:Pancreas | group:Panc1 cells | group:CON 14:0-AMP 4.4623 7.0619 7.6765 0.5068 4.1507 5.6150 4.3959 4.0304 6.6213 10.3886 7.7069 11.7389 15:0-AMP 0.3110 0.1353 0.0767 1.8328 0.0583 0.7549 15:1-AMP 16:0-AMP 10.1284 29.1957 42.0732 32.8324 34.9038 3.7370 23.3448 42.4540 69.5667 17.0620 28.4452 16:1n10-AMP 2.2876 2.8550 3.0047 2.2904 2.0519 1.9852 1.8467 1.9897 2.2245 7.6939 8.2083 9.9682 16:1n7-AMP 26.0992 34.2443 43.3817 23.7674 29.5487 28.0779 23.9899 24.8281 27.3413 12.5143 12.6024 14.3256 16:1n9-AMP 3.5291 4.3573 3.5163 4.1790 3.1475 3.3548 3.0245 3.2711 5.4234 2.6309 2.1883 3.5564 17:0-AMP 1.4111 17:1-AMP 3.0100 4.2763 4.4500 4.5367 4.9101 4.8025 2.6645 2.9520 2.9691 4.0094 2.7295 3.2358 18:0-AMP 0.6659 49.5023 3.6273 18:1n10-AMP 31.5100 30.2474 18.2654 51.7756 28.6863 23.2702 33.8928 37.6829 28.7856 39.1504 39.0951 68.8487 18:1n7-AMP 61.9747 79.0523 84.3075 62.3559 66.5389 92.5726 85.3544 73.8483 73.8719 58.9356 49.0764 51.6111 18:1n9-AMP 76.7310 117.8263 101.3354 142.1195 127.3654 128.7485 112.7502 115.6057 120.8206 118.3814 114.4969 114.0015 18:1n9t-AMP 1.4484 1.8227 1.4654 2.0103 1.9842 1.9647 1.7490 1.8928 2.3328 3.3836 3.3598 3.8127 18:2n6-AMP 5.5231 8.5975 9.5705 8.0529 9.8789 9.8450 6.4388 6.5397 8.3155 8.6694 5.9273 6.1912 18:2n6t-AMP 0.6363 1.0537 0.9947 0.9282 1.0102 1.0348 0.7001 1.2140 1.1397 1.7823 1.8555 1.9713 18:3n3-AMP 0.0972 0.2372 0.2734 0.1646 0.2580 0.2188 0.1787 0.1633 0.2434 0.0376 0.0132 0.0083 18:3n6-AMP 0.0784 0.1031 0.0798 0.0439 0.0850 0.0691 0.0431 0.0828 0.0542 0.1508 0.1127 0.1518 19:0-AMP 65.7956 77.6806 78.6142 94.8786 92.0788 103.1629 80.0915 70.2426 66.8488 140.3558 97.6512 105.2461 19:1-AMP 1.9422 2.7668 2.8361 3.0307 3.1393 2.9072 2.8967 3.0540 3.5692 3.5560 2.7245 2.4514 20:0-AMP 0.2392 0.2197 0.5114 0.5719 20:1-AMP 13.5366 17.6285 18.5385 12.4548 14.5682 14.6634 19.1678 23.7120 30.7186 18.0924 18.7539 18.7432 20:2-AMP 1.5469 2.1782 2.4540 2.3289 2.3069 2.4383 2.3677 2.6532 2.7431 7.8021 7.2563 6.9391 20:3-AMP 3.3923 4.8655 5.1076 6.7755 7.0477 6.6502 4.2962 4.4925 5.1393 8.7718 6.6699 6.5623 20:4-AMP 18.0621 25.1771 24.4620 26.6974 28.5781 28.1924 22.8624 21.0620 24.5223 39.3880 29.2013 32.3896 20:5-AMP 1.2914 1.9934 2.1601 1.8416 2.0108 2.3203 1.6193 1.3855 1.8617 1.0341 0.6362 0.9196 21:0-AMP 0.0914 22:0-AMP 0.4359 0.4800 0.1221 0.0289 0.2583 0.1156 0.5732 0.2893 0.8312 22:1-AMP 3.5546 3.9159 3.9117 0.8190 2.5862 2.5350 5.1815 5.0369 6.9636 2.9434 3.7407 4.7446 22:2-AMP 0.7844 0.8656 0.8556 0.3924 0.5976 1.2273 1.4729 1.7438 2.3766 2.9710 3.0133 22:4-AMP 3.4019 5.6114 4.6594 5.6030 5.8636 5.8090 4.9181 5.2002 5.2239 13.2699 8.4772 9.3489 22:5n3-AMP 11.1360 17.4505 16.5165 17.7397 20.6048 19.6198 14.7555 14.1160 16.6682 9.9892 6.9066 7.5398 22:5n6-AMP 0.5766 0.9446 0.7403 0.8240 0.9492 0.9379 0.8211 0.9617 0.9019 10.1567 7.2150 6.7640 22:6-AMP 13.4933 20.3990 19.1546 24.4512 23.5291 23.0439 19.0421 17.8526 20.1259 50.4623 39.3323 40.9488 23:0-AMP 0.1245 0.4163 24:0-AMP 1.1442 1.6766 1.3290 0.8482 1.4998 1.3513 1.2644 0.9932 1.8265 0.9151 1.3806 24:1-AMP 3.3373 4.0730 3.1675 1.2604 2.5467 2.5759 5.1024 4.4236 6.8254 1.0599 1.2763 2.0182 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant 14:0-AMP 2.27 15:0-AMP 3.04 15:1-AMP 2.01 16:0-AMP 4.02 16:1n10-AMP 2.88 16:1n7-AMP 2.65 16:1n9-AMP 2.73 17:0-AMP 5.31 17:1-AMP 3.49 18:0-AMP 7.06 18:1n10-AMP 4.61 18:1n7-AMP 4.46 18:1n9-AMP 4.54 18:1n9t-AMP 4.98 18:2n6-AMP 3.22 18:2n6t-AMP 3.64 18:3n3-AMP 2.39 18:3n6-AMP 2.56 19:0-AMP 8.27 19:1-AMP 5.79 20:0-AMP 8.6 20:1-AMP 7.59 20:2-AMP 5.26 20:3-AMP 3.96 20:4-AMP 3.28 20:5-AMP 2.47 21:0-AMP 8.84 22:0-AMP 9.06 22:1-AMP 8.62 22:2-AMP 8.21 22:4-AMP 4.85 22:5n3-AMP 3.71 22:5n6-AMP 4.28 22:6-AMP 3.25 23:0-AMP 9.28 24:0-AMP 9.48 24:1-AMP 9.02 METABOLITES_END #END