#METABOLOMICS WORKBENCH jgengatharan_20240730_104423 DATATRACK_ID:5052 STUDY_ID:ST003358 ANALYSIS_ID:AN005500 PROJECT_ID:PR002085 VERSION 1 CREATED_ON July 30, 2024, 11:25 am #PROJECT PR:PROJECT_TITLE Altered sphingolipid biosynthetic flux and lipoprotein trafficking contribute to PR:PROJECT_TITLE trans fat-induced atherosclerosis PR:PROJECT_SUMMARY The goal of the project is to determine the role of sphingolipid metabolism in PR:PROJECT_SUMMARY atherosclerosis induced by dietary trans fat. We analyzed lipid metabolites in PR:PROJECT_SUMMARY Huh7 cells following various fatty acid treatments, with specific focus on cis PR:PROJECT_SUMMARY and trans unsaturated fatty acids. Additionally, we analyzed lipid metabolites PR:PROJECT_SUMMARY in plasma and liver of Ldlr-/- mice fed high-fat diets enriched in cis or trans PR:PROJECT_SUMMARY fatty acids in the presence or absence of myriocin, a pharmacological inhibitor PR:PROJECT_SUMMARY of SPT, the initial rate-limiting enzyme of sphingolipid biosynthesis. PR:INSTITUTE Salk Institute for Biological Studies PR:LAST_NAME Gengatharan PR:FIRST_NAME Jivani PR:ADDRESS 10010 N Torrey Pines Rd, La Jolla, CA, 92037, USA PR:EMAIL jivani14@gmail.com PR:PHONE (858) 453-4100 #STUDY ST:STUDY_TITLE Secretion of sphingomyelin from Huh7 cells treated with various fatty acids ST:STUDY_TITLE including of oleate-d9 and elaidate-d17. ST:STUDY_SUMMARY We analyzed sphingomyelin (SM) in fresh and spent media from Huh7 cells treated ST:STUDY_SUMMARY with BSA-EtOH, BSA-palmitate, BSA-stearate, BSA-oleate-d9 or BSA-elaidate-d17. ST:STUDY_SUMMARY We aimed to determine the secretory flux of SM induced by various fatty acid ST:STUDY_SUMMARY treatments. ST:INSTITUTE Salk Institute for Biological Studies ST:LAST_NAME Gengatharan ST:FIRST_NAME Jivani ST:ADDRESS 10010 N Torrey Pines Rd, La Jolla, CA, 92037, USA ST:EMAIL jivani14@gmail.com ST:PHONE (858) 453-4100 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_STRAIN_DETAILS Huh7 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - Huh7freshmedia_C8SL2_EtOH1 Sample source:Huh7 cells | Condition:BSA-EtOH fresh media RAW_FILE_NAME(Raw file name)=Huh7freshmedia_C8SL2_EtOH1.d SUBJECT_SAMPLE_FACTORS - Huh7freshmedia_C8SL2_palm1 Sample source:Huh7 cells | Condition:BSA-palmitate fresh media RAW_FILE_NAME(Raw file name)=Huh7freshmedia_C8SL2_palm1.d SUBJECT_SAMPLE_FACTORS - Huh7freshmedia_C8SL2_palm2 Sample source:Huh7 cells | Condition:BSA-palmitate fresh media RAW_FILE_NAME(Raw file name)=Huh7freshmedia_C8SL2_palm2.d SUBJECT_SAMPLE_FACTORS - Huh7freshmedia_C8SL2_stear1 Sample source:Huh7 cells | Condition:BSA-stearate fresh media RAW_FILE_NAME(Raw file name)=Huh7freshmedia_C8SL2_stear1.d SUBJECT_SAMPLE_FACTORS - Huh7freshmedia_C8SL2_stear2 Sample source:Huh7 cells | Condition:BSA-stearate fresh media RAW_FILE_NAME(Raw file name)=Huh7freshmedia_C8SL2_stear2.d SUBJECT_SAMPLE_FACTORS - Huh7freshmedia_C8SL2_d9oleic1 Sample source:Huh7 cells | Condition:BSA-oleate-d9 fresh media RAW_FILE_NAME(Raw file name)=Huh7freshmedia_C8SL2_d9oleic1.d SUBJECT_SAMPLE_FACTORS - Huh7freshmedia_C8SL2_d9oleic2 Sample source:Huh7 cells | Condition:BSA-oleate-d9 fresh media RAW_FILE_NAME(Raw file name)=Huh7freshmedia_C8SL2_d9oleic2.d SUBJECT_SAMPLE_FACTORS - Huh7freshmedia_C8SL2_d17elaidic1 Sample source:Huh7 cells | Condition:BSA-elaidate-d17 fresh media RAW_FILE_NAME(Raw file name)=Huh7freshmedia_C8SL2_d17elaidic1.d SUBJECT_SAMPLE_FACTORS - Huh7freshmedia_C8SL2_d17elaidic2 Sample source:Huh7 cells | Condition:BSA-elaidate-d17 fresh media RAW_FILE_NAME(Raw file name)=Huh7freshmedia_C8SL2_d17elaidic2.d SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_C8SL2_EtOH1 Sample source:Huh7 cells | Condition:BSA-EtOH spent media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_C8SL2_EtOH1.d SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_C8SL2_EtOH2 Sample source:Huh7 cells | Condition:BSA-EtOH spent media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_C8SL2_EtOH2.d SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_C8SL2_EtOH3 Sample source:Huh7 cells | Condition:BSA-EtOH spent media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_C8SL2_EtOH3.d SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_C8SL2_palm1 Sample source:Huh7 cells | Condition:BSA-palmitate spent media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_C8SL2_palm1.d SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_C8SL2_palm2 Sample source:Huh7 cells | Condition:BSA-palmitate spent media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_C8SL2_palm2.d SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_C8SL2_palm3 Sample source:Huh7 cells | Condition:BSA-palmitate spent media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_C8SL2_palm3.d SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_C8SL2_stear1 Sample source:Huh7 cells | Condition:BSA-stearate spent media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_C8SL2_stear1.d SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_C8SL2_stear2 Sample source:Huh7 cells | Condition:BSA-stearate spent media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_C8SL2_stear2.d SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_C8SL2_stear3 Sample source:Huh7 cells | Condition:BSA-stearate spent media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_C8SL2_stear3.d SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_C8SL2_d9oleic1 Sample source:Huh7 cells | Condition:BSA-oleate-d9 spent media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_C8SL2_d9oleic1.d SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_C8SL2_d9oleic2 Sample source:Huh7 cells | Condition:BSA-oleate-d9 spent media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_C8SL2_d9oleic2.d SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_C8SL2_d9oleic3 Sample source:Huh7 cells | Condition:BSA-oleate-d9 spent media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_C8SL2_d9oleic3.d SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_C8SL2_d17elaidic1 Sample source:Huh7 cells | Condition:BSA-elaidate-d17 spent media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_C8SL2_d17elaidic1.d SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_C8SL2_d17elaidic2 Sample source:Huh7 cells | Condition:BSA-elaidate-d17 spent media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_C8SL2_d17elaidic2.d SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_C8SL2_d17elaidic3 Sample source:Huh7 cells | Condition:BSA-elaidate-d17 spent media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_C8SL2_d17elaidic3.d #COLLECTION CO:COLLECTION_SUMMARY 0.75 ml of fresh media or 1.5 ml of spent media was evaporated under vacuum at CO:COLLECTION_SUMMARY 4°C and resuspended into 0.1 mL of H2O. Concentrated media was spiked with the CO:COLLECTION_SUMMARY following internal standards: 20 pmol sphinganine-d7 (Avanti Polar Lipids, Cat# CO:COLLECTION_SUMMARY 860658), deoxysphinganine-d3 (Avanti Polar Lipids, Cat# 860474), 100 pmol CO:COLLECTION_SUMMARY d18:0-d7/13:0 dihydroceramide (Avanti Polar Lipids, Cat# 330726), 200 pmol CO:COLLECTION_SUMMARY d18:1-d7/15:0 ceramide (Avanti Polar Lipids, Cat# 860681), 100 pmol CO:COLLECTION_SUMMARY d18:1-d7/15:0 glucosylceramide (Avanti Polar Lipids, Cat# 330729), 100 pmol CO:COLLECTION_SUMMARY d18:1-d7/15:0 lactosylceramide (Avanti Polar Lipids, Cat# 330727), 200 pmol CO:COLLECTION_SUMMARY sphingosine-d7 (Avanti Polar Lipids, Cat# 860657). CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY Huh7 cells were treated with 1) 100 µM BSA-EtOH, 2) 100 µM BSA-palmitate, 3) TR:TREATMENT_SUMMARY 100 µM BSA-stearate , 4) 100 µM BSA-oleate-d9 or 5) 100 µM BSA-elaidate-d17 TR:TREATMENT_SUMMARY for 48 hours in delipidated media. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 0.75 ml of fresh media or 1.5 ml of spent media was evaporated under vacuum at SP:SAMPLEPREP_SUMMARY 4°C and resuspended into 0.1 mL of H2O. Concentrated media was spiked with the SP:SAMPLEPREP_SUMMARY following internal standards: 20 pmol sphinganine-d7 (Avanti Polar Lipids, Cat# SP:SAMPLEPREP_SUMMARY 860658), deoxysphinganine-d3 (Avanti Polar Lipids, Cat# 860474), 100 pmol SP:SAMPLEPREP_SUMMARY d18:0-d7/13:0 dihydroceramide (Avanti Polar Lipids, Cat# 330726), 200 pmol SP:SAMPLEPREP_SUMMARY d18:1-d7/15:0 ceramide (Avanti Polar Lipids, Cat# 860681), 100 pmol SP:SAMPLEPREP_SUMMARY d18:1-d7/15:0 glucosylceramide (Avanti Polar Lipids, Cat# 330729), 100 pmol SP:SAMPLEPREP_SUMMARY d18:1-d7/15:0 lactosylceramide (Avanti Polar Lipids, Cat# 330727), 200 pmol SP:SAMPLEPREP_SUMMARY sphingosine-d7 (Avanti Polar Lipids, Cat# 860657). 0.5 mL methanol, 0.5 mL H2O, SP:SAMPLEPREP_SUMMARY and 1 mL chloroform were added directly. Samples were vortexed for 5 min and SP:SAMPLEPREP_SUMMARY centrifuged for 5 min at 4 ˚C at 15,000g. The organic phase was collected and 2 SP:SAMPLEPREP_SUMMARY μL of formic acid was added to the remaining polar phase which was re-extracted SP:SAMPLEPREP_SUMMARY with 1 mL of chloroform. Combined organic phases were dried under nitrogen. SP:SAMPLEPREP_SUMMARY After dried extracts for media were resuspended in 60 µl Buffer B, 5 µL of SP:SAMPLEPREP_SUMMARY sample was injected. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1260 Infinity II CH:COLUMN_NAME Peeke Scientific Spectra C8SR (150 x 3.0 mm, 3μm) CH:SOLVENT_A 100% water; 0.2% formic acid; 2 mM ammonium formate CH:SOLVENT_B 100% methanol; 0.2% formic acid; 1 mM ammonium formate CH:FLOW_GRADIENT 0 min, 82% B; 3 min, 82% B; 4 min, 90% B; 18 min, 99% B; 25 min, 99% B; 27 min, CH:FLOW_GRADIENT 82% B; 30 min, 82% B CH:FLOW_RATE 0.5 mL/min CH:COLUMN_TEMPERATURE 40˚C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6460 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Sphingolipid species were analyzed by multiple reaction monitoring of the MS:MS_COMMENTS transition from precursor to product ions at associated optimized collision MS:MS_COMMENTS energies, and fragmentor voltages using Agilent Masshunter. The m/z values of MS:MS_COMMENTS the precursor and product ions are provided in the metabolite metadata section. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples Huh7freshmedia_C8SL2_EtOH1 Huh7freshmedia_C8SL2_palm1 Huh7freshmedia_C8SL2_palm2 Huh7freshmedia_C8SL2_stear1 Huh7freshmedia_C8SL2_stear2 Huh7freshmedia_C8SL2_d9oleic1 Huh7freshmedia_C8SL2_d9oleic2 Huh7freshmedia_C8SL2_d17elaidic1 Huh7freshmedia_C8SL2_d17elaidic2 Huh7spentmedia_C8SL2_EtOH1 Huh7spentmedia_C8SL2_EtOH2 Huh7spentmedia_C8SL2_EtOH3 Huh7spentmedia_C8SL2_palm1 Huh7spentmedia_C8SL2_palm2 Huh7spentmedia_C8SL2_palm3 Huh7spentmedia_C8SL2_stear1 Huh7spentmedia_C8SL2_stear2 Huh7spentmedia_C8SL2_stear3 Huh7spentmedia_C8SL2_d9oleic1 Huh7spentmedia_C8SL2_d9oleic2 Huh7spentmedia_C8SL2_d9oleic3 Huh7spentmedia_C8SL2_d17elaidic1 Huh7spentmedia_C8SL2_d17elaidic2 Huh7spentmedia_C8SL2_d17elaidic3 Factors Sample source:Huh7 cells | Condition:BSA-EtOH fresh media Sample source:Huh7 cells | Condition:BSA-palmitate fresh media Sample source:Huh7 cells | Condition:BSA-palmitate fresh media Sample source:Huh7 cells | Condition:BSA-stearate fresh media Sample source:Huh7 cells | Condition:BSA-stearate fresh media Sample source:Huh7 cells | Condition:BSA-oleate-d9 fresh media Sample source:Huh7 cells | Condition:BSA-oleate-d9 fresh media Sample source:Huh7 cells | Condition:BSA-elaidate-d17 fresh media Sample source:Huh7 cells | Condition:BSA-elaidate-d17 fresh media Sample source:Huh7 cells | Condition:BSA-EtOH spent media Sample source:Huh7 cells | Condition:BSA-EtOH spent media Sample source:Huh7 cells | Condition:BSA-EtOH spent media Sample source:Huh7 cells | Condition:BSA-palmitate spent media Sample source:Huh7 cells | Condition:BSA-palmitate spent media Sample source:Huh7 cells | Condition:BSA-palmitate spent media Sample source:Huh7 cells | Condition:BSA-stearate spent media Sample source:Huh7 cells | Condition:BSA-stearate spent media Sample source:Huh7 cells | Condition:BSA-stearate spent media Sample source:Huh7 cells | Condition:BSA-oleate-d9 spent media Sample source:Huh7 cells | Condition:BSA-oleate-d9 spent media Sample source:Huh7 cells | Condition:BSA-oleate-d9 spent media Sample source:Huh7 cells | Condition:BSA-elaidate-d17 spent media Sample source:Huh7 cells | Condition:BSA-elaidate-d17 spent media Sample source:Huh7 cells | Condition:BSA-elaidate-d17 spent media Lac-Cer d18:1-d7/15:0 5080 7318 7766 6898 5673 5657 6458 6429 7020 5478 4606 5955 6498 4887 4410 6288 4523 6021 6518 5845 5011 3974 4245 4516 SM d30:0 3973 2952 2366 1338 408 1092 2260 1665 1989 4250 3619 5492 1024 1146 1044 SM d30:1 2656 3980 3800 1467 2379 3922 1877 2379 1610 10213 3514 8804 3857 3812 2666 4916 3271 3766 7931 12142 10109 3075 3405 2031 SM d32:0 3810 4237 3162 3708 4650 3268 3079 3637 7385 60081 48957 53892 27428 30277 26738 41893 47471 43243 61627 91931 83150 21523 24710 24241 SM d32:1 52498 52046 59762 56456 62939 57739 58205 53089 71036 266390 208484 210192 158538 171591 153790 173658 195614 175780 207072 260542 235297 113046 112733 111843 SM d32:2 12167 13020 18693 12798 14113 14906 14066 13508 11767 29032 21167 20549 17160 18646 14308 16282 16655 17990 23122 24410 26341 12100 13651 13020 SM d34:0 34643 48995 55057 37371 39414 38575 40136 38349 84245 246657 199606 226465 283323 296498 264737 276153 284680 276121 282729 312834 308590 123475 118508 132035 SM d34:1 611301 737646 781012 639954 643236 585225 561410 531593 708513 1948212 1469208 1672911 2088090 2293111 2084545 1837338 1954220 1918362 1742791 2010057 1823998 1072800 1169298 1120877 SM d34:2 178960 198564 208994 202924 206535 210319 198168 200040 223788 511743 407543 442207 511167 547658 502297 423570 507717 463510 455734 550446 510275 320589 337504 338484 SM d34:2-d34 95141 104811 110318 SM d36:0 10908 17119 14001 11454 14145 12639 12403 10966 43279 26108 16232 27947 54855 22883 39655 111564 111998 116833 30594 40997 39331 21255 24840 25226 SM d36:1 61612 78829 90603 54231 59130 63475 65984 66969 99466 140559 102557 113618 188836 185640 167317 239253 248228 254679 155511 165280 168720 128837 119141 143726 SM d36:2 55505 49620 55959 56668 56855 41362 41891 44391 50497 98103 79247 79818 96600 97042 100715 122276 129673 101469 94443 122362 91359 62947 67336 67632 SM d36:2-d17 101069 109697 99297 SM d36:2-d9 67604 80427 78244 SM d36:3 2911 2718 2955 3802 1339 9301 7975 8490 9347 6965 6575 10841 8021 8871 36824 52546 45340 4045 5605 6299 SM d36:3-d17 45546 34771 41636 SM d36:3-d34 1420665 1567767 1571827 SM d38:0 2361 1701 3346 4069 1048 3174 4174 1499 16908 17229 5209 13726 9841 14950 7053 42920 40672 38833 6179 8646 5506 7515 5528 6866 SM d38:1 8736 9080 11425 10175 6782 10295 9339 12169 33784 30516 24702 32493 37622 42928 31463 71304 80815 83468 33804 45949 37880 25174 21922 23368 SM d38:2 3192 4132 2959 3466 4598 4152 4245 3242 6296 17642 6597 16406 16272 18114 13437 9178 12884 14897 13838 14748 12378 10863 8993 8175 SM d38:2-d17 15392 13974 13640 SM d38:2-d9 42782 72771 50612 SM d38:3-d17 6452 8895 4787 SM d38:3-d34 148930 158219 166952 SM d38:3-d9 8259 9449 5673 SM d40:1 37777 52599 55202 41605 42029 52432 45181 49411 71821 157900 110754 132209 153688 185255 145837 176514 154635 132888 131752 177133 142088 108624 115156 109910 SM d40:2 47172 63063 65866 52633 62885 56945 53723 46963 61805 214475 146658 166792 154068 156277 145103 157606 163823 154642 133339 144747 152126 114057 116643 115382 SM d40:2-d17 10144 10739 7926 SM d40:2-d9 28151 28738 27493 SM d40:3-d17 6415 8073 8180 SM d40:3-d34 57545 59487 66056 SM d42:1 54890 73117 76009 53765 61456 58323 54322 65741 83947 186725 157283 166279 170891 169257 147761 182138 176620 181314 176599 186171 173101 143316 130183 131095 SM d42:2 77190 90537 106720 87307 97487 90834 93512 77685 115158 358597 273013 294533 289458 319116 286813 277494 279770 281174 287737 330997 279315 221036 231280 209511 SM d42:2-d17 4222 4295 7372 SM d42:2-d9 124834 150285 156991 SM d42:3 276774 285749 311925 297756 306027 301735 294450 254960 296091 734737 564103 647786 579396 604216 561924 572970 588599 543794 583307 702858 635337 470057 495224 497336 SM d42:3-d17 23934 21211 18472 SM d42:3-d34 62402 64770 70704 SM d42:3-d9 44926 33207 24779 SM d44:2 1760 2832 3551 2499 4075 2045 2470 2103 6261 12188 7318 9815 6738 11657 8320 14902 15615 15390 9559 8795 10205 4616 5988 5227 SM d44:2-d9 6232 11407 8173 SM d44:3 13704 9862 11817 11502 11809 12243 13116 8601 12686 31730 25538 28295 21015 26959 30339 23842 26241 23144 28336 26372 24469 22079 24360 26777 SM d44:3-d17 4051 5347 4739 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Precursor Ion Product Ion Lac-Cer d18:1-d7/15:0 855.7 271.2 SM d30:0 649.5 184 SM d30:1 647.5 184 SM d32:0 677.6 184 SM d32:1 675.5 184 SM d32:2 673.5 184 SM d34:0 705.6 184 SM d34:1 703.6 184 SM d34:2 701.6 184 SM d34:2-d34 735.6 184 SM d36:0 733.6 184 SM d36:1 731.6 184 SM d36:2 729.6 184 SM d36:2-d17 746.6 184 SM d36:2-d9 738.6 184 SM d36:3 727.6 184 SM d36:3-d17 744.6 184 SM d36:3-d34 761.6 184 SM d38:0 761.7 184 SM d38:1 759.6 184 SM d38:2 757.6 184 SM d38:2-d17 774.6 184 SM d38:2-d9 766.6 184 SM d38:3-d17 772.6 184 SM d38:3-d34 789.6 184 SM d38:3-d9 764.6 184 SM d40:1 787.7 184 SM d40:2 785.7 184 SM d40:2-d17 802.7 184 SM d40:2-d9 794.7 184 SM d40:3-d17 800.6 184 SM d40:3-d34 817.6 184 SM d42:1 815.7 184 SM d42:2 813.7 184 SM d42:2-d17 830.7 184 SM d42:2-d9 822.7 184 SM d42:3 811.7 184 SM d42:3-d17 828.7 184 SM d42:3-d34 845.7 184 SM d42:3-d9 820.7 184 SM d44:2 841.7 184 SM d44:2-d9 850.7 184 SM d44:3 839.7 184 SM d44:3-d17 856.7 184 METABOLITES_END #END