#METABOLOMICS WORKBENCH weitang_20241026_153558 DATATRACK_ID:5316 STUDY_ID:ST003595 ANALYSIS_ID:AN005903 PROJECT_ID:PR002223 VERSION 1 CREATED_ON November 26, 2024, 5:52 pm #PROJECT PR:PROJECT_TITLE Trifluoroacetate reduces plasma lipid levels and the development of PR:PROJECT_TITLE atherosclerosis in mice PR:PROJECT_SUMMARY Trifluoroacetate (TFA) has been assumed to be an innocuous counterion (to PR:PROJECT_SUMMARY cationic amino acid side chains) present in countless synthetic bioactive PR:PROJECT_SUMMARY peptides and a few FDA-approved therapeutics. We show here that TFA is in fact PR:PROJECT_SUMMARY bioactive and causes dramatic biological effects in multiple strains of mice and PR:PROJECT_SUMMARY cultured human and rat liver cells. In high-fat diet (HFD)-fed low-density PR:PROJECT_SUMMARY lipoprotein receptor-null (LDLr-/-) mice, TFA reduces the levels of plasma PR:PROJECT_SUMMARY cholesterol, triglycerides, and the development of atherosclerotic lesions PR:PROJECT_SUMMARY following either oral or intraperitoneal administration. These physiological PR:PROJECT_SUMMARY effects were observed with TFA alone, or with TFA present as a counterion of a PR:PROJECT_SUMMARY variety of short, unrelated synthetic peptide sequences. Mechanistic PR:PROJECT_SUMMARY investigations including RNA-seq, confocal microscopy, western blotting, PR:PROJECT_SUMMARY metabolomics, proteomics, pharmacokinetics, and biochemical assays indicated PR:PROJECT_SUMMARY that TFA induces peroxisome proliferation by activating peroxisome PR:PROJECT_SUMMARY proliferator-activated receptor (PPAR)-alpha. We confirmed that TFA also caused PR:PROJECT_SUMMARY peroxisome proliferation and downstream phenotypic effects in cultured human and PR:PROJECT_SUMMARY rat liver cells, wild-type C57/Bl mice, and apolipoprotein E-null (apoE-/-) PR:PROJECT_SUMMARY mice, leading to anti-atherosclerotic effects in the latter strain. Given that PR:PROJECT_SUMMARY TFA is a counterion in many peptides employed in early research and development PR:PROJECT_SUMMARY settings, these findings raise the possibility that TFA may be confounding or PR:PROJECT_SUMMARY contributing to phenotypic changes observed in many studies involving peptides. PR:PROJECT_SUMMARY Although our studies suggest that TFA or its analogues might have therapeutic PR:PROJECT_SUMMARY applications, it should be noted that TFA is also a persistent environmental PR:PROJECT_SUMMARY contaminant that is found at high levels in humans relative to other PR:PROJECT_SUMMARY polyfluoroalkyl substances (PFAS), and is a major metabolite following treatment PR:PROJECT_SUMMARY of patients with common inhaled anesthetics, suggesting that the biological PR:PROJECT_SUMMARY effects reported here could have other implications for human health. PR:INSTITUTE Scripps Research PR:LAST_NAME Tang PR:FIRST_NAME Wei PR:ADDRESS 10550 North Torrey Pines Road, La Jolla, California, 92037, USA PR:EMAIL wtang@scripps.edu PR:PHONE 858-784-2711 #STUDY ST:STUDY_TITLE Trifluoroacetate reduces plasma lipid levels and the development of ST:STUDY_TITLE atherosclerosis in mice ST:STUDY_SUMMARY Trifluoroacetate (TFA) has been assumed to be an innocuous counterion (to ST:STUDY_SUMMARY cationic amino acid side chains) present in countless synthetic bioactive ST:STUDY_SUMMARY peptides and a few FDA-approved therapeutics. We show here that TFA is in fact ST:STUDY_SUMMARY bioactive and causes dramatic biological effects in multiple strains of mice and ST:STUDY_SUMMARY cultured human and rat liver cells. In high-fat diet (HFD)-fed low-density ST:STUDY_SUMMARY lipoprotein receptor-null (LDLr-/-) mice, TFA reduces the levels of plasma ST:STUDY_SUMMARY cholesterol, triglycerides, and the development of atherosclerotic lesions ST:STUDY_SUMMARY following either oral or intraperitoneal administration. These physiological ST:STUDY_SUMMARY effects were observed with TFA alone, or with TFA present as a counterion of a ST:STUDY_SUMMARY variety of short, unrelated synthetic peptide sequences. Mechanistic ST:STUDY_SUMMARY investigations including RNA-seq, confocal microscopy, western blotting, ST:STUDY_SUMMARY metabolomics, proteomics, pharmacokinetics, and biochemical assays indicated ST:STUDY_SUMMARY that TFA induces peroxisome proliferation by activating peroxisome ST:STUDY_SUMMARY proliferator-activated receptor (PPAR)-alpha. We confirmed that TFA also caused ST:STUDY_SUMMARY peroxisome proliferation and downstream phenotypic effects in cultured human and ST:STUDY_SUMMARY rat liver cells, wild-type C57/Bl mice, and apolipoprotein E-null (apoE-/-) ST:STUDY_SUMMARY mice, leading to anti-atherosclerotic effects in the latter strain. Given that ST:STUDY_SUMMARY TFA is a counterion in many peptides employed in early research and development ST:STUDY_SUMMARY settings, these findings raise the possibility that TFA may be confounding or ST:STUDY_SUMMARY contributing to phenotypic changes observed in many studies involving peptides. ST:STUDY_SUMMARY Although our studies suggest that TFA or its analogues might have therapeutic ST:STUDY_SUMMARY applications, it should be noted that TFA is also a persistent environmental ST:STUDY_SUMMARY contaminant that is found at high levels in humans relative to other ST:STUDY_SUMMARY polyfluoroalkyl substances (PFAS), and is a major metabolite following treatment ST:STUDY_SUMMARY of patients with common inhaled anesthetics, suggesting that the biological ST:STUDY_SUMMARY effects reported here could have other implications for human health. ST:INSTITUTE Scripps Research ST:LAST_NAME Tang ST:FIRST_NAME Wei ST:ADDRESS 10550 North Torrey Pines Road, La Jolla, California, 92037, USA ST:EMAIL wtang@scripps.edu ST:PHONE 858-784-2711 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 - PBS1 Sample source:plasma | Treatment:Control RAW_FILE_NAME(Raw file name)=rp_pos_PBS1_P1-A-4_01_16083.d SUBJECT_SAMPLE_FACTORS - PBS2 Sample source:plasma | Treatment:Control RAW_FILE_NAME(Raw file name)=rp_pos_PBS2_P1-A-6_01_16087.d SUBJECT_SAMPLE_FACTORS - PBS3 Sample source:plasma | Treatment:Control RAW_FILE_NAME(Raw file name)=rp_pos_PBS3_P1-A-8_01_16091.d SUBJECT_SAMPLE_FACTORS - PBS4 Sample source:plasma | Treatment:Control RAW_FILE_NAME(Raw file name)=rp_pos_PBS4_P1-B-1_01_16095.d SUBJECT_SAMPLE_FACTORS - PBS5 Sample source:plasma | Treatment:Control RAW_FILE_NAME(Raw file name)=rp_pos_PBS5_P1-B-3_01_16099.d SUBJECT_SAMPLE_FACTORS - PBS6 Sample source:plasma | Treatment:Control RAW_FILE_NAME(Raw file name)=rp_pos_PBS6_P1-B-5_01_16105.d SUBJECT_SAMPLE_FACTORS - PBS7 Sample source:plasma | Treatment:Control RAW_FILE_NAME(Raw file name)=rp_pos_PBS7_P1-B-7_01_16109.d SUBJECT_SAMPLE_FACTORS - PBS8 Sample source:plasma | Treatment:Control RAW_FILE_NAME(Raw file name)=rp_pos_PBS8_P1-B-9_01_16113.d SUBJECT_SAMPLE_FACTORS - PBS9 Sample source:plasma | Treatment:Control RAW_FILE_NAME(Raw file name)=rp_pos_PBS9_P1-C-2_01_16117.d SUBJECT_SAMPLE_FACTORS - PBS10 Sample source:plasma | Treatment:Control RAW_FILE_NAME(Raw file name)=rp_pos_PBS10_P1-C-4_01_16121.d SUBJECT_SAMPLE_FACTORS - TFA1 Sample source:plasma | Treatment:TFA RAW_FILE_NAME(Raw file name)=rp_pos_TFA1_P1-A-3_01_16081.d SUBJECT_SAMPLE_FACTORS - TFA2 Sample source:plasma | Treatment:TFA RAW_FILE_NAME(Raw file name)=rp_pos_TFA2_P1-A-5_01_16085.d SUBJECT_SAMPLE_FACTORS - TFA3 Sample source:plasma | Treatment:TFA RAW_FILE_NAME(Raw file name)=rp_pos_TFA3_P1-A-7_01_16089.d SUBJECT_SAMPLE_FACTORS - TFA4 Sample source:plasma | Treatment:TFA RAW_FILE_NAME(Raw file name)=rp_pos_TFA4_P1-A-9_01_16093.d SUBJECT_SAMPLE_FACTORS - TFA5 Sample source:plasma | Treatment:TFA RAW_FILE_NAME(Raw file name)=rp_pos_TFA5_P1-B-2_01_16097.d SUBJECT_SAMPLE_FACTORS - TFA6 Sample source:plasma | Treatment:TFA RAW_FILE_NAME(Raw file name)=rp_pos_TFA6_P1-B-4_01_16103.d SUBJECT_SAMPLE_FACTORS - TFA7 Sample source:plasma | Treatment:TFA RAW_FILE_NAME(Raw file name)=rp_pos_TFA7_P1-B-6_01_16107.d SUBJECT_SAMPLE_FACTORS - TFA8 Sample source:plasma | Treatment:TFA RAW_FILE_NAME(Raw file name)=rp_pos_TFA8_P1-B-8_01_16111.d SUBJECT_SAMPLE_FACTORS - TFA9 Sample source:plasma | Treatment:TFA RAW_FILE_NAME(Raw file name)=rp_pos_TFA9_P1-C-1_01_16115.d SUBJECT_SAMPLE_FACTORS - TFA10 Sample source:plasma | Treatment:TFA RAW_FILE_NAME(Raw file name)=rp_pos_TFA10_P1-C-3_01_16119.d #COLLECTION CO:COLLECTION_SUMMARY Blood was collected from female LDLr-/- mice treated by daily oral gavage for CO:COLLECTION_SUMMARY two weeks with a 200 µmol/kg TFA daily dose (n=10) or PBS vehicle (n=10). CO:COLLECTION_SUMMARY Samples were obtained by retro-orbital puncture using a heparinized capillary CO:COLLECTION_SUMMARY tube and then transferred to a K2EDTA collection tube. Plasma was immediately CO:COLLECTION_SUMMARY separated by centrifuging the blood at 5000 rpm for 10 minutes at 4°C. CO:SAMPLE_TYPE Blood (plasma) #TREATMENT TR:TREATMENT_SUMMARY Female LDLr-/- mice were treated for two weeks with daily oral gavage of either TR:TREATMENT_SUMMARY 200 µmol/kg TFA (n=10) or a PBS vehicle (n=10). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Plasma samples (100 µL) from female LDLr-/- mice treated by daily oral gavage SP:SAMPLEPREP_SUMMARY for two weeks with a 200 µmol/kg TFA daily dose (n=10) or PBS vehicle (n=10) SP:SAMPLEPREP_SUMMARY were extracted with cold MeOH (400 µL) and analyzed individually by reversed SP:SAMPLEPREP_SUMMARY phase LC-MS, in both positive and negative mode. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Waters ACQUITY UPLC BEH C18 (100 x 1 mm , 1.7 um, 130A) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% Acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 0-2min, 1%B; 2-12min, 1%-99%B; 12-15min, 99%B; 15-15.1min, 99%-1%B; 15.1-18min, CH:FLOW_GRADIENT 1%B CH:FLOW_RATE 0.15 mL/min CH:COLUMN_TEMPERATURE 25 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker Impact II MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Untargeted metabolomics were performed on an Agilent Technologies 1290 Infinity MS:MS_COMMENTS II system with an Waters ACQUITY UPLC BEH C18 Column, 130A, 1.7 um, 1 mm X 100 MS:MS_COMMENTS mm, coupled online to a Bruker Impact II QTOF mass spectrometer with MS:MS_COMMENTS electrospray ionization (ESI) source. The source dry gas temperature was set to MS:MS_COMMENTS 200°C at a flow of 8 L/min. The capillary voltage was set to 4000 V for MS:MS_COMMENTS positive mode and 5000 V for negative mode and the nebulizer operated at 29 psi. MS:CAPILLARY_VOLTAGE 4000 V MS:DRY_GAS_FLOW 8 l/min MS:DRY_GAS_TEMP 200 MS:NEBULIZER 29 psi MS:MS_RESULTS_FILE ST003595_AN005903_Results.txt UNITS:intensity Has m/z:Yes Has RT:Yes RT units:Minutes #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS intensity MS_METABOLITE_DATA_START Samples PBS1 PBS2 PBS3 PBS4 PBS5 PBS6 PBS7 PBS8 PBS9 PBS10 TFA1 TFA2 TFA3 TFA4 TFA5 TFA6 TFA7 TFA8 TFA9 TFA10 Factors Sample source:plasma | Treatment:Control Sample source:plasma | Treatment:Control Sample source:plasma | Treatment:Control Sample source:plasma | Treatment:Control Sample source:plasma | Treatment:Control Sample source:plasma | Treatment:Control Sample source:plasma | Treatment:Control Sample source:plasma | Treatment:Control Sample source:plasma | Treatment:Control Sample source:plasma | Treatment:Control Sample source:plasma | Treatment:TFA Sample source:plasma | Treatment:TFA Sample source:plasma | Treatment:TFA Sample source:plasma | Treatment:TFA Sample source:plasma | Treatment:TFA Sample source:plasma | Treatment:TFA Sample source:plasma | Treatment:TFA Sample source:plasma | Treatment:TFA Sample source:plasma | Treatment:TFA Sample source:plasma | Treatment:TFA pantothenic acid 389708.4153 361659.3991 424050.771 312069.7116 359596.9404 519775.3651 394217.2933 285212.0289 385793.8428 407339.936 825258.1191 585664.4188 639193.4057 737358.0059 577863.9958 734607.725 650550.6438 792153.8123 677114.9743 668778.3448 Linoelaidic Acid methyl ester 64455.47029 89251.344 56751.912 59208.8328 51841.1016 62539.14 67892.775 75579.8904 62517.5325 54700.6425 38135.664 25315.92 47396.99571 45758.655 37513.908 48268.46771 30474.90189 31979.0856 26371.7025 66549.0996 Deoxycorticosterone 28215.3595 18357.696 42919.9272 22120.81341 21715.12353 45133.35382 23692.71207 36437.32448 23816.916 26527.98 59516.0653 58937.11513 40874.53535 43182.01628 25546.7844 46236.82758 39042.78404 49246.78696 31026.57157 45827.78945 Dodecanoylcarnitine 44971.6595 97464.79875 80733.43674 57497.63085 52484.42647 104716.552 55534.28667 100445.441 57215.58544 73596.432 41335.42461 45075.39318 56268.6105 73230.49345 38871.9708 39704.04237 33734.33647 49583.09814 31113.50545 42745.84721 LysoPC(15:0) 818958.9359 734167.2326 972045.648 908657.1278 783168.1868 1018081.999 837168.7144 1225117.274 701754.4983 986248.8311 597207.7303 615708.9656 555792.2531 584569.7447 504626.0163 526309.9224 589508.8664 607982.5183 446615.8276 597103.8875 PC(O-16:0/0:0) 1798436.555 1615204.335 1666816.06 1944539.149 1720009.126 1748477.4 1853263.481 1892791.906 1809667.747 1900946.94 1090301.184 985599.72 1013693.075 1036302.229 979680.9154 1012337.003 1078489.579 1035343.808 927285.8832 1248273.974 PC(O-18:1/0:0) 2090044.142 1827920.943 2117310.048 2147154.252 1946043.302 2653339.084 2184222.482 2459802.121 2081835.967 2205287.58 1348154.732 1223932.996 1176459.765 1231377.079 1159142.139 1138870.526 1309319.869 1257407.716 1092967.273 1489429.91 PC(19:1/0:0) 482904.804 390973.3186 631374.912 462023.6797 489758.0756 841191.7952 532674.1216 770747.904 451735.704 580995.7053 370372.8 397704.5268 304205.5236 350052.9969 340658.8344 281876.7343 340454.7062 327362.5212 291519.5598 395260.9843 PC(O-18:0/0:0) 1830748.707 1500021.432 1939281.624 1923133.641 1764514.178 2088900.652 2141415.701 2358607.743 2331328.168 1954778.72 1161335.448 1062634.104 927840.2335 1050907.718 930401.1533 963644.8596 1036530.735 932144.3582 809110.7675 1143340.26 PC(19:0/0:0) 507920.616 456665.8325 655944.965 527930.189 524520.724 731963.9462 598232.7825 756811.857 501263.581 631509.7608 359257.4507 338891.2485 307074.6 317277.813 314195.079 291537.794 318485.7655 307016.3535 244596.8502 374628.1845 PC(20:0/0:0) 2091568.489 1643697.72 2646462.672 1900346.963 2048040.106 2594113.343 2323246.273 2631301.638 2228362.208 2065164.106 1674018.66 1636030.553 1418490.934 1513379.672 1469868.612 1214706.341 1333921.425 1218142.912 1176406.268 1544322.152 PC(O-20) 1216631.547 1253235.312 1293140.52 1253029.044 1111045.55 1434830.019 1684582.768 1653128.665 2023435.885 1225008.344 1009144.961 905214.1932 802051.9945 862615.0255 700368.8321 763830.1117 791264.27 763689.6428 572838.35 1013975.496 LysoPC(22:5) 402767.1657 397718.5443 478298.016 261330.9995 381953.4196 541477.5617 406887.6548 498219.1011 351378.5585 388732.5471 709796.4952 621108.432 549472.2046 610207.0814 566180.1554 670230.0376 592651.273 617526.4811 534108.0622 772666.8441 PC(17:0/0:0) 61572.93825 57821.19235 70096.32 62431.182 63998.704 69723.784 64901.9268 82384.13425 54681.98713 72583.236 45837.288 42179.8248 41179.04375 40930.068 43567.848 40899.936 42027.77775 37202.02602 35924.49648 46526.494 PC(17:0/0:0)2 4146940.963 4155606.467 3822039.749 3489315.933 3335339.594 4006587.903 4518850.648 6544242.988 3184209.189 3946921.069 2058472.083 3107317.657 2727575.507 3032112.247 1956216.285 2092389.663 2122837.994 2907484.186 2380483.354 3324236.435 PC(22:1/0:0) 443034.5233 407509.0663 501315.696 496550.4 451009.3275 575762.974 512361.5625 567078.7875 472783.155 518834.7675 360434.503 331617.0757 303867.648 366556.056 310539.2337 306091.3425 313330.3908 311657.1745 269593.26 378496.1455 PC(33:2) 114355.584 105171.1872 128503.9831 132638.704 118334.5234 125230.2739 100669.5411 161310.54 87499.09387 109964.7731 81765.05847 69328.1053 82679.51238 84226.34123 68892.63566 74474.628 65252.817 69552.44205 66964.14208 74443.7804 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name MW PubChem ID pantothenic acid 219 6613 Linoelaidic Acid methyl ester 295 5362793 Deoxycorticosterone 346 6166 Dodecanoylcarnitine 344 168381 LysoPC(15:0) 482 24779458 PC(O-16:0/0:0) 482 162126 PC(O-18:1/0:0) 85335863 PC(19:1/0:0) 520 42607444 PC(O-18:0/0:0) 510 2733532 PC(19:0/0:0) 538 24779472 PC(20:0/0:0) 552 24779473 PC(O-20) LysoPC(22:5) 570 53480475 PC(17:0/0:0) 510 24779463 PC(33:2) 744 52922715 PC(17:0/0:0)2 510 24779463 PC(22:1/0:0) 902 21308787 METABOLITES_END #END