#METABOLOMICS WORKBENCH weitang_20241026_153558 DATATRACK_ID:5316 STUDY_ID:ST003595 ANALYSIS_ID:AN005904 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 NEGATIVE 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_AN005904_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 308570.9279 290642.6459 330958.1572 259901.766 293580.7456 416279.8846 330000.8117 244874.2633 316166.4549 346556.4165 662188.9205 484580.124 523012.9256 578431.275 477543.4055 615796.7475 529036.1685 669152.1928 566888.579 567108.3563 3-Hydroxydodecanoic acid 31663.53474 158392.2497 74785.78491 55154.36606 48582.30429 75968.00857 62252.9478 114082.3762 84322.65234 67426.0752 51487.31029 42345.42309 77390.67549 63205.022 43007.90149 34770.09228 37340.77286 49677.06103 29475.36709 37162.46716 LysoPC(15:0) 142070.6179 140745.6656 167352.036 155858.9162 152574.1864 169734.8076 147990.159 208541.0653 143260.8791 201569.4752 93345.78186 100162.7839 92118.354 106253.7902 88511.24688 105200.4589 113714.1841 115941.2451 93768.4431 118075.003 PC(O-16:0/0:0) 181705.686 177597.198 157663.175 202702.129 172226.5862 146092.0032 186857.8747 175763.714 211221.0644 181939.3488 110531.0582 106054.2945 112191.7478 99815.3904 107184.9919 109780.7938 107330.1461 116502.6192 99085.45536 122488.7136 PC(O-18:1/0:0) 438420.7767 392241.8348 415584.4628 467956.6756 416615.568 514855.4678 457164.3927 477463.5094 451399.9369 449729.7905 289713.2431 257031.8045 262399.128 264263.3421 268203.336 260528.9384 280501.3325 281187.4467 251874.1211 323273.2402 PC(19:1/0:0) 136496.448 112555.0482 185253.768 127319.13 133462.5743 238521.5806 155718.2686 214087.1585 135447.7703 161866.0062 103876.2667 116285.928 91271.07378 95034.98492 89841.00462 83323.06523 93603.576 93605.81954 83819.96444 115624.1272 PC(O-18:0/0:0) 319750.1217 265293.1659 346937.713 337517.5732 294459.036 345173.8184 365103.5937 372593.9061 416859.9136 318621.1377 197463.3386 193844.8977 175131.7904 185756.7835 162110.4417 179160.1492 166294.8334 165205.6821 144799.6346 213212.641 PC(19:0/0:0) 113261.2157 95689.746 145999.1311 119410.344 120028.8797 147178.214 130138.0152 148345.8855 107531.8841 125801.2512 74663.18191 74749.4075 68677.533 74676.359 69695.3865 62258.216 67029.8112 70726.6032 54503.84832 75050.3952 PC(20:0/0:0) 275811.84 240150.3903 369761.0958 254299.4922 261623.1616 315742.2435 306831.539 303505.5388 313509.716 291053.549 203087.0671 224918.7621 207458.9797 201567.8567 195363.5447 170548.2522 159878.6494 165973.8825 156981.0115 225354.04 PC(O-20) 62139.43909 52826.50286 71210.80364 63862.14545 57398.13273 58289.16571 70796.75236 56072.78182 84702.89455 50975.69455 39115.72364 44760.52286 37678.10073 45287.54836 28616.56364 40321.09091 31281.39 29617.54435 26326.05183 42868.09309 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name MW PubChem ID pantothenic acid 219 6613 3-Hydroxydodecanoic acid 216 94216 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) METABOLITES_END #END