#METABOLOMICS WORKBENCH AF_20230407_103033 DATATRACK_ID:3851 STUDY_ID:ST002549 ANALYSIS_ID:AN004197 PROJECT_ID:PR001639 VERSION 1 CREATED_ON April 7, 2023, 1:28 pm #PROJECT PR:PROJECT_TITLE Lipidomic analysis of serum from mice with Toxoplasma gondii infection PR:PROJECT_SUMMARY Cachexia is a life-threatening disease characterized by chronic, inflammatory PR:PROJECT_SUMMARY muscle wasting and systemic metabolic impairment. Despite its high prevalence, PR:PROJECT_SUMMARY there are no efficacious therapies for cachexia. Mice chronically infected with PR:PROJECT_SUMMARY the protozoan parasite Toxoplasma gondii represent a novel animal model PR:PROJECT_SUMMARY recapitulating the chronic kinetics of cachexia. To understand how perturbations PR:PROJECT_SUMMARY to metabolic tissue homeostasis influence circulating metabolite availability we PR:PROJECT_SUMMARY used mass spectrometry analysis. Despite the significant reduction in PR:PROJECT_SUMMARY circulating triacylglycerides, nonesterified fatty acids, and glycerol, PR:PROJECT_SUMMARY sphingolipid long-chain bases and a subset of phosphatidylcholines (PCs) were PR:PROJECT_SUMMARY significantly increased in the sera of mice with T. gondii infection-induced PR:PROJECT_SUMMARY cachexia. In addition, the TCA cycle intermediates -ketoglutarate, 2- PR:PROJECT_SUMMARY hydroxyglutarate, succinate, fumarate, and malate were highly depleted in PR:PROJECT_SUMMARY cachectic mouse sera. Sphingolipids and their de novo synthesis precursors PCs PR:PROJECT_SUMMARY are the major components of the mitochondrial membrane and regulate PR:PROJECT_SUMMARY mitochondrial function consistent with a causal relationship in the energy PR:PROJECT_SUMMARY imbalance driving T. gondii-induced chronic cachexia. PR:INSTITUTE University of Virginia PR:LAST_NAME Feng PR:FIRST_NAME Tzu-Yu PR:ADDRESS 345 Crispell Dr. PR:EMAIL ttf4ye@virginia.edu PR:PHONE 702-217-4454 #STUDY ST:STUDY_TITLE Lipidomic analysis of serum from mice with Toxoplasma gondii infection ST:STUDY_SUMMARY Cachexia is a life-threatening disease characterized by chronic, inflammatory ST:STUDY_SUMMARY muscle wasting and systemic metabolic impairment. Despite its high prevalence, ST:STUDY_SUMMARY there are no efficacious therapies for cachexia. Mice chronically infected with ST:STUDY_SUMMARY the protozoan parasite Toxoplasma gondii represent a novel animal model ST:STUDY_SUMMARY recapitulating the chronic kinetics of cachexia. To understand how perturbations ST:STUDY_SUMMARY to metabolic tissue homeostasis influence circulating metabolite availability we ST:STUDY_SUMMARY used mass spectrometry analysis. Despite the significant reduction in ST:STUDY_SUMMARY circulating triacylglycerides, nonesterified fatty acids, and glycerol, ST:STUDY_SUMMARY sphingolipid long-chain bases and a subset of phosphatidylcholines (PCs) were ST:STUDY_SUMMARY significantly increased in the sera of mice with T. gondii infection-induced ST:STUDY_SUMMARY cachexia. In addition, the TCA cycle intermediates -ketoglutarate, 2- ST:STUDY_SUMMARY hydroxyglutarate, succinate, fumarate, and malate were highly depleted in ST:STUDY_SUMMARY cachectic mouse sera. Sphingolipids and their de novo synthesis precursors PCs ST:STUDY_SUMMARY are the major components of the mitochondrial membrane and regulate ST:STUDY_SUMMARY mitochondrial function consistent with a causal relationship in the energy ST:STUDY_SUMMARY imbalance driving T. gondii-induced chronic cachexia. ST:INSTITUTE University of Virginia ST:LAST_NAME Feng ST:FIRST_NAME Tzu-Yu ST:ADDRESS 345 Crispell Dr. ST:EMAIL ttf4ye@virginia.edu ST:PHONE 702-217-4454 #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 - Uninfected-1 Factor:Uninfected RAW_FILE_NAME=2020_10_28_Ewald_Plasma_SLs_27 SUBJECT_SAMPLE_FACTORS - Uninfected-2 Factor:Uninfected RAW_FILE_NAME=2020_10_28_Ewald_Plasma_SLs_28 SUBJECT_SAMPLE_FACTORS - Uninfected-3 Factor:Uninfected RAW_FILE_NAME=2020_10_28_Ewald_Plasma_SLs_29 SUBJECT_SAMPLE_FACTORS - Uninfected-4 Factor:Uninfected RAW_FILE_NAME=2020_10_28_Ewald_Plasma_SLs_30 SUBJECT_SAMPLE_FACTORS - Uninfected-5 Factor:Uninfected RAW_FILE_NAME=2020_10_28_Ewald_Plasma_SLs_31 SUBJECT_SAMPLE_FACTORS - Uninfected-6 Factor:Uninfected RAW_FILE_NAME=2020_10_28_Ewald_Plasma_SLs_32 SUBJECT_SAMPLE_FACTORS - Uninfected-7 Factor:Uninfected RAW_FILE_NAME=2020_10_28_Ewald_Plasma_SLs_33 SUBJECT_SAMPLE_FACTORS - Uninfected-8 Factor:Uninfected RAW_FILE_NAME=2020_10_28_Ewald_Plasma_SLs_34 SUBJECT_SAMPLE_FACTORS - Uninfected-9 Factor:Uninfected RAW_FILE_NAME=2020_10_28_Ewald_Plasma_SLs_35 SUBJECT_SAMPLE_FACTORS - Toxoplasma infected-1 Factor:Infected RAW_FILE_NAME=2020_10_28_Ewald_Plasma_SLs_36 SUBJECT_SAMPLE_FACTORS - Toxoplasma infected-2 Factor:Infected RAW_FILE_NAME=2020_10_28_Ewald_Plasma_SLs_37 SUBJECT_SAMPLE_FACTORS - Toxoplasma infected-3 Factor:Infected RAW_FILE_NAME=2020_10_28_Ewald_Plasma_SLs_38 SUBJECT_SAMPLE_FACTORS - Toxoplasma infected-4 Factor:Infected RAW_FILE_NAME=2020_10_28_Ewald_Plasma_SLs_39 SUBJECT_SAMPLE_FACTORS - Toxoplasma infected-5 Factor:Infected RAW_FILE_NAME=2020_10_28_Ewald_Plasma_SLs_40 SUBJECT_SAMPLE_FACTORS - Toxoplasma infected-6 Factor:Infected RAW_FILE_NAME=2020_10_28_Ewald_Plasma_SLs_41 SUBJECT_SAMPLE_FACTORS - Toxoplasma infected-7 Factor:Infected RAW_FILE_NAME=2020_10_28_Ewald_Plasma_SLs_43 SUBJECT_SAMPLE_FACTORS - Toxoplasma infected-8 Factor:Infected RAW_FILE_NAME=2020_10_28_Ewald_Plasma_SLs_44 SUBJECT_SAMPLE_FACTORS - Toxoplasma infected-9 Factor:Infected RAW_FILE_NAME=2020_10_28_Ewald_Plasma_SLs_45 #COLLECTION CO:COLLECTION_SUMMARY At 7 weeks post-infection, isoflurane-anesthetized mice were retro-orbitally CO:COLLECTION_SUMMARY bled and sera was flash frozen and sent to the National Institute of Health CO:COLLECTION_SUMMARY (NIH) West Coast Metabolomics Center (UC Davis) for untargeted mass spectrometry CO:COLLECTION_SUMMARY analysisusing the primary metabolism assay (ALEXCIS GCTOF-MS) or the complex CO:COLLECTION_SUMMARY lipids (CSH-QTOF MS) assay. Detected meatbolites were identified based on CO:COLLECTION_SUMMARY retention time and mass spectra from MassBank of North America, curated by the CO:COLLECTION_SUMMARY NIH West Coast Metabolomics Center , and reported as raw peak heights (Table S1 CO:COLLECTION_SUMMARY and S3). The raw peak heights from each analytical platform were normalized to CO:COLLECTION_SUMMARY the average peak heights of the identified metabolites in uninfected group. The CO:COLLECTION_SUMMARY resulting data were analyzed for fold-change and multiple unpaired t-test and CO:COLLECTION_SUMMARY visualized using volcano plots to identify the differential expression of CO:COLLECTION_SUMMARY metabolites in response to T. gondii-induced cachexia. CO:SAMPLE_TYPE Blood (serum) #TREATMENT TR:TREATMENT_SUMMARY To generate cysts for infection, 8-10 week female CBA/J mice were infected with TR:TREATMENT_SUMMARY 10 Me49 bradyzoite cysts by intraperitoneal injection. 4–8 weeks following TR:TREATMENT_SUMMARY infection, mice were euthanized by CO2 inhalation and cysts were harvest from TR:TREATMENT_SUMMARY brains homogenate passed through a 70 μm filter. Homegenate was washed 3 times TR:TREATMENT_SUMMARY in PBS, stained with dolichos biflorus agglutinin conjugated to FITC (Vector TR:TREATMENT_SUMMARY labs) at a 1:500 dilution. The number of cysts were determined by counting TR:TREATMENT_SUMMARY FITCpositive cysts at 20x magnification using an EVOS FL imaging system (Thermo TR:TREATMENT_SUMMARY Fisher). For experimental infections 10–14-week-old male C57BL/6 mice were TR:TREATMENT_SUMMARY infected with 10 Me49 bradyzoite cysts by intraperitoneal infection resuspended TR:TREATMENT_SUMMARY in 200 Μl PBS per mouse using a 5G 5/8” tuberculin syringe. Prior to TR:TREATMENT_SUMMARY infection, mice were cross-housed on dirty, wood chip bedding for two weeks to TR:TREATMENT_SUMMARY normalize commensal microbiota and limit the effect of eating corn husk bedding TR:TREATMENT_SUMMARY on dietary metabolites. At experimental endpoints, mice were fasted for 4 hours TR:TREATMENT_SUMMARY and isoflurane anaesthetized to isolate sera via retro-orbital bleed and/or TR:TREATMENT_SUMMARY euthanized by CO2 asphyxiation to harvest tissues for weighing and histological TR:TREATMENT_SUMMARY analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY At 7 weeks post-infection, isoflurane-anesthetized mice were retro-orbitally SP:SAMPLEPREP_SUMMARY bled and sera was flash frozen and sent to the National Institute of Health SP:SAMPLEPREP_SUMMARY (NIH) West Coast Metabolomics Center (UC Davis) for untargeted mass spectrometry SP:SAMPLEPREP_SUMMARY analysisusing the primary metabolism assay (ALEXCIS GCTOF-MS) or the complex SP:SAMPLEPREP_SUMMARY lipids (CSH-QTOF MS) assay. Detected meatbolites were identified based on SP:SAMPLEPREP_SUMMARY retention time and mass spectra from MassBank of North America, curated by the SP:SAMPLEPREP_SUMMARY NIH West Coast Metabolomics Center , and reported as raw peak heights (Table S1 SP:SAMPLEPREP_SUMMARY and S3). The raw peak heights from each analytical platform were normalized to SP:SAMPLEPREP_SUMMARY the average peak heights of the identified metabolites in uninfected group. The SP:SAMPLEPREP_SUMMARY resulting data were analyzed for fold-change and multiple unpaired t-test and SP:SAMPLEPREP_SUMMARY visualized using volcano plots to identify the differential expression of SP:SAMPLEPREP_SUMMARY metabolites in response to T. gondii-induced cachexia. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Low pH polar CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um) CH:SOLVENT_A 60% water/40% acetonitrile CH:SOLVENT_B 90% isopropanol/10% methanol CH:FLOW_GRADIENT 30% B for 0.5min, a linear gradient to 40% B for 3min, 60% B over 3min, then 85% CH:FLOW_GRADIENT B for 4.5min CH:FLOW_RATE 0.4mL/min CH:COLUMN_TEMPERATURE 65 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Xevo-TQ-S MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE Other MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS Lipid extraction and analysis for targeted sphingolipid analysis was done using MS:MS_COMMENTS liquid chromatography-electrospray ionization-tandem mass spectrometry MS:MS_COMMENTS (LC-ESI-MS/MS)69. Lipids were extracted from sera using an azeotrophic mix of MS:MS_COMMENTS isopropanol:water:ethyl acetate (3:1:6; v:v:v). Internal standards (10 pmol of MS:MS_COMMENTS d17 long-chain bases and C12 acylated sphingolipids) were added to samples at MS:MS_COMMENTS the onset of the extraction procedure. Extracts were separated on a Waters MS:MS_COMMENTS Iclass Acquity UPLC chromatography system. Mobile phases were (A) 60:40 MS:MS_COMMENTS water:acetonitrile and (B) 90:10 isopropanol:methanol with both mobile phases MS:MS_COMMENTS containing 5 mM ammonium formate and 0.1% formic acid. A Waters C18 CSH 2.1 mm MS:MS_COMMENTS ID × 10 cm column maintained at 65°C was used for the separation of the MS:MS_COMMENTS sphingoid bases, 1-phosphates, and acylated sphingolipids. The eluate was MS:MS_COMMENTS analyzed with an inline Waters TQ-S mass spectrometer using multiple reaction MS:MS_COMMENTS monitoring. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS pmol/mL of plasma MS_METABOLITE_DATA_START Samples Toxoplasma infected-1 Toxoplasma infected-2 Toxoplasma infected-3 Toxoplasma infected-4 Toxoplasma infected-5 Toxoplasma infected-6 Toxoplasma infected-7 Toxoplasma infected-8 Toxoplasma infected-9 Uninfected-1 Uninfected-2 Uninfected-3 Uninfected-4 Uninfected-5 Uninfected-6 Uninfected-7 Uninfected-8 Uninfected-9 Factors Factor:Infected Factor:Infected Factor:Infected Factor:Infected Factor:Infected Factor:Infected Factor:Infected Factor:Infected Factor:Infected Factor:Uninfected Factor:Uninfected Factor:Uninfected Factor:Uninfected Factor:Uninfected Factor:Uninfected Factor:Uninfected Factor:Uninfected Factor:Uninfected Sph 123.96 130.23 82.67 41.34 122.48 58.61 43.85 49.39 29.44 45.88 26.74 72.20 62.20 61.32 44.73 65.14 58.13 36.70 dhSph 161.42 155.47 94.55 48.12 147.86 66.76 46.79 62.52 29.68 51.82 38.13 81.30 76.01 72.50 55.01 70.28 75.09 38.59 S1P 4048.32 4207.97 3880.85 3623.65 3938.66 4375.00 4082.47 3496.77 2365.45 2782.51 3042.28 3332.13 2954.61 2886.61 3346.80 3005.36 3553.26 3279.96 dhS1P 2038.09 1708.26 1664.26 1124.11 1771.59 1726.96 1651.66 1590.24 1030.52 1076.49 1118.87 1366.54 1093.12 1000.26 916.17 1095.88 1327.46 1199.91 HexSph 1.958 1.435 1.793 0.641 1.350 0.891 1.498 1.358 1.390 1.253 1.329 1.317 1.837 0.965 1.470 2.056 1.597 1.338 LysoSM 1.757 1.652 2.143 2.631 1.516 1.820 1.879 2.231 2.460 2.338 1.800 1.515 1.554 2.036 1.385 1.222 1.881 1.637 Cer C16 621.70 749.05 691.52 626.16 562.85 613.96 691.86 590.92 474.33 684.81 516.03 729.22 576.47 503.40 580.21 619.37 530.95 472.28 Cer C18 26.14 58.59 55.54 26.16 38.84 48.85 41.64 36.68 27.60 39.39 19.62 44.46 18.01 26.21 23.03 20.38 24.17 13.26 Cer C20 191.90 156.03 218.06 187.32 181.56 165.91 251.39 108.51 137.62 250.65 149.62 282.84 186.08 186.61 127.74 214.04 151.56 131.17 Cer C22 1895.44 1535.59 2107.59 2413.10 1628.54 1186.32 1704.46 999.38 1299.64 1931.80 2323.01 2602.25 2325.24 2537.86 2112.33 3205.89 2172.93 2021.25 Cer C22:1 147.60 128.58 150.71 175.61 71.56 67.23 88.83 59.99 84.71 327.82 152.14 617.59 171.85 195.49 76.28 231.47 166.48 109.83 Cer C24 3179.05 3045.30 3839.18 3557.35 3194.36 2799.46 2862.87 2519.44 2601.32 2745.23 4219.86 3361.08 4663.96 3848.05 4043.47 5390.13 4708.62 3447.87 Cer C24:1 3660.55 2845.13 3791.65 4252.38 3301.90 2722.86 2426.92 3235.18 2545.98 4464.09 4148.72 5456.69 5072.10 4075.28 2883.68 4903.33 4340.43 2883.49 dhC16 24.09 66.88 27.78 62.65 29.80 21.35 24.71 14.96 14.73 20.84 36.94 25.12 32.95 40.96 33.04 38.46 18.11 55.33 dhC18 9.31 13.70 9.73 12.91 12.69 9.23 17.52 6.44 5.95 14.71 11.56 10.22 6.44 10.00 7.17 10.42 13.30 12.50 dhC20 12.99 20.67 11.89 28.90 18.18 8.10 17.56 10.70 8.57 11.03 38.75 27.26 15.35 22.92 17.19 29.24 13.53 25.96 dhC22 121.86 286.41 128.67 395.65 140.08 76.75 136.14 71.34 68.87 158.16 387.69 221.81 201.76 327.00 221.59 343.73 206.97 338.06 dhC24 152.78 376.59 154.97 444.93 208.03 138.05 240.42 105.68 108.56 204.45 459.55 259.58 285.56 375.95 255.97 402.22 298.20 373.36 dhC24:1 190.14 314.20 171.04 459.04 249.93 130.54 150.35 149.31 96.50 225.97 452.80 332.94 261.76 383.02 259.12 425.98 349.86 310.86 Hex C16 204.78 254.67 316.51 123.12 242.96 173.23 229.71 354.68 177.57 144.35 128.45 144.30 118.04 119.47 100.61 118.98 115.78 121.01 Hex C18 10.69 12.80 18.23 18.81 21.36 19.32 15.60 12.85 8.89 18.18 15.37 23.37 12.24 12.52 11.81 13.93 17.62 18.42 Hex C20 32.32 37.86 55.15 53.32 45.01 36.39 48.03 38.15 24.85 75.93 41.38 84.82 40.97 50.60 26.34 40.81 40.37 52.23 Hex C22 346.21 392.64 470.35 604.17 376.17 283.19 355.94 336.25 234.39 667.33 542.84 731.59 497.02 584.14 412.99 581.91 465.93 617.12 Hex C22:1 13.51 16.64 23.80 29.38 9.88 6.55 13.15 17.35 10.54 83.43 27.77 99.10 23.61 28.75 9.38 25.89 22.71 19.18 Hex C24 253.21 299.46 363.16 309.31 286.02 232.53 220.56 316.48 160.51 303.19 324.92 285.92 339.27 311.29 278.10 356.17 362.72 323.34 Hex C24:1 447.63 493.27 627.54 555.05 549.99 375.27 337.60 681.53 290.03 715.39 490.03 644.30 513.98 433.63 347.65 458.36 443.09 447.34 SpM C16 28851.28 31772.10 35527.77 30556.34 31705.21 28803.61 30457.26 31577.58 21780.00 29629.99 27009.16 29059.37 24531.52 29505.26 26580.32 23954.23 26577.60 27265.30 SpM C18 2963.69 3003.39 3755.13 3534.88 3418.81 3225.72 3093.70 3452.18 2341.87 3147.90 3133.91 3077.43 2640.39 3431.31 3030.03 2860.72 2995.11 3193.51 SpM C20 2335.41 2474.78 3224.94 2524.55 2827.16 2537.68 2729.11 1958.32 2064.51 2800.07 2507.97 2547.35 1997.94 2781.94 2357.75 2331.77 2448.41 2713.39 SpM C20:1 520.93 541.06 707.86 666.32 622.16 641.92 613.67 439.94 424.97 739.23 508.87 803.17 452.17 676.40 541.58 538.18 569.46 672.84 SpM C22 14908.14 18205.48 20866.54 18396.66 15991.00 13842.50 15902.16 10095.63 10638.83 18341.33 17893.61 16020.28 14506.84 20737.74 18145.84 22114.65 19226.69 20916.44 SpM C22:1 4824.59 6253.39 6916.28 7262.87 4646.99 4643.07 4953.63 3035.69 3474.67 8036.88 5484.71 9415.53 5077.72 7221.94 5332.38 6434.06 6416.44 6831.36 SpM C24 12255.75 16388.18 14343.94 12851.81 12578.13 12126.58 12344.32 10129.03 9251.83 17748.77 15402.53 13119.39 13840.59 15143.74 14497.51 19641.49 16372.90 14766.31 SpM C24:1 21737.80 26504.30 30018.03 28282.99 26254.92 25175.42 19193.32 23391.07 15946.56 27794.51 23380.89 27278.89 23585.64 26574.89 21423.32 24763.91 28947.75 25498.32 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Sph dhSph S1P dhS1P HexSph LysoSM Cer C16 Cer C18 Cer C20 Cer C22 Cer C22:1 Cer C24 Cer C24:1 dhC16 dhC18 dhC20 dhC22 dhC24 dhC24:1 Hex C16 Hex C18 Hex C20 Hex C22 Hex C22:1 Hex C24 Hex C24:1 SpM C16 SpM C18 SpM C20 SpM C20:1 SpM C22 SpM C22:1 SpM C24 SpM C24:1 METABOLITES_END #END