#METABOLOMICS WORKBENCH boryana_ptrva_20240110_210727 DATATRACK_ID:4565 STUDY_ID:ST003073 ANALYSIS_ID:AN005031 PROJECT_ID:PR001914 VERSION 1 CREATED_ON February 9, 2024, 11:00 am #PROJECT PR:PROJECT_TITLE Optimized Mass Spectrometry Detection of Thyroid Hormones and Polar Metabolites PR:PROJECT_TITLE in Rodent Cerebrospinal Fluid PR:PROJECT_SUMMARY Thyroid hormones (TH) are required for brain development and function. PR:PROJECT_SUMMARY Cerebrospinal fluid (CSF), which bathes the brain and spinal cord, contains TH PR:PROJECT_SUMMARY as free hormone, or as bound to transthyretin (TTR). Tight TH level regulation PR:PROJECT_SUMMARY in the central nervous system is essential for de-velopmental gene expression PR:PROJECT_SUMMARY that governs neurogenesis, myelination, and synaptogenesis. This integrated PR:PROJECT_SUMMARY function of TH highlights the importance of developing precise and reliable PR:PROJECT_SUMMARY methods for assessing TH levels in CSF. We report an optimized liquid PR:PROJECT_SUMMARY chromatography-mass spectrome-try (LC-MS) based method to measure TH in rodent PR:PROJECT_SUMMARY CSF and serum, applicable to both fresh and frozen samples. Using this new PR:PROJECT_SUMMARY method, we find distinct differences in CSF TH in pregnant dams vs. non-pregnant PR:PROJECT_SUMMARY adults and in embryonic vs. adult CSF. Further, targeted LC-MS metabolic PR:PROJECT_SUMMARY pro-filing uncovers distinct central carbon metabolism in the CSF of these PR:PROJECT_SUMMARY populations. TH detection and metabolite profiling of related metabolic pathways PR:PROJECT_SUMMARY open new avenues of rigorous research into CSF TH and will inform future studies PR:PROJECT_SUMMARY on metabolic alterations in CSF during normal de-velopment. PR:INSTITUTE Boston Childrens Hospital PR:DEPARTMENT Patholody PR:LABORATORY Kanarek Lab PR:LAST_NAME Petrova PR:FIRST_NAME Boryana PR:ADDRESS 300 Longwood Av PR:EMAIL boryana.petrova@childrens.harvard.edu PR:PHONE 6173557433 #STUDY ST:STUDY_TITLE Investigation of 3,5,3’-L-triiodothyronine (T3) and ST:STUDY_TITLE 3,5,3’5’-L-tetraiodothyronine (T4) by targeted LC-MS analysis from mouse ST:STUDY_TITLE adult or embryonic CSF and from adult serum. ST:STUDY_SUMMARY To maximize discovery from samples with limited amounts, we optimized a method ST:STUDY_SUMMARY to detect the thyroid hormones ( 3,5,3’-L-triiodothyronine (T3) and ST:STUDY_SUMMARY 3,5,3’5’-L-tetraiodothyronine (T4)) as well as polar metabolites from ST:STUDY_SUMMARY central carbon metabolism from mouse adult or embryonic CSF and from adult mouse ST:STUDY_SUMMARY serum. Samples were ran on reverse phase and HILIC chromatography respectively. ST:STUDY_SUMMARY For T3 and T4 analysis we utilized targeted analysis using information from ST:STUDY_SUMMARY previously ran synthetic standards. We interrogated relative changes between ST:STUDY_SUMMARY fresh and frozen adult (male or female) CSF compared to fresh and frozen ST:STUDY_SUMMARY embryonic CSF. This data presents the levels of T3 and T4 as interrogated by ST:STUDY_SUMMARY reverse phase chromatography ST:INSTITUTE Boston Childrens Hospital ST:DEPARTMENT Pathology ST:LABORATORY Kanarek Lab ST:LAST_NAME Petrova ST:FIRST_NAME Boryana ST:ADDRESS 300 Longwood Av ST:EMAIL boryana.petrova@childrens.harvard.edu ST:PHONE 6173557433 ST:NUM_GROUPS 7 ST:TOTAL_SUBJECTS 19 ST:NUM_MALES 8 ST:NUM_FEMALES 6 ST:STUDY_COMMENTS embryos also investigated. samples split for fresh and frozen conditions #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 - RF377 Sample source:Adult Mock | Storage condition:Frozen | Sex:Male Combined label=Mock-Frozen Male; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF377 SUBJECT_SAMPLE_FACTORS - RF378 Sample source:Adult Mock | Storage condition:Frozen | Sex:Female Combined label=Mock Frozen-Famale; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF378 SUBJECT_SAMPLE_FACTORS - RF379 Sample source:Embryonic Mock | Storage condition:Frozen | Sex:- Combined label=Mock-Frozen-Embryo; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF379 SUBJECT_SAMPLE_FACTORS - RF380 Sample source:Adult Mock | Storage condition:Fresh | Sex:Male Combined label=Mock-Fresh-Male; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF380 SUBJECT_SAMPLE_FACTORS - RF381 Sample source:Adult Mock | Storage condition:Fresh | Sex:Female Combined label=Mock-Fresh-Female; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF381 SUBJECT_SAMPLE_FACTORS - RF382 Sample source:Embryonic Mock | Storage condition:Fresh | Sex:- Combined label=Mock-Fresh-Embryo; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF382 SUBJECT_SAMPLE_FACTORS - RF383 Sample source:Adult CSF | Storage condition:Frozen | Sex:Male Combined label=Male-Frozen-1; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF383 SUBJECT_SAMPLE_FACTORS - RF384 Sample source:Adult CSF | Storage condition:Frozen | Sex:Male Combined label=Male Frozen 3; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF384 SUBJECT_SAMPLE_FACTORS - RF385 Sample source:Adult CSF | Storage condition:Frozen | Sex:Male Combined label=Male-Frozen-4; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF385 SUBJECT_SAMPLE_FACTORS - RF386 Sample source:Adult CSF | Storage condition:Frozen | Sex:Male Combined label=Male-Frozen-6 (small pellet); Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF386 SUBJECT_SAMPLE_FACTORS - RF387 Sample source:Adult CSF | Storage condition:Frozen | Sex:Female Combined label=Female-Frozen-1; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF387 SUBJECT_SAMPLE_FACTORS - RF388 Sample source:Adult CSF | Storage condition:Frozen | Sex:Female Combined label=Female-Frozen-2; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF388 SUBJECT_SAMPLE_FACTORS - RF389 Sample source:Adult CSF | Storage condition:Frozen | Sex:Female Combined label=Frmale-Frozen-3; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF389 SUBJECT_SAMPLE_FACTORS - RF390 Sample source:Adult CSF | Storage condition:Frozen | Sex:Female Combined label=Female-Frozen-4; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF390 SUBJECT_SAMPLE_FACTORS - RF391 Sample source:Embryonic CSF | Storage condition:Frozen | Sex:- Combined label=Embryo-Frozen-1; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF391 SUBJECT_SAMPLE_FACTORS - RF392 Sample source:Embryonic CSF | Storage condition:Frozen | Sex:- Combined label=Embryo-Frozen-2; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF392 SUBJECT_SAMPLE_FACTORS - RF393 Sample source:Embryonic CSF | Storage condition:Frozen | Sex:- Combined label=Embryo-Frozen-3; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF393 SUBJECT_SAMPLE_FACTORS - RF394 Sample source:Embryonic CSF | Storage condition:Frozen | Sex:- Combined label=Embryo-Frozen-4; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF394 SUBJECT_SAMPLE_FACTORS - RF395 Sample source:Embryonic CSF | Storage condition:Frozen | Sex:- Combined label=Embryo-Frozen-5; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF395 SUBJECT_SAMPLE_FACTORS - RF396 Sample source:Adult CSF | Storage condition:Fresh | Sex:Male Combined label=Male-Fresh-1+4+6; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF396 SUBJECT_SAMPLE_FACTORS - RF397 Sample source:Adult CSF | Storage condition:Fresh | Sex:Male Combined label=Male-Fresh-2; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF397 SUBJECT_SAMPLE_FACTORS - RF398 Sample source:Adult CSF | Storage condition:Fresh | Sex:Male Combined label=Male-Fresh-5; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF398 SUBJECT_SAMPLE_FACTORS - RF399 Sample source:Adult CSF | Storage condition:Fresh | Sex:Male Combined label=Male-Fresh-8; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF399 SUBJECT_SAMPLE_FACTORS - RF400 Sample source:Adult CSF | Storage condition:Fresh | Sex:Female Combined label=Femal-Fresh-4; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF400 SUBJECT_SAMPLE_FACTORS - RF401 Sample source:Adult CSF | Storage condition:Fresh | Sex:Female Combined label=Female-Fresh-5; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF401 SUBJECT_SAMPLE_FACTORS - RF402 Sample source:Adult CSF | Storage condition:Fresh | Sex:Female Combined label=Female-Fresh-6; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF402 SUBJECT_SAMPLE_FACTORS - RF403 Sample source:Adult CSF | Storage condition:Fresh | Sex:Female Combined label=Female-Fresh-1 (small pellet); Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF403 SUBJECT_SAMPLE_FACTORS - RF404 Sample source:Embryonic CSF | Storage condition:Fresh | Sex:- Combined label=Embryo-Fresh-1; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF404 SUBJECT_SAMPLE_FACTORS - RF405 Sample source:Embryonic CSF | Storage condition:Fresh | Sex:- Combined label=Embryo-Fresh-2; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF405 SUBJECT_SAMPLE_FACTORS - RF406 Sample source:Embryonic CSF | Storage condition:Fresh | Sex:- Combined label=Embryo-Fresh-3; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF406 SUBJECT_SAMPLE_FACTORS - RF407 Sample source:Embryonic CSF | Storage condition:Fresh | Sex:- Combined label=Embryo-Fresh-4; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF407 SUBJECT_SAMPLE_FACTORS - RF408 Sample source:Embryonic CSF | Storage condition:Fresh | Sex:- Combined label=Embryo-Fresh-5; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF408 SUBJECT_SAMPLE_FACTORS - RF409 Sample source:Adult Serum | Storage condition:Frozen | Sex:Male Combined label=Male-1-Serum-Frozen; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF409 SUBJECT_SAMPLE_FACTORS - RF410 Sample source:Adult Serum | Storage condition:Frozen | Sex:Male Combined label=Male-2-Serum-Frozen; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF410 SUBJECT_SAMPLE_FACTORS - RF411 Sample source:Adult Serum | Storage condition:Fresh | Sex:Male Combined label=Male-1-Serum-Fresh; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF411 SUBJECT_SAMPLE_FACTORS - RF412 Sample source:Adult Serum | Storage condition:Fresh | Sex:Male Combined label=Male-2-Serum-Fresh; Genotype=wild type; RAW_FILE_NAME=20220920_QEF2_C18_T3T4readjusted_RF412 #COLLECTION CO:COLLECTION_SUMMARY Animals The Boston Children’s Hospital IACUC approved all experiments CO:COLLECTION_SUMMARY involving mice in this study. Adult CD-1 male and female mice and pregnant CD-1 CO:COLLECTION_SUMMARY dams were obtained from Charles River Laboratories. Sprague Dawley rats were CO:COLLECTION_SUMMARY purchased from Charles River Laboratories. Animals were housed in a temperature- CO:COLLECTION_SUMMARY and humidity-controlled room (70 ± 3 °F, 35–70% humidity) on a 12 h CO:COLLECTION_SUMMARY light/12 h dark cycle (7 a.m. on 7 p.m. off) and had free access to food and CO:COLLECTION_SUMMARY water. All animals younger than postnatal day 10 were al-located into groups CO:COLLECTION_SUMMARY based solely on the gestational age without respect to sex (both males and CO:COLLECTION_SUMMARY females were included). For studies involving rodents older than 10 days, sex CO:COLLECTION_SUMMARY was treated as a variable. Serum and CSF collection CSF was collected from the CO:COLLECTION_SUMMARY cisterna magna of adult (≥ 3 months old) or embryonic (E14.5) wild-type CD-1 CO:COLLECTION_SUMMARY mice. Independent samples (n) were defined as independent animals for adult CO:COLLECTION_SUMMARY samples, and as a CSF pool from all embryos in a single litter for embryonic CSF CO:COLLECTION_SUMMARY samples. Samples were maintained on ice, then spun 1000 × g for 10 min at CO:COLLECTION_SUMMARY 4 °C. The supernatant was collected and used for analysis. Blood was CO:COLLECTION_SUMMARY collected from cardiac puncture, allowed to clot for 10 minutes at room CO:COLLECTION_SUMMARY temperature, spun at 500 × g for 10 min at room temperature. The CO:COLLECTION_SUMMARY supernatant serum was collected and used for analysis. CO:SAMPLE_TYPE Cerebrospinal fluid CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY No treatments were performed. All animals were CD-1 wild type. We compared TR:TREATMENT_SUMMARY different groups based on samples storage condition (fresh, frozen, or 24h TR:TREATMENT_SUMMARY stored), animal sex (female or male), or type of biofluid (serum, adult CSF, and TR:TREATMENT_SUMMARY embryonic CSF) #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Sample preparation for LC-MS analysis of thyroid hormone metabolites from CSF SP:SAMPLEPREP_SUMMARY and serum in parallel to analysis for polar metabolites. Per condition, SP:SAMPLEPREP_SUMMARY 5–10 μL of CSF or serum was extracted in 4:6:3 chloroform:methanol:water SP:SAMPLEPREP_SUMMARY mixture supplemented with isotopically labeled T3 and T4 (at 100 nM, Cambridge SP:SAMPLEPREP_SUMMARY Isotope Laboratories, CLM-7185-C and CLM-8931-PK) and isotopically labeled 17 SP:SAMPLEPREP_SUMMARY amino acids (at 1/5000, Cambridge Isotope Laboratories, MSK-A2-1.2) and SP:SAMPLEPREP_SUMMARY isotopically labeled reduced glutathione (at 1 µM, Cambridge Isotope SP:SAMPLEPREP_SUMMARY Laboratories and CNLM-6245-10). After centrifugation for 10 min at maximum SP:SAMPLEPREP_SUMMARY speed on a benchtop centrifuge (Eppendorf) the top, hydrophilic layer was SP:SAMPLEPREP_SUMMARY transferred to a new tube, dried using a nitrogen dryer (ThermoFisher SP:SAMPLEPREP_SUMMARY Scientific, TS-18826) and reconstituted in 20 µL 70% ac-etonitrile SP:SAMPLEPREP_SUMMARY (supplemented with QReSS, Cambridge Isotope Laboratories, MSK-QRESS-KIT) by SP:SAMPLEPREP_SUMMARY brief vortexing. Extracted metabolites were spun again and cleared supernatant SP:SAMPLEPREP_SUMMARY was transferred to LC-MS micro vials. The protocol was used for both fresh and SP:SAMPLEPREP_SUMMARY frozen CSF and serum samples. A small amount of each sample was also pooled and SP:SAMPLEPREP_SUMMARY serially diluted 3- and 10-fold to be used as quality controls throughout the SP:SAMPLEPREP_SUMMARY run of each batch. Serum and CSF sample pools were kept separate and serum and SP:SAMPLEPREP_SUMMARY CSF sample sets were run consecutively on our chromatography to avoid SP:SAMPLEPREP_SUMMARY interspersing the run of two different matrixes. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Sigma Aldrich Ascentis Express C18 (150 × 2.1 mm, 2.7 um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid. CH:FLOW_GRADIENT The chromatographic gradient was run at a flow rate of 0.250 mL min−1 as CH:FLOW_GRADIENT follows: 0–5 min: gradient was held at 5% B; 2–12.1 min: linear gradient of CH:FLOW_GRADIENT 5% to 95% B; 12.1–17.0 min: 95% B; 17.1–21.0 min: gradient was returned to CH:FLOW_GRADIENT 5% B CH:FLOW_RATE 0.25 mL/min CH:COLUMN_TEMPERATURE 30 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS MS Data Acquisition Conditions for Targeted Analysis of Polar Metabolites and MS:MS_COMMENTS Thyroid Hormones MS data acquisition was performed using a Q Exactive Orbitrap MS:MS_COMMENTS benchtop orbitrap mass spectrometer equipped with an Ion Max source and a HESI MS:MS_COMMENTS II probe (Thermo Fisher Scientific, San Jose, CA, USA). A narrow scan in MS:MS_COMMENTS positive mode at m/z = 600–800 was used for more specific detection of TH. The MS:MS_COMMENTS resolution was set at 70,000, the AGC target was 5 × 105, and the max IT was MS:MS_COMMENTS 100 msec. HESI conditions were as follows: sheath gas flow rate: 40 units; Aug MS:MS_COMMENTS gas flow rate: 10 units; sweet gas flow rate: 0; spray voltage: 3.5 kV (pos), MS:MS_COMMENTS 2.8 kV (neg); capillary temperature: 380 °C; S-lens RF: 60; Aux gas heater MS:MS_COMMENTS temperature: 420 °C. Targeted Metabolomics Data Analysis Relative MS:MS_COMMENTS quantification of polar metabolites was performed with TraceFinder 5.1 (Thermo MS:MS_COMMENTS Fisher Scientific, Waltham, MA, USA) using a 5 ppm mass tolerance and MS:MS_COMMENTS referencing an in-house library of chemical standards (see associated MS:MS_COMMENTS Supplemental Dataset S1). We routinely queried 266 compounds (40 internal MS:MS_COMMENTS standards and 226 metabolites). Pooled samples and fractional dilutions were MS:MS_COMMENTS prepared as quality controls and injected at the beginning and end of each run. MS:MS_COMMENTS In addition, pooled samples were interspersed throughout the run to control for MS:MS_COMMENTS technical drift in signal quality as well as to serve to assess the coefficient MS:MS_COMMENTS of variability (CV) for each metabolite. Data from TraceFinder were further MS:MS_COMMENTS consolidated and normalized with an in-house R script, freely accessible at MS:MS_COMMENTS github MS:MS_COMMENTS (https://github.com/FrozenGas/KanarekLabTraceFinderRScripts/blob/main/MS_data_script_v2.4_20221018.R). MS:MS_COMMENTS Briefly, this script performs the following normalization and quality control MS:MS_COMMENTS steps: (1) extracts and combines the peak areas from TraceFinder output.csvs; MS:MS_COMMENTS (2) calculates and normalizes to an averaged factor from all mean-centered MS:MS_COMMENTS chromatographic peak areas of isotopically labeled amino acid and QReSS internal MS:MS_COMMENTS standards within each sample; (3) filters out low-quality metabolites based on MS:MS_COMMENTS user-inputted cut-offs calculated from pool reinjections and pool dilutions; (4) MS:MS_COMMENTS calculates and normalizes for biological material amounts based on the total MS:MS_COMMENTS integrated peak area values of high-confidence metabolites. In this study, the MS:MS_COMMENTS linear correlation between the dilution factor and the peak area cut-offs is set MS:MS_COMMENTS to RSQ > 0.95 and the coefficient of variation (CV) < 30%. Finally, data were MS:MS_COMMENTS log transformed and Pareto scaled within the MetaboAnalyst-based statistical MS:MS_COMMENTS analysis platform [42] to generate PCA, PLSDA, volcano plots, and heatmaps. MS:MS_COMMENTS Individual metabolite bar plots and statistics were calculated in Excel (v16.81) MS:MS_COMMENTS and GraphPad Prism (v.10). Data Analysis for TH Levels Data analysis for T3 and MS:MS_COMMENTS T4 levels was performed with TraceFinder 5.1 as described above, referencing MS:MS_COMMENTS empirically determined retention times using standards. Isotopically labeled T3 MS:MS_COMMENTS and T4 internal standards (Cambridge Isotope Laboratories, Tewksbury, MA, USA; MS:MS_COMMENTS CLM-7185-C and CLM-8931-PK) were used to normalize for technical signal MS:MS_COMMENTS variability and matrix effects. Equal volumes of samples were compared per MS:MS_COMMENTS condition. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS a.u. MS_METABOLITE_DATA_START Samples RF377 RF378 RF379 RF380 RF381 RF382 RF383 RF384 RF385 RF386 RF387 RF388 RF389 RF390 RF391 RF392 RF393 RF394 RF395 RF396 RF397 RF398 RF399 RF400 RF401 RF402 RF403 RF404 RF405 RF406 RF407 RF408 RF409 RF410 RF411 RF412 Factors Sample source:Adult Mock | Storage condition:Frozen | Sex:Male Sample source:Adult Mock | Storage condition:Frozen | Sex:Female Sample source:Embryonic Mock | Storage condition:Frozen | Sex:- Sample source:Adult Mock | Storage condition:Fresh | Sex:Male Sample source:Adult Mock | Storage condition:Fresh | Sex:Female Sample source:Embryonic Mock | Storage condition:Fresh | Sex:- Sample source:Adult CSF | Storage condition:Frozen | Sex:Male Sample source:Adult CSF | Storage condition:Frozen | Sex:Male Sample source:Adult CSF | Storage condition:Frozen | Sex:Male Sample source:Adult CSF | Storage condition:Frozen | Sex:Male Sample source:Adult CSF | Storage condition:Frozen | Sex:Female Sample source:Adult CSF | Storage condition:Frozen | Sex:Female Sample source:Adult CSF | Storage condition:Frozen | Sex:Female Sample source:Adult CSF | Storage condition:Frozen | Sex:Female Sample source:Embryonic CSF | Storage condition:Frozen | Sex:- Sample source:Embryonic CSF | Storage condition:Frozen | Sex:- Sample source:Embryonic CSF | Storage condition:Frozen | Sex:- Sample source:Embryonic CSF | Storage condition:Frozen | Sex:- Sample source:Embryonic CSF | Storage condition:Frozen | Sex:- Sample source:Adult CSF | Storage condition:Fresh | Sex:Male Sample source:Adult CSF | Storage condition:Fresh | Sex:Male Sample source:Adult CSF | Storage condition:Fresh | Sex:Male Sample source:Adult CSF | Storage condition:Fresh | Sex:Male Sample source:Adult CSF | Storage condition:Fresh | Sex:Female Sample source:Adult CSF | Storage condition:Fresh | Sex:Female Sample source:Adult CSF | Storage condition:Fresh | Sex:Female Sample source:Adult CSF | Storage condition:Fresh | Sex:Female Sample source:Embryonic CSF | Storage condition:Fresh | Sex:- Sample source:Embryonic CSF | Storage condition:Fresh | Sex:- Sample source:Embryonic CSF | Storage condition:Fresh | Sex:- Sample source:Embryonic CSF | Storage condition:Fresh | Sex:- Sample source:Embryonic CSF | Storage condition:Fresh | Sex:- Sample source:Adult Serum | Storage condition:Frozen | Sex:Male Sample source:Adult Serum | Storage condition:Frozen | Sex:Male Sample source:Adult Serum | Storage condition:Fresh | Sex:Male Sample source:Adult Serum | Storage condition:Fresh | Sex:Male 3,5,3’-L-triiodothyronine 7798.4164 7010.98903 10374.3618 12280.8962 7408.42237 15211.0644 16491.831 22321.9549 5889.79765 14281.0002 10352.106 8665.56812 8594.34228 8827.26479 373394.699 232050.25 344687.228 440688.173 303772.743 13978.4949 13229.8489 48863.6649 6927.50729 9508.70428 9412.62374 7278.39272 15912.8301 356896.356 436778.112 339880.944 304540.165 363436.675 79986.1724 95641.9977 57299.2961 50168.5061 3,5,3’5’-L-tetraiodothyronine 16990.9192 12720.3077 NA 153365.659 16843.4988 NA 291104.987 180982.823 32690.3207 230798.299 167576.78 170785.073 151111.783 152098.627 114213.875 96177.2154 91453.9111 22382.1654 30247.6007 188563.769 254621.98 274282.697 268012.162 181123.297 93709.3032 107055.383 117170.45 70588.9884 336067.793 925097.83 63255.1561 222686.025 1327435.93 8190690.32 4459162.86 5784880.41 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name 3,5,3’-L-triiodothyronine 3,5,3’5’-L-tetraiodothyronine METABOLITES_END #END