#METABOLOMICS WORKBENCH NicholasDevanney_20230120_133842 DATATRACK_ID:3695 STUDY_ID:ST002451 ANALYSIS_ID:AN003999 PROJECT_ID:PR001580 VERSION 1 CREATED_ON January 20, 2023, 4:22 pm #PROJECT PR:PROJECT_TITLE APOE modulates microglial immunometabolism in response to age, amyloid PR:PROJECT_TITLE pathology, and inflammatory challenge PR:PROJECT_SUMMARY The E4 allele of Apolipoprotein E (APOE) is associated with both metabolic PR:PROJECT_SUMMARY dysfunction and a heightened pro-inflammatory response – two findings that may PR:PROJECT_SUMMARY be intrinsically linked through the concept of immunometabolism. Here, we PR:PROJECT_SUMMARY combined bulk, single-cell, and spatial transcriptomics with cell-specific and PR:PROJECT_SUMMARY spatially resolved metabolic analyses to systematically address the role of APOE PR:PROJECT_SUMMARY across age, neuroinflammation, and AD pathology. RNAseq highlighted PR:PROJECT_SUMMARY immunometabolic changes across the APOE4 glial transcriptome, specifically in PR:PROJECT_SUMMARY subsets of metabolically distinct microglia enriched in the E4 brain during PR:PROJECT_SUMMARY aging or following an inflammatory challenge. E4 microglia display increased PR:PROJECT_SUMMARY Hif1α expression, a disrupted TCA cycle, and are inherently pro-glycolytic, PR:PROJECT_SUMMARY while spatial transcriptomics and MALDI mass spectrometry imaging highlight an PR:PROJECT_SUMMARY E4-specific response to amyloid that is characterized by widespread alterations PR:PROJECT_SUMMARY in lipid metabolism. Taken together, our findings emphasize a central role for PR:PROJECT_SUMMARY APOE in regulating microglial immunometabolism. PR:INSTITUTE University of Kentucky PR:DEPARTMENT Physiology PR:LABORATORY Lance Johnson; Josh Morganti PR:LAST_NAME Devanney PR:FIRST_NAME Nicholas PR:ADDRESS Physiology, 760 Press Ave, Healthy Kentucky Research Bldg, Rm152, Lexington, PR:ADDRESS Kentucky, 40508, USA PR:EMAIL Nicholas.Devanney@uky.edu PR:PHONE 8593238083 PR:PROJECT_COMMENTS Part 2 of 3 #STUDY ST:STUDY_TITLE APOE modulates microglial immunometabolism in response to age, amyloid ST:STUDY_TITLE pathology, and inflammatory challenge (Part 2 of 3) ST:STUDY_SUMMARY The E4 allele of Apolipoprotein E (APOE) is associated with both metabolic ST:STUDY_SUMMARY dysfunction and a heightened pro-inflammatory response – two findings that may ST:STUDY_SUMMARY be intrinsically linked through the concept of immunometabolism. Here, we ST:STUDY_SUMMARY combined bulk, single-cell, and spatial transcriptomics with cell-specific and ST:STUDY_SUMMARY spatially resolved metabolic analyses to systematically address the role of APOE ST:STUDY_SUMMARY across age, neuroinflammation, and AD pathology. RNAseq highlighted ST:STUDY_SUMMARY immunometabolic changes across the APOE4 glial transcriptome, specifically in ST:STUDY_SUMMARY subsets of metabolically distinct microglia enriched in the E4 brain during ST:STUDY_SUMMARY aging or following an inflammatory challenge. E4 microglia display increased ST:STUDY_SUMMARY Hif1α expression, a disrupted TCA cycle, and are inherently pro-glycolytic, ST:STUDY_SUMMARY while spatial transcriptomics and MALDI mass spectrometry imaging highlight an ST:STUDY_SUMMARY E4-specific response to amyloid that is characterized by widespread alterations ST:STUDY_SUMMARY in lipid metabolism. Taken together, our findings emphasize a central role for ST:STUDY_SUMMARY APOE in regulating microglial immunometabolism. ST:INSTITUTE University of Kentucky ST:DEPARTMENT Physiology ST:LABORATORY Lance Johnson; Josh Morganti ST:LAST_NAME Devanney ST:FIRST_NAME Nicholas ST:ADDRESS Physiology, 760 Press Ave, Healthy Kentucky Research Bldg, Rm152, Lexington, ST:ADDRESS Kentucky, 40508, USA ST:EMAIL Nicholas.Devanney@uky.edu ST:PHONE 8593238083 ST:STUDY_COMMENTS Part 2 of 3 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN APOE-targeted replacement mice homozygous for human E3 SU:GENOTYPE_STRAIN (B6.129P2-Apoe^tm2(APOE*3)Mae N8, Taconic #1548-F) or human E4 (B6.129P2- SU:GENOTYPE_STRAIN Apoe^tm3(APOE*4)Mae N8, Taconic #1549-F) alleles SU:AGE_OR_AGE_RANGE P0-P3 SU:GENDER Pooled #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 - 47_3_1 Group:E3_control | APOE genotype:E3/E3 | Treatment:Control RAW_FILE_NAME=47_3_1.CDF SUBJECT_SAMPLE_FACTORS - 47_3_2 Group:E3_control | APOE genotype:E3/E3 | Treatment:Control RAW_FILE_NAME=47_3_2.CDF SUBJECT_SAMPLE_FACTORS - 47_3_3 Group:E3_control | APOE genotype:E3/E3 | Treatment:Control RAW_FILE_NAME=47_3_3.CDF SUBJECT_SAMPLE_FACTORS - 47_3_4 Group:E3_control | APOE genotype:E3/E3 | Treatment:Control RAW_FILE_NAME=47_3_4.CDF SUBJECT_SAMPLE_FACTORS - 47_3_9 Group:E3_control | APOE genotype:E3/E3 | Treatment:Control RAW_FILE_NAME=47_3_9.CDF SUBJECT_SAMPLE_FACTORS - 47_3_10 Group:E3_control | APOE genotype:E3/E3 | Treatment:Control RAW_FILE_NAME=47_3_10.CDF SUBJECT_SAMPLE_FACTORS - 47_3_11 Group:E3_control | APOE genotype:E3/E3 | Treatment:Control RAW_FILE_NAME=47_3_11.CDF SUBJECT_SAMPLE_FACTORS - 47_3_12 Group:E3_control | APOE genotype:E3/E3 | Treatment:Control RAW_FILE_NAME=47_3_12.CDF SUBJECT_SAMPLE_FACTORS - 47_3_5 Group:E3_proinflammatory | APOE genotype:E3/E3 | Treatment:Proinflammatory RAW_FILE_NAME=47_3_5.CDF SUBJECT_SAMPLE_FACTORS - 47_3_6 Group:E3_proinflammatory | APOE genotype:E3/E3 | Treatment:Proinflammatory RAW_FILE_NAME=47_3_6.CDF SUBJECT_SAMPLE_FACTORS - 47_3_7 Group:E3_proinflammatory | APOE genotype:E3/E3 | Treatment:Proinflammatory RAW_FILE_NAME=47_3_7.CDF SUBJECT_SAMPLE_FACTORS - 47_3_8 Group:E3_proinflammatory | APOE genotype:E3/E3 | Treatment:Proinflammatory RAW_FILE_NAME=47_3_8.CDF SUBJECT_SAMPLE_FACTORS - 47_3_13 Group:E3_proinflammatory | APOE genotype:E3/E3 | Treatment:Proinflammatory RAW_FILE_NAME=47_3_13.CDF SUBJECT_SAMPLE_FACTORS - 47_3_14 Group:E3_proinflammatory | APOE genotype:E3/E3 | Treatment:Proinflammatory RAW_FILE_NAME=47_3_14.CDF SUBJECT_SAMPLE_FACTORS - 47_3_15 Group:E3_proinflammatory | APOE genotype:E3/E3 | Treatment:Proinflammatory RAW_FILE_NAME=47_3_15.CDF SUBJECT_SAMPLE_FACTORS - 47_3_16 Group:E3_proinflammatory | APOE genotype:E3/E3 | Treatment:Proinflammatory RAW_FILE_NAME=47_3_16.CDF SUBJECT_SAMPLE_FACTORS - 47_4_1 Group:E4_control | APOE genotype:E4/E4 | Treatment:Control RAW_FILE_NAME=47_4_1.CDF SUBJECT_SAMPLE_FACTORS - 47_4_2 Group:E4_control | APOE genotype:E4/E4 | Treatment:Control RAW_FILE_NAME=47_4_2.CDF SUBJECT_SAMPLE_FACTORS - 47_4_3 Group:E4_control | APOE genotype:E4/E4 | Treatment:Control RAW_FILE_NAME=47_4_3.CDF SUBJECT_SAMPLE_FACTORS - 47_4_4 Group:E4_control | APOE genotype:E4/E4 | Treatment:Control RAW_FILE_NAME=47_4_4.CDF SUBJECT_SAMPLE_FACTORS - 47_4_9 Group:E4_control | APOE genotype:E4/E4 | Treatment:Control RAW_FILE_NAME=47_4_9.CDF SUBJECT_SAMPLE_FACTORS - 47_4_10 Group:E4_control | APOE genotype:E4/E4 | Treatment:Control RAW_FILE_NAME=47_4_10.CDF SUBJECT_SAMPLE_FACTORS - 47_4_12 Group:E4_control | APOE genotype:E4/E4 | Treatment:Control RAW_FILE_NAME=47_4_12.CDF SUBJECT_SAMPLE_FACTORS - 47_4_6 Group:E4_proinflammatory | APOE genotype:E4/E4 | Treatment:Proinflammatory RAW_FILE_NAME=47_4_6.CDF SUBJECT_SAMPLE_FACTORS - 47_4_7 Group:E4_proinflammatory | APOE genotype:E4/E4 | Treatment:Proinflammatory RAW_FILE_NAME=47_4_7.CDF SUBJECT_SAMPLE_FACTORS - 47_4_8 Group:E4_proinflammatory | APOE genotype:E4/E4 | Treatment:Proinflammatory RAW_FILE_NAME=47_4_8.CDF SUBJECT_SAMPLE_FACTORS - 47_4_13 Group:E4_proinflammatory | APOE genotype:E4/E4 | Treatment:Proinflammatory RAW_FILE_NAME=47_4_13.CDF SUBJECT_SAMPLE_FACTORS - 47_4_14 Group:E4_proinflammatory | APOE genotype:E4/E4 | Treatment:Proinflammatory RAW_FILE_NAME=47_4_14.CDF SUBJECT_SAMPLE_FACTORS - 47_4_15 Group:E4_proinflammatory | APOE genotype:E4/E4 | Treatment:Proinflammatory RAW_FILE_NAME=47_4_15.CDF SUBJECT_SAMPLE_FACTORS - 47_4_16 Group:E4_proinflammatory | APOE genotype:E4/E4 | Treatment:Proinflammatory RAW_FILE_NAME=47_4_16.CDF #COLLECTION CO:COLLECTION_SUMMARY Primary microglia were plated at 7x10^6 cells/well in 6-well plates (VWR CO:COLLECTION_SUMMARY #10062-894) and incubated at 5% CO2 37°C. Upon reaching confluence, cells were CO:COLLECTION_SUMMARY washed with warm, sterile phosphate-buffered saline (PBS; Thomas CO:COLLECTION_SUMMARY #QZY-11666789001-4L) to remove traces of non-13C media and then incubated in CO:COLLECTION_SUMMARY glucose- and sodium pyruvate-free DMEM (Thermo #11966-025) containing 2mM CO:COLLECTION_SUMMARY GlutaMAX (Thermo #35050-061), 1% penicillin/streptomycin, and 10mM universally CO:COLLECTION_SUMMARY labelled 13C-glucose (Cambridge Isotope Laboratories # CLM-1396-PK) for two CO:COLLECTION_SUMMARY hours with either a pro-inflammatory cocktail of 20ng/ml interferon-γ (IFNγ, CO:COLLECTION_SUMMARY R&D Systems #485-MI-100) and 50ng/ml tumor necrosis factor α (TNFα, R&D CO:COLLECTION_SUMMARY Systems #410-MT-025) or vehicle control. Cells were then removed from the CO:COLLECTION_SUMMARY incubator and washed with warm 0.9% NaCl solution. Culture plates were placed on CO:COLLECTION_SUMMARY a bed of crushed dry ice and 1mL of ice cold 50% methanol (HPLC-grade, Sigma CO:COLLECTION_SUMMARY #A456-4) was added to quench cellular metabolic activity followed by a 10 minute CO:COLLECTION_SUMMARY incubation at -80°C to ensure cell lysis. After removing from the freezer, CO:COLLECTION_SUMMARY cells were detached with a cell scraper (VWR #10062-906) and the entire contents CO:COLLECTION_SUMMARY collected into a microcentrifuge tube, vortexed briefly, and placed on ice until CO:COLLECTION_SUMMARY all samples were collected. The tubes were then placed on a Disruptor Genie Cell CO:COLLECTION_SUMMARY Disruptor Homogenizer (Scientific Industries) for 5 min at 3,000 rpm. Tubes were CO:COLLECTION_SUMMARY then centrifuged at 20,000 x g for 10 min at 4 °C. The supernatant containing CO:COLLECTION_SUMMARY polar metabolites was isolated to a new tube and stored at -80C until use. CO:SAMPLE_TYPE Brain #TREATMENT TR:TREATMENT_SUMMARY APOE3/3 and APOE4/4 primary microglia were treated for 2 hours with a TR:TREATMENT_SUMMARY pro-inflammatory cocktail of 20ng/ml interferon-γ (IFNγ, R&D Systems TR:TREATMENT_SUMMARY #485-MI-100) and 50ng/ml tumor necrosis factor α (TNFα, R&D Systems TR:TREATMENT_SUMMARY #410-MT-025) or vehicle control. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The supernatant fraction containing polar metabolites was thawed gently on ice SP:SAMPLEPREP_SUMMARY and dried at 10-3 mbar using a CentriVap vacuum concentrator (LabConco) to SP:SAMPLEPREP_SUMMARY evaporate methanol. The dried polar metabolite pellet was derivatized by a SP:SAMPLEPREP_SUMMARY two-step methoxyamine protocol first by addition of 70µL methoxyamine HCl SP:SAMPLEPREP_SUMMARY (Sigma-Aldrich #226904-5G) in pyridine (20 mg/mL; Sigma-Aldrich #TS25730) to SP:SAMPLEPREP_SUMMARY each pellet followed by 90 min dry heat incubation at 30°C. Samples were then SP:SAMPLEPREP_SUMMARY centrifuged at 20,000 x g for 10 minutes after which 50µL of each sample was SP:SAMPLEPREP_SUMMARY transferred to an amber V-shaped glass chromatography vial (Agilent #5184-3554) SP:SAMPLEPREP_SUMMARY containing 80µL N-methyl-trimethylsilyl-trifluoroacetamide (MSTFA; ThermoFisher SP:SAMPLEPREP_SUMMARY #TS48915) and gently vortexed followed by 30 min dry heat incubation at 37°C. SP:EXTRACTION_METHOD 50% ice-cold methanol SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_DERIVATIZATION MSTFA #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 8890 CH:COLUMN_NAME Agilent HP5-MS (30m x 0.25mm, 0.25 um) CH:SOLVENT_A N/A CH:SOLVENT_B N/A CH:FLOW_GRADIENT N/A CH:FLOW_RATE 0.581 mL/min CH:COLUMN_TEMPERATURE 60°C, rising at 10°C/min to 325°C, and held for 10 min #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 5977B MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS Metabolites were identified and quantified using DExSI v1.11 MS:MS_COMMENTS (https://github.com/DExSI/DExSI) #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Fractional Enrichment (adjusted for natural abundance) MS_METABOLITE_DATA_START Samples 47_3_1 47_3_10 47_3_11 47_3_12 47_3_13 47_3_14 47_3_15 47_3_16 47_3_2 47_3_3 47_3_4 47_3_5 47_3_6 47_3_7 47_3_8 47_3_9 47_4_1 47_4_10 47_4_12 47_4_13 47_4_14 47_4_15 47_4_16 47_4_2 47_4_3 47_4_4 47_4_6 47_4_7 47_4_8 47_4_9 Factors Group:E3_control | APOE genotype:E3/E3 | Treatment:Control Group:E3_control | APOE genotype:E3/E3 | Treatment:Control Group:E3_control | APOE genotype:E3/E3 | Treatment:Control Group:E3_control | APOE genotype:E3/E3 | Treatment:Control Group:E3_proinflammatory | APOE genotype:E3/E3 | Treatment:Proinflammatory Group:E3_proinflammatory | APOE genotype:E3/E3 | Treatment:Proinflammatory Group:E3_proinflammatory | APOE genotype:E3/E3 | Treatment:Proinflammatory Group:E3_proinflammatory | APOE genotype:E3/E3 | Treatment:Proinflammatory Group:E3_control | APOE genotype:E3/E3 | Treatment:Control Group:E3_control | APOE genotype:E3/E3 | Treatment:Control Group:E3_control | APOE genotype:E3/E3 | Treatment:Control Group:E3_proinflammatory | APOE genotype:E3/E3 | Treatment:Proinflammatory Group:E3_proinflammatory | APOE genotype:E3/E3 | Treatment:Proinflammatory Group:E3_proinflammatory | APOE genotype:E3/E3 | Treatment:Proinflammatory Group:E3_proinflammatory | APOE genotype:E3/E3 | Treatment:Proinflammatory Group:E3_control | APOE genotype:E3/E3 | Treatment:Control Group:E4_control | APOE genotype:E4/E4 | Treatment:Control Group:E4_control | APOE genotype:E4/E4 | Treatment:Control Group:E4_control | APOE genotype:E4/E4 | Treatment:Control Group:E4_proinflammatory | APOE genotype:E4/E4 | Treatment:Proinflammatory Group:E4_proinflammatory | APOE genotype:E4/E4 | Treatment:Proinflammatory Group:E4_proinflammatory | APOE genotype:E4/E4 | Treatment:Proinflammatory Group:E4_proinflammatory | APOE genotype:E4/E4 | Treatment:Proinflammatory Group:E4_control | APOE genotype:E4/E4 | Treatment:Control Group:E4_control | APOE genotype:E4/E4 | Treatment:Control Group:E4_control | APOE genotype:E4/E4 | Treatment:Control Group:E4_proinflammatory | APOE genotype:E4/E4 | Treatment:Proinflammatory Group:E4_proinflammatory | APOE genotype:E4/E4 | Treatment:Proinflammatory Group:E4_proinflammatory | APOE genotype:E4/E4 | Treatment:Proinflammatory Group:E4_control | APOE genotype:E4/E4 | Treatment:Control Pyruvic acid 73.7033551 27.86065381 37.13190722 46.60241407 49.60560957 27.51108433 79.12872878 52.61921617 43.70983361 44.56509299 85.15340672 62.48673442 39.58435399 52.65937904 44.44812968 67.31029927 54.82112392 35.79953096 30.73494682 50.11426557 33.56302892 55.66063718 36.54578033 60.86801665 61.33121599 61.3855051 65.11339702 28.72169818 59.89044304 36.8315281 Pyruvic acid_13C_1 -3.358709209 -2.802629989 -3.735056162 -4.687458911 -4.989773155 -2.767328851 -2.957435275 -1.310175914 -4.397100684 -4.482832618 -3.688409145 -2.25206784 -3.982049423 -2.570921485 -1.887401392 -4.475415015 -5.514656465 -3.601132515 -3.091784008 -5.041396782 -1.071776551 -5.59916197 -0.609209819 -6.122857151 -3.585328885 -1.373230413 0.391565915 -2.889052794 -4.064479709 -3.704935679 Pyruvic acid_13C_2 -1.160930359 -0.920432651 1.317814312 -1.53969548 -1.638871286 -0.908925935 -3.117854401 -0.60379387 -1.444375972 -1.472460658 -3.304188122 0.107118299 -1.308079719 -2.014175819 -0.903553912 -0.865213395 -1.811508039 -1.183017325 -1.015679102 -1.655871291 -1.340793078 2.469336166 -1.516215732 -2.011271449 -0.404117526 -0.208114677 -2.849762035 -0.949033035 -2.176123653 -1.216883918 Pyruvic acid_13C_3 30.81628447 75.86240883 65.28533463 59.62474032 57.02303487 76.16517046 26.94656089 49.29475362 62.13164304 61.39020029 21.83919054 39.65821512 65.70577515 51.92571826 58.34282562 38.03032914 52.50504058 68.98461888 73.37251629 56.58300251 68.84954071 47.46918863 65.57964522 47.26611195 42.65823042 40.19583999 37.3447991 75.11638765 46.35016032 68.09029149 Lactic acid 87.26464779 73.95039846 71.30749528 90.2547858 82.49027196 46.93854414 50.48690722 77.34200502 79.35094021 49.60481566 62.85763244 65.57993946 48.01605425 31.78803455 75.19876801 61.75693055 58.81813922 66.34121843 76.66644061 50.2011948 42.42137828 0 46.1971792 51.76042928 69.46822876 31.92762084 45.07075014 36.86427538 51.69579421 33.15856952 Lactic acid_13C_1 2.991679698 2.589703002 1.858712424 1.808041831 2.47163255 -3.247081089 2.646005121 1.216157751 0.291267923 3.677230311 1.348353663 2.097852314 -0.006227943 -0.689814092 1.84462771 4.446564495 1.288096847 1.097642002 1.277536423 -2.40014844 2.701893918 0 0.830031311 -8.805564236 1.766116547 2.859035883 0.634307895 5.160658398 1.446005475 1.257349268 Lactic acid_13C_2 1.543802079 1.080209758 2.409470698 0.468622322 2.062567865 3.867444237 -1.69806231 0.35244832 0.728970275 2.639298949 2.278899325 2.797463733 0.571240745 3.766102476 -0.020486254 1.858255784 5.00585294 3.410805654 1.242313606 4.496746199 1.993703097 0 2.599822661 -2.883676419 0.713319972 5.810959938 7.293100395 3.277510556 -0.315646309 4.537943707 Lactic acid_13C_3 8.199870434 22.37968878 24.4243216 7.468550043 12.97552762 52.44109271 48.56514997 21.08938891 19.62882159 44.07865508 33.51511457 29.52474449 51.41893295 65.13567707 22.97709053 31.93824917 34.88791099 29.15033392 20.81370936 47.70220744 52.88302471 0 50.37296683 59.92881137 28.05233472 59.40238334 47.00184157 54.69755566 47.17384662 61.04613751 beta-Alanine 104.8478489 98.0143368 86.96643088 104.6203335 0 104.4669112 0 96.89985937 102.9537124 104.7219461 102.2481643 90.10250061 96.40837093 101.7423714 105.9048007 104.6685171 100.3531361 99.4356538 104.1810278 96.84067924 101.091937 0 101.2166826 102.7118343 102.3140065 103.7284279 102.5008095 98.66935636 0 102.8291472 beta-Alanine_13C_1 -3.291342254 2.645256602 11.92578935 -3.123459221 0 -2.764495878 0 3.490846708 -3.286877098 -4.029668635 -1.161525885 8.924562261 6.289985945 -0.082303331 -4.170376828 -4.18878763 0.197894841 -0.222947515 -2.809140346 2.566411342 -0.648558943 0 -2.74199868 -0.985299901 -2.381826486 -3.864674259 -4.088762586 2.782763172 0 -2.305318589 beta-Alanine_13C_2 -2.523670298 -1.684321856 -1.037140276 -0.938757335 0 -2.350989893 0 -1.170867848 -1.874344558 -1.308658839 -1.549654694 -3.193886548 -1.538227688 -2.710704107 -2.608475466 -1.386419271 -0.647353247 -1.384384297 -1.521568077 -0.225821568 -0.406024174 0 0.23739432 -1.956110506 -0.074977924 -1.054591057 1.038776957 -2.389261815 0 -1.139393169 beta-Alanine_13C_3 0.967163629 1.024728457 2.144920046 -0.558116923 0 0.648574527 0 0.780161767 2.207509291 0.616381408 0.463016275 4.166823673 -1.160129182 1.050636024 0.874051616 0.90668985 0.09632232 2.171678011 0.14968063 0.818730989 -0.03735385 0 1.287921804 0.229576073 0.142797944 1.190837387 0.549176139 0.937142286 0 0.615564519 Leucine 101.7699823 104.1697619 104.0853458 104.0455805 105.0242299 104.1997523 105.5425041 103.6190315 103.3457533 101.498168 103.3391695 102.7543606 104.0091128 103.2549544 103.7685138 103.462805 103.4639132 103.8604531 103.4366289 103.7003741 103.5527211 0 103.9231387 103.9836018 104.3564673 103.7724516 102.6248022 103.9861619 0 103.2064071 Leucine_13C_1 -1.737444355 -2.004293589 -1.928850653 -2.12735763 -2.605532863 -2.015750053 -3.045302794 -1.68389297 -2.969848107 -1.638638216 -2.381215094 -1.659418132 -1.9809969 -2.026673372 -1.893862753 -1.674711198 -1.714164886 -2.046572335 -1.823316946 -1.849477664 -1.682864117 0 -1.962636442 -2.511271889 -1.785848883 -1.818513479 -2.60753976 -2.066899697 0 -1.081104431 Leucine_13C_2 -3.532517248 -3.627716409 -3.863968229 -3.741542574 -3.983555622 -3.664361072 -3.964739052 -3.600595498 -3.734150126 -3.518606988 -3.471521899 -3.977626787 -3.999050714 -3.600807372 -3.553880616 -3.522835593 -3.618766059 -3.619123154 -3.820096143 -3.844146398 -3.786165633 0 -3.706520023 -3.754059465 -3.465911431 -3.708315403 -3.88699993 -3.922544477 0 -3.644559695 Leucine_13C_3 1.470418949 0.603161426 0.738149859 1.196174918 0.495256241 0.682466686 0.896063639 0.675633957 1.953791722 1.92867312 1.121959483 2.186276972 1.148383291 1.763340855 0.887072924 0.838575142 1.165166926 0.922135088 1.012197802 1.239079697 1.179132141 0 0.781264283 0.943771017 0.204925041 1.047082469 2.098490535 1.234887002 0 1.036486823 Leucine_13C_4 0.106642935 0.077332203 0.074544175 0.028694084 0.102226804 0.116414661 0.066041238 0.069254657 -0.038289204 0.112431854 0.209865234 0.0505112 0.043106051 -0.028086771 0.092881316 0.051458904 0.025957753 0.048371847 0.048013688 0.028534661 0.031349904 0 0.064416493 0.052616428 0.106309772 0.040317249 -0.045677688 0.032392365 0 0.037538576 Leucine_13C_5 0.85624172 0.803094478 0.85551557 0.434458534 1.067862285 0.694285062 0.559357507 0.951346831 0.708342128 0.839208304 0.874287138 0.558338452 0.605199423 0.619207855 0.563446801 0.931550772 0.570985454 0.841115421 1.078519452 0.800369272 0.778628759 0 0.86347979 1.223491225 0.646329565 0.629389039 1.998051344 0.725088708 0 0.492263694 Leucine_13C_6 1.066675655 -0.021339998 0.039263446 0.163992141 -0.100486774 -0.012807602 -0.053924618 -0.030778466 0.734400285 0.778763908 0.307455641 0.087557705 0.174246088 0.018064372 0.135828576 -0.086843071 0.106907643 -0.006379946 0.068053264 -0.074733637 -0.072802151 0 0.036857189 0.061850849 -0.062271389 0.037588562 -0.181126742 0.010914184 0 -0.047032034 Isoleucine 101.9907682 102.5844664 102.8402718 103.3208254 99.53960634 102.9327788 101.612481 100.290906 102.8456484 102.3779486 102.7295771 102.1068577 103.7581996 101.9119262 102.5484065 102.0336112 103.3656901 102.6395036 100.4790521 102.4203981 103.0597524 0 102.3626626 101.316002 103.1057644 102.4048616 99.23355042 101.096932 0 102.4630617 Isoleucine_13C_1 -0.945601903 -1.254275389 -0.991334502 -1.211224084 -0.788622366 -1.113200589 -1.69647858 -0.368028517 -1.1894752 -1.080714222 -0.723515562 -0.701095796 -1.204831898 -0.062449288 -0.806065986 -0.632330072 -1.357781684 -1.102931906 -0.644484421 -0.013733266 -0.992279751 0 -0.422146701 -0.629209164 -1.157655631 -0.694441146 -0.171321216 -0.490342734 0 -1.157168333 Isoleucine_13C_2 -3.504635669 -3.426734369 -3.382187302 -3.847859836 -3.725020581 -3.336885353 -3.740290196 -3.3527681 -3.517516608 -3.434728131 -3.586685324 -3.251603527 -3.412985947 -3.528403381 -3.312977686 -3.461895812 -3.440825767 -3.383485905 -3.525660893 -3.961507079 -3.308217055 0 -3.478180557 -3.464280328 -3.385167633 -3.295943207 -3.970280981 -3.450348454 0 -3.361968345 Isoleucine_13C_3 0.757464562 0.996386991 0.422652082 0.715769555 3.943705448 0.571007393 3.114402777 1.866979632 1.029318457 0.9793726 0.486663358 0.625537234 0.432851536 0.657219185 0.633057422 0.815818643 0.460039589 0.987820228 2.386367563 0.511542716 0.697357079 0 0.81909755 1.647414037 0.456856338 0.657044854 3.548914077 1.671348758 0 0.881309347 Isoleucine_13C_4 0.110727488 0.032805539 0.091251832 0.07481929 -0.221059342 0.067225466 -0.144503966 -0.008450834 0.033331255 0.090297876 0.08422213 0.134800545 0.08274634 0.065514664 0.060210302 0.049513813 0.081745666 0.084251156 -0.007610968 0.095875683 0.054463 0 0.049456345 0.025472123 0.079493436 0.057199329 -0.176626099 0.024148707 0 0.040428785 Isoleucine_13C_5 0.960076439 0.976936352 1.070202075 1.045460657 1.372297896 0.913367305 0.573267303 1.573906738 0.761073733 0.868935141 0.66708048 0.986332239 0.381932112 0.981323791 0.875154049 1.188788281 0.894489614 0.8565303 1.338741829 1.022656802 0.54044899 0 0.738462588 1.118828707 0.909520075 0.891976561 1.685998105 1.208476061 0 1.249489358 Isoleucine_13C_6 0.631200892 0.090414433 -0.050855967 -0.097790995 -0.120907397 -0.034292985 0.281121627 -0.002544932 0.037619982 0.198888181 0.342657865 0.099171638 -0.037911743 -0.025131122 0.002215448 0.00649394 -0.003357508 -0.081687489 -0.026405259 -0.075232921 -0.051524621 0 -0.069351832 -0.014227404 -0.008810955 -0.020697948 -0.150234303 -0.060214373 0 -0.115152521 Serine 101.5154132 105.5089008 102.7568977 106.8310101 100.5660028 99.78460371 101.2868614 102.3184569 100.9134759 101.1424005 103.5167351 99.19348573 102.42884 103.0371456 104.3780818 107.7952236 104.171074 104.345086 104.5321842 101.4727838 102.1567318 0 102.8440766 101.9328942 102.344812 104.9206097 112.64239 103.4570228 0 102.2262471 Serine_13C_1 -0.71792468 -2.544466954 -1.52052433 -1.774035915 1.361956416 2.067420451 2.329299381 -0.498534477 3.129852595 -0.018669973 1.074290594 4.894568269 0.991136524 -1.458951881 -1.728614497 -5.373354597 -1.96630987 -1.941487092 0.428894504 1.834747035 0.667610349 0 -1.384810769 0.836487078 1.644150192 -2.258012123 -4.676350843 0.394942092 0 0.876226961 Serine_13C_2 -3.127375172 -3.263361631 -1.175279196 -5.042778442 -2.540834668 -2.436332254 -2.940317917 -1.789185508 -4.803484875 -1.19148374 -4.962757687 -3.271425326 -2.827503523 -1.984685003 -3.257026394 -2.55426608 -2.900088025 -2.942812297 -5.909706205 -3.400644033 -3.342407784 0 -2.065395248 -2.061787955 -3.857889279 -3.011765623 -8.795341381 -3.930325385 0 -3.04174704 Serine_13C_3 2.329886675 0.298927765 -0.061094131 -0.014195782 0.612875484 0.584308091 -0.675842815 -0.030736892 0.76015643 0.067753195 0.371731986 -0.816628677 -0.592473031 0.406491265 0.607559128 0.132397055 0.695323881 0.539213424 0.948627527 0.093113214 0.518065631 0 0.606129423 -0.707593342 -0.13107291 0.349168091 0.8293022 0.078360502 0 -0.060726982 Glycine 98.71711475 108.004888 117.3179427 107.9714457 0 107.560433 0 121.873843 0 103.0790483 105.5093737 0 0 100.8613032 104.3187169 100.107439 100.7952682 102.9022176 113.1908016 117.3834214 104.0608575 0 96.69202273 118.8051046 99.50104119 97.27012135 0 0 0 101.5808253 Glycine_13C_1 0.163619623 -5.548538196 -7.505098805 -0.879404461 0 -5.937378067 0 -14.22103263 0 -5.23764873 1.41961156 0 0 0.391127912 0.954236564 1.222836849 1.507608676 1.140603403 -7.013865353 -11.90647945 1.969783552 0 2.588955455 -15.87759738 2.884070232 4.298420241 0 0 0 -0.36062279 Glycine_13C_2 1.119265626 -2.456349833 -9.812843848 -7.092041268 0 -1.623054939 0 -7.6528104 0 2.158600408 -6.928985266 0 0 -1.252431098 -5.272953428 -1.330275815 -2.30287683 -4.042821048 -6.176936268 -5.476941904 -6.030641077 0 0.719021816 -2.927507182 -2.385111422 -1.568541591 0 0 0 -1.220202508 Fumaric acid 81.56300713 71.26757919 77.56558178 92.71515882 77.42151365 55.77200326 66.54226198 63.02341853 89.5394271 69.48937696 80.33206215 66.66199929 85.05387698 64.71336117 59.70653845 76.37662346 74.06598433 67.15910229 75.34728074 63.4911913 56.96116072 82.15704318 57.81521089 65.74437905 71.78362807 77.77413853 80.9563996 60.89219998 61.37988647 81.07437205 Fumaric acid_13C_1 7.390014683 -0.639393956 -0.032560839 -9.313019716 -0.335804346 4.38548958 8.959511498 5.5812103 4.097866277 10.38820683 -1.652418177 -6.877113282 2.44969634 2.523520871 16.32226939 4.75924643 3.063558142 4.917293928 -0.947588897 2.458558028 11.94014641 2.276089413 6.812234169 -1.288376567 0.153620624 3.524069189 -13.78470531 3.901640949 0.170693364 -0.101793894 Fumaric acid_13C_2 16.18170686 11.00939213 20.24812303 10.51773279 17.34461129 27.1707465 19.60513383 22.27600115 -0.617127473 11.35127492 17.33245319 29.34221171 -0.296950569 26.74366146 13.34157114 18.29377383 20.41974359 25.31568526 19.51994607 22.75916732 18.79719956 12.77475568 27.08588405 24.43056756 26.25060283 17.68260657 42.14535733 21.08958719 29.11752518 16.82590565 Fumaric acid_13C_3 -4.196115962 -2.51782076 4.99313391 8.559339465 8.724181081 11.90691616 8.226378542 6.151376713 8.630169317 12.06233934 6.808780848 16.26423548 15.63920214 2.23848154 14.55481198 2.855480599 5.318055869 6.022866476 9.554373209 16.18242734 13.64607834 4.942010584 6.878963891 16.10454405 -1.511933544 3.321648402 -6.774544342 9.896288076 10.61785078 4.622692895 Fumaric acid_13C_4 -0.938612708 20.88024339 -2.774277881 -2.479211354 -3.154501679 0.764844496 -3.333285851 2.967993305 -1.650335224 -3.291198053 -2.820878013 -5.391333209 -2.845824898 3.780974958 -3.925190958 -2.285124328 -2.867341933 -3.414947957 -3.474011123 -4.891343989 -1.344585029 -2.149898853 1.407706993 -4.991114082 3.324082029 -2.302462688 -2.542507278 4.2202838 -1.285955784 -2.421176702 Asparatic acid 71.67503376 70.87580693 0 69.55266774 0 59.20422824 0 56.628093 71.63247659 66.37484913 69.38087812 0 0 58.74361949 60.3932639 70.11974111 68.70032683 65.15347446 68.91486019 57.84235616 61.9458018 0 59.95966036 63.12232842 64.90282891 64.64924315 57.88915467 0 0 67.29461489 Asparatic acid_13C_1 13.56663118 14.2663441 0 15.36635049 0 17.64296657 0 18.60532672 14.94660074 15.20047928 14.70366671 0 0 13.18248566 14.36172365 15.19962997 14.41084814 16.35715616 14.53354779 18.22723488 15.26868264 0 19.99202112 17.71202966 16.80472069 17.75537255 19.38933891 0 0 16.10481141 Asparatic acid_13C_2 12.46486722 13.53341187 0 13.71493786 0 19.8436988 0 19.96313425 11.69792486 16.66768786 15.99584784 0 0 25.25227727 19.62355237 12.94851032 14.82974225 16.11301879 15.82677327 20.19259759 18.19717448 0 17.95386191 16.85848714 15.13768989 14.19711532 19.04239974 0 0 13.31792135 Asparatic acid_13C_3 4.45598149 2.692333431 0 3.481623967 0 4.407857211 0 7.531096168 3.700752189 4.257200127 1.46148634 0 0 6.410311403 8.20669923 2.940144975 3.723941951 4.155881612 2.916672232 5.814406502 6.535947712 0 4.256253347 4.525525922 4.721649931 4.768603143 6.865423312 0 0 4.599003382 Asparatic acid_13C_4 -2.162513654 -1.367896339 0 -2.115580063 0 -1.098750831 0 -2.727650138 -1.97775437 -2.500216395 -1.541879011 0 0 -3.588693817 -2.585239152 -1.208026376 -1.664859178 -1.779531027 -2.191853478 -2.076595133 -1.947606638 0 -2.161796737 -2.218371145 -1.566889424 -1.370334159 -3.186316633 0 0 -1.316351029 Pyroglutamic acid 83.49582698 87.56410165 85.81384007 0 73.84660231 0 85.11041417 84.13149907 96.9888751 91.54387528 93.43547114 86.55952555 90.3240664 89.60089582 94.94296955 86.94650723 81.51662545 0 0 82.84602981 78.56455643 74.29836423 80.32017759 86.47961618 85.07285331 81.90916236 85.57312725 85.58185173 85.24452701 89.70713531 Pyroglutamic acid_13C_1 2.919125301 5.74061286 7.081739498 0 5.722717278 0 3.3683239 4.385837353 2.24535341 1.560682457 2.032279302 7.249817278 3.805374708 3.962881666 3.208900138 4.363825681 4.829156449 0 0 4.564410222 3.714755006 3.862562717 6.6310251 5.006678752 4.368210389 4.819925402 -1.593383742 2.661703356 4.242707922 6.011219023 Pyroglutamic acid_13C_2 4.562776203 2.845152242 7.5791923 0 16.61811941 0 7.239509633 6.078403351 0.646896541 8.584709744 2.05300167 8.679432831 8.913308171 3.94340886 1.043024078 8.270712772 8.302621368 0 0 7.796422713 8.579762243 11.58510935 8.625078092 5.5262584 7.908556983 9.929460704 14.17221771 6.957334499 6.792647327 4.150350347 Pyroglutamic acid_13C_3 2.358341697 5.570822091 -0.741184697 0 2.775381955 0 3.959512536 3.715747529 0.479238004 -1.061867059 1.817916817 -2.972915405 -2.756517734 2.312736209 0.315240365 -0.474774429 4.201556275 0 0 2.056219823 5.863932953 4.331985372 3.460406278 3.936988019 1.508760973 3.963567185 2.435586252 2.290448853 1.725958143 0.0142871 Pyroglutamic acid_13C_4 -0.996624102 -1.444586036 0.367948198 0 0.171953368 0 0.907759152 1.978887741 -0.356437938 -0.747773105 1.034036241 0.414642805 -0.500563191 0.528931473 0.662487054 0.853345174 1.337244424 0 0 2.962670372 3.548308033 6.680065695 1.031006352 -0.650156065 0.774127102 -0.20024772 -0.281075074 1.653594129 2.172895497 0.087808307 Pyroglutamic acid_13C_5 7.66055392 -0.276102809 -0.10153537 0 0.865225679 0 -0.585519393 -0.290375043 -0.003925116 0.120372683 -0.372705171 0.069496941 0.214331642 -0.348854031 -0.17262119 0.040383577 -0.187203971 0 0 -0.225752936 -0.271314663 -0.758087367 -0.067693412 -0.299385282 0.367491244 -0.421867936 -0.306472397 0.85506743 -0.178735895 0.029199912 Glutamatic acid 55.94960516 49.4534222 57.44484697 74.78942588 0 43.94385106 0 44.61920968 77.99879356 49.5020371 57.36022365 0 0 52.02104583 43.94299703 51.81181642 46.21968017 52.44124278 48.90199637 53.56035098 50.10468205 0 50.87390145 0 51.35707375 51.39721401 0 0 0 52.64427807 Glutamatic acid_13C_1 10.37142106 15.80212885 14.80615827 -1.953248243 0 11.31762495 0 11.48268316 -3.352720828 12.78597077 12.50438499 0 0 10.092284 13.34565418 12.29604759 12.96642974 10.8850487 11.7648295 -0.168350168 12.2320595 0 12.97788358 0 14.32822001 14.24454763 0 0 0 9.563881441 Glutamatic acid_13C_2 23.51246469 19.33487282 32.25948866 20.16826808 0 26.31665956 0 24.58733266 20.08802568 24.34952818 17.78951174 0 0 22.35382871 26.77519502 22.57684544 26.27880515 21.48204327 21.84793199 37.38598102 22.8957374 0 19.94240711 0 22.91236266 20.25544232 0 0 0 25.91980146 Glutamatic acid_13C_3 9.128074074 13.03106006 -2.057661261 11.43266887 0 12.05300232 0 16.66284613 9.161894265 10.3662254 10.95333507 0 0 14.9538483 12.02044487 10.56651133 12.13454238 12.40036334 15.54236317 5.436179982 11.2682269 0 13.93598595 0 9.17658851 11.80461919 0 0 0 10.65893129 Glutamatic acid_13C_4 2.554304192 4.553073742 -2.740230004 -3.905062383 0 6.727193273 0 3.785959612 -3.494589623 1.7918052 3.112107314 0 0 2.599214757 4.307179923 2.678406717 3.134277166 5.000195386 4.311491069 5.634118967 5.758460033 0 4.517492707 0 2.163323018 3.279261895 0 0 0 2.954958464 Glutamatic acid_13C_5 -1.515869167 -2.174557668 0.287397366 -0.532052201 0 -0.358331153 0 -1.138031227 -0.401403049 1.204433354 -1.71956277 0 0 -2.02022161 -0.391471025 0.0703725 -0.733734603 -2.208893472 -2.368612093 -1.848280788 -2.259165881 0 -2.247670797 0 0.062432044 -0.981085047 0 0 0 -1.741850726 Citric acid 67.74204978 40.26285324 43.48395931 41.76954897 31.58715775 28.71299122 45.49139185 39.60233121 54.73390277 49.95122381 71.69781933 51.27326294 46.81478844 39.53035036 47.14303172 58.8372589 42.45186176 35.26143306 36.23985295 36.64218109 32.06068706 33.26227571 32.6520809 43.97928607 47.26520503 45.08578877 41.38292662 27.28814296 0 39.30682324 Citric acid_13C_1 4.273924469 9.84286539 8.729842825 6.481335376 9.0801078 5.292402163 5.087132604 1.363479995 -0.433972583 2.947923576 -6.548178987 5.300021374 -2.358473146 4.110907393 4.54508559 -0.744251373 3.875406486 7.621494528 6.462556845 3.70095607 6.185950345 6.988804713 5.100073356 5.077373497 3.383679874 2.962500865 3.804799678 7.046902698 0 6.308068023 Citric acid_13C_2 25.06850326 32.56897026 34.21922841 41.21537272 42.9243448 43.92270385 33.82984474 41.51784155 38.76629065 40.79904876 32.64756673 35.00892614 44.4249235 37.2351322 34.14759241 32.75772305 37.03970463 43.3141438 45.51032768 39.3831324 38.59142434 38.74140087 43.54991509 39.66345866 36.53490817 40.01306759 36.22744036 44.82020602 0 37.20951812 Citric acid_13C_3 1.304514719 10.94554319 10.36784718 5.703351423 5.864547123 13.24906261 10.19603763 9.16937441 -1.023792903 2.011233871 -0.77542761 4.818578675 5.807876965 9.956976935 8.778156473 7.235070484 10.06123313 7.317582007 6.004292575 13.84401146 10.40504921 9.898094917 8.910998248 10.1521094 11.24591593 6.233963501 9.941433849 7.069264908 0 11.21078422 Citric acid_13C_4 3.136238817 8.341645735 6.668197591 7.117904017 12.67187439 6.098919511 8.343987164 9.138467162 13.22660394 8.091910092 5.615391367 8.904614069 8.927798882 8.513479938 6.073761418 2.080977138 8.896212351 6.807051572 6.610565685 6.214262544 12.90619233 9.949932349 8.306175433 2.806083097 1.4950482 6.140556454 12.22818419 12.99525052 0 7.155224498 Citric acid_13C_5 -0.630997726 -0.221034305 -1.636898687 -1.753709655 0.651329922 4.622587467 -0.373456088 2.020662168 -3.13540745 -2.191925972 -1.586826754 -4.137854681 -1.322807851 0.691845455 2.265897542 0.238769312 -0.580207269 3.144188354 2.26688342 4.154134364 1.934963674 3.650428458 2.045961991 -0.003436868 0.903138186 2.604586011 -2.053329038 3.378232788 0 -0.250675835 Citric acid_13C_6 -0.894233324 -1.740843512 -1.832176635 -0.533802853 -2.779361794 -1.898666824 -2.5749379 -2.812156493 -2.133624427 -1.609414139 -1.050344078 -1.167548517 -2.294106785 -0.03869228 -2.953525151 -0.405547508 -1.744211088 -3.465893322 -3.094479153 -3.938677932 -2.084266953 -2.490937013 -0.565205017 -1.674873855 -0.827895388 -3.040463181 -1.531455656 -2.597999895 0 -0.939742258 Urea 100.0452885 93.12513553 90.64811566 92.42939774 93.22676928 91.30935616 92.07389472 91.492632 101.1804975 96.41649805 96.36193662 100.1163032 95.64155789 93.80108271 91.87947873 93.87729884 96.58247235 90.78805866 92.51539416 90.76750932 92.37657214 93.11003746 91.83454811 92.39316849 94.53764465 91.39587582 95.89081932 95.15296134 88.41080152 89.67285627 Urea_13C_1 -0.04528847 6.874864466 9.351884342 7.570602255 6.773230718 8.690643836 7.926105277 8.507367998 -1.180497484 3.583501947 3.638063383 -0.116303163 4.358442113 6.198917291 8.120521268 6.122701156 3.417527651 9.21194134 7.484605841 9.232490676 7.623427856 6.889962542 8.165451893 7.606831511 5.462355353 8.604124176 4.109180681 4.847038656 11.58919848 10.32714373 Ethanolamine 112.0852779 112.2397261 110.8229425 111.2200226 111.7355842 111.5140925 111.090961 111.6558372 111.3584295 111.2431896 112.1622569 111.7141994 110.0909956 111.5171265 110.1795207 110.9897587 109.2637733 111.6963814 111.9581174 111.9165457 112.1613838 112.1998764 111.7110452 112.3971408 111.4059187 111.6987614 111.5640464 111.1397655 109.1309631 111.6000927 Ethanolamine_13C_1 -8.808460423 -8.619972963 -7.496510539 -8.047968081 -8.650411459 -8.168818851 -8.220912604 -8.508679089 -8.670129543 -8.190107973 -8.819988378 -9.102679667 -6.999986438 -8.308522417 -7.230864216 -7.807267886 -6.798201511 -8.427787255 -8.59807054 -8.670736359 -8.988279622 -8.713518975 -8.284277035 -9.378935325 -8.191760715 -8.158086761 -8.294283697 -7.479576834 -6.736791112 -8.453585505 Ethanolamine_13C_2 -3.276817524 -3.619753163 -3.326431929 -3.172054472 -3.085172739 -3.345273685 -2.870048389 -3.147158137 -2.688299962 -3.053081628 -3.342268526 -2.611519781 -3.091009205 -3.208604105 -2.94865651 -3.182490852 -2.465571788 -3.268594165 -3.360046876 -3.245809361 -3.173104161 -3.486357388 -3.42676812 -3.018205451 -3.214157996 -3.540674677 -3.269762698 -3.66018871 -2.394171984 -3.146507181 Glyoxylic acid 0 99.03304712 99.34219351 108.1321663 96.02341333 96.7210309 99.33014753 95.17383181 109.2068269 101.8331573 104.675082 99.70004272 96.5100926 100.5855224 99.11158218 100.4909753 99.95382564 94.039535 99.52151454 94.23852406 97.12171689 95.10866261 100.2484722 101.1624016 101.1940824 97.26484914 100.7472694 99.55827629 95.07011642 96.91235853 Glyoxylic acid_13C_1 0 5.586940858 5.26301082 -3.946883609 5.271805673 6.614529273 3.961614471 7.65333181 -5.072664634 2.653003619 -0.324581164 4.887909518 6.286569632 3.960198202 5.504633999 4.059327248 4.622150684 7.284490259 2.581681382 9.181192831 3.400577112 8.121200608 3.228238437 2.103796587 1.061858824 5.33930767 -0.203222967 3.205380071 8.573584174 3.754656219 Glyoxylic acid_13C_2 0 -4.619987976 -4.605204328 -4.185282699 -1.295219003 -3.335560169 -3.291761997 -2.827163616 -4.134162278 -4.486160931 -4.350500787 -4.587952241 -2.796662236 -4.5457206 -4.616216178 -4.550302533 -4.575976328 -1.324025263 -2.103195918 -3.419716893 -0.522294003 -3.229863222 -3.476710608 -3.266198155 -2.255941197 -2.604156815 -0.544046473 -2.763656357 -3.643700597 -0.667014745 Malic acid 117.0438346 76.50712106 79.56391139 87.38004252 82.64397606 73.25577967 68.36825597 69.05601473 122.8189112 89.09022888 89.49796554 72.90599301 75.53358193 67.52289794 72.03483839 86.73494442 77.70848946 67.59372198 75.92346489 75.02639427 68.27187827 69.67450165 67.92535941 72.45174433 63.23458727 76.82550638 92.89058589 73.19084831 71.94290433 75.84311121 Malic acid_13C_1 -4.564623426 4.95422177 8.048119447 12.7700839 16.65576558 8.013387591 14.79357374 9.310428084 -31.32236441 -22.72029622 14.42220911 16.86467247 12.0648425 21.20953604 12.40800725 1.842429605 15.56745798 18.86021447 17.75038027 12.67580127 13.01180631 18.18661262 15.44486474 19.50866955 21.31346606 19.53543848 -1.796248491 5.862644034 9.884687979 9.079403734 Malic acid_13C_2 -15.08374911 24.68362157 13.61003483 5.489311573 -8.219553274 24.34178378 6.756043528 17.88894753 11.27996141 19.83673234 0.268235745 21.19484745 17.55677996 10.64976704 16.63919826 6.989758338 6.936489669 16.2329592 15.66194327 8.699706388 18.87016807 15.95779517 12.53269242 18.42085801 14.28792785 12.03331184 16.40279155 19.91465006 9.127344579 18.46579588 Malic acid_13C_3 1.565098598 -2.808240081 1.867343744 -5.349241466 3.733484858 -2.081227041 15.89050214 9.280668853 -1.58820841 22.23874297 -4.299839159 -9.562672488 -2.578379871 4.118816151 2.698770894 8.080046869 2.214029547 0.645270517 -8.349478291 2.352750784 4.347275448 -0.929305882 8.950979259 -9.210765166 5.495648764 -7.694356907 -6.247607456 5.938219542 14.80771499 -0.134307431 Malic acid_13C_4 1.039439297 -3.336724313 -3.089409416 -0.290196529 5.186326771 -3.529723992 -5.808375383 -5.536059204 -1.188299817 -8.445407977 0.111428759 -1.402840439 -2.576824522 -3.501017165 -3.780814797 -3.647179228 -2.426466652 -3.332166177 -0.986310143 1.245347287 -4.501128096 -2.889603563 -4.853895836 -1.170506727 -4.331629943 -0.699899798 -1.249521491 -4.906361946 -5.762651882 -3.254003395 Phenylalanine 104.2988898 104.2830198 102.5126978 102.33804 96.07785186 101.5376718 97.95188384 101.2929632 102.3560445 103.1696187 101.1708835 100.4789414 99.89202253 103.3684452 102.2991705 100.7252814 102.9427062 103.0179721 103.2678523 97.67234709 101.0712013 104.0363725 101.4141166 99.62368108 100.1197312 101.8441902 103.2591071 100.5417442 0 101.0642247 Phenylalanine_13C_1 -0.78540551 -1.208427357 -0.278154348 -0.834293475 1.715054348 0.910764278 1.501205981 0.48811343 0.853942713 -0.603285336 0.195659784 0.891805346 0.882465488 -1.599876181 0.987218509 1.293586672 -0.274266152 -0.37867345 -0.004611382 1.374105337 0.852320296 -3.048720131 1.359544405 0.564005839 1.403316789 0.426049651 -1.184668844 0.20535546 0 1.00516074 Phenylalanine_13C_2 -4.116466124 -3.919708005 -4.090347256 -4.033382849 -3.558995121 -4.051558335 -4.067189958 -4.002676557 -4.185818348 -4.087514267 -4.079064427 -4.113838567 -4.089436518 -4.005931311 -3.853171286 -3.848609031 -4.03007394 -4.101606231 -4.145260773 -4.044528125 -3.987075687 -3.902531456 -4.064749745 -4.050053723 -4.145400108 -3.966460659 -4.038836408 -3.91647714 0 -3.89689663 Phenylalanine_13C_3 0.223455124 0.22277252 0.203792763 0.221268535 0.087020888 0.154236873 0.13803282 0.167232519 0.167170141 0.215459049 0.186049775 0.162396356 0.161596642 0.247796977 0.204185439 0.122011798 0.197479967 0.207966885 0.196448603 0.141602512 0.151586741 0.295539196 0.137614045 0.171293624 0.145320046 0.165517619 0.234252234 0.172114468 0 0.137348934 Phenylalanine_13C_4 0.129051319 0.184271655 0.129216481 0.126326743 0.116045537 0.130241789 0.131925998 0.127879077 0.134449455 0.128494711 0.341779971 0.132218766 0.131427456 0.123978372 0.163452496 0.209495286 0.305649435 0.129380451 0.131480177 0.130961075 0.128065481 0.117932866 0.146146338 0.129565225 0.134207945 0.126587221 0.125809844 0.124586565 0 0.20391453 Phenylalanine_13C_5 -0.013992622 -0.019186206 -0.013326504 -0.013794566 -0.008649027 -0.011686581 -0.011234687 -0.01201522 -0.012354256 -0.013691123 -0.03180913 -0.012083823 -0.012027359 -0.014558672 -0.01703438 -0.017928561 -0.029036107 -0.013476834 -0.013201941 -0.011321401 -0.011490818 -0.015837507 -0.012502521 -0.012249539 -0.011595235 -0.011882087 -0.014171843 -0.012003691 0 -0.017913725 Phenylalanine_13C_6 -0.003618782 -0.005395026 -0.003665172 0.14400971 0.891168176 0.086957813 0.587187235 0.034110675 0.485796224 0.051413118 -0.010498935 -0.003836134 -0.003796345 -0.003414997 -0.004747468 -0.006374138 -0.009334329 -0.00365353 -0.003737451 0.586298997 -0.003713883 -0.003181642 0.132805383 -0.00372575 -0.003916843 0.29746094 -0.003473491 0.37164436 0 -0.006178757 Phenylalanine_13C_7 0.208423262 0.341498117 0.626974439 1.718395516 1.491293932 0.490771407 1.765151211 0.695784553 0.177353357 0.535096308 1.194331921 1.927178021 2.325513422 1.001772403 0.02160458 0.092684535 0.470395813 0.438053974 0.289196889 2.121691805 1.219661424 0.702153053 0.561109218 1.724622373 1.616361981 0.874735877 1.092482393 1.899084714 0 0.998390566 Phenylalanine_13C_8 -0.018576412 -0.030466714 0.42333503 0.013053918 0.661093439 0.037389632 -0.182225699 0.322502115 0.034478196 0.102731027 0.208104745 -0.173041629 -0.035880502 0.140234556 0.068397884 0.317637624 0.121364403 0.077932494 0.122536327 0.972314584 -0.089180808 -0.062996513 0.097073325 0.542123874 0.176102634 0.082653097 -0.022091403 -0.035373116 0 0.21845095 Phenylalanine_13C_9 0.078239914 0.15162124 0.489476816 0.320376471 2.528115971 0.715211305 2.18526326 0.886106169 -0.01106197 0.501677781 0.824562799 0.710260285 0.748115182 0.741553624 0.130923754 1.112214416 0.30511475 0.626104144 0.159297225 1.056528131 0.668625925 1.881269602 0.228842996 1.310736993 0.565871634 0.161148168 0.551590373 0.649324159 0 0.293498719 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Quantified m/z PubChem ID KEGG ID HMDB Pyruvic acid 174 1060 C00022 HMDB0000243 Pyruvic acid_13C_1 175 1060 C00022 HMDB0000243 Pyruvic acid_13C_2 176 1060 C00022 HMDB0000243 Pyruvic acid_13C_3 177 1060 C00022 HMDB0000243 Lactic acid 219 107689 C00186 HMDB0000190 Lactic acid_13C_1 220 107689 C00186 HMDB0000190 Lactic acid_13C_2 221 107689 C00186 HMDB0000190 Lactic acid_13C_3 222 107689 C00186 HMDB0000190 beta-Alanine 116 239 C00099 HMDB0000056 beta-Alanine_13C_1 117 239 C00099 HMDB0000056 beta-Alanine_13C_2 118 239 C00099 HMDB0000056 beta-Alanine_13C_3 119 239 C00099 HMDB0000056 Leucine 86 6106 C00123 HMDB0000687 Leucine_13C_1 87 6106 C00123 HMDB0000687 Leucine_13C_2 88 6106 C00123 HMDB0000687 Leucine_13C_3 89 6106 C00123 HMDB0000687 Leucine_13C_4 90 6106 C00123 HMDB0000687 Leucine_13C_5 91 6106 C00123 HMDB0000687 Leucine_13C_6 92 6106 C00123 HMDB0000687 Isoleucine 86 6306 C00407 HMDB0000172 Isoleucine_13C_1 87 6306 C00407 HMDB0000172 Isoleucine_13C_2 88 6306 C00407 HMDB0000172 Isoleucine_13C_3 89 6306 C00407 HMDB0000172 Isoleucine_13C_4 90 6306 C00407 HMDB0000172 Isoleucine_13C_5 91 6306 C00407 HMDB0000172 Isoleucine_13C_6 92 6306 C00407 HMDB0000172 Serine 116 5951 C00716 HMDB0062263 Serine_13C_1 117 5951 C00716 HMDB0062263 Serine_13C_2 118 5951 C00716 HMDB0062263 Serine_13C_3 119 5951 C00716 HMDB0062263 Glycine 276 750 C00037 HMDB0000123 Glycine_13C_1 277 750 C00037 HMDB0000123 Glycine_13C_2 278 750 C00037 HMDB0000123 Fumaric acid 245 444972 C00122 HMDB0000134 Fumaric acid_13C_1 246 444972 C00122 HMDB0000134 Fumaric acid_13C_2 247 444972 C00122 HMDB0000134 Fumaric acid_13C_3 248 444972 C00122 HMDB0000134 Fumaric acid_13C_4 249 444972 C00122 HMDB0000134 Asparatic acid 160 5960 C00049 HMDB0000191 Asparatic acid_13C_1 161 5960 C00049 HMDB0000191 Asparatic acid_13C_2 162 5960 C00049 HMDB0000191 Asparatic acid_13C_3 163 5960 C00049 HMDB0000191 Asparatic acid_13C_4 164 5960 C00049 HMDB0000191 Pyroglutamic acid 258 7405 C01879 HMDB0000267 Pyroglutamic acid_13C_1 259 7405 C01879 HMDB0000267 Pyroglutamic acid_13C_2 260 7405 C01879 HMDB0000267 Pyroglutamic acid_13C_3 261 7405 C01879 HMDB0000267 Pyroglutamic acid_13C_4 262 7405 C01879 HMDB0000267 Pyroglutamic acid_13C_5 263 7405 C01879 HMDB0000267 Glutamatic acid 246 33032 C00025 HMDB0000148 Glutamatic acid_13C_1 247 33032 C00025 HMDB0000148 Glutamatic acid_13C_2 248 33032 C00025 HMDB0000148 Glutamatic acid_13C_3 249 33032 C00025 HMDB0000148 Glutamatic acid_13C_4 250 33032 C00025 HMDB0000148 Glutamatic acid_13C_5 251 33032 C00025 HMDB0000148 Citric acid 273 311 C00158 HMDB0000094 Citric acid_13C_1 274 311 C00158 HMDB0000094 Citric acid_13C_2 275 311 C00158 HMDB0000094 Citric acid_13C_3 276 311 C00158 HMDB0000094 Citric acid_13C_4 277 311 C00158 HMDB0000094 Citric acid_13C_5 278 311 C00158 HMDB0000094 Citric acid_13C_6 279 311 C00158 HMDB0000094 Urea 189 1176 C00086 HMDB0000294 Urea_13C_1 190 1176 C00086 HMDB0000294 Ethanolamine 174 700 C00189 HMDB0000149 Ethanolamine_13C_1 175 700 C00189 HMDB0000149 Ethanolamine_13C_2 176 700 C00189 HMDB0000149 Glyoxylic acid 134 760 C00048 HMDB0000119 Glyoxylic acid_13C_1 135 760 C00048 HMDB0000119 Glyoxylic acid_13C_2 136 760 C00048 HMDB0000119 Malic acid 233 525 C03668 HMDB0000744 Malic acid_13C_1 234 525 C03668 HMDB0000744 Malic acid_13C_2 235 525 C03668 HMDB0000744 Malic acid_13C_3 236 525 C03668 HMDB0000744 Malic acid_13C_4 237 525 C03668 HMDB0000744 Phenylalanine 120 6140 C00079 HMDB0000159 Phenylalanine_13C_1 121 6140 C00079 HMDB0000159 Phenylalanine_13C_2 122 6140 C00079 HMDB0000159 Phenylalanine_13C_3 123 6140 C00079 HMDB0000159 Phenylalanine_13C_4 124 6140 C00079 HMDB0000159 Phenylalanine_13C_5 125 6140 C00079 HMDB0000159 Phenylalanine_13C_6 126 6140 C00079 HMDB0000159 Phenylalanine_13C_7 127 6140 C00079 HMDB0000159 Phenylalanine_13C_8 128 6140 C00079 HMDB0000159 Phenylalanine_13C_9 129 6140 C00079 HMDB0000159 METABOLITES_END #END