#METABOLOMICS WORKBENCH cwalker_bcm_20200127_072952 DATATRACK_ID:1909 STUDY_ID:ST001309 ANALYSIS_ID:AN002179 PROJECT_ID:PR000890 VERSION 1 CREATED_ON January 29, 2020, 2:04 pm #PROJECT PR:PROJECT_TITLE Metabolomic profiling after early-life exposure to an endocrine disrupting PR:PROJECT_TITLE chemical in the liver. PR:PROJECT_TYPE Targeted MS analysis PR:PROJECT_SUMMARY Metabolic profiling in the liver (240 days post-natal) after early-life exposure PR:PROJECT_SUMMARY to an endocrine disrupting chemical. PR:INSTITUTE Baylor College of Medicine PR:LAST_NAME Walker PR:FIRST_NAME Cheryl PR:ADDRESS 1 Baylor Plaza, Houston, TX, 77030, USA PR:EMAIL Cheryl.walker@bcm.edu PR:PHONE 713-798-8219 #STUDY ST:STUDY_TITLE Metabolite expression in liver after early life exposure to an endocrine ST:STUDY_TITLE disruptor at 240 days postnatal (part-I) ST:STUDY_TYPE Metabolite expression after chemical exposure versus control. ST:STUDY_SUMMARY Our early-life environment has a profound influence on developing organs that ST:STUDY_SUMMARY impact metabolic function and determines disease susceptibility across the ST:STUDY_SUMMARY life-course. Using a rat model for exposure to an endocrine disrupting chemical ST:STUDY_SUMMARY (EDC), we show that early-life exposure causes metabolic dysfunction in ST:STUDY_SUMMARY adulthood and reprograms histone marks in the developing liver to accelerate ST:STUDY_SUMMARY acquisition of an adult epigenomic signature. This epigenomic reprogramming ST:STUDY_SUMMARY persists long after the initial exposure, but many reprogrammed genes remain ST:STUDY_SUMMARY transcriptionally silent with their impact on metabolism not revealed until a ST:STUDY_SUMMARY later life exposure to a Western-style diet. Diet-dependent metabolic disruption ST:STUDY_SUMMARY was largely driven by reprogramming of the Early Growth Response 1 (EGR1) ST:STUDY_SUMMARY transcriptome and production of metabolites in pathways linked to cholesterol, ST:STUDY_SUMMARY lipid and one-carbon metabolism. These findings demonstrate the importance of ST:STUDY_SUMMARY epigenome: environment interactions, which early in life accelerate epigenomic ST:STUDY_SUMMARY aging, and later in adulthood unlock metabolically restricted epigenetic ST:STUDY_SUMMARY reprogramming to drive metabolic dysfunction. ST:INSTITUTE Baylor College of Medicine ST:DEPARTMENT Molecular and Cellular Biology ST:LABORATORY Center for Precision Environmental Health ST:LAST_NAME Walker ST:FIRST_NAME Cheryl ST:ADDRESS 1 Baylor Plaza, Houston, TX, 77030, USA ST:EMAIL Cheryl.walker@bcm.edu ST:PHONE 713-798-8219 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 10 ST:NUM_MALES 10 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Rattus norvegicus SU:TAXONOMY_ID 10116 SU:GENOTYPE_STRAIN Sprague Dawley SU:AGE_OR_AGE_RANGE 240 days SU:GENDER Male SU:ANIMAL_ANIMAL_SUPPLIER Harlan SU:ANIMAL_HOUSING polycarbonate-free caging SU:ANIMAL_LIGHT_CYCLE 14-hr light and 10-hr dark SU:ANIMAL_FEED Phytoestrogen Reduced II 18-5 (Ziegler Bros, Inc) or D09100301 (Research Diets, SU:ANIMAL_FEED Inc) #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 - HFD-VEH13 Treatment:vehicle Day of Liver Harvest Post-Birth=240; BPA_exposure=0 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-VEH15 Treatment:vehicle Day of Liver Harvest Post-Birth=240; BPA_exposure=0 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-VEH18 Treatment:vehicle Day of Liver Harvest Post-Birth=240; BPA_exposure=0 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-VEH12 Treatment:vehicle Day of Liver Harvest Post-Birth=240; BPA_exposure=0 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-VEH14 Treatment:vehicle Day of Liver Harvest Post-Birth=240; BPA_exposure=0 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-BPA12 Treatment:BPA Day of Liver Harvest Post-Birth=240; BPA_exposure=50 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-BPA13 Treatment:BPA Day of Liver Harvest Post-Birth=240; BPA_exposure=50 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-BPA14 Treatment:BPA Day of Liver Harvest Post-Birth=240; BPA_exposure=50 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-BPA16 Treatment:BPA Day of Liver Harvest Post-Birth=240; BPA_exposure=50 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-BPA18 Treatment:BPA Day of Liver Harvest Post-Birth=240; BPA_exposure=50 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. #COLLECTION CO:COLLECTION_SUMMARY Liver tissue was harvested on post-natal day 240 after challenge with CO:COLLECTION_SUMMARY Western-style diet.Tissue was snap-frozen in liquid nitrogen. CO:SAMPLE_TYPE Liver #TREATMENT TR:TREATMENT_SUMMARY Neonatal rats were treated with vehicle (sesame oil) or bisphenol A (BPA; 50 TR:TREATMENT_SUMMARY µg/kg dissolved in sesame oil) orally via pipette tip on post-natal days 1, 3, TR:TREATMENT_SUMMARY and 5. Littermates were randomly assigned to the treatment groups. BPA was TR:TREATMENT_SUMMARY obtained from the National Institute of Environmental Health Sciences (NIEHS). TR:TREATMENT_SUMMARY The dose and route of administration recapitulates human exposure to BPA. At day TR:TREATMENT_SUMMARY 180, adult rats in both treatment groups were fed a diet high in fat (40% kcal), TR:TREATMENT_SUMMARY fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days TR:TREATMENT_SUMMARY (D09100301, Research Diets, Inc). Rats were fasted overnight prior to tissue TR:TREATMENT_SUMMARY collection. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were extracted from crushed liver samples and a mouse liver pool was SP:SAMPLEPREP_SUMMARY used for quality control. Twenty-five mg of crushed liver was used for the SP:SAMPLEPREP_SUMMARY metabolic extraction. The extraction step started with the addition of 750 µL SP:SAMPLEPREP_SUMMARY ice-cold methanol:water (4:1) containing 20 µL spiked internal standards to SP:SAMPLEPREP_SUMMARY each tissue sample. Ice-cold chloroform and water were added in a 3:1 ratio for SP:SAMPLEPREP_SUMMARY a final proportion of 1:4:3:1 water:methanol:chloroform:water. The organic SP:SAMPLEPREP_SUMMARY (methanol and chloroform) and aqueous layers were mixed, dried and resuspended SP:SAMPLEPREP_SUMMARY with 50:50 methanol: water. The extract was deproteinized using a 3kDa molecular SP:SAMPLEPREP_SUMMARY filter (Amicon ultracel-3K Membrane; Millipore Corporation, Billerica, MA) and SP:SAMPLEPREP_SUMMARY the filtrate was dried under vacuum (Genevac EZ-2plus; Gardiner, Stone Ridge, SP:SAMPLEPREP_SUMMARY NY). Prior to mass spectrometry, the dried extracts were re-suspended in SP:SAMPLEPREP_SUMMARY identical volumes of injection solvent composed of 1:1 water: methanol and were SP:SAMPLEPREP_SUMMARY subjected to liquid chromatography-mass spectrometry. Fifty µl of sample was SP:SAMPLEPREP_SUMMARY used for preparation. Internal standards were spiked into the samples. Then it SP:SAMPLEPREP_SUMMARY was processed through a 3 kDa filter. After that, 50 µl of sample was diluted SP:SAMPLEPREP_SUMMARY with 450 µl solvent (methanol: water = 50:50 v/v) and subjected to LC/MS SP:SAMPLEPREP_SUMMARY analysis. The injection volume was 10 µl. For internal standards, SP:SAMPLEPREP_SUMMARY high-performance liquid chromatography (HPLC)-grade acetonitrile, methanol, and SP:SAMPLEPREP_SUMMARY water were procured from Burdick & Jackson (Morristown, NJ). Mass SP:SAMPLEPREP_SUMMARY spectrometry-grade formic acid was purchased from Sigma-Aldrich (St Louis, MO). SP:SAMPLEPREP_SUMMARY Calibration solution containing multiple calibrants in a solution of SP:SAMPLEPREP_SUMMARY acetonitrile, trifluroacetic acid, and water was purchased from Agilent SP:SAMPLEPREP_SUMMARY Technologies (Santa Clara, CA). Metabolites and internal standards, including SP:SAMPLEPREP_SUMMARY N-acetyl Aspartic acid-d3, Tryptophan-15N2, Sarcosine-d3, Glutamic acid-d5, SP:SAMPLEPREP_SUMMARY Thymine-d4, Gibberellic acid, Trans-Zeatine, Jasmonic acid, 15N Anthranilic SP:SAMPLEPREP_SUMMARY acid, and Testosterone-d3, were purchased from Sigma-Aldrich (St. Louis, MO). SP:SAMPLEPREP_SUMMARY Three LC- MS methods were used to separate metabolites. Method A: In ESI SP:SAMPLEPREP_SUMMARY positive mode the HPLC column was waters X-bridge amide 3.5 µm, 4.6 x 100 mm SP:SAMPLEPREP_SUMMARY (Waters, Milford, MA). Mobile phase A and B were 0.1% formic acid in water and SP:SAMPLEPREP_SUMMARY acetonitrile, respectively. Gradient flow: 0-3 min 85% B; 3-12 min 30% B, 12-15 SP:SAMPLEPREP_SUMMARY min 2% B, 16 min 95%B, followed by re-equilibration till the end of the gradient SP:SAMPLEPREP_SUMMARY 23 min to the initial starting condition of 85% B. Flow rate of the solvents SP:SAMPLEPREP_SUMMARY used for the analysis is 0.3 ml/min. Injection volume was 10 µL. Method B: In SP:SAMPLEPREP_SUMMARY ESI negative mode the HPLC column was waters X-bridge amide 3.5 µm, 4.6 x 100 SP:SAMPLEPREP_SUMMARY mm (Waters, Milford, MA). Mobile phase A and B were 20 mM ammonium acetate in SP:SAMPLEPREP_SUMMARY water with pH 9.0 and 100% acetonitrile, respectively. Gradient flow: 0-3 min SP:SAMPLEPREP_SUMMARY 85% B, 3-12 min 30% B, 12-15 min 2% B, 15-16 min 85% B followed by SP:SAMPLEPREP_SUMMARY re-equilibration till the end of the gradient 23 min to the initial starting SP:SAMPLEPREP_SUMMARY condition of 85% B. Flow rate of the solvents used for analysis is 0.3 ml/min. SP:SAMPLEPREP_SUMMARY Injection volume was 10 µL. Method C: In ESI positive mode the HPLC column was SP:SAMPLEPREP_SUMMARY Luna 3 µM NH2 100 A0 Chromatography column (Phenomenex, Torrance, CA). Mobile SP:SAMPLEPREP_SUMMARY phase A and B were 20 mM ammonium acetate in water with pH 9.0 and 100% SP:SAMPLEPREP_SUMMARY acetonitrile, respectively. Gradient flow: 0-3 min 85% B, 3-12 min 30% B, 12-15 SP:SAMPLEPREP_SUMMARY min 2% B, 15-16 min 85% B followed by re-equilibration till the end of the SP:SAMPLEPREP_SUMMARY gradient 23 min to the initial starting condition of 85% B. Flow rate of the SP:SAMPLEPREP_SUMMARY solvents used for analysis is 0.3 ml/min. Injection volume was 10 µL. For data SP:SAMPLEPREP_SUMMARY acquisition through LC/MS analysis, 10 µL of suspended samples were injected SP:SAMPLEPREP_SUMMARY and analyzed using a 6495 triple quadrupole mass spectrometer (Agilent SP:SAMPLEPREP_SUMMARY Technologies, Santa Clara, CA) coupled to a HPLC system (Agilent Technologies, SP:SAMPLEPREP_SUMMARY Santa Clara, CA) via Multiple reaction monitoring (MRM). Source parameters were SP:SAMPLEPREP_SUMMARY as follows: Gas temperature- 250°C; Gas flow- 14 l/min; Nebulizer - 20psi; SP:SAMPLEPREP_SUMMARY Sheath gas temperature - 350°C; Sheath gas flow- 12 l/min; Capillary - 3000 V SP:SAMPLEPREP_SUMMARY positive and 3000 V negative; Nozzle voltage- 1500 V positive and 1500 V SP:SAMPLEPREP_SUMMARY negative. Approximately 8–11 data points were acquired per detected SP:SAMPLEPREP_SUMMARY metabolite. The data acquired using Agilent mass hunter software and data was SP:SAMPLEPREP_SUMMARY analyzed using mass hunter quantitative analysis software. SP:SAMPLEPREP_PROTOCOL_FILENAME Targeted.MS.method.pdf;unbiased.liver.MS.method.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 6495 QQQ CH:COLUMN_NAME Waters XBridge Amide (100 x 4.6mm, 3.5um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6495 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS For data acquisition through LC/MS analysis, 10 µL of suspended samples were MS:MS_COMMENTS injected and analyzed using a 6495 triple quadrupole mass spectrometer (Agilent MS:MS_COMMENTS Technologies, Santa Clara, CA) coupled to a HPLC system (Agilent Technologies, MS:MS_COMMENTS Santa Clara, CA) via Multiple reaction monitoring (MRM). Source parameters were MS:MS_COMMENTS as follows: Gas temperature- 250°C; Gas flow- 14 l/min; Nebulizer - 20psi; MS:MS_COMMENTS Sheath gas temperature - 350°C; Sheath gas flow- 12 l/min; Capillary - 3000 V MS:MS_COMMENTS positive and 3000 V negative; Nozzle voltage- 1500 V positive and 1500 V MS:MS_COMMENTS negative. Approximately 8–11 data points were acquired per detected MS:MS_COMMENTS metabolite. The data acquired using Agilent mass hunter software and data was MS:MS_COMMENTS analyzed using mass hunter quantitative analysis software. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak intensity MS_METABOLITE_DATA_START Samples HFD-VEH13 HFD-VEH15 HFD-VEH18 HFD-VEH12 HFD-VEH14 HFD-BPA12 HFD-BPA13 HFD-BPA14 HFD-BPA16 HFD-BPA18 Factors Treatment:vehicle Treatment:vehicle Treatment:vehicle Treatment:vehicle Treatment:vehicle Treatment:BPA Treatment:BPA Treatment:BPA Treatment:BPA Treatment:BPA trehalose-6-Phosphate 13585.17948 18844.51835 60268.60113 8724.744716 19624.76487 6603.560576 8016.884755 9129.429103 6148.970126 10524.14903 shikimate 36108.2828 63019.95884 114212.8506 46150.04826 39121.04536 112640.9434 54125.31143 27896.43732 41884.15577 22639.59748 Octanoic acid 5018.518706 5639.914857 5873.077847 5974.951612 4790.500285 5496.06238 3618.431298 6495.416783 4930.581802 4087.528326 anthranilate 48186.15204 50842.5427 47049.19542 56902.63522 49145.7506 71373.62116 53459.71342 63413.26577 59053.56527 64974.70001 p-aminobenzoate 48186.15204 50842.5427 47049.19542 56902.63522 49145.7506 71373.62116 53459.71342 63413.26577 59053.56527 64974.70001 S-ribosyl-L-homocysteine_neg 20675.8139 26458.81165 10041.2936 9319.052253 11042.62886 5634.044459 17270.86139 21464.53244 17987.33681 12457.27708 inosine 6984903.157 6167027.013 7267897.552 6876153.877 8524654.999 6315892.421 5984907.459 6650734.309 6309532.359 7250274.347 Uric acid 418781.3646 349137.9424 436698.5342 434565.5992 241748.0117 544937.2285 456862.574 417504.8292 438392.7254 274314.2946 allantoin 29448.89635 40294.46014 25408.46146 29779.95515 38661.06253 35132.97871 21341.69841 21729.43552 27212.21904 32533.31863 succinate 112713.0683 54622.9343 197821.6704 194595.8658 64078.79862 102384.6946 201362.831 129615.4988 143945.0445 94079.41599 taurine 1306492.688 3159319.43 1263793.883 1901721.742 1305782.664 1576403.099 1107271.169 1585970.101 1625890.149 1596774.085 lactate 2640230.881 2640317.386 2607412.199 2630004.077 2662692.164 2366624.701 2302251.526 1784196.104 2519542.696 1993527.629 Glucuronic acid 16885.56563 16610.21623 14935.70889 14848.01076 18552.81694 13602.15179 11520.59736 14189.20592 16650.32518 19328.53673 Malic acid 5515009.813 6088263.101 5306916.513 5164819.12 5616570.728 4529798.072 4792329.905 4627828.945 5177815.624 4135088.445 UDP-N-acetyl-glucosamine 329412.2766 284286.7093 210085.5067 273456.1752 225086.3748 147319.8959 211524.5613 152341.0165 228275.1799 151113.5715 dGDP_neg 305857.186 350435.2117 238969.9909 309403.9467 243203.3889 201189.8332 255458.2218 283872.7532 292569.2655 296315.4349 PEP 36097.66332 44967.08288 55200.98342 27425.88736 86236.05099 50495.08195 20679.42157 16501.98304 31016.37754 17288.3665 sn-glycerol-3-phosphate 7047779.316 5267301.495 6281177.546 7825418.392 6850294.553 5545268.271 5254728.77 5049479.455 5902122.492 5173301.016 glutathione disulfide_neg 1608284.267 1789976.631 1530211.493 1670995.344 1597914.975 1622264.368 996985.3062 1450361.272 1293558.986 1600362.911 UDP-D-glucuronate 113107.4021 130623.6155 77510.68153 65572.72019 73762.57994 14124.61972 51008.95626 48697.66615 100158.7995 38657.41089 UDP-D-glucose 1409287.198 556519.4219 630129.9108 1110431.053 928673.5334 197835.4132 314767.9051 680839.1446 766323.7285 728413.5053 FBP/GBP 20836.67698 19027.15856 17261.73217 20041.03656 29401.90085 10808.4339 12618.01942 11796.05525 13972.655 12066.35221 D-sedoheptulose-1-7-phosphate 354687.1457 245864.4551 423097.6732 305091.2033 574590.3354 461676.4359 326512.6215 208353.554 351327.2292 150270.8374 Frctose-6-Phosphate 124058.185 111190.9407 101391.1936 82665.58787 193598.6556 121720.0736 102036.0606 56214.78764 77584.96908 56844.5416 Glucose-6-Phosphate 90634.64497 55726.07783 56999.90195 55592.56999 123044.676 52920.58831 73350.75751 25919.72327 50973.13799 29546.83521 hexose-phosphate 183687.0838 129545.439 152919.7879 104908.0883 269969.1978 169447.5283 162916.6231 73025.37193 112929.4747 78899.91082 NAD+_neg 282750.4218 106232.8142 183480.7309 191422.2919 179728.4995 111108.3232 130430.4674 195721.7447 183978.1964 178130.4386 octulose-monophosphate (O8P-O1P) 7944.274629 8915.583677 6832.145536 6545.237216 8323.529794 9054.387236 8281.75772 4122.29635 6690.729112 4580.603865 Malonate (Propanedioate) 255610.5358 250804.1488 170471.9075 212892.321 181091.9462 143341.8069 152999.6768 204215.4184 199439.2485 177584.8729 Ketoglutarate 209983.6979 181172.8406 187935.8623 206075.4231 190880.0918 201753.9052 188168.3787 202356.5827 203584.5113 176028.1531 acetylphosphate 20193.74445 17172.66833 19676.19719 17633.1712 22160.16019 18708.21498 16315.90081 15107.24592 15609.4318 16351.45173 ADP_neg 305814.96 350435.2117 238972.1494 309403.9467 243188.8567 201189.8332 255458.2218 283872.7532 292569.2655 296315.4349 Maltose 424107.3133 281363.8192 553985.3279 338519.9194 890833.5735 497487.7924 416961.3015 79678.91736 286539.2967 97729.01838 6-phospho-D-gluconate 37396.76427 16359.43198 22639.04455 18721.97832 42531.48633 6916.323352 47761.09717 4376.965243 15533.55672 6056.217877 MALTOTETRAOSE 877149.9886 272807.2459 997583.6977 449918.5441 2104884.057 456661.7346 590551.454 28869.03014 275187.6583 31589.79401 L-Anthranilic aicd (internal standard) 44022.5138 47527.55497 39501.56649 43744.2963 44438.19865 37672.58448 38522.88451 48910.24767 43927.51479 54314.22861 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name trehalose-6-Phosphate shikimate Octanoic acid anthranilate p-aminobenzoate S-ribosyl-L-homocysteine_neg inosine Uric acid allantoin succinate taurine lactate Glucuronic acid Malic acid UDP-N-acetyl-glucosamine dGDP_neg PEP sn-glycerol-3-phosphate glutathione disulfide_neg UDP-D-glucuronate UDP-D-glucose FBP/GBP D-sedoheptulose-1-7-phosphate Frctose-6-Phosphate Glucose-6-Phosphate hexose-phosphate NAD+_neg octulose-monophosphate (O8P-O1P) Malonate (Propanedioate) Ketoglutarate acetylphosphate ADP_neg Maltose 6-phospho-D-gluconate MALTOTETRAOSE L-Anthranilic aicd (internal standard) METABOLITES_END #END