#METABOLOMICS WORKBENCH ramkhattri_20221031_115523 DATATRACK_ID:3544 STUDY_ID:ST002356 ANALYSIS_ID:AN003847 PROJECT_ID:PR001513 VERSION 1 CREATED_ON November 22, 2022, 8:15 pm #PROJECT PR:PROJECT_TITLE Isolated murine skeletal muscles utilize pyruvate over glucose for PR:PROJECT_TITLE oxidation-Part 1 PR:PROJECT_TYPE Study of the different substrate by isolated skeletal muscle at room temperature PR:PROJECT_TYPE via C-13 isotopomer analysis PR:PROJECT_SUMMARY The goal of this study was to determine the differential utilization of PR:PROJECT_SUMMARY substrates in isolated murine skeletal muscle, and to evalute how isopotomer PR:PROJECT_SUMMARY anlaysis provided insight into skeletal muscle metabolism. PR:INSTITUTE University of Florida PR:DEPARTMENT Applied Physiology and Kinesiology PR:LABORATORY Rm 42 and Rm 43 PR:LAST_NAME Khattri PR:FIRST_NAME Ram PR:ADDRESS 1864 Stadium RD, Gainesville, FL, 32611, USA PR:EMAIL rbk11@ufl.edu PR:PHONE 3307856045 PR:FUNDING_SOURCE This work was supported by grants from the Southeastern Center for Integrated PR:FUNDING_SOURCE Metabolomics (SECIM) (ERB), NIH AR U54 AR052646 (Physiological Assessment Core, PR:FUNDING_SOURCE ERB), and Wellstone Muscular Dystrophy Cooperative Research Center Grant (NIAMS: PR:FUNDING_SOURCE U54AR052646/P50 AR052646). The AMRIS Facility is supported by the National PR:FUNDING_SOURCE Science Foundation Cooperative Agreement No. DMR-1644779 and the State of PR:FUNDING_SOURCE Florida. PR:CONTRIBUTORS Ram B. Khattri, Jason Puglise, Terence E. Ryan, Glenn A. Walter, Matthew E. PR:CONTRIBUTORS Merritt, Elisabeth R. Barton #STUDY ST:STUDY_TITLE Isolated murine skeletal muscles utilize pyruvate over glucose for ST:STUDY_TITLE oxidation-Part 1 ST:STUDY_TYPE Study of the different substrate by isolated skeletal muscle at room temperature ST:STUDY_TYPE via C-13 isotopomer analysis ST:STUDY_SUMMARY Preclinical studies of muscle contractile function often employ ex vivo ST:STUDY_SUMMARY preparations of the soleus and/or extensor digitorum longus (EDL) muscles which ST:STUDY_SUMMARY are relatively easy to prepare and represent slow and fast fiber properties, ST:STUDY_SUMMARY respectively. Therefore, the current study sought to examine the utility of this ST:STUDY_SUMMARY preparation for understanding the metabolic fuel utilization in isolated resting ST:STUDY_SUMMARY mouse muscles at room temperature. 13C-labeling in both muscle types was ST:STUDY_SUMMARY performed using three fuels: glucose, pyruvate, and acetate, followed by ST:STUDY_SUMMARY NMR-based metabolomics analyses. Incubating 13C-labeled substrates in the ST:STUDY_SUMMARY isolated skeletal muscles makes it possible to examine TCA cycle flux and ST:STUDY_SUMMARY substrate selection by these muscles. ST:INSTITUTE University of Florida ST:DEPARTMENT Applied Physiology and Kinesiology ST:LABORATORY Rm 42 and Rm 43 ST:LAST_NAME Khattri ST:FIRST_NAME Ram ST:ADDRESS 1864 Stadium RD, Gainesville, FL, 32611, USA ST:EMAIL rbk11@ufl.edu ST:PHONE 3307856045 ST:NUM_GROUPS 4 ST:TOTAL_SUBJECTS 18 ST:STUDY_COMMENTS Southeastern Center for Integrated Metabolomics (SECIM) (ERB), NIH AR U54 ST:STUDY_COMMENTS AR052646 (Physiological Assessment Core, ERB), and Wellstone Muscular Dystrophy ST:STUDY_COMMENTS Cooperative Research Center Grant (NIAMS: U54AR052646/P50 AR052646). The AMRIS ST:STUDY_COMMENTS Facility is supported by the National Science Foundation Cooperative Agreement ST:STUDY_COMMENTS No. DMR-1644779 and the State of Florida. #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6J SU:AGE_OR_AGE_RANGE 16±3 weeks SU:ANIMAL_ANIMAL_SUPPLIER Jackson Labs (Stock # 000664) SU:ANIMAL_HOUSING Housed in a temperature of 22 oC SU:ANIMAL_LIGHT_CYCLE 12-hour light/12-hour dark SU:ANIMAL_FEED Ad libitum chow diet food SU:ANIMAL_WATER free access to food and water (3-5 animals per cage). #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 - Glucose_EDL_1D_DecON&OFF-1 Group:Glucose_EDL RAW_FILE_NAME=Glucose_EDL_1D_DecON&OFF-1.raw SUBJECT_SAMPLE_FACTORS - Glucose_EDL_1D_DecON&OFF-2 Group:Glucose_EDL RAW_FILE_NAME=Glucose_EDL_1D_DecON&OFF-2.raw SUBJECT_SAMPLE_FACTORS - Glucose_EDL_1D_DecON&OFF-3 Group:Glucose_EDL RAW_FILE_NAME=Glucose_EDL_1D_DecON&OFF-3.raw SUBJECT_SAMPLE_FACTORS - Glucose_EDL_1D_DecON&OFF-4 Group:Glucose_EDL RAW_FILE_NAME=Glucose_EDL_1D_DecON&OFF-4.raw SUBJECT_SAMPLE_FACTORS - Glucose_Soleus_1D_DecON&OFF-1 Group:Glucose_Soleus RAW_FILE_NAME=Glucose_Soleus_1D_DecON&OFF-1.raw SUBJECT_SAMPLE_FACTORS - Glucose_Soleus_1D_DecON&OFF-2 Group:Glucose_Soleus RAW_FILE_NAME=Glucose_Soleus_1D_DecON&OFF-2.raw SUBJECT_SAMPLE_FACTORS - Glucose_Soleus_1D_DecON&OFF-3 Group:Glucose_Soleus RAW_FILE_NAME=Glucose_Soleus_1D_DecON&OFF-3.raw SUBJECT_SAMPLE_FACTORS - Glucose_Soleus_1D_DecON&OFF-4 Group:Glucose_Soleus RAW_FILE_NAME=Glucose_Soleus_1D_DecON&OFF-4.raw SUBJECT_SAMPLE_FACTORS - Pyruvate_EDL_1D_DecON&OFF-1 Group:Pyruvate_EDL RAW_FILE_NAME=Pyruvate_EDL_1D_DecON&OFF-1.raw SUBJECT_SAMPLE_FACTORS - Pyruvate_EDL_1D_DecON&OFF-2 Group:Pyruvate_EDL RAW_FILE_NAME=Pyruvate_EDL_1D_DecON&OFF-2.raw SUBJECT_SAMPLE_FACTORS - Pyruvate_EDL_1D_DecON&OFF-3 Group:Pyruvate_EDL RAW_FILE_NAME=Pyruvate_EDL_1D_DecON&OFF-3.raw SUBJECT_SAMPLE_FACTORS - Pyruvate_Soleus_1D_DecON&OFF-1 Group:Pyruvate_Soleus RAW_FILE_NAME=Pyruvate_Soleus_1D_DecON&OFF-1.raw SUBJECT_SAMPLE_FACTORS - Pyruvate_Soleus_1D_DecON&OFF-2 Group:Pyruvate_Soleus RAW_FILE_NAME=Pyruvate_Soleus_1D_DecON&OFF-2.raw SUBJECT_SAMPLE_FACTORS - Pyruvate_Soleus_1D_DecON&OFF-3 Group:Pyruvate_Soleus RAW_FILE_NAME=Pyruvate_Soleus_1D_DecON&OFF-3.raw #COLLECTION CO:COLLECTION_SUMMARY Mice were anesthetized (using a combination of xylazine (80mg/kg) and ketamine CO:COLLECTION_SUMMARY (10mg/kg)) to allow removal of soleus and extensor digitorum longus (EDL) CO:COLLECTION_SUMMARY muscles. Upon removal, muscles were incubated at 22 oC in Ringer/MEM solution CO:COLLECTION_SUMMARY gas equilibrated with 95/5% O2/CO2 with appropriate 13C labeled substrates in a CO:COLLECTION_SUMMARY perfusion chamber routinely used for isolated muscle mechanics for 30 minutes. CO:COLLECTION_SUMMARY These included the following: 5.5 mM [U-13C6] glucose; 5.5 mM [U-13C3] pyruvate, CO:COLLECTION_SUMMARY or 16.5 mM [13C2] labeled Na-acetate. Following incubation, muscles were quickly CO:COLLECTION_SUMMARY removed, blotted, and then rapidly frozen in liquid nitrogen for subsequent NMR CO:COLLECTION_SUMMARY analysis. CO:COLLECTION_PROTOCOL_COMMENTS Mice were anesthetized (using a combination of xylazine (80mg/kg) and ketamine CO:COLLECTION_PROTOCOL_COMMENTS (10mg/kg)) to allow removal of soleus and extensor digitorum longus (EDL) CO:COLLECTION_PROTOCOL_COMMENTS muscles. Upon removal, muscles were incubated at 22 oC in Ringer/MEM solution CO:COLLECTION_PROTOCOL_COMMENTS gas equilibrated with 95/5% O2/CO2 with appropriate 13C labeled substrates in a CO:COLLECTION_PROTOCOL_COMMENTS perfusion chamber routinely used for isolated muscle mechanics for 30 minutes. CO:COLLECTION_PROTOCOL_COMMENTS These included the following: 5.5 mM [U-13C6] glucose; 5.5 mM [U-13C3] pyruvate, CO:COLLECTION_PROTOCOL_COMMENTS or 16.5 mM [13C2] labeled Na-acetate. Following incubation, muscles were quickly CO:COLLECTION_PROTOCOL_COMMENTS removed, blotted, and then rapidly frozen in liquid nitrogen for subsequent NMR CO:COLLECTION_PROTOCOL_COMMENTS analysis. N=4 muscles were pooled into a single biological replicate of 30-50 mg CO:COLLECTION_PROTOCOL_COMMENTS tissue to afford detectable levels of substrates in the NMR analysis. CO:SAMPLE_TYPE Muscle CO:COLLECTION_METHOD Mice were anesthetized (using a combination of xylazine (80mg/kg) and ketamine CO:COLLECTION_METHOD (10mg/kg)) to allow removal of soleus and extensor digitorum longus (EDL) CO:COLLECTION_METHOD muscles. CO:COLLECTION_LOCATION University of Florida, Applied Physiology and Kinesiology, MBI, 1149 Newell Dr, CO:COLLECTION_LOCATION Gainesville, FL 32610 CO:STORAGE_CONDITIONS -80℃ CO:COLLECTION_VIALS cryovials CO:STORAGE_VIALS cryovials #TREATMENT TR:TREATMENT_SUMMARY Mice were anesthetized (using a combination of xylazine (80mg/kg) and ketamine TR:TREATMENT_SUMMARY (10mg/kg)) to allow removal of soleus and extensor digitorum longus (EDL) TR:TREATMENT_SUMMARY muscles. Upon removal, muscles were incubated at 22 oC in Ringer/MEM solution TR:TREATMENT_SUMMARY gas equilibrated with 95/5% O2/CO2 with appropriate 13C labeled substrates in a TR:TREATMENT_SUMMARY perfusion chamber routinely used for isolated muscle mechanics for 30 minutes. TR:TREATMENT_SUMMARY These included the following: 5.5 mM [U-13C6] glucose; 5.5 mM [U-13C3] TR:TREATMENT_SUMMARY pyruvate, or 16.5 mM [13C2] labeled Na-acetate. Following incubation, muscles TR:TREATMENT_SUMMARY were quickly removed, blotted, and then rapidly frozen in liquid nitrogen for TR:TREATMENT_SUMMARY subsequent NMR analysis. N=4 muscles were pooled into a single biological TR:TREATMENT_SUMMARY replicate of 30-50 mg tissue to afford detectable levels of substrates in the TR:TREATMENT_SUMMARY NMR analysis. TR:ANIMAL_VET_TREATMENTS none TR:ANIMAL_ANESTHESIA a combination of xylazine (80mg/kg) and ketamine (10mg/kg)) TR:ANIMAL_FASTING non-fasted TR:ANIMAL_ENDP_EUTHANASIA Euthanasia was carried out by thoracotomy followed by cervical dislocation. TR:ANIMAL_ENDP_TISSUE_COLL_LIST Skeletal muscle (soleus and EDL) TR:ANIMAL_ENDP_TISSUE_PROC_METHOD Mice were anesthetized (using a combination of xylazine (80mg/kg) and ketamine TR:ANIMAL_ENDP_TISSUE_PROC_METHOD (10mg/kg)) to allow removal of soleus and extensor digitorum longus (EDL) TR:ANIMAL_ENDP_TISSUE_PROC_METHOD muscles. Upon removal, muscles were incubated at 22 oC in Ringer/MEM solution TR:ANIMAL_ENDP_TISSUE_PROC_METHOD gas equilibrated with 95/5% O2/CO2 with appropriate 13C labeled substrates in a TR:ANIMAL_ENDP_TISSUE_PROC_METHOD perfusion chamber routinely used for isolated muscle mechanics for 30 minutes. TR:ANIMAL_ENDP_TISSUE_PROC_METHOD These included the following: 5.5 mM [U-13C6] glucose; 5.5 mM [U-13C3] pyruvate, TR:ANIMAL_ENDP_TISSUE_PROC_METHOD or 16.5 mM [13C2] labeled Na-acetate. Following incubation, muscles were quickly TR:ANIMAL_ENDP_TISSUE_PROC_METHOD removed, blotted, and then rapidly frozen in liquid nitrogen for subsequent NMR TR:ANIMAL_ENDP_TISSUE_PROC_METHOD analysis. N=4 muscles were pooled into a single biological replicate of 30-50 mg TR:ANIMAL_ENDP_TISSUE_PROC_METHOD tissue to afford detectable levels of substrates in the NMR analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY "Perchloric acid (PCA) or acetonitrile:isopropanol:water (3:3:2) extractions SP:SAMPLEPREP_SUMMARY were performed for all samples to isolate metabolites. The latter method was SP:SAMPLEPREP_SUMMARY more efficient in sample recovery due to the reduced number of steps in the SP:SAMPLEPREP_SUMMARY procedure but did not affect the proportion of metabolites. For PCA extraction, SP:SAMPLEPREP_SUMMARY isolated muscle samples were homogenized with a FASTPREP-24 (MP Biomedicals, SP:SAMPLEPREP_SUMMARY Solon, Ohio, USA) with 6% (v/v) ice cold PCA and centrifuged with 13.2 K rpm at SP:SAMPLEPREP_SUMMARY 4 oC. The solid muscle portion was washed again with the 6% (v/v) ice cold PCA SP:SAMPLEPREP_SUMMARY followed by centrifugation (13.2 K rpm) at 4 oC. The supernatant (combined) SP:SAMPLEPREP_SUMMARY obtained was further neutralized with 5M potassium hydroxide and centrifuged SP:SAMPLEPREP_SUMMARY again maintaining 13.2 K rpm speed at 4 oC. The resulting supernatants were then SP:SAMPLEPREP_SUMMARY lyophilized (Thermo-Scientific, Dallas, USA). The pH of the dried powder was SP:SAMPLEPREP_SUMMARY adjusted to 7.2 after dissolving it in 200 μL of ultra-pure water using 1M SP:SAMPLEPREP_SUMMARY sodium hydroxide and 1 M hydrochloric acid. The pH-adjusted solution was further SP:SAMPLEPREP_SUMMARY centrifuged, the resulting supernatant was dried and the powder was used to SP:SAMPLEPREP_SUMMARY prepare the NMR sample. For acetonitrile:isopropanol:water extraction, SP:SAMPLEPREP_SUMMARY homogenization of isolated muscle samples was carried out in 1 mL SP:SAMPLEPREP_SUMMARY acetonitrile:isopropanol:water (3:3:2, v:v:v) ice cold mixture with a SP:SAMPLEPREP_SUMMARY FASTPREP-24 (MP Biomedicals, Solon, Ohio, USA) and centrifuged at 4 oC in SP:SAMPLEPREP_SUMMARY separate vials. Resultant supernatants were further lyophilized till dryness SP:SAMPLEPREP_SUMMARY (Thermo-Scientific, Dallas, USA). The dried powder was further dissolved in 1 mL SP:SAMPLEPREP_SUMMARY of Acetonitrile:Water (1:1, v:v) mixture, vortexed well for ~5 minutes. The SP:SAMPLEPREP_SUMMARY resultant solution was further centrifuged, the supernatant obtained was further SP:SAMPLEPREP_SUMMARY dried and the powder was used to prepare the NMR sample. The centrifugation SP:SAMPLEPREP_SUMMARY speed for each step used was 13.2K rpm. Each NMR sample consisted of 50 mM SP:SAMPLEPREP_SUMMARY phosphate buffer (pH 7), 2 mM EDTA, 0.02% of NaN3 with 0.5 mM of DSS as a SP:SAMPLEPREP_SUMMARY standard internal reference in deuterated environment. 1H NMR spectra were taken SP:SAMPLEPREP_SUMMARY at 25oC using a 600 MHz Bruker Avance II Console equipped with a TCI CryoProbe SP:SAMPLEPREP_SUMMARY that utilized Bruker Topspin 4 software (Bruker BioSpin Corporation, Billerica, SP:SAMPLEPREP_SUMMARY MA, USA). The first slice of a NOESY pulse sequence (noesypr1d) was used to SP:SAMPLEPREP_SUMMARY acquire proton NMR. Fractional enrichment for glutamate, lactate and alanine SP:SAMPLEPREP_SUMMARY were determined using 13C decoupling ON/OFF 1H proton spectra as well as 1D SP:SAMPLEPREP_SUMMARY NOESY spectra. To determine enrichements, a standard zgig pulse sequence was SP:SAMPLEPREP_SUMMARY adapted to allow 13C decoupling during the acquistion period (1.36 s) to remove SP:SAMPLEPREP_SUMMARY the satellites. Total enrichment was measured by taking a ratio of the SP:SAMPLEPREP_SUMMARY metabolite peak heights in the decoupling on/off experiments. NOESY spectra were SP:SAMPLEPREP_SUMMARY collected with a 1 s relaxation delay (d1), and a 4 s acqusition time (at), in SP:SAMPLEPREP_SUMMARY accordance with Chenomx recommendations for producing quantitative estimates of SP:SAMPLEPREP_SUMMARY concentration. Using the Chenomx quantification and the fractional enrichments, SP:SAMPLEPREP_SUMMARY a final concentration of the metabolites was calculated. Conventional 1H SP:SAMPLEPREP_SUMMARY decoupled 13C spectra were acquired using a 600 MHz Agilent with a specially SP:SAMPLEPREP_SUMMARY designed 1.5 mm superconducting (HTS) probe at 30oC. " SP:SAMPLEPREP_PROTOCOL_FILENAME Isolated_muscle_Procedures.docx SP:PROCESSING_METHOD Lyophilization and Homogenization SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD Perchloric acid (PCA) or acetonitrile:isopropanol:water (3:3:2) extractions SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION In 35 microliter of 50 mM phosphate buffer (pH 7.2) with 2 mM EDTA, 0.5 mM DSS SP:SAMPLE_RESUSPENSION and 0.2% sodium azide for aqueous phase samples. SP:SAMPLE_SPIKING 0.5 mM of DSS for aqueous phase samples #ANALYSIS AN:DATA_FORMAT fid, 1r #NMR NM:INSTRUMENT_NAME Bruker 600 MHz NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:FIELD_FREQUENCY_LOCK Deuterium NM:STANDARD_CONCENTRATION 0.5mM DSS NM:SPECTROMETER_FREQUENCY 600 MHz NM:NMR_PROBE 5 mm CPTXI 1H/D-13C/15N Z-GRD Z44866/0026 NM:NMR_SOLVENT Phosphate buffer (pH 7.2) + 2 mM EDTA + 0.5 mM DSS + 0.2% of sodium azide in NM:NMR_SOLVENT deuterated environment NM:NMR_TUBE_SIZE 1.5 mm NMR tube NM:SHIMMING_METHOD Topshim NM:PULSE_SEQUENCE zgig1d NM:WATER_SUPPRESSION none NM:PULSE_WIDTH 90-degree NM:RECEIVER_GAIN 25.4 NM:OFFSET_FREQUENCY 4.77 ppm NM:CHEMICAL_SHIFT_REF_CPD DSS NM:TEMPERATURE 298.2 oK NM:NUMBER_OF_SCANS 32 NM:DUMMY_SCANS 8 NM:ACQUISITION_TIME 1.15s NM:RELAXATION_DELAY 2.85 s NM:SPECTRAL_WIDTH 7142.9 Hz NM:NUM_DATA_POINTS_ACQUIRED 32768 NM:REAL_DATA_POINTS 65536 NM:LINE_BROADENING 0.22 Hz NM:ZERO_FILLING 65,536 points NM:APODIZATION Exponential NM:BASELINE_CORRECTION_METHOD Spline NM:CHEMICAL_SHIFT_REF_STD 0 ppm for DSS #NMR_METABOLITE_DATA NMR_METABOLITE_DATA:UNITS mM NMR_METABOLITE_DATA_START Samples Glucose_EDL_1D_DecON&OFF-1 Glucose_EDL_1D_DecON&OFF-2 Glucose_EDL_1D_DecON&OFF-3 Glucose_EDL_1D_DecON&OFF-4 Glucose_Soleus_1D_DecON&OFF-1 Glucose_Soleus_1D_DecON&OFF-2 Glucose_Soleus_1D_DecON&OFF-3 Glucose_Soleus_1D_DecON&OFF-4 Pyruvate_EDL_1D_DecON&OFF-1 Pyruvate_EDL_1D_DecON&OFF-2 Pyruvate_EDL_1D_DecON&OFF-3 Pyruvate_Soleus_1D_DecON&OFF-1 Pyruvate_Soleus_1D_DecON&OFF-2 Pyruvate_Soleus_1D_DecON&OFF-3 Factors Group:Glucose_EDL Group:Glucose_EDL Group:Glucose_EDL Group:Glucose_EDL Group:Glucose_Soleus Group:Glucose_Soleus Group:Glucose_Soleus Group:Glucose_Soleus Group:Pyruvate_EDL Group:Pyruvate_EDL Group:Pyruvate_EDL Group:Pyruvate_Soleus Group:Pyruvate_Soleus Group:Pyruvate_Soleus Glutamate 0.576 1.638 0.937 0.461 1.961 6.188 3.019 2.088 0.525 0.265 0.675 0.970 0.833 1.327 Lactate 35.414 85.909 43.529 20.094 15.812 53.517 14.314 7.949 39.765 11.259 34.762 16.711 7.172 16.287 Alanine 1.998 3.972 2.033 1.325 2.302 6.123 3.091 1.862 1.966 1.104 1.905 2.309 1.914 2.884 Leucine 0.098 2.715 1.253 0.240 0.119 2.716 1.289 0.247 0.128 0.145 1.347 0.115 0.184 1.563 Isoleucine 0.055 1.512 0.776 0.198 0.088 1.966 0.708 0.223 0.055 0.132 0.891 0.050 0.151 1.055 Valine 0.120 1.500 0.898 0.210 0.124 1.771 0.682 0.241 0.093 0.147 0.824 0.089 0.165 0.985 Aspartate 0.525 4.424 1.977 0.316 0.944 5.629 3.084 0.994 0.455 0.099 2.199 0.347 0.163 2.311 Taurine 48.191 90.697 43.322 36.574 44.451 104.267 41.670 41.453 46.729 33.127 58.302 36.655 31.919 57.577 Creatine 29.064 59.450 31.039 20.280 18.309 49.402 20.105 15.165 27.533 16.977 36.287 15.617 12.104 25.255 ATP/AMP 7.662 12.069 6.417 4.514 4.601 9.587 3.729 3.885 7.482 3.354 4.151 4.254 2.590 6.418 Lactate/Alanine 17.722 21.628 21.415 15.170 6.868 8.741 4.631 4.269 20.228 10.198 1.607 7.238 3.746 2.280 NMR_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Glutamate Lactate Alanine Leucine Isoleucine Valine Aspartate Taurine Creatine ATP/AMP Lactate/Alanine METABOLITES_END #END