#METABOLOMICS WORKBENCH ryant_20201130_110104 DATATRACK_ID:2334 STUDY_ID:ST001615 ANALYSIS_ID:AN002650 PROJECT_ID:PR001039 VERSION 1 CREATED_ON December 3, 2020, 11:07 am #PROJECT PR:PROJECT_TITLE Unique metabolomic profile of skeletal muscle in chronic limb threatening PR:PROJECT_TITLE ischemia PR:PROJECT_TYPE NMR PR:PROJECT_SUMMARY This project is focused on a cross-sectional analysis of non-PAD controls and PR:PROJECT_SUMMARY CLTI patients undergoing either a vascular intervention or undergoing limb PR:PROJECT_SUMMARY amputation was performed and involved a detailed assessment of the limb muscle PR:PROJECT_SUMMARY metabolome using HR-MAS and solution state NMR spectroscopy. It was hypothesized PR:PROJECT_SUMMARY that patients undergoing limb amputation would present with altered muscle PR:PROJECT_SUMMARY metabolite features compared with non-PAD controls. PR:INSTITUTE University of Florida PR:DEPARTMENT Applied Physiology and Kinesiology PR:LABORATORY Rm 42 and Rm 43 PR:LAST_NAME Ryan PR:FIRST_NAME Terence PR:ADDRESS University of Florida, Applied Physiology and Kinesiology, 1864 stadium RD, PR:ADDRESS Gainesville, FL 32611 PR:EMAIL ryant@ufl.edu PR:PHONE 352-294-1700 PR:FUNDING_SOURCE National Institutes of Health and the National Heart, Lung, and Blood, Institute PR:FUNDING_SOURCE numbers R01-HL149704 (to T.E.R.) and R01-HL148597 (to S.T.S.); as well as the PR:FUNDING_SOURCE American Heart Association grant number 18CDA34110044 (to T.E.R.). A portion of PR:FUNDING_SOURCE this work was supported by pilot funds (awarded to T.E.R.) from The University PR:FUNDING_SOURCE of Florida Claude D. Pepper Older Americans Independence Center P30AG028740. (if PR:FUNDING_SOURCE applicable) PR:PROJECT_COMMENTS PAD metabolomic study via NMR. A detailed assessment of the limb muscle PR:PROJECT_COMMENTS metabolome using semi-solid and solution state NMR spectroscopy. PR:PUBLICATIONS Journal of Clinical Medicine PR:CONTRIBUTORS Ram B. Khattri, Kyoungrae Kim, Trace Thome, Zachary R. Salyers, Kerri A. PR:CONTRIBUTORS O’Malley, Scott A. Berceli, Salvatore T. Scali, Terence E. Ryan* #STUDY ST:STUDY_TITLE Unique metabolomic profile of skeletal muscle in chronic limb threatening ST:STUDY_TITLE ischemia (organic phase samples) ST:STUDY_TYPE NMR ST:STUDY_SUMMARY This project is focused on a cross-sectional analysis of non-PAD controls and ST:STUDY_SUMMARY CLTI patients undergoing either a vascular intervention or undergoing limb ST:STUDY_SUMMARY amputation was performed and involved a detailed assessment of the limb muscle ST:STUDY_SUMMARY metabolome using HR-MAS and solution state NMR spectroscopy. It was hypothesized ST:STUDY_SUMMARY that patients undergoing limb amputation would present with altered muscle ST:STUDY_SUMMARY metaolite features compared with non-PAD controls. ST:INSTITUTE University of Florida ST:DEPARTMENT Applied Physiology and Kinesiology ST:LABORATORY Rm 42 and Rm 43 ST:LAST_NAME Ryan ST:FIRST_NAME Terence ST:ADDRESS University of Florida, Applied Physiology and Kinesiology, 1864 stadium RD, ST:ADDRESS Gainesville, FL 32611 ST:EMAIL ryant@ufl.edu ST:PHONE 352-294-1700 ST:NUM_GROUPS 3 ST:TOTAL_SUBJECTS 30 ST:NUM_MALES 25 ST:NUM_FEMALES 5 ST:STUDY_COMMENTS PAD metabolomic study via NMR. A detailed assessment of the limb muscle ST:STUDY_COMMENTS metabolome using semi-solid and solution state NMR spectroscopy ST:PUBLICATIONS Journal of Clinical Medicine #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:AGE_OR_AGE_RANGE 55-82 years SU:GENDER Male and female SU:HUMAN_RACE unknown SU:HUMAN_ETHNICITY unknown SU:HUMAN_MEDICATIONS Asprin, ACE inhibitor, statins, cilostazol SU:HUMAN_SMOKING_STATUS Controls = 4, CLTI Pre-surgery = 7, and CLTI Amputation = 9 #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 - CLTI_Amputation1a Group:CLTI_Amputation RAW_FILE_NAME=CLTI_Amputation1a.raw SUBJECT_SAMPLE_FACTORS - CLTI_Amputation2a Group:CLTI_Amputation RAW_FILE_NAME=CLTI_Amputation2a.raw SUBJECT_SAMPLE_FACTORS - CLTI_Amputation3a Group:CLTI_Amputation RAW_FILE_NAME=CLTI_Amputation3a.raw SUBJECT_SAMPLE_FACTORS - CLTI_Amputation4a Group:CLTI_Amputation RAW_FILE_NAME=CLTI_Amputation4a.raw SUBJECT_SAMPLE_FACTORS - CLTI_Amputation5a Group:CLTI_Amputation RAW_FILE_NAME=CLTI_Amputation5a.raw SUBJECT_SAMPLE_FACTORS - CLTI_Amputation6a Group:CLTI_Amputation RAW_FILE_NAME=CLTI_Amputation6a.raw SUBJECT_SAMPLE_FACTORS - CLTI_Amputation7a Group:CLTI_Amputation RAW_FILE_NAME=CLTI_Amputation7a.raw SUBJECT_SAMPLE_FACTORS - CLTI_Amputation8a Group:CLTI_Amputation RAW_FILE_NAME=CLTI_Amputation8a.raw SUBJECT_SAMPLE_FACTORS - CLTI_Amputation9a Group:CLTI_Amputation RAW_FILE_NAME=CLTI_Amputation9a.raw SUBJECT_SAMPLE_FACTORS - CLTI_Amputation10a Group:CLTI_Amputation RAW_FILE_NAME=CLTI_Amputation10a.raw SUBJECT_SAMPLE_FACTORS - CLTI_Pre_surgery1a Group:CLTI_Pre_surgery RAW_FILE_NAME=CLTI_Pre_surgery1a.raw SUBJECT_SAMPLE_FACTORS - CLTI_Pre_surgery2a Group:CLTI_Pre_surgery RAW_FILE_NAME=CLTI_Pre_surgery2a.raw SUBJECT_SAMPLE_FACTORS - CLTI_Pre_surgery5a Group:CLTI_Pre_surgery RAW_FILE_NAME=CLTI_Pre_surgery5a.raw SUBJECT_SAMPLE_FACTORS - CLTI_Pre_surgery6a Group:CLTI_Pre_surgery RAW_FILE_NAME=CLTI_Pre_surgery6a.raw SUBJECT_SAMPLE_FACTORS - CLTI_Pre_surgery7a Group:CLTI_Pre_surgery RAW_FILE_NAME=CLTI_Pre_surgery7a.raw SUBJECT_SAMPLE_FACTORS - CLTI_Pre_surgery9a Group:CLTI_Pre_surgery RAW_FILE_NAME=CLTI_Pre_surgery9a.raw SUBJECT_SAMPLE_FACTORS - Control2a Group:Control RAW_FILE_NAME=Control2a.raw SUBJECT_SAMPLE_FACTORS - Control3a Group:Control RAW_FILE_NAME=Control3a.raw SUBJECT_SAMPLE_FACTORS - Control4a Group:Control RAW_FILE_NAME=Control4a.raw SUBJECT_SAMPLE_FACTORS - Control5a Group:Control RAW_FILE_NAME=Control5a.raw SUBJECT_SAMPLE_FACTORS - Control6a Group:Control RAW_FILE_NAME=Control6a.raw SUBJECT_SAMPLE_FACTORS - Control7a Group:Control RAW_FILE_NAME=Control7a.raw SUBJECT_SAMPLE_FACTORS - Control8a Group:Control RAW_FILE_NAME=Control8a.raw SUBJECT_SAMPLE_FACTORS - Control10a Group:Control RAW_FILE_NAME=Control10a.raw #COLLECTION CO:COLLECTION_SUMMARY Gastrocnemius muscle specimens were obtained from ten older adult non-PAD CO:COLLECTION_SUMMARY controls (Control), ten patients with critical limb ischemia (CLI) undergoing CO:COLLECTION_SUMMARY surgical intervention (CLTI Pre-surgery), and ten CLI patients undergoing limb CO:COLLECTION_SUMMARY amputation (CLTI Amputation). Five pre-surgery patients underwent bypass CO:COLLECTION_SUMMARY interventions and five underwent endovascular procedures. Muscle specimens were CO:COLLECTION_SUMMARY collected within the confines of the operating rooms (CLI patients) or via CO:COLLECTION_SUMMARY percutaneous muscle biopsy using sterile procedures. A portion of the muscle was CO:COLLECTION_SUMMARY quickly trimmed of fat/connective tissue and snap frozen in liquid nitrogen for CO:COLLECTION_SUMMARY metabolomics analysis. CO:SAMPLE_TYPE Muscle CO:COLLECTION_METHOD Muscle specimens were collected within the confines of the operating rooms (CLI CO:COLLECTION_METHOD patients) or via percutaneous muscle biopsy using sterile procedures. A portion CO:COLLECTION_METHOD of the muscle was quickly trimmed of fat/connective tissue and snap frozen in CO:COLLECTION_METHOD liquid nitrogen for metabolomics analysis. CO:STORAGE_CONDITIONS -80℃ CO:STORAGE_VIALS cryovials #TREATMENT TR:TREATMENT_SUMMARY This was a prospective cohort study that examined the metabolomic profile of TR:TREATMENT_SUMMARY skeletal muscle from CLTI patients undergoing surgerical intervention or TR:TREATMENT_SUMMARY amputation, as well as a cohort of non-PAD controls. CLTI patients exhibited TR:TREATMENT_SUMMARY severe symptomology (Rutherford Classification 3-6), with high incidence of TR:TREATMENT_SUMMARY common PAD risk factors including hypertension, hyperlipidemia, coronary artery TR:TREATMENT_SUMMARY disease, and diabetes.Ten patients with critical limb ischemia (CLI) undergoing TR:TREATMENT_SUMMARY surgical intervention (CLTI Pre-surgery), and ten CLI patients undergoing limb TR:TREATMENT_SUMMARY amputation (CLTI Amputation). Five pre-surgery patients underwent bypass TR:TREATMENT_SUMMARY interventions and five underwent endovascular procedures. Muscle specimens were TR:TREATMENT_SUMMARY collected within the confines of the operating rooms (CLI patients) or via TR:TREATMENT_SUMMARY percutaneous muscle biopsy using sterile procedures. TR:HUMAN_FASTING non-fasted TR:HUMAN_ENDP_CLINICAL_SIGNS N/A #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Extraction. Both polar and non-polar metabolites were extracted from the SP:SAMPLEPREP_SUMMARY gastrocnemius muscle specimens using FOLCH extraction. In brief, wet weigh of SP:SAMPLEPREP_SUMMARY the frozen tissues were determined and immediately homogenized in 1 mL of SP:SAMPLEPREP_SUMMARY ice-cold methanol using a PowerLyzer 24 Homogenizer (QIAGEN Group, Hilden, SP:SAMPLEPREP_SUMMARY Germany). All enzymatic activities are halted once the sample was homogenized in SP:SAMPLEPREP_SUMMARY the methanol. Homogenization was followed by centrifugation (13.2K r.p.m., 4 oC, SP:SAMPLEPREP_SUMMARY 30 minutes) and supernatant was transferred into a new glass vial consisting a SP:SAMPLEPREP_SUMMARY mixture of 3 mL of ice-cold chloroform and methanol (2:1 v/v) ratio. The cold SP:SAMPLEPREP_SUMMARY mixture was vortexed for several minutes and left in an ice bath 15 minutes to SP:SAMPLEPREP_SUMMARY allow for phase separation. Next, 1 mL of ice-cold 0.9% saline was added to the SP:SAMPLEPREP_SUMMARY mixture followed by vigorous mixing. The mixture was again left in the ice bath SP:SAMPLEPREP_SUMMARY for 45 minutes for phase separation. The upper methanol/water layer was SP:SAMPLEPREP_SUMMARY transferred to a new falcon tube. To the lower chloroform layer, 1 mL of SP:SAMPLEPREP_SUMMARY ice-cold 0.9% saline was added and all steps were followed as mentioned above. SP:SAMPLEPREP_SUMMARY Following a 45-minute incubation, upper methanol/water layer was again SP:SAMPLEPREP_SUMMARY transferred to the previous falcon tube and dried using a Labconco freeze drier SP:SAMPLEPREP_SUMMARY (Labconco Corporation, MO, USA). The chloroform layer was dried under a stream SP:SAMPLEPREP_SUMMARY of nitrogen gas. The dried samples (both aqueous and organic phases) were stored SP:SAMPLEPREP_SUMMARY at -80 oC until resuspension for NMR experiments. Lyophilized organic phase SP:SAMPLEPREP_SUMMARY sample were re-suspended in 80 µL of CDCl3 along with 10 mM of pyrazine. All SP:SAMPLEPREP_SUMMARY samples were loaded in 1.7 mm NMR tube to acquire spectra. SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION Deuterated chloroform (80 microliter) with 10 mM pyrazine was used to re-suspend SP:SAMPLE_RESUSPENSION organic phase samples. SP:SAMPLE_SPIKING 10 mM of pyrazine for organic phase samples. #ANALYSIS AN:DATA_FORMAT fid, 1r #NMR NM:INSTRUMENT_NAME Bruker Avance Neo 600 MHz/54mm console NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:FIELD_FREQUENCY_LOCK Deuterium NM:STANDARD_CONCENTRATION 10mM pyrazine NM:SPECTROMETER_FREQUENCY 600.2328273 MHz NM:NMR_PROBE 1.7 mm TXI CryoProbe NM:NMR_SOLVENT Deuterated chloroform NM:NMR_TUBE_SIZE 1.7 mm O.D. NM:SHIMMING_METHOD Topshim NM:PULSE_SEQUENCE noesypr1d NM:WATER_SUPPRESSION none NM:PULSE_WIDTH 90-degree NM:RECEIVER_GAIN 101 NM:CHEMICAL_SHIFT_REF_CPD CDCl3 at 7.26 ppm and pyrazine at 8.61 ppm NM:TEMPERATURE 25 o C NM:NUMBER_OF_SCANS 128 scans NM:DUMMY_SCANS 8 NM:ACQUISITION_TIME 4 s NM:RELAXATION_DELAY 1 s NM:SPECTRAL_WIDTH 7142.9 Hz NM:NUM_DATA_POINTS_ACQUIRED 28571 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 7.26ppm for CDCl3 #NMR_METABOLITE_DATA NMR_METABOLITE_DATA:UNITS A.U. NMR_METABOLITE_DATA_START Samples CLTI_Amputation1a CLTI_Amputation2a CLTI_Amputation3a CLTI_Amputation4a CLTI_Amputation5a CLTI_Amputation6a CLTI_Amputation7a CLTI_Amputation8a CLTI_Amputation9a CLTI_Amputation10a CLTI_Pre_surgery1a CLTI_Pre_surgery2a CLTI_Pre_surgery5a CLTI_Pre_surgery6a CLTI_Pre_surgery7a CLTI_Pre_surgery9a Control2a Control3a Control4a Control5a Control6a Control7a Control8a Control10a Factors Group:CLTI_Amputation Group:CLTI_Amputation Group:CLTI_Amputation Group:CLTI_Amputation Group:CLTI_Amputation Group:CLTI_Amputation Group:CLTI_Amputation Group:CLTI_Amputation Group:CLTI_Amputation Group:CLTI_Amputation Group:CLTI_Pre_surgery Group:CLTI_Pre_surgery Group:CLTI_Pre_surgery Group:CLTI_Pre_surgery Group:CLTI_Pre_surgery Group:CLTI_Pre_surgery Group:Control Group:Control Group:Control Group:Control Group:Control Group:Control Group:Control Group:Control C18-CH3 chol+V26C1C1:W25 2.714041568 2.505950482 3.319406373 3.188474262 2.704978038 3.074472658 2.797740242 3.710180502 1.607834751 3.06792606 2.244804577 2.363162949 2.543305963 3.185292817 3.437542525 2.353380253 2.238660449 1.708933109 1.918673926 1.899426647 2.096208669 2.740330267 1.507798502 2.420962339 CH3-protons 368.8836615 374.2847304 343.5151808 349.9381038 141.7790904 531.5804148 399.7925022 228.7786528 92.31654578 646.0032584 737.500607 620.9208463 244.5379919 181.221034 380.5336032 282.4544822 300.8408581 76.95000272 170.7987152 142.2055761 135.3437887 152.4311938 282.3657315 123.61555 C19-CH3 Chol 10.14325014 10.01120228 10.56855559 9.280217434 3.840617024 13.30996851 9.378840179 8.112021443 2.202381707 17.93696818 17.83191403 13.89713123 3.846828281 4.172201081 7.430344221 6.581953649 9.013446641 2.177849695 2.668400507 2.464515706 2.832968864 3.761590142 7.100257967 3.496630225 CH2n chol 294.3170626 322.0096512 272.252103 352.9859101 218.6316986 428.0540119 426.7218723 230.1589942 166.3593249 483.0326103 589.3042625 461.8404083 438.1115566 355.5962827 526.8400508 292.556382 248.475611 141.1412704 296.8791359 263.0163829 239.8529979 287.3815193 269.7529788 220.7052698 CH2n aliphatic chains 481.0012761 547.5080907 437.0304092 616.2762498 480.8736536 1039.03141 832.341339 385.6210388 335.4144958 1008.396513 1212.197206 1243.019287 924.1961482 657.2446763 1170.000538 681.3266816 358.3928383 267.3022409 688.4987584 558.8119361 474.3776902 550.6272985 474.8330179 373.4883774 Cholesterol2 9.26262472 10.46965538 8.993140078 8.213110917 1.712339595 14.60546646 9.348651905 5.743567038 1.393150366 20.38148244 20.83061253 15.59156922 2.728164122 1.423576825 6.173155702 6.812621635 9.620999484 1.169062267 2.046295631 1.590609454 1.309533849 2.006062275 8.59012663 2.259324337 CH2-CH2-COO-beta 106.009607 116.6087043 103.9634412 123.2977231 78.7831896 196.0326217 157.3264891 76.75550045 53.51290745 211.5848809 263.9079369 311.7415002 142.093278 105.4857808 189.6560301 122.0744132 86.81871147 41.15341228 102.2002449 88.47092822 72.41272095 88.57455851 100.9151986 64.28426614 CH2-CH=CH-CH2-alpha 133.052001 116.3706745 80.37788313 111.7766414 118.0339775 212.5026439 158.5594274 142.6658711 77.68090613 194.4958609 219.333571 239.0576202 178.5321206 153.6053034 373.7044027 142.5426762 99.41359267 84.11113662 181.4050808 113.3021647 109.5122154 259.522595 88.56229495 82.54831242 CH2COO-alpha 48.04342649 59.46530258 43.44530649 70.46807876 62.72420074 120.1273219 99.55880354 43.18736952 41.78689341 107.3363747 138.8650152 146.2157202 116.5152879 88.90023806 155.1940422 76.57018604 30.85121705 31.15852467 86.40526673 71.929937 57.42699188 67.28055624 50.50922606 43.10692086 CH-CH2-CH=CH 32.16559249 31.77504401 24.73504449 28.36325651 30.08357866 60.42234718 43.06633571 28.64054329 20.96239035 53.47266532 50.24823766 51.21265244 39.79368384 40.13105102 63.05616756 36.16399224 32.21671677 20.89259914 39.77981468 27.17728665 26.74992265 39.47632093 27.16106406 30.67306857 N+CH33 17.73764039 10.84874206 6.941277351 10.14602645 3.581055335 12.80292097 16.69603471 17.73186378 10.36697413 18.81011611 6.232727864 3.195145777 13.19153849 15.93263325 16.39629368 14.08852186 17.95796467 7.358125085 10.84225729 0.345419149 13.83186663 13.86319731 5.437822063 15.96011342 Cholesterol3 2.077118487 1.366053447 0.483969354 0.924178565 3.47782494 0.606684053 3.995929359 3.246006421 1.441639936 1.706575916 1.71005134 0.578939535 1.587894105 2.238353569 1.412122147 0.339591817 1.076736785 0.907436231 0.563355704 0.441398184 0.062591145 4.803650849 1.747908709 1.498989899 3CH2 glycerophospholipids 10.21870662 7.639336432 4.648316269 6.734740177 5.077463228 5.792214789 10.34535206 9.848529984 6.272037754 9.067953294 2.022102022 1.436413655 5.020714061 9.560796631 10.59644692 7.243032036 12.3015915 5.853662157 7.843017207 2.758890792 7.827804542 11.7122127 5.234660974 9.790812169 CH2_TG1 15.96444416 19.10765378 13.39464446 22.4489794 21.26283368 39.08513536 34.89396913 15.31329752 14.46887051 35.50512225 44.8833641 47.91093633 37.50251072 30.07413781 49.67939109 24.05536085 9.319265509 9.065159241 26.39779145 22.36659325 17.74155177 24.19432603 18.03168792 15.15817628 CH2_TG2 16.33432251 18.40078541 12.85923706 21.33873591 16.3894927 31.51402901 27.47415835 13.14509987 12.17350639 28.1922278 41.28882646 45.1343581 34.53404825 25.42492231 49.44839263 23.32200466 7.46195952 9.180144394 26.27109417 21.56176859 19.26245007 17.74837085 12.70242217 11.92523473 1CH phospholipids+TG 4.20554211 3.225017168 1.984972041 2.622649235 0.865468791 0.258241001 2.198916173 2.009079946 1.291898695 1.513756952 2.998600749 2.164251238 3.165311039 1.795262363 4.435622008 2.923569654 4.878605345 1.864423371 2.747682779 1.121792524 2.333438797 2.112047809 1.416635068 2.096994302 CH phospholipid 3.920071327 2.671951217 2.062125046 2.469215091 0.542658997 0.362693274 0.851326657 1.697583439 1.46447666 1.143507574 1.528868527 1.014665088 3.138856393 1.912474676 4.73334409 3.195408492 4.555699942 2.172526755 3.424698059 1.679915588 3.156282331 2.942829764 0.905218213 2.67929626 CH TG 6.718454183 8.236728237 5.94418048 10.22785735 9.16246456 17.23915153 13.65915945 4.879001989 5.738506174 14.32298616 20.64487653 23.18523326 17.83348322 13.24803113 23.90190921 11.04920773 2.4998946 4.059844574 12.71547139 11.30923045 8.535500592 9.143773809 6.578462187 5.391403724 CH=CH 65.92872587 70.08734594 56.31030349 75.43886986 68.98144336 142.2768693 100.9708586 56.0517196 49.99474763 120.3394215 135.0359894 154.9569476 117.814748 99.17942742 167.0881909 96.70153136 56.06493789 44.2843357 101.657178 76.61632426 70.95772079 77.67132139 61.26816156 61.8154775 Aromatic protons1 0.361886917 0.36777323 0.198390126 0.273512745 0.078490979 0.142355291 0.507886885 0.175110984 0.163274425 0.387888217 0.345068487 0.438715848 0.770492446 0.206377597 0.22273964 0.445408414 0.28088295 1.082006723 0.520190081 0.336642018 0.341297238 3.04923615 0.675543015 0.394333963 Aromatic protons2 2.781696362 3.463287722 1.991516212 2.449058548 0.029040623 2.67623299 2.207985184 1.959438273 0.132193675 5.310349767 3.753276716 4.169100807 0.334636033 0.040677396 2.083612572 2.232269716 2.812469655 0.078211386 0.064621037 0.048902917 0.004626087 0.046774765 2.414274405 0.061170452 NMR_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name C18-CH3 chol+V26C1C1:W25 CH3-protons C19-CH3 Chol CH2n chol CH2n aliphatic chains Cholesterol2 CH2-CH2-COO-beta CH2-CH=CH-CH2-alpha CH2COO-alpha CH-CH2-CH=CH N+CH33 Cholesterol3 3CH2 glycerophospholipids CH2_TG1 CH2_TG2 1CH phospholipids+TG CH phospholipid CH TG CH=CH Aromatic protons1 Aromatic protons2 METABOLITES_END #END