#METABOLOMICS WORKBENCH jkempa_20210119_173517 DATATRACK_ID:2402 STUDY_ID:ST001667 ANALYSIS_ID:AN002719 VERSION 1 CREATED_ON 12-15-2022 #PROJECT PR:PROJECT_TITLE NMR and MS Metabolomics Reveal a Distinct Metabolic State of Ricin-induced PR:PROJECT_TITLE Hypoglycemia PR:PROJECT_TYPE Targeted NMR and Untargeted MS PR:PROJECT_SUMMARY Ricin toxin is a ribosome inactivating protein. Due to its toxic and chemical PR:PROJECT_SUMMARY properties, ricin is a potential agent of bioterrorism but has also been studied PR:PROJECT_SUMMARY for therapeutic use in immunotoxins. Previous research by our group has PR:PROJECT_SUMMARY demonstrated lethal hypoglycemia associated with ricin toxicity. Research PR:PROJECT_SUMMARY efforts have focused on better understanding this ricin-induced metabolic state PR:PROJECT_SUMMARY of hypoglycemia to aid in better understanding the systemic effects of PR:PROJECT_SUMMARY hypoglycemia, and the use of ricin in immunotherapy. Here, we have used a mouse PR:PROJECT_SUMMARY model to characterize liver metabolome changes associated with hypoglycemia PR:PROJECT_SUMMARY induced by two conditions. Mice were challenged with intraperitoneal injections PR:PROJECT_SUMMARY of ricin at high and low doses for 2 hrs, 8 hrs, and overnight. To understand PR:PROJECT_SUMMARY how ricin-induced hypoglycemia differs from that of fasting, another group of PR:PROJECT_SUMMARY mice had food withheld for 8-hours and overnight. 1H NMR-based metabolomics was PR:PROJECT_SUMMARY performed on polar molecules extracted from mouse livers, with metabolites PR:PROJECT_SUMMARY annotated using Chenomx software. MetaboAnalyst was employed for multivariate PR:PROJECT_SUMMARY statistical analysis. In this study, similar decreases in blood glucose in mice PR:PROJECT_SUMMARY were observed following injection of a lethal dose of ricin and with overnight PR:PROJECT_SUMMARY fasting. NMR analyses identified 59 polar metabolites present in mice livers PR:PROJECT_SUMMARY from all treatments. Multivariate statistical analyses were used to evaluate PR:PROJECT_SUMMARY global metabolic state differences. Results from these analyses indicated that PR:PROJECT_SUMMARY the profiled liver metabolomes for mice subjected to the two conditions differ PR:PROJECT_SUMMARY significantly at both 8 hours and overnight. Additionally, ricin treatment with PR:PROJECT_SUMMARY a lethal dose reveal a progression of metabolic changes over time, from 2 to 22 PR:PROJECT_SUMMARY hours. Additionally, mass spectrometry supported these findings, and NMR PR:PROJECT_SUMMARY analyses revealed key metabolites that contribute to these differences. While PR:PROJECT_SUMMARY both ricin and fasting induce hypoglycemia, the metabolic states resulting from PR:PROJECT_SUMMARY these two conditions are different. Further analyses may give insights into PR:PROJECT_SUMMARY mechanisms of ricin toxicity, specific metabolic pathways that are altered, and PR:PROJECT_SUMMARY potential treatments for hypoglycemia. We propose to extend these studies to PR:PROJECT_SUMMARY insulin-induced hypoglycemia. PR:INSTITUTE Montana State University PR:DEPARTMENT Chemistry & Biochemistry PR:LABORATORY Copié PR:LAST_NAME Kempa PR:FIRST_NAME Jake PR:ADDRESS 103 Chemistry and Biochemistry Building, Bozeman, Montana, 59717, USA PR:EMAIL jkempa97@gmail.com PR:PHONE 4067997200 PR:DOI http://dx.doi.org/10.21228/M8NM4H #STUDY ST:STUDY_TITLE LC-MS metabolomics analysis of ricin-induced and fasting hypoglycemia (part-II) ST:STUDY_SUMMARY Mice were subjected to ricin exposure or fasting conditions for 2 hours, 8 ST:STUDY_SUMMARY hours, or an overnight period. Following treatment, livers were removed and ST:STUDY_SUMMARY metabolites were extracted and analyzed by LC-MS. ST:INSTITUTE Montana State University ST:LAST_NAME Kempa ST:FIRST_NAME Jake ST:ADDRESS 103 Chemistry and Biochemistry Building, Bozeman, Montana, 59717, USA ST:EMAIL jkempa97@gmail.com ST:PHONE 4067997200 ST:SUBMIT_DATE 2021-01-19 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 1--1 Treatment:Control | Timepoint:Overnight RAW_FILE_NAME=1--1 SUBJECT_SAMPLE_FACTORS - 1--10 Treatment:Control | Timepoint:Overnight RAW_FILE_NAME=1--10 SUBJECT_SAMPLE_FACTORS - 1--11 Treatment:Control | Timepoint:Overnight RAW_FILE_NAME=1--11 SUBJECT_SAMPLE_FACTORS - 1--12 Treatment:Control | Timepoint:Overnight RAW_FILE_NAME=1--12 SUBJECT_SAMPLE_FACTORS - 1--13 Treatment:Control | Timepoint:Overnight RAW_FILE_NAME=1--13 SUBJECT_SAMPLE_FACTORS - 1--14 Treatment:Control | Timepoint:Overnight RAW_FILE_NAME=1--14 SUBJECT_SAMPLE_FACTORS - 1--15 Treatment:Control | Timepoint:Overnight RAW_FILE_NAME=1--15 SUBJECT_SAMPLE_FACTORS - 1--16 Treatment:Control | Timepoint:Overnight RAW_FILE_NAME=1--16 SUBJECT_SAMPLE_FACTORS - 1--2 Treatment:Control | Timepoint:Overnight RAW_FILE_NAME=1--2 SUBJECT_SAMPLE_FACTORS - 1--3 Treatment:Control | Timepoint:Overnight RAW_FILE_NAME=1--3 SUBJECT_SAMPLE_FACTORS - 1--4 Treatment:Control | Timepoint:Overnight RAW_FILE_NAME=1--4 SUBJECT_SAMPLE_FACTORS - 1--5 Treatment:Control | Timepoint:Overnight RAW_FILE_NAME=1--5 SUBJECT_SAMPLE_FACTORS - 1--6 Treatment:Control | Timepoint:Overnight RAW_FILE_NAME=1--6 SUBJECT_SAMPLE_FACTORS - 1--7 Treatment:Control | Timepoint:Overnight RAW_FILE_NAME=1--7 SUBJECT_SAMPLE_FACTORS - 1--8 Treatment:Control | Timepoint:Overnight RAW_FILE_NAME=1--8 SUBJECT_SAMPLE_FACTORS - 1--9 Treatment:Control | Timepoint:Overnight RAW_FILE_NAME=1--9 SUBJECT_SAMPLE_FACTORS - 7--1 Treatment:Fast | Timepoint:8h RAW_FILE_NAME=7--1 SUBJECT_SAMPLE_FACTORS - 7--10 Treatment:Fast | Timepoint:8h RAW_FILE_NAME=7--10 SUBJECT_SAMPLE_FACTORS - 7--2 Treatment:Fast | Timepoint:8h RAW_FILE_NAME=7--2 SUBJECT_SAMPLE_FACTORS - 7--3 Treatment:Fast | Timepoint:8h RAW_FILE_NAME=7--3 SUBJECT_SAMPLE_FACTORS - 7--4 Treatment:Fast | Timepoint:8h RAW_FILE_NAME=7--4 SUBJECT_SAMPLE_FACTORS - 7--5 Treatment:Fast | Timepoint:8h RAW_FILE_NAME=7--5 SUBJECT_SAMPLE_FACTORS - 7--6 Treatment:Fast | Timepoint:8h RAW_FILE_NAME=7--6 SUBJECT_SAMPLE_FACTORS - 7--7 Treatment:Fast | Timepoint:8h RAW_FILE_NAME=7--7 SUBJECT_SAMPLE_FACTORS - 7--8 Treatment:Fast | Timepoint:8h RAW_FILE_NAME=7--8 SUBJECT_SAMPLE_FACTORS - 7--9 Treatment:Fast | Timepoint:8h RAW_FILE_NAME=7--9 SUBJECT_SAMPLE_FACTORS - 2--1 Treatment:Fast | Timepoint:Overnight RAW_FILE_NAME=2--1 SUBJECT_SAMPLE_FACTORS - 2--10 Treatment:Fast | Timepoint:Overnight RAW_FILE_NAME=2--10 SUBJECT_SAMPLE_FACTORS - 2--11 Treatment:Fast | Timepoint:Overnight RAW_FILE_NAME=2--11 SUBJECT_SAMPLE_FACTORS - 2--12 Treatment:Fast | Timepoint:Overnight RAW_FILE_NAME=2--12 SUBJECT_SAMPLE_FACTORS - 2--13 Treatment:Fast | Timepoint:Overnight RAW_FILE_NAME=2--13 SUBJECT_SAMPLE_FACTORS - 2--14 Treatment:Fast | Timepoint:Overnight RAW_FILE_NAME=2--14 SUBJECT_SAMPLE_FACTORS - 2--15 Treatment:Fast | Timepoint:Overnight RAW_FILE_NAME=2--15 SUBJECT_SAMPLE_FACTORS - 2--16 Treatment:Fast | Timepoint:Overnight RAW_FILE_NAME=2--16 SUBJECT_SAMPLE_FACTORS - 2--2 Treatment:Fast | Timepoint:Overnight RAW_FILE_NAME=2--2 SUBJECT_SAMPLE_FACTORS - 2--3 Treatment:Fast | Timepoint:Overnight RAW_FILE_NAME=2--3 SUBJECT_SAMPLE_FACTORS - 2--4 Treatment:Fast | Timepoint:Overnight RAW_FILE_NAME=2--4 SUBJECT_SAMPLE_FACTORS - 2--5 Treatment:Fast | Timepoint:Overnight RAW_FILE_NAME=2--5 SUBJECT_SAMPLE_FACTORS - 2--6 Treatment:Fast | Timepoint:Overnight RAW_FILE_NAME=2--6 SUBJECT_SAMPLE_FACTORS - 2--7 Treatment:Fast | Timepoint:Overnight RAW_FILE_NAME=2--7 SUBJECT_SAMPLE_FACTORS - 2--8 Treatment:Fast | Timepoint:Overnight RAW_FILE_NAME=2--8 SUBJECT_SAMPLE_FACTORS - 2--9 Treatment:Fast | Timepoint:Overnight RAW_FILE_NAME=2--9 SUBJECT_SAMPLE_FACTORS - 5--1 Treatment:High Dose Ricin | Timepoint:2h RAW_FILE_NAME=5--1 SUBJECT_SAMPLE_FACTORS - 5--10 Treatment:High Dose Ricin | Timepoint:2h RAW_FILE_NAME=5--10 SUBJECT_SAMPLE_FACTORS - 5--2 Treatment:High Dose Ricin | Timepoint:2h RAW_FILE_NAME=5--2 SUBJECT_SAMPLE_FACTORS - 5--3 Treatment:High Dose Ricin | Timepoint:2h RAW_FILE_NAME=5--3 SUBJECT_SAMPLE_FACTORS - 5--4 Treatment:High Dose Ricin | Timepoint:2h RAW_FILE_NAME=5--4 SUBJECT_SAMPLE_FACTORS - 5--5 Treatment:High Dose Ricin | Timepoint:2h RAW_FILE_NAME=5--5 SUBJECT_SAMPLE_FACTORS - 5--6 Treatment:High Dose Ricin | Timepoint:2h RAW_FILE_NAME=5--6 SUBJECT_SAMPLE_FACTORS - 5--7 Treatment:High Dose Ricin | Timepoint:2h RAW_FILE_NAME=5--7 SUBJECT_SAMPLE_FACTORS - 5--8 Treatment:High Dose Ricin | Timepoint:2h RAW_FILE_NAME=5--8 SUBJECT_SAMPLE_FACTORS - 5--9 Treatment:High Dose Ricin | Timepoint:2h RAW_FILE_NAME=5--9 SUBJECT_SAMPLE_FACTORS - 9--1 Treatment:High Dose Ricin | Timepoint:8h RAW_FILE_NAME=9--1 SUBJECT_SAMPLE_FACTORS - 9--10 Treatment:High Dose Ricin | Timepoint:8h RAW_FILE_NAME=9--10 SUBJECT_SAMPLE_FACTORS - 9--2 Treatment:High Dose Ricin | Timepoint:8h RAW_FILE_NAME=9--2 SUBJECT_SAMPLE_FACTORS - 9--3 Treatment:High Dose Ricin | Timepoint:8h RAW_FILE_NAME=9--3 SUBJECT_SAMPLE_FACTORS - 9--4 Treatment:High Dose Ricin | Timepoint:8h RAW_FILE_NAME=9--4 SUBJECT_SAMPLE_FACTORS - 9--5 Treatment:High Dose Ricin | Timepoint:8h RAW_FILE_NAME=9--5 SUBJECT_SAMPLE_FACTORS - 9--6 Treatment:High Dose Ricin | Timepoint:8h RAW_FILE_NAME=9--6 SUBJECT_SAMPLE_FACTORS - 9--7 Treatment:High Dose Ricin | Timepoint:8h RAW_FILE_NAME=9--7 SUBJECT_SAMPLE_FACTORS - 9--8 Treatment:High Dose Ricin | Timepoint:8h RAW_FILE_NAME=9--8 SUBJECT_SAMPLE_FACTORS - 9--9 Treatment:High Dose Ricin | Timepoint:8h RAW_FILE_NAME=9--9 SUBJECT_SAMPLE_FACTORS - 6--10 Treatment:High Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=6--10 SUBJECT_SAMPLE_FACTORS - 6--11 Treatment:High Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=6--11 SUBJECT_SAMPLE_FACTORS - 6--12 Treatment:High Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=6--12 SUBJECT_SAMPLE_FACTORS - 6--13 Treatment:High Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=6--13 SUBJECT_SAMPLE_FACTORS - 6--14 Treatment:High Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=6--14 SUBJECT_SAMPLE_FACTORS - 6--15 Treatment:High Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=6--15 SUBJECT_SAMPLE_FACTORS - 6--16 Treatment:High Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=6--16 SUBJECT_SAMPLE_FACTORS - 6--17 Treatment:High Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=6--17 SUBJECT_SAMPLE_FACTORS - 6--18 Treatment:High Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=6--18 SUBJECT_SAMPLE_FACTORS - 6--19 Treatment:High Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=6--19 SUBJECT_SAMPLE_FACTORS - 6--20 Treatment:High Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=6--20 SUBJECT_SAMPLE_FACTORS - 6--3 Treatment:High Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=6--3 SUBJECT_SAMPLE_FACTORS - 6--4 Treatment:High Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=6--4 SUBJECT_SAMPLE_FACTORS - 6--5 Treatment:High Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=6--5 SUBJECT_SAMPLE_FACTORS - 6--6 Treatment:High Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=6--6 SUBJECT_SAMPLE_FACTORS - 6--7 Treatment:High Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=6--7 SUBJECT_SAMPLE_FACTORS - 6--8 Treatment:High Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=6--8 SUBJECT_SAMPLE_FACTORS - 3--1 Treatment:Low Dose Ricin | Timepoint:2h RAW_FILE_NAME=3--1 SUBJECT_SAMPLE_FACTORS - 3--10 Treatment:Low Dose Ricin | Timepoint:2h RAW_FILE_NAME=3--10 SUBJECT_SAMPLE_FACTORS - 3--2 Treatment:Low Dose Ricin | Timepoint:2h RAW_FILE_NAME=3--2 SUBJECT_SAMPLE_FACTORS - 3--3 Treatment:Low Dose Ricin | Timepoint:2h RAW_FILE_NAME=3--3 SUBJECT_SAMPLE_FACTORS - 3--4 Treatment:Low Dose Ricin | Timepoint:2h RAW_FILE_NAME=3--4 SUBJECT_SAMPLE_FACTORS - 3--5 Treatment:Low Dose Ricin | Timepoint:2h RAW_FILE_NAME=3--5 SUBJECT_SAMPLE_FACTORS - 3--6 Treatment:Low Dose Ricin | Timepoint:2h RAW_FILE_NAME=3--6 SUBJECT_SAMPLE_FACTORS - 3--7 Treatment:Low Dose Ricin | Timepoint:2h RAW_FILE_NAME=3--7 SUBJECT_SAMPLE_FACTORS - 3--8 Treatment:Low Dose Ricin | Timepoint:2h RAW_FILE_NAME=3--8 SUBJECT_SAMPLE_FACTORS - 3--9 Treatment:Low Dose Ricin | Timepoint:2h RAW_FILE_NAME=3--9 SUBJECT_SAMPLE_FACTORS - 8--1 Treatment:Low Dose Ricin | Timepoint:8h RAW_FILE_NAME=8--1 SUBJECT_SAMPLE_FACTORS - 8--2 Treatment:Low Dose Ricin | Timepoint:8h RAW_FILE_NAME=8--2 SUBJECT_SAMPLE_FACTORS - 8--3 Treatment:Low Dose Ricin | Timepoint:8h RAW_FILE_NAME=8--3 SUBJECT_SAMPLE_FACTORS - 8--4 Treatment:Low Dose Ricin | Timepoint:8h RAW_FILE_NAME=8--4 SUBJECT_SAMPLE_FACTORS - 8--5 Treatment:Low Dose Ricin | Timepoint:8h RAW_FILE_NAME=8--5 SUBJECT_SAMPLE_FACTORS - 8--6 Treatment:Low Dose Ricin | Timepoint:8h RAW_FILE_NAME=8--6 SUBJECT_SAMPLE_FACTORS - 8--7 Treatment:Low Dose Ricin | Timepoint:8h RAW_FILE_NAME=8--7 SUBJECT_SAMPLE_FACTORS - 8--8 Treatment:Low Dose Ricin | Timepoint:8h RAW_FILE_NAME=8--8 SUBJECT_SAMPLE_FACTORS - 8--9 Treatment:Low Dose Ricin | Timepoint:8h RAW_FILE_NAME=8--9 SUBJECT_SAMPLE_FACTORS - 4--1 Treatment:Low Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=4--1 SUBJECT_SAMPLE_FACTORS - 4--10 Treatment:Low Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=4--10 SUBJECT_SAMPLE_FACTORS - 4--2 Treatment:Low Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=4--2 SUBJECT_SAMPLE_FACTORS - 4--3 Treatment:Low Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=4--3 SUBJECT_SAMPLE_FACTORS - 4--4 Treatment:Low Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=4--4 SUBJECT_SAMPLE_FACTORS - 4--5 Treatment:Low Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=4--5 SUBJECT_SAMPLE_FACTORS - 4--6 Treatment:Low Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=4--6 SUBJECT_SAMPLE_FACTORS - 4--7 Treatment:Low Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=4--7 SUBJECT_SAMPLE_FACTORS - 4--8 Treatment:Low Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=4--8 SUBJECT_SAMPLE_FACTORS - 4--9 Treatment:Low Dose Ricin | Timepoint:Overnight RAW_FILE_NAME=4--9 #COLLECTION CO:COLLECTION_SUMMARY Livers were removed from mice immediately following sacrifice and stored at -80 CO:COLLECTION_SUMMARY C. Before metabolite extraction, livers were homogenized into powder with mortar CO:COLLECTION_SUMMARY and pestle over liquid nitrogen. CO:SAMPLE_TYPE Liver #TREATMENT TR:TREATMENT_SUMMARY Mice were injected via intraperitoneal (IP) route with either ricin or saline in TR:TREATMENT_SUMMARY 10 microliter per g body weight. Mice in all but the fasted group were given TR:TREATMENT_SUMMARY food and water ad libitum. Fasted mice were transferred to new cages with fresh TR:TREATMENT_SUMMARY bedding where they had food withheld while given water ad libitum. Mice were TR:TREATMENT_SUMMARY sacrificed at 2h (ricin), 8h (ricin and fasted), and overnight (16-22h, control, TR:TREATMENT_SUMMARY ricin, fasted). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Microcentrifuge tubes with 30 mg of frozen liver tissue had 500 uL of ice-cold SP:SAMPLEPREP_SUMMARY acetone and three Zirconia beads added to them. Tissues were lysed and SP:SAMPLEPREP_SUMMARY homogenized, then sonicated and centrifuged. The supernatant was transferred to SP:SAMPLEPREP_SUMMARY a new tube and 500 uL of ice-cold MeOH/H2O was added to the pellet. The pellet SP:SAMPLEPREP_SUMMARY was sonicated and centrifuged again. The supernatant was combined with the SP:SAMPLEPREP_SUMMARY acetone fraction and vacuum dried. Dried pellets were resuspended in SP:SAMPLEPREP_SUMMARY acetonitrile/H2O with 1% Formic Acid, vortexed, sonicated, and centrifuged. 180 SP:SAMPLEPREP_SUMMARY uL of eluate was used for LCMS analysis. #CHROMATOGRAPHY CH:INSTRUMENT_NAME Agilent 1290 CH:COLUMN_NAME Waters Acquity BEH HILIC (150 x 2.1mm,1.7um) CH:COLUMN_PRESSURE 600 bar CH:COLUMN_TEMPERATURE 40 CH:FLOW_GRADIENT 100% B, 55% B, 20% B, 100%B CH:FLOW_RATE 0.400 mL/min CH:RETENTION_TIME 25 min CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:CHROMATOGRAPHY_TYPE HILIC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6530 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:MS_COMMENTS mzML formatting acquired in ProteoWizard msconvert, Feature analysis performed MS:MS_COMMENTS in XCMS Online v3.7.1 MS:ION_MODE POSITIVE MS:MS_RESULTS_FILE ST001667_AN002719_Results.txt UNITS:Intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END