{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001614","ANALYSIS_ID":"AN002649","VERSION":"1","CREATED_ON":"December 1, 2020, 2:09 pm"},

"PROJECT":{"PROJECT_TITLE":"NMR and MS Metabolomics Reveal a Distinct Metabolic State of Ricin-induced Hypoglycemia","PROJECT_TYPE":"Targeted NMR and Untargeted MS","PROJECT_SUMMARY":"Ricin toxin is a ribosome inactivating protein. Due to its toxic and chemical properties, ricin is a potential agent of bioterrorism but has also been studied for therapeutic use in immunotoxins. Previous research by our group has demonstrated lethal hypoglycemia associated with ricin toxicity. Research efforts have focused on better understanding this ricin-induced metabolic state of hypoglycemia to aid in better understanding the systemic effects of hypoglycemia, and the use of ricin in immunotherapy. Here, we have used a mouse model to characterize liver metabolome changes associated with hypoglycemia induced by two conditions. Mice were challenged with intraperitoneal injections of ricin at high and low doses for 2 hrs, 8 hrs, and overnight. To understand how ricin-induced hypoglycemia differs from that of fasting, another group of mice had food withheld for 8-hours and overnight. 1H NMR-based metabolomics was performed on polar molecules extracted from mouse livers, with metabolites annotated using Chenomx software. MetaboAnalyst was employed for multivariate statistical analysis. In this study, similar decreases in blood glucose in mice were observed following injection of a lethal dose of ricin and with overnight fasting. NMR analyses identified 59 polar metabolites present in mice livers from all treatments. Multivariate statistical analyses were used to evaluate global metabolic state differences. Results from these analyses indicated that the profiled liver metabolomes for mice subjected to the two conditions differ significantly at both 8 hours and overnight. Additionally, ricin treatment with a lethal dose reveal a progression of metabolic changes over time, from 2 to 22 hours. Additionally, mass spectrometry supported these findings, and NMR analyses revealed key metabolites that contribute to these differences. While both ricin and fasting induce hypoglycemia, the metabolic states resulting from these two conditions are different. Further analyses may give insights into mechanisms of ricin toxicity, specific metabolic pathways that are altered, and potential treatments for hypoglycemia. We propose to extend these studies to insulin-induced hypoglycemia.","INSTITUTE":"Montana State University","DEPARTMENT":"Chemistry & Biochemistry","LABORATORY":"Copié","LAST_NAME":"Kempa","FIRST_NAME":"Jake","ADDRESS":"103 Chemistry and Biochemistry Building, Bozeman, Montana, 59717, USA","EMAIL":"jkempa97@gmail.com","PHONE":"4067997200"},

"STUDY":{"STUDY_TITLE":"NMR metabolomics analysis of ricin-induced and fasting hypoglycemia (part-I)","STUDY_TYPE":"Targeted NMR","STUDY_SUMMARY":"Mice were subject to ricin exposure or fasting conditions for 2 hours, 8 hours, or an overnight time period. Following treatment, livers were removed and metabolites were extracted and analyzed by NMR.","INSTITUTE":"Montana State Universty","DEPARTMENT":"Chemistry & Biochemistry","LABORATORY":"Copié","LAST_NAME":"Kempa","FIRST_NAME":"Jake","ADDRESS":"103 Chemistry and Biochemistry Building","EMAIL":"jkempa97@gmail.com","PHONE":"4067997200","NUM_GROUPS":"9","TOTAL_SUBJECTS":"107","NUM_MALES":"54","NUM_FEMALES":"53"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090","GENOTYPE_STRAIN":"BALB/c","AGE_OR_AGE_RANGE":"70-74","GENDER":"Male and female","ANIMAL_ANIMAL_SUPPLIER":"Montana State University Animal Resource Center","ANIMAL_FEED":"PicoLab Rodent Diet 20 5053"},
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{
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{
"Subject ID":"-",
"Sample ID":"6--8",
"Factors":{"Treatment":"High Dose Ricin","Timepoint":"Overnight"},
"Additional sample data":{"RAW_FILE_NAME":"6--8"}
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{
"Subject ID":"-",
"Sample ID":"6--10",
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"Subject ID":"-",
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"Subject ID":"-",
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{
"Subject ID":"-",
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{
"Subject ID":"-",
"Sample ID":"6--17",
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"Additional sample data":{"RAW_FILE_NAME":"6--17"}
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{
"Subject ID":"-",
"Sample ID":"6--18",
"Factors":{"Treatment":"High Dose Ricin","Timepoint":"Overnight"},
"Additional sample data":{"RAW_FILE_NAME":"6--18"}
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{
"Subject ID":"-",
"Sample ID":"6--19",
"Factors":{"Treatment":"High Dose Ricin","Timepoint":"Overnight"},
"Additional sample data":{"RAW_FILE_NAME":"6--19"}
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{
"Subject ID":"-",
"Sample ID":"6--20",
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],
"COLLECTION":{"COLLECTION_SUMMARY":"Livers were removed from mice immediately following sacrificed, and stored at -80° C. Before metabolite extraction, livers were homogenized into powder with mortar and pestle over liquid nitrogen.","SAMPLE_TYPE":"Liver","STORAGE_CONDITIONS":"-80℃"},

"TREATMENT":{"TREATMENT_SUMMARY":"Mice were injected via intraperitoneal (IP) route with either ricin or saline in 10 µL per g body weight. Mice in all but the fasted group were given food and water ad libitum. Fasted mice were transferred to new cages with fresh bedding where they had food withheld while given water ad libitum. Mice were sacrificed at 2hr (ricin), 8 hr (ricin and fasted), and overnight (16-22 hr, control, ricin, fasted).","TREATMENT_ROUTE":"Intraperitoneal","TREATMENT_DOSE":"0.6 µg/kg body weight or 30 µg/kg body weight","TREATMENT_VEHICLE":"saline"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Frozen livers were homogenized in liquid nitrogen using a mortar and pestle. Liver powder was weighed (60-80 mg) into 2-mL microcentrifuge tubes. 900 µL of ice-cold 1:2 MeOH/CHCl3 was added to the liver tissue and tissue was mixed into suspension and lysed using a FastPrep-24TM 5G homogenizer using the S. aureus setting of 6.0 meters/second speed for 2 x 40 seconds. Tubes were placed back on ice and 200 µL of ice-cold H2O was added, before repeating the lysis step. Samples were then placed at -20 ºC for approximately 45 minutes to precipitate protein, then centrifuged at 14,000 g for 10 minutes. 250 µL of upper polar phase were transferred to a 1.5 mL Eppendorf tube and dried overnight using a Savant™ Universal SpeedVac™ vacuum system with no heat. Dried metabolite mixtures were re-suspended in 600 µL of NMR buffer containing 0.25 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS), 90%H2O/10% D2O, 25 mM sodium phosphate, pH 7."},

"ANALYSIS":{"LABORATORY_NAME":"Copie Lab"},

"NM":{"INSTRUMENT_NAME":"Bruker Avance III Solution NMR Spectrometer","INSTRUMENT_TYPE":"FT-NMR","NMR_EXPERIMENT_TYPE":"1D-1H","SPECTROMETER_FREQUENCY":"600.13 MHz (1H Larmor Frequency)","NMR_PROBE":"5 mm liquid-helium-cooled TCI cryoprobe with Z-gradient","NMR_SOLVENT":"0.25 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS), 90%H2O/10% D2O, 25 mM sodium phosphate, pH 7","NMR_TUBE_SIZE":"5 mm","SHIMMING_METHOD":"topshim","PULSE_SEQUENCE":"zgesgp","NUMBER_OF_SCANS":"256","NUM_DATA_POINTS_ACQUIRED":"64K"},

"NMR_METABOLITE_DATA":{
"Units":"µM/mg liver tissue",

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}

}