Summary of Study ST001614

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench,, where it has been assigned Project ID PR001037. The data can be accessed directly via it's Project DOI: 10.21228/M8NM4H This work is supported by NIH grant, U2C- DK119886.


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Study IDST001614
Study TitleNMR metabolomics analysis of ricin-induced and fasting hypoglycemia (part-I)
Study TypeTargeted NMR
Study SummaryMice 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.
Montana State University
DepartmentChemistry & Biochemistry
Last NameKempa
First NameJake
Address103 Chemistry and Biochemistry Building
Submit Date2020-09-23
Num Groups9
Total Subjects107
Num Males54
Num Females53
Raw Data AvailableYes
Raw Data File Type(s)d
Analysis Type DetailNMR
Release Date2022-11-29
Release Version1
Jake Kempa Jake Kempa application/zip

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Project ID:PR001037
Project DOI:doi: 10.21228/M8NM4H
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
Last Name:Kempa
First Name:Jake
Address:103 Chemistry and Biochemistry Building, Bozeman, Montana, 59717, USA


Subject ID:SU001691
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


Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Treatment Timepoint
SA1370581--13Control Overnight
SA1370591--12Control Overnight
SA1370601--14Control Overnight
SA1370611--16Control Overnight
SA1370621--2Control Overnight
SA1370631--10Control Overnight
SA1370641--15Control Overnight
SA1370651--11Control Overnight
SA1370661--4Control Overnight
SA1370671--3Control Overnight
SA1370681--6Control Overnight
SA1370691--5Control Overnight
SA1370701--7Control Overnight
SA1370711--8Control Overnight
SA1370721--9Control Overnight
SA1370737--8Fast 8h
SA1370747--9Fast 8h
SA1370757--10Fast 8h
SA1370767--7Fast 8h
SA1370777--2Fast 8h
SA1370787--6Fast 8h
SA1370797--3Fast 8h
SA1370807--1Fast 8h
SA1370817--4Fast 8h
SA1370827--5Fast 8h
SA1370832--10Fast Overnight
SA1370842--11Fast Overnight
SA1370852--12Fast Overnight
SA1370862--14Fast Overnight
SA1370872--9Fast Overnight
SA1370882--16Fast Overnight
SA1370892--13Fast Overnight
SA1370902--15Fast Overnight
SA1370912--3Fast Overnight
SA1370922--1Fast Overnight
SA1370932--8Fast Overnight
SA1370942--4Fast Overnight
SA1370952--2Fast Overnight
SA1370962--7Fast Overnight
SA1370972--6Fast Overnight
SA1370982--5Fast Overnight
SA1370995--7High Dose Ricin 2h
SA1371005--8High Dose Ricin 2h
SA1371015--6High Dose Ricin 2h
SA1371025--2High Dose Ricin 2h
SA1371035--1High Dose Ricin 2h
SA1371045--10High Dose Ricin 2h
SA1371055--9High Dose Ricin 2h
SA1371065--3High Dose Ricin 2h
SA1371075--4High Dose Ricin 2h
SA1371085--5High Dose Ricin 2h
SA1371099--2High Dose Ricin 8h
SA1371109--1High Dose Ricin 8h
SA1371119--7High Dose Ricin 8h
SA1371129--8High Dose Ricin 8h
SA1371139--9High Dose Ricin 8h
SA1371149--6High Dose Ricin 8h
SA1371159--10High Dose Ricin 8h
SA1371169--5High Dose Ricin 8h
SA1371179--3High Dose Ricin 8h
SA1371189--4High Dose Ricin 8h
SA1371196--5High Dose Ricin Overnight
SA1371206--4High Dose Ricin Overnight
SA1371216--6High Dose Ricin Overnight
SA1371226--3High Dose Ricin Overnight
SA1371236--17High Dose Ricin Overnight
SA1371246--16High Dose Ricin Overnight
SA1371256--15High Dose Ricin Overnight
SA1371266--18High Dose Ricin Overnight
SA1371276--19High Dose Ricin Overnight
SA1371286--20High Dose Ricin Overnight
SA1371296--14High Dose Ricin Overnight
SA1371306--13High Dose Ricin Overnight
SA1371316--8High Dose Ricin Overnight
SA1371326--10High Dose Ricin Overnight
SA1371336--11High Dose Ricin Overnight
SA1371346--12High Dose Ricin Overnight
SA1371356--7High Dose Ricin Overnight
SA1371363--4Low Dose Ricin 2h
SA1371373--3Low Dose Ricin 2h
SA1371383--2Low Dose Ricin 2h
SA1371393--1Low Dose Ricin 2h
SA1371403--5Low Dose Ricin 2h
SA1371413--6Low Dose Ricin 2h
SA1371423--9Low Dose Ricin 2h
SA1371433--8Low Dose Ricin 2h
SA1371443--7Low Dose Ricin 2h
SA1371453--10Low Dose Ricin 2h
SA1371468--3Low Dose Ricin 8h
SA1371478--7Low Dose Ricin 8h
SA1371488--8Low Dose Ricin 8h
SA1371498--9Low Dose Ricin 8h
SA1371508--6Low Dose Ricin 8h
SA1371518--5Low Dose Ricin 8h
SA1371528--1Low Dose Ricin 8h
SA1371538--2Low Dose Ricin 8h
SA1371548--4Low Dose Ricin 8h
SA1371554--1Low Dose Ricin Overnight
SA1371564--2Low Dose Ricin Overnight
SA1371574--7Low Dose Ricin Overnight
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Collection ID:CO001684
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 ID:TR001704
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

Sample Preparation:

Sampleprep ID:SP001697
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 ID:AN002649
Laboratory Name:Copie Lab
Analysis Type:NMR
Num Factors:9
Num Metabolites:60
Units:µM/mg liver tissue


NMR ID:NM000186
Analysis ID:AN002649
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