Summary of Study ST001891

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001191. The data can be accessed directly via it's Project DOI: 10.21228/M8RM4F This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST001891
Study TitleSmall molecule signatures of mice lacking T-cell p38 alternate activation, a model for immunosuppression conditions, after exposure to total body radiation (part I)
Study SummaryIntroduction Novel biodosimetry assays are needed in the event of radiological/nuclear emergencies for both immediate triage and identifying delayed effects of acute radiation exposure. Genetically engineered mouse models are used to assess how genotypic variation in the general population may affect post-irradiation classification performance. Here, we used a mouse model that lacks the T-cell receptor specific alternative p38 pathway (p38αβY323F, double knock-in [DKI] mice) to determine how attenuated autoimmune and inflammatory responses may affect dose reconstruction. Objectives To determine if deficient alternative p38 activation differentially affects biofluid metabolic signatures post-irradiation compared to wild-type (WT). Methods Untargeted global metabolomics was used to assess biofluid signatures between WT and DKI mice (8 – 10 weeks old) after exposure to total body radiation (0, 2, or 7 Gy). Urine was analyzed in the first week (1, 3, and 7 d) and serum at 1 d. Spectral features of interest were identified using the machine learning algorithm Random Forests and MetaboLyzer. Validated metabolite panels were constructed and classification performance was assessed by determining the area under the receiver operating characteristic curve (AUROC). Results A multidimensional scaling plot showed excellent separation of IR exposed groups in WT with slightly dampened responses in DKI mice. For both urine and serum, excellent sensitivity and specificity (AUROC > 0.90) was observed for 0 Gy vs. 7 Gy groups irrespective of genotype using identical metabolite panels. Similarly, excellent to fair classification (AUROC > 0.75) was observed for ≤ 2 Gy vs. 7 Gy post-irradiation mice for both genotypes, however, model performance declined (AUROC < 0.75) between genotypes post-irradiation. Conclusion Overall, these results suggest less influence of the alternative p38 activation pathway for dose reconstruction compared to other radiosensitive genotypes.
Institute
Georgetown University
Last NamePannkuk
First NameEvan
Address3970 Reservoir Rd, NW New Research Building E504
Emailelp44@georgetown.edu
Phone2026875650
Submit Date2021-07-23
Raw Data AvailableYes
Raw Data File Type(s)raw(Waters)
Analysis Type DetailLC-MS
Release Date2022-07-06
Release Version1
Evan Pannkuk Evan Pannkuk
https://dx.doi.org/10.21228/M8RM4F
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001191
Project DOI:doi: 10.21228/M8RM4F
Project Title:Small molecule signatures of mice lacking T-cell p38 alternate activation, a model for immunosuppression conditions, after exposure to total body radiation
Project Summary:Introduction Novel biodosimetry assays are needed in the event of radiological/nuclear emergencies for both immediate triage and identifying delayed effects of acute radiation exposure. Genetically engineered mouse models are used to assess how genotypic variation in the general population may affect post-irradiation classification performance. Here, we used a mouse model that lacks the T-cell receptor specific alternative p38 pathway (p38αβY323F, double knock-in [DKI] mice) to determine how attenuated autoimmune and inflammatory responses may affect dose reconstruction. Objectives To determine if deficient alternative p38 activation differentially affects biofluid metabolic signatures post-irradiation compared to wild-type (WT). Methods Untargeted global metabolomics was used to assess biofluid signatures between WT and DKI mice (8 – 10 weeks old) after exposure to total body radiation (0, 2, or 7 Gy). Urine was analyzed in the first week (1, 3, and 7 d) and serum at 1 d. Spectral features of interest were identified using the machine learning algorithm Random Forests and MetaboLyzer. Validated metabolite panels were constructed and classification performance was assessed by determining the area under the receiver operating characteristic curve (AUROC). Results A multidimensional scaling plot showed excellent separation of IR exposed groups in WT with slightly dampened responses in DKI mice. For both urine and serum, excellent sensitivity and specificity (AUROC > 0.90) was observed for 0 Gy vs. 7 Gy groups irrespective of genotype using identical metabolite panels. Similarly, excellent to fair classification (AUROC > 0.75) was observed for ≤ 2 Gy vs. 7 Gy post-irradiation mice for both genotypes, however, model performance declined (AUROC < 0.75) between genotypes post-irradiation. Conclusion Overall, these results suggest less influence of the alternative p38 activation pathway for dose reconstruction compared to other radiosensitive genotypes.
Institute:Georgetown University
Last Name:Pannkuk
First Name:Evan
Address:3970 Reservoir Rd, NW New Research Building E504
Email:elp44@georgetown.edu
Phone:2026875650
Publications:https://meridian.allenpress.com/radiation-research/article-abstract/197/6/613/478727/Small-Molecule-Signatures-of-Mice-Lacking-T-cell

Subject:

Subject ID:SU001969
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Gender:Male
Species Group:Mammals

Factors:

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

mb_sample_id local_sample_id Irradiation Genotype Collection_time
SA1758361522Gy p38 1d
SA1758371182Gy p38 1d
SA1758381532Gy p38 1d
SA1758392452Gy p38 1d
SA1758401172Gy p38 1d
SA1758411202Gy p38 1d
SA1758421162Gy p38 1d
SA1758431192Gy p38 1d
SA175844422Gy p38 3d
SA17584592Gy p38 3d
SA175846412Gy p38 3d
SA1758471632Gy p38 3d
SA1758481642Gy p38 3d
SA1758493212Gy p38 3d
SA175850152Gy p38 3d
SA175851162Gy p38 3d
SA175852962Gy p38 7d
SA175853952Gy p38 7d
SA1758541742Gy p38 7d
SA175855492Gy p38 7d
SA175856942Gy p38 7d
SA1758571752Gy p38 7d
SA175858482Gy p38 7d
SA175859932Gy p38 7d
SA1758602852Gy p38 pre
SA175861252Gy p38 pre
SA175862202Gy p38 pre
SA1758631452Gy p38 pre
SA1758642062Gy p38 pre
SA175865622Gy p38 pre
SA1758662052Gy p38 pre
SA175867632Gy p38 pre
SA175816832Gy WT 1d
SA1758171262Gy WT 1d
SA1758181272Gy WT 1d
SA175819672Gy WT 1d
SA175820662Gy WT 1d
SA1758211302Gy WT 3d
SA1758222362Gy WT 3d
SA175823462Gy WT 3d
SA1758241122Gy WT 3d
SA1758252962Gy WT 3d
SA175826512Gy WT 7d
SA175827992Gy WT 7d
SA1758281842Gy WT 7d
SA175829752Gy WT 7d
SA1758301852Gy WT 7d
SA175831132Gy WT pre
SA1758322442Gy WT pre
SA1758331482Gy WT pre
SA175834142Gy WT pre
SA175835872Gy WT pre
SA175888347Gy p38 1d
SA1758892137Gy p38 1d
SA1758902477Gy p38 1d
SA1758912467Gy p38 1d
SA1758922357Gy p38 1d
SA1758932227Gy p38 1d
SA1758942147Gy p38 1d
SA175895907Gy p38 1d
SA1758961217Gy p38 1d
SA1758971287Gy p38 1d
SA1758981597Gy p38 1d
SA1758991547Gy p38 1d
SA175900697Gy p38 1d
SA1759012617Gy p38 3d
SA175902727Gy p38 3d
SA1759032947Gy p38 3d
SA175904127Gy p38 3d
SA1759051327Gy p38 3d
SA1759063417Gy p38 3d
SA1759071657Gy p38 3d
SA1759082547Gy p38 3d
SA1759091667Gy p38 3d
SA1759101737Gy p38 3d
SA1759112237Gy p38 3d
SA1759121677Gy p38 3d
SA1759133287Gy p38 7d
SA1759142837Gy p38 7d
SA1759152267Gy p38 7d
SA1759161777Gy p38 7d
SA1759172847Gy p38 7d
SA1759181767Gy p38 7d
SA1759191387Gy p38 7d
SA1759202307Gy p38 7d
SA1759211397Gy p38 7d
SA1759223297Gy p38 7d
SA175923507Gy p38 7d
SA1759243017Gy p38 7d
SA1759252087Gy p38 pre
SA1759262077Gy p38 pre
SA1759271157Gy p38 pre
SA175928647Gy p38 pre
SA175929267Gy p38 pre
SA175930887Gy p38 pre
SA1759312397Gy p38 pre
SA175932317Gy p38 pre
SA175933337Gy p38 pre
SA175934327Gy p38 pre
SA1759351147Gy p38 pre
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Collection:

Collection ID:CO001962
Collection Summary:Urine was collected after irradiation
Sample Type:Urine

Treatment:

Treatment ID:TR001981
Treatment Summary:WT C57Bl/6 mice (C57BL/6NCrl strain code #027) were obtained from Charles River Laboratories (Frederick, MD) and DKI mice were kindly provided by the Laboratory of Immune Cell Biology, National Cancer Institute (P.I. Jonathan D. Ashwell, M.D.) (Jirmanova et al. 2011). Animals were bred/irradiated (12 h light / 12 h dark cycle conditions) at Georgetown University and water and food (PicoLab Rodent Diet 20 #5053) were provided ad libitum according to Georgetown University Institutional Animal Care and Use Committee (GUACUC) protocols (2016-1152). Before irradiation and biofluid collection the mice were acclimated to metabolic cages for 24 h. Male mice that were 8 – 10 weeks old were exposed to a total body ionization (TBI) x-ray dose (~1.67 Gy/min; X-Rad 320, Precision X-Ray Inc, Branford, CT; filter, 0.75 mm tin/ 0.25 mm copper/1.5 mm aluminum) of 0, 2, or 7 Gy. All urine samples were collected over a 24 h period in a metabolic cage pre-irradiation and at days 1, 3, and 7 d post-irradiation (Figure S1). Blood for metabolomics was collected at 1 d via cheek bleed from the submandibular vein and serum was separated in a BD microtainer serum separator tube and centrifuged for 10 min (10,000 x g, 4°C). Serum samples from sham-irradiated mice were used as a control (Figure S1). All biofluids were flash frozen and stored at -80°C until further use. Seven days post-irradiation, blood was collected in a dipotassium EDTA Tube (BD Cat #365974) via the facial vein from each animal and subjected to a complete blood count by VRL Diagnostics (Gaithersburg, MD, http://www.vrlsat.com/) (Figure S2).

Sample Preparation:

Sampleprep ID:SP001975
Sampleprep Summary:Biofluids were prepared as previously described (Pannkuk et al. 2018;2020). Urine (20 μl) was deproteinated with 50% acetonitrile (80 μl) containing internal standards (2 μM debrisoquine sulfate, 30 μM 4-nitrobenzoic acid), incubated on ice for 10 min, vortexed for 30 seconds, and centrifuged for 10 min (10,000 x g, 4°C). Serum (5 μl) was prepared as above but was deproteinated with 66% acetonitrile (195 μl). A quality control (QC) sample was prepared by mixing 1 μl from each sample and prepared as above.
Processing Storage Conditions:-80℃

Combined analysis:

Analysis ID AN003070 AN003071
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Waters Acquity Waters Acquity
Column Waters Acquity BEH C18 (50 x 2.1mm,1.7um) Waters Acquity BEH C18 (50 x 2.1mm,1.7um)
MS Type ESI ESI
MS instrument type QTOF QTOF
MS instrument name Waters Synapt G2 Waters Synapt G2
Ion Mode POSITIVE NEGATIVE
Units peak area peak area

Chromatography:

Chromatography ID:CH002272
Chromatography Summary:Mobile phases consisted of the following: solvent A (water/0.1% formic acid [FA]), solvent B (ACN/0.1% FA), solvent C (isopropanol [IPA]/ACN (90:10)/0.1% FA). The gradient for urine was (solvent A and B) 4.0 min 5% B, 4.0 min 20% B, 5.1 min 95% B, and 1.9 min 5% B at a flow rate of 0.5 ml/min. The gradient for serum was (solvent A, B, and C) 4.0 min 98:2 A:B, 4.0 min 40:60 A:B, 1.5 min 2:98 A:B, 2.0 min 2:98 A:C, 0.5 min 50:50 A:C, and 1.0 min 98:2 A:B at a flow rate of 0.5 ml/min.
Instrument Name:Waters Acquity
Column Name:Waters Acquity BEH C18 (50 x 2.1mm,1.7um)
Flow Gradient:The gradient for urine was (solvent A and B) 4.0 min 5% B, 4.0 min 20% B, 5.1 min 95% B, and 1.9 min 5% B at a flow rate of 0.5 ml/min. The gradient for serum was (solvent A, B, and C) 4.0 min 98:2 A:B, 4.0 min 40:60 A:B, 1.5 min 2:98 A:B, 2.0 min 2:98 A:C, 0.5 min 50:50 A:C, and 1.0 min 98:2 A:B at a flow rate of 0.5 ml/min.
Flow Rate:0.5 ml/min
Solvent A:100% water; 0.1% formic acid
Solvent B:solvent B:100% acetonitrile; 0.1% formic acid solvent C:90% isopropanol/10% acetonitrile; 0.1% formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS002857
Analysis ID:AN003070
Instrument Name:Waters Synapt G2
Instrument Type:QTOF
MS Type:ESI
MS Comments:Negative and positive electrospray ionization (ESI) data-independent modes were used for data acquisition with leucine enkephalin ([M+H]+ = 556.2771, [M-H]- = 554.2615) as Lock-Spray®. Operating conditions for ESI were: capillary voltage 2.75 kV, cone voltage 30 V, desolvation temperature 500°C, desolvation gas flow 1000 L/Hr.
Ion Mode:POSITIVE
  
MS ID:MS002858
Analysis ID:AN003071
Instrument Name:Waters Synapt G2
Instrument Type:QTOF
MS Type:ESI
MS Comments:Negative and positive electrospray ionization (ESI) data-independent modes were used for data acquisition with leucine enkephalin ([M+H]+ = 556.2771, [M-H]- = 554.2615) as Lock-Spray®. Operating conditions for ESI were: capillary voltage 2.75 kV, cone voltage 30 V, desolvation temperature 500°C, desolvation gas flow 1000 L/Hr.
Ion Mode:NEGATIVE
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