Summary of study ST000121

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 PR000108. The data can be accessed directly via it's Project DOI: 10.21228/M8GW2Z This work is supported by NIH grant, U2C- DK119886.

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Study IDST000121
Study TitleImpact of anesthesia and euthanasia on metabolomics of mammalian tissues: studies in a C57BL/6J mouse model
Study TypeAnesthesia effect study
Study SummaryWe examined the effect of several commonly-used methods of anesthesia and euthanasia for collection of skeletal muscle, liver, heart, adipose and serum of C57BL/6J mice. The data revealed tissue-specific impacts of different anesthesia and euthanasia strategies. Based on these findings, we present a more optimal collection strategy mammalian tissues and recommend that rodent tissues intended for metabolomics studies be collected under anesthesia rather than post-euthanasia.
Institute
University of Michigan
DepartmentInternal Medicine
LaboratoryEvans Lab / Burant Lab
Last NameEvans
First NameCharles
Address6321 Brehm Tower, 1000 Wall Street, Ann Arbor MI 48105
Emailchevans@umich.edu
Phone734-232-8177
Submit Date2014-11-21
Num Groups6
Total Subjects49
Raw Data AvailableNo
Analysis Type DetailLC-MS
Release Date2015-02-01
Release Version1
Charles Evans Charles Evans
https://dx.doi.org/10.21228/M8GW2Z
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000108
Project DOI:doi: 10.21228/M8GW2Z
Project Title:Impact of anesthesia and euthanasia on metabolomics of mammalian tissues: studies in a C57BL/6J mouse model
Project Type:Anesthesia effect study
Project Summary:We examined, for the first time using untargeted and targeted metabolomics, the effect of several commonly-used methods of anesthesia and euthanasia for collection of skeletal muscle, liver, heart, adipose and serum of C57BL/6J mice. The data revealed tissue-specific impacts of different anesthesia and euthanasia strategies. Based on these findings, we present a more optimal collection strategy mammalian tissues and recommend that rodent tissues intended for metabolomics studies be collected under anesthesia rather than post-euthanasia.
Institute:University of Michigan
Department:Internal Medicine
Laboratory:Evans lab / Burant lab
Last Name:Evans
First Name:Charles
Address:6321 Brehm Tower, 1000 Wall St.
Email:chevans@umich.edu
Phone:734-232-8177
Funding Source:National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health

Subject:

Subject ID:SU000140
Subject Type:Animal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57BL/6J
Age Or Age Range:20 weeks
Weight Or Weight Range:26.8 +/- 2.2 g
Gender:Male
Animal Animal Supplier:Jackson Labs (Bar Harbor, ME)
Animal Housing:8 / cage
Animal Light Cycle:12 h / 12 h light/dark
Animal Feed:Standard chow, ad lib.
Animal Water:Ad lib. Access
Species Group:Mammal

Factors:

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

mb_sample_id local_sample_id Tissue Anesthesia / Euthanasia method
SA006504F14Epididymal white adipose (fat) Carbon Dioxide
SA006505F16Epididymal white adipose (fat) Carbon Dioxide
SA006506F17Epididymal white adipose (fat) Carbon Dioxide
SA006507F13Epididymal white adipose (fat) Carbon Dioxide
SA006508F15Epididymal white adipose (fat) Carbon Dioxide
SA006509F12Epididymal white adipose (fat) Carbon Dioxide
SA006510F09Epididymal white adipose (fat) Carbon Dioxide
SA006511F10Epididymal white adipose (fat) Carbon Dioxide
SA006512F11Epididymal white adipose (fat) Carbon Dioxide
SA006513F23Epididymal white adipose (fat) Cervical Dislocation
SA006514F24Epididymal white adipose (fat) Cervical Dislocation
SA006515F22Epididymal white adipose (fat) Cervical Dislocation
SA006516F18Epididymal white adipose (fat) Cervical Dislocation
SA006517F25Epididymal white adipose (fat) Cervical Dislocation
SA006518F19Epididymal white adipose (fat) Cervical Dislocation
SA006519F20Epididymal white adipose (fat) Cervical Dislocation
SA006520F21Epididymal white adipose (fat) Cervical Dislocation
SA006521F40Epididymal white adipose (fat) Continuous isoflurane
SA006522F35Epididymal white adipose (fat) Continuous isoflurane
SA006523F37Epididymal white adipose (fat) Continuous isoflurane
SA006524F38Epididymal white adipose (fat) Continuous isoflurane
SA006525F41Epididymal white adipose (fat) Continuous isoflurane
SA006526F39Epididymal white adipose (fat) Continuous isoflurane
SA006527F34Epididymal white adipose (fat) Continuous isoflurane
SA006528F36Epididymal white adipose (fat) Continuous isoflurane
SA006529F05Epididymal white adipose (fat) Isoflurane overdose
SA006530F06Epididymal white adipose (fat) Isoflurane overdose
SA006531F07Epididymal white adipose (fat) Isoflurane overdose
SA006532F04Epididymal white adipose (fat) Isoflurane overdose
SA006533F08Epididymal white adipose (fat) Isoflurane overdose
SA006534F03Epididymal white adipose (fat) Isoflurane overdose
SA006535F01Epididymal white adipose (fat) Isoflurane overdose
SA006536F02Epididymal white adipose (fat) Isoflurane overdose
SA006537F28Epididymal white adipose (fat) Ketamine
SA006538F26Epididymal white adipose (fat) Ketamine
SA006539F29Epididymal white adipose (fat) Ketamine
SA006540F27Epididymal white adipose (fat) Ketamine
SA006541F30Epididymal white adipose (fat) Ketamine
SA006542F33Epididymal white adipose (fat) Ketamine
SA006543F32Epididymal white adipose (fat) Ketamine
SA006544F31Epididymal white adipose (fat) Ketamine
SA006545F46Epididymal white adipose (fat) Pentobarbital
SA006546F47Epididymal white adipose (fat) Pentobarbital
SA006547F49Epididymal white adipose (fat) Pentobarbital
SA006548F45Epididymal white adipose (fat) Pentobarbital
SA006549F43Epididymal white adipose (fat) Pentobarbital
SA006550F42Epididymal white adipose (fat) Pentobarbital
SA006551F48Epididymal white adipose (fat) Pentobarbital
SA006552F44Epididymal white adipose (fat) Pentobarbital
SA006553G17Gastrocnemius (skeletal muscle) Carbon Dioxide
SA006554G13Gastrocnemius (skeletal muscle) Carbon Dioxide
SA006555G12Gastrocnemius (skeletal muscle) Carbon Dioxide
SA006556G11Gastrocnemius (skeletal muscle) Carbon Dioxide
SA006557G09Gastrocnemius (skeletal muscle) Carbon Dioxide
SA006558G16Gastrocnemius (skeletal muscle) Carbon Dioxide
SA006559G15Gastrocnemius (skeletal muscle) Carbon Dioxide
SA006560G14Gastrocnemius (skeletal muscle) Carbon Dioxide
SA006561G10Gastrocnemius (skeletal muscle) Carbon Dioxide
SA006562G18Gastrocnemius (skeletal muscle) Cervical Dislocation
SA006563G23Gastrocnemius (skeletal muscle) Cervical Dislocation
SA006564G24Gastrocnemius (skeletal muscle) Cervical Dislocation
SA006565G22Gastrocnemius (skeletal muscle) Cervical Dislocation
SA006566G21Gastrocnemius (skeletal muscle) Cervical Dislocation
SA006567G19Gastrocnemius (skeletal muscle) Cervical Dislocation
SA006568G20Gastrocnemius (skeletal muscle) Cervical Dislocation
SA006569G25Gastrocnemius (skeletal muscle) Cervical Dislocation
SA006570G34Gastrocnemius (skeletal muscle) Continuous isoflurane
SA006571G39Gastrocnemius (skeletal muscle) Continuous isoflurane
SA006572G40Gastrocnemius (skeletal muscle) Continuous isoflurane
SA006573G38Gastrocnemius (skeletal muscle) Continuous isoflurane
SA006574G37Gastrocnemius (skeletal muscle) Continuous isoflurane
SA006575G35Gastrocnemius (skeletal muscle) Continuous isoflurane
SA006576G36Gastrocnemius (skeletal muscle) Continuous isoflurane
SA006577G41Gastrocnemius (skeletal muscle) Continuous isoflurane
SA006578G08Gastrocnemius (skeletal muscle) Isoflurane overdose
SA006579G02Gastrocnemius (skeletal muscle) Isoflurane overdose
SA006580G01Gastrocnemius (skeletal muscle) Isoflurane overdose
SA006581G03Gastrocnemius (skeletal muscle) Isoflurane overdose
SA006582G04Gastrocnemius (skeletal muscle) Isoflurane overdose
SA006583G06Gastrocnemius (skeletal muscle) Isoflurane overdose
SA006584G07Gastrocnemius (skeletal muscle) Isoflurane overdose
SA006585G05Gastrocnemius (skeletal muscle) Isoflurane overdose
SA006586G30Gastrocnemius (skeletal muscle) Ketamine
SA006587G31Gastrocnemius (skeletal muscle) Ketamine
SA006588G29Gastrocnemius (skeletal muscle) Ketamine
SA006589G28Gastrocnemius (skeletal muscle) Ketamine
SA006590G26Gastrocnemius (skeletal muscle) Ketamine
SA006591G27Gastrocnemius (skeletal muscle) Ketamine
SA006592G32Gastrocnemius (skeletal muscle) Ketamine
SA006593G33Gastrocnemius (skeletal muscle) Ketamine
SA006594G47Gastrocnemius (skeletal muscle) Pentobarbital
SA006595G48Gastrocnemius (skeletal muscle) Pentobarbital
SA006596G49Gastrocnemius (skeletal muscle) Pentobarbital
SA006597G42Gastrocnemius (skeletal muscle) Pentobarbital
SA006598G46Gastrocnemius (skeletal muscle) Pentobarbital
SA006599G45Gastrocnemius (skeletal muscle) Pentobarbital
SA006600G43Gastrocnemius (skeletal muscle) Pentobarbital
SA006601G44Gastrocnemius (skeletal muscle) Pentobarbital
SA006602H16Heart Carbon Dioxide
SA006603H17Heart Carbon Dioxide
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Collection:

Collection ID:CO000124
Collection Summary:4 tissues plus blood serum were collected and immediately following euthanasia or anesthesia induction, frozen in liquid nitrogen and stored at -80C until analysis
Sample Type:Skeletal muscle (gastrocnemius), Liver, Heart, Epididymal white adipose (fat), blood serum
Collection Method:Surgical dissection
Collection Time:As soon as possible following anesthesia/euthanasia (all tissues collected within 3 minutes)
Storage Conditions:-80 C

Treatment:

Treatment ID:TR000142
Treatment Summary:Mice were prepared or tissue collection by one of 6 modes of anesthesia or euthanasia, then tissues were rapidly surgically collected and frozen
Animal Anesthesia:One of 6 methods: 1) Cervical dislocation, 2) Carbon dioxide inhalation, 3) Isoflurane overdose, 4) continuous isoflurane, 5) ketamine, 6) pentobarbital
Animal Fasting:5 hours
Animal Endp Euthanasia:All tissue collection were terminal studies; in methods 1-3, animals were euthanized prior to tissue collection; in methods 4-6, animals were under deep anesthesia while tissues were collected.

Sample Preparation:

Sampleprep ID:SP000137
Sampleprep Summary:Frozen tissue samples were pulverized in a liquid nitrogen-chilled mortar and pestle, then sonicated in cold extraction solvent (7:2:1 methanol:chloroform:water) at an approximate ratio of 30 mg tissue (wet mass) /1 mL of solvent. After allowing samples to rest on ice 5 minutes, they were centrifuged to pellet precipitated protein and cell debris; the supernatant was placed in autosampler vials for direct LC-MS analysis. For serum, extraction solvent (1:1:1 methanol:acetonitrile:acetone) was added to serum at a 4:1 solvent:serum ratio, vortexed to mix, then centrifuged and supernatant recovered for LC-MS analysis.
Sampleprep Protocol Filename:PROTOCOLS_mouse_anesthesia.pdf
Processing Method:Cryo-pulverization + sonication in extraction solvent

Combined analysis:

Analysis ID AN000203
Analysis type MS
Chromatography type HILIC
Chromatography system Agilent 1200
Column Phenomenex Luna NH2 (150 x 2.1mm, 3um)
MS Type ESI
MS instrument type TOF
MS instrument name Agilent 6220 TOF
Ion Mode NEGATIVE
Units Peak area

Chromatography:

Chromatography ID:CH000135
Chromatography Summary:Polar metabolites were analyzed by HILIC-MS using an aminopropyl stationary phase
Instrument Name:Agilent 1200
Column Name:Phenomenex Luna NH2 (150 x 2.1mm, 3um)
Column Pressure:150 bar
Column Temperature:25 C
Flow Gradient:80-0% B over 20 minutes, hold 4 minutes, return to 80%B
Flow Rate:0.25 mL/min
Sample Injection:25 uL
Solvent A:5 mM ammonium acetate in water, adjusted to pH 9.9 using ammonium hydroxide
Solvent B:Acetonitrile
Analytical Time:32 min
Chromatography Type:HILIC

MS:

MS ID:MS000166
Analysis ID:AN000203
Instrument Name:Agilent 6220 TOF
Instrument Type:TOF
MS Type:ESI
Ion Mode:NEGATIVE
Capillary Voltage:3500
Collision Gas:N2
Dry Gas Flow:10 L/min
Dry Gas Temp:350 C
Gas Pressure:20 psig
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