Summary of Study ST002426

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 PR001513. The data can be accessed directly via it's Project DOI: 10.21228/M8471X 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 IDST002426
Study TitleIsolated murine skeletal muscles utilize pyruvate over glucose for oxidation:Part 2
Study TypeStudy of the different substrate by isolated skeletal muscle at room temperature via C-13 isotopomer analysis
Study SummaryPreclinical studies of muscle contractile function often employ ex vivo preparations of the soleus and/or extensor digitorum longus (EDL) muscles which are relatively easy to prepare and represent slow and fast fiber properties, respectively. Therefore, the current study sought to examine the utility of this preparation for understanding the metabolic fuel utilization in isolated resting mouse muscles at room temperature. 13C-labeling in both muscle types was performed using three fuels: glucose, pyruvate, and acetate, followed by NMR-based metabolomics analyses. Incubating 13C-labeled substrates in the isolated skeletal muscles makes it possible to examine TCA cycle flux and substrate selection by these muscles.
Institute
University of Florida
DepartmentApplied Physiology and Kinesiology
LaboratoryRm 42 and Rm 43
Last NameKhattri
First NameRam
Address1864 Stadium RD, Gainesville, FL, 32611, USA
Emailrbk11@ufl.edu
Phone3307856045
Submit Date2022-10-31
Num Groups4
Total Subjects18
Num MalesNA
Num FemalesNA
PublicationsMetabolomics journal (submitted)
Raw Data AvailableYes
Raw Data File Type(s)fid
Analysis Type DetailNMR
Release Date2023-05-01
Release Version1
Ram Khattri Ram Khattri
https://dx.doi.org/10.21228/M8471X
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001513
Project DOI:doi: 10.21228/M8471X
Project Title:Isolated murine skeletal muscles utilize pyruvate over glucose for oxidation-Part 1
Project Type:Study of the different substrate by isolated skeletal muscle at room temperature via C-13 isotopomer analysis
Project Summary:The goal of this study was to determine the differential utilization of substrates in isolated murine skeletal muscle, and to evalute how isopotomer anlaysis provided insight into skeletal muscle metabolism.
Institute:University of Florida
Department:Applied Physiology and Kinesiology
Laboratory:Rm 42 and Rm 43
Last Name:Khattri
First Name:Ram
Address:1864 Stadium RD, Gainesville, FL, 32611, USA
Email:rbk11@ufl.edu
Phone:3307856045
Funding Source:This work was supported by grants from the Southeastern Center for Integrated Metabolomics (SECIM) (ERB), NIH AR U54 AR052646 (Physiological Assessment Core, ERB), and Wellstone Muscular Dystrophy Cooperative Research Center Grant (NIAMS: U54AR052646/P50 AR052646). The AMRIS Facility is supported by the National Science Foundation Cooperative Agreement No. DMR-1644779 and the State of Florida.
Contributors:Ram B. Khattri, Jason Puglise, Terence E. Ryan, Glenn A. Walter, Matthew E. Merritt, Elisabeth R. Barton

Subject:

Subject ID:SU002515
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57BL/6J
Age Or Age Range:16±3 weeks
Gender:Not applicable
Animal Animal Supplier:Jackson Labs (Stock # 000664)
Animal Housing:Housed in a temperature of 22 oC
Animal Light Cycle:12-hour light/12-hour dark
Animal Feed:Ad libitum chow diet food
Animal Water:free access to food and water (3-5 animals per cage).

Factors:

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

mb_sample_id local_sample_id Group
SA242751Glucose_EDL_13CNMR-1Glucose_EDL
SA242752Glucose_EDL_13CNMR-4Glucose_EDL
SA242753Glucose_EDL_13CNMR-3Glucose_EDL
SA242754Glucose_EDL_13CNMR-2Glucose_EDL
SA242755Glucose_Soleus_13CNMR-3Glucose_Soleus
SA242756Glucose_Soleus_13CNMR-4Glucose_Soleus
SA242757Glucose_Soleus_13CNMR-1Glucose_Soleus
SA242758Glucose_Soleus_13CNMR-2Glucose_Soleus
SA242759Pyruvate_EDL_13CNMR-3Pyruvate_EDL
SA242760Pyruvate_EDL_13CNMR-2Pyruvate_EDL
SA242761Pyruvate_EDL_13CNMR-1Pyruvate_EDL
SA242762Pyruvate_Soleus_13CNMR-3Pyruvate_Soleus
SA242763Pyruvate_Soleus_13CNMR-2Pyruvate_Soleus
SA242764Pyruvate_Soleus_13CNMR-1Pyruvate_Soleus
Showing results 1 to 14 of 14

Collection:

Collection ID:CO002508
Collection Summary:Mice were anesthetized (using a combination of xylazine (80mg/kg) and ketamine (10mg/kg)) to allow removal of soleus and extensor digitorum longus (EDL) muscles. Upon removal, muscles were incubated at 22 oC in Ringer/MEM solution gas equilibrated with 95/5% O2/CO2 with appropriate 13C labeled substrates in a perfusion chamber routinely used for isolated muscle mechanics for 30 minutes. These included the following: 5.5 mM [U-13C6] glucose; 5.5 mM [U-13C3] pyruvate, or 16.5 mM [13C2] labeled Na-acetate.  Following incubation, muscles were quickly removed, blotted, and then rapidly frozen in liquid nitrogen for subsequent NMR analysis. N=4 muscles were pooled into a single biological replicate of 30-50 mg tissue to afford detectable levels of substrates in the NMR analysis.
Sample Type:Muscle
Collection Location:University of Florida, Applied Physiology and Kinesiology, MBI, 1149 Newell Dr, Gainesville, FL 32610
Storage Conditions:-80℃
Collection Vials:cryovials
Storage Vials:cryovials

Treatment:

Treatment ID:TR002527
Treatment Summary:Mice were anesthetized (using a combination of xylazine (80mg/kg) and ketamine (10mg/kg)) to allow removal of soleus and extensor digitorum longus (EDL) muscles. Upon removal, muscles were incubated at 22 oC in Ringer/MEM solution gas equilibrated with 95/5% O2/CO2 with appropriate 13C labeled substrates in a perfusion chamber routinely used for isolated muscle mechanics for 30 minutes. These included the following: 5.5 mM [U-13C6] glucose; 5.5 mM [U-13C3] pyruvate, or 16.5 mM [13C2] labeled Na-acetate.  Following incubation, muscles were quickly removed, blotted, and then rapidly frozen in liquid nitrogen for subsequent NMR analysis. N=4 muscles were pooled into a single biological replicate of 30-50 mg tissue to afford detectable levels of substrates in the NMR analysis.
Animal Vet Treatments:none
Animal Anesthesia:a combination of xylazine (80mg/kg) and ketamine (10mg/kg))
Animal Fasting:non-fasted
Animal Endp Euthanasia:Euthanasia was carried out by thoracotomy followed by cervical dislocation.
Animal Endp Tissue Coll List:Skeletal muscle (soleus and EDL)

Sample Preparation:

Sampleprep ID:SP002521
Sampleprep Summary:Perchloric acid (PCA) or acetonitrile:isopropanol:water (3:3:2) extractions were performed for all samples to isolate metabolites. The latter method was more efficient in sample recovery due to the reduced number of steps in the procedure but did not affect the proportion of metabolites. For PCA extraction, isolated muscle samples were homogenized with a FASTPREP-24 (MP Biomedicals, Solon, Ohio, USA) with 6% (v/v) ice cold PCA and centrifuged with 13.2 K rpm at 4 oC. The solid muscle portion was washed again with the 6% (v/v) ice cold PCA followed by centrifugation (13.2 K rpm) at 4 oC. The supernatant (combined) obtained was further neutralized with 5M potassium hydroxide and centrifuged again maintaining 13.2 K rpm speed at 4 oC. The resulting supernatants were then lyophilized (Thermo-Scientific, Dallas, USA). The pH of the dried powder was adjusted to 7.2 after dissolving it in 200 μL of ultra-pure water using 1M sodium hydroxide and 1 M hydrochloric acid. The pH-adjusted solution was further centrifuged, the resulting supernatant was dried and the powder was used to prepare the NMR sample. For acetonitrile:isopropanol:water extraction, homogenization of isolated muscle samples was carried out in 1 mL acetonitrile:isopropanol:water (3:3:2, v:v:v) ice cold mixture with a FASTPREP-24 (MP Biomedicals, Solon, Ohio, USA) and centrifuged at 4 oC in separate vials. Resultant supernatants were further lyophilized till dryness (Thermo-Scientific, Dallas, USA). The dried powder was further dissolved in 1 mL of Acetonitrile:Water (1:1, v:v) mixture, vortexed well for ~5 minutes. The resultant solution was further centrifuged, the supernatant obtained was further dried and the powder was used to prepare the NMR sample. The centrifugation speed for each step used was 13.2K rpm. Each NMR sample consisted of 50 mM phosphate buffer (pH 7), 2 mM EDTA, 0.02% of NaN3 with 0.5 mM of DSS as a standard internal reference in deuterated environment. 1H NMR spectra were taken at 25oC using a 600 MHz Bruker Avance II Console equipped with a TCI CryoProbe that utilized Bruker Topspin 4 software (Bruker BioSpin Corporation, Billerica, MA, USA). The first slice of a NOESY pulse sequence (noesypr1d) was used to acquire proton NMR. Fractional enrichment for glutamate, lactate and alanine were determined using 13C decoupling ON/OFF 1H proton spectra as well as 1D NOESY spectra. To determine enrichements, a standard zgig pulse sequence was adapted to allow 13C decoupling during the acquistion period (1.36 s) to remove the satellites. Total enrichment was measured by taking a ratio of the metabolite peak heights in the decoupling on/off experiments. NOESY spectra were collected with a 1 s relaxation delay (d1), and a 4 s acqusition time (at), in accordance with Chenomx recommendations for producing quantitative estimates of concentration. Using the Chenomx quantification and the fractional enrichments, a final concentration of the metabolites was calculated. Conventional 1H decoupled 13C spectra were acquired using a 600 MHz Agilent with a specially designed 1.5 mm superconducting (HTS) probe at 30oC.
Sampleprep Protocol Filename:Isolated_muscle_Procedures.docx
Processing Method:Lyophilization and Homogenization
Processing Storage Conditions:-80℃
Extraction Method:Perchloric acid (PCA) or acetonitrile:isopropanol:water (3:3:2) extractions
Extract Storage:-80℃
Sample Resuspension:In 35 microliter of 50 mM phosphate buffer (pH 7.2) with 2 mM EDTA, 0.5 mM DSS and 0.2% sodium azide for aqueous phase samples.
Sample Spiking:0.5 mM DSS

Analysis:

Analysis ID:AN003949
Laboratory Name:McKnight Brain Institute
Analysis Type:NMR
Acquisition Date:11/17/2017 to 05/04/2021
Software Version:Varian
Operator Name:Ram Khattri
Detector Type:Agilent 600 MHz
Data Format:fid, 1r
Num Factors:4
Num Metabolites:2
Units:For Fc3, % unit

NMR:

NMR ID:NM000261
Analysis ID:AN003949
Instrument Name:Agilent 600 MHz
Instrument Type:FT-NMR
NMR Experiment Type:1D-13C
Field Frequency Lock:13C
Standard Concentration:0.5mM DSS
Spectrometer Frequency:600 MHz
NMR Probe:HTS-1A
NMR Solvent:Phosphate buffer (pH 7.2) + 2 mM EDTA + 0.5 mM DSS + 0.2% of sodium azide in deuterated environment
NMR Tube Size:1.5 mm NMR tube
Shimming Method:Varian
Pulse Sequence:s2pul
Water Suppression:WALTZ-16
Pulse Width:45 degree
Receiver Gain:60
Chemical Shift Ref Cpd:DSS
Temperature:30oC
Number Of Scans:6000 to 30000 scans
Dummy Scans:8
Acquisition Time:1.5 s
Relaxation Delay:1.5 s
Spectral Width:36764.7 Hz
Num Data Points Acquired:55147
Real Data Points:65536
Line Broadening:0.5 Hz
Zero Filling:65,536 points
Apodization:Exponential
Baseline Correction Method:Whittaker Smoother
Chemical Shift Ref Std:0 ppm for DSS
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