Summary of study ST001008

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 PR000681. The data can be accessed directly via it's Project DOI: 10.21228/M8NH4G This work is supported by NIH grant, U2C- DK119886.

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

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Study IDST001008
Study TitleMulti-Platform Physiologic and Metabolic Phenotyping Reveals Microbial Toxicity (part II)
Study SummaryThe gut microbiota are susceptible to modulation by environmental stimuli and therefore can serve as biological sensors. Recent evidence suggests that xenobiotics can disrupt the interaction between the microbiota and host. Here, we describe an approach that combines in vitro microbial incubation (isolated cecal contents from mice), flow cytometry, and mass spectrometry- and 1H NMR-based metabolomics to evaluate xenobiotic-induced microbial toxicity. Tempol, a stabilized free radical scavenger known to remodel the microbial community structure and function in vivo, was studied to assess its direct effects on the gut microbiota. Microbiota were isolated from mouse cecum and were exposed to tempol for 4 h under strict anaerobic condition. The flow cytometry data suggested short term exposure of the microbiota to tempol is associated with disrupted membrane physiology as well as compromised metabolic activity. Mass spectrometry and NMR metabolomics revealed that tempol exposure significantly disrupted microbial metabolic activity, specifically indicated by changes in short chain fatty acids, branched chain amino acids, amino acids, nucleotides, glucose, and oligosaccharides. In addition, a mouse study with tempol (5 days gavage) showed similar microbial physiologic and metabolic changes, indicating the in vitro approach reflected in vivo conditions. Our results, through evaluation of microbial viability, physiology and metabolism, and comparison of in vitro and in vivo exposures with tempol, suggests that physiologic and metabolic phenotyping provides unique insight into gut microbiota toxicity.
Institute
Pennsylvania State University
Last NameNichols
First NameRobert
Address650 toftrees ave Apt #108, State College, Pa 16802
Emailrgn5011@psu.edu
Phone7247662694
Submit Date2018-07-15
Raw Data AvailableYes
Raw Data File Type(s).raw
Analysis Type DetailLC-MS
Release Date2018-08-27
Release Version1
Robert Nichols Robert Nichols
https://dx.doi.org/10.21228/M8NH4G
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000681
Project DOI:doi: 10.21228/M8NH4G
Project Title:Multi-Platform Physiologic and Metabolic Phenotyping Reveals Microbial Toxicity
Project Summary:The gut microbiota are susceptible to modulation by environmental stimuli and therefore can serve as biological sensors. Recent evidence suggests that xenobiotics can disrupt the interaction between the microbiota and host. Here, we describe an approach that combines in vitro microbial incubation (isolated cecal contents from mice), flow cytometry, and mass spectrometry- and 1H NMR-based metabolomics to evaluate xenobiotic-induced microbial toxicity. Tempol, a stabilized free radical scavenger known to remodel the microbial community structure and function in vivo, was studied to assess its direct effects on the gut microbiota. Microbiota were isolated from mouse cecum and were exposed to tempol for 4 h under strict anaerobic condition. The flow cytometry data suggested short term exposure of the microbiota to tempol is associated with disrupted membrane physiology as well as compromised metabolic activity. Mass spectrometry and NMR metabolomics revealed that tempol exposure significantly disrupted microbial metabolic activity, specifically indicated by changes in short chain fatty acids, branched chain amino acids, amino acids, nucleotides, glucose, and oligosaccharides. In addition, a mouse study with tempol (5 days gavage) showed similar microbial physiologic and metabolic changes, indicating the in vitro approach reflected in vivo conditions. Our results, through evaluation of microbial viability, physiology and metabolism, and comparison of in vitro and in vivo exposures with tempol, suggests that physiologic and metabolic phenotyping provides unique insight into gut microbiota toxicity.
Institute:Pennsylvania State University
Last Name:Nichols
First Name:Robert
Address:650 toftrees ave Apt #108, State College, Pa 16802
Email:rgn5011@psu.edu
Phone:7247662694

Subject:

Subject ID:SU001047
Subject Type:Other
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

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

mb_sample_id local_sample_id Treatment
SA063432C1Control
SA063433C6Control
SA063434C5Control
SA063435C2Control
SA063436C3Control
SA063437C4Control
SA063438H6High Dose Tempol
SA063439H5High Dose Tempol
SA063440H2High Dose Tempol
SA063441H4High Dose Tempol
SA063442H1High Dose Tempol
SA063443H3High Dose Tempol
SA063444L5Low Dose Tempol
SA063445L6Low Dose Tempol
SA063446L4Low Dose Tempol
SA063447L3Low Dose Tempol
SA063448L1Low Dose Tempol
SA063449L2Low Dose Tempol
SA063450M5Medium Dose Tempol
SA063451M4Medium Dose Tempol
SA063452M6Medium Dose Tempol
SA063453M3Medium Dose Tempol
SA063454M1Medium Dose Tempol
SA063455M2Medium Dose Tempol
SA063456P6PH4
SA063457P5PH4
SA063458P3PH4
SA063459P1PH4
SA063460P2PH4
SA063461P4PH4
Showing results 1 to 30 of 30

Collection:

Collection ID:CO001041
Collection Summary:6-week-old wild-type male C57BL/6J mice (Jackson Laboratory, Bar Harbor, Maine) were transferred into anaerobic chamber (Coy Laboratory Products, Inc., Grass Lake, MI) following CO2 asphyxiation. All the following procedures were performed under strict anaerobic conditions with an oxygen level below 20 ppm. Cecal contents were collected
Sample Type:Cecum

Treatment:

Treatment ID:TR001061
Treatment Summary:The cecal content suspension was treated with tempol at a final concentration 0.01 mg/mL, 0.1 mg/mL and 1 mg/mL, following a brief centrifugation and incubation at 37 °C for 4 h in dark.

Sample Preparation:

Sampleprep ID:SP001054
Sampleprep Summary:600 µL of bacteria suspension after 4h incubation was centrifuged (700 g, 4 °C for 1 min) and supernatants were transferred to a new tube, centrifuged (6000 g, 4 °C for 3 min) and washed 3 times with PBS. After the final centrifugation, 1 mL cold 50% aqueous methanol containing 1 µM chlorpropamide and 1.0 mm diameter zirconia/silica beads (BioSpec, Bartlesville, OK) were added to the microbial pellet, followed with homogenization (6500 rpm, 1 cycle, 60 s). The sample was freeze-thawed three times with liquid nitrogen to break apart tough microbial cell wall. Then the sample was centrifuged (max speed, 4 °C, and 10 min), supernatants were transferred to a new EP tube, dried down and resuspended in 200 µL 3% aqueous methanol. After a final spin (max speed, 4 °C, and 10 min), 150 µL supernatants were transferred to autosampler for LC-MS analysis.
Sampleprep Protocol ID:LC sample prep.docx
Sampleprep Protocol Filename:LC_sample_prep.docx

Combined analysis:

Analysis ID AN001650
Analysis type MS
Chromatography type Reversed phase
Chromatography system Thermo Fisher Orbitrap Exactive plus
Column Phenomenex (Torrance, CA) Hydro-RP C18 column
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Plus Orbitrap
Ion Mode NEGATIVE
Units ppm

Chromatography:

Chromatography ID:CH001161
Chromatography Summary:Metabolomics profiling was performed with a Dionex Ultimate 3000 quaternary high-performance liquid chromatography (HPLC) pump, column compartment and autosampler coupled Exactive plus Orbitrap mass spectrometer controlled by Xcalibur 2.2 software (Thermo Fisher Scientific, Waltham, MA). LC-MS was run with a modified ion pairing reversed phase negative ion electrospray ionization method (61). A total volume of 10 µL sample is injected and separated on a Phenomenex (Torrance, CA) Hydro-RP C18 column (100 × 2.1 mm, 2.5 µm particle size) using a water/methanol gradient with tributylamine and acetic acid added to the aqueous mobile phase to enhance separation. The HPLC column is maintained at flow rate of 200 µL/min with the temperature of 30 °C. Solvents and gradient are as follows: Solvent A is 3% aqueous methanol with 10 mM tributylamine and 15 mM acetic acid; solvent B is 100% methanol. The gradient is 0 min, 0% B; 5 min, 20% B; 7.5 min, 20% B; 13 min, 55% B; 15.5 min, 95% B; 18.5 min, 95% B; 19 min, 0% B; and 25 min, 0% B. The Exactive plus is operated in negative ion mode at maximum resolving power (140,000), and scanned from m/z 72 -1000 for the first 90 sec and then from m/z 85-1000 for the remainder of the chromatographic run for the detection of small molecule metabolites. The automatic gain control target is 3E6 with a maximum injection time of 100 us, the nitrogen sheath gas flow rate is set at 35, the auxillary gas at 10 and the sweep gas at 1. The capillary voltage is 3.2 kV and both the capillary and heater set at 200 °C, the S-lens was 55.
Instrument Name:Thermo Fisher Orbitrap Exactive plus
Column Name:Phenomenex (Torrance, CA) Hydro-RP C18 column
Chromatography Type:Reversed phase

MS:

MS ID:MS001525
Analysis ID:AN001650
Instrument Name:Thermo Q Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
Ion Mode:NEGATIVE
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