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.

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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(Thermo)
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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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
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