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
| Study ID | ST001008 |
| Study Title | Multi-Platform Physiologic and Metabolic Phenotyping Reveals Microbial Toxicity (part II) |
| Study 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 |
| rgn5011@psu.edu | |
| Phone | 7247662694 |
| Submit Date | 2018-07-15 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2018-08-27 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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 |
| Species Group: | Mammals |
Factors:
Subject type: Other; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Treatment |
|---|---|---|
| SA063432 | C1 | Control |
| SA063433 | C6 | Control |
| SA063434 | C5 | Control |
| SA063435 | C2 | Control |
| SA063436 | C3 | Control |
| SA063437 | C4 | Control |
| SA063438 | H6 | High Dose Tempol |
| SA063439 | H5 | High Dose Tempol |
| SA063440 | H2 | High Dose Tempol |
| SA063441 | H4 | High Dose Tempol |
| SA063442 | H1 | High Dose Tempol |
| SA063443 | H3 | High Dose Tempol |
| SA063444 | L5 | Low Dose Tempol |
| SA063445 | L6 | Low Dose Tempol |
| SA063446 | L4 | Low Dose Tempol |
| SA063447 | L3 | Low Dose Tempol |
| SA063448 | L1 | Low Dose Tempol |
| SA063449 | L2 | Low Dose Tempol |
| SA063450 | M5 | Medium Dose Tempol |
| SA063451 | M4 | Medium Dose Tempol |
| SA063452 | M6 | Medium Dose Tempol |
| SA063453 | M3 | Medium Dose Tempol |
| SA063454 | M1 | Medium Dose Tempol |
| SA063455 | M2 | Medium Dose Tempol |
| SA063456 | P6 | PH4 |
| SA063457 | P5 | PH4 |
| SA063458 | P3 | PH4 |
| SA063459 | P1 | PH4 |
| SA063460 | P2 | PH4 |
| SA063461 | P4 | PH4 |
| 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 |
| 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 Temperature: | 30 |
| Flow Gradient: | 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. |
| Flow Rate: | 200 µL/min |
| Solvent A: | 97% water/3% methanol; 15 mM acetic acid; 10 mM tributylamine |
| Solvent B: | 100% methanol |
| 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 |
