Summary of Study ST001007

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	ST001007
Study Title	Multi-Platform Physiologic and Metabolic Phenotyping Reveals Microbial Toxicity (part-I)
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
Email	rgn5011@psu.edu
Phone	7247662694
Submit Date	2018-07-13
Raw Data File Type(s)	fid
Analysis Type Detail	NMR
Release Date	2018-08-27
Release Version	1

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Sample Preparation:

Sampleprep ID:	SP001053
Sampleprep Summary:	The microbiota suspension saved after 4h incubation was used for 1H NMR spectroscopy. 1 mL of microbiota suspension was centrifuged at low speed (700 g, 4 °C for 1 min) to pellet down large particles. The maximum supernatant volume was transferred to a new tube, centrifuged at high speed (6000 g, 4 °C for 3 min) to pellet down bacteria. The microbial pellet was washed two times with PBS. After the third wash, 1 mL of pre-cooled methanol:H2O (v/v = 2:1) and 1.0 mm diameter zirconia/silica beads (BioSpec, Bartlesville, OK) were added to the microbial pellet, followed by homogenization (6500 rpm, 1 cycle, 60 s) using the Precellys tissue homogenizer (Bertin Technologies, Rockville, MD). The homogenized sample was freeze-thawed three times with liquid nitrogen and 37°C water bath, then was homogenized again and sonicated for 15 min at 250W with Branson 1510 Ultrasonic Cleaner (Branson Ultrasonics, Danbury, CT) to rupture microbial cell walls and release intracellular metabolites. The sample was centrifuged (11180g, 4 °C, and 10 min) and the supernatants was transferred to a new 2 mL tube. Another 1 mL methanol:H2O (v/v = 2:1) was added to the pellets and the extraction procedure was repeated. All supernatants were combined, dried down and reconstituted in 600 μL of PBS (K2HPO4/NaH2PO4, 0.1M, pH 7.4, containing 50% D2O and 0.005% TSP-d4 as internal standard). Following centrifugation (13 000g, 4 °C, 10min), 550 μL of each extract was transferred into a 5 mm NMR tube for analysis.

Sampleprep ID:

SP001053

Sampleprep Summary:

The microbiota suspension saved after 4h incubation was used for 1H NMR spectroscopy. 1 mL of microbiota suspension was centrifuged at low speed (700 g, 4 °C for 1 min) to pellet down large particles. The maximum supernatant volume was transferred to a new tube, centrifuged at high speed (6000 g, 4 °C for 3 min) to pellet down bacteria. The microbial pellet was washed two times with PBS. After the third wash, 1 mL of pre-cooled methanol:H2O (v/v = 2:1) and 1.0 mm diameter zirconia/silica beads (BioSpec, Bartlesville, OK) were added to the microbial pellet, followed by homogenization (6500 rpm, 1 cycle, 60 s) using the Precellys tissue homogenizer (Bertin Technologies, Rockville, MD). The homogenized sample was freeze-thawed three times with liquid nitrogen and 37°C water bath, then was homogenized again and sonicated for 15 min at 250W with Branson 1510 Ultrasonic Cleaner (Branson Ultrasonics, Danbury, CT) to rupture microbial cell walls and release intracellular metabolites. The sample was centrifuged (11180g, 4 °C, and 10 min) and the supernatants was transferred to a new 2 mL tube. Another 1 mL methanol:H2O (v/v = 2:1) was added to the pellets and the extraction procedure was repeated. All supernatants were combined, dried down and reconstituted in 600 μL of PBS (K2HPO4/NaH2PO4, 0.1M, pH 7.4, containing 50% D2O and 0.005% TSP-d4 as internal standard). Following centrifugation (13 000g, 4 °C, 10min), 550 μL of each extract was transferred into a 5 mm NMR tube for analysis.