Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA240964

Local Sample ID:	2001
Subject ID:	SU002496
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU002496
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
2001	SA240964	FL030298	Capsule Type 1	Treatment

Collection:

Collection ID:	CO002489
Collection Summary:	The CapScan sampling devices (Envivo Bio Inc, San Carlos CA) were constructed with a coating designed to dissolve at a specific pH to take advantage of the pH gradient of the human intestine. After the coating dissolved, a compressed elastic bladder expanded to pull in 400 µL of luminal contents through a oneway valve. This valve remained sealed until recovery from stool. The pH coating of each capsule type Page 13/30 dissolved at pH 5.5 (type 1), 6 (type 2), or 7.5 (types 3 and 4). Type 4 also had a time delay to target the distal ileum or ascending colon. Four sampling capsules were swallowed 3 hours after lunch or dinner across 2 days (Figure 1A). Subjects were instructed to maintain their normal diet, record the time of any food or drink consumed over the testing period, and to not consume caffeinated beverages after lunch on sampling days. Detailed guidelines are provided in Supplemental Material. Stool was collected and immediately frozen at -20 °C until stool was thawed and filled capsule devices were retrieved. Liquid sample was removed from each bladder using a hypodermic needle. An aliquot of each sample was used for 16S rRNA gene sequencing while another aliquot was centrifuged at 10,000 rcf for 3 min, and the supernatant was used for metabolomics analysis.
Sample Type:	Intestine

Treatment:

Treatment ID:	TR002508
Treatment Summary:	We aimed to comprehensively study metabolomic differences among luminal samples from the upper intestinal tract of 15 healthy subjects to better understand the extent of spatial and temporal variation and to gauge the prospects of integrating metabolome and microbiome data. Volunteers swallowed sets of 4 sampling devices per sampling timepoint. These ingestible sampling devices were comprised of a collapsed collection bladder capped by a one-way valve in a capsule treated with pH-sensitive coatings. The four types of capsules differed only in their enteric coating which dissolved at pH 5.5 (capsule 1), pH 6 (capsule 2), and pH 7.5 (capsules 3 and 4) (Figure 1A). The thickness and pH-responsiveness of the coating enabled sampling at specific locations of the intestinal tract after entry into the duodenum. The devices did not contain any electronics beyond a passive radio frequency identification chip for tracking purposes. Once the coatings dissolved, an elastic collection bladder expanded and collected up to 400 µL of luminal contents through vacuum suction. The one-way valve prevented loss of sample and contamination from downstream fluids. Stool samples were frozen at -20 °C and all capsules were recovered from the stool prior to analysis. Liquid contents were retrieved from capsules using hypodermic needles. Aliquots of the raw sample were used for 16S ribosomal RNA microbiome analyses and the supernatants from centrifugated samples were used for metabolomic studies.

Treatment ID:

TR002508

Treatment Summary:

We aimed to comprehensively study metabolomic differences among luminal samples from the upper intestinal tract of 15 healthy subjects to better understand the extent of spatial and temporal variation and to gauge the prospects of integrating metabolome and microbiome data. Volunteers swallowed sets of 4 sampling devices per sampling timepoint. These ingestible sampling devices were comprised of a collapsed collection bladder capped by a one-way valve in a capsule treated with pH-sensitive coatings. The four types of capsules differed only in their enteric coating which dissolved at pH 5.5 (capsule 1), pH 6 (capsule 2), and pH 7.5 (capsules 3 and 4) (Figure 1A). The thickness and pH-responsiveness of the coating enabled sampling at specific locations of the intestinal tract after entry into the duodenum. The devices did not contain any electronics beyond a passive radio frequency identification chip for tracking purposes. Once the coatings dissolved, an elastic collection bladder expanded and collected up to 400 µL of luminal contents through vacuum suction. The one-way valve prevented loss of sample and contamination from downstream fluids. Stool samples were frozen at -20 °C and all capsules were recovered from the stool prior to analysis. Liquid contents were retrieved from capsules using hypodermic needles. Aliquots of the raw sample were used for 16S ribosomal RNA microbiome analyses and the supernatants from centrifugated samples were used for metabolomic studies.

Sample Preparation:

Sampleprep ID:	SP002502
Sampleprep Summary:	For all non-targeted analyses, 10 µL of intestinal lumen samples were subjected to a modified biphasic water, methanol, and methyl tert-butyl ether extraction 78 to separate polar and non-polar metabolites. The polar and non-polar phases were divided into multiple aliquots in 96-well plates, dried by rotary vacuum, and frozen until further analysis. Homogenized stool samples were prepared using an analogous extraction procedure with modification for bead-homogenization and extraction in microcentrifuge tubes to account for the solid nature of the sample. Targeted bile acid analysis was performed using aqueous phase of the described biphasic extraction. Targeted SCFA analysis used an acidified water and MTBE extraction followed by MTBSTFA derivatization 79 . For detailed sample preparation methods see Supplemental Material.

Sampleprep ID:

SP002502

Sampleprep Summary:

For all non-targeted analyses, 10 µL of intestinal lumen samples were subjected to a modified biphasic water, methanol, and methyl tert-butyl ether extraction 78 to separate polar and non-polar metabolites. The polar and non-polar phases were divided into multiple aliquots in 96-well plates, dried by rotary vacuum, and frozen until further analysis. Homogenized stool samples were prepared using an analogous extraction procedure with modification for bead-homogenization and extraction in microcentrifuge tubes to account for the solid nature of the sample. Targeted bile acid analysis was performed using aqueous phase of the described biphasic extraction. Targeted SCFA analysis used an acidified water and MTBE extraction followed by MTBSTFA derivatization 79 . For detailed sample preparation methods see Supplemental Material.

Combined analysis:

Analysis ID	AN003924
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Thermo Vanquish
Column	HILIC BEH Amide
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive HF-X Orbitrap
Ion Mode	NEGATIVE
Units	Peak Height

Chromatography:

Chromatography ID:	CH002903
Instrument Name:	Thermo Vanquish
Column Name:	HILIC BEH Amide
Column Temperature:	45
Flow Gradient:	100%B to 30% B over 15 minutes
Flow Rate:	400 ul/min
Solvent A:	100% water; 0.1% formic acid 10mM ammonium formate
Solvent B:	95% acetonitrile/5% water; 0.1% formic acid; 10 mM ammonium formate
Chromatography Type:	HILIC

MS:

MS ID:	MS003662
Analysis ID:	AN003924
Instrument Name:	Thermo Q Exactive HF-X Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	See associated paper
Ion Mode:	NEGATIVE