Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA241649

Local Sample ID:	1418
Subject ID:	SU002500
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU002500
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
1418	SA241649	FL030317	Capsule Type 1	Treatment

Collection:

Collection ID:	CO002493
Collection Summary:	Fifteen healthy subjects were enrolled in this study, and each swallowed at least 17 devices over the course of three days. Sample size was chosen to assess general variation across human intestinal tracts. Daily instructions included the following guidelines: record all foods and time they were consumed throughout the day; if you work out, do so in the morning; eat breakfast and lunch as usual; swallow a set of four devices three hours after lunch with up to 2/3 cup water; do not eat or drink anything for at least two hours after swallowing devices; if hungry after two hours, snack lightly (up to 200 calories); do not drink any caffeinated beverages after lunch until the next morning; collect all stool starting six hours after swallowing this set of devices until 48 hours after swallowing the next set of devices; eat dinner as usual at least six hours after lunch; swallow a set of four CapScan devices three hours after dinner with 2/3 cup water; after swallowing this set, do not eat or drink anything until the morning. Alcohol consumption and diet contents were not restricted. All ingested devices were recovered, and no adverse events were reported during the study. In total 274 capsule devices provided sufficient material for metabolomics analysis, and 225 provided sufficient volume or number of sequencing reads (>2500) for genomic analysis. Every bowel movement during the study was immediately frozen by the subject at -20 °C. Subject 1 provided additional samples for assessment of replicability and blooming. A total of 333 intestinal, and stool samples were analyzed with metabolomics methods.
Sample Type:	Intestine

Treatment:

Treatment ID:	TR002512
Treatment Summary:	The capsule sampling device (CapScan®, Envivo® Bio Inc, San Carlos, CA) consists of a one-way valve capping a hollow elastic collection bladder5 . The device is prepared for packaging by evacuating the collection bladder, folding it in half, and packaging the folded device inside a dissolvable capsule measuring 6.5 mm in diameter and 23 mm in length, onto which an enteric coating is applied. The capsule and the enteric coating prevent contamination of the collection bladder from oral-pharyngeal and gastric microbes during ingestion. When the device reaches the target pH, the enteric coating and capsule disintegrate. The target pH is pH 5.5 for type 1, pH 6 for type 2, and pH 7.5 for type 3 and type 4, with type 4 also having a time-delay coating to bias collection toward the ascending colon. After the enteric coating disintegrates, the collection bladder unfolds and expands into a tube 6 mm in diameter and 33 mm in length, thereby drawing in up to 400 µL of gut luminal contents through the one-way valve. The one-way valve maintains the integrity of the sample collected inside the collection bladder as the device moves through the colon and is exposed to stool. In this study, subjects concurrently ingested sets of 4 capsules, each with distinct coatings to target the proximal to medial regions of the small intestine (coating types 1 and 2) and more distal regions (coating types 3 and 4). After sampling, the devices were passed in the stool into specimen-collection containers and immediately frozen. After completion of sampling, the stool was thawed, and the devices were retrieved by study staff. The elastic collection bladders were rinsed in 70% isopropyl alcohol and punctured with a sterile hypodermic needle attached to a 1-mL syringe for sample removal. Samples were transferred into microcentrifuge tubes and the pH was measured with an InLab Ultra Micro ISM pH probe (Mettler Toledo). A 40-µL aliquot was spun down for 3 min at 10,000 rcf, and its supernatant was used for metabolomics analysis. The rest of the sample was frozen until being thawed for DNA extraction.

Treatment ID:

TR002512

Treatment Summary:

The capsule sampling device (CapScan®, Envivo® Bio Inc, San Carlos, CA) consists of a one-way valve capping a hollow elastic collection bladder5 . The device is prepared for packaging by evacuating the collection bladder, folding it in half, and packaging the folded device inside a dissolvable capsule measuring 6.5 mm in diameter and 23 mm in length, onto which an enteric coating is applied. The capsule and the enteric coating prevent contamination of the collection bladder from oral-pharyngeal and gastric microbes during ingestion. When the device reaches the target pH, the enteric coating and capsule disintegrate. The target pH is pH 5.5 for type 1, pH 6 for type 2, and pH 7.5 for type 3 and type 4, with type 4 also having a time-delay coating to bias collection toward the ascending colon. After the enteric coating disintegrates, the collection bladder unfolds and expands into a tube 6 mm in diameter and 33 mm in length, thereby drawing in up to 400 µL of gut luminal contents through the one-way valve. The one-way valve maintains the integrity of the sample collected inside the collection bladder as the device moves through the colon and is exposed to stool. In this study, subjects concurrently ingested sets of 4 capsules, each with distinct coatings to target the proximal to medial regions of the small intestine (coating types 1 and 2) and more distal regions (coating types 3 and 4). After sampling, the devices were passed in the stool into specimen-collection containers and immediately frozen. After completion of sampling, the stool was thawed, and the devices were retrieved by study staff. The elastic collection bladders were rinsed in 70% isopropyl alcohol and punctured with a sterile hypodermic needle attached to a 1-mL syringe for sample removal. Samples were transferred into microcentrifuge tubes and the pH was measured with an InLab Ultra Micro ISM pH probe (Mettler Toledo). A 40-µL aliquot was spun down for 3 min at 10,000 rcf, and its supernatant was used for metabolomics analysis. The rest of the sample was frozen until being thawed for DNA extraction.

Sample Preparation:

Sampleprep ID:	SP002506
Sampleprep Summary:	Sample preparation was performed using a biphasic extraction64 with water, methanol, and methyl tert-butyl ether to separate polar and non-polar metabolites. Capsule device supernatant and stool samples were prepared separately because device samples were liquid and stool samples were solid. For each supernatant sample, 10 µL were aliquoted into one well of a deep sample preparation 96-well plate in a pre-determined randomized order. Samples were extracted one 96-well plate at a time and all steps were carried out at 4 °C unless otherwise specified. Between every 10 experimental samples, a method blank and external QC sample were prepared. Blanks used 10 µL of LC-MS grade water instead of sample, and QC samples used 10 µL of a pooled sample of human gastrointestinal tract contents from unrelated studies. One hundred seventy microliters of methanol containing SPLASH LIPIDOMIX Mass Spec Standard (Avanti) were added to each well and the plate was heat-sealed with foil, shaken vigorously for 30 s at room temperature, unsealed, and 490 µL of methyl tert-butyl ether (MTBE) were added. The plate was then heat-sealed again, vortexed vigorously for 30 s at room temperature, and shaken for 5 min on an orbital shaker. The foil seal was removed and 150 µL of LC-MS grade water were added to each well. The plate was vortexed for 30 s at room temperature and centrifuged at 2400 rpm for 12 min. The foil was removed from the deep-well plate and two 180-µL aliquots of the top phase were transferred to two 96-well Vanquish LC plate using a 12-channel pipette. Two 50-µL aliquots of the aqueous phase were then transferred to two other 96-well Vanquish LC plates. All 96-well plates were dried completely under vacuum at room temperature, heat sealed with foil, and stored at -80 °C until further analysis. Each stool sample was prepared by mixing with spatula and 5±1 mg were transferred to a 2-mL microcentrifuge tube. Two hundred twenty-five microliters of methanol containing SPLASH LIPIDOMIX Mass Spec Standard (Avanti) were added to all microcentrifuge tubes and the tubes were vortexed for 10 s at room temperature. Seven hundred fifty microliters of MTBE and two 3-mm stainless steel balls were added to each tube and samples were homogenized in a Geno/Grinder (SPEX) at 1500 rpm for 1 min. One hundred eighty-eight microliters of water were added to each tube and each tube was vortexed for 30 s at room temperature. Tubes were centrifuged at 14,000 rcf for 2 min at room temperature. Two aliquots of 180 µL of the organic phase were transferred to two 96-well plates. Two 50-µL aliquots of the aqueous phase were transferred to two 96-well plates. All plates were dried completely in a rotary vacuum evaporator, heat-sealed with foil, and stored at -80 °C until further analysis.

Sampleprep ID:

SP002506

Sampleprep Summary:

Sample preparation was performed using a biphasic extraction64 with water, methanol, and methyl tert-butyl ether to separate polar and non-polar metabolites. Capsule device supernatant and stool samples were prepared separately because device samples were liquid and stool samples were solid. For each supernatant sample, 10 µL were aliquoted into one well of a deep sample preparation 96-well plate in a pre-determined randomized order. Samples were extracted one 96-well plate at a time and all steps were carried out at 4 °C unless otherwise specified. Between every 10 experimental samples, a method blank and external QC sample were prepared. Blanks used 10 µL of LC-MS grade water instead of sample, and QC samples used 10 µL of a pooled sample of human gastrointestinal tract contents from unrelated studies. One hundred seventy microliters of methanol containing SPLASH LIPIDOMIX Mass Spec Standard (Avanti) were added to each well and the plate was heat-sealed with foil, shaken vigorously for 30 s at room temperature, unsealed, and 490 µL of methyl tert-butyl ether (MTBE) were added. The plate was then heat-sealed again, vortexed vigorously for 30 s at room temperature, and shaken for 5 min on an orbital shaker. The foil seal was removed and 150 µL of LC-MS grade water were added to each well. The plate was vortexed for 30 s at room temperature and centrifuged at 2400 rpm for 12 min. The foil was removed from the deep-well plate and two 180-µL aliquots of the top phase were transferred to two 96-well Vanquish LC plate using a 12-channel pipette. Two 50-µL aliquots of the aqueous phase were then transferred to two other 96-well Vanquish LC plates. All 96-well plates were dried completely under vacuum at room temperature, heat sealed with foil, and stored at -80 °C until further analysis. Each stool sample was prepared by mixing with spatula and 5±1 mg were transferred to a 2-mL microcentrifuge tube. Two hundred twenty-five microliters of methanol containing SPLASH LIPIDOMIX Mass Spec Standard (Avanti) were added to all microcentrifuge tubes and the tubes were vortexed for 10 s at room temperature. Seven hundred fifty microliters of MTBE and two 3-mm stainless steel balls were added to each tube and samples were homogenized in a Geno/Grinder (SPEX) at 1500 rpm for 1 min. One hundred eighty-eight microliters of water were added to each tube and each tube was vortexed for 30 s at room temperature. Tubes were centrifuged at 14,000 rcf for 2 min at room temperature. Two aliquots of 180 µL of the organic phase were transferred to two 96-well plates. Two 50-µL aliquots of the aqueous phase were transferred to two 96-well plates. All plates were dried completely in a rotary vacuum evaporator, heat-sealed with foil, and stored at -80 °C until further analysis.

Combined analysis:

Analysis ID	AN003930
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Vanquish
Column	Waters Acquity UPLC CSH C18 column (100 mm length × 2.1 mm i.d.; 1.7-µm particle size)
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:	CH002909
Instrument Name:	Thermo Vanquish
Column Name:	Waters Acquity UPLC CSH C18 column (100 mm length × 2.1 mm i.d.; 1.7-µm particle size)
Column Temperature:	65
Flow Gradient:	15% B from 0 to 0.6 min, 30% B by 2 min, 48% B by 2.5 min, 82% B by 11 min, 99% B from 11.5 to 12 min, and 15% B from 12.1 to 14.2 min
Flow Rate:	600 µL/min
Solvent A:	90% acetonitrile/10% water; 10 mM ammonium acetate
Solvent B:	80% isopropanol/20% acetonitrile; 10 mM ammonium acetate
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003668
Analysis ID:	AN003930
Instrument Name:	Thermo Q Exactive HF-X Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	HESI source conditions are as follows: sheath gas flow 55, auxiliary gas flow 15, sweep gas flow 3, capillary temperature 275°C, S-lens RF level 50, auxiliary gas heater temperature 450 °C, and needle voltage 3500 V and -3500 V for positive and negative ionization mode, respectively. DDA MS/MS spectra were acquired for the top 4 ions. MS scans were collected with 60k resolving power from 120-1700 m/z, AGC target of 106 ions, and maximum accumulation time of 100 ms. MS/MS spectra were collected with 15k resolving power, 1 Da isolation window, normalized collision energy of 20, 30, 60, 2 s dynamic exclusion window, 8×103 AGC target, and 50 ms maximum accumulation time. Spectra were stored in centroid mode. Three rounds of iterative exclusion MS/MS were acquired for each pooled QC sample.
Ion Mode:	NEGATIVE