Summary of study ST001521

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 PR001024. The data can be accessed directly via it's Project DOI: 10.21228/M8B984 This work is supported by NIH grant, U2C- DK119886.

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Study IDST001521
Study TitlePlasma metabolites of known identity profiled using hybrid nontargeted methods (part-III)
Study SummaryWe determined the effect of diet on the composition and metabolic function of human gut microbiome using a controlled feeding experiment with three divergent diets (vegan, omnivore, and an exclusive enteral nutrition diet (EEN) devoid of dietary fiber) in the Food And Resulting Microbial Metabolites (FARMM) study. The study included an antibiotic and polyethylene glycol (Abx/PEG) intervention to dynamically assess the effect of diet on the reconstitution of the gut microbiota and its associated fecal and plasma metabolome. Samples from thirty healthy volunteers between the ages of 18 and 60, 10 mper diet group were analyzed. Self-reported vegans were required to have followed a vegan diet for a minimum of 6 months prior to enrollment. Key exclusion criteria included inflammatory bowel disease, celiac disease, or other chronic intestinal disorders; prior bowel resection surgery other than appendectomy; baseline bowel frequency less than every 2 days or greater than 3 times daily; creatinine concentration greater than the upper limit of normal; diabetes mellitus; currently smoking; body mass index (BMI) <18.5 or >35; and use of antibiotics and probiotics in the prior 6 months. The 10 vegans continued to follow their usual diet as outpatients. All participants completed the Diet History Questionnaire II (DHQ II), a food frequency questionnaire developed by the Risk Factor Monitoring and Methods Branch of the National Cancer Institute. The vegan participants also completed three 24 hour diet recalls with a dietitian in the week prior to starting antibiotics. We randomly assigned the 20 omnivores to receive an omnivore diet or EEN (Modulen® IBD) while residing in an inpatient research unit. The macronutrient composition of Modulen® is protein 36g, fat 47g, and carbohydrate 110g per 1000 Kcal. The two omnivore diets were engineered to have a similar composition to EEN. All the subjects consumed the menu A on day 11 and menu B on days 4 and 14. Diets for omnivores were constructed to provide the expected total calories required per day for the participant to maintain their current weight and were adjusted if there was weight gain or loss of more than 2.5 pounds. On days 6, 7, and 8, inpatients participants received vancomycin 500mg orally every 6 hours and neomycin 1000mg orally every 6 hours. On day 7, participants consumed 4L of polyethylene glycol (PEG) based bowel purgative (GoLytely®). Participants in the omnivore and EEN arms left the inpatient research unit only under the direct supervision of a research staff member. The vegan outpatients reported to the hospital twice on days 6,7 and 8 to receive antibiotics and to consume PEG on day 7. The first stool sample of each day of the inpatient groups was collected, aliquoted and frozen immediately at -80oC. Blood was collected on days 1, 5, 9, 12 and 15 from which plasma aliquots were immediately isolated and frozen at -80oC. The outpatient participants following the vegan diet also had blood collected on days 1, 5, 9, 12, and 15 from which plasma aliquots were immediately isolated and frozen at -80oC. Among these participants, the first stool of the day was collected at home daily and kept on ice packs until it was brought to the research unit where it could be aliquoted and frozen. Samples from vegans were received for aliquoting within 24 hours and on average within 4 hours (Wu et al., 2010). Day 0 stool was not collected from vegans since their diet did not change. The aliquoted amounts for all samples ranged from 500 mg to 1 g. The aliquots were taken from different areas of the sample. Remaining sample was then collected into one residual 50 ml conical tube with a tongue depressor. Any remaining stool was discarded. The University of Pennsylvania Institutional Review Board (IRB) approved the research protocol.
Institute
Broad Institute of MIT and Harvard
Last NameClish
First NameClary
Address415 Main Street, Cambridge, MA, 02142, USA
Emailclary@broadinstitute.org
Phone617-714-7654
Submit Date2020-11-05
Num Groups3
Total Subjects30
Num Males20
Num Females10
Study Commentsplasma samples collected at baseline and 3-4 timepoints
Raw Data AvailableYes
Raw Data File Type(s).raw
Analysis Type DetailLC-MS
Release Date2021-04-01
Release Version1
Clary Clish Clary Clish
https://dx.doi.org/10.21228/M8B984
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:


Project:

Project ID:PR001024
Project DOI:doi: 10.21228/M8B984
Project Title:Role of Diet in the Reconstitution of the Human Gut Microbiome and its Metabolome
Project Type:Observational study
Project Summary:We studied the impact of three divergent diets, vegan, omnivore, and a synthetic enteral nutrition (EEN) diet lacking fiber, on the human gut microbiome and its metabolome in a longitudinal analysis that included a microbiota depletion intervention. Hybrid nontargeted LC-MS methods were used to profile stool and plasma metabolites.
Institute:Broad Institute of MIT and Harvard
Last Name:Clish
First Name:Clary
Address:415 Main Street, Cambridge, MA, 02142, USA
Email:clary@broadinstitute.org
Phone:617-714-7654
Funding Source:Crohn’s & Colitis Foundation, P30 DK 050306, PennCHOP Microbiome Program, and the Penn Center for Nutritional Science and Medicine
Contributors:Ceylan Tanes, Kyle Bittinger, Yuan Gao, Elliot S. Friedman, Lisa Nessel, Unmesha Roy Paladhi, Lillian Chau, Erika Panfen, Michael A. Fischbach, Jonathan Braun, Ramnik J. Xavier, Clary B. Clish, Hongzhe Li, Frederic D. Bushman, James D. Lewis, Gary D. Wu

Subject:

Subject ID:SU001595
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Age Or Age Range:20-60
Weight Or Weight Range:BMI range: 20-35
Gender:Male and female
Human Race:White; American Indian/Alaskan Native; Black/African American
Human Ethnicity:Hispanic or Latino; Non-Hispanic or Latino
Human Trial Type:Intervention trial
Human Exclusion Criteria:Key exclusion criteria included inflammatory bowel disease, celiac disease, or other chronic intestinal disorders; prior bowel resection surgery other than appendectomy; baseline bowel frequency less than every 2 days or greater than 3 times daily; creatinine concentration greater than the upper limit of normal; diabetes mellitus; currently smoking; body mass index (BMI) <18.5 or >35; and use of antibiotics and probiotics in the prior 6 months

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Study_Diet Sex Time
SA1280509024-2-PEModulen Female Baseline
SA1280609016-2-PEModulen Female Baseline
SA1280709034-2-PEModulen Female Baseline
SA1280809038-2-PEModulen Female Baseline
SA1280519024-2-PBModulen Female Day 12
SA1280619016-2-PBModulen Female Day 12
SA1280719034-2-PBModulen Female Day 12
SA1280819038-2-PBModulen Female Day 12
SA1280529024-2-PAModulen Female Day 15
SA1280629016-2-PAModulen Female Day 15
SA1280729034-2-PAModulen Female Day 15
SA1280829038-2-PAModulen Female Day 15
SA1280539024-2-PDModulen Female Day 5
SA1280639016-2-PDModulen Female Day 5
SA1280739034-2-PDModulen Female Day 5
SA1280839038-2-PDModulen Female Day 5
SA1280549024-2-PCModulen Female Day 9
SA1280649016-2-PCModulen Female Day 9
SA1280749034-2-PCModulen Female Day 9
SA1280849038-2-PCModulen Female Day 9
SA1280359003-2-PEModulen Male Baseline
SA1280409009-2-PEModulen Male Baseline
SA1280459029-2-PEModulen Male Baseline
SA1280559025-2-PEModulen Male Baseline
SA1280659013-2-PEModulen Male Baseline
SA1280759032-2-PEModulen Male Baseline
SA1280369003-2-PBModulen Male Day 12
SA1280419009-2-PBModulen Male Day 12
SA1280469029-2-PBModulen Male Day 12
SA1280569025-2-PBModulen Male Day 12
SA1280669013-2-PBModulen Male Day 12
SA1280769032-2-PBModulen Male Day 12
SA1280379003-2-PAModulen Male Day 15
SA1280429009-2-PAModulen Male Day 15
SA1280479029-2-PAModulen Male Day 15
SA1280579025-2-PAModulen Male Day 15
SA1280679013-2-PAModulen Male Day 15
SA1280779032-2-PAModulen Male Day 15
SA1280389003-2-PDModulen Male Day 5
SA1280439009-2-PDModulen Male Day 5
SA1280489029-2-PDModulen Male Day 5
SA1280589025-2-PDModulen Male Day 5
SA1280689013-2-PDModulen Male Day 5
SA1280789032-2-PDModulen Male Day 5
SA1280399003-2-PCModulen Male Day 9
SA1280449009-2-PCModulen Male Day 9
SA1280499029-2-PCModulen Male Day 9
SA1280599025-2-PCModulen Male Day 9
SA1280699013-2-PCModulen Male Day 9
SA1280799032-2-PCModulen Male Day 9
SA128085QPP09NA NANA
SA128086QPP10NA NANA
SA128087QPP06NA NANA
SA128088QPP08NA NANA
SA128089QPP07NA NANA
SA128090QPP03NA NANA
SA128091QPP05NA NANA
SA128092QPP04NA NANA
SA128093QPP02NA NANA
SA128094QPP01NA NANA
SA1281009027-3-PEVegan Female Baseline
SA1281059014-3-PEVegan Female Baseline
SA1281159026-3-PEVegan Female Baseline
SA1281409031-3-PEVegan Female Baseline
SA1281019027-3-PBVegan Female Day 12
SA1281069014-3-PBVegan Female Day 12
SA1281169026-3-PBVegan Female Day 12
SA1281419031-3-PBVegan Female Day 12
SA1281029027-3-PAVegan Female Day 15
SA1281079014-3-PAVegan Female Day 15
SA1281179026-3-PAVegan Female Day 15
SA1281429031-3-PAVegan Female Day 15
SA1281039027-3-PDVegan Female Day 5
SA1281089014-3-PDVegan Female Day 5
SA1281189026-3-PDVegan Female Day 5
SA1281439031-3-PDVegan Female Day 5
SA1281049027-3-PCVegan Female Day 9
SA1281099014-3-PCVegan Female Day 9
SA1281199026-3-PCVegan Female Day 9
SA1281449031-3-PCVegan Female Day 9
SA1280959002-3-PEVegan Male Baseline
SA1281109008-3-PEVegan Male Baseline
SA1281209004-3-PEVegan Male Baseline
SA1281259017-3-PEVegan Male Baseline
SA1281269035-3-PEVegan Male Baseline
SA1281279022-3-PEVegan Male Baseline
SA1280969002-3-PBVegan Male Day 12
SA1281119008-3-PBVegan Male Day 12
SA1281219004-3-PBVegan Male Day 12
SA1281289022-3-PBVegan Male Day 12
SA1281299017-3-PBVegan Male Day 12
SA1281309035-3-PBVegan Male Day 12
SA1280979002-3-PAVegan Male Day 15
SA1281129008-3-PAVegan Male Day 15
SA1281229004-3-PAVegan Male Day 15
SA1281319017-3-PAVegan Male Day 15
SA1281329035-3-PAVegan Male Day 15
SA1281339022-3-PAVegan Male Day 15
SA1280989002-3-PDVegan Male Day 5
SA1281139008-3-PDVegan Male Day 5
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Collection:

Collection ID:CO001590
Collection Summary:The first stool sample of each day of the inpatient groups was collected, aliquoted and frozen immediately at -80oC. Blood was collected on days 1, 5, 9, 12 and 15 from which plasma aliquots were immediately isolated and frozen at -80oC. The outpatient participants following the vegan diet also had blood collected on days 1, 5, 9, 12, and 15 from which plasma aliquots were immediately isolated and frozen at -80oC. Among these participants, the first stool of the day was collected at home daily and kept on ice packs until it was brought to the research unit where it could be aliquoted and frozen. Samples from vegans were received for aliquoting within 24 hours and on average within 4 hours (Wu et al., 2010). Day 0 stool was not collected from vegans since their diet did not change. The aliquoted amounts for all samples ranged from 500 mg to 1 g. The aliquots were taken from different areas of the sample. Remaining sample was then collected into one residual 50 ml conical tube with a tongue depressor. Any remaining stool was discarded. The University of Pennsylvania Institutional Review Board (IRB) approved the research protocol.
Sample Type:Blood (plasma)
Storage Conditions:-80℃

Treatment:

Treatment ID:TR001610
Treatment Summary:The 10 vegan participants continued to follow their usual diet as outpatients. All participants completed the Diet History Questionnaire II (DHQ II), a food frequency questionnaire developed by the Risk Factor Monitoring and Methods Branch of the National Cancer Institute. The vegan participants also completed three 24 hour diet recalls with a dietitian in the week prior to starting antibiotics. 20 omnivores were randomly assigned to receive an omnivore diet or EEN (Modulen® IBD) while residing in an inpatient research unit. The macronutrient composition of Modulen® is protein 36g, fat 47g, and carbohydrate 110g per 1000 Kcal. The two omnivore diets were engineered to have a similar composition to EEN. All the subjects consumed the menu A on day 11 and menu B on days 4 and 14. Diets for omnivores were constructed to provide the expected total calories required per day for the participant to maintain their current weight and were adjusted if there was weight gain or loss of more than 2.5 pounds. On days 6, 7, and 8, inpatients participants received vancomycin 500mg orally every 6 hours and neomycin 1000mg orally every 6 hours. On day 7, participants consumed 4L of polyethylene glycol (PEG) based bowel purgative (GoLytely®). Participants in the omnivore and EEN arms left the inpatient research unit only under the direct supervision of a research staff member. The vegan outpatients reported to the hospital twice on days 6,7 and 8 to receive antibiotics and to consume PEG on day 7.

Sample Preparation:

Sampleprep ID:SP001603
Sampleprep Summary:Plasma samples were thawed on ice and aliquoted for metabolite profiling analyses. Plasma samples were thawed and aliquoted for each LC-MS method. LC-MS samples were prepared for four profiling methods as follows: HILIC-pos: Metabolites were extracted by adding 90 μL of 74.9:24.9:0.2 v/v/v acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards (valine-d8, Isotec; and phenylalanine-d8, Cambridge Isotope Laboratories; Andover, MA) to a 10 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, 4°C) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis. C8-pos: Lipids were extracted by adding 190 μL of isopropanol containing 1-dodecanoyl-2-tridecanoyl-sn-glycero-3-phosphocholine as an internal standard (Avanti Polar Lipids; Alabaster, AL) to a 10 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, ambient temperature) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis. HILIC-neg: Metabolites were extracted by adding 120 μL of 80% methanol containing inosine-15N4, thymine-d4 and glycocholate-d4 internal standards (Cambridge Isotope Laboratories; Andover, MA) to a 30 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, 4°C) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis. C18-neg: Metabolites were extracted by adding 90 μL of methanol containing PGE2-d4 as an internal standard (Cayman Chemical Co.; Ann Arbor, MI) to a 30 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, 4°C) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis.

Combined analysis:

Analysis ID AN002533 AN002534 AN002535 AN002536
Analysis type MS MS MS MS
Chromatography type HILIC Reversed phase HILIC Reversed phase
Chromatography system Shimadzu Nexera X2 Shimadzu Nexera X2 Shimadzu Nexera X2 Shimadzu Nexera X2
Column Waters Atlantis HILIC (150 x 2 mm, 3 μm) Waters Acquity BEH C8 (100 x 2.1mm, 1.7um) Phenomenex Luna NH2 (150 x 2.1mm, 3um) Waters Acquity T3 (150 x 2.1 mm, 1.7 um)
MS Type ESI ESI ESI ESI
MS instrument type Orbitrap Orbitrap Orbitrap Orbitrap
MS instrument name Thermo Exactive Plus Orbitrap Thermo Exactive Plus Orbitrap Thermo Q Exactive Plus Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE POSITIVE NEGATIVE NEGATIVE
Units unitless peak areas unitless peak areas unitless peak areas unitless peak areas

Chromatography:

Chromatography ID:CH001853
Chromatography Summary:HILIC-pos: high resolution and accurate mass profiling of polar metabolites using HILIC and positive ion mode full scan MS
Instrument Name:Shimadzu Nexera X2
Column Name:Waters Atlantis HILIC (150 x 2 mm, 3 μm)
Column Temperature:30 ℃
Flow Gradient:linear
Flow Rate:250 uL/min
Injection Temperature:4
Solvent A:10 mM Ammonium formate/0.1% Formic acid in water
Solvent B:0.1% Formic acid in Acetonitrile
Chromatography Type:HILIC
  
Chromatography ID:CH001854
Chromatography Summary:C8-pos: high resolution and accurate mass profiling of polar and nonpolar lipids using reversed phase C8 chromatography and positive ion mode full scan MS
Instrument Name:Shimadzu Nexera X2
Column Name:Waters Acquity BEH C8 (100 x 2.1mm, 1.7um)
Column Temperature:40 ℃
Flow Gradient:linear
Flow Rate:450 uL/min
Solvent A:10 mM Ammonium Acetate in 95:5:0.1, v:v:v Water: Methanol: Formic Acid
Solvent B:0.1% Formic Acid in Methanol
Chromatography Type:Reversed phase
  
Chromatography ID:CH001855
Chromatography Summary:HILIC-neg: high resolution and accurate mass profiling of polar metabolites using HILIC and negative ion mode full scan MS
Instrument Name:Shimadzu Nexera X2
Column Name:Phenomenex Luna NH2 (150 x 2.1mm, 3um)
Column Temperature:30 ℃
Flow Gradient:linear
Flow Rate:400 uL/min
Injection Temperature:4
Solvent A:20 mM Ammonium Acetate, 20 mM Ammonium Hydroxide in water
Solvent B:10 mM Ammonium Hydroxide in 25% Methanol/75% Acetonitrile
Chromatography Type:HILIC
  
Chromatography ID:CH001856
Chromatography Summary:C18-neg: high resolution and accurate mass profiling of metabolites of intermediate polarity using reversed phase C18 chromatography and negative ion mode full scan MS
Instrument Name:Shimadzu Nexera X2
Column Name:Waters Acquity T3 (150 x 2.1 mm, 1.7 um)
Column Temperature:45 ℃
Flow Gradient:linear
Flow Rate:450 uL/min
Injection Temperature:4
Solvent A:0.01% Formic acid in water
Solvent B:0.01% Acetic acid in Acetonitrile
Chromatography Type:Reversed phase

MS:

MS ID:MS002351
Analysis ID:AN002533
Instrument Name:Thermo Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:MS analyses were carried out using electrospray ionization in the positive ion mode using full scan analysis over m/z 70-800 at 70,000 resolution and 3 Hz data acquisition rate. Additional MS settings were: ion spray voltage, 3.5 kV; capillary temperature, 350°C; probe heater temperature, 300°C; sheath gas, 40; auxiliary gas, 15; and S-lens RF level 40. All raw data were processed using Progenesis QI software (NonLinear Dynamics) for feature alignment, nontargeted signal detection, and signal integration. Targeted processing of a subset of known metabolites was conducted using TraceFinder software (Thermo Fisher Scientific; Waltham, MA). Compound identities were confirmed using reference standards
Ion Mode:POSITIVE
  
MS ID:MS002352
Analysis ID:AN002534
Instrument Name:Thermo Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:MS analyses were carried out using electrospray ionization in the positive ion mode using full scan analysis over m/z 200-1100 at 70,000 resolution and 3 Hz data acquisition rate. Additional MS settings were: ion spray voltage, 3.0 kV; capillary temperature, 300°C; probe heater temperature, 300°C; sheath gas, 50; auxiliary gas, 15; and S-lens RF level 60. All raw data were processed using Progenesis QI software (NonLinear Dynamics) for feature alignment, nontargeted signal detection, and signal integration. Targeted processing of a subset of known lipids was conducted using TraceFinder software (Thermo Fisher Scientific; Waltham, MA). Lipid identities were confirmed using reference standards representative of different lipid classes and previously characterized reference samples. Lipids were denoted by headgroup and total acyl carbon number and double bond content.
Ion Mode:POSITIVE
  
MS ID:MS002353
Analysis ID:AN002535
Instrument Name:Thermo Q Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:MS analyses were carried out using electrospray ionization in the negative ion mode using full scan analysis over m/z 60-750 at 70,000 resolution and 3 Hz data acquisition rate. Additional MS settings were: ion spray voltage, -3.0 kV; capillary temperature, 350°C; probe heater temperature, 325°C; sheath gas, 55; auxiliary gas, 10; and S-lens RF level 40. All raw data were processed using Progenesis QI software (NonLinear Dynamics) for feature alignment, nontargeted signal detection, and signal integration. Targeted processing of a subset of known metabolites was conducted using TraceFinder software (Thermo Fisher Scientific; Waltham, MA). Compound identities were confirmed using reference standards.
Ion Mode:NEGATIVE
  
MS ID:MS002354
Analysis ID:AN002536
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:MS analyses were carried out using electrospray ionization in the negative ion mode using full scan analysis over m/z 70-850 at 70,000 resolution and 3 Hz data acquisition rate. Additional MS settings were: ion spray voltage, -3.5 kV; capillary temperature, 320°C; probe heater temperature, 300°C; sheath gas, 45; auxiliary gas, 10; and S-lens RF level 60All raw data were processed using Progenesis QI software (NonLinear Dynamics) for feature alignment, nontargeted signal detection, and signal integration. Targeted processing of a subset of known metabolites was conducted using TraceFinder software (Thermo Fisher Scientific; Waltham, MA). Compound identities were confirmed using reference standards.
Ion Mode:NEGATIVE
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