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MB Sample ID: SA128079

Local Sample ID:9032-2-PC
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

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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:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
9032-2-PCSA128079FL015922ModulenStudy_Diet
9032-2-PCSA128079FL01592253Age
9032-2-PCSA128079FL015922MaleSex
9032-2-PCSA128079FL015922WhiteRace
9032-2-PCSA128079FL015922Day 9Time

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 2mm,3um) 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,1.7um)
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 2mm,3um)
Column Temperature:30 ℃
Flow Gradient:linear
Flow Rate:250 uL/min
Injection Temperature:4
Solvent A:100% water; 0.1% formic acid; 10 mM ammonium formate
Solvent B:100% acetonitrile; 0.1% formic acid
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:95% water/5% methanol; 0.1% formic acid; 10 mM ammonium acetate
Solvent B:100% methanol; 0.1% formic acid
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:100% water; 20 mM ammonium acetate, 20 mM ammonium hydroxide
Solvent B:25% methanol/75% acetonitrile; 10 mM ammonium hydroxide
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,1.7um)
Column Temperature:45 ℃
Flow Gradient:linear
Flow Rate:450 uL/min
Injection Temperature:4
Solvent A:100% water; 0.01% formic acid
Solvent B:100% acetonitrile; 0.01% acetic acid
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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