Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA209296

Local Sample ID:	136
Subject ID:	SU002266
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

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Subject:

Subject ID:	SU002266
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
136	SA209296	FL025539	Arabinoxylan	Fiber
136	SA209296	FL025539	20	Timepoint
136	SA209296	FL025539	F	Sex

Collection:

Collection ID:	CO002259
Collection Summary:	The study is a longitudinal, randomized crossover design in which 18 consented participants (8 men and 10 women) had their diets periodically supplemented with two fibers, arabinoxylan (AX) and long-chain inulin, and a mixture of fibers consisting of equal parts AX, LCI, acacia gum, glucomannans, and resistant starch. Participants were randomized to consume either AX or LCI first, and the mixed fibers were always administered last. For each of the fiber cycles, blood, urine and stool samples were collected at seven timepoints: baseline, end of week one, end of week two, end of week three, day 3 after end of supplementation and day 10 after end of supplementation. Blood was fractionated into plasma, serum and peripheral blood mononucleotide cells (PBMCs). Lipidomics was performed on the plasma fraction of the blood. Once all samples were in the correct aliquots, they were stored at -80C. Samples were only thawed when prepared for analysis for each of the respective omics assay which were all performed within 5 years of collecting the samples.
Sample Type:	Blood (plasma)

Treatment:

Treatment ID:	TR002278
Treatment Summary:	Participants went through 3 cycles of fiber supplementation, each cycle was three weeks long with weekly increasing doses of 10 g/day during the first week, 20g/day during the second week and 30 g/day during the third week. Randomized for the first two cycles, fibers tested were chicory inulin (99%>5 dp; range 2-60dp; average >23 dp) and arabinoxylan (psyllium husks powder Now Foods) and a mix of 5 fibers during the third cycle. The fiber mix included equal amounts of inulin, arabinoxylan, glucomannan (Now Foods), resistant starch (Hi Maize from Honeyville), and acacia fiber (Now Foods). Washout period between the cycles was from 6-10 weeks. Fiber was provided in 10 g sachets and participants were instructed to resuspend content of the sachet in at least 8 oz of water and drink one with breakfast for the first week, one with breakfast and one with dinner during the second week, and one with each meal (breakfast, lunch and dinner) during the third week.

Treatment ID:

TR002278

Treatment Summary:

Participants went through 3 cycles of fiber supplementation, each cycle was three weeks long with weekly increasing doses of 10 g/day during the first week, 20g/day during the second week and 30 g/day during the third week. Randomized for the first two cycles, fibers tested were chicory inulin (99%>5 dp; range 2-60dp; average >23 dp) and arabinoxylan (psyllium husks powder Now Foods) and a mix of 5 fibers during the third cycle. The fiber mix included equal amounts of inulin, arabinoxylan, glucomannan (Now Foods), resistant starch (Hi Maize from Honeyville), and acacia fiber (Now Foods). Washout period between the cycles was from 6-10 weeks. Fiber was provided in 10 g sachets and participants were instructed to resuspend content of the sachet in at least 8 oz of water and drink one with breakfast for the first week, one with breakfast and one with dinner during the second week, and one with each meal (breakfast, lunch and dinner) during the third week.

Sample Preparation:

Sampleprep ID:	SP002272
Sampleprep Summary:	Lipids were extracted using a modified biphasic separation protocol with methyl tertiary-butyl ether (MTBE)(Matyash et al., 2008). Pipetting was performed using Rainin BioClean Ultra pipette tips. In 2 ml Protein LoBind Eppendorf tubes (cat# 022431102), 40 μl of neat plasma were mixed with 260 μl ice-cold MeOH and briefly vortexed to denature proteins. Next, 40 μL of an isotopically labeled standard lipid stock that approximates the blood plasma lipid composition was added to each sample (SCIEX, cat# 5040156, LPISTDKIT-101), which was prepared according to the instruction in Lipidomics Workflow Manager (LWM). The mix was briefly vortexed. Next, 1000 μL ice-cold MTBE was added after which samples were vortexed for 10 seconds followed by shaking at 4ºC for 30 min. Phase separation was induced by adding 250 μL of HPLC grade water. Samples were subsequently vortexed for 60 seconds. Next, samples were centrifuged at 16,000 g for 5 min at room temperature. 750 μL of the upper organic (MTBE) phase were transferred to a new 2 mL tube. This MTBE lipid fraction was dried down in a nitrogen blower for about 2 hours. For storage at -20ºC, 200 μL of MeOH was added to dried-down samples.

Sampleprep ID:

SP002272

Sampleprep Summary:

Lipids were extracted using a modified biphasic separation protocol with methyl tertiary-butyl ether (MTBE)(Matyash et al., 2008). Pipetting was performed using Rainin BioClean Ultra pipette tips. In 2 ml Protein LoBind Eppendorf tubes (cat# 022431102), 40 μl of neat plasma were mixed with 260 μl ice-cold MeOH and briefly vortexed to denature proteins. Next, 40 μL of an isotopically labeled standard lipid stock that approximates the blood plasma lipid composition was added to each sample (SCIEX, cat# 5040156, LPISTDKIT-101), which was prepared according to the instruction in Lipidomics Workflow Manager (LWM). The mix was briefly vortexed. Next, 1000 μL ice-cold MTBE was added after which samples were vortexed for 10 seconds followed by shaking at 4ºC for 30 min. Phase separation was induced by adding 250 μL of HPLC grade water. Samples were subsequently vortexed for 60 seconds. Next, samples were centrifuged at 16,000 g for 5 min at room temperature. 750 μL of the upper organic (MTBE) phase were transferred to a new 2 mL tube. This MTBE lipid fraction was dried down in a nitrogen blower for about 2 hours. For storage at -20ºC, 200 μL of MeOH was added to dried-down samples.

Combined analysis:

Analysis ID	AN003570	AN003571
Analysis type	MS	MS
Chromatography type	Flow induction analysis	Flow induction analysis
Chromatography system	Shimazdu LC-30AD	Shimazdu LC-30AD
Column	none	none
MS Type	ESI	ESI
MS instrument type	QTRAP	QTRAP
MS instrument name	ABI Sciex 5500 QTrap	ABI Sciex 5500 QTrap
Ion Mode	UNSPECIFIED	UNSPECIFIED
Units	nmol/g	nmol/g

Chromatography:

Chromatography ID:	CH002640
Chromatography Summary:	The Lipidyzer (SCIEX), a QTRAP system with SelexION ion mobility, was used for targeted profiling as described previously (Contrepois et al., 2018).DMS separates lipids based on the principle that each lipid class has a different head group dipole moment and thus mobility in the DMS aperture (Schneider et al., 2010).
Instrument Name:	Shimazdu LC-30AD
Column Name:	none
Flow Rate:	8 μL/min
Solvent A:	50% dichloromethane/50% methanol; 10 mM ammonium acetate
Chromatography Type:	Flow induction analysis

MS:

MS ID:	MS003327
Analysis ID:	AN003570
Instrument Name:	ABI Sciex 5500 QTrap
Instrument Type:	QTRAP
MS Type:	ESI
MS Comments:	The Lipidyzer (SCIEX), a QTRAP system with SelexION ion mobility, was used for targeted profiling as described previously (Contrepois et al., 2018). In brief, flow injection analysis was performed with a LC-30AD (Shimazdu) operating at 8 μL/min (50 μL injection volume) using a running solution that consisted of 10 mM ammonium acetate in dichloromethane:MeOH (50:50). DMS separates lipids based on the principle that each lipid class has a different head group dipole moment and thus mobility in the DMS aperture (Schneider et al., 2010). The lipid molecular species were identified and quantified using multiple reaction monitoring (MRM) and positive/negative switching. Two acquisition methods were employed covering 10 lipid classes across positive and negative mode. Method 1 had SelexION voltages turned on while Method 2 had SelexION voltages turned off. Method 1 employed an isocratic flow of 8 μL/min for 7.9 min, followed by a 2 minute wash at 30 μL/min. Method 2 employed an isocratic flow of 8 μL/min for 6 minutes, followed by 2 minute wash at 30 μL/min. Each lipid was acquired throughout 20 cycles. Lipid classes targeted in positive mode: SM, DAG, CE, CER, and TAG. Lipid classes targeted in negative mode: LPE, LPC, PC, PE, and FFA. Lipids were quantified using the LWM software, which compares endogenous lipids to the known concentrations of structurally most similar spiked-in lipid standards and reports all detected lipids in nmol/g. Data analysis. Data were downloaded from the Lipidyzer LWM and merged and processed in R. In brief, Excel files (LWM output) were read with the “loadWorkbook” package. From all samples, lipid concentrations determined in a blank control (sample processed in parallel without the addition of cells) were subtracted to correct for background signals. The data set was further filtered accepting only lipid species that detected in at least 25% of all samples. Missing values were imputed by drawing from a random distribution.
Ion Mode:	UNSPECIFIED

MS ID:	MS003328
Analysis ID:	AN003571
Instrument Name:	ABI Sciex 5500 QTrap
Instrument Type:	QTRAP
MS Type:	ESI
MS Comments:	The Lipidyzer (SCIEX), a QTRAP system with SelexION ion mobility, was used for targeted profiling as described previously (Contrepois et al., 2018). In brief, flow injection analysis was performed with a LC-30AD (Shimazdu) operating at 8 μL/min (50 μL injection volume) using a running solution that consisted of 10 mM ammonium acetate in dichloromethane:MeOH (50:50). DMS separates lipids based on the principle that each lipid class has a different head group dipole moment and thus mobility in the DMS aperture (Schneider et al., 2010). The lipid molecular species were identified and quantified using multiple reaction monitoring (MRM) and positive/negative switching. Two acquisition methods were employed covering 10 lipid classes across positive and negative mode. Method 1 had SelexION voltages turned on while Method 2 had SelexION voltages turned off. Method 1 employed an isocratic flow of 8 μL/min for 7.9 min, followed by a 2 minute wash at 30 μL/min. Method 2 employed an isocratic flow of 8 μL/min for 6 minutes, followed by 2 minute wash at 30 μL/min. Each lipid was acquired throughout 20 cycles. Lipid classes targeted in positive mode: SM, DAG, CE, CER, and TAG. Lipid classes targeted in negative mode: LPE, LPC, PC, PE, and FFA. Lipids were quantified using the LWM software, which compares endogenous lipids to the known concentrations of structurally most similar spiked-in lipid standards and reports all detected lipids in nmol/g. Data analysis. Data were downloaded from the Lipidyzer LWM and merged and processed in R. In brief, Excel files (LWM output) were read with the “loadWorkbook” package. From all samples, lipid concentrations determined in a blank control (sample processed in parallel without the addition of cells) were subtracted to correct for background signals. The data set was further filtered accepting only lipid species that detected in at least 25% of all samples. Missing values were imputed by drawing from a random distribution.
Ion Mode:	UNSPECIFIED