Summary of Study ST001369

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 PR000922. The data can be accessed directly via it's Project DOI: 10.21228/M8HH5M This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST001369
Study TitleGrass pollen sublingual immunotherapy treatment induces transcriptomic and metabolic changes due to AIT treatment
Study Summary47 patients were enrolled in a double-blind, placebo-controlled, multicenter trial using with GRAZAX® (Phleum pretense) during 2 years of therapy (T2). Immunological assays such as sIgE, sIgG4 and ISAC were carried out to the 31 patients who finished the trial. Additionally, serum and PBMCs samples from these samples were analyzed by metabolomics and transcriptomics, respectively. Based on their sensitization level, 22 patients were grouped in Mono and Poli groups, excluding epithelial allergic patients. Individuals were studied based on their treatment in Active and Placebo and their sensitization level. For metabolomics, samples were analyzed by Liquid and Gas Chromatography coupled to Mass Spectrometry (LC-MS and GC-MS, respectively).
Institute
Institute of Applied Molecular Medicine;The Centre of Metabolomics and Bioanalysis
Last NameObeso Montero
First NameDavid
AddressAvda. Monteprincipe s/n 28668 Boadilla del Monte, Madrid, España
Emaildavid.obesomontero@beca.ceu.es
PhoneTlf: 91 372 47 00 ext. 4662
Submit Date2020-03-27
Num Groups2 main groups: Active and Placebo, and 2 subgroups: Monosensitized and Polisensitized patients.
Total Subjects22
Study Commentshttps://www.ceu.es;http://www.metabolomica.uspceu.es
Raw Data AvailableYes
Raw Data File Type(s)d
Analysis Type DetailGC-MS/LC-MS
Release Date2020-09-29
Release Version1
David Obeso Montero David Obeso Montero
https://dx.doi.org/10.21228/M8HH5M
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR000922
Project DOI:doi: 10.21228/M8HH5M
Project Title:Metabolomics analysis of serum samples from patients during 2 years of IAT trial, double blind placebo controled study
Project Type:Study the changes produced by the use of AIT treatment or placebo
Project Summary:47 patients were enrolled in a double-blind, placebo-controlled, multicenter trial using with GRAZAX® (Phleum pretense) during 2 years of therapy (T2). Immunological assays such as sIgE, sIgG4 and ISAC were carried out to the 31 patients who finished the trial. Additionally, serum and PBMCs samples from these samples were analyzed by metabolomics and transcriptomics, respectively. Based on their sensitization level, 22 patients were grouped in Mono and Poli groups, excluding epithelial allergic patients. Individuals were studied based on their treatment in Active and Placebo and their sensitization level.
Institute:Institute of Applied Molecular Medicine;The Centre of Metabolomics and Bioanalysis
Last Name:Barber Hernández
First Name:Domingo
Address:Avda. Monteprincipe s/n 28668 Boadilla del Monte, Madrid, España
Email:domingo.barberhernandez@ceu.es
Phone:Tlf: 91 372 47 00 ext. 4662

Subject:

Subject ID:SU001443
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

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

mb_sample_id local_sample_id Treatment Sensitization
SA099421P28_t0Active Mono
SA099422P27_t0Active Mono
SA099423P1_t0Active Mono
SA099424P27_T2Active Mono
SA099425P28_t2Active Mono
SA099426P17_t0Active Mono
SA099427P17_t2Active Mono
SA099428P1_t2Active Mono
SA099429P11_t2Active Poli
SA099430P15_t0Active Poli
SA099431P13_t0Active Poli
SA099432P12_t0Active Poli
SA099433P11_t0Active Poli
SA099434P12_t2Active Poli
SA099435P13_t2Active Poli
SA099436P15_t2Active Poli
SA099437P22_t2Placebo Mono
SA099438P16_t2Placebo Mono
SA099439P25_T2Placebo Mono
SA099440P26_t2Placebo Mono
SA099441P30_t0Placebo Mono
SA099442P22_t0Placebo Mono
SA099443P21_t0Placebo Mono
SA099444P21_t2Placebo Mono
SA099445P16_t0Placebo Mono
SA099446P26_t0Placebo Mono
SA099447P25_t0Placebo Mono
SA099448P30_t2Placebo Mono
SA099449P3_t0Placebo Poli
SA099450P4_t0Placebo Poli
SA099451P24_t2Placebo Poli
SA099452P8_t0Placebo Poli
SA099453P23_t2Placebo Poli
SA099454P9_t2Placebo Poli
SA099455P24_t0Placebo Poli
SA099456P23_t0Placebo Poli
SA099457P20_t0Placebo Poli
SA099458P14_t0Placebo Poli
SA099459P3_t2Placebo Poli
SA099460P4_t2Placebo Poli
SA099461P9_t0Placebo Poli
SA099462P14_t2Placebo Poli
SA099463P8_t2Placebo Poli
SA099464P20_t2Placebo Poli
Showing results 1 to 44 of 44

Collection:

Collection ID:CO001438
Collection Summary:For immunological analyses and metabolomics, serum samples were obtained and stored at -20°C until analysis. In the case of transcriptomics, Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and stored at -20°C in Buffer RLT until analysis.
Sample Type:Blood (serum)
Storage Conditions:-20℃

Treatment:

Treatment ID:TR001458
Treatment Summary:Subjects were randomized (1:1) during autumn 2013 to receive either active treatment with GRAZAX® (Phleum pratense, 75,000SQ‐T tablets ALK, Hørsholm, Denmark) or placebo as Investigational Medical Products (IMP) during 2 years. Twenty-five patients were assigned to daily sublingual administration of GRAZAX®, while the rest received placebo tablets that were similar to the active IMP with regard to appearance, smell and taste.

Sample Preparation:

Sampleprep ID:SP001451
Sampleprep Summary:For LC-MS, proteins were removed adding 300 µL of cold (-20 °C) methanol:ethanol (1:1) to 100 µL of sample. Samples were then vortex-mixed and stored on ice for 5 min. Supernatant containing the metabolites was separated from the pellet by centrifugation (16,000× g for 20 min at 4 °C), and put into a LC vial for analysis. For GC-MS, serum samples were first deproteinized using cold acetonitrile (ACN) in a 3:1 proportion (120 μL of ACN were added to 40 μL of serum). Samples were kept on ice for 5 minutes. Afterwards, the metabolites were separated by centrifugation (10 min at 15,400 x g and 4°C). 100 µL of the resulting supernatant were transferred to a GC vial with insert and were evaporated to dryness for 2 hours at 30°C (SpeedVac Concentrator, Thermo Fisher Scientific, Waltham, MA, USA). Afterwards, a methoximation was performed by addition of 10 µL of O-methoxyamine hydrochloride 15mg/mL in pyridine, with the purpose of protecting the reactive oxygen groups in the metabolites. The mixture was vigorously vortex-mixed (1 minute each vial), ultrasonicated (3 times, 20 seconds each) and further vortexed another 2 minutes. Then, the samples were left in darkness at room temperature for 16 h for the completion of the methoximation reaction. Finally, the samples were derivatized by the addition of 10 µL of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS).
Processing Method:Protein precipitation and metabolite extraction
Processing Storage Conditions:On ice

Combined analysis:

Analysis ID AN002283 AN002284 AN002285
Analysis type MS MS MS
Chromatography type Reversed phase Reversed phase GC
Chromatography system Agilent 1200 Agilent 1200 Agilent 7890A
Column Discovery HS C18 (150 x 2.1mm,3.0um) Supelco,Sigma Aldrich,Germany),with a guard Discovery HS C18 (2 cm x 2.1mm,3um; Supelco,Sigma Aldrich,Germany), Discovery HS C18 (150 x 2.1mm,3.0um) Supelco,Sigma Aldrich,Germany) GC-Column DB5-MS (30 m length, 0.25 mm i.d., 0.25 μm film 95% dimethyl/5% diphenylpolysiloxane) with an integrated precolumn (10 m J&W, Agilent)
MS Type ESI ESI EI
MS instrument type QTOF QTOF Single quadrupole
MS instrument name Agilent 6520 QTOF Agilent 6520 QTOF Agilent 5975C
Ion Mode POSITIVE NEGATIVE POSITIVE
Units peak area peak area peak area

Chromatography:

Chromatography ID:CH001677
Chromatography Summary:For LC-MS, 10 μL of sample were injected into a Discovery HS C18 column (2.1 mm × 150 mm, 3.0 μm; Supelco, Sigma Aldrich, Germany), with a guard column Discovery® HS C18 (2 cm × 2.1 mm, 3 μm; Supelco, Sigma Aldrich, Germany), both maintained at 40 °C. The flow rate was set at 0.6 mL/min. The gradient elution involved a mobile phase consisting of (A) 0.1% formic acid (FA) in water and (B) 0.1% FA in acetonitrile (ACN). The initial condition was set at 25% B, increasing to 95% B in 35 min, followed by re-equilibration for 1 min, finally it was held for 9 min in initial conditions. Flow rate was set at 0.6 mL/min, and 10 μL of samples were injected. The electrospray source ionization (ESI) data were acquired in positive and negative ion mode, respectively. The capillary voltage was set at 3,500 for ESI (+) and 4,000V for ESI (−). The drying gas flow rate was 10.5 L/min at 330 °C and gas nebulizer at 52 psi; fragmentor voltage was 175 V; skimmer and octopole radio frequency voltage (OCT RF Vpp) were set to 65 and 750 V. Data were collected in the centroid mode at a scan rate of 1.2 spectra per second. Mass spectrometry detection was performed in full scan from 100 to 1200 m/z for both, positive and negative ESI mode. The reference m/z ions were purine (121.0508) and HP-0921 (922.0097) for ESI (+), whereas TFA NH4 (119.0363) and HP-0921 (966.0007) for ESI (−). These masses were continuously infused into the system to allow constant mass correction. Samples were analyzed in separate runs.
Instrument Name:Agilent 1200
Column Name:Discovery HS C18 (150 x 2.1mm,3.0um) Supelco,Sigma Aldrich,Germany),with a guard Discovery HS C18 (2 cm x 2.1mm,3um; Supelco,Sigma Aldrich,Germany),
Column Temperature:40
Flow Gradient:The initial condition was set at 25% B, increasing to 95% B in 35 min, followed by re-equilibration for 1 min, finally it was held for 9 min in initial conditions
Flow Rate:0.6ml/min
Injection Temperature:4
Sample Injection:10 μL
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Analytical Time:45 min
Capillary Voltage:3500 for ESI (+)
Chromatography Type:Reversed phase
  
Chromatography ID:CH001678
Chromatography Summary:For LC-MS, 10 μL of sample were injected into a Discovery HS C18 column (2.1 mm × 150 mm, 3.0 μm; Supelco, Sigma Aldrich, Germany), with a guard column Discovery® HS C18 (2 cm × 2.1 mm, 3 μm; Supelco, Sigma Aldrich, Germany), both maintained at 40 °C. The flow rate was set at 0.6 mL/min. The gradient elution involved a mobile phase consisting of (A) 0.1% formic acid (FA) in water and (B) 0.1% FA in acetonitrile (ACN). The initial condition was set at 25% B, increasing to 95% B in 35 min, followed by re-equilibration for 1 min, finally it was held for 9 min in initial conditions. Flow rate was set at 0.6 mL/min, and 10 μL of samples were injected. The electrospray source ionization (ESI) data were acquired in positive and negative ion mode, respectively. The capillary voltage was set at 3,500 for ESI (+) and 4,000V for ESI (−). The drying gas flow rate was 10.5 L/min at 330 °C and gas nebulizer at 52 psi; fragmentor voltage was 175 V; skimmer and octopole radio frequency voltage (OCT RF Vpp) were set to 65 and 750 V. Data were collected in the centroid mode at a scan rate of 1.2 spectra per second. Mass spectrometry detection was performed in full scan from 100 to 1200 m/z for both, positive and negative ESI mode. The reference m/z ions were purine (121.0508) and HP-0921 (922.0097) for ESI (+), whereas TFA NH4 (119.0363) and HP-0921 (966.0007) for ESI (−). These masses were continuously infused into the system to allow constant mass correction. Samples were analyzed in separate runs.
Instrument Name:Agilent 1200
Column Name:Discovery HS C18 (150 x 2.1mm,3.0um) Supelco,Sigma Aldrich,Germany)
Column Temperature:40
Flow Gradient:The initial condition was set at 25% B, increasing to 95% B in 35 min, followed by re-equilibration for 1 min, finally it was held for 9 min in initial conditions
Flow Rate:0.6ml/min
Injection Temperature:4
Sample Injection:10 μL
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Analytical Time:45 min
Capillary Voltage:4000V for ESI (−)
Chromatography Type:Reversed phase
  
Chromatography ID:CH001679
Chromatography Summary:GC-MS analysis was performed by a GC system (Agilent Technologies 7890A) equipped with an autosampler (Agilent 7693) coupled to a mass spectrometer with triple-Axis detector (5975C, Agilent). Two microliters (2 μL) of the derivatized sample were injected through a GC-Column DB5-MS (30 m length, 0.25 mm i.d., 0.25 μm film 95% dimethyl/5% diphenylpolysiloxane) with an integrated precolumn (10 m J&W, Agilent). Carrier gas (He) flow rate was set at 1 mL/min and injector temperature at 250 °C. Split ratio was fixed from 1:5 to 1:10 with 3 to 10 mL/min. He split flow into a Restek 20782 (Bellefonte, PA, USA) deactivated glass-wool split liner. The temperature gradient was programmed as follows: the initial oven temperature was set at 60 ºC (held for 1 min), increased to 325 ºC at 10 ºC/min rate (within 26.5 min) and hold 325 ºC for 10 min. The total run time was 37.5 min. A cool-down period was applied for 10 min before the next injection. Detector transfer line, filament source and the quadrupole temperature were set at 280 ºC, 230 ºC and 150 ºC, respectively. MS detection was performed with electron ionization (EI) mode at -70 eV. The mass spectrometer was operated in scan mode over a mass range of m/z 50-600 at a rate of 2.7 scan/s. Several IS injections, a standard mix of fatty acid methyl esters (FAME C8-C30), extraction blanks and 3 QCs samples were injected at the beginning of analysis, following QCs injections every 5 experimental samples and 1 QC injection at the end of worklist.
Instrument Name:Agilent 7890A
Column Name:GC-Column DB5-MS (30 m length, 0.25 mm i.d., 0.25 μm film 95% dimethyl/5% diphenylpolysiloxane) with an integrated precolumn (10 m J&W, Agilent)
Column Temperature:As Oven
Injection Temperature:250
Internal Standard:Standard mix of fatty acid methyl esters (FAME C8-C30)
Sample Injection:2 μL
Analytical Time:37.5 min
Oven Temperature:The initial oven temperature was set at 60 ºC (held for 1 min), increased to 325 ºC at 10 ºC/min rate (within 26.5 min) and hold 325 ºC for 10 min.
Transferline Temperature:280
Chromatography Type:GC

MS:

MS ID:MS002127
Analysis ID:AN002283
Instrument Name:Agilent 6520 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:LC−MS analysis was performed on an Agilent HPLC system (1200 series, Agilent Technologies, Waldbronn, Germany), equipped with a degasser, two binary pumps, and a thermostated autosampler coupled with quadrupole-time of flight analyzer (Q-TOF), LC-MS (6520) system (Agilent Technologies, Waldbronn, Germany)
Ion Mode:POSITIVE
Capillary Voltage:3,500 for ESI (+)
Dry Gas Flow:10.5 L/min
Dry Gas Temp:330 °C
Fragment Voltage:175 V
Nebulizer:52 psi
Octpole Voltage:750V
  
MS ID:MS002128
Analysis ID:AN002284
Instrument Name:Agilent 6520 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:LC−MS analysis was performed on an Agilent HPLC system (1200 series, Agilent Technologies, Waldbronn, Germany), equipped with a degasser, two binary pumps, and a thermostated autosampler coupled with quadrupole-time of flight analyzer (Q-TOF), LC-MS (6520) system (Agilent Technologies, Waldbronn, Germany)
Ion Mode:NEGATIVE
Capillary Voltage:4,000V for ESI (−)
Dry Gas Flow:10.5 L/min
Dry Gas Temp:330 °C
Fragment Voltage:175 V
Nebulizer:52 psi
Octpole Voltage:750V
  
MS ID:MS002129
Analysis ID:AN002285
Instrument Name:Agilent 5975C
Instrument Type:Single quadrupole
MS Type:EI
MS Comments:GC-MS analysis was performed by a GC system (Agilent Technologies 7890A) equipped with an autosampler (Agilent 7693) coupled to a mass spectrometer with triple-Axis detector (5975C, Agilent).
Ion Mode:POSITIVE
Helium Flow:1 mL/min
Ionization Energy:-70 eV
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