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MB Sample ID: SA094612

Local Sample ID:HFD-BPA13
Subject ID:SU001383
Subject Type:Mammal
Subject Species:Rattus norvegicus
Taxonomy ID:10116
Genotype Strain:Sprague Dawley
Age Or Age Range:240 days
Gender:Male
Animal Animal Supplier:Harlan
Animal Housing:polycarbonate-free caging
Animal Light Cycle:14-hr light and 10-hr dark
Animal Feed:Phytoestrogen Reduced II 18-5 (Ziegler Bros, Inc) or D09100301 (Research Diets, Inc)

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Subject:

Subject ID:SU001383
Subject Type:Mammal
Subject Species:Rattus norvegicus
Taxonomy ID:10116
Genotype Strain:Sprague Dawley
Age Or Age Range:240 days
Gender:Male
Animal Animal Supplier:Harlan
Animal Housing:polycarbonate-free caging
Animal Light Cycle:14-hr light and 10-hr dark
Animal Feed:Phytoestrogen Reduced II 18-5 (Ziegler Bros, Inc) or D09100301 (Research Diets, Inc)

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
HFD-BPA13SA094612FL013498BPATreatment

Collection:

Collection ID:CO001378
Collection Summary:Liver tissue was harvested on post-natal day 240 after challenge with Western-style diet.Tissue was snap-frozen in liquid nitrogen.
Sample Type:Liver

Treatment:

Treatment ID:TR001398
Treatment Summary:Neonatal rats were treated with vehicle (sesame oil) or bisphenol A (BPA; 50 µg/kg dissolved in sesame oil) orally via pipette tip on post-natal days 1, 3, and 5. Littermates were randomly assigned to the treatment groups. BPA was obtained from the National Institute of Environmental Health Sciences (NIEHS). The dose and route of administration recapitulates human exposure to BPA. At day 180, adult rats in both treatment groups were fed a diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days (D09100301, Research Diets, Inc). Rats were fasted overnight prior to tissue collection.

Sample Preparation:

Sampleprep ID:SP001391
Sampleprep Summary:Metabolites were extracted from crushed liver samples and a mouse liver pool was used for quality control. Twenty-five mg of crushed liver was used for the metabolic extraction. The extraction step started with the addition of 750 µL ice-cold methanol:water (4:1) containing 20 µL spiked internal standards to each tissue sample. Ice-cold chloroform and water were added in a 3:1 ratio for a final proportion of 1:4:3:1 water:methanol:chloroform:water. The organic (methanol and chloroform) and aqueous layers were mixed, dried and resuspended with 50:50 methanol: water. The extract was deproteinized using a 3kDa molecular filter (Amicon ultracel-3K Membrane; Millipore Corporation, Billerica, MA) and the filtrate was dried under vacuum (Genevac EZ-2plus; Gardiner, Stone Ridge, NY). Prior to mass spectrometry, the dried extracts were re-suspended in identical volumes of injection solvent composed of 1:1 water: methanol and were subjected to liquid chromatography-mass spectrometry. Fifty µl of sample was used for preparation. Internal standards were spiked into the samples. Then it was processed through a 3 kDa filter. After that, 50 µl of sample was diluted with 450 µl solvent (methanol: water = 50:50 v/v) and subjected to LC/MS analysis. The injection volume was 10 µl. For internal standards, high-performance liquid chromatography (HPLC)-grade acetonitrile, methanol, and water were procured from Burdick & Jackson (Morristown, NJ). Mass spectrometry-grade formic acid was purchased from Sigma-Aldrich (St Louis, MO). Calibration solution containing multiple calibrants in a solution of acetonitrile, trifluroacetic acid, and water was purchased from Agilent Technologies (Santa Clara, CA). Metabolites and internal standards, including N-acetyl Aspartic acid-d3, Tryptophan-15N2, Sarcosine-d3, Glutamic acid-d5, Thymine-d4, Gibberellic acid, Trans-Zeatine, Jasmonic acid, 15N Anthranilic acid, and Testosterone-d3, were purchased from Sigma-Aldrich (St. Louis, MO). Three LC- MS methods were used to separate metabolites. Method A: In ESI positive mode the HPLC column was waters X-bridge amide 3.5 µm, 4.6 x 100 mm (Waters, Milford, MA). Mobile phase A and B were 0.1% formic acid in water and acetonitrile, respectively. Gradient flow: 0-3 min 85% B; 3-12 min 30% B, 12-15 min 2% B, 16 min 95%B, followed by re-equilibration till the end of the gradient 23 min to the initial starting condition of 85% B. Flow rate of the solvents used for the analysis is 0.3 ml/min. Injection volume was 10 µL. Method B: In ESI negative mode the HPLC column was waters X-bridge amide 3.5 µm, 4.6 x 100 mm (Waters, Milford, MA). Mobile phase A and B were 20 mM ammonium acetate in water with pH 9.0 and 100% acetonitrile, respectively. Gradient flow: 0-3 min 85% B, 3-12 min 30% B, 12-15 min 2% B, 15-16 min 85% B followed by re-equilibration till the end of the gradient 23 min to the initial starting condition of 85% B. Flow rate of the solvents used for analysis is 0.3 ml/min. Injection volume was 10 µL. Method C: In ESI positive mode the HPLC column was Luna 3 µM NH2 100 A0 Chromatography column (Phenomenex, Torrance, CA). Mobile phase A and B were 20 mM ammonium acetate in water with pH 9.0 and 100% acetonitrile, respectively. Gradient flow: 0-3 min 85% B, 3-12 min 30% B, 12-15 min 2% B, 15-16 min 85% B followed by re-equilibration till the end of the gradient 23 min to the initial starting condition of 85% B. Flow rate of the solvents used for analysis is 0.3 ml/min. Injection volume was 10 µL. For data acquisition through LC/MS analysis, 10 µL of suspended samples were injected and analyzed using a 6495 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA) coupled to a HPLC system (Agilent Technologies, Santa Clara, CA) via Multiple reaction monitoring (MRM). Source parameters were as follows: Gas temperature- 250°C; Gas flow- 14 l/min; Nebulizer - 20psi; Sheath gas temperature - 350°C; Sheath gas flow- 12 l/min; Capillary - 3000 V positive and 3000 V negative; Nozzle voltage- 1500 V positive and 1500 V negative. Approximately 8–11 data points were acquired per detected metabolite. The data acquired using Agilent mass hunter software and data was analyzed using mass hunter quantitative analysis software.
Sampleprep Protocol Filename:Targeted.MS.method.pdf
unbiased.liver.MS.method.pdf

Combined analysis:

Analysis ID AN002178 AN002179 AN002180
Analysis type MS MS MS
Chromatography type HILIC HILIC HILIC
Chromatography system Agilent 6495 QQQ Agilent 6495 QQQ Agilent 6495 QQQ
Column Waters XBridge Amide (100 x 4.6mm,3.5um) Waters XBridge Amide (100 x 4.6mm,3.5um) Phenomenex Luna NH2 (150 x 2.1mm,3um)
MS Type ESI ESI ESI
MS instrument type Triple quadrupole Triple quadrupole Triple quadrupole
MS instrument name Agilent 6495 QQQ Agilent 6495 QQQ Agilent 6495 QQQ
Ion Mode POSITIVE NEGATIVE POSITIVE
Units peak intensity peak intensity peak intensity

Chromatography:

Chromatography ID:CH001593
Instrument Name:Agilent 6495 QQQ
Column Name:Waters XBridge Amide (100 x 4.6mm,3.5um)
Chromatography Type:HILIC
  
Chromatography ID:CH001594
Instrument Name:Agilent 6495 QQQ
Column Name:Waters XBridge Amide (100 x 4.6mm,3.5um)
Chromatography Type:HILIC
  
Chromatography ID:CH001595
Instrument Name:Agilent 6495 QQQ
Column Name:Phenomenex Luna NH2 (150 x 2.1mm,3um)
Chromatography Type:HILIC

MS:

MS ID:MS002025
Analysis ID:AN002178
Instrument Name:Agilent 6495 QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:For data acquisition through LC/MS analysis, 10 µL of suspended samples were injected and analyzed using a 6495 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA) coupled to a HPLC system (Agilent Technologies, Santa Clara, CA) via Multiple reaction monitoring (MRM). Source parameters were as follows: Gas temperature- 250°C; Gas flow- 14 l/min; Nebulizer - 20psi; Sheath gas temperature - 350°C; Sheath gas flow- 12 l/min; Capillary - 3000 V positive and 3000 V negative; Nozzle voltage- 1500 V positive and 1500 V negative. Approximately 8–11 data points were acquired per detected metabolite. The data acquired using Agilent mass hunter software and data was analyzed using mass hunter quantitative analysis software.
Ion Mode:POSITIVE
  
MS ID:MS002026
Analysis ID:AN002179
Instrument Name:Agilent 6495 QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:For data acquisition through LC/MS analysis, 10 µL of suspended samples were injected and analyzed using a 6495 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA) coupled to a HPLC system (Agilent Technologies, Santa Clara, CA) via Multiple reaction monitoring (MRM). Source parameters were as follows: Gas temperature- 250°C; Gas flow- 14 l/min; Nebulizer - 20psi; Sheath gas temperature - 350°C; Sheath gas flow- 12 l/min; Capillary - 3000 V positive and 3000 V negative; Nozzle voltage- 1500 V positive and 1500 V negative. Approximately 8–11 data points were acquired per detected metabolite. The data acquired using Agilent mass hunter software and data was analyzed using mass hunter quantitative analysis software.
Ion Mode:NEGATIVE
  
MS ID:MS002027
Analysis ID:AN002180
Instrument Name:Agilent 6495 QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:For data acquisition through LC/MS analysis, 10 µL of suspended samples were injected and analyzed using a 6495 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA) coupled to a HPLC system (Agilent Technologies, Santa Clara, CA) via Multiple reaction monitoring (MRM). Source parameters were as follows: Gas temperature- 250°C; Gas flow- 14 l/min; Nebulizer - 20psi; Sheath gas temperature - 350°C; Sheath gas flow- 12 l/min; Capillary - 3000 V positive and 3000 V negative; Nozzle voltage- 1500 V positive and 1500 V negative. Approximately 8–11 data points were acquired per detected metabolite. The data acquired using Agilent mass hunter software and data was analyzed using mass hunter quantitative analysis software.
Ion Mode:POSITIVE
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