Summary of Study ST001106

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 PR000740. The data can be accessed directly via it's Project DOI: 10.21228/M81D57 This work is supported by NIH grant, U2C- DK119886.

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Study IDST001106
Study TitleLipidomics of Newborn Heart Tissue Exposed to Excess Maternal Cortisol in Late Gestation (part-1)
Study SummaryCardiac tissue from newborn hearts from animals exposed to excess maternal cortisol in late gestation and untreated was compared via MS lipidomic analysis
Institute
University of Florida
DepartmentBiochemsitry & Molecular Biology
Last NameWalejko
First NameJacquelyn
AddressR3-226 Academic Research Building, Department of Biochemistry and Molecular Biology, PO Box 100245, Gainesville, FL 32610-0245
Emailjwalejko@ufl.edu
Phonena
Submit Date2018-11-30
Num Groups2
Total Subjects12
Raw Data AvailableYes
Raw Data File Type(s)fid
Analysis Type DetailLC-MS
Release Date2019-03-06
Release Version1
Jacquelyn Walejko Jacquelyn Walejko
https://dx.doi.org/10.21228/M81D57
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000740
Project DOI:doi: 10.21228/M81D57
Project Title:Chronic Maternal Cortisol Excess During Late Gestation Leads to Metabolic Alterations in the Newborn Heart
Project Summary:Our laboratory has previously shown in an ovine model of pregnancy that abnormal elevations in maternal cortisol during late gestation lead to increased fetal cardiac arrhythmias and mortality during peripartum. Furthermore, transcriptomic analysis of the fetal heart suggested alterations in TCA cycle intermediates and lipid metabolites in animals exposed to excess cortisol in utero. Therefore, we utilized a sheep model of pregnancy to determine how chronic increases in maternal cortisol alter maternal and fetal serum prior to birth and neonatal cardiac metabolites and lipids at term. Ewes were either infused with 1 mg/kg/day of cortisol starting at gestational day 115 (n=9), or untreated (n=6). Serum was collected from the mother and fetus (125 d-birth), and hearts were collected following birth. Proton nuclear magnetic resonance (1H-NMR) spectroscopy was conducted to measure metabolic profiles of newborn heart specimens as well as fetal and maternal serum specimens. Mass spectrometry was conducted to measure lipid profiles of newborn heart specimens. We observed alterations in amino acid and TCA cycle metabolism as well as lipid and glycerophospholipid metabolism in newborn hearts after excess maternal cortisol in late gestation. In addition, we observed alterations in amino acid and TCA cycle metabolites in fetal but not in maternal serum during late gestation. These results suggest that fetal exposure to excess maternal cortisol alters placental and fetal metabolism prior to birth and limits normal cardiac metabolic maturation, which may contribute to increased risk of peripartum cardiac arrhythmias observed in these animals, or later life cardiomyopathies.
Institute:University of Florida
Department:Pharmacodynamics
Last Name:Keller-Wood
First Name:Maureen
Address:1345 SW Archer Rd, PO 100487, Gainesville, FL, 32610
Email:kellerwd@cop.ufl.edu
Phone:NA

Subject:

Subject ID:SU001151
Subject Type:Mammal
Subject Species:Ovis aries
Taxonomy ID:9940
Age Or Age Range:within 6 hours of birth

Factors:

Subject type: Mammal; Subject species: Ovis aries (Factor headings shown in green)

mb_sample_id local_sample_id Treatment
SA075241767Control
SA0752421577Control
SA0752431610-16Control
SA075244753Control
SA075245770Control
SA075246734Control
SA0752471576Cortisol
SA0752483843Cortisol
SA075249717Cortisol
SA075250729Cortisol
SA075251706Cortisol
SA075252802Cortisol
Showing results 1 to 12 of 12

Collection:

Collection ID:CO001145
Collection Summary:Immediately following birth, the lambs were euthanized with an overdose of Euthasol (pentobarbital sodium and phenytoin sodium; Virbac AH Inc), and heart tissue was collected from the right ventricle (RV), left ventricle (LV), and intraventricular septum (IVS) and immediately frozen in liquid nitrogen. Tissue samples were stored at -80 °C until data collection.
Sample Type:Cardiac tissue
Collection Method:Heart tissue was collected under sterile conditions and immediately frozen in liquid nitrogen
Collection Location:University of Florida
Storage Conditions:-80℃
Collection Vials:Cryovials
Storage Vials:Cryovials

Treatment:

Treatment ID:TR001165
Treatment Summary:The treatment protocol for this study was previous published in Antolic, A., et al. (2018). Chronic maternal hypercortisolemia in late gestation alters fetal cardiac function at birth. Am J Physiol Regul Integr Comp Physiol 314(3): R342-R352.
Treatment Compound:Hydrocortisone sodium succinate in sodium phosphate (Solu-Cortef; Pfizer, New York, NY, USA)
Treatment Dose:1 mg/kg/day

Sample Preparation:

Sampleprep ID:SP001158
Sampleprep Summary:Homogenized right ventricle tissue samples (100 mg from 7 fetuses and 7 newborns) were used for lipid extraction via the Folch method. Briefly, 2 mL of internal standard mixture was added to each sample before adding 1.2 mL of 2:1 chloroform/methanol solution (HPLC-grade). Samples were incubated at 4 °C for 20 min with occasional vortexing. Following incubation, 200 mL of water (HPLC-grade) was added and samples were incubated at 4 °C for 10 min. Samples were centrifuged at 2,000 g for 5 min at 4 °C, and the resulting chloroform layer was removed and transferred into a clean tube. The extraction process was repeated with 400 mL 2:1 chloroform/methanol (HPLC-grade) and the resulting chloroform layers were combined. Samples were dried under a nitrogen stream at 30 °C and stored at -80 °C until reconstitution. Samples were reconstituted with 200 mL of isopropanol and 2 mL injection standard mixture. Ammonium acetate and all analytical grade solvents (formic acid, chloroform, and methanol) were purchased from Fisher Scientific (Waltham, MA, USA). All mobile phase solvents were Fisher Optima LC/MS-grade (acetonitrile, isopropanol, and water). Organ: Heart, Organ Specification: Right Ventricle
Processing Method:50 mg of tissue was weighed and added to tubes with three 3mm glass beads, two 5mm steal beads, and 0.7mm zirconia beads (“2 squirts”). HPLC-water (1mL) was added to each tube before homogenization at 1800 rpm for 30 seconds for 5 rounds. Samples were incubated at 4C for 20 min between rounds. Following homogenization, samples were centrifuged at 2,000g for 10 min at 4C to pellet tissue debris before transferring supernatant to a clean tube. Homogenates were stored at -80C.
Extraction Method:2 mL of internal standard mixture was added to each sample before adding 1.2 mL of 2:1 chloroform/methanol solution (HPLC-grade). Samples were incubated at 4 °C for 20 min with occasional vortexing. Following incubation, 200 mL of water (HPLC-grade) was added and samples were incubated at 4 °C for 10 min. Samples were centrifuged at 2,000 g for 5 min at 4 °C, and the resulting chloroform layer was removed and transferred into a clean tube. The extraction process was repeated with 400 mL 2:1 chloroform/methanol (HPLC-grade) and the resulting chloroform layers were combined. Samples were dried under a nitrogen stream at 30 °C (Organomation Associated MultiVap) and stored at -80 °C until reconstitution.
Sample Resuspension:Samples were reconstituted with 200 mL of isopropanol and 2 mL injection standard mixture and vortexed to mix. Fifty uL of reconstituted sample was transferred to a glass LC vial for analysis.
Sample Spiking:Internal standards: lysophosphatidylcholine (17:0), phosphatidylcholine (17:0/17:0), phosphatidylglycerol (14:0/14:0), phosphatidylethanolamine (15:0/15:0), phosphatidylserine (14:0/14:0), triglyceride (15:0/15:0/15:0). Injection Standards: lysophosphatidylcholine (19:0), phosphatidylcholine (19:0/19:0), phosphatidylglycerol (17:0/17:0), phosphatidylethanolamine (17:0/17:0), phosphatidylserine (17:0/17:0), triglyceride (17:0/17:0/17:0).

Combined analysis:

Analysis ID AN001799 AN001800
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Thermo Dionex Ultimate 3000 Thermo Dionex Ultimate 3000
Column Waters Acquity BEH C18 (50 x 2.1mm,1.7um) Waters Acquity BEH C18 (50 x 2.1mm,1.7um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE NEGATIVE
Units Relative peak area Relative peak area

Chromatography:

Chromatography ID:CH001270
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:Waters Acquity BEH C18 (50 x 2.1mm,1.7um)
Column Temperature:4C
Flow Gradient:80% A and 20% B at 0 min; 80% A and 20% B at 1 min; 70% A and 30% B at 3 min; 55% A and 45% B at 4 min; 40% A and 60% B at 6 min; 35% A and 65% B at 8 min; 35% A and 65% B at 10 min; 10% A and 90% B at 15 min; 2% A and 98% B at 17 min; 2% A and 98% B at 18 min; 80% A and 20% B at 19 min; 80% A and 20% B at 23 min
Flow Rate:0.5mL/min
Sample Injection:2 uL
Solvent A:40% water/60% acetonitrile; 0.1% formic acid; 10 mM ammonium formate
Solvent B:90% isopropanol/8% acetonitrile/2% water; 0.1% formic acid; 10 mM ammonium formate
Analytical Time:18 min
Chromatography Type:Reversed phase

MS:

MS ID:MS001660
Analysis ID:AN001799
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
Ion Mode:POSITIVE
Capillary Temperature:250C
Ion Spray Voltage:1500
Ionization:Heated electrospray
Resolution Setting:70,000
Scanning Range:200-2200 m/z
  
MS ID:MS001661
Analysis ID:AN001800
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
Ion Mode:NEGATIVE
Capillary Temperature:250C
Ion Spray Voltage:1500
Ionization:Heated electrospray
Resolution Setting:70,000
Scanning Range:200-2200 m/z
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