{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001106","ANALYSIS_ID":"AN001800","VERSION":"1","CREATED_ON":"December 4, 2018, 7:55 pm"},

"PROJECT":{"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"},

"STUDY":{"STUDY_TITLE":"Lipidomics of Newborn Heart Tissue Exposed to Excess Maternal Cortisol in Late Gestation (part-1)","STUDY_SUMMARY":"Cardiac 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","DEPARTMENT":"Biochemsitry & Molecular Biology","LAST_NAME":"Walejko","FIRST_NAME":"Jacquelyn","ADDRESS":"R3-226 Academic Research Building, Department of Biochemistry and Molecular Biology, PO Box 100245, Gainesville, FL 32610-0245","EMAIL":"jwalejko@ufl.edu","PHONE":"na","NUM_GROUPS":"2","TOTAL_SUBJECTS":"12"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Ovis aries","TAXONOMY_ID":"9940","AGE_OR_AGE_RANGE":"within 6 hours of birth"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"802",
"Sample ID":"802",
"Factors":{"Treatment":"Cortisol"},
"Additional sample data":{"Batch":"2"}
},
{
"Subject ID":"734",
"Sample ID":"734",
"Factors":{"Treatment":"Control"},
"Additional sample data":{"Batch":"1"}
},
{
"Subject ID":"770",
"Sample ID":"770",
"Factors":{"Treatment":"Control"},
"Additional sample data":{"Batch":"1"}
},
{
"Subject ID":"729",
"Sample ID":"729",
"Factors":{"Treatment":"Cortisol"},
"Additional sample data":{"Batch":"1"}
},
{
"Subject ID":"706",
"Sample ID":"706",
"Factors":{"Treatment":"Cortisol"},
"Additional sample data":{"Batch":"1"}
},
{
"Subject ID":"767",
"Sample ID":"767",
"Factors":{"Treatment":"Control"},
"Additional sample data":{"Batch":"2"}
},
{
"Subject ID":"717",
"Sample ID":"717",
"Factors":{"Treatment":"Cortisol"},
"Additional sample data":{"Batch":"1"}
},
{
"Subject ID":"753",
"Sample ID":"753",
"Factors":{"Treatment":"Control"},
"Additional sample data":{"Batch":"2"}
},
{
"Subject ID":"1610-16",
"Sample ID":"1610-16",
"Factors":{"Treatment":"Control"},
"Additional sample data":{"Batch":"3"}
},
{
"Subject ID":"3843",
"Sample ID":"3843",
"Factors":{"Treatment":"Cortisol"},
"Additional sample data":{"Batch":"1"}
},
{
"Subject ID":"1577",
"Sample ID":"1577",
"Factors":{"Treatment":"Control"},
"Additional sample data":{"Batch":"3"}
},
{
"Subject ID":"1576",
"Sample ID":"1576",
"Factors":{"Treatment":"Cortisol"},
"Additional sample data":{"Batch":"3"}
}
],
"COLLECTION":{"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_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"},

"SAMPLEPREP":{"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)."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Normal phase","INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","COLUMN_NAME":"Waters Acquity BEH C18 (50 x 2.1mm, 1.7um)","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","COLUMN_TEMPERATURE":"4C","SOLVENT_A":"Acetonitrile:H2O (60:40) with 10mM ammonium formate and 0.1% formic acid","SOLVENT_B":"Isopropanol:acetonitrile:H2O (90:8:2) with 10mM ammonium formate and 0.1% formic acid","SAMPLE_INJECTION":"2 uL","ANALYTICAL_TIME":"18 min"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","LABORATORY_NAME":"Timothy Garrett","OPERATOR_NAME":"Jeremy Koelmel"},

"MS":{"MS_COMMENTS":"-","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"},

"MS_METABOLITE_DATA":{
"Units":"Relative peak area",

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}

}