Summary of Study ST001140

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 PR000761. The data can be accessed directly via it's Project DOI: 10.21228/M89Q32 This work is supported by NIH grant, U2C- DK119886.

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Study IDST001140
Study TitleChanges in the Canine Plasma Lipidome after Short- and Long-Term Excess Glucocorticoid Exposure
Study SummaryGlucocorticoids (GCs) are widely used in veterinary and human medicine. Chromic endogenous or iatrogenic GC overexposure impairs metabolic function and can result in diverse side-effects, including Cushing’s syndrome. This study examines the effects of experimentally induced short-term and long-term GC excess (induced by prednisolone and tetracosactide, respectively) on the plasma lipidome of Beale dogs. Both, long- and short-term GC resulted in significant changes of the plasma lipidome.
Institute
National University of Singapore;University of Zurich
DepartmentSingapore Lipidomics Incubator (SLING);Vetsuisse Faculty, University of Zurich
LaboratorySingapore Lipidomics Incubator (SLING), National University of Singapore
Last NameBurla
First NameBo
Address28 Medical Drive, Singapore 117456, Singapore
Emailbo.burla@nus.edu.sg
Phone+6565166683
Submit Date2019-01-19
Num Groups2
Total Subjects14
Num Males9
Num Females5
Raw Data AvailableYes
Raw Data File Type(s)d
Analysis Type DetailLC-MS
Release Date2019-03-06
Release Version1
Bo Burla Bo Burla
https://dx.doi.org/10.21228/M89Q32
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000761
Project DOI:doi: 10.21228/M89Q32
Project Title:Changes in the Canine Plasma Lipidome after Short- and Long-Term Excess Glucocorticoid Exposure
Project Summary:Glucocorticoids (GCs) are widely used in veterinary and human medicine. Chromic endogenous or iatrogenic GC overexposure impairs metabolic function and can result in diverse side-effects, including Cushing’s syndrome. This study examines the effects of experimentally induced short-term and long-term GC excess (induced by prednisolone and tetracosactide, respectively) on the plasma lipidome of Beale dogs. Both, long- and short-term GC resulted in significant changes of the plasma lipidome.
Institute:National University of Singapore;University of Zurich
Department:Singapore Lipidomics Incubator (SLING);Vetsuisse Faculty, University of Zurich
Laboratory:Singapore Lipidomics Incubator (SLING)
Last Name:Burla
First Name:Bo
Address:28 Medical Drive, Singapore 117456, Singapore
Email:bo.burla@nus.edu.sg
Phone:+6565166683

Subject:

Subject ID:SU001204
Subject Type:Mammal
Subject Species:Canis lupus familiaris
Taxonomy ID:9615
Genotype Strain:Beagle
Age Or Age Range:8 to 83 months
Weight Or Weight Range:12.2 to 18.9 kg
Gender:Male and female
Animal Animal Supplier:In-house breeding
Animal Feed:Standard pellet/kibble maintenance diet

Factors:

Subject type: Mammal; Subject species: Canis lupus familiaris (Factor headings shown in green)

mb_sample_id local_sample_id TreatmentGroup TreatmentDuration SamplingTimePoint
SA078299Prednisolone-d0-P5Prednisolone 0d before
SA078300Prednisolone-d0-P6Prednisolone 0d before
SA078301Prednisolone-d0-P1Prednisolone 0d before
SA078302Prednisolone-d0-P4Prednisolone 0d before
SA078303Prednisolone-d0-P7Prednisolone 0d before
SA078304Prednisolone-d0-P8Prednisolone 0d before
SA078305Prednisolone-d0-P2Prednisolone 0d before
SA078306Prednisolone-d0-P3Prednisolone 0d before
SA078307Prednisolone-d4-P4Prednisolone 4d after
SA078308Prednisolone-d4-P1Prednisolone 4d after
SA078309Prednisolone-d4-P7Prednisolone 4d after
SA078310Prednisolone-d4-P8Prednisolone 4d after
SA078311Prednisolone-d4-P2Prednisolone 4d after
SA078312Prednisolone-d4-P3Prednisolone 4d after
SA078313Prednisolone-d4-P6Prednisolone 4d after
SA078314Prednisolone-d4-P5Prednisolone 4d after
SA078315Tetracosactide-w00-T6Tetracosactide 00w before
SA078316Tetracosactide-w00-T5Tetracosactide 00w before
SA078317Tetracosactide-w00-T4Tetracosactide 00w before
SA078318Tetracosactide-w00-T2Tetracosactide 00w before
SA078319Tetracosactide-w00-T1Tetracosactide 00w before
SA078320Tetracosactide-w00-T3Tetracosactide 00w before
SA078321Tetracosactide-w25-T5Tetracosactide 25w after
SA078322Tetracosactide-w25-T6Tetracosactide 25w after
SA078323Tetracosactide-w25-T1Tetracosactide 25w after
SA078324Tetracosactide-w25-T2Tetracosactide 25w after
SA078325Tetracosactide-w25-T3Tetracosactide 25w after
SA078326Tetracosactide-w25-T4Tetracosactide 25w after
Showing results 1 to 28 of 28

Collection:

Collection ID:CO001198
Collection Summary:Blood was collected from the jugular vein after overnight fasting right before start and at the end of the treatments (12 h after the last prednisolone administration and 25 weeks after start of tetracosactide infusion, respectively). Plasma was prepared lithium heparin anticoagulated blood (Vacuette Greiner Bio-One, Germany) that centrifuged immediately after collection (1,862 g, 10 min, 4°C). Plasma samples were stored at −80°C until lipidomic analysis.
Collection Protocol Comments:Blood collected after overnight fasting
Sample Type:Blood (plasma)
Collection Method:Venepuncture
Collection Location:Jugular vein
Collection Frequency:Before and after treatment
Storage Conditions:-80℃
Collection Vials:VACUETTE® lithium heparin tubes (Greiner Bio-One, Germany)
Collection Tube Temp:Room temperature
Additives:Lithium heparin

Treatment:

Treatment ID:TR001219
Treatment Summary:In the short-term prednisolone group, dogs were treated with 50 mg prednisolone (Streuli Pharma AG, Switzerland) orally twice daily for 3 consecutive days. For the long-term tetracosactide treatment, ALZET osmotic pumps (Durect Corporation, USA) subcutaneously delivering tetracosactide (synthetic ACTH; Bachem AG, Switzerland) were implanted into the dorsolateral neck. New pumps were implanted every 4 weeks. Total infusion time was 25 weeks, with a starting dose of 1.3–1.95 µg/kg/d tetracosactide, which was gradually increased to a final dose of 6–10 µg/kg/d.

Sample Preparation:

Sampleprep ID:SP001212
Sampleprep Summary:Plasma lipids were extracted using a single-phase butanol/methanol extraction method (Alshehry et al, Metabolites 2015 with modifications). After thawing on ice, a 10 µL aliquot of each plasma sample was transferred into a 2 mL polypropylene tube (Eppendorf, Germany). Subsequently, 1 µL BHT (2,6-di-tert-butyl-4-methylphenol, 10 mmol/L in ethanol) and 90 µL of 1-butanol:methanol (1:1, v/v) containing internal standards was added to each sample. Following internal standards were added at 50 ng/mL final concentration in the extraction solvent: ceramide d18:1/17:0 (Avanti 860517P), Glucosylceramide (Matreya 1533), Lactosylceramide d18:1/16:0 D3 (Matreya 1534), LPC 20:0 (Avanti 855777P), LPE 14:0 (Avanti 856735P), PI 12:0/13:0 (Avanti LM-1500), PE 14:0/14:0 (Avanti 850745P), PS 14:0/14:0 (Avanti 840033P), SM d18:1/12:0 (Avanti 860583P), and at 100 ng/mL: DG 12:0_12:0 (Avanti 800812P), PC 14:0/14:0 (Avanti 850345P), TAG 16:0_16:0_16:0 d5 (CDN Isotopes D5815). Samples were then vortexed (30 sec) and sonicated in an ultrasound water bath for 30 min (20°C). After centrifugation (14,000 g, 10 min, 4°C), 90 µL of supernatant were transferred into 1.5 mL polypropylene tubes (Sarstedt, Germany) and dried under a nitrogen stream at 37°C. Dried extracts were stored at −80°C until LC-MS analysis. Dried samples were reconstituted with 90 µL 1-butanol:methanol (1:1, v/v) and sonication in an ultrasound water for 10 min. After centrifugation for 10 min at 20,800 g (4°C), supernatants (80 µL) were transferred into autosampler vials with glass inserts (Agilent Technologies, USA). For the analysis of sphingosine-1-phosphate (S1P), lipid extracts were derivatized according to the method described by Narayanaswamy et al. (Anal. Chem., 2014). To 50 µL lipid extract (see above), 50 µL 13C2D2–S1P d18:1 internal standard solution (Toronto Research Chemicals, Canada; 20 ng/mL in 1-butanol:methanol 1:1 [v/v]) was added. Derivatization was performed by adding 20 µL TMS-diazomethane (2 mol/L in hexanes; Acros Organics, Thermo Fisher Scientific, USA) and subsequent incubation for 20 min at 25°C and 700 rpm (Thermomixer, Eppendorf, Germany). To stop the reaction and inactivate TMS, 1 µL acetic acid 100% (glacial) was added. After centrifugation for 10 min at 20,800 g (7°C), supernatants were transferred into autosampler vials for LC-MS analysis. Process Quality Control (PQC) samples were generated by pooling equal volumes of plasma samples within an experimental group. For Blank samples, plasma was omitted. PQC and Blank were processed together with the study plasma samples with the same procedures.
Extraction Method:Liquid–liquid extraction using butanol:methanol
Extract Storage:-80℃

Combined analysis:

Analysis ID AN001870 AN001871 AN001872 AN001873
Analysis type MS MS MS MS
Chromatography type Reversed phase Reversed phase HILIC Reversed phase
Chromatography system Agilent 1290 Infinity Agilent 1290 Infinity Agilent 1290 Infinity Agilent 1100
Column Agilent Zorbax RRHD Eclipse Plus C18 (50 x 2.1mm,1.8um,95 Å) Agilent Zorbax RRHD Eclipse Plus C18 (50 x 2.1mm,1.8um,95 Å) Waters Acquity BEH HILIC (100 x 2.1mm,1.7um,130 Å) Agilent Zorbax Eclipse XDB C18 (150 x 3.0mm,1.8um)
MS Type ESI ESI ESI ESI
MS instrument type Triple quadrupole Triple quadrupole Triple quadrupole Triple quadrupole
MS instrument name Agilent 6460 QQQ Agilent 6495 QQQ Agilent 6490 QQQ ABI Sciex 4000 QTrap
Ion Mode POSITIVE POSITIVE POSITIVE POSITIVE
Units µmol/L µmol/L µmol/L µmol/L

Chromatography:

Chromatography ID:CH001351
Instrument Name:Agilent 1290 Infinity
Column Name:Agilent Zorbax RRHD Eclipse Plus C18 (50 x 2.1mm,1.8um,95 Å)
Column Temperature:40
Flow Gradient:20% B: 2 min, 20% - 60% B: 2 - 7 min, 60% - 100% B: 7 - 9 min, 20% B: 9 - 10.8 min
Flow Rate:0.4 mL/min
Sample Injection:2 µL
Solvent A:60% acetonitrile/40% water; 10 mM ammonium formate
Solvent B:90% isopropanol/10% acetonitrile; 10 mM ammonium formate
Chromatography Type:Reversed phase
  
Chromatography ID:CH001352
Instrument Name:Agilent 1290 Infinity
Column Name:Agilent Zorbax RRHD Eclipse Plus C18 (50 x 2.1mm,1.8um,95 Å)
Column Temperature:40
Flow Gradient:20% B: 2 min, 20% - 60% B: 2 - 7 min, 60% - 100% B: 7 - 9 min, 20% B: 9 - 10.8 min
Flow Rate:0.4 mL/min
Sample Injection:1 µL
Solvent A:60% acetonitrile/40% water; 10 mM ammonium formate
Solvent B:90% isopropanol/10% acetonitrile; 10 mM ammonium formate
Chromatography Type:Reversed phase
  
Chromatography ID:CH001353
Instrument Name:Agilent 1290 Infinity
Column Name:Waters Acquity BEH HILIC (100 x 2.1mm,1.7um,130 Å)
Column Temperature:60
Flow Gradient:99.9% B: 0 - 5 min; 40% B: 5 - 5.5 min; 10% B: 5.5 - 6.6 min and 99.9% B: 6.6 - 9.6 min (total run time 9.6 min)
Flow Rate:0.4 mL/min
Sample Injection:2 µL
Solvent A:50% water/50% acetonitrile; 25 mM ammonium formate, pH 4.6
Solvent B:95% acetonitrile/5% water; 25 mM ammonium formate, pH 4.6
Chromatography Type:HILIC
  
Chromatography ID:CH001354
Instrument Name:Agilent 1100
Column Name:Agilent Zorbax Eclipse XDB C18 (150 x 3.0mm,1.8um)
Column Temperature:25
Flow Gradient:Isocratic (25 min)
Flow Rate:0.128 mL/min
Sample Injection:10 µL
Solvent A:50% chloroform/50% methanol; 2 mM ammonium acetate
Chromatography Type:Reversed phase

MS:

MS ID:MS001726
Analysis ID:AN001870
Instrument Name:Agilent 6460 QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Phospholipids, cholesteryl esters and diacylglycerols were measured with an Agilent 6460 triple quadrupole mass spectrometer in dynamic MRM mode. The ESI source settings were: polarity: positive, dry gas temperature 300°C, dry gas flow 5L/min, nebulizer pressure: 45 psi, sheath gas temperature: 250°C, sheath gas flow: 11 L/min, capillary voltage: 3500 V, noozle: 500. MRM transitions with collision energies are detailed in the attached protocol. Data were processed with Agilent MassHunter QQQ Quantitative Analysis (version B.08). For plasmalogen PE (PE-P), transitions with the fatty acid as product were used as quantifiers, and those with the head group as qualifiers. Plasmalogen PCs (PC-P), ether PCs (PC-O) and odd-chain fatty acid PCs were distinguished based on retention time (Alshehry et al., Circulation, 2016; Huynh et al, Cell Chem. Biol. 2019). Normalised peak areas were calculated by dividing the peak areas of the analyte with the corresponding class-specific internal standard. Relative abundance was obtained by multiplying the normalised peak areas with the molar concentration of the corresponding internal standard. Lipid species with a median peak area in the PQC samples below 250 or less than 5 times of the highest signal in Blank samples were excluded. Additionally, the coefficient of variation (CV) of the normalised peak area was calculated for each lipid species in the PQC samples of each experimental group. Species with a CV higher than 25% in any of the two groups were excluded from subsequent evaluation.
Ion Mode:POSITIVE
Capillary Voltage:3500 V
Collision Gas:Nitrogen
Dry Gas Flow:5 L/min
Dry Gas Temp:300°C
Fragment Voltage:135 V
Nebulizer:45 psi
  
MS ID:MS001727
Analysis ID:AN001871
Instrument Name:Agilent 6495 QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Sphingolipids were measured with an Agilent 6495A triple quadrupole mass spectrometer in dynamic MRM mode. The ESI source settings were: polarity: positive, dry gas temperature 200°C, dry gas flow 12 L/min, nebulizer pressure: 25 psi, sheath gas temperature: 250°C, sheath gas flow: 12 L/min, capillary voltage: 3500 V, noozle: 500, iFunnel high pressure RF: 80, iFunnel high pressure RF: 40. MRM transitions with collision energies are detailed in the attached protocol. Data were processed with Agilent MassHunter QQQ Quantitative Analysis (version B.08). Transitions of precursor ions with water loss were used as qualifiers. Normalised peak areas were calculated by dividing the peak areas of the analyte with the corresponding class-specific internal standard. Relative abundance was obtained by multiplying the normalised peak areas with the molar concentration of the corresponding internal standard. Lipid species with a median peak area in the PQC samples below 250 or less than 5 times of the highest signal in Blank samples were excluded. Additionally, the coefficient of variation (CV) of the normalised peak area was calculated for each lipid species in the PQC samples of each experimental group. Species with a CV higher than 25% in any of the two groups were excluded from subsequent evaluation.
Ion Mode:POSITIVE
Capillary Voltage:3500 V
Collision Gas:Nitrogen
Dry Gas Flow:12 L/min
Dry Gas Temp:200°C
Fragment Voltage:135 V
Nebulizer:25 psi
  
MS ID:MS001728
Analysis ID:AN001872
Instrument Name:Agilent 6490 QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Derivatized S1P species were measured with an Agilent 6490 triple quadrupole mass spectrometer in MRM mode. The ESI source settings were: polarity: positive, dry gas temperature 200°C, dry gas flow 12 L/min, nebulizer pressure: 25 psi, sheath gas temperature: 400°C, sheath gas flow: 12 L/min, capillary voltage: 3500 V, noozle: 500, iFunnel high pressure RF: 200, iFunnel high pressure RF: 110. MRM transitions with collision energies are detailed in the attached protocol. Data were processed with Agilent MassHunter QQQ Quantitative Analysis (version B.08). The m/z 60 fragments were used as quantifiers, the m/z 103 fragments as qualifiers. Normalised peak areas were calculated by dividing the peak areas of the S1P species with the internal standard. Relative abundance was obtained by multiplying the normalised peak areas with the molar concentration of the corresponding internal standard. S1P species with a median peak area in the PQC samples below 250 or less than 5 times of the highest signal in Blank samples were excluded. Additionally, the coefficient of variation (CV) of the normalised peak area was calculated for each lipid species in the PQC samples of each experimental group. Species with a CV higher than 25% in any of the two groups were excluded from subsequent evaluation.
Ion Mode:POSITIVE
Capillary Voltage:3500 V
Collision Gas:Nitrogen
Dry Gas Flow:12 L/min
Dry Gas Temp:200°C
Fragment Voltage:135 V
Nebulizer:25 psi
  
MS ID:MS001729
Analysis ID:AN001873
Instrument Name:ABI Sciex 4000 QTrap
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Triacylglycerol species were measured with a Sciex 4000 QTRAP mass spectrometer operated in single-ion monitoring (SIM) mode at unit resolution. The ESI source settings were: polarity: positive, electrospray voltage: 5 kV, source temperature: 250°C, drying gas: nitrogen, gas 1 flow: 40 units, gas 2 flow: 30 units and curtain gas flow: 10 units. MRM transitions with collision energies are detailed in the attached protocol. Raw data were processed with Sciex Analyst (Version 1.6.2). Normalised peak areas were calculated by dividing the peak areas of the TG species with the internal standard. Relative abundance was obtained by multiplying the normalised peak areas with the molar concentration of the internal standard. Lipid species with a median peak area in the PQC samples below 250 or less than 5 times of the highest signal in Blank samples were excluded. Additionally, the coefficient of variation (CV) of the normalised peak area was calculated for each lipid species in the PQC samples of each experimental group. Species with a CV higher than 25% in any of the two groups were excluded from subsequent evaluation.
Ion Mode:POSITIVE
Ion Spray Voltage:5 kV
Source Temperature:250°C
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