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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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℃
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