C17 0 ceramide

Ceramides and diacylglycerols were measured according to the methods described by Blachnio-Zabielska et al. [35 (link), 36 (link)]. Briefly, lipids were extracted from ~20 mg of tissue by the use of the extraction mixture composed of isopropanol : water : ethyl acetate (35 : 5 : 60; v : v : v). Quantitative measurement of ceramides and diacylglycerols was made using an Agilent 6460 triple quadrupole mass spectrometer. Both ceramides and diacylglycerols were analyzed using positive ion electrospray ionization (ESI) source with multiple reaction monitoring (MRM). The chromatographic separation was performed using an Agilent 1290 Infinity Ultra Performance Liquid Chromatography (UPLC). The analytical column was a reverse-phase Zorbax SB-C8, 2.1 × 150 mm, 1.8 μm (Agilent, Santa Clara, CA). Chromatographic separation was conducted in binary gradient using 2 mM ammonium formate, 0.15% formic acid in methanol as Solvent A, and 1.5 mM ammonium formate; 0.1% formic acid in water as Solvent B at the flow rate of 0.4 mL/min. C17:0-ceramide and 1,3-dipentadecanoyl-rac-glycerol (Avanti Polar Lipids, Alabaster, AL) were used as internal standards for ceramides and diacylglycerols, respectively. The HPLC grade methanol and water as well as formic acid and ammonium formate were obtained from Sigma-Aldrich (St. Louis, MO).

Total lipids were extracted from skeletal muscle homogenates (Folch et al., 1957) with non‐lipid impurities removed by washing with 0.88% (w/v) KCl. C17:0 ceramide (Avanti Polar Lipids Inc., Alabaster, AL, USA) was added as an internal standard. Ceramides were isolated using solid‐phase extraction chromatography (100 mg, 3 mL silica columns, Biotage, Uppsala, Sweden) and eluted in 5‐mL chloroform/ ethyl acetate (1:1) prior to quantification by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) as described previously (Mcilroy et al., 2016). All analyses were performed on a Thermo TSQ Quantum Ultra triple quadrupole mass spectrometer interfaced to a Thermo Accela 1250 UHPLC system in positive ion mode. Individual ceramide species were separated on a Kinetex C8 LC column (100 x 2.1mm x 2.6µm, Phenomenex, Macclesfield, UK) using an acetonitrile/ water gradient and detected through multiple reaction monitoring (MRM). The data were processed using Thermo Xcalibur 2.1 Quan Browser software.

Plasma was obtained from anticoagulated whole blood by subjecting it to centrifugation at about 250g for 10 minutes at 4°C, followed by collection of plasma, which was stored at –80°C until analysis. To extract lipids, frozen tissues were bead homogenized in Tris buffer using a FastPrep FP120 cell disrupter (Thermo Fisher Scientific). Plasma and tissue homogenates were spiked with internal standard and extracted as described (66 (link)). Internal standard mixture contained sphingosine, C17:0 ceramide, D-erythro-sphingosine-d7-1-P, C12:0 ceramide-1-P, sphingomyelin (d18:1/C12:0), and lyso-sphingomyelin (d17:1) (Avanti Polar Lipids). Targeted metabolomics was performed using single reaction monitoring with an Agilent Technologies 6490 triple-quadrupole LC-MS/MS instrument coupled with a 1290 Infinity UPLC system as described (66 (link)).