Glucosyl β ceramide

Sphingolipids were extracted from sera according to previously published paper [22 (link)]. Briefly, 100 μL of serum for each sample was mixed with 1.5 mL of a 0.01% (w/v) BHT, methanol/chloroform 2:1 solution, fortified with internal standards: sphingomyelin (d18:1/12:0), ceramide (d18:1/12:0) and glucosyl (β)ceramide (d18:1/12:0) (AVANTI polar lipids, Alabaster, AL, US). Mixes were extracted overnight at 48 °C under shaking. After extraction, 0.15 mL of KOH 1 M was added and samples were incubated at 37 °C for two hours. Solutions were neutralized with 0.15 mL of acetic acid 1 M and dried under a nitrogen stream. Sphingolipids were resuspended in methanol, transferred to a clean tube and dried using speedvac. Samples were resuspended in 0.15 mL of methanol and after centrifugation at 10,000× g for 3 min, supernatants were stored in glass vial at −80 °C.

LC-MS grade solvents and reagents as water, methanol, ammonium formate, formic acid, and acetate acid, as well as 3,5-Di-tert-4-butylhydroxytoluene (BHT), were from Sigma-Aldrich (Saint Louis, MO, USA). Ethanol and HPLC analytical grade chloroform were, respectively, from J.T. Baker (Center Valley, PA, USA) and Carlo Erba (Cornaredo, MI, Italy). Potassium hydroxide was from Merk Millipore (Burlington, MA, USA). Sphingosine (d17:1), sphinganine-1-phosphate (d17:1), ceramide (d18:1/12:0), sphingomyelin (d18:1/12:0) and glucosyl (β)ceramide (d18:1/12:0) were from Avanti Polar Lipids (Alabaster, AL, USA).