Sphingolipid analysis

The content of S1P, dhS1P, sphingosine, dihydrosphingosine, ceramide, and dihydroceramide was determined as previously described in detail [21]. Briefly, lipids were extracted from samples in the presence of internal standards (C17-sphingosine and C17-S1P, Avanti Polar Lipids, Alabaster, AL). An aliquot of the lipid extract was transferred to a fresh tube with pre-added N-palmitoyl-D-erythro-sphingosine (C17 base, Avanti Polar Lipids) as an internal standard, and then subjected to alkaline hydrolysis to deacylate ceramide and dihydroceramide to sphingosine and dihydrosphingosine, respectively. The amounts of S1P and dhS1P were determined indirectly after dephosphorylation to sphingosine and dihydrosphingosine, respectively, with the use of alkaline phosphatase. Free sphingosine and dihydrosphingosine, dephosphorylated sphingoid base-1-phosphates, and sphingoid bases released from ceramide and dihydroceramide were then converted to their o-phthalaldehyde derivatives and analyzed using a UPLC system (Nexera, Shimadzu Corp., Kioto, Japan) equipped with a fluorescence detector (RF-20Axs) and a C18 reversed-phase column (Reproshell ODS-1, 2.7 μm, 125 × 3 mm, Dr Maisch, Ammerbuch, Germany). The isocratic eluent composition of acetonitrile:water (84:16, v/v) and a flow rate of 0.4 mL/min were used. Column temperature was maintained at 30°C.

For sphingomyelin analysis, lipids were extracted from erythrocytes and platelets by the method of Folch. Next, sphingomyelin was separated by thin-layer chromatography using the method described by Mahadevappa et al. [22]. The gel bands corresponding to the sphingomyelin standard were scrapped off the plates and transferred into fresh tubes containing pentadecanoic acid as an internal standard. Sphingomyelin fatty acids were then transmethylated in the presence of 14% boron trifluoride in methanol at 100°C for 90 min. The resulting methyl esters of palmitic, stearic, arachidic, behenic and lignoceric acid were analyzed by means of gas-liquid chromatography using a Hewlett-Packard 5890 Series II system equipped with a double flame ionization detector and Agilent (Santa Clara, CA) CP-Sil 88 capillary column (100 m, 0.25 mm i.d.). The content of sphingomyelin is presented as the sum of all analyzed individual fatty acid methyl esters.

Hemoglobin concentration was determined using Drabkin’s reagent kit (Sigma, Schnelldorf, Germany). Protein concentration was measured with the BCA protein assay kit (Sigma). Bovine serum albumin (fatty acid free, Sigma) was used as a standard.