We focused on sphingolipids that carried a saturated fatty acid acylated to the sphingoid backbone, including palmitic acid (16:0 [16 carbons, 0 double bonds]), stearic acid (18:0), arachidic acid (20:0), behenic acid (22:0), and lignoceric acid (24:0). The sphingolipids were measured on baseline fasting plasma samples that had been stored at −70
°C with the following method. Lipids were extracted using organic protein precipitation in a mixture of methyl
tert-butyl ether, methanol, and isopropanol. For each sample, 10 µL was pipetted into the appropriate well of a 96–deep well polypropylene microtiter plate (Masterblock; Greiner Bio-One, cat. no. 780270). In a chemical fume hood, 190 µL of precipitation solvent was added to each well using a multichannel pipet. The plate was sealed with a MicroLiter silicone cap mat with sprayed-on PTFE barrier (Wheaton, cat. no. 07–0061N), placed in a plastic Ziploc bag, and mixed on a multitube vortex (VWR) for 5 min at speed 10. Subsequently, in a fume hood, a 10-μm glass filter plate (Captiva; Agilent, cat. no. A596401000) was placed above a new Masterblock plate. Using a multichannel pipet, the samples were transferred from the precipitation plate into the filter plate and allowed to flow through using gravity (approximately 50 µL flows through the membrane filter in each well). The filter plate was carefully removed and discarded. To each sample in the new Masterblock plate, 450 µL of 65% methanol/25% isopropanol (v:v) was added and mixed by pipetting up and down 10 times with an electronic multichannel pipet. The plate was sealed and then a volume of 5 µL was injected using an autosampler (samples were cooled at 8°C) and resolved using reversed-phase chromatography at 50°C on an Acquity UPLC Protein BEH C4 Column, 300Å, 1.7 µm, 2.1 mm × 50 mm analytical column (Waters, cat. no. 186004495) equipped with an Acquity UPLC Protein BEH C4 VanGuard Pre-column, 300Å, 1.7 µm, 2.1 mm × 5 mm guard column (Waters, cat. no. 186004623). Mobile phases were Optima water/0.2% formic acid (buffer A) and 60% acetonitrile/40% isopropanol/0.2% formic acid (buffer B). A linear gradient from 49% to 79% buffer B over 8.4 min at 0.4 mL/min was used to resolve the analytes. Analytes were introduced to the mass spectrometer (Sciex 6500) and analyzed using optimized mass spectrometric parameters for each compound.
Internal standards were included in the precipitation solvent at a concentration of 19.4 nmol/L (Ceramide/Sphingolipid Internal Standard Mixture I, 25 μmol/L; Avanti Polar Lipids, LM-6002), which controls for variability in extraction efficiency, pipetting, and ion suppression. Chromatographic peak areas of the endogenous analytes and the internal standards were quantified using SkyLine software (14 (
link)). Each peak area for each endogenous sphingolipid was divided by the sum of the peak area of five internal standards (ceramide C12 [CerC12], CerC25, glucosyl ceramide C12 [GluCerC12], lactosyl ceramide C12 [LacCerC12], and sphingomyelin 12 [SM12]), which was called the peak area ratio. The peak area ratio for each sphingolipid was then divided by the mean peak area ratio in the single point calibrator in the batch (precipitated and analyzed 5 times in each batch, spread across the plate). The single point calibrator was a pooled EDTA-anticoagulated plasma sample made from discarded de-identified clinical samples from the clinical laboratory at the University of Washington Medical Center. Additional details on the sphingolipid measurements and quality control procedures are provided in the
Supplementary Data. The coefficients of variation for each sphingolipid species are shown in
Table 1.
In total we measured 22 sphingolipid species. This report is restricted to the 15 species with coefficient of variation ≤21% over the whole study period. It includes five ceramides: ceramide with 16:0 (Cer-16), 18:0 (Cer-18), 20:0 (Cer-20), 22:0 (Cer-22), and a composite concentration of Cer-24 computed as the sum of the concentrations of two species of ceramides with 24:0 having the distinct “d181” and “d182” sphingoid backbones. It also includes six sphingomyelins, SM-14, SM-16, SM-18, SM-20, SM-22, and SM-24; three glucosyl ceramides, GluCer-16, GluCer-22, and GluCer-24; and one lactosyl ceramide, LacCer-16. Simplified relationships between the sphingolipid classes that were measured are shown in
Fig. 1.
Lemaitre R.N., Yu C., Hoofnagle A., Hari N., Jensen P.N., Fretts A.M., Umans J.G., Howard B.V., Sitlani C.M., Siscovick D.S., King I.B., Sotoodehnia N, & McKnight B. (2018). Circulating Sphingolipids, Insulin, HOMA-IR, and HOMA-B: The Strong Heart Family Study. Diabetes, 67(8), 1663-1672.
