Quantification of Ceramide, Sphingosine and S1P in Mouse Liver

Lipid extracts were prepared from liver homogenates by the classic Folch method (Folch et al., 1957) using chloroform/methanol (2:1). The lipid extract was then dried under nitrogen gas and re-dissolved in a 2% Igepal solution. The ceramide, sphingosine, and S1P levels were measured as previously described (He et al., 2009, 2017). Briefly, to determine the total ceramide content the 2% Igepal lipid extract was resuspended in a hydrolysis buffer containing 0.2 mg/ml of recombinant AC. This completely hydrolyzed the ceramide in the extract to sphingosine. The produced sphingosine was then derivatized into a fluorescent product after being reacted with a fluorogenic compound, NDA, and analyzed using an Acquity H-Class UPLC system (Waters) monitored at excitation and emission wavelengths of 252 and 483 nm, respectively. Quantification of the fluorescent sphingosine was calculated using the Waters Empower software according to a standard curve derived from commercial sphingosine. For quantification of sphingosine, the same procedure was used except that the ceramide hydrolysis step was excluded (i.e., no recombinant AC was added). In this way endogenous sphingosine present in the lipid extract could be derivatized directly with NDA and quantified by UPLC. For S1P quantification the same procedure was used since the derivatized sphingosine and S1P peaks could be readily separated and individually quantified by UPLC.