Quantitative Analysis of Sphingomyelin by High-Performance Liquid Chromatography after Enzymatic Hydrolysis

Abstract

Sphingomyelin is the most abundant sphingolipid in mammalian cells and is mostly present in the plasma membrane. A new analytical method using high-performance liquid chromatography (HPLC) was developed to quantify sphingomyelin in mouse plasma and tissues, 3T3-L1 cells, rat aortic smooth muscle cells, and HT-29 cells. Sphingomyelin and dihydrosphingomyelin, an internal standard, were separated by high-performance thin-layer chromatography and simultaneously hydrolyzed with sphingolipid ceramideN-deacylase and sphingomyelinase to release sphingosine and dihydrosphingosine, respectively. Sphingomyelin content was measured by HPLC followingo-phthalaldehyde derivatization. Sphingomyelin concentrations in 3T3-L1 cells, rat aortic smooth muscle cells, and HT-29 cells were60.10±0.24,62.69±0.08, and58.38±0.37 pmol/μg protein, respectively, whereas those in brain, kidney, and liver of ICR mice were55.60±0.43,43.75±0.21, and22.26±0.14 pmol/μg protein. The sphingomyelin concentration in mouse plasma was407.40±0.31 μM. The limits of detection and quantification for sphingomyelin were 5 and 20 pmol, respectively, in the HPLC analysis with fluorescence detection. This sensitivity was sufficient for analyzing sphingomyelin in biological samples. In conclusion, this analytical method is a sensitive and specific technique for quantifying sphingomyelin and was successfully applied to diverse biological samples with excellent reproducibility.