Analysis of Molecular Species of Cellular Sphingomyelins and Ceramides

Abstract

Ceramides (CER) have been proposed to be the intracellular mediators of responses to such agents as interferon-γ (IFN-γ), dexamethasone, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β, and vitamin D₃. These agents induce hydrolysis of the plasma membrane sphingomyelin (SM) by a sphingomyelinase, followed by downstream activation of signaling kinases and nuclear translocation of NF-kB, with the final effect of induction of cell differentiation and/or apoptotic death (1). The growing interest in the SM-CER-dependent cell signaling makes an accurate and simultaneous determination of molecular species (MS) of both SM and CER desirable. Numerous MS are found in SM and CER, and these occur in characteristic proportions in different species, organs, subcellular organelles, and developmental stages. More than half of the fatty acids (FA) in CER contain a hydroxyl group at the 2 carbon position, the other half being non-hydroxy FA. In Chapter 22 the methods for extraction and separation of lipids from cellular samples, the separation of classes of phospholipids (PLs), and finally, the molecular species (MS) analysis are described. Separation of SM and ceramides CER follow very similar pathways (e.g., hydrolysis of SM to CER is similar to hydrolysis of PLs to diacylglycerol [DAG]). However, as seen in Fig. 1, SM consists of an N-acyl-fatty acid linked to a long-chain hydrocarbon and phosphorylcholine. The FA position of the molecule varies as in PLs, but with more major long-chain (C20–C24) saturated and monounsaturated FAs present than in PLs.