The Lipid Library

SPHINGOMYELIN AND RELATED LIPIDS

STRUCTURE, OCCURRENCE, BIOSYNTHESIS and ANALYSIS

1.  Structure and Occurrence of Sphingomyelin

Sphingomyelin (or ceramide phosphorylcholine) consists of a ceramide unit with a phosphorylcholine moiety attached to position 1. It is thus the sphingolipid analogue of phosphatidylcholine. The d18:1/16:0 molecular species is illustrated as an example.

It is a ubiquitous component of animal cell membranes, where it is by far the most abundant sphingolipid. Indeed, it can comprise as much as 50% of the lipids in certain tissues, though it is usually lower in concentration than phosphatidylcholine. For example, it makes up about 10% of the lipids of brain. It is the single most abundant lipid in erythrocytes of most ruminant animals, where it replaces phosphatidylcholine entirely. In this instance, there is known to be a highly active phospholipase A that breaks down the glycerophospholipids, but not sphingomyelin. Like phosphatidylcholine, sphingomyelin tends to be in greatest concentration in the plasma membrane, and especially in the outer leaflet, of cells.

Sphingomyelin does not appear to occur in plants or microorganisms, and its evolutionary significance is a matter for speculation.

Sphingosine is usually the most abundant long-chain base constituent, together with sphinganine and C₂₀ homologues, although other bases can be present, especially in ruminant animals. Sphinganine is the major sphingoid base in sphingomyelin human lens membranes, where the relevant molecular species can comprise more than half of the total phospholipids. Typically, the fatty acids are very-long-chain saturated and monounsaturated, including odd-numbered components. In contrast to glycosphingolipids, 2-hydroxy acids are only rarely present, but they are found in kidney and skin sphingomyelin, for example. The absolute proportions of each fatty acid and sphingoid base can vary markedly between tissues and species, and some of the variability in compositions can be seen from the data in Tables 1 and 2.

This only hints at the potential complexity, as there can be variability within tissues. For example, about 60% of the fatty acids of the sphingomyelin of the grey matter of human brain consist of stearic acid (18:0), while lignoceric (24:0) and nervonic (24:1) acids make up 60% of the corresponding lipid of white matter. Polyunsaturated fatty acids such as arachidonic acid are rarely present, although they have sometimes been mistakenly identified in the literature. Exceptions are the sphingomyelins of testes and spermatozoa, which contain very-long-chain polyunsaturated fatty acids (up to 34 carbon atoms), the major components being 28:4(n-6) and 30:5(n-6), a proportion of which have hydroxyl groups in position 2.

2.  Biosynthesis, Metabolism and Function of Sphingomyelin

The biosynthesis of sphingomyelin is distinct from that of phosphatidylcholine. Indeed, it involves transfer of phosphorylcholine from phosphatidylcholine to ceramide, liberating diacylglycerols, and catalysed by a ceramide choline-phosphotransferase (sphingomyelin synthase). The reaction takes place in the plasma membrane and Golgi, with distinct integral enzymes in each organelle. A specific ceramide transport molecule (CERT) is important to the reaction (see our web page on ceramides) in that it transfers ceramide from the endoplasmic reticulum to the Golgi in a non-vesicular manner.

The nature of the molecular species of sphingomyelins produced differs appreciably from that of the ceramide precursors, suggesting considerable substrate specificity for the sphingomyelin synthases. The reaction can be reversible, using sphingomyelin to generating ceramide for specific signalling functions. It is evident that it forms a link between the sphingolipid signalling pathway (pro-apoptotic - see below) and that of glycerolipids via the mitogenic diacylglycerols.

An alternative pathway of sphingomyelin synthesis has been demonstrated in which ceramide is first converted to ethanolamine phosphorylceramide via transfer of the head group from phosphatidylethanolamine, followed by stepwise methylation of the ethanolamine moiety. However, the physiological significance of this pathway has yet to be established.

It was long thought that the only function of sphingomyelin was to serve as a substitute for phosphatidylcholine as a building block of membranes, i.e. by forming a stable and chemically resistant outer leaflet of the plasma membrane lipid bilayer. This is one of its functions certainly. The apparent similarity between phosphatidylcholine and sphingomyelin is superficial, and there are great differences in the hydrogen bonding capacities and physical properties of the two lipids. For example, sphingomyelin has an amide bond at position 2 and a hydroxyl on position 3 of the sphingoid base that can both participate in hydrogen bonding, while the trans double bond also appears to assist intermolecular interactions in membranes. With phosphatidylcholine, in contrast, the two ester carbonyl groups can only act as hydrogen acceptors. The degree of unsaturation of the alkyl moieties in each lipid is very different, and this gives them very different packing properties in membranes.

It is now recognized that sphingomyelin and cholesterol have a high affinity for each other and are usually located together in specific sub-domains or 'rafts' of membranes and on the surface of lipoprotein particles. Indeed, evidence has accumulated to suggest that sphingomyelin and cholesterol metabolism are closely integrated, and in particular that sphingomyelin may control the distribution of cholesterol in cells. They are most abundant in the same membranes, i.e. plasma membrane and Golgi as opposed to intracellular organelle membranes such as mitochondria (cancer cells may be an exception), although sphingomyelin can be transported through the cytosol. Other sphingolipids, such as the neutral glycosphingolipids, also promote raft formation but do not co-localize with cholesterol.

In addition to its function in membranes, sphingomyelin serves as a precursor for ceramides, long-chain bases and sphingosine-1-phosphate, together with many other biologically important sphingolipids, as part of the 'sphingomyelin cycle'. Some of these have functions as intracellular messengers, and others are essential membrane constituents. These molecular relationships are illustrated only briefly below, as most are discussed in detail here on other pages on this site dealing with the relevant metabolites.

In particular in animals, sphingomyelin is the primary source of the ceramide (and its subsequent metabolites) that is required to trigger apoptosis and other metabolic changes. It performs this function in most cellular organelles, including the nucleus and even mitochondria, where it is a rather minor component.

3.  Sphingomyelin Catabolism

The key enzymes for the degradation of sphingomyelin to ceramides in most tissues are sphingomyelinases, which are similar in function to phospholipase C. There are a number of such enzymes with differing pH optima that appear to operate in different regions of the cell with potentially distinct biochemical roles. For example, there is an acid sphingomyelinase in the outer leaflet of the plasma membrane and a neutral sphingomyelinase on the inner leaflet, but the acid sphingomyelinase in lysosomes has received most study. A diverse range of factors activate the enzymes, including chemotherapeutic agents, tumor necrosis factor-alpha, 1,25-dihydroxy-vitamin D₃, endotoxin, gamma-interferon, interleukins, nerve growth factor, and most conditions known to induce cellular stress. In that they generate ceramides and other sphingolipid metabolites that have important signalling functions, sphingomyelinases are believed to function as regulators of signalling mechanisms, especially in the nucleus of the cell. The lysosomal acid sphingomyelinase may be involved in recycling of sphingolipid constituents.

The type A and B forms of Niemann-Pick disease are lysosomal lipid storage disorders that are a consequence of a deficiency of acid sphingomyelinase, which causes an accumulation of sphingomyelin in cells and tissues. The resulting lack of ceramide production may be involved in the pathology of the disease. It is noteworthy that membranes containing ceramides have a much lower binding capacity for cholesterol, so sphingomyelin degradation may play a part in cholesterol homeostasis. Conversely, increasing sphingomyelin levels, as in Niemann-Pick disease, result in elevated cholesterol concentrations.

Intriguingly, there is a sphingomyelinase in the bacterium Pseudomonas aeruginosa that can also act as a sphingomyelin synthase in vitro.

Although there is no known nutritional requirement for sphingolipids such as sphingomyelin, they are a component of any diet containing egg, meat or dairy products. Thus, it has been estimated that per capita sphingolipid consumption in the United States, for example, is of the order of 0.3-0.4 g/d. As sphingolipids are the main polar lipid constituents of milk, they may be especially important as minor but significant nutrients for infants. From animal experiments, there is evidence that feeding sphingolipids inhibits colon carcinogenesis, reduces serum LDL cholesterol but elevates HDL concentrations, and may alleviate some of the symptoms of inflammatory bowel disease.

In contrast to the glycerolipids, sphingolipids are not hydrolysed by pancreatic enzymes. Rather, sphingomyelin in the diet is hydrolysed by an alkaline sphingomyelinase to ceramide and thence by a neutral ceramidase to free fatty acids and sphingosine in the brush border of the intestines. The sphingosine is absorbed, some is re-esterified and the remainder is converted to palmitic acid and acylated into the triacylglycerol component of chylomicrons. In the process, some of the hydrolysis intermediates may have signalling functions in the intestines. The alkaline sphingomyelinase in particular is unusual in that is very different in its structure and other properties from intracellular enzymes with a related function. It is believed to have a role in the production of sphingolipid metabolites within the intestines and colon especially, which may influence a number of disease states. In addition, it has phospholipase C activity towards platelet-activating factor and lysophosphatidylcholine. By reducing the level of endogenous sphingomyelin in the membranes of intestinal cells, it is believed to reduce the uptake of dietary cholesterol.

4.  Sphingolipids Closely Related to Sphingomyelin

An unusual sphingolipid, 3-O-acyl-D-erythro-sphingomyelin, has been found in plasma of the newborn pig and infant (but not in that of adults). In this instance, position 3 of the sphingosine residue is linked to an additional fatty acid (C₁₆ or C₁₈) via an ester bond (alkali-labile).

Sphingosine phosphorylcholine or lyso-sphingomyelin is found at trace levels only in tissues. For example, in plasma it is present at concentrations of about 50 nM, and there is evidence that it is metabolized very rapidly. It is formed by the action of a sphingomyelin deacylase in skin, and probably by a similar route in some other tissues, including heart, blood vessels, skin, brain and the immune system.

Sphingosine phosphorylcholine is believed to be involved in many cellular processes, including the promotion of proliferation and differentiation, although it can also inhibit the growth of cancer cells. It is produced under physiological and pathological conditions and activates various signalling cascades. It has been reported to have many similar functions to sphingosine 1-phosphate. Indeed, the activities of the two lipids may not be easily distinguished as sphingosine phosphorylcholine can be converted to sphingosine 1-phosphate by the action of the plasma enzyme autotaxin. No specific cellular receptors for sphingosine phosphorylcholine have been positively identified.

Sphingolipids have been found in a species of earthworm with phosphorylcholine linked to the carbohydrate moiety of mono- and digalactosylceramides.

5.  Ceramide Phosphorylethanolamine and Other Ceramide-Containing Phospholipids

Ceramide phosphorylethanolamine, the sphingolipid analogue of phosphatidylethanolamine, is a component of the lipids of insects, some fresh water invertebrates and many species of bacteria (where it is often accompanied by ceramide phosphorylglycerol). For example, it is one of the main sphingolipid in Drosophila. Recently, it has been characterized comprehensively in three species of plant fungal pathogens (Oomycetes); the fatty acid and long-chain bases components vary with species, and for example one contains phytosphingosine and another an unusual branched-trienoic base.

Ceramide phosphorylethanolamine is synthesised by a similar mechanism to sphingomyelin, i.e. by transfer of phosphorylethanolamine from phosphatidylethanolamine to ceramide. A phosphonolipid analogue is found in certain organisms.

Ceramide phosphorylglycerol has long been known as a constituent of the membranes of anaerobic bacteria of the genus Bacteriodes. More recently, an unusual form of it was identified is the most abundant lipid in the oral pathogen Porphyromonas gingivalis (illustrated).

In this instance, it has spinganine or an iso-methyl-branched sphinganine as the long-chain base with an amide linkage to 3-hydroxy-iso-methylhexadecanoic acid, the hydroxyl group of which is esterified to iso-methyltetradecanoic acid. It is believed to make a significant contribution to the virulence of the organism in dental decay.

Further novel sphingolipids isolated from a cyanobacterium, Scytonema julianum, are ceramide phosphoglycolipids with an additional fatty acid with an ester link to position 3 of the sphingoid base. In addition, some species contain fatty acids in an estolide linkage, i.e. with an acetyl group esterified to an ω-1 hydroxyl of a long-chain fatty acid.

Ceramide-1-phosphate and ceramide phosphorylinositol are particularly important sphingo-phospholipids and as such have their own web pages here.

6.  Analysis

Sphingomyelin is readily isolated from animal tissues by adsorption chromatography (TLC and HPLC), although peaks or bands can split into two or three poorly resolved fractions. This is due in part to the changes in hydrophobicity resulting from the wide range of chain lengths in the fatty acid constituents, and in part to the presence of 2-hydroxy acids. Molecular species of the intact lipid can be resolved by reversed-phase HPLC, but a more usual approach is to hydrolyse to the less polar ceramides with the enzyme phospholipase C. Then, the ceramides can be analysed either by reversed-phase HPLC or by high-temperature GC. Nowadays, direct-inlet mass spectrometric methods (electrospray, fast-atom bombardment, etc) are being used increasingly for the analysis of sphingomyelin and other sphingo-phospholipids. The amide bond is resistant to mild alkaline hydrolysis, so special methods are required for analysis of the fatty acid and sphingoid base components.

Suggested Reading

• Christie, W.W. Lipid Analysis (3rd edition). (Oily Press, Bridgwater) (2003).
• Duan, R.-D. Alkaline sphingomyelinase: an old enzyme with novel implications. Biochim. Biophys. Acta, 1761, 281-291 (2006).
• Harder, T. Formation of functional cell membrane domains: the interplay of lipid- and protein-mediated interactions. Phil. Trans. Roy. Soc. Lond. B, 358, 863-868 (2003).
• Merrill, A.H. and Sandhoff, K. Sphingolipids: metabolism and cell signalling. In: Biochemistry of Lipids, Lipoproteins and Membranes. pp. 373-407 (Vance, D.E. and Vance, J. (editors), Elsevier, Amsterdam) (2002).
• Nilsson, A. and Duan, R.-D. Absorption and lipoprotein transport of sphingomyelin. J. Lipid Res., 47, 154-171 (2006).
• Nixon, G.F., Mathieson, F.A. and Hunter, I. The multi-functional role of sphingosylphosphorylcholine. Prog. Lipid Res., 47, 62-75 (2008).
• Ramstedt, B. and Slotte, J.P. Membrane properties of sphingomyelins. FEBS Letts, 531, 33-37 (2002).
• Ridgway, N.D., Byers, D.M., Cook, H.W. and Storey, M.K. Integration of phospholipid and sterol metabolism in mammalian cells. Prog. Lipid Res., 38, 337-360 (1999).
• Vesper, H., Schmelz, E.M., Nikolova-Karakashian, M.N., Dillehay, D.L., Lynch, D.V. and Merrill, A.H. Sphingolipids in food and the emerging importance of sphingolipids to nutrition. J. Nutr., 129, 1239-1250 (1999).

Updated: 5/3/2008 Credits/disclaimer	Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland