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Sphingomyelin and Related Lipids



1.  Structure and Occurrence of Sphingomyelin

Sphingomyelin (or ceramide phosphocholine) 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.

Structural formula of sphingomyelin

It is a ubiquitous component of animal cell membranes, from mammals to nematodes to protozoa, where it is by far the most abundant sphingolipid. Indeed, it can comprise as much as 50% or more 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 and 70% of the phospholipids of the human lens. 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, but it is also abundant in the nucleus where it is the main phospholipid associated with chromatin.

Sphingomyelin does not appear to occur in plants or fungi, which produce ceramide phosphoinositol instead, or in bacteria with rare exceptions, 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. In contrast, sphinganine is the major sphingoid base in the sphingomyelin of human lens membranes, linked mainly to 16:0. Typically, the fatty acids are very-long-chain saturated and monounsaturated, including odd-numbered components. In comparison to the glycosphingolipids, 2-hydroxy acids are only rarely present, but they are found in testes, spermatozoa, 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.

Table 1. Fatty acid compositions of sphingomyelin (wt % of the total) in some animal tissues.
Fatty acids
16:0 18:0 18:1 20:0 22:0 22:1 23:0 23:1 24:0 24:1
 
Egg 66 10 1 4 6 1 2 - 5 3
Bovine brain 3 42 - 6 7 3 3 3 6 27
Cow's milk 14 3 1 1 22 - 32 - 19 5
Adapted from Ramstedt, B. et al. Analysis of natural and synthetic sphingomyelins using high-performance thin-layer chromatography. Eur. J. Biochem., 266, 997-1002 (1999).

Table 2. Long-chain base compositions of sphingomyelin (wt % of the total) in some animal tissues.
Sphingoid base
d16:0*d17:0d17:1d17:1-methyld18:0d18:1d19:0
 
Egg 7 93
Bovine brain 19 81
Cow's milk 9 15 8 11 10 44 3
Also from Ramstedt, B. et al. Eur. J. Biochem., 266, 997-1002 (1999).
* d = dihydroxy base

Palmitic acid (16:0) is the most common fatty acid component of sphingomyelin in peripheral cells of mammals, while stearic acid (18:0) is more abundant in that of neural tissue, but 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. Approximately 100 molecular species of sphingomyelin have been detected in human plasma. Although polyunsaturated fatty acids such as arachidonic acid are rarely present, 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) with a proportion having 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, synthesised in the endoplasmic reticulum, with liberation of diacylglycerols. The reaction is catalysed by a ceramide choline-phosphotransferase (sphingomyelin synthase or SMS) and takes place primarily in the Golgi but also in the plasma membrane, with two related enzymes each with six transmembrane domains and their N- and C-termini facing the cytosol, i.e. SMS1 and SMS2. Both enzymes are present in the Golgi, but only SMS2 is in the plasma membrane (facing the extra-cellular space in this instance) and may be necessary for raft formation (see below). SMS2 is also present in the membranes of nuclei from rat liver cells.

Biosynthesis of sphingomyelin

A specific ceramide transport molecule (CERT) is important to the reaction with SMS1 (see our web page on ceramides) in that it transfers ceramide from the cytosolic surface of the endoplasmic reticulum to the trans-Golgi in an ATP-dependent and non-vesicular manner. Much of the sphingomyelin produced in the Golgi is delivered to the apical plasma membrane by vesicular transport.

SMS2 in the plasma membrane is not dependent on CERT-mediated ceramide delivery, but is believed to convert ceramide produced locally by a sphingomyelinase back to sphingomyelin; this may be an important protective mechanism for the cell. The location of the enzymes explains the enrichment of sphingomyelin in specific membranes and the sidedness, i.e. the luminal trans-Golgi and the outer leaflet of the plasma membrane, while ceramide reaching the cis-Golgi is utilized for synthesis of glucosylceramide.

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 sphingomyelin biosynthesis forms a link between the sphingolipid signalling pathway (pro-apoptotic - see below) and that of glycerolipids via the mitogenic diacylglycerol by-products, although the importance of this production relative to that via phosphatidylinositol is not known.

An alternative pathway of sphingomyelin synthesis has been demonstrated in the endoplasmic reticulum in which ceramide is first converted to ceramide phosphoethanolamine (see below) 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.

Scottish thistleIt 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. While this is certainly one of its functions, 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, both of which can 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 act only as hydrogen acceptors. The degree of unsaturation of the alkyl moieties in each lipid is very different, and this gives them dissimilar packing properties in membranes.

It is now recognized that sphingomyelin and cholesterol have a high affinity for each other via strong van der Waals interactions, and they are usually located together in specific sub-domains or 'rafts' of membranes and on the surface of lipoprotein particles. Thus, saturated sphingomyelin forms a liquid-ordered phase with cholesterol or a gel phase with saturated ceramides. Indeed, evidence has accumulated to suggest that sphingomyelin and cholesterol metabolism are closely integrated, and in particular that the sphingomyelin concentration 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.

Sphingomyelin per se is generally considered to be a relatively inert molecule, although modern molecular biology methods are uncovering potential regulatory functions via interactions with specific proteins. For example, it has been shown to inhibit the activity of phospholipase A2α, a key enzyme in eicosanoid production. Sphingomyelin in the plasma membrane may be essential for the internalization of transferrin and thence of iron into cells, and it appears to be required for the activity of a number of membrane-bound proteins, including those of certain ion channels and receptors. As the most abundant sphingolipid in the nucleus, it is intimately involved in chromatin assembly and dynamics as well as being an integral component of the nuclear matrix. A single molecular species of sphingomyelin with a C18 acyl chain binds specifically to a coat protein designated 'p24' to enable it to form membrane vesicles. In addition, sphingomyelin is selectively recognized and acts as a receptor for the pore-forming toxins actinoporins, which are produced by sea anemones.

As well as its role in membranes, it 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' (also termed the ‘sphingolipid’ or ‘ceramide’ cycles depending on the context). Some of these metabolites have functions as intra- and inter-cellular messengers, and others are essential membrane constituents. The sphingomyelin cycle extends to other sphingolipids via the action of sphingomyelinases and enzymes such as glycosylhydrolases and glycosyltransferases in cells to produce innumerable new oligoglycosylceramides. These molecular relationships are illustrated only briefly below, as most are discussed in detail on other pages on this site dealing with each lipid class.

The sphingomyelin cycle

In particular in animals, sphingomyelin is a major source of ceramides in most cellular organelles, including the nucleus and even mitochondria, via the action of sphingomyelinases (see next section), and in addition to being a source of other sphingolipids these are required to trigger apoptosis and other metabolic changes. As ceramides do not mix well with glycerophospholipids and cholesterol, this conversion results in the formation of new membrane domains enriched in ceramide that exclude cholesterol and so differ in composition from other sphingolipid rafts. This has profound effects on membrane function, especially of the plasma membrane, in that different proteins may be recruited or excluded depending on their relative affinities for cholesterol and ceramides.

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 and may alleviate some of the symptoms of inflammatory bowel disease. On the other hand, plasma sphingomyelin levels are considered to be an independent risk factor for atherosclerosis.


3.  Sphingomyelin Catabolism

Catabolism of sphingomyelin - sphingomyelinasesThe key enzymes for the degradation of sphingomyelin to ceramides in most tissues are sphingomyelinases (phosphodiesterases),which are similar in function to phospholipase C and generate ceramide with its multiple signalling properties as the main product. There are at least five such enzymes with different pH optima and metal ion requirements that operate in different regions of the cell with potentially distinct biochemical roles. For example, there is an acid sphingomyelinase (pH optimum ca. 5) in the endo-lysosomes, and different neutral sphingomyelinases in the plasma membrane, endoplasmic reticulum, Golgi and mitochondria. It should not be forgotten that the other product of the reaction is phosphocholine, which has importance as a nutrient.

The lysosomal acid sphingomyelinase is expressed ubiquitously and has a key house-keeping role in maintaining normal membrane turnover and remodelling of the sphingolipid constituents. Under resting conditions, acid sphingomyelinase is stored inside lysosomes, but upon stimulation it undergoes vesicular transport to the plasma membrane, where it docks with a specific protein and is exposed onto the outer leaflet. It then generates ceramide by hydrolysis of sphingomyelin and initiates the train of events that leads to apoptosis.

There is a related secreted acid sphingomyelinase (Zn++ dependent), which can be transported to the outer membrane of the cell and is especially important in endothelial cells of the human coronary artery. This enzyme is produced by the same gene but differs from the lysosomal enzyme as it requires Zn2+ ions for activation and has a different glycosylation pattern. It can also operate at neutral pH and has multiple functions in that it is involved in many aspects of cellular signalling as well as in membrane sphingomyelin turnover.

Neutral sphingomyelinases (pH optima 7.4), of which four quite distinct enzymes are known, are located in membranes such as the Golgi and plasma membranes with one in mitochondria, where they have signalling functions by generating ceramides and thence other biologically active sphingolipids. Neutral sphingomyelinases-2 and 4 are ubiquitously expressed in tissues, but the former appears to be especially important in brain and nervous tissue, where it is required for the secretion of hypothalamic releasing hormones, although it is relevant to many cellular functions and physiological processes in most other tissues. Neutral sphingomyelinases-3 is found mainly in the plasma membrane of bone and cartilage, where it is vital for the process of mineralization. It is also important in striated and cardiac muscle.

Scottish thistleA 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, especially in relation to inflammation. In that they generate ceramides and other sphingolipid metabolites that have important signalling functions from by far the most abundant sphingolipid in animal tissues, sphingomyelinases are believed to function as regulators of signalling mechanisms, especially in the nucleus of the cell. Thus, they have a much wider metabolic role than simply catabolism of sphingomyelin.

In contrast to the glycerolipids, sphingolipids are not hydrolysed by pancreatic enzymes. Rather, sphingomyelin in the diet is hydrolysed in the brush border of the intestines by an alkaline sphingomyelinase to ceramide and thence by a neutral ceramidase to free fatty acids and sphingosine. 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 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. For example, it appears to in inhibit colon cancer by generating ceramides. In addition, alkaline sphingomyelinase has phospholipase C activity towards the pro-inflammatory metabolite platelet-activating factor and towards lysophosphatidylcholine with potentially further beneficial effects. By reducing the level of endogenous sphingomyelin and increasing that of ceramides in the membranes of intestinal cells, it is believed to reduce the uptake of dietary cholesterol. Intriguingly, there is a sphingomyelinase in the bacterium Pseudomonas aeruginosa that can also act as a sphingomyelin synthase in vitro.

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 with a resulting accumulation of sphingomyelin in cells and tissues; a consequent lack of ceramide production may be involved in the pathology of the disease. Increasing sphingomyelin levels in turn result in elevated cholesterol concentrations. 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. Type C Niemann-Pick disease differs from the A and B forms and is caused by defects in two distinct cholesterol-binding proteins (NPC1 and NPC2).


4.  Sphingosine phosphocholine


Formula - sphingosine phosphocholineSphingosine phosphocholine or 'sphingosine phosphorylcholine' or 'lyso-sphingomyelin' is found at trace levels only in tissues but has important biological properties. For example, it is present at concentrations of about 50 to 130 nM in plasma in association with the high-density lipoproteins, although the levels are elevated in Niemann-Pick disease type C and in patients with the metabolic syndrome. It is formed by the action of a sphingomyelin deacylase in skin (where in excess it may have a role in atopic dermatitis), and probably by a similar route in some other tissues, including heart, blood vessels, brain and the immune system. There is evidence that it is metabolized very rapidly.

Sphingosine phosphorylcholine is a multi-functional lipid, produced under physiological and pathological conditions, that activates various signalling cascades affecting many cellular processes. By its effects upon cellular proliferation and differentiation, it is believed to stimulate the progression of many types of cancer. For example, it promotes the invasion of breast cancer cells. In addition, it has potent anti-inflammatory properties, and reduces the level of organ dysfunction caused by lipopolysaccharides in rats in vivo, for example. While sphingosine phosphcholine has been reported to have many similar functions to sphingosine 1-phosphate, the activities of the two lipids may not be easily distinguished as the former can be converted to sphingosine 1-phosphate by the action of the plasma enzyme autotaxin. Although it is believed to function as an intracellular second messenger or as an extracellular agent through G protein coupled receptors, none appears to have been identified unequivocally to date.


5.  Other 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).

Formula of 3-O-acyl-sphingomyelin

A phospholipase D in the venom of the brown spider converts sphingomyelin to a cyclic phosphate by an intramolecular transphosphatidylation reaction with the potential to disrupt membranes. Sphingolipids have been found in a species of earthworm with phosphorylcholine linked to the carbohydrate moiety of mono- and digalactosylceramides (see our web page on ceramide phosphoglycosides).


6.  Ceramide Phosphoethanolamine and Other Sphingophospholipids

Ceramide phosphoethanolamine, 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 phosphoglycerol), but it is present at trace levels only in mammalian cells (300 to 1,500-fold below those of sphingomyelin). As an example, it is one of the main sphingolipid in Drosophila melanogaster, where d14:1 and d16:1 are the main long-chain bases. The insect species, Manduca sexta, contains tetradecasphing-4,6-dienine as a major sphingoid base component, together with 18:0, 20:0, 22:0 and 24:0 fatty acids. In Sphingobacterium spiritivorum, the type species of genus Sphingobacterium, ceramide phosphoethanolamine is accompanied by ceramide phosphoinositol and ceramide phosphomannose, with branched-chain base (mainly iso-17:0) and fatty acid (mainly iso-15:0) components. Ceramide phosphoethanolamine has also been fully characterized 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. A phosphonolipid analogue is found in certain organisms. In addition to ceramide phosphoethanolamine, the protozoan parasite Trypanosoma brucei contains sphingomyelin and ceramide phosphoinositol.

Formula of ceramide phosphoethanolamine

In insects, CDP-ethanolamine is the donor of the head group for ceramide phosphoethanolamine synthesis (akin to phospholipid biosynthesis by the Kennedy pathway). However, in mammalian cells, it is produced at low levels in the endoplasmic reticulum mainly by all members of the sphingomyelin synthase family, which are bifunctional. However, there is also a distinctive synthase, termed 'SMS related protein' because of its structural similarity to the sphingomyelin synthase, which is a mono-functional enzyme and is active in brain especially. Although the lipid never accumulates in membranes, the process may assist in the removal of any excess of potentially toxic ceramides. No other function for the lipid in mammalian tissues is known.

Ceramide phosphoglycerol has long been known as a constituent of the membranes of anaerobic bacteria of the genus Bacteriodes. Unusual forms of it, including dihydroceramide phosphoglycerol and a form with two fatty acid components (illustrated), are the most abundant lipids in the oral Gram-negative pathogen Porphyromonas gingivalis.

Formula of ceramide phosphoglycerol

In this instance, it has sphinganine (dihydrosphingosine) 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 phosphoinositol are particularly important sphingo-phospholipids and as such have their own web pages here.


7.  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. As with other sphingolipids, the amide bond is resistant to mild alkaline hydrolysis, so special methods are required for analysis of the fatty acid and sphingoid base components. Molecular species of the intact lipid can be resolved by reversed-phase HPLC, but another useful approach is to hydrolyse to the less polar ceramides with the enzyme phospholipase C, after which the ceramides can be analysed either by reversed-phase HPLC or by high-temperature GC. Nowadays, mass spectrometric methods in conjunction with HPLC or direct-inlet (‘shotgun’) methods (electrospray ionization, especially) are being used increasingly for the analysis of sphingomyelin and other sphingolipids.


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Lipid listings Credits/disclaimer Updated: March 20th, 2017 Author: William W. Christie LipidWeb icon
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