Sphingomyelin and Related Sphingophospholipids

Sphingomyelin or ceramide 1-phosphocholine is a sphingophospholipid that consists of a ceramide unit with a phosphorylcholine moiety attached to position 1 of the sphingoid base component. It is thus the sphingolipid analogue of phosphatidylcholine, and like that lipid it is zwitterionic, although the similarities between the two do not extend much further in spite of a metabolic relationship. Sphingomyelin is primarily of animal origin and is a ubiquitous component of all animal cell membranes from mammals to nematodes (and a few protozoa), where it is by far the most abundant sphingolipid. As a representative molecular species, d18:1/16:0 is illustrated.

Structural formula of sphingomyelin

1. Occurrence and Composition of Sphingomyelin

Sphingomyelin 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, where it is a major constituent of myelin, and 70% of the phospholipids of the human lens. Like phosphatidylcholine, sphingomyelin tends to be in greatest concentration in the plasma membrane of cells (up to 20%), the endocytic recycling compartment and trans Golgi network. It is also abundant in the nucleus where it is the main phospholipid associated with chromatin, but there is very little in the endoplasmic reticulum (2 to 4%) and even less in mitochondria. In human erythrocyte membranes, all the sphingomyelin is in the outer leaflet, as is ~90% of that in the plasma membrane of nucleated cells. All lipoprotein fractions in plasma contain appreciable amounts of sphingomyelin with a higher proportion in the VLDL/LDL. Sphingomyelin is the single most abundant lipid in erythrocytes of most ruminant animals, where it replaces phosphatidylcholine entirely, but in this instance, there is known to be a phospholipase A that rapidly breaks down the glycerophospholipids, but not sphingomyelin.

Sphingomyelin is not synthesised in plants or fungi, which produce small amounts of the sphingophospholipid ceramide phosphoinositol and related lipids instead, or in bacteria, and its evolutionary significance is a matter for speculation. On the other hand, many bacteria and viruses utilize sphingomyelin or its metabolism in their animal hosts for growth and viability.

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 detected and then in small amounts, but they are found in sphingomyelin from testes, spermatozoa, kidney and skin. Approximately 100 molecular species of sphingomyelin have been identified in human plasma. 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.

	Fatty acids
Table 1. Fatty acid compositions of sphingomyelin (wt % of the total) in some animal tissues.
	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); DOI.

	Sphingoid base
Table 2. Long-chain base compositions of sphingomyelin (wt % of the total) in some animal tissues.
	d16:0*	d17:0	d17:1	d17:1-methyl	d18:0	d18:1	d19:0

Egg					7	93
Bovine brain					19	81
Cow's milk	9	15	8	11	10	44	3
From Ramstedt, B. et al. Eur. J. Biochem., 266, 997-1002 (1999); DOI. * 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 (discussed here..). In human brain, about 60% of the fatty acids of the sphingomyelin of the grey matter consists of stearic acid (18:0), while lignoceric (24:0) and nervonic (24:1) acids make up 60% of the corresponding lipid of white matter, although this is dependent on the stage of development. During the first two years of life, the 18:0 concentration in sphingomyelin of white matter decreases from 82% to 33%, while the proportions of 24:0 and 24:1 increase, but such a pronounced shift from long-chain to very-long-chain species is not observed in the cerebral cortex. Although polyunsaturated fatty acids such as arachidonic acid are rarely present, they have sometimes been mistakenly identified in publications. 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. The nematode Caenorhabditis elegans, widely studied as a primitive animal model, contains an isomethyl branched sphingoid base, esterified with saturated, long or very-long-chain fatty acids, often with methyl branches.

2. Biosynthesis, Metabolism and Function of Sphingomyelin

The biosynthesis of sphingomyelin differs from that of phosphatidylcholine and indeed depends upon it, as it involves transfer of phosphorylcholine from phosphatidylcholine to ceramide synthesised in the endoplasmic reticulum, with liberation of 1,2-diacyl-sn-glycerols. The reaction is catalysed by a ceramide choline-phosphotransferase (sphingomyelin synthase or SMS) and takes place primarily on the luminal side of the trans 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 the formation of raft domains (see below), while SMS2 is present in the membranes of nuclei from rat liver cells. It is noteworthy that in the absence of ceramide, both SMS1 and 2 have phospholipase C activity and so may regulate the steady-state levels of phosphatidylcholine and diacylglycerols as well as that of sphingomyelin.

Figure 1. Biosynthesis of sphingomyelin.

A ceramide transport molecule (CERT) is necessary for 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 then delivered to the apical plasma membrane by a vesicular transport mechanism. Sphingomyelin synthesis is regulated through CERT metabolism by interactions with other classes of lipids. Diacylglycerols formed in the reaction activate protein kinase D, which phosphorylates multiple sites on CERT to cause a conformational change that reduces ceramide flux to the Golgi, and together with further reaction with phosphoinositides and oxysterol-binding protein 1 (OSBP1), a lipid transfer protein for cholesterol, serve to repress sphingomyelin biosynthesis.

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, possibly as a protective mechanism for the cell. The location of the enzymes explains the enrichment of sphingomyelin in certain membranes and their 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. As the nature of the molecular species of sphingomyelins produced differs appreciably from that of the ceramide precursors, the sphingomyelin synthases must have considerable substrate specificity. The reaction is reversible and uses sphingomyelin to generate ceramide for signalling purposes.

Although the physiological significance has yet to be established, 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.

Membrane properties: Scottish thistle 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, where it may limit the ingress of oxygen and thence oxidation of adjacent unsaturated acyl chains. While this is certainly one purpose, 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. 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 appears to assist intermolecular interactions in membranes, features that may be of great significance during myelin formation. Indeed, the first five carbon atoms of the sphingoid base in sphingolipids constitute a distinctive feature that has been termed the ‘sphingoid motif’, which facilitates a relatively large number of molecular interactions with other membrane lipids, via hydrogen-bonding, charge-pairing, and hydrophobic and van der Waals forces. 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, so they pack in very different ways in membranes.

It is now recognized that sphingomyelin and other sphingolipids have a strong tendency to interact with proteins and cholesterol, often by strong van der Waals interactions and hydrogen bonding, to form transient nano-domains in membranes known as 'rafts' (discussed in greater detail in a separate web page) and on the surface of lipoprotein particles. Initially, there was a view that saturated sphingomyelin formed a liquid-ordered phase with cholesterol or a gel phase with saturated ceramides to lead to lateral segregation within the membrane, and that sphingomyelin and cholesterol metabolism were closely integrated, with the suggestion that the sphingomyelin concentration might control the distribution of cholesterol in cells. Conversely, it has been suggested that cholesterol may regulate the synthesis of sphingomyelin, especially those molecular species containing very-long-chain fatty acids. On the other hand, the understanding of the mechanism of raft formation in membranes has changed substantially in recent years, and while an interaction with cholesterol is certainly important, it may not be the major factor in vivo. Ceramide can displace cholesterol from its association with sphingomyelin, when formed in membranes by hydrolysis of the latter.

Other functions: Sphingomyelin is generally considered to be a relatively inert molecule, although modern molecular biology methods are uncovering potential regulatory interactions with particular proteins. For example, it has been shown to inhibit 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 operation of many 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 actinoporins, which are pore-forming toxins produced by sea anemones. It accumulates during differentiation of brown adipocytes and takes part in the regulation of UCP1-mediated nonshivering thermogenesis.

There is a characteristic binding site for sphingomyelin on the amyloid beta-peptide (Aβ) in brain, and there is evidence from studies in vitro that this may promote the aggregation of these proteins in Alzheimer's disease. In turn, this leads to depletion of brain sphingomyelin by stimulation of acid sphingomyelinase with disruption of many protein–lipid interactions and thence of down-stream signalling pathways. In contrast, the ganglioside GM1 may have a protective role towards peptide Aβ aggregation.

As well as its role in membranes, sphingomyelin serves as a precursor for ceramides, long-chain bases, sphingosine-1-phosphate and many other sphingolipids, as part of the 'sphingomyelin cycle' (sometimes termed the ‘sphingolipid’ or ‘ceramide’ cycles depending on the context). Some of these metabolites are 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 web pages on this site dealing with each lipid class.

Figure 2. The sphingomyelin cycle.

In particular, 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 as well as 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, and it may influence disease states such as cancer.

Sphingomyelin synthesis give rise to sn‑1,2-diacylglycerols as a by-product, and aside from feedback regulation of sphingomyelin synthesis, these are central to many metabolic and signalling pathways, not least as pro-survival mediators. As the latter have been shown to compete against ceramide-induced apoptosis, it has been suggested that sphingomyelin synthase is critical for cell growth and survival as it is a regulator of the balance between the pro-apoptotic and mitogenic mediators. This is a further link between glycerolipid and sphingolipid signalling pathways. Although the extent of diacylglycerol production relative to that from phosphatidylinositol is not known, it is possible that it is significant locally at the external leaflet of the plasma membrane.

2‑Hydroxyoleic acid suppresses the growth and induces autophagy in cancer cells by stimulating the synthesis of sphingomyelin and increasing the amount of this lipid in the plasma membrane. On the other hand, plasma sphingomyelin levels are believed to be an independent risk factor for atherosclerosis, possibly as a result of its ability to retain cholesterol in cells and the arterial wall with consequent diminished reverse cholesterol transfer via HDL.

Chlamydiae (widespread bacterial pathogens) acquire sphingomyelin from the Golgi apparatus and plasma membrane of their hosts, and this is necessary for the viability and growth of the organisms. Other pathogenic bacteria, notably Pseudomonas aeruginosa and Neisseria gonorrhoeae, can hijack sphingomyelin catabolic enzymes with deleterious effects upon the host. Likewise, human immunodeficiency virus (HIV) and the hepatitis C virus utilize host sphingomyelin for their own nefarious purposes.

Nutrition: Although there is no known nutritional requirement for sphingomyelin and other sphingolipids, they are a component of any diet containing egg, meat and dairy products. Thus, it has been estimated that per capita sphingolipid consumption in the United States is of the order of 0.3-0.4 g/d. As sphingolipids constitute an appreciable proportion of the polar lipid constituents of milk, including that of humans, they may be significant if minor nutrients for infants, and benefits towards myelin formation and brain development have been claimed. Sphingomyelin per se is poorly absorbed in the small intestine and is broken down by hydrolases and further catabolized (see next section). From animal experiments, there is nonetheless evidence that dietary sphingolipids can reduce the intestinal absorption of cholesterol and other lipids, leading to reductions in serum lipid concentrations. Feeding sphingolipids inhibits colon carcinogenesis and may alleviate some of the symptoms of inflammatory bowel disease.

3. Sphingomyelin Catabolism

Intestinal digestion: In contrast to the glycerolipids, dietary sphingolipids are not hydrolysed by pancreatic enzymes only. Rather, most of the sphingomyelin in the diet is hydrolysed in the brush border of the intestines by an alkaline sphingomyelinase (at a pH of 8.5–9 optimally) to ceramide and thence by a neutral ceramidase to free fatty acids and sphingosine. Some of this enzyme is present in liver from which it is secreted in bile into the intestinal lumen where it can hydrolyse sphingomyelin and other phospholipids with the aid of bile salts. The sphingosine released at the brush border is absorbed, some is re-N-acylated to form ceramides, and the remainder is converted via sphingosine-1-phosphate to palmitic acid, which is esterified into the triacylglycerol component of chylomicrons. In the process, some of these sphingolipid intermediates may act as signalling mediators and be anti-inflammatory in intestinal cells.

The alkaline sphingomyelinase is unusual in that is very different in its structure and actions from related intracellular enzymes; it is part of the (ecto)nucleotidepyrophosphatase-phosphodiesterase protein family (NPP) that includes autotaxin, and it is classed as anectoenzyme because it can cross the plasma membrane through either the N- or C-terminal transmembrane domain, with much of the enzyme located outside the cells. The enzyme is believed to have a role in the production of sphingolipid metabolites within the intestines and colon, which may influence several disease states. For example, it appears to in inhibit colon cancer by generating ceramides. 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. In addition, alkaline sphingomyelinase acts as a phospholipase C towards the pro-inflammatory metabolite platelet-activating factor and towards lysophosphatidylcholine with further beneficial effects.

Catabolism of sphingomyelin - sphingomyelinases

Figure 3. Catabolism/hydrolysis of sphingomyelin by sphingomyelinases.

Catabolism in other tissues: The main enzymes for the degradation of sphingomyelin to ceramides in most tissues are also sphingomyelinases (phosphodiesterases), which can be compared to phospholipase C and generate ceramides with their innumerable signalling properties as the main product (further catabolism is via sphingosine-1-phosphate ). There are many such enzymes with different pH optima and metal ion requirements that operate in different regions of the cell with potentially distinct biochemical roles. Thus, there is an acid sphingomyelinase 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 (pH optimum ca. 5) is expressed ubiquitously and has a house-keeping role in maintaining normal membrane turnover and remodelling of the sphingolipid constituents, in particular those of lipoproteins. The enzyme contains several Zn²⁺-binding motifs and zinc is an essential cofactor. While other lysosomal sphingolipid hydrolases require a saposin protein for maximum efficiency, the acid sphingomyelinase incorporates a built-in N‑terminal saposin domain so does not require an external activator. 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 ceramides for signalling purposes by hydrolysis of sphingomyelin and initiates the train of events that leads to apoptosis and other metabolic events. There are reports that acid sphingomyelinase, by acting at the plasma membrane to produce ceramides, regulates the localization and trafficking of palmitoylated proteins from the Golgi, and it may facilitate bacteria-host interactions. Experiments in vitro have demonstrated that the enzyme can be considered as a phospholipase C that operates against a wide range of phospholipids, including ceramide-1-phosphate and glycerophospholipids, including the unique lysosomal phospholipid bis(monoacylglycero)phosphate.

There is a related secreted acid sphingomyelinase (Zn²⁺-dependent), which can be transported to the outer membrane of the cell including endothelial cells of the human coronary artery. Although this enzyme is produced by the same gene, it has a different glycosylation pattern but also requires Zn²⁺ ions for activation. It can operate at neutral pH and takes part in many aspects of cellular signalling as well as in membrane sphingomyelin turnover.

Neutral sphingomyelinases (pH optima 7.4), of which four are known, are located in membranes of the endoplasmic reticulum, Golgi and plasma membrane with one in mitochondria (MA-NSM), where they act as signalling mediators by generating ceramides and thence other biologically active sphingolipids. Human NSM-1 has 423 amino acid residues and a molecular weight of 47.6 kDa; it has two putative transmembrane domains in the C‑terminus and resides mainly in the nucleus and endoplasmic reticulum. It has a broad specificity for choline phospholipids, but it prefers sphingomyelin and may not have a significant role in cellular signalling. Scottish thistle In contrast, NSM‑2 which is located in the Golgi apparatus and plasma membrane is activated by phosphatidylserine and is relevant to ceramide signalling, especially in brain and nervous tissue, where it is required for the secretion of hypothalamic releasing hormones, although it is necessary for many physiological processes in most other tissues. Dysregulation of NMS-2 is reported to be a factor in many inflammation-related pathologies. Neutral sphingomyelinases-3 is found mainly in the plasma membrane of bone and cartilage, where it is vital for the process of mineralization, and it is present in striated and cardiac muscle, but little seems to be known of why the mitochondrial enzyme is necessary. While losses, mutation and poor expression of the gene encoding neutral sphingomyelinase have been observed in several cancers, exposure to ionizing irradiation was found to lead to rapid hydrolysis of sphingomyelin to ceramide by this enzyme and thence to cancer cell death.

A diverse range of mediators induce the enzymes, including chemotherapeutic agents, tumour necrosis factor-alpha, 1,25‑dihydroxy-vitamin D₃, endotoxin, gamma-interferon, interleukins, nerve growth factor and most conditions known to induce cellular stress in relation to inflammation. As they utilize by far the most abundant sphingolipid in animal tissues to generate ceramides and other sphingolipid metabolites that are vital for signalling, sphingomyelinases are believed to serve 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.

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 and smaller amounts of other sphingolipids, including gangliosides, in cells and tissues and in the monocyte/macrophage system to form the so-called "foam cells” that characterize the disease. Increasing sphingomyelin levels in turn result in elevated cholesterol concentrations. A consequent lack of ceramide production may influence the pathology of the disease, and as membranes containing ceramides have a much lower binding capacity for cholesterol, sphingomyelin degradation may play a part in cholesterol homeostasis. Type C Niemann-Pick disease differs from the A and B forms and is caused primarily by defects in two cholesterol-binding proteins (NPC1 and NPC2), although variants of the sphingomyelin phosphodiesterase 1 (SMPD1) have been implicated also.

Bacterial sphingomyelinases are known that lyse red blood cells, although intriguingly, there is a sphingomyelinase in the bacterium Pseudomonas aeruginosa that can act as a sphingomyelin synthase in vitro at least.

4. Sphingosine phosphocholine

Formula - sphingosine phosphocholine Sphingosine phosphocholine or 'sphingosine phosphorylcholine' or 'lyso-sphingomyelin', i.e., lacking a fatty acid component, is found at trace levels only in tissues but has distinctive biological properties. It is released when platelets are activated and is present at concentrations of about 50 to 130 nM in plasma in association with the high-density lipoproteins mainly, from which it is believed to exert an influence upon the cardiovascular system. In skin, sphingosine phosphocholine is formed by the action of a sphingomyelin deacylase, where in excess it may have a role in atopic dermatitis, and it is probably produced by an analogous route in some other tissues including heart, blood vessels, brain and the immune system. There is evidence that it is metabolized very rapidly in tissues, possibly by a choline-specific glycerophosphodiester phosphodiesterase.

Sphingosine phosphorylcholine is a multi-functional lipid produced under physiological and pathological conditions that triggers various signalling cascades affecting many cellular processes. It may act as an extracellular agent through G protein-coupled receptors (sphingosine-1-phosphate receptors S1P_1-5, GPR12 - but at low affinity) or as a second messenger to mediate intracellular Ca²⁺ release in various human tissues. By its effects upon cellular proliferation and differentiation, it is believed to stimulate the progression of many types of cancer, and it promotes the invasion of breast cancer cells. In contrast, it is a potent anti-inflammatory agent and reduces the level of organ dysfunction caused by bacterial lipopolysaccharide toxins in rats in vivo, although some pro-inflammatory actions have been described. While sphingosine phosphocholine has been reported to act in much the same way as sphingosine 1‑phosphate, the parts played by 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, which is responsible for the biosynthesis of lysophosphatidic acid.

The levels of sphingosine phosphocholine are elevated in Niemann-Pick disease type C for which it is a biomarker, and they increase also in patients with the metabolic syndrome. In the former, it occurs together with an unusual lipid with some superficial structural and presumably functional similarity, now known to be N‑palmitoyl-O-phosphocholineserine, which was originally termed "lysosphingomyelin 509” in publications, although it is not in fact a sphingolipid and might be better classified as a lipoamino acid.

Formula - N-palmitoyl-O-phosphocholineserine

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 human infants (but not in that of adults) in which position 3 of the sphingosine residue is linked to an additional fatty acid (C₁₆ or C₁₈) via an ester bond (alkali-labile), although this was no longer observed after 4 weeks of age. The cyanobacterium Scytonema julianum contains sphingomyelin with an acetyl group esterified to an ω-1 hydroxyl of a long-chain fatty acid.

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 rather than directly to ceramide of mono- and digalactosylceramides (see our web page on monoglycosylceramides).

6. Ceramide Phosphoethanolamine and Other Sphingophospholipids

Formula of ceramide phosphoethanolamine Ceramide phosphoethanolamine, the sphingolipid analogue of phosphatidylethanolamine, is a component of the lipids of insects, some fresh water invertebrates and a few species of bacteria of the genus Bacteroides of the human gut biome (where it is often accompanied by ceramide phosphoglycerol and/or ceramide phosphoinositol), but it is present at trace levels only in mammalian cells (300 to 1,500-fold below those of sphingomyelin). In Drosophila melanogaster, it is one of the main sphingolipids, where d14:1 and d16:1 are the main long-chain bases, and it has an important role in the functional equivalent of myelin to ensheath axons where it replaces galactosylceramide, a major component of myelin in mammals. It is apparently able to do this because the two lipids have comparable physical properties. There are obvious differences in the charged state, but both lipids have a high phase transition temperature and tight packing, and neither is miscible with cholesterol, so they do not form raft domains.

In insects, CDP-ethanolamine is the donor of the head group for ceramide phosphoethanolamine synthesis in a mechanism like that for phospholipid biosynthesis by the Kennedy pathway and quite different from that for sphingomyelin. It serves to modulate circadian rhythm via neural–glial coupling in Drosophila, while a deficiency leads to arrhythmic locomotor behaviour and a shortened lifespan. In mammalian cells, ceramide phosphoethanolamine is produced at very low levels in the endoplasmic reticulum mainly by all members of the sphingomyelin synthase family, which are bifunctional, and it never accumulates in membranes. A further synthase has been termed 'SMS related protein' because of its structural similarity to the sphingomyelin synthase, and is most active in brain, but mainly it acts as a phosphatidylethanolamine phospholipase C.

In Sphingobacterium spiritivorum, the type species of the bacterial 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 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 some organisms such as marine invertebrates. As well as ceramide phosphoethanolamine, the protozoan parasite Trypanosoma brucei contains sphingomyelin and ceramide phosphoinositol. Ceramide-1-phosphate and ceramide phosphoinositol are sufficiently distinctive sphingophospholipids to have their own web pages here.

Ceramide phosphoglycerol has long been known as a constituent of the membranes of anaerobic bacteria of the genus Bacteroides. Unusual forms of it, including dihydroceramide phosphoglycerol and a form with two fatty acid components in an estolide linkage (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. Among many other unusual lipids, this organism contains the cardiolipin analogue bis(phosphodihydroceramide) glycerol. Bacteroides ovatus found human gut microbiota contains related lipids in a highly complex lipidome, which includes ceramide phosphate linked to lipoamino acids (glycerolipid analogues are known), while unique inositol-bis-(phosphoryldihydroceramide (or bis-dihydroceramide phosphoryl-inositol (DHC-PIP-DHC)) was characterized from B. vulgatus. Ceramide phosphoglycerate and diphosphoglycerate have been identified in genetically modified, Gram-negative bacteria, Caulobacter crescentus, deficient in lipid A.

Ceramide phosphorylmannose has been identified and characterized in the lipids of the bacterium Sphingobacterium spiritivorum, the type species of genus. Like the other sphingolipids in this species, the ceramide unit contains 15‑methylhexadecasphinganine and 13‑methyltetradecanoic acid primarily for which there is an absolute requirement when these lipids stimulate murine macrophages via a Toll-like receptor to effect bacterial clearance.

Structural formula of ceramide phosphorylmannose

In addition to acetylated sphingomyelin, the cyanobacterium Scytonema julianum contains an acylacetylglycerol phosphoacetylated glycolipid that exhibits a biological activity like that of platelet-activating factor. A second type of glycosphingophospholipid is known in which glycosphingolipids are further phosphorylated, i.e., where the ceramide is linked directly to carbohydrate moieties not via phosphate, are known from earth worms and the model nematode C. elegans, and these are discussed in relation to their parent glycosylceramides.

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 occasional 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 with electrospray ionization are being used increasingly for the analysis of sphingomyelin and other sphingolipids.

	© Author: William W. Christie
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