Long-Chain (Sphingoid) Bases

Long-chain/sphingoid bases are the characteristic and defining structural unit of the sphingolipids, which are important structural and signalling lipids of animals and plants and of a few bacterial species (see our Introduction to the topic). These are long-chain aliphatic amines, containing two or three hydroxyl groups, and often a distinctive trans-double bond in position 4. To be more precise, they are 2‑amino-1,3-dihydroxy-alkanes or alkenes with (2S,3R)‑erythro stereochemistry, often with various further structural modifications in the alkyl chain. They are important for the physical and biological properties of all complex sphingolipids, but free sphingoid bases are also bioactive and interact with specific receptors and target molecules. As discussed below, the mechanisms for biosynthesis of sphingoid bases and of the N-acylated form (ceramides) are intimately linked.

1.   Structures and Occurrence

In animal tissues, the most common or abundant of the sphingoid bases is sphingosine ((2S,3R,4E)-2-amino-4-octadecene-1,3-diol) or sphing-4E-enine, i.e., with a C18 aliphatic chain, hydroxyl groups in positions 1 and 3 and an amine group in position 2; the double bond in position 4 has the trans (or E) configuration. This was first characterized in 1947 by Professor Herbert Carter, who was also the first to propose the term “sphingolipides” for those lipids containing sphingosine. It is usually accompanied by the saturated analogue dihydrosphingosine (or sphinganine).

Structures of sphingoid bases

For shorthand purposes, a nomenclature like that for fatty acids can be used; the chain length and number of double bonds are denoted in the same manner with the prefix 'd' or 't' to designate di- and trihydroxy bases, respectively. Thus, sphingosine is denoted as d18:1 and phytosphingosine is t18:0. The position of the double bond may be indicated by a superscript, i.e., 4-sphingenine is d18:1Δ4t or 4E-d18:1. While alternative nomenclatures are occasionally seen in publications, they are not recommended.

Scottish thistleThe number of different long-chain bases that has been found in animals, plants and microorganisms now amounts to over one hundred, and many of these may occur in a single tissue or organism, but almost always as part of a complex lipid with an N-acyl-linked fatty acid and often phosphate or carbohydrate functional groups, as opposed to in the free form. The aliphatic chains can contain from 14 to as many as 28 carbon atoms, and most often they are saturated, monounsaturated or diunsaturated, with double bonds of either the cis or trans configuration. For example, the main dienoic long-chain base (sphingadienine) in human plasma is D-erythro-1,3-dihydroxy-2-amino-4-trans,14-cis-octadecadiene, and this is especially abundant in kidney, with more in women than in men. It is not present in zebra fish, widely used as a model species. Forms with three double bonds, such as sphinga-4E,8E,10E-trienine, sometimes with a methyl group in position 9, have been found the sphingolipids of some marine invertebrates and in a dinoflagellate. In addition, long-chain bases can have branched chains with methyl substituents in the omega‑1 (iso), omega‑2 (anteiso) or other positions, hydroxyl groups in positions 4, 5 or 6, ethoxy groups in position 3, and even a cyclopropane ring in the aliphatic chain in some organisms. N-Methyl, N,N-dimethyl and N,N,N-trimethyl derivatives of sphingoid bases have been detected in mouse brain.

As an example, the compositions of long-chain bases of sphingomyelins of some animal tissues are listed in Table 2 of our web page on sphingomyelins, where the main C18 components are accompanied by small amounts of C16 to C19 dihydroxy bases, although the latter attain higher proportions in tissues of ruminant animals. In gangliosides from human brain and intestinal tissues, eicosasphingosine (2S,3R,4E-d20:1) occurs in appreciable concentrations with variable amounts in different regions and membranes. However, human skin contains an especially wide range of isomers, including saturated, monoenoic and 6-hydroxy bases and phytosphingosines from C16 to C28 in chain-length. Shorter-chain bases are found in many insect species, and in the fruit fly, Drosophila melanogaster, which is widely used as a model species in genetic and metabolic experiments, the main components are C14 bases. In contrast to higher animals, nematodes such as Caenorhabditis elegans produce C17 iso-methyl-branched sphingoid bases, which are essential for normal sphingolipid function in the organism.

The long-chain base composition of individual lipids can vary markedly between species, tissues, organelles, and even different membranes within a single organelle. For example, the data in Table 1 is perhaps from an extreme example, but it illustrates that remarkable differences that can exist among lipids in one cellular component (rat liver mitochondria). Only part of the data from the paper cited is listed, but it illustrates that 3-keto-sphinganine, produced in the first step of sphingosine biosynthesis (see below) and normally a minor component of sphingolipids - often not detectable, can vary from 28 to 100% of the sphingoid bases depending on the lipid class and membrane within the organelle.

Table 1. Long chain base composition of some lipid components of mitochondria from rat liver.
  Base (%)
d18:1 d18:0-3keto t21:1 (phyto) Unidentified
  Ceramidesa 18 28 53 -
  Glucosylceramidesa 3 95 - 3
  Lactosylceramidesb 100
a whole mitochondria; b mitochondrial inner membrane
Data from Ardail, D. et al. FEBS Letts, 488, 160-164 (2001).

Phytosphingosine or 4D-hydroxy-sphinganine ((2S,3R,4R)-2-amino-octadecanetriol) is a common long-chain base of mainly plant origin. It is a saturated C18-trihydroxy compound, although unsaturated analogues, for example with a trans (or occasionally a cis (Z)) double bond in position 8, i.e., dehydrophytosphingosine or 4D‑hydroxy-8-sphingenine, tend to be much more abundant (see Table 2 of our web page on ceramide monohexosides for tabulated data on two plant species other than Arabidopsis). In many plant species, there are lipid class preferences also, and dihydroxy long-chain bases are more enriched in glucosylceramides than in glycosylinositolphosphoceramides, for example. This is true in the model plant Arabidopsis thaliana, where the data listed for whole tissue is probably representative largely of the latter lipid (Table 2 below)

Table 2. Sphingolipid long-chain base composition of whole tissue and glucosylceramides from Arabidopsis thaliana.
  Base (%)
  t18:1 (8Z) t18:1 (8E) t18:0 d18:1 (8Z) d18:1 (8E) d18:0
  Whole tissue 12 70 13 4 1
  Glucosylceramides 44 22 5 28 2
Data from Sperling, P. et al. Plant Physiol. Biochem., 43, 1032-1038 (2005)

Other plant long-chain bases have double bonds in position 4, which can be of either the cis or trans configuration, although trans-isomers are by far the more common, while the base d18:2Δ4E,8Z/E is relatively abundant in most plant species. In A. thaliana and related species, Δ4 long-chain bases are found mainly in the flowers and pollen and then exclusively as a component of the glucosylceramides. In general outwith Brassica species, the composition is dependent on species, but typically it is composed of up to eight different C18‑sphingoid bases, with variable geometry of the double bond in position 8, i.e., (E/Z)-sphing-8-enine (d18:1Δ8), (4E,8E/Z)-sphinga-4,8-dienine (d18:2Δ4,8) and (8E/Z)-4-hydroxy-8-sphingenine (t18:1Δ8); d18:1Δ4, d18:0 and t18:0 tend to be present in small amounts only.

Phytosphingosine is not restricted to plants but is found in significant amounts in intestinal cells and skin of animals, with much smaller relative proportions in kidney. Although non-mammalian sphingoid bases in general tend to be poorly absorbed from the intestines, a small proportion of the phytosphingosine and related sphingoid bases found in animal tissues may enter via the food chain.

Yeasts and fungi tend to have distinctive and characteristic long-chain base compositions. For example, filamentous fungi have 9-methyl-4E,8E-sphingadienine as the main sphingoid base in the glucosylceramides but not in the ceramide phosphoinositol glycosides, while yeasts contain mainly the saturated C18 bases sphinganine and phytosphingosine, although some trans-4/8-unsaturated forms are usually present. Only a few bacterial species synthesise sphingolipids, but the family Bacteroidetes, which is abundant in the human gut is an important exception; they usually contain saturated (and branched) long-chain bases. Other pathogenic bacteria may utilize sphingolipids and sphingoid bases from their hosts.

Structure of 9-methylsphinga-4,8-dienine

Sphingoid bases are surface-active amphiphiles with critical micellar concentrations of about 20 μM in aqueous solutions; they probably exist in the gel phase at physiological temperatures. In that they bear a small positive charge at neutral pH, they are unusual amongst lipids, although their pKa (9.1) is lower than in simple amines because of intra-molecular hydrogen bonding. Together with their relatively high solubility (> 1μM), this enables them to cross membranes or move between membranes with relative ease. In so doing, they increase the permeability of membranes to small solutes. In esterified form in complex lipids, they participate in the formation of ordered lipid domains in membranes such as rafts.

The complex sphingolipids are discussed elsewhere in these web pages, but in most the sphingoid base is linked via the amine group to a fatty acid, including very-long-chain saturated or monoenoic and 2-hydroxy components, i.e., to form ceramides, which can be attached a polar head group, such as phosphate or a carbohydrate, via the primary hydroxyl moiety. An important exception is sphingosine-1-phosphate, which is not acylated and has signalling functions in cells akin to those of lysophospholipids.

2.   Biosynthesis and Metabolism

Sphinganine biosynthesis: The basic mechanism for the biosynthesis of sphinganine involves condensation of palmitoyl-coenzyme A with L-serine, catalysed by the membrane-bound enzyme serine palmitoyltransferase, requiring pyridoxal 5’-phosphate as a cofactor, which binds to a specific lysine residue on the enzyme. The reaction occurs on the cytosolic side of the endoplasmic reticulum in animal, plant, and yeast cells with formation of 3-keto-sphinganine as illustrated. This is believed to be the key regulatory or rate-limiting step in sphingolipid biosynthesis and is conserved in all organisms studied to date. Elimination of this enzyme is embryonically fatal in mammals and fruit flies. In mammals, serine palmitoyltransferase is a heterotrimer composed of two main subunits, designated SPTLC1 with either SPTLC2 or SPTLC3 (sometimes termed SPTLC2a and SPTLC2b, respectively). SPTLC1 is essential for activity, and it is ubiquitously expressed as is SPTLC2, while SPTLC3 is present in a relatively limited range of tissues and is most abundant in skin and placental tissue. In addition, there are two small subunits ssSPTA and ssSPTB (again other nomenclatures exist), which differ in a single amino acid residue, and may have regulatory functions; the active site is at the interface between the two main subunits. ssSPTA is essential for serine palmitoyltransferase function during development and hematopoiesis.

Addition of either of the two small subunits to the complexes changes the substrate preferences substantially and enables the synthesis of the wide range of homologues found in nature. In mammals, the SPTLC1-SPTLC2 complex forms C18 sphingoid bases specifically (with some C19, and C20), while the combination of SPTLC1 and SPTLC3 gives a broader product spectrum, including an anteiso-methylbranched-C18 isomer (from anteiso-methyl-palmitate as the precursor). Such branched bases are synthesised to a limited extent in human skin, but they are the main forms in lower invertebrates such as C. elegans. The activity of the serine palmitoyltransferase is governed by negative feedback and partly by orosomucoid (ORM-like or ORMDL) proteins, three in mammals (ORMDL1 to 3) and two in yeast (Orm1/2), which are ubiquitously expressed trans-membrane proteins located in the endoplasmic reticulum. The availability of serine is also an important factor.

Biosynthesis of sphinganine

The second step in sphinganine biosynthesis is reduction of the keto group to a hydroxyl in an NADPH-dependent manner by a specific 3‑ketodihydrosphingosine reductase ('3KSR'), also on the cytosolic side of the endoplasmic reticulum, a step that must occur rapidly as the intermediate is rarely encountered in tissues. The enzymes are presumed to be in similar subcellular locations in plant cells.

In plants, serine palmitoyltransferase is a heterodimer composed of LCB1 and LCB2 subunits with some homology to the mammalian enzymes, while in the yeast Saccharomyces cerevisiae, there are three subunits: Lcb1, Lcb2, and Tsc3. In the few bacteria that synthesise sphingoid bases, serine palmitoyltransferase is a water-soluble homodimer. The enzyme in the apicomplexan parasite Toxoplasma gondii is a homodimer also in contrast to other eukaryotes, but it is located in the endoplasmic reticulum.

Sphingosine biosynthesis via ceramide: Free sphinganine formed in this way is rapidly N-acylated by acyl-coA to form dihydroceramides by dihydroceramide synthases, which in animals are located primarily on the endoplasmic reticulum, presumably on the cytoplasmic surface. Animals and plants have multiple isoforms of this enzyme, for which the abbreviated term ‘ceramide synthase’ is now widely applied as they utilize most other sphingoid bases, such as those produced by hydrolysis of sphingolipids, as substrates. They are unique gene products with each located on a different chromosome and with considerable variation in the expression of the enzymes in different cell types within each tissue. Each isoenzyme has distinct specificities for the chain-length of the fatty acyl-CoA moieties but to a limited extent only for the base, suggesting that ceramides containing different fatty acids have differing roles in cellular physiology. All these enzymes have six membrane spanning regions, but the only substantial difference is in an 11-residue sequence in a loop between the last two putative transmembrane domains. As ceramides are central to all elements of sphingolipid biochemistry, there is much more discussion of their occurrence, biosynthesis and metabolism in a web page dedicated to this lipid.

Humans and mice have six ceramide synthases, which utilize subsets of acyl-CoAs and thus producing ceramides with specific acyl chain lengths. Of these, ceramide synthase 2 is most abundant and is specific for coA esters of very-long-chain fatty acids (C20 to C26); it is most active in lung, liver, and kidney. Ceramide synthase 1 is specific for 18:0 and is located mainly in brain with lower levels in skeletal muscle and testes. Ceramide synthase 3 is responsible for the unusual ceramides of skin and testes and uses C26-CoA and higher including polyunsaturated-CoAs with the latter tissue, while ceramide synthase 4 (skin, liver, heart, adipose tissue, and leukocytes) uses C18 to C22-CoAs. Ceramide synthases 5 (lung epithelia and brain gray and white matter) generates C16 (mainly) and C18 ceramides, and ceramide synthase 6 (intestine, kidney, and lymph nodes) produces C14 and C16 ceramides. However, hydroxylation and the presence or otherwise of double bonds in the acyl-coAs do not appear to influence the specificity of the ceramide synthases. Also, the expression of mRNA expression for ceramide synthases does not always correlate with the fatty acid composition of sphingolipids in a particular tissue, suggesting that other factors are involved in determining which molecular species are formed. One such is acyl-coenzyme A-binding protein (ACBP), which facilitates the synthesis of ceramides containing very-long fatty acids and stimulates ceramide synthases 2 and 3 especially.

Biosynthesis of long-chain bases via ceramide

Insertion of the trans-double bond in position 4 to produce sphingosine occurs only after the sphinganine has been esterified in this way to form a ceramide as illustrated above, with desaturation occurring at the cytosolic surface of the endoplasmic reticulum also. The desaturases were first characterized in plants, and this subsequently simplified the isolation of the appropriate enzymes in humans and other organisms. Two dihydroceramide desaturases have now been identified in animals and designated 'DEGS1 and DEGS2'. Both enzymes insert trans double bonds in position 4, but DEGS2 is a dual function enzyme that also acts as a hydroxylase to generate phytoceramides, i.e., to add a hydroxyl group on position 4. Distribution of the enzymes in tissues is very different, with DEGS1 expressed ubiquitously but highest in liver, Harderian gland, kidney, and lung. DEGS2 expression is largely restricted to skin, intestine, and kidney, where phytoceramides are more important. A considerable family of Δ4-sphingolipid desaturases has now been identified, and an early study by Stoffel and colleagues demonstrated that Δ4-desaturation involves first syn-removal of the C(4)- HR and then the C(5)-HS hydrogens. This appears to have been the first evidence that desaturases in general operate in this stepwise fashion.

The enzyme responsible for the insertion of the cis-14 double bond into sphinga-4-trans,14-cis-dienine is the fatty acid desaturase 3 (FADS3), which utilizes ceramides containing sphingosine as the precursor. The only other known activity of this enzyme is to insert a cis-double bond in position 13 of the CoA ester of vaccenic acid (11t-18:1) to produce the conjugated diene 11t,13c-18:2.

Scottish thistleSynthesis of sphingoid bases de novo is essential in most organisms and inhibition of the biosynthetic pathways affects growth and viability. However, this can be tissue specific, as deletion of the liver-specific SPTLC2 in mice, was found to have no effect on liver function, while a comparable deletion of adipocyte-specific SPTLC1 caused major tissue defects. Presumably, the latter tissue is unable to take up enough sphingolipid from the circulation to remedy the problem. Deficiencies in SPTLC3 are related to dermal pathologies, and genetic variant of SPTLC3 are associated with dyslipidemia and atherosclerosis. The essentiality of sphingoid base synthesis in plants has been demonstrated in a similar manner in studies with mutants in which specific enzymes have been deleted.

Phytosphingosine and plant ceramides: Phytosphingosine is formed from sphinganine, produced as above, by hydroxylation in position 4, possibly via the free base in plants, although it can be formed both from sphinganine and a ceramide substrate in yeasts. A single sphinganine C4‑hydroxylase is present in yeast, but Arabidopsis has two such enzymes (SBH1 and 2), which are critical for growth and viability. Much remains to be learned of the processes involved, but it is known that the enzyme responsible is closely related to a Δ4 desaturase. Indeed, it has been shown that there are bifunctional Δ4‑desaturase/Δ4-hydroxylases in Candida albicans and mammals, especially in keratinocytes (DEGS2 discussed above) with which either 4‑hydroxylation or Δ4‑desaturation is initiated by removal of the proR C-4 hydrogen. Sphinganine linked to ceramide is the substrate for 4-hydroxylation in intestinal cells.

In Arabidopsis thaliana leaves, 90% of the sphingoid bases are phytosphingosine with a Δ8‑double bond. In plants in general, in addition to Δ4‑desaturation, two distinct types (20 gene products) of sphingoid Δ8-desaturase have been characterized that catalyse the introduction of a double bond at position 8,9 of phytosphingosine. These are evolutionarily distinct from the Δ4‑desaturases. One type produces the trans (E)-8 isomer mainly and the other mostly the cis (Z)-8 isomer, with overall the trans-isomer tending to predominate but dependent upon plant species. It appears that the trans isomer is formed when the hydrogen on carbon 8 is removed first, and the cis when carbon 9 is the point of attack. While the main group of Δ8-desaturases requires a 4‑hydroxysphinganine moiety as substrate, the second does not.

In Arabidopsis, three different isoforms of ceramide synthase have been identified and denoted LOH1, LOH2 and LOH3. Phytosphingosine is used efficiently by LOH1 and LOH3 (class II synthases), but only LOH2 (class I synthase) uses sphinganine efficiently; LOH2 and 3 prefer unsaturated long-chain bases. Marked fatty acid specificity is also observed with LOH2 showing almost completely specific for palmitoyl-CoA and dihydroxy bases, while LOH1 shows greatest activity for 24:0- and 26:0-CoAs and trihydroxy bases; none utilize unsaturated acyl-CoA esters efficiently. In plants, fatty acid desaturases and hydroxylases are also closely related, and sphingolipid fatty acid α-hydroxylation is believed to occur on the ceramide, as opposed to the free acyl chain. It is believed that the Δ8‑desaturase utilizes ceramide as the substrate and the channels the products selectively into the synthesis of complex sphingolipids, while Δ4‑desaturation channels ceramides for synthesis of glucosylceramide. The free ceramide pool of leaves has a very different content of sphingoid bases in comparison with complex sphingolipids and consists of roughly equimolar amounts of t18:1Δ8E and t18:0.

It has been established that long-chain bases with 4-hydroxyl groups are necessary for the viability of the filamentous fungus Aspergillus nidulans and for growth in plants such as A. thaliana. The presence of an 8E double bond confers aluminium tolerance to yeasts and plants, and it is important for chilling resistance in tomatoes. However, a trans-4 double bond in the sphingoid base does not appear to be essential for growth and development in Arabidopsis.

Scottish thistle Fungal sphingoid bases: Fungi produce trans Δ8-isomers only, but Δ4- and Δ8-desaturases do not occur in the widely studied yeast S. cerevisiae. In the biosynthesis of sphingoid bases in fungi, the double bonds in positions 4 and 8 and the methyl group in position 9 are inserted sequentially into the sphinganine portion of a ceramide, the last by means of an S‑adenosylmethionine-dependent methyltransferase similar to plant and bacterial cyclopropane fatty acid synthases. In S. cerevisiae the ceramide synthase is a heteromeric protein complex, containing three subunits, Lag1, Lac1, and Lip1, of which the first two are homologous proteins that feature eight transmembrane domains. In the yeast Pichia pastoris, there is a distinct ceramide synthase, which utilizes dihydroxy sphingoid bases and C16/C18 acyl-coenzyme A as substrates to produce ceramides. The long-chain-base components of the ceramide are then desaturated in situ by a Δ4‑desaturase and the fatty acid components are hydroxylated in position 2. Further desaturation of the long-chain base component by a Δ8-(trans)- desaturase occurs before the methyl group in position 9 is introduced by an S-adenosylmethionine-dependent sphingolipid C-9 methyltransferase. As a final step a trans-double bond may be introduced into position 3 of the fatty acid component. These ceramides are used exclusively for the production of glucosylceramides, and it is believed that a separate ceramide synthase encoded by a different gene produces the ceramide precursors for ceramide phosphorylinositol mannosides.

Viral sphingoid bases: The genome of an important marine virus (EhV) encodes for a novel serine palmitoyltransferase, which hijacks the metabolism of algal hosts to produce unusual hydroxylated C17 sphingoid bases; these accumulate in lytic cells of infected algae such as the important bloom-forming species Emiliania huxleyi. While this may seem a rather esoteric topic, viruses constitute a high proportion of the marine biome, and their control of the growth of algal blooms has global consequences.

Unesterified sphingosine: A cycle of reactions occurs in tissues by which sphingoid bases are incorporated via ceramide intermediates into sphingolipids (see the web pages on individual sphingolipids), which are utilized for innumerable functions, before being broken down again to their component parts. It is worth noting that all the free sphingosine in tissues must arise by this route, in particular by the action of ceramidases on ceramides. Five such ceramidases are known with differing pH optima and varying subcellular locations. The levels of free sphingoids and their capacities to function as lipid mediators (see below) are controlled mainly by enzymic re‑acylation to form ceramides, although some is acted upon by sphingosine kinases to produce sphingosine-1-phosphate.

Free sphingosine production from ceramide

Free sphingoid bases are absorbed by enterocytes following digestion of dietary sphingolipids in animals (including some from gut microorganisms), and while some of this is converted to complex sphingolipids, much is catabolized with eventual formation of palmitic acid.

Catabolism of sphingosine and other long-chain bases occurs after conversion to sphingosine-1-phosphate and analogues as discussed in our web page on this metabolite. In yeasts, an alternative means of detoxification has been reported in which an excess of phytosphingosine is first acetylated and then converted to a vinyl ether prior to export from the cells.

3.   1-Deoxy-Sphingoid Bases

A few rare genetic disorders have been found in which there is a defect in one of the subunits of the serine palmitoyl transferase or one of the regulatory proteins (ORM), or of ceramide synthesis. The most important of these result in the formation of distinctive sphingoid base analogues that lack the hydroxyl group on carbon 1. 1-Deoxysphinganine (2-amino,3-hydroxy-octadecane) is formed at low levels in animal cells because of condensation of palmitoyl-CoA with L‑alanine catalysed by the serine palmitoyl transferase, especially when cells are depleted in L-serine. Unsaturated 1-deoxy-sphingosine is also produced and has a cis-double bond in position 14 and not a trans-double bond in position 4 as might have been expected. The desaturase responsible for this is the FADS3 desaturase, which introduces the 14-cis double bond into sphingadiene (see above). Reaction with glycine produces 1-desoxymethylsphinganine ((2R)‑1‑aminoheptadecan-2-ol or M17:0). In yeasts, one subunit of the serine palmitoyltransferase promotes alanine utilization. Both types of deoxy-base are N-acylated to ceramide analogues subsequently by ceramide synthases, and these are hydrolysed by ceramidases.

Formulae of deoxy- and desoxymethyl-sphingoid bases

Deoxy bases have no known biological function and can have harmful effects. In a rare genetic disorder (hereditary sensory and autonomic neuropathy type 1, HSAN1), similar lipids are formed in a reaction with alanine and glycine because of defects in the sub-units of the serine palmitoyltransferase, and they also accumulate in vitro in cells treated with the fungal toxin fumonisin B1 (see below). Elevated concentrations have been reported in human serum and visceral adipose tissue in patients with type 2 diabetes, and in various tissues of aged mice. Deoxysphingoid bases are neurotoxic in that they cause mitochondrial fragmentation and dysfunction, but promising preliminary results have been obtained with dietary supplements of serine to “compete out” this deleterious activity. They also interfere with the survival of pancreatic β‑cells and insulin production; plasma levels are prospective biomarkers for the risk of the development of type 2 diabetes. In contrast, a 1‑deoxy analogue termed ‘enigmol’, which can be administered orally, has shown promise against colon and prostate cancer cells in culture, and various analogues are under evaluation in phase I clinical trials. 1-Deoxy-sphingosine appears to be less toxic than the saturated analogue.

Ceramides formed from deoxysphinganine are present in normal cells tissues at low levels, especially the liver, and in many foods, but they are not usually noticed because they are swamped by the much larger amounts of conventional ceramides. For example, commensal bacteria from the Bacteroidetes phylum produce some deoxy-sphingolipids, which can be absorbed by intestinal cells. Lacking a terminal hydroxyl group, deoxy-bases cannot form complex ceramide derivatives such as the glycosphingolipids or sphingomyelin. For the same reason, they cannot be catabolized by the normal pathway via sphingosine 1-phosphate, although degradation can be accomplished slowly by the action of cytochrome P4504F enzymes involving hydroxylation and desaturation reactions that produce a number of intermediate metabolites. The toxicity of these bases in different cell types may depend on the degradative capacity of the latter.

4.   Biological Functions of Unesterified Sphingoid Bases

The primary function of sphingoid bases is to serve as a basic component of ceramides and complex sphingolipids, where variations in their compositions can influence the physical and biological properties of these lipids. Independently of this in their free (unesterified) form, they are important mediators of many cellular events even though they are present at low levels only in tissues (typically 25 and 50 nM in plasma), with intracellular levels determined by hydrolysis by ceramidases or by the action of sphingosine kinases (sphingosine-1-phosphate production). In animal cells, they inhibit protein kinase C indirectly, possibly by a mechanism involving interference with the binding of activators of the enzyme, such as diacylglycerols or phorbol esters. In addition, sphingoid bases are known to be potent inhibitors of cell growth, although they stimulate cell proliferation and DNA synthesis. They are involved in the process of apoptosis in a manner distinct from that of ceramides by binding to specific proteins and regulating their phosphorylation. While sphingosine does not appear to participate in raft formation in membranes, it may rigidify pre-existing gel domains in mixed bilayers, although any such effects will be dependent on local concentrations and pH. It should be noted that some of the biological effects observed experimentally may be due to conversion to sphingosine-1-phosphate.

Free sphingosine has been implicated in various pathological conditions, and for example, plasma sphingosine levels are increased in hyperthyroidism and in patients with type 2 diabetes. Lysosomal storage of the lipid is an initiating factor in Niemann Pick type C disease, a neurodegenerative disorder, where it causes a change in calcium release leading to a buildup of cholesterol and sphingolipids. In the human adrenal cortex, sphingosine produced in situ by the acid ceramidase has a function in steroid production by serving as a ligand for steroidogenic factor 1 at the cell nucleus, which controls the transcription of genes involved in the conversion of cholesterol to steroid hormones. Unesterified sphingoid bases may have a protective role against cancer of the colon in humans. Thus, N,N‑dimethylsphingosine and dihydrosphingosine, like the deoxysphingoid bases, are known to induce cell death in a variety of different types of malignant cells. There is evidence that sphingadienes of plant and animal origin inhibit colorectal cancer in mouse models by reducing sphingosine-1-phosphate levels. In consequence, synthetic analogues of long-chain bases are being tested for their pharmaceutical properties.

With some bacterial pathogens, sphingosine is reported have bactericidal activity due to binding of the protonated NH2 group to the negatively–charged lipid cardiolipin in bacterial plasma membranes promoting rapid permeabilization of the membrane and thence bacterial cell death. For example, free sphingoid bases derived from ceramide in skin have antimicrobial properties and are important mediators of host epithelial defence against bacterial invasion. Similarly, they inhibit the effects of bacterial pathogens in pulmonary infections. Free phytosphingosine has anti-proliferative and anti-inflammatory effects and is especially important for the maintenance of skin homeostasis. Sphinganine may have functions in various disease states that differ from those of sphingosine, including effects upon cardiovascular disease, and liver and kidney diseases, while impaired 3-ketosphinganine metabolism also has implications for human pathology. N,N‑Dimethylsphingosine is of further interest in that it inhibits protein kinase C, sphingosine kinase and many other enzyme systems, while it induces mechanical hypersensitivity to neuropathic pain.

Free sphingosine is believed to have a signalling role in plants by controlling pH gradients across membranes, and synthesis of sphingoid bases is stimulated by infection with bacterial pathogens as part of the immune response. They are secreted in root exudates where they inhibit pathogenic fungi in soils. In addition, free long chain bases (and the balance with the 1-phosphate derivatives) are essential for the regulation of apoptosis in plants.

5.   Unusual Marine Sphingoid Bases and Fungal Toxins

Some plants and animals, especially marine organisms, synthesise long-chain bases lacking the hydroxyl group in position 1 or 2, i.e., 1- or 2‑deoxy-sphingoid bases. In fact, 1-deoxysphinganine per se was first found in a marine organism where it was given the trivial name 'spisulosine'. Among the more unusual of these deoxy bases are the C28 α,ω- or two-headed-sphingoid base-like compounds, such as calyxinin and oceanin (and their β-glycosides) found in sponges. Some bacterial species of the family Bacteroidetes produce sulfonolipids (capnoids), which have structural similarities to the deoxy bases.

Formulae of calyxin and oceanin

Myriocin or 2-amino-3,4-dihydroxy-2-(hydroxymethyl)-14-oxo-eicos-6-enoic acid from the thermophilic fungus Isaria sinclairii is is a potent inhibitor of serine palmitoyltransferase, the first step in sphingosine biosynthesis, and powerful immunosuppressants. A family of six similar molecules from Aspergillus fumigatus and termed sphingofungins has analogous properties. Biosynthesis of the last is very different from that of the sphingoid bases and involves the condensation of a C18 polyketide with the uncommon substrate aminomalonate. Via a programme of structural modification, a drug termed ‘fingolimod’ has been developed from myriocin for the treatment of multiple sclerosis, as is discussed further in our web page on sphingosine-1-phosphate.

Formula of myriocin and sphingofungin B

Fungal toxins that have structural similarities to deoxysphingoid bases, e.g., fumonisin B1 with propane-1,2,3-tricarboxylic acid groups esterified at the C14 and C15 positions (illustrated), are found in maize and other crop plants where they are a cause of disease and can cause cell death. By inhibiting the dihydroceramide synthases, they disturb the synthesis of both the structural and signalling sphingolipids, such that there is reduced formation of dihydroceramides, ceramides and complex sphingolipids, but elevated levels of sphinganine, sphingosine and their 1-phosphates, together with formation of novel 1-deoxy-sphingoid bases. When ingested by humans and other animals, fumonisin can exacerbate or cause several disease states, including oesophageal cancer, non-alcoholic steatohepatitis and diabetes, which can in consequence be classified among the “sphingolipidoses”.

Formula of fumonisin B1

6.   Analysis

The first step in the analysis of the sphingoid bases of sphingolipids is hydrolysis of any glycosidic linkage or phosphate bonds as well as the amide bond to the fatty acyl group. This should be accomplished by a procedure in which the minimum degradation or rearrangement of the bases occurs, such as artefactual O- or N‑methylation. While many analysts claim that base-catalysed hydrolysis causes least disruption, rapid acid-catalysed methods are often preferred for convenience. Subsequently, the bases are best analysed by gas chromatography after derivatization to reduce their polarity. Analysis of long-chain bases in intact sphingolipids by liquid chromatography-mass spectrometry methods now appears to be a valuable alternative.

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Lipid listings Contact/credits/disclaimer Updated: June 22nd, 2022 Author: William W. Christie LipidWeb icon