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Simple N-Acylamides and Lipoamino Acids

Many simple fatty amides occur naturally, some of which have profound biological functions. Various amine and fatty acid moieties are involved, and the latter are often saturated or monoenoic in nature. Of these, anandamide or N-arachidonoylethanolamide is of special importance as an endocannabinoid and it is most appropriately discussed under that heading, together with oleamide and N-acyldopamines, which interact with the same receptors.

In addition to these, many simple fatty acyl-amino acid conjugates (lipoamino acids) are present in animal, plant and bacterial tissues and have important biological properties that are now being revealed and are discussed below. Fatty acid conjugates with short peptides, such as glutathione, are discussed here, but proteolipids in which fatty acids are linked covalently to proteins have their own web page. Similarly, bacterial lipopeptides are important natural products, but as these are different in structure and function and constitute a substantial topic in their own right, they are discussed separately in this web site, as are some of the taurolipids and the cysteinyl leukotrienes, which could be considered to be lipoamino acids. Fatty amides are produced synthetically in industry in large amounts (> 300,000 tons per annum) for use as ingredients of detergents, lubricants, inks and many other products, but these are not considered here.

1.   Long-Chain N-Acylethanolamides in Animals

Long-chain N-acylethanolamides are ubiquitous trace constituents of animal and human cells, tissues and body fluids, with important pharmacological properties. For example, in rat plasma, the concentrations of palmitoyl-, oleoyl- and arachidonoylethanolamides were found to be 17, 8 and 5 pmol/ml, respectively, and somewhat higher concentrations are reported in brain and other tissues. Similar lipids have been found in fish, molluscs, slime moulds, and certain bacteria also. The fatty acyl ethanolamides other than anandamide are the subject of this section, and indeed they are the most abundant components of this lipid class; their biological properties are now being explored. Although most do not appear to interact with the cannabinoid receptors, they may potentiate the activity of endocannabinoids by minimizing their degradation.

For example, there is evidence for a signalling system resembling that of the endocannabinoids, which involves N-palmitoylethanolamide and depends on receptors other than CB1/CB2 for endocannabinoids. This lipid was first identified in egg yolk more than 50 years ago, and its anti-inflammatory properties were recognized immediately. However, little more was done until there was a resurgence of interest in recent years, during which it has also been shown to have anti-inflammatory, analgesic and neuroprotective actions. It is produced in most mammalian tissues, although most interest has been on its occurrence in the central nervous system. In particular, it has been shown to have neuroprotective properties in mast cell-mediated models of stroke, spinal cord injury, traumatic brain injury and Parkinson disease. It is believed that the effects on inflammation and inflammatory pain are mediated mainly through actions upon peroxisome proliferator-activated receptor-α (PPARα), although other mechanisms have been postulated especially synergistic effects with other acyl amides as part of a complex web of direct and indirect interactions. More recently, both N‑palmitoyl- and N‑oleoylethanolamides have been show to bind to specific G‑protein coupled receptors designated GPR55 and TPRV1. N‑Palmitoylethanolamide is undergoing clinical trials for the relief of chronic pain.

Formula of N-oleoylethanolamide

N-Oleoylethanolamide is an endogenous regulator of food intake, and may have some potential as an anti-obesity drug. It is believed to act as a local satiety signal rather than as a blood-borne hormone. For example, food intake was found to be inhibited in rats following intraperitoneal injection and even after oral administration. Under normal physiological conditions, oleic acid from dietary fat is transported into enterocytes in the small intestinal by a fatty acid translocase, and some is converted to oleoylethanolamide and acts as a sensor for ingestion of fat. The effect is highly specific, as linoleoylethanolamide has no such action, although it is produced in tissues in significant amounts and may have other biological effects. Here also the effects are mediated by binding with high affinity to PPARα (and not to receptors CB1/2 so it is not considered to be an endocannabinoid), especially in the enterocytes in the intestinal brush border. This stimulates the vagal nerve via the capsaicin receptor, leading to increased lipolysis and β-oxidation of fats. Oleoylethanolamide has anti-inflammatory and anti-oxidant properties also. While oleoyl- and palmitoylethanolamides do not activate cannabinoid receptors directly, they can enhance the activity of anandamide by inhibiting its inactivation by fatty acid amide hydrolase ('entourage effects').

Scottish thistleBasal levels of acylethanolamides are especially high in the gut. Anandamide and N-oleoylethanolamide are selectively decreased and increased in rat intestine during food deprivation and re-feeding through remodelling of the original acyl donor phospholipids. However, they have opposing effects upon lipogenesis. These products of phospholipid metabolism are thus in a state of dynamic equilibrium as part of the normal system of redistribution of molecular species in phospholipids. Indeed, there is increasing evidence that the balance between the various N-acylethanolamides is important for the correct functioning of innumerable biological systems, with an imbalance leading to pathological conditions. In adipose tissue, oleoylethanolamide reduces the triacylglycerol content by stimulating lipolysis and elevating the circulating levels of unesterified fatty acids and glycerol. Recent research suggests that this lipid is an important factor that fuels the growth of cells in chronic lymphocytic leukemia and may be involved in drug resistance and the wasting effects of the disease. In addition, it has been demonstrated that oleoylethanolamide by acting as a PPARα agonist has a novel effect in enhancing memory consolidation through noradrenergic activation of specific regions of the brain. It may have an influence on sleep patterns and the effects of stress.

Recently, the isomeric N-cis-vaccenoylethanolamide (i.e. of 11-18:1) was shown to be the most abundant 18:1 fatty acylethanolamide in rat plasma and the second most abundant in human plasma, although its biological properties are as yet unknown. As there has been a potential for confusion with N-oleoylethanolamide in certain studies, some re-appraisal of the latter may be necessary.

N-Stearoylethanolamide is an immunomodulator and it induces apoptosis of glioma cells. It down-regulates the expression of liver stearoyl-CoA desaturase-1 mRNA, an anorexic effect, and also has marked anti-inflammatory properties. By activating the nuclear receptor PPARα, it is able to regulate lipid metabolism.

N-Docosahexaenoylethanolamide or 'synaptamide' is present in brain tissue in amounts comparable to anandamide. While it has sometimes been considered to be an endocannabinoid, it has recently been found to have its own receptor, i.e. the orphan G-protein-coupled receptor GPR110 (ADGRF1). Thus, synaptamide binds specifically to GPR110 and triggers cAMP production and signalling with low nM potency, with the effect of inducing neurogenesis, neuritogenesis and synaptogenesis in developing neurons, a further mechanism by which DHA promotes brain development and function. Synaptamide may also have neuroprotective effects after conversion to oxygenated metabolites, which may regulate leukocyte motility. There is a report that its anti-inflammatory effects are due to inhibition of eicosanoid production by COX-2. Various oxygenated metabolites with anti-inflammatory and organ-protective properties have been described.

In some stress situations, increased levels of saturated and mono-unsaturated ethanolamides are produced and in others there is selective stimulation of anandamide synthesis. N-Acylethanolamides in human reproductive fluids may help to regulate many physiological and pathological processes in the reproductive system. Saturated and monoenoic N-acylethanolamides may also function as intracellular messengers by activating specific kinases and interacting with the signalling pathways mediated by ceramide, with which they have some structural similarities; some of these effects may be specific to particular tissues.

Biosynthesis and catabolism. N-oleoyl- and N-palmitoylethanolamide are produced by the same general biosynthetic mechanisms in animals as described on our web page for the endocannabinoid anandamide with N-acyl-phosphatidylethanolamines as key intermediates. They are catabolized by the fatty acid amide hydrolase, although a lysosomal enzyme that is highly specific for N-palmitoylethanolamide has been characterized (N-acylethanolamine-hydrolysing acid amidase). Inhibition of the latter enzyme is seen as a promising pharmaceutical target with the potential for anti-inflammatory and analgesic actions.

2.   Long-Chain N-Acylserotonins in Animals

Serotonin or 5-hydroxytryptamine per se is a monoamine neurotransmitter derived from tryptophan, and is found mainly in the gastrointestinal tract, platelets and the central nervous system of animals, where it is popularly known as a contributor to feelings of well-being. A number of N-acylserotonins (16:0, 18:0, 18:1 and 20:4, 20:5 and 22:6) have been detected in intestinal tissue from the rat, pig and humans, especially in the jejunum and ileum where they are believed to regulate intestinal function. In particular, N-docosahexaenoylserotonin in human intestinal tissue is a potent anti-inflammatory mediator that may be relevant to intestinal inflammatory conditions such as Crohn's disease and ulcerative colitis. N-Arachidonoylserotonin has been found in brain where it is reported to have an analgesic effect; it is an inhibitor of fatty acid amide hydrolase and binds to the vanilloid TRPV1 receptor. Of the other acylserotonins, N-palmitoylserotonin is reported to have anti-allergy properties and to improve memory in animal models. In fact, such lipids with saturated acyl groups were first detected in the wax layer of green coffee beans.

Formula of N-palmitoylserotonin

An arylalkylamine N-acyltransferase has been characterized from Drosophila melanogaster that catalyses the formation of long-chain N-acylserotonins from CoA esters and serotonin. Little appears to be known of their biosynthesis in higher animals.

3.   N-Acylamides in Plants

N-Acylethanolamides are minor but ubiquitous components of plant tissues, and they are especially abundant in desiccated seeds. The fatty acids are representative of those in plants with up to three double bonds, and with 12 to 18 carbon atoms. For example, oleoylethanolamide is present naturally at low levels in such food products as oatmeal, nuts and cocoa powder (up to 2 μg/g). In this instance, the precursor N‑acylphosphatidylethanolamine is synthesised by a different mechanism from that in animals, i.e. by direct acylation of phosphatidylethanolamine by an N‑acyl phosphatidylethanolamine synthase. N-acylethanolamides are released from this by the action of two (but not all) isoforms of phospholipase D in response to stress situations.

As in humans, it appears that such compounds have a variety of biological functions in plants with a highly conserved role in cell signalling, although research is still at an early stage in comparison to that with animals. They appear to block essential processes that are part of the transition from seed germination to seedling growth, although some of their effects may be beneficial. For example, N-linoleoylethanolamine disrupts root development in seedlings, while N-linolenoylethanolamine inhibits chloroplast development; oxylipins formed from these by the action of lipoxygenases have similar effects. N-lauroylethanolamine also has negative effects on seedling growth and flower senescence, and N-myristoylethanolamine functions in plant defence against pathogen attack and inhibits stomatal closure. In addition, further metabolites of N-lauroylethanolamine have been characterized in which glucose is attached to the free hydroxyl of the ethanolamine moiety together with mono- or dimalonylglucosides; these may represent a mechanism for modulating the biological activities of N-Acylethanolamides in plants. As in animals, a fatty acid amide hydrolase (FAAH) that degrades N-acylethanolamides in vivo is present in Arabidopsis, and this is also believed to be an important regulatory factor in plants.

Formula of affininWhile these simple derivatives have attracted most recent interest because of their similarity to animal lipids, more than 300 different N-acylamides have been identified from eight families of plants and some fungi with as many as 200 different fatty acids and many different amine moieties including propyl, isopropyl, butyl and often isobutyl moieties. The fatty acid moieties are distinctive, and fall into two groups, those with double bonds only and those with double and triple bonds. For example, affinin has a 10-carbon fatty acid attached to isobutylamine, i.e. it is N‑isobutyl-2E,6Z,8E-decatrienamide; it has been shown to have insecticidal activity. The biosynthetic mechanism is believed to be quite different from that of the N-acylethanolamines, and the amine moiety may be derived from amino acids. Important biological functions are slowly being revealed, including toxicity to insect predators, bacteria and fungi, while promoting plant growth.

N-Acyl ureas are produced by fungi, often in the form of symmetrical molecular species with palmitoleic, oleic or linoleic acids as the fatty acid constituents. They interact with the human immune system, possibly alerting the host to the presence of a fungal infection.

4.   Simple Lipoamino Acids from Animal Cells

N-acetyl derivatives of amino acids are minor but ubiquitous components of animal tissues, and may simply be a means of excreting or detoxifying excesses of particular amino acids or fatty acid metabolites. In addition, more than 50 different long-chain fatty acid conjugates with amino acids have been found in rat brain, some of which have important biological properties although those of most have yet to be determined.

N-Acylglycine derivatives of short-chain fatty acids (C2 to C12) have long been recognized as minor constituents of urine and blood, and their compositions in the former especially may have some relation to metabolic diseases. They are formed in the liver as a detoxification mechanism for removal of excess acyl-coenzyme A esters, although it is possible also that they function as intercellular messengers via cell surface receptors.

N-Palmitoylglycine is produced in most tissues, but especially skin and spinal cord, and it has a role in sensory neuronal signalling by acting as a modulator of calcium influx and nitric oxide production. N-Oleoylglycine was first detected in mouse neuroblastoma cells, and it is now known to have a regulatory effect on body temperature and locomotion. It is a precursor for oleamide by oxidative cleavage in a reaction catalyzed by peptidylglycine α-amidating monooxygenase (other acylglycines may react similarly). N-Arachidonoylglycine is present in bovine and rat brain as well as other tissues at low levels; it is synthesised from anandamide by at least two pathways in mammalian cells, of which one involves sequential oxidation of N-acylethanolamine by alcohol dehydrogenase and aldehyde dehydrogenase while in the second arachidonoyl-CoA and glycine combine in a reaction catalysed by glycine N-acyltransferase-like 3. Other glycine N-acyltransferases are responsible for the formation of oleoylglycine and for short-chain fatty acylglycines.

Biosynthesis of N-arachidonoyl glycine/serine

In a possible third pathway, cytochrome C catalyses the formation of arachidonoylglycine from arachidonoyl-CoA and glycine, in the presence of hydrogen peroxide. The same pathway is believed to operate for N-arachidonoylserine, as is illustrated in the figure. N-Arachidonylglycine has been shown to suppress inflammatory pain. It does not bind to the CB1 receptor for endocannabinoids, but it is a ligand for other receptors and may have a role in regulating tissue levels of anandamide by inhibiting the fatty acid amide hydrolase. It is a substrate for cyclooxygenase-2 (COX-2), producing glycine conjugates of prostaglandins, and it may divert the biosynthetic pathway from the pro-inflammatory PGE2 towards the anti-inflammatory J prostaglandins

N-Acylserines have been detected at trace levels in bovine brain with the palmitoyl and stearoyl forms being most abundant, but with unknown functions. N-Arachidonoylserine is also present. While it does not bind strongly to cannabinoid receptors, it does have a potent vasodilatory effect on rat arteries in vitro, and it activates certain calcium channels in neurons, amongst other biological effects. N-Oleoylserine stimulates osteoblast proliferation, but has not been detected in brain. At least three other arachidonyl amino acids, i.e. of γ‑aminobutyric acid, alanine and asparagine, occur naturally and also inhibit pain, suggesting that such biomolecules may be integral to pain regulation and perhaps have other functions in mammals. Like oleoylglycine, they can be converted to primary fatty amides in vitro, and it is possible that this also occurs in vivo.

N-Acylaspartates with 16:0, 18:2 and 20:4 fatty acid moieties attached to the amino group of aspartic acid that may inhibit Hedgehog signalling have been isolated from animal tissues. N-(17-Hydroxy)-linolenoyl-L-glutamine (volicitin), N-(17-hydroxy)-linoleoyl-L-glutamic acid and related lipoamino acids have been found in insect larvae. Their presence in oral secretions elicits a defense response in plants.

The N-arachidonoyl amino acid and vanilloid derivatives are minimally oxidized by COX-2, but they are good substrates for the 12S- and 15S‑lipoxygenases. However, it is not yet clear whether this leads to inactivation of these lipids or rather converts them to new bioactive compounds. While the fatty acid amide hydrolase will cleave the N-acyltaurines and N-arachidonoylglycine to the corresponding fatty acid and amino acid, the other N-acyl amino acids are not affected and their catabolic fate is uncertain.

5.  Lipid-Glutathione Adducts (Animal)

Glutathione is the tripeptide, γ-L-glutamyl-L-cysteinylglycine, and is most abundant thiol-containing small molecule (3 to 4 mM) in animal cells, where it is located mainly in the cytosol. It has a major defensive role in combating oxidative stress, for example by undergoing oxidation to glutathione disulfide while reducing lipid (and other) hydroperoxides to hydroxides. It also reacts with lipid oxidation products, including peroxy-fatty acids and unsaturated aldehydes, to produce lipid-glutathione adducts, facilitated by the action of glutathione S-transferases. For example, bioactive eicosanoids and α,β-unsaturated aldehydes (e.g. trans,trans-2,4-decadienal) and malondialdehyde can be deactivated or detoxified by conversion to inactive glutathione conjugates.

In contrast, the eicosanoid 5-hydroperoxyeicosapentaenoic acid (5-HPETE) is converted to the glutathione adduct as an intermediate in the biosynthesis of the cysteinyl leukotrienes, as described elsewhere on this website.

6.  Simple Lipoamino Acids from Bacteria

Formula of an ornithine lipidOrnithine lipids: A variety of lipoamino acids have been isolated from bacterial species, of which the best know is probably the zwitterionic N-acyl-ornithine derivative illustrated, which is widely distributed among prokaryotes, but especially Gram-negative bacteria and other eubacteria, where it is located predominantly on the outer membrane. Although it is normally a minor component, it can assume major proportions when phosphate is limiting or in response to stress in some species. Ornithine lipids contain a non-hydroxy fatty acid with an estolide linkage to a 3-hydroxy acid (often but not always C16 or C18) and thence via an amide bond to the α-amino group of ornithine. It may be relevant that such fatty acid linkages are also seen in the bacterial endotoxin lipid A.

In most bacteria, biosynthesis of ornithine lipids occurs in two steps via sequential acyl-ACP-dependent acylation of ornithine by two different acyltransferases. The first N-acyltransferase transfers a 3-hydroxy fatty acyl residue from acyl carrier protein to the α-amino group of ornithine forming a lyso-ornithine lipid, which is then acylated by an O-acyltransferase to produce the final ornithine lipid. However, at least one species contains a bifunctional acyltransferase in which the N-terminal domain is responsible for the O-acyltransferase reaction, whereas the C-terminal domain carries out the N-acyltransferase reaction. In some bacterial species, either the ester- or amide-linked fatty acid has a hydroxyl group in position 2, and analogous lipids in which the ornithine moiety is hydroxylated are known, with both modifications inserted post synthesis of the basic lipid. In the nodule bacterium Mesorhizobium loti, the ornithine moiety is surprisingly mainly of the D-configuration. Mono, di- and trimethylornithine lipids are formed by sequential methylation of the ornithine moiety by a specific N-methyl transferase requiring S-adenosylmethionine in Planctomycetes.

Rhodobacter sphaeroides contains both ornithine and related glutamine lipids, while cerilipin characterized from Gluconobacter cerinus has an ornithine-containing lipid core and an additional amide-linked taurine moiety. Structurally related lipids with lysine, serine, glycine, glutamine and taurine residues occur in microorganisms such as the gliding bacterium Cytophaga (Flavobacterium) johnsonae and the Gram-negative marine species Cyclobacterium marinus. They include one containing a glycine-serine dipeptide linked to branched chain acids and termed 'flavolipin'. Biosynthesis of these lipaminoacids is by a similar mechanism to that of the ornithine lipids.

Formulae of flavolipin and cerilipin

It appears that such lipoamino acids have a variety of different functions in bacteria depending upon species, and it does not seem possible to generalize. For example, they have been implicated in temperature and stress tolerance in some species, while in others they may be recognized by plant defense systems or be essential for symbiotic relationships. In Gram-negative bacteria, they may stabilize the outer membrane by counteracting the negative charge of the lipopolysaccharides. Some of these lipoamino acids have interesting and potentially useful pharmacological properties.

N-Acyl-L-homoserine lactones are produced by a number of Gram-negative bacteria. The fatty acid components can vary in chain length from C4 to C18, sometimes with one or two double bonds (in position 2 and/or more central positions), with hydroxyl or keto groups in position 3, and/or with methyl branches. The photosynthetic bacterium Rhodopseudomonas palustris contains p-coumaroyl-homoserine lactone.

Formula for an N-acyl-L-homoserine lactone

They are used in a form of intercellular signalling termed 'quorum sensing', which controls gene expression in response to population density, resulting in coordinate regulation of a range of group-level behaviours, including production of secondary metabolites and virulence factors, bioluminescence and biofilm formation, i.e. when these signal molecules reach a threshold concentration in a particular environment, they bind to their intracellular receptor/activator proteins (LuxR-type) to induce the expression of relevant genes. It is hoped that drugs will be found to de-activate these lipids and reduce the virulence of bacteria. In contrast, certain plants appear to be able to detect quorum sensing signals with the potential to permit them to alter the outcomes to their own benefit via the action of oxylipins and salicylic acid. Also, plants can hydrolyse N-acyl-L-homoserine lactones with fatty acid amide hydrolases to generate L‑homoserine, which encourages plant growth. Other quorum sensing molecules include the lipidic 2-heptyl-4-quinolone and 2-heptyl-3-hydroxy-4(1H)-quinolone.

Biosynthesis of homoserine lactones involves reaction of an acyl carrier protein-linked fatty acid, such as a 3-oxo isomer illustrated, with S-adenosylmethionine (SAM) and catalysed by an acyl-homoserine lactone (AHL) synthase. The mechanism is believed to begin with nucleophilic attack on the 1-carbonyl carbon of the fatty acyl moiety by the amine of SAM, followed by nucleophilic attack on the γ-carbon of SAM by its own carboxylate oxygen to produce the lactone.

Biosynthesis of an N-acyl-homoserine lactone

Other bacterial lipoamino acids: Simple N-acyl derivatives amino acids (without a secondary fatty acid constituent) also occur in bacteria, including N-acylleucine (or isoleucine) derivatives in Deleya marina, N-acyl-D-asparagine in Bacillus pumilus and N-acylserine in Serratia sp. On the other hand, lipoamino acid forms of increasing complexity have been characterized, including molecules with a long-chain alcohol moiety linked to the carboxyl group of the amino acid (such as siolipin A from Streptomyces species). The more complex microbial lipopeptides have their own web page.

8.   Analysis

The main problems in the analysis of simple lipoamino acids relate to the low levels at which they occur naturally, and there is a concern that artefactually high results might be obtained because of the physiological effects of sampling methods. Until recently, high-performance liquid chromatography with fluorescent detection or gas chromatography-mass spectrometry with selected ion monitoring were most used for the purpose, but liquid chromatography allied to tandem mass spectrometry with electrospray ionization would probably be the preferred method now.

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Lipid listings Credits/disclaimer Updated: June 11th, 2017 Author: William W. Christie LipidWeb icon