The Lipid Library

ANANDAMIDE, OLEAMIDE AND OTHER SIMPLE FATTY AMIDES

STRUCTURE, OCCURRENCE, BIOLOGY AND ANALYSIS

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. In nature, fatty acids are linked to the complex sphingolipids via amide bonds. However, here we are concerned only with those simple fatty amides that occur naturally, some of which have profound biological functions.

1.   Anandamide and Other Long-Chain N-Acylethanolamides

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. Somewhat higher concentrations are reported in brain and other tissues. Similar lipids have also been found in fish, molluscs, slime moulds, and certain bacteria.

Of these, anandamide or N-arachidonoylethanolamide has attracted special interest, because of its marked biological activities ('ananda' means inner bliss and tranquility in Sanskrit). Like the pharmacologically active compounds in marijuana or cannabis (from Cannabis sativa), it exerts its effects through binding to and activating specific cannabinoid receptors, designated 'CB1' and 'CB2', both of which are membrane-bound G-proteins. CB1 is found in the central nervous system and in some other organs, including the heart, uterus, testis and small intestine, while the CB2 receptor is found in the periphery of the spleen and other cells associated with immunochemical functions, but not in brain. Like 2-arachidonoyl-glycerol, discussed elsewhere on this website, anandamide is an endogenous cannabinoid or 'endocannabinoid'.

Anandamide is synthesised upon demand from phospholipid precursors in cell membranes in response to a rise in intra-cellular calcium levels. Although direct N-acylation of ethanolamine is possible, the main mechanism for the biosynthesis of anandamide and related amides requires as a first step the production of N-acyl-phosphatidylethanolamine, a lipid that is normally present in animal tissues at very low levels only other than during injury. Unusual transacylase reactions, rather than hydrolysis and re-synthesis via CoA esters, are involved, and the reaction is Ca²⁺-dependent and energy-independent. It seems that only 1-O-acyl groups of phospholipids such as phosphatidylcholine can serve as acyl donors and that much of the acyl transfer is intramolecular, with production of a hypothetical intermediate, 2-O-acyl-sn-glycero-3-phospho-(N-acyl)-ethanolamine (or N-acyl lysoPE). The free sn-1 hydroxy group is then subjected to re-acylation, presumably by the same transacylase system. A second mechanism involves this transacylase catalysing direct transfer of the fatty acids of position 1 of phosphatidylcholine (or of other phospholipids such as cardiolipin) to N-acylate phosphatidylethanolamine. A surprising feature of this reaction is the fact that the arachidonic acid levels in position 1 of phospholipids are usually very low (typically <0.3%), other than in testis.

The second step in the biosynthesis of anandamide and related amides is hydrolysis of the N-acyl-phosphatidylethanolamine by a phosphodiesterase that is specific for this lipid. It has a similar function to phospholipase D, but it differs from all others of this type in its amino acid sequence. In addition to anandamide, phosphatidic acid is formed, and this also has messenger functions.

A related pathway that has been described involves double O-deacylation of N-acyl-phosphatidylethanolamine by phospholipases A/B prior to the action of the phosphodiesterase on the resulting glycerophospho-N-arachidonoylethanolamine. Indeed, it has recently been suggested that this may be the major route to anandamide. The intermediate glycerophospho-N-acylethanolamines have been detected in mouse brain.

A third pathway is known in which N-acyl-phosphatidylethanolamine is again the main precursor, but is acted upon by phospholipase C to release a phospho-anandamide, which is then de-phosphorylated to anandamide by a specific phosphatase. This may be the main route when anandamide is produced in response to bacterial endotoxins.

In rat brain, the endogenous precursor of anandamide, N-arachidonoyl phosphatidylethanolamine, contains alkenyl groups (16:0, 18:0, 18:1) in position sn-1 and mono- (18:1) and polyunsaturated (20:4, 22:4, 22:6) acyl groups in position sn-2 of the glycerol backbone.

Anandamide and other endocannabinoids are highly lipophilic and have a tendency to remain in the membrane, where they can diffuse to encounter membrane-bound enzymes and receptors. However, they are also able to diffuse into the cytoplasm, where they are transported by a high-affinity, saturable anandamide transporter, a process that may be facilitated by the fatty acid amide hydrolase (see below), to act on presynaptic cannabinoid receptors. In plasma, anandamide binds reversibly to serum albumin and is presumably transported to other tissues in this form.

The key to the activity of anandamide is that it binds to and activates both the central (CB1) and peripheral (CB2) cannabinoid receptors, and thereby elicits virtually all of the known effects of cannabis (though it may not be as important as 2-arachidonoylglycerol in this respect). Thus, as with the bioactive constituents of marijuana, the endocannabinoids produce neurobehavioral effects and may have important signalling roles in the central nervous system, especially in the perception of pain, anxiety and fear, in the regulation of body temperature and in the control of appetite. As in many other membrane associated processes, lipid rafts and caveolae serve as important platforms for regulation of the endocannabinoid system, and especially in the modulation of binding and signalling of the CB1 receptor.

Anandamide is believed to have important anti-inflammatory and anti-cancer properties, and, it affects the cardiovascular system by inducing profound decreases in blood pressure and heart rate. In addition, it is an anabolic regulator of metabolism in that it increases the intake of food, promotes the storage of lipid, and decreases the expenditure of energy. Some of these effects appear to be independent of the two main receptors, and anandamide is known to bind to a number of other proteins including the peroxisome proliferator-activated receptors (PPARα and PPARγ). There are suggestions that modulation of anandamide levels in the gut has potential for treatment of inflammatory bowel disease and colon cancer.

Macrophages generate anandamide in response to the presence of bacterial endotoxin, and it is involved in the pathology of septic shock and cirrhosis of the liver. In addition, anandamide derived from macrophages has anti-inflammatory effects both in the peripheral and central nervous system.

It has been demonstrated that anandamide can be converted by cellular systems in vitro to ethanolamides of the prostaglandins PGE₂, PGD₂ and PGF_2α by the action of the enzyme cyclooxygenase-2. For cyclo-oxygenation to occur, there is an essential requirement for the hydroxyl-group of anandamide. The biological importance of these novel lipids is only now being explored.

O-Arachidonoyl ethanolamine, i.e. with an ester instead of an amide linkage to arachidonic acid, and termed ‘virodhamine’, has been isolated from brain tissues. It acts as an agonist at the CB2 receptor.

Other N-acylethanolamides. The biological effects of the other fatty acyl ethanolamide derivatives are less clear, although they are by far the most abundant components of this lipid class. Most do not appear to interact with cannabinoid receptors, but they may have a role in minimizing the effects of cellular damage. For example, there is evidence for an additional endocannabinoid signalling system that involves N-palmitoylethanolamide and depends on receptors other than CB1 and CB2. This lipid was first identified more than 50 years ago, but has only recently been shown to have beneficial properties in experimental models of inflammation and inflammatory pain. It is believed that these effects are mediated through actions upon peroxisome proliferator-activated receptor-α (PPARα).

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 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. Here also the effects are mediated by binding with high affinity to PPARα (and not to receptors CB1/2 so it is not 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. 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. In addition, it has recently been demonstrated that that oleoylethanolamide, again by acting as a PPAR-α agonist, has a novel effect in enhancing memory consolidation through noradrenergic activation of specific regions of the brain. It may also have an influence on sleep patterns and the effects of stress.

N-Stearoylethanolamide is a pro-apoptotic agent, and it down-regulates the expression of liver stearoyl-CoA desaturase-1 mRNA, an anorexic effect.

Basal 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 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 it has some structural similarities. Some of these effects may be specific to particular tissues.

N-Acylethanolamides are also 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). It appears that such compounds have a variety of biological functions in plants, but research is still at a relatively early stage.

N-oleoyl- and N-palmitoylethanolamide are produced by the same general biosynthetic mechanisms as for anandamide Above).

Catabolism. There is currently great interest in the potential use of endocannabinoids for therapeutic purposes, such as the alleviation of inflammation, asthma and some forms of chronic pain, and as anti-tumour drugs. In vivo, the concentrations of all of these amides in many animal species are controlled by a single hydrolytic enzyme, i.e. a fatty acid amide hydrolase, which is an integral membrane protein (primarily in the perinuclear membranes). However, a second enzyme of this type has been found in humans and other primates that is absent in mice and rats, and there is a palmitoylethanolamide-preferring acid amidase. There are believed to be active transport systems for anandamide from the plasma membrane to other tissues, although the detailed mechanisms are poorly understood. Once it enters a cell, it is rapidly degraded. The products, arachidonic acid and ethanolamine, may then have further signalling functions. Because of their role in terminating amide signalling, amide hydrolases are the subject of intensive study and are targets for potential drug therapies. For example, there is evidence that by inhibiting hydrolase activity and increasing the concentration of anandamide the growth of certain tumor cells is inhibited.

2.   Oleamide

cis-9,10-Octadecenamide or 'oleamide' is a primary fatty acid amide. It was first isolated from the cerebrospinal fluid of sleep-deprived cats, and has been characterized and identified as the signalling molecule responsible for causing sleep. For example, it induced physiological sleep when injected directly into the brain of rats. Although other fatty acid primary amides in addition to cis-9,10-octadecenoamide are present naturally in the cerebrospinal fluid of animals, only linoleamide is known to be biologically active, for example in increasing Ca²⁺ flux.

A rather unusual mechanism is suggested for the biosynthesis of oleamide, involving the enzyme cytochrome c and oleoyl-CoA and ammonium ions as the substrates, with hydrogen peroxide as an essential cofactor.

In addition to its sleep-inducing properties, oleamide has other neurological activities including regulation of memory processes, decreasing body temperature and locomotive activity, stimulating Ca²⁺ release, modulation or activation of a number of receptors, and effects on the perception of pain. Unlike anandamide, it does not interact with the CB1 receptor, for example. As with the N-acylethanolamines, the concentration of oleamide is controlled by the specific fatty acid amide hydrolase in vivo, but it is not known how these simple molecules avoid hydrolysis by the innumerable proteases, lipases and amidases present in brain.

3.   N-Arachidonoyldopamine and Related Amides

More recently, N-arachidonoyldopamine has been detected as an endogenous component of mammalian nervous tissue with distinctive biological effects. For example, it interacts with the same receptor (vanilloid type 1) as capsaicin, the active ingredient of chili peppers, with which it has some structural similarity. It has thus been termed a ‘vanilloid’ or ‘endovanilloid’. In addition, it binds to the CB1 receptor and shows cannabimimetic effects.

The N-oleoyl analogue has characteristic biological properties and interacts with the same receptors as N-arachidonoyldopamine. While the N-palmitoyl and N-stearoyl derivatives of dopamine do not interact with these receptors to a significant extent, they appear to act together with N-arachidonoyldopamine and anandamide to enhance calcium mobilization. N-acetyldopamine is also present in many animal tissues. The mechanisms for biosynthesis and catabolism of these lipids are not fully elucidated.

Oxidized derivatives of arachidonic acid (including hexanoic acid) and docosahexaenoic acid linked to dopamine may be involved in the pathogenesis of Parkinson’s disease. N-Hexanoyl dopamine is highly cytotoxic.

4.   Simple Lipoamino Acids

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.

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 may have some relation to metabolic disease. It is possible that they function as intercellular messengers via cell surface receptors. N-Oleoylglycine was first detected in mouse neuroblastoma cells, and it is now known to have a regulatory effect on body temperature and locomotion. As in the biosynthesis of oleamide, cytochrome c catalyses the formation of oleoylglycine from oleoyl-CoA and glycine, in the presence of hydrogen peroxide. However, other biosynthetic routes are possible.

N-Arachidonoylglycine is present in bovine and rat brain as well as other tissues at low levels. It is synthesised from anandamide by two pathways in mammalian cells. One route involves oxidation of anandamide via an alcohol dehydrogenase, proceeding via an aldehyde intermediate.

In the second pathway, hydrolysis of anandamide by the fatty acid amide hydrolase is followed by conjugation of the released arachidonic acid with glycine by the pathway proposed for oleoylamide and N-oleoylglycine.

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 has been suggested that simple lipoamino acids or fatty acid-amino acid conjugates of which N-arachidonylglycine is the considered to be the prototype should be termed 'elmiric' acids. More than forty such compounds have now been isolated from animal tissues.

N-Arachidonylserine has been detected at trace levels in bovine brain. It does not bind strongly to cannabinoid receptors, but it does have a potent vasodilatory effect on rat arteries in vitro amongst other biological effects. At least three other arachidonyl amino acids, 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. They can be converted to primary fatty amides in vitro, and it is possible that this also occurs in vivo.

A range of fatty N-acyltaurines have been isolated from both the central nervous system and peripheral tissues (see also our web-page dealing with other taurolipids). In the former, the fatty acyl groups are largely long-chain saturated, but in liver and kidney, arachidonoyl and docosahexaenoyl species predominate. In kidney, these compounds have been shown to activate receptors that control calcium channels. Like the other biologically active amides in animals, the levels of these metabolites are controlled by the activity of the fatty acid amide hydrolase.

The N-arachidonoyl amino acid and vanilloid derivatives are minimally oxidized by cyclooxygenase-2 (COX-2), but are good substrates for the 12S- and 15S-lipoxygenases. It is not yet clear whether this leads to inactivation of these lipids or rather converts them to new bioactive compounds. Arachidonoyltaurine has been shown to be an excellent substrate for lipoxygenases, but the functions of the resulting hydroxyeicosatetraenoyltaurines have yet to be determined. 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.

N-Linolenoyl-L-glutamine, N-linolenoyl-L-glutamic acid and related lipoamino acids have been found in insect larvae. Their presence in oral secretions elicits a defense response in plants.

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. It is normally a minor component, but can assume major proportions when phosphate is limiting in some species. Ornithine lipids contain a normal fatty acid with an estolide linkage to a 3-hydroxy acid and thence via an amide bond to ornithine. It may be relevant that such fatty acid linkages are also seen in the bacterial endotoxin lipid A. In some bacterial species, the ester-linked fatty acid has a hydroxyl group in position 2.

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. Related lipids with lysine, serine, glycine and taurine residues occur with structures of the same type, including one containing a glycine-serine dipeptide linked to branched chain acids and termed ‘flavolipin’. Some of these lipoamino acids have interesting and potentially useful pharmacological properties.

Simple N-acyl derivatives (without a secondary fatty acid constituent) also occur in bacteria, including N-acyl leucine (or isoleucine) derivatives in Deleya marina and N-acyl D-asparagine in Bacillus pumilus. N-Acyl-L-homoserine lactones are produced by a number of gram-negative bacteria. They are used for a form of intercellular signalling termed quorum sensing, which is believed to be associated with their pathogenic or communal behaviour. The fatty acid components can vary in chain length from C₄ to C₁₈, sometimes with double bonds at the 7 or 8 position and with hydroxyl or keto groups in position 3.

On the other hand, other 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) Microbial lipopeptides have their own webpage.

5.   Analysis

The main problems in the analysis of N-acylethanolamines and other simple amides relate to the low levels at which they occur naturally. There is a concern that artefactually high results can be obtained because of the physiological effects of sampling methods. However, sensitive methods that utilize high-performance liquid chromatography with fluorescent detection or gas chromatography-mass spectrometry with selected ion monitoring are available for the actual measurements. Liquid chromatography allied to tandem mass spectrometric methods has also proved of value. For a list of references on analysis, see our literature survey of analytical methods.

Recommended Reading

• Chapman, K.D. Occurrence, metabolism, and prospective functions of N-acylethanolamines in plants. Prog. Lipid Res., 43, 302-327 (2004).
• Driscoll, W.J., Chaturvedi, S. and Mueller, G.P. Oleamide synthesizing activity from rat kidney: identification as cytochrome c. J. Biol. Chem., 282, 22353-22363 (2007).
• Farrell, E.K. and Merkler, D.J. Biosynthesis, degradation and pharmacological importance of the fatty acid amides. Drug Discovery Today, 13, 558-568 (2008).
• Fowler, C.J. The contribution of cyclooxygenase-2 to endocannabinoid metabolism and action. Brit. J. Pharmacol., 152, 594-601 (2007).
• Hohmann, A.G. and Suplita, R.L. Endocannabinoid mechanisms of pain modulation. AAPS J., 8, E693-E708 (2006)
• Huang, S.M. and 12 others. Identification of a new class of molecules, the arachidonyl amino acids, and characterization of one member that inhibits pain. J. Biol. Chem., 276, 42639-42644 (2001).
• Kokotos, G. Endocannabinoids. In: Bioactive Lipids. pp. 245-264. (edited by A. Nicolaou and G. Kokotos, The Oily Press, Bridgwater) (2004).
• McKinney, M.K. and Cravatt, B.F. Structure and function of fatty acid amide hydrolase. Annu. Rev. Biochem., 74, 411-432 (2005).
• Pacher, P., Bátkai, S. and Kunos, G. The endocannabinoid system as an emerging target of pharmacotherapy. Pharmacol. Rev., 58, 389-462 (2006).
• Schmid, H.H.O. and Berdyshev, E.V. Cannabinoid receptor-inactive N-acylethanolamines and other fatty acid amides: metabolism and function. Prostaglandins, Leukotrienes, Essential Fatty Acids, 66, 363-376 (2002).
• Tan, B., Bradshaw, H.B., Rimmerman, N., Srinivasan, H., Yu, Y.W., Krey, J.F., Monn, M.F., Chen, J.S.-C., Hu, S.S.-J., Pickens, S.R. and Walker, J.M. Targeted lipidomics: discovery of new fatty acyl amides. AAPS J., 8, E461-E465 (2006).
• Thabuis, C., Tissot-Favre, D., Bezelgues, J.-B., Martin, J.-C., Cruz-Hernandez, C., Dionisi, F. and Destaillats, F. Biological functions and metabolism of oleoylethanolamide. Lipids, 43, 887-894 (2008).

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