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. Simple fatty acid-amino acid conjugates (lipoamino acids) are discussed on a separate web page.
1. Anandamide and Related Endocannabinoids 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 (see below). As with 2-arachidonoyl-glycerol, discussed elsewhere on this website, anandamide has been termed an endogenous cannabinoid or 'endocannabinoid'.
Anandamide is synthesised upon demand from phospholipid precursors in cell membranes in almost all cells and tissues of the body 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 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. In neurons, there is evidence that 1,2-diarachidonoyl-phosphatidylcholine, produced by a specific acylation of 2-arachidonoyl-lysophosphatidylcholine, is the key intermediate. This reaction is the key to the specificity of the process, since subsequent reactions are independent of the N-acyl substituent.
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 second pathway has been described that does not use the specific phosphodiesterase. Rather it involves either single or double O-deacylation of N-acyl- or N-alkenyl-phosphatidylethanolamine catalysed by phospholipases prior to the action of specific phospholipase Ds on the resulting lysoglycerophospho- or glycerophospho-N-arachidonoylethanolamines. Indeed, it has been suggested that this may be the major route to anandamide from plasmalogens in brain, where the intermediate glycerophospho-N-acylethanolamines have been detected in a mouse model. In rodent brain, the endogenous precursor of anandamide is mainly the plasmalogen form of N-arachidonoyl phosphatidylethanolamine (N-acylplasmenylethanolamines), and 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.
Yet another 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. Further biosynthetic pathways to anandamide are known to exist and the regulation and relative importance of these are obviously complex.
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. It appears that cytoplasmic lipid droplets (‘adiposomes’) may act as a reservoir, although they are also an active site for metabolism. In plasma, anandamide binds reversibly to serum albumin and is presumably transported to other tissues in this form.
Anadamide exerts its effects at nanomolar to sub-micromolar concentrations mainly through binding to and activating specific cannabinoid receptors, especially those designated ‘CB1’ and ‘CB2’, both of which are membrane-bound G-proteins (a large and diverse family of receptors with a characteristic membrane spanning structure whose main function is to convert extracellular stimuli into intracellular signals). 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 spleen and other cells associated with immunochemical functions, but not in brain. 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. N-dihomo-γ-linolenoylethanolamine, N-eicosa-5,8,11-trienoylethanolamine, N-eicosapentaenoylethanolamine and N-docosahexaenoylethanolamine are other N-acylethanolamides that bind to the CB1 and CB2 receptors.
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 has important anti-inflammatory and anti-cancer properties both in vivo and in vitro in animal models. 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. It is also involved in the regulation of body temperature, locomotion, feeding and anxiety. 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. Anandamide is present in the reproductive fluids of both males and females and is believed to be important in reproduction.
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 can induce apoptosis in a number of cell types
It has been demonstrated that anandamide (and the other endocannabinoid 2-arachidonoylglycerol) can be converted by cellular systems in vitro to ethanolamides of the prostaglandins PGE2, PGD2 and PGF2α (‘prostamides’) by the action of the enzyme cyclooxygenase-2 (COX-2), but interestingly not by COX-1. For cyclo-oxygenation to occur, there is an essential requirement for the hydroxyl-group of anandamide. In addition, anandamide is a substrate for the action of lipoxygenases and of enzymes of the cytochrome P450 family.
The biological importance of these novel lipids is now being actively explored, and there is evidence that COX-2 metabolites induce apoptosis of cancer cells. The prostamides do not bind to either the cannabinoid or prostanoid receptors. However, 12(S)-hydroxy-eicosa-5Z,8Z,10E,14Z-tetraenoyl-N-(2-hydroxyethyl)amine binds to both CB receptors with an affinity similar to that of anandamide per se. PGE2-ethanolamide is extremely stable in human plasma, and mobilizes calcium in cell preparations in vitro at picomolar concentrations.
O-Arachidonoylethanolamine, 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 a full agonist for the CB2 receptor and is a partial agonist for the CB1 receptor. It has yet to be determined how it is synthesised, stored or degraded, but inter-conversion with anandamide can occur.
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 present in most tissues other than skeletal muscle and heart, i.e. a fatty acid amide hydrolase (‘FAAH’), which is an integral membrane protein (primarily in the perinuclear membranes). It belongs to a large family of enzymes that share a highly conserved 130 amino acid motif termed the ‘amidase signature’ sequence and is well conserved in the primary structure. However, a second enzyme of this type (‘FAAH-2’) has been found in humans and other primates that is absent in mice and rats. The two enzymes are found in different tissues, with the second being specific to heart and ovary, where perhaps surprisingly it is located on the surface of cytoplasmic lipid droplets.
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. Also, administration of inhibitors has beneficial effects against inflammatory pain.
2. Other Long-Chain 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. They may potentiate the activity of endocannabinoids by minimizing their degradation.
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 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 was also shown to have anticonvulsant and antiproliferative effects. 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. More recently, both N-palmitoyl- and N-oleoylethanolamides have been show to bind to specific G-protein coupled receptors. N-Palmitoylethanolamide is undergoing clinical trials for the relief of chronic pain. It is catabolized by the fatty acid amide hydrolase and by an N-acylethanolamine amidase, specific for amides having less than 18 carbon atoms.
N-Docosahexaenoylethanolamide is present in brain tissue in amounts comparable to anandamide. It may have neuroprotective effects in this form or after conversion to oxygenated metabolites. The latter are believed to regulate leukocyte motility.
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. It also has anto-inflammatory and anti-oxidant properties. 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.
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 adipose tissue, oleoylethanolamide reduces the triacylglycerol content by stimulating lipolysis and elevating the circulating levels of unesterified fatty acids and glycerol.
In addition, it has been demonstrated that oleoylethanolamide 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 have an influence on sleep patterns and the effects of stress.
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.
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-oleoyl- and N-palmitoylethanolamide are produced by the same general biosynthetic mechanisms in animals as for anandamide (see above). They are catabolized by the fatty acid amide hydrolase similarly, although a lysosomal enzyme that is highly specific for N-palmitoylethanolamide has been characterized (N-acylethanolamine-hydrolysing acid amidase).
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. It is an agonist for the CB1 receptor, which may be a mediator for its biological activity
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. 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.
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.
4. N-Arachidonoyldopamine and Other Biologically Active Amides
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.
Biosynthesis is believed to occur mainly by conjugation of dopamine with arachidonic acid, catalysed by a fatty acid amide hydrolase (not via the CoA ester)), although there are suggestions that some might be derived from arachidonoyltyrosine.
The N-oleoyl analogue has characteristic biological properties of its own but 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.
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 and pig, especially in the jejunum and ileum where they are believed to regulate intestinal function. 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. In fact, such lipids with saturated acyl groups were first detected in the wax layer of green coffee beans.
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.
5. N-Acylamides in Plants
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). In this instance, the precursor N-acyl phosphatidylethanolamine 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. The biochemistry and function of these compounds in plants are discussed in much greater detail on this site here...
It appears that such compounds have a variety of biological functions in plants. It appears that such compounds have a variety of biological functions in plants For example, N-linoleoylethanolamine is involved in the regulation of seed germination, N-lauroylethanolamine influences the elongation of main and lateral roots and root hair formation, seedling growth and flower senescence, and N-myristoylethanolamine functions in plant defence against pathogen attack and also inhibits stomatal closure. However, research is still at an early stage in comparison to that with animals.
In addition, structurally related N-acylamides have been identified in a few families of plants and some fungi in which the amine moiety contains propyl, isopropyl, butyl or often isobutyl moieties. The fatty acid moieties are also distinctive, sometimes with only ten carbons. Affinin is N-isobutyl-2E,6Z,8E-decatrienamide, for example. The biosynthetic mechanism is believed to be quite different from that of the N-acylethanolamines; the amine moiety may be derived from amino acids. Important biological functions are slowly being revealed.
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 spectrometry is now proving of particular value. For a list of references on analysis, see our literature survey of analytical methods.
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James Hutton Institute (and Mylnefield Lipid Analysis), Invergowrie, Dundee (DD2 5DA), Scotland.
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