Fatty Acids: Polyunsaturated with Methylene-Interrupted Double Bonds

1. Structure and Nomenclature

Structural formula for methylene-interrupted double bondsThe lipids of all higher organisms contain appreciable quantities of polyunsaturated fatty acids ('PUFA') with methylene-interrupted double bonds, i.e. with two or more double bonds of the cis-configuration separated by a single methylene group; the term ‘homo-allylic’ may also be used to describe this molecular feature. Most bacteria of terrestrial origin, other than Cyanobacteria, cannot produce polyunsaturated fatty acids, but many bacterial families of marine origin can do so. In higher plants, the number of double bonds in fatty acids seldom exceeds three, but in algae and animals there can be up to six (very rarely more in some marine organisms). Two principal families of polyunsaturated fatty acids occur in nature that are derived biosynthetically from linoleic (9‑cis,12‑cis-octadecadienoic) and α-linolenic (9-cis,12-cis,15-cis-octadecatrienoic) acids.

Structural formulae of linoleic and linolenic acids

In the shorthand nomenclature, these precursor fatty acids are designated 9c,12c-18:2 and 9c,12c,15c-18:3, respectively. The number before the colon specifies the number of carbon atoms, and that after the colon, the number of double bonds. It is useful for biochemists and nutritionists to denote the position of the terminal double bond in the form (n-x), where n is the chain-length of the fatty acid and x is the number of carbon atoms from the last double bond, if it is assumed that all the other double bonds are methylene-interrupted. Thus linoleate and α-linolenate are 18:2(n-6) and 18:3(n-3), respectively (18:2ω6 and 18:3ω3 in the older literature), and they are the precursors for the omega-6 or (n-6) and omega-3 or (n-3) families of polyunsaturated fatty acids, respectively.

Both of the parent fatty acids can be synthesised in plants, but not in the tissues of higher animals, and they are therefore essential dietary components (see below). Polyunsaturated fatty acids of both families can be found in most lipid classes, but they are especially important as constituents of the phospholipids, where they appear to confer distinctive properties to the membranes, in particular by decreasing their rigidity. The exception is the sphingolipids, where they are rarely detected in other than trace amounts. In both animals and plants, they are key precursors of many classes of oxylipins with vital signalling and metabolic functions.

2. The n-6 family of Polyunsaturated Fatty Acids

Linoleic acid is a ubiquitous component of plant lipids, and of all the seed oils of commercial importance. For example, corn, sunflower and soybean oils usually contain over 50% of linoleate, and safflower oil contains up to 75%. Although all the linoleate in tissues of higher animals must be acquired from the diet, it is usually the most abundant dienoic fatty acid in mammals (and in most lipid classes), typically at levels of 15 to 25%, although it can amount to as much as 75% of the total fatty acids of heart cardiolipin. It is also a significant component of fish oils, although fatty acids of the n-3 family tend to predominate in this instance.

Analogues of linoleic acid with trans-double bonds are occasionally found in seed oils. For example, 9c,12t-18:2 is reported from Dimorphotheca and Crepis species, and 9t,12t-18:2 is found in Chilopsis linearis.

Scottish thistleThe remaining members of the n-6 family of fatty acids are synthesised from linoleate in animal and plant tissues by a sequence of elongation and desaturation reactions as described below. These metabolites can function as essential fatty acids also. Shorter-chain components may be produced by alpha or beta-oxidation.

γ-Linolenic acid ('GLA' or 6-cis,9-cis,12-cis-octadecatrienoic acid or 18:3(n-6)) is usually a minor component of animal tissues in quantitative terms (< 1%), as it is rapidly converted to higher metabolites. It is found in a few seed oils, and those of evening primrose, borage and blackcurrant have some commercial importance. Evening primrose oil contains about 10% GLA, and is widely used both as a nutraceutical and a medical/veterinary product.

11-cis,14-cis-Eicosadienoic acid (20:2(n-6)) is a common minor component of animal tissues. 8-cis,11-cis,14-cis-Eicosatrienoic acid (dihomo-γ-linolenic acid or 20:3(n-6)) is the immediate precursor of arachidonic acid, and of a family of eicosanoids (PG1 prostaglandins), but it does not accumulate to a significant extent in animal tissue lipids and is typically about 1-2% of the phospholipid fatty acids.

Structural formula of arachidonic acidArachidonic acid (5-cis,8-cis,11-cis,14-cis-eicosatetraenoic acid or 20:4(n-6)) is the most important metabolite of linoleic acid in animal tissues, both in quantitative and biological terms. It is often the most abundant polyunsaturated component of the phospholipids, and can comprise as much as 40% of the fatty acids of phosphatidylinositol. As such, it has an obvious role in regulating the physical properties of membranes, but the free acid is also involved in the mechanism by which apoptosis is regulated, and it has other signalling functions in cells, especially in the central nervous system. Meat is the main dietary source in humans. While some arachidonate is found in all fish oils, polyunsaturated fatty acids of the n-3 family tend to be present in much larger amounts. Arachidonic acid is sometimes detected as a constituent of mosses, liverworts, lichens and ferns, but there appears to be only one definitive report of its occurrence in a higher plant (Agathis robusta). The fungus Mortierella alpina is a commercial source of arachidonate via a fermentation process.

Several families of eicosanoids are derived from arachidonate, including prostaglandins (PG2 series), thromboxanes, leukotrienes, and lipoxins, with phosphatidylinositol being the primary source. These have an enormous range of essential biological functions that are discussed in elsewhere in these web pages. In addition, 2-arachidonoylglycerol and anandamide (N-arachidonoylethanolamine) have important biological properties as endocannabinoids, although they are minor lipids in tissues in quantitative terms.

4,7,10,13,16-Docosapentaenoic acid (22:5(n-6)) is usually a relatively minor component of animal lipids, but it is the main C22 polyunsaturated fatty acid in the phospholipids of testes, where it can amount to 70% of the lysobisphosphatidic acid in this tissue, for example. In this instance, C22 fatty acids of the n-3 family are present at relatively low levels, in contrast to most other lipids of reproductive tissues.

Other fatty acids of the n-6 family that are found in animal tissues include 22:3(n-6) and 22:4(n-6), and the latter, 7,10,13,16-docosatetraenoic or adrenic acid, is a significant component of the phospholipids of the adrenal glands and of testes. Tetra- and pentaenoic fatty acids of the n-6 family from C24 to C30 have been found in testes, where they are essential for male fertility and sperm maturation, while even longer homologues occur in retina. Very-long-chain (VLC) fatty acids of this type were first reported from human brain in patients with the rare inherited disorder, Zellweger's syndrome, but it is now established that such fatty acids with up to 38 carbon atoms and with from 3 to 6 methylene-interrupted double bonds are present at low levels in the brains of normal young humans, with 34:4(n-6) and 34:5(n-6) tending to predominate. In ceramides and sphingomyelin of rat testes, 28:4(n‑6) and 30:5(n‑6) fatty acids are the major VLC-PUFAs found, while 32:3(n‑6) and 32:4(n‑6) are abundant in the lipids of human spermatozoa.

The most highly unsaturated fatty acid of the n-6 family to have been characterized are 28:7(n-6) (4,7,10,13,16,19,22-octacosaheptaenoate), which has been found in the lipids of marine dinoflagellates and herring muscle, and 4,7,10,13,16,19,22,25,28-tetratriacontanonaenoic acid (34:9(n‑6)) from the freshwater crustacean species Bathynella natans.

3. The n-3 family of Polyunsaturated Fatty Acids

α-Linolenic acid (9-cis,12-cis,15-cis-octadecatrienoic acid or 18:3(n-3)) is a major component of the leaves and especially of the photosynthetic apparatus of algae and higher plants, where most of it is synthesised. It can amount to 65% of the total fatty acids of linseed oil, where its relative susceptibility to oxidation has practical commercial value in paints and related products. In contrast, soybean and rapeseed oils have up to 7% of linolenate, and this reduces the value of these oils for cooking purposes. In animal tissue lipids, α-linolenic acid tends to be a minor component (<1%), the exception being grass-eating non-ruminants such as the horse or goose, where it can amount to as much as 10% of the adipose tissue lipids. α-Linolenic acid is the biosynthetic precursor of oxylipins such as the jasmonates in plants, which appear to have functions that parallel those of the eicosanoids in animals.

As with linoleate, the remaining members of the n-3 family of fatty acids are synthesised from α-linolenate in animal and plant tissues by a sequence of elongation and desaturation reactions as described below, while shorter-chain components may also be produced by alpha or beta-oxidation. They also are essential fatty acids. For example, the elongation product 11,14,17-eicosatrienoic acid (20:3(n-3)) can usually be detected in the phospholipids of animal tissues but rarely at above 1% of the total, although somewhat higher concentrations may be found in fish oils.

When platinum salts (e.g. cisplatin) are administered to cancer patients, synthesis of hexadeca-4,7,10,13-tetraenoic acid or 16:4(n-3) is induced and confers systemic resistance to this and a broad range of other DNA-damaging chemotherapeutic agents. The mechanism involves activation of the G protein-coupled receptor 120 (GPR120 or FFAR4) on splenic macrophages by the free acid to produce chemo-protective lysophosphatidylcholines.

Stearidonic acid (6,9,12,15-octadecatetraenoic or 18:4(n-3)) is occasionally found in plants as a minor component, and it occurs in algae and fish oils. 3,6,9,12,15-Octadecapentaenoic acid or 18:5(n-3) is a significant component of the lipids of dinoflagellates, and it can enter the marine food chain from this source. 8,11,14,17-Eicosatetraenoic acid (20:4(n-3)) is found in most fish oils and as a minor component of animal phospholipids. It is frequently encountered in algae and in terrestrial bryophytes (mosses, liverworts and hornworts), together with other long-chain polyunsaturated fatty acids of the n‑3 family, but there are few definitive reports in higher plants.

Structural formula of eicosapentaenoic acid5,8,11,14,17-Eicosapentaenoic acid ('EPA' or 20:5(n-3)) is one of the most important fatty acids of the n-3 family. It occurs widely in algae and in fish oils, which are major commercial sources, but there are few definitive reports of its occurrence in higher plants. It is a key constituent of the phospholipids in animal tissues, especially in brain, and it is the precursor of the PG3 series of prostaglandins and of resolvins, which have anti-inflammatory effects (see the appropriate web pages for further discussion). However, it may have biological activities in its own right, including anti-cancer effects, by promoting the expression of genes that suppress tumours. There is currently great interest in the role of EPA in alleviating the symptoms of neurological disorders such as schizophrenia.

7,10,13,16,19-Docosapentaenoic acid (22:5(n-3)) is an important constituent of fish oils, and it is usually present in animal phospholipids at a level of 2 to 5%. It is the most second most abundant n-3 polyunsaturated fatty acid in the brain, so it may be especially beneficial for the elderly and for early-life development. While it has received relatively little study, it is known that it can be retro-converted to EPA, and that it reacts with lipoxygenases to form distinctive oxylipins, such as the specialized pro-resolving mediators involved in the resolution of inflammation.

Structural formula of docosahexaenoic acid4,7,10,13,16,19-Docosahexaenoic acid ('DHA' or 22:6(n-3)) is usually the end point of α-linolenic acid metabolism in animal tissues. It is a major component of fish oils, especially from tuna eyeballs, and of animal phospholipids, those of brain synapses and retina containing particularly high proportions. Indeed, there is some evidence that increased levels of this fatty acid are correlated with improved cognitive and behavioural function in the development of the human infant, although this is controversial. DHA is not present in higher plants, but it is found in high concentrations in many species of algae, especially those of marine origin, where it may be the major source of the DHA and EPA in fish. Via the food chain, fish contribute substantially to the levels of these fatty acids in terrestrial systems including humans.

As with the n-6 family, very-long-chain fatty acids (C24 to C38) of the n-3 family occur in the retina, brain, skin, testes and sperm, where they are derived biosynthetically by elongation of the C20 and C22 polyunsaturated precursors by the elongase ELOV4. 20:5(n-3) in retina is preferentially elongated in comparison to 22:6(n‑3). Although DHA is preferentially esterified to position sn-2 of phospholipids, the longer-chain analogues are esterified to position sn-1 in retina. They are precursors of the oxylipins known as elovanoids (protectin analogues).

Other fatty acids of the n-3 family that are found in nature include 22:3(n-3) from animal tissues and 16:3(n-3), which is a common constituent of leaf lipids (see our web pages on mono- and digalactosyldiacylglycerols). 16:4(n-3), 16:4(n-3), 21:5(n-3), 24:5(n-3) and 24:6(n-3) are occasionally present in marine organisms, including fish. Trace levels of highly unsaturated fatty acids of the n-3 family (suggested to be 38:7(n-3) to 44:12(n-3)) have been reported from the brains of patients with genetic impairments of peroxisome function, but the most highly unsaturated fatty acids of the n‑3 family to have been characterized from samples of normal origin are 4,7,10,13,16,19,22,25-octacosaoctaenoate (28:8(n-3)) from marine dinoflagellates and 34:10 from a fish oil concentrate.

Like 16:4(n-3) discussed above, EPA and DHA and probably other polyunsaturated fatty acids of the n-3 family activate the GPR120 receptor/sensor to cause broad anti-inflammatory effects in macrophages. For example, when activated by these fatty acids, GPR120 mediates potent insulin sensitizing and anti-diabetic effects in vivo by repressing macrophage-induced tissue inflammation. While n-3 fatty acids appear to be of special importance, other fatty acids, including palmitoleate and linoleate, are also agonists for this receptor and the precise molecular mechanisms behind its actions remain to be elucidated.

Biological Effects of DHA: Of these fatty acids, DHA appears to have special properties, both as a precursor of other metabolites and in esterified form as a component of membrane lipids. It is not a substrate for the prostaglandin synthase-cyclooxygenase enzymes, and indeed it inhibits them. However, via the action of lipoxygenases, it is the precursor of the docosanoids, such as the resolvins, protectins and maresins (or specialized pro-resolving mediators), which are analogous to the eicosanoids but have potent anti-inflammatory and immuno-regulatory actions. DHA per se may ameliorate autoimmune inflammation directly, for example by activating the GPR120 signalling pathway in dendritic cells. It has been demonstrated to have beneficial effects upon inflammatory disorders of the intestine and in reducing the risk of colon cancer, which may be mediated through associations with specific signalling proteins in membranes. DHA is believed to have specific effects on gene transcription that regulate a number of proteins involved in fatty acid synthesis and desaturation.

Scottish thistleThe concentration of DHA in tissues has been correlated with a number of human disease states, and while it is essential to many functions of the brain there appear to be no organ where it is not required for some key purpose. Particular attention has been given to its role in the retina, where it is a major component of the phospholipids in the photoreceptor outer segment membranes and is necessary for optimum retinal function. In this instance, its primary role is to maintain the disc shape in photoreceptor cells; cellular membrane containing DHA in the phospholipids are more flexible than those containing arachidonic acid and other fatty acids, and they may also increase the stability and function of rhodopsin. In some cases, sight defects have been ameliorated with DHA supplementation. The preferred substrate for uptake by the brain is DHA esterified to lysophosphatidylcholine, as this can cross the blood-brain barrier more easily than the free acid. DHA is intimately involved with phosphatidylserine metabolism in neuronal tissue. Similarly, N‑docosahexaenoylethanolamide or 'synaptamide' is a significant component of brain tissue and is an important signalling molecule that induces neurogenesis, neuritogenesis and synaptogenesis in developing neurons. This is a further mechanism by which DHA promotes brain development and function. During spermatogenesis, DHA-containing phospholipids provide membranes in spermatids with the physicochemical properties needed for normal cellular processes.

During phospholipid biosynthesis, DHA is esterified to form a phosphatidic acid precursor by a specific lysophosphatidic acid acyltransferase 3 (LPAAT3). As a phospholipid constituent, it has profound effects on the properties of all membranes, modulating their structure and function. In such an environment, DHA is believed to be more compact than more saturated chains with an average length of 8.2Å at 41°C compared to 14.2Å for oleic chains. This is the result of the adoption of a conformation with pronounced twists of the chain, which reduce the distance between the ends. The methyl group with its extra bulk is located in the interior region. In mixed-chain phospholipids, a further consequence is a marked increase in the conformational disorder of the saturated chain. There appears to be an incompatibility between the rigid structure of cholesterol and the highly flexible chains of DHA, promoting the lateral segregation of membranes into PUFA-rich/cholesterol-poor and PUFA-poor/cholesterol-rich regions. The latter may ultimately become the membrane microdomains known as rafts. PUFA-rich/cholesterol-poor membrane microdomains are technically less easy to study than rafts, but they may also contain particular proteins and have important biological functions. It has been proposed that changes in the conformation of signalling proteins when they move between these very different domains may have the potential to modulate cell function in a manner that may explain some of the health benefits of dietary consumption of DHA.

4. The (n-9) and Other Families of Polyunsaturated Fatty Acids

Oleate can be chain elongated and desaturated in animal tissues with 5,8,11-eicosatrienoic acid (20:3(n-9) or 'Mead's acid') as the most important product, but this only accumulates in tissues when the animals are suffering from essential fatty acid deficiency (see below). In this condition, 18:2(n‑9), 20:2(n‑9) and 22:3(n‑9) are other fatty acids of this family that may be found at low levels.

9,12-Hexadecadienoic acid (16:2(n-4)) is found in marine microorganisms and is presumably the biosynthetic precursor of other fatty acids with an n‑4 terminal structure, i.e. 18:2(n-4), 20:2(n-4), 16:3(n-4) and 18:3(n-4). Fatty acids of an n-1 family, also found in marine organisms, are believed to be derived biosynthetically by further desaturation (Δ15) of 6,9,12-hexadecatrienoic acid (16:3(n-4)). The main naturally occurring fatty acids of this type are 16:4(n-1) and 18:4(n-1), but 18:5(n-1) has also been detected. Similarly, trace amounts of polyunsaturated fatty acids of an n-7 family are occasionally encountered in marine organisms and are presumably metabolites of 9-16:1. 5,8-Octadecadienoic acid (‘sebaleic’ acid), a unique component of human skin wax, is derived by elongation and desaturation of 6-16:1.

5. Biosynthesis of Linoleic and Linolenic Acids in Plants

Linoleic and α-linolenic acids are synthesised in plant tissues from oleic acid by the introduction of double bonds between the existing double bond and the terminal methyl group by the sequential action of Δ12- and Δ15-desaturases.

Biosynthesis of alpha-linolenic acid in plants

However, this depiction of the process is over simplistic as many of the steps occur when the fatty acids are linked to glycerolipids, and they require transfer across membranes between different cellular compartments. As described in our web page dealing with saturated fatty acids most fatty acid synthesis occurs in the plastids, with palmitoyl-ACP and then stearoyl-ACP as the primary products of fatty acid synthases. The latter is desaturated to form oleoyl-ACP, which must be hydrolysed to oleic acid (see our web page on monoenes) and then converted to oleoyl-CoA for transport across the plastid envelope. 1-Acyl,2-oleoyl-phosphatidylcholine (PC) is formed by an acyl transferases, and this is the main substrate for the membrane-bound Δ12‑desaturase in the endoplasmic reticulum with formation of 1-acyl,2-linoleoyl-PC. How the diacylglycerol moiety of the latter is transferred back into the plastid in not yet known; it may be that a phospholipid transfer protein is able to transport the intact PC across the membrane or hydrolysis to form diacylglycerols (DAG) may occur for transport. The next certain product in the plastid is linoleoyl-monogalactosyldiacylglycerol (MGDG), and this is the substrate for the Δ15-desaturase with formation of α-linolenoyl-MGDG. As more plant species are investigated, further desaturases with variations in their substrates and sub-cellular distributions continue to be discovered. Those plants that produce significant amounts of 16:3(n-3) add further complications to the problem, and it is evident that much remains to be learned of the overall process.

Biosynthesis of linoleate and linolenate in leaves of plants

In fact, two distinct desaturases have been characterized that can insert the Δ12-double bond, i.e. a plastidial enzyme (FAD6), which uses the terminal methyl group as a reference point and is an ω6-desaturase as it introduces the double bond six carbons from the terminal carbon, and secondly an extra-plastidial oleate Δ12-desaturase (FAD2) that is selective for C12,13-desaturation independently of chain length. The latter is related closely to an enzyme in the seeds of castor oil (Ricinus communis) that converts oleate to (R)-12-hydroxystearate. Indeed, whether the product is a hydroxyl group or a double bond may depend on the nature of only four amino acid residues. Less is known of the desaturase (FAD3) that converts linoleate to α-linolenate, but it is argued that it should be considered as an ω3- rather than as a Δ15-enzyme. It also has much in common with hydroxylase enzymes. Lipid-bound fatty acids synthesised in this way in plastids are believed to be hydrolysed to the free fatty acids for transport out of this organelle to the endoplasmic reticulum where they are rapidly esterified for incorporation into other membranes.

Infrequently in plants, a double bond is inserted between an existing double bond and the carboxyl group as in the biosynthesis of γ-linolenic acid in evening primrose and borage seed oils. In this instance, the double bond in position 6 is inserted after those in positions 9 and 12. Plant Δ6‑desaturases contain the required donor of reduced equivalents, cytochrome b5, physically fused to the N-terminus; it is sometimes termed a 'front-end' desaturase.

Biosynthesis of gamma-linolenic acid in plants

Algae are capable of synthesising C20 and C22 polyunsaturated fatty acids of both the n-6 and n-3 families. Obviously, they contain a much wider range of desaturases and elongases than are present in higher plants, and it is apparent that these are distributed between plastids and extra-plastid cellular compartments with involvement of various lipid substrates. Both eukaryotic-like (C20/C20, C18/C18) and prokaryotic-like (C18/C16, C20/C16) species of complex lipids are formed (see our web page on plant galactolipids). Longer-chain polyunsaturated fatty acids can also be synthesised by the fungus, Mortierella alpina and some mosses.

6. Biosynthesis of the n-6 Family of Polyunsaturated Fatty Acids in Animals

In the tissues of higher animals, additional double bonds can only be inserted between an existing double bond and the carboxyl group. The linoleic acid, which is the primary precursor molecule for the n-6 family of fatty acids, must come from the diet. Biosynthesis of polyunsaturated fatty acids requires a sequence of desaturation and chain elongation steps, as illustrated below, and the various enzymes require the acyl-coenzyme A esters as substrates not intact lipids (unlike plants), with the liver as the main organ involved in the process.

Biosynthesis of the n-6 family of polyunsaturated fatty acids

The first step is believed to be rate limiting and involves desaturation with the introduction of a double bond in position 6 by the desaturase FADS2 to form γ-linolenic acid. Chain elongation by a two-carbon unit gives 20:3(n-6), which is converted to arachidonic acid by a Δ5-desaturase (FADS1). This is the main end product of the process. However, two further chain-elongation steps yield first 22:4(n-6) and then 24:4(n-6), which can be further desaturated by a Δ6‑desaturase to 24:5(n-6). At least three elongases, designated ELOVL2, 4, and 5, have been characterized of which ELOVL4 is especially important in the retina and ELOVL5 in liver. ELOVL4 is responsible for the formation of very-long-chain polyunsaturated fatty acids (up to C38) in brain, retina and testes (see our web pages on saturated fatty acids for a more detailed discussion of elongases). All the enzymes to this stage are located in the endoplasmic reticulum of the cell, but the last fatty acid must be transferred to the peroxisomes for retro-conversion (β-oxidation) to 22:5(n-6). However, a recent study reports direct desaturation of 22:4(n-6) to 22:5(n-6) by a desaturase produced by the FADS2 gene in human cells in vitro. The relative importance of the two pathways is not known.

The marine parasitic protozoon Perkinus marinus (and at least three other unrelated unicellular organisms) synthesises arachidonic acid by an alternative pathway in which elongation of linoleic to 11,14-eicosadienoic acid is followed by sequential desaturation by Δ8- and Δ5-desaturases. These enzymes are now known to be present in mammals, although the extent of their participation in synthesis of polyunsaturated fatty acids in vivo is uncertain. Linoleate and its longer-chain metabolites can in fact be synthesised by some primitive invertebrates, including many species of insects, nematodes (Caenorhabditis elegans) and pulmonates (air-breathing slugs and snails).

Desaturases: The Δ5- and Δ6-desaturases, FADS1 and FADS2, respectively, are membrane bound enzymes with cytochrome b5-like domain physically fused to the N‑terminus and with three histidine-enriched boxes, characteristic of membrane desaturases, and two membrane spanning regions. They use molecular oxygen and an electron transport system as described in our web page dealing with the biosynthesis of monoenoic fatty acids. FADS2 consists of 444 amino acids and has a molecular weight of 52.2 kDa, and it is expressed in the brain, liver, lungs, heart, and other tissues in humans. It is the rate-limiting enzyme in the biosynthesis of polyunsaturated fatty acids, and it is also responsible for the biosynthesis of sapienic acid (6-16:1) in skin. Mice modified genetically to lack this enzyme can produce 5-cis,11-cis,14-cis-eicosatrienoic (sapienic) acid instead of arachidonic acid.

While FADS2 is vital for the healthy functioning of human metabolism, it is expressed abnormally in many different malignant cancers, and this expression is significantly correlated with tumor proliferation, cell migration and invasion, together with a poor prognosis for the progression of the disease. Membrane fluidity and the transmission of signals is affected, and the production of proinflammatory factors such as eicosanoids is promoted to the detriment of human health.

7. Biosynthesis of the n-3 family of Polyunsaturated Fatty Acids in Animals

Again, the α-linolenic acid, which is the primary precursor molecule for the n-3 family of fatty acids in tissues of higher animals, must come from the diet. The main pathway to the formation of docosahexaenoic acid (22:6(n-3)) requires a sequence of chain elongation and desaturation steps (Δ5 and Δ6 desaturases), as illustrated below, with acyl-coenzyme A esters as substrates. Thus, α-linolenic acid is sequentially desaturated and elongated, with double bonds being inserted between existing double bonds and the carboxyl group, as far as 24:6(n-3), starting again with a Δ6‑desaturase as illustrated.

Biosynthesis of the n-3 family of polyunsaturated fatty acids

The final steps of what has been termed the ‘Sprecher’ pathway (after Prof. Howard Sprecher of Ohio State University) involve retro-conversion of 24:6(n‑3), i.e. removal of the first two carbon atoms by a process of β-oxidation, and take place in the peroxisomes of the cell (as in the case of the n‑6 family of fatty acids) to generate 22:6(n-3). However, as with the n-6 family, a recent study reports direct desaturation of 22:5(n-3) to 22:6(n-3) by a Δ4‑desaturase produced by the FAD2 gene in human cells in vitro, with evidence suggesting that the process may occur in mitochondria. This enzyme can also act as a Δ6- or Δ8‑desaturase depending on the substrate. Again, the relative importance of the two pathways for the synthesis of 22:6(n-3) in rodents and humans has still to be determined. The reverse reaction, i.e. synthesis of 24:6(n-3) from 22:6(n-3), does occur in rats, as does retroconversion of 22:6(n-3) to 20:5(n-3).

A single gene in the rabbitfish codes for an enzyme that performs both Δ5 and Δ4 desaturation to synthesize 22:6(n-3) directly. Similarly, Δ4-, Δ5- and Δ8‑desaturases have been found in certain micro-algae of marine origin (e.g. Pavlova salina), suggesting that a more direct route to DHA may exist in these organisms, including desaturation of 22:5(n-3). Thraustochytrium, a unicellular marine protist, used as a commercial source of DHA, has both a Δ4‑desaturation-dependent pathway (aerobic) and a polyketide synthase-like pathway (anaerobic - see below). On the other hand, marine invertebrates such as molluscs and cephalopods appear to lack some of the key desaturases and elongases and produce polyunsaturated fatty acids of both the (n-3) and (n-6) families by unconventional routes.

All the various intermediates may be found in tissues, especially those of fish, but eicosapentaenoic (20:5(n-3)), docosapentaenoic (22:5(n-3)) and docosahexaenoic (22:6(n-3)) acids tend to be by far the most abundant. In human tissues, the measured rates of conversion of α-linolenic acid to longer-chain metabolites are very low, suggesting that a high proportion of the latter must come from the diet (meat, eggs and fish) in normal circumstances. The rate of DHA synthesis in particular is so low from this precursor that it has been argued that dietary supplementation is essential to maintain sufficient levels in brain and retina, although vegans do not appear to suffer any deficiency symptoms, and there is a school of thought that DHA is utilized rapidly for other purposes and that the actual rate of synthesis is higher than has been reported. In addition, much of the α-linolenic acid in the diet is directed to long-term storage in adipose tissue, and may be made available only slowly for DHA synthesis, thus leading to misleading experimental data. During the natural processes of turnover and renewal of cell membranes in retinal cells, mechanisms exist to ensure that DHA is conserved.

Efficient use of DHA in brain and reproductive tissues for synthesis of phospholipids to support membrane structures requires a specific acylcoA synthetase (ACLS6). In addition, DHA crosses the blood-brain barrier much more effectively esterified to lysophosphatidylcholine than in the form of the free fatty acid.

In mice, the fatty acid elongase ELOVL2 has been shown to be important for the synthesis of endogenous DHA in the liver by elongating EPA to the C22 and C24 metabolites, and it may be a factor in the control of lipogenesis de novo and many other aspects of lipid metabolism. Much of the DHA synthesised in the liver is esterified initially to phosphatidylethanolamine, but then it is exported via the plasma triacylglycerols to the brain. Similarly, ELOVL2 activity is indispensable for photoreceptor function in the retina. As with the n-6 family, the fatty acid elongase designated ELOVL4 is responsible for the biosynthesis of the very-long-chain polyunsaturated fatty acids (up to C38) of the n-3 family found in the retina, brain, testis and skin.

In contrast to higher plants and mammals, the nematode C. elegans and many related species possess all of the enzymes required for the synthesis of 20:4(n-6) and 20:5(n-3) fatty acids de novo. In this instance, an unusual bifunctional Δ12/Δ15 desaturase is utilized in the synthesis of the linoleate/linolenate precursors. Many primitive marine invertebrates have an omega-3 desaturase also, and so they are able to synthesise omega‑3 polyunsaturated fatty acids from omega-6 precursors, including arachidonic acid, and in particular the marine copepod Tigriopus californicus has been demonstrated to possess all the desaturases (including Δ4) and elongases necessary for the biosynthesis of docosahexaenoic acid de novo.

8. Biosynthesis of Polyunsaturated Fatty Acids by a Polyketide Route

With acetyl-CoA as the primary precursor, the synthesis of 22:6(n-3) by the route described above involves approximately 30 distinct enzymes and 70 reactions. However, a very different and much simpler pathway has been found in marine bacteria, especially Shewanella species. The conventional view of polyketides is of secondary metabolites consisting of multiple building blocks of ketide groups (–CH2–CO–), which are synthesised by a polyketide synthase and often lead to polar cyclic structures. The enzymes involved in the synthesis of polyunsaturated fatty acids in these organisms have a modular organization similar to that of fatty acid synthases with which they have some structural homology, and for example, in marine bacteria such as Moritella marina, a polyketide synthase-like mega-enzyme contains at least four proteins designated PfaA, PfaB, PfaC and PfaD. Like the fatty acid synthase in bacteria, this enzyme system uses acyl carrier protein as a covalent attachment for chain synthesis and proceeds in iterative cycles adding C2 units and double bonds, but in contrast to the elongation-desaturation pathway, the double bonds are introduced during the process of fatty acid synthesis. Thus, aerobic desaturation is not required for introducing double bonds into the existing acyl chain, and this is sometimes termed an ‘anaerobic’ pathway, although a similar pathway has been found in aerobic organisms such as the micro alga Schizochytrium sp. Much remains to be learned of this process in relation to EPA and DHA synthesis, but it is evident that it proceeds via different intermediates from the well-established aerobic pathway. Which pathway is involved can be suggested from analysis of the fatty acid composition as illustrated.

Intermediates in the polyketide pathway to DHA

It is believed that as the chain elongates ketones groups formed initially are reduced to hydroxyls, and this is followed by dehydration reactions to introduce the double bonds. For this purpose, six active centres are required: 3-ketoacyl synthase, 3-ketoacyl-ACP-reductase, dehydrase, enoyl reductase, dehydratase/2-trans 3-cis isomerase, dehydratase/2-trans, and 2-cis isomerase. These take part in a sequence of reactions involving chain elongation, trans-double bond formation and isomerization to the cis-conformation for the production of fatty acids of both the omega-3 and omega-6 families, ultimately of (DHA or 22:6(n-3)) and docosapentaenoic (DPA or 22:5(n-6)) acids as shown. In marine Gammaproteobacteria, the polyketide/fatty acid synthase mechanism is encoded by a set of five genes, which are regulated by a novel transcriptional regulator.

Polyketide pathways for biosynthesis of omega-3 and omega-6 fatty acids

Similar reactions occur is some terrestrial bacteria such as the myxobacterial genus Aetherobacter, but the biosynthetic pathways differ somewhat from those in marine organisms in terms of gene organization and the structures of the enzyme components of the PUFA synthases. In contrast to the aerobic method of fatty acid synthesis, polyketide biosynthesis pathways can modify the intermediates in the growing polyketone chain by re-arranging the order and combinations of the various enzymes to produce many different final products including antibiotics, toxins and pigments.

9. Catabolism

Polyunsaturated fatty acids of all families are broken down in animal tissues to produce energy by a multi-step process of β-oxidation. This is discussed in our web page on carnitines. In addition, some very-long-chain fatty acids are oxidized in peroxisomes or 'microbodies, especially in the kidney and liver; the products are medium-chain fatty acids, which are transported to mitochondria for further oxidation. In plants, glyoxysomes in germinating seeds can break down fatty acids rapidly to acetyl-CoA, while β-oxidation occurs in leaves mainly in peroxisomes but also in mitochondria. All polyunsaturated fatty acids with methylene-interrupted double bonds are susceptible to autoxidation through the action of Reactive Oxygen Species (ROS) in tissues, the higher the degree of unsaturation the greater the reactivity. However, for convenience, this process is discussed in the web page on isoprostanes (with links to other relevant pages). While autoxidation is of great importance in non-biological conditions, for example during cooking or storage of foods, these web pages are devoted to metabolism in living tissues. Polyunsaturated fatty acids can also be converted enzymatically to various oxygenated derivatives by many different oxidases, for example to the eicosanoids and related metabolites or oxylipins (see the Introduction to this topic).

10. Essential Fatty Acids

Most fatty acids have important or even vital properties that are not easily replaced. For example, many different fatty acids in the unesterified (free) state, but especially the polyunsaturated components, interact with multiple G protein-coupled receptors for free fatty acids (FFAR) on cell surfaces and have important roles in the regulation of nutrition (see our web page on free fatty acids). Other key functions of specific fatty acids, both saturated and unsaturated, require an esterified state.

However, as discussed briefly above, linoleic and α‑linolenic acids cannot be synthesised in the tissues of higher animals and must be obtained from the diet, i.e. mainly from plants via the food chain (some invertebrates are an exception as discussed above). On a global scale, the longer-chain polyunsaturated fatty acids are produced primarily from photosynthetic marine microalgae, heterotrophic protists and bacteria, before entering the food chain of fish and thence of other animals. Of course, linoleic and α‑linolenic acids serve as precursors of the longer-chain metabolites in animals also, but the biosynthetic rates are low. There is an absolute requirement for these 'essential fatty acids' for growth, reproduction and good health. Young animals deprived of them in the diet rapidly display adverse effects, including diminished growth, liver and kidney damage, and dermatitis, and these can result eventually in death. One key biochemical parameter is the 'triene-tetraene' ratio, i.e. the ratio of 20:3(n‑9) to 20:4(n‑6) fatty acids in plasma; levels greater than 0.4 reflect essential fatty acid deficiency. While it takes longer for the effects to become apparent in older animals, which may have substantial stores of essential fatty acids in their body fats, symptoms do appear eventually, and the effects of essential fatty acid deficiency have been seen in human infants and on adults on parenteral nutrition or with certain genetic disorders.

The absolute requirements for essential fatty acids are dependent on a number of factors, including species and sex (females appear to have a higher requirement for n-3 fatty acids), but they are usually considered to be a minimum of 1 to 2% for linoleate, and somewhat less for α‑linolenate. In contrast, the requirement for α‑linolenate in fish is higher than for linoleate. For some years it was believed that cats lacked a Δ6‑desaturase and had an absolute requirement for arachidonic acid especially in their diet, i.e. they were obligate carnivores, but this now known not to be true although the enzyme activity can be low.

Scottish thistleIt has sometimes been argued that linoleate and α‑linolenate per se may in fact be less important than the longer-chain polyunsaturated fatty acids in animal biology, but there is an absolute requirement for linoleate for the proper functioning of ceramides in skin, while octadecanoid oxylipins derived from linoleate have important functions in skin, adipose tissue and other organs. On the other hand, the consensus appears to be that the essentiality of α‑linolenic acid may reside in the polyunsaturated fatty acids formed from it.

It is evident that arachidonic, eicosapentaenoic, docosahexaenoic acids and other polyunsaturated fatty acids of the n-6 and n-3 families each have distinct functions, some of which are discussed briefly above, which make them essential for healthy animal metabolism. They are precursors of eicosanoids and other oxylipins, including prostaglandins (PG1, PG2 and PG3 series), thromboxanes, leukotrienes, and lipoxins, and of docosanoids, including resolvins, protectins and maresins (specialized pro-resolving mediators), which have a variety of vital biological properties. In addition, n-3 polyunsaturated fatty acids per se may exert beneficial effects by regulatory actions in signalling processes, especially in T-cells, for example by modulating the activities of membrane receptors or by influencing gene transcription. In addition, arachidonic acid is an essential component of the endocannabinoids.

Polyunsaturated fatty acids confer distinctive attributes on the complex lipids that may be required for their function in membranes, where their highly flexible nature affects membrane biophysical properties such as fluidity, flexibility and thickness, and this may in turn influence innumerable biological events. On the other hand, there are suggestions that excessive amounts of polyunsaturated fatty acids in membranes and tissues, including those of the n-3 family, have the potential to cause harm, because the propensity of all such fatty acids for oxidation can lead to potentially toxic levels of hydroperoxides in tissues. Such effects are discussed in relation to specific lipids in other web pages on this website, and for example see that on oxidized phospholipids.

Although the actual requirement for polyunsaturated fatty acids is relatively low, general nutritional advice for the human diet until recently was that they should comprise a substantial part of the daily intake. Over the last 30 years, there has been a large increase in the consumption of linoleic acid because of an increased use of vegetable oils rich in this fatty acid in the diet. By comparison, the intake of n-3 polyunsaturated fatty acids has been reduced because of a relative decrease in the consumption of fish and vegetables. The consequence is that the ratio of n‑6 to n‑3 fatty acids in the diet is of the order of 20:1, whereas it was probably closer to 2:1 in historical times in western countries. It is now generally believed that the relative proportion of n‑3 to n‑6 polyunsaturated fatty acids in the diet should be increased, if not the total amounts. There appears to be evidence that dietary fish oils are beneficial to brain development in young children, but not as might be anticipated in the management of neurodegenerative diseases in the elderly.

An expert nutritional panel has recommended that infant formulae should contain both arachidonic and docosahexaenoic acids at levels of at least 0.5 to 0.64% of the total calories each, although the optimum concentrations are not known. Indeed, it is not sufficient to consider simply the minimum levels required, as these fatty acids are necessary for innumerable aspects of the healthy development of the neonate and infant; suboptimal levels can impact the development of the immune system and increase the risk of allergic diseases and respiratory illness. The Mediterranean diet, which is now often recommended by nutritionists, contains high proportions of oleic acid (18:1(n-9)) to balance out the polyunsaturates. However, detailed discussion of such contentious topics is best left to nutritional experts of whom I am not one.

Recommended Reading

I can also recommend the Chapter on fatty acid biosynthesis in the book - Gurr, M.I., Harwood, J.L., Frayn, K.N., Murphy, D.J. and Michell, R.H. Lipids: Biochemistry, Biotechnology and Health (6th Edition). (Wiley-Blackwell) (2016).

Lipid listings Credits/disclaimer Updated: September 29th, 2021 Author: William W. Christie LipidWeb icon