Methylene-Interrupted Double Bonds
1. Structure and Nomenclature
The 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’ is occasionally used to describe this molecular feature.
In higher plants, the number of double bonds in fatty acids only rarely exceeds three, but in algae and animals there can be up to six. 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.
In the shorthand nomenclature, these 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. The position of the terminal double bond can be denoted 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, assuming 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).
Both of the parent fatty acids can be synthesised in plants, but not in animal tissues, and they are therefore essential dietary components (see below). Polyunsaturated fatty acids 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.
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 animal tissues 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.
The 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. The intermediates 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). However, it does not accumulate to a significant extent in animal tissue lipids, and is typically about 1-2% of the phospholipid fatty acids.
Arachidonic 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.
Meat is the main dietary source in humans. While arachidonate is present in all fish oils, polyunsaturated fatty acids of the (n-3) families tend to be present in much larger amounts. Arachidonic acid is frequently found as a constituent of mosses, liverworts 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. 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 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). The last of these, 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, and even longer homologues occur in retina. Those in testes are known to be essential for male fertility and sperm maturation. Very-long-chain 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 brain of normal young humans, with 34:4(n-6) and 34:5(n-6) tending to predominate. The function of these is not known.
The most highly unsaturated fatty acids 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. α-Linolenic acid is the biosynthetic precursor of jasmonates in plants, which appear to have functions that parallel those of the eicosanoids in animals.
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.
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 are essential fatty acids.
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. Somewhat higher concentrations may be found in fish oils.
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 mosses, but rarely in higher plants.
5,8,11,14,17-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 an important constituent of the phospholipids in animal tissues, especially in brain, and it is the precursor of the PG3 series of prostaglandins and resolvins, which have anti-inflammatory effects (see the appropriate web page for further discussion). There is currently great interest in the role of this acid in 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%. A lack of availability of the pure fatty acid has meant that it has been relatively little studied, but it is known that it can be retro-converted to EPA although desaturation to DHA does not appear to occur. It reacts with lipoxygenases to form distinctive metabolites.
4,7,10,13,16,19-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. Dietary supplements may also benefit the elderly. DHA is not present in higher plants, but it is found in high concentrations in many species of algae, especially those of marine origin. Indeed, it has been argued that micro-algae in aquatic systems are the major source of DHA and EPA in fish, and via the food chain contribute substantially to the levels of these fatty acids in terrestrial systems including humans.
DHA 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, termed 'resolvins' or 'protectins', which are analogous to the eicosanoids but have potent anti-inflammatory and immuno-regulatory actions.
The concentration of DHA in tissues has been correlated with a number of human disease states, and it is essential to many neurological functions. Particular attention has been given to its role in the retina where it is a major structural component of the photoreceptor outer segment membranes. For example, it binds strongly to specific sites on rhodopsin, the primary light receptor in the eye, modifying its stability and activity. It affects signalling mechanisms involved in photo-transduction, enhancing activation of membrane-bound retinal proteins, and it may be involved in rhodopsin regeneration. In some cases, sight defects have been ameliorated with DHA supplementation. It is intimately involved with phosphatidylserine metabolism in neuronal tissue.
DHA is believed to have specific effects on gene transcription that regulate a number of proteins involved in fatty acid synthesis and desaturation, for example. 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.
As a phospholipid constituent, it has profound effects on the properties of 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 8.2 Å at 41°C compared to 14.2 Å for oleic chains. This is the result of 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 distinctive 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.
As with the (n-6) family, very-long-chain fatty acids (C24 to C38) of the (n-3) families occur in the retina, brain and sperm. They are derived biosynthetically by elongation of the C20 and C22 polyunsaturated precursors. 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. Heptaenoic fatty acids of the (n-3) family (38:7(n-3) and 40:7(n-3)) have been reported from brains of patients with a defined genetic defect, but the most highly unsaturated fatty acid of the (n-3) family yet found is 4,7,10,13,16,19,22,25-octacosaoctaenoate (28:8(n-3)) from marine dinoflagellates.
4. The (n-9) Family 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. This only accumulates in tissues when the animals are suffering from essential fatty acid deficiency (see below). Other fatty acids of this family that may also be found at low levels include 18:2(n-9), 20:2(n-9) and 22:3(n-9).
5. Other Families of Polyunsaturated Fatty Acids
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. 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.
6. Biosynthesis of Linoleic and Linolenic Acids
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.
The main substrate for the Δ12 desaturase is 1-acyl,2-oleoyl-phosphatidylcholine in the endoplasmic reticulum of the cell (although other lipids may also be substrates in chloroplasts). The newly formed linoleate is then transferred by a variety of mechanisms to other lipids. Phosphatidylcholine can also be the substrate for further desaturation, but in leaf tissue in a number of plant species it appears that most α-linolenate is formed by desaturation of linoleic acid linked to monogalactosyldiacylglycerols. 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.
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 oxidation 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 know 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.
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. These processes are discussed in much more detail in a web page in the plant biochemistry section of this site.
7. Biosynthesis of the (n-6) Family of Polyunsaturated Fatty Acids
In animal tissues, 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 chain elongation and desaturation steps, as illustrated below, and the various enzymes require the acyl-coenzyme A esters as substrates, not intact lipids (unlike plants). The liver is the main organ involved in the process.
The first step is believed to be rate limiting and involves desaturation with the introduction of a double bond in position 6 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. 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. 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).
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.
8. Biosynthesis of the (n-3) Family of Polyunsaturated Fatty Acids
Again, the α-linolenic acid, which is the primary precursor molecule for the (n-3) family of fatty acids in animal tissues, 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 elongated and desaturated, with double bonds being inserted between existing double bonds and the carboxyl group, as far as 24:6(n-3). The final steps of what has been termed the ‘Sprecher’ pathway involve retro-conversion, 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).
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 rates of conversion of α-linoleic acid to longer-chain metabolites is 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 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. During the natural processes of turnover and renewal of cell membranes in retinal cells, mechanisms exist to ensure that DHA is conserved.
The fatty acid elongase, designated ELOVL4, is responsible for the biosynthesis of the very-long-chain polyunsaturated fatty acids of the (n-3) family found in the retina.
Δ4, Δ5 and Δ8 desaturase has been found in certain micro-algae of marine origin (e.g. Pavlova salina), suggesting that a more direct route to DHA may exist in this instance, including desaturation of 22:5(n-3). It has also been shown that the mammalian Δ6 desaturase can operate as a Δ8 desaturase with 20:3(n-3) as substrate.
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, an entirely different and much simpler pathway catalysed by a polyketide synthase has been found in marine microbes. 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. This is an enzyme system similar to the fatty acid synthase in bacteria in that it uses acyl carrier protein as a covalent attachment for chain synthesis and proceeds in iterative cycles. However, the double bonds are introduced during the process of fatty acid synthesis in contrast to the elongation-desaturation pathway. Much remains to be learned of this process in relation to DHA synthesis, but it is believed that as the chain elongates the ketone groups are reduced to hydroxyls, and this is followed by dehydration reactions to introduce the double bonds. Thus, aerobic desaturation is not required for introducing double bonds into the existing acyl chain, and it is sometimes termed an ‘anaerobic’ pathway, although it can occur under aerobic conditions.
In contrast to higher plants and mammals, the nematode Caenorhabditis elegans possesses all of the enzymes required for the synthesis of 20:4(n-6) and 20:5(n-3) fatty acids de novo, feats that can also be accomplished by the fungus Mortierella alpina and some mosses and red algae.
9. Essential Fatty Acids
As discussed briefly above, linoleic and linolenic acids cannot be synthesised in animal tissues and must be obtained from the diet, i.e. ultimately from plants. There is an absolute requirement for these 'essential fatty acids' for growth, reproduction and good health. Young animals deprived of these fatty acids in the diet rapidly display adverse effects, including diminished growth, liver and kidney damage, and dermatitis; these eventually result in death. A 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. 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, but symptoms will appear eventually. The effects of essential fatty acid deficiency have been seen in human infants, on adults on parenteral nutrition or with certain genetic disorders. The absolute requirements are dependent on a number of factors, including species and sex (females appear to have a higher requirement for (n-3) fatty acids), but are usually considered to be a minimum of 1 to 2% for linoleate, and somewhat less for 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 appears not to be the case.
Linoleate and linolenate per se may in fact be less important than their longer-chain metabolites in animal biology. It is evident that arachidonic, eicosapentaenoic and docosahexaenoic acids each have distinct functions, some of which are discussed briefly above, which make them essential for healthy animal metabolism. They are precursors of eicosanoids, including prostaglandins (PG1, PG2 and PG3 series), thromboxanes, leukotrienes, and lipoxins, and docosanoids, including resolvins and protectins, which have a variety of important 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. Polyunsaturated fatty acids confer distinctive attributes on the complex lipids that may be required for their function in membranes, and in esterified form they can also have important biological properties. For example, arachidonic acid is an essential component of the endocannabinoids. On the other hand, there are suggestions that excessive amounts of polyunsaturated fatty acids in tissues, including those of the n-3 family, have the potential to cause harm.
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. A consensus is emerging that the proportion of n-3 polyunsaturated fatty acids in the diet should be increased. On the other hand, it is recognized that the propensity of all such fatty acids for oxidation can lead to potentially harmful levels of hydroperoxides in tissues, so higher relative proportions of dietary oleate are now often advised. However, detailed discussion of such a contentious topic is not possible here.
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Updated: December 5, 2013