The Lipid Library

FATTY ACIDS: BRANCHED-CHAIN

STRUCTURE, OCCURRENCE AND BIOSYNTHESIS

Branched-chain fatty acids are common constituents of the lipids of bacteria and animals, although they are rarely found in the integral lipids of higher plants. Normally, the fatty acyl chain is saturated and the branch is a methyl-group. However, unsaturated branched-chain fatty acids are found in marine animals, and branches other than methyl may be present in microbial lipids. The most common branched chain fatty acids are mono-methyl-branched, but di- and poly-methyl-branched fatty acids are also known. Their main function in membranes may be to increase the fluidity of lipids as an alternative to double bonds, which are more liable to oxidation. The following discussion is not intended to be comprehensive.

Saturated iso- and anteiso-Methyl-Branched Fatty Acids

iso-Methyl branched fatty acids have the branch point on the penultimate carbon (one from the end), while anteiso-methyl-branched fatty acids have the branch point on the ante-penultimate carbon atom (two from the end) as illustrated. The common range of fatty acids of this kind with a single branch point only in a saturated chain are discussed in this section.

Fatty acids with structures of this type and with 10 to more than 30 carbons in the acyl chain are found in nature, but those most often encountered have 14 to 18 carbons. They are common constituents of bacteria but are rarely found in other microorganisms. Via the food chain, they can be found in animal tissues, especially those of marine animals and ruminants. However, they can also be synthesised in animal tissues per se. In bacteria, their content and composition can often be used as taxonomic markers, and in bacilli, for example, some species have fatty acids with the iso-structure only, while others have the anteiso-structure.

These fatty acids are produced biosynthetically via the conventional mechanisms for the synthesis of saturated fatty acids in bacteria (see the appropriate web pages), except that the nature of the primer molecule differs.

Thus instead of acetyl-coA, 2-methylpropanyl-CoA (derived from the amino acid valine) is the primer for the biosynthesis of iso-branched fatty acids, while 2-methylbutanyl-CoA (derived from leucine) is the primer for anteiso-fatty acids. One consequence of this is that iso-methyl fatty acids tend to have an even number of carbons in total, although the chain-length is odd-numbered, sometimes leading to confusion in the informal nomenclatures that may be used in scientific publications. In contrast, anteiso-methyl fatty acids tend to have an odd number of carbons in total, while the chain-length is even-numbered. However, because of alpha-oxidation processes, odd-numbered iso-methyl acids and even-numbered anteiso-methyl acids are also found in tissues.

In animal tissues, the biosynthesis of these fatty acids de novo is normally a very minor process and is believed to involve the same mechanism as above. However, it can occur at a significant rate in some instances. For example, lanolin, the waxy material produced as a protective coating for the fleece of sheep, contains a high proportion of iso and anteiso fatty acids from C₁₀ to C₃₄ in chain-length (see the web page on waxes). One anteiso-branched fatty acid, 18-methyl-eicosanoic acid, constitutes up 60% of the total fatty acids esterified directly to wool via thiol ester bonds, and it comprises 40% or more of the same lipid in all mammalian hairs examined to date. Triacylglycerols containing isovaleric (3-methylbutyric) acid are important constituents of the blubber and melon oils of the Beluga whale, and an alkyldiacylglycerol containing this acid occurs in rabbit harderian gland. There is recent evidence that iso-methyl fatty acids are essential for the development of the free-living nematode Caenorhabditis elegans. Branched-chain acids have also been implicated in some human disease states.

In most mammalian tissues, branched-chain fatty acids of this type rarely make up more that 1-2% of the total, and are probably derived mainly from bacteria in the intestines or from consumption of such fatty acids in dairy products or meat from ruminant animals. Similarly fish oils usually contain 1-2% iso- and anteiso-fatty acids of chain-length C₁₄ to C₁₈, which are presumed to be derived from the marine food chain.

In higher plants, 14-methylhexadecanoic occurs at a level of 0.5 to 1% in seed oils from the family Pinaceae, where it appears to be a useful taxonomic marker. iso-/anteiso-Methyl-branched fatty acids are major components of plant surface waxes, however (see the appropriate web pages).

Neo fatty acids, which can be considered as having a terminal tertiary butyl group or with two iso-methyl groups, have been found in certain microorganisms, algae, plants and marine invertebrates. For example, 13,13-dimethyltetradecanoic acid or ‘neopalmitic acid’ illustrated is a minor component of bark and resins from conifers and other plants, and it has been found in the shell, chitin and chitosan of a species of crab.

Saturated Mid-Chain Methyl-Branched Fatty Acids

10-R-Methyloctadecanoic acid or tuberculostearic acid is a major component of the lipids of the tubercle bacillus and related bacterial species. Indeed its presence in bacterial cultures and sputum from patients is used in the diagnosis of tuberculosis. It is also found in Corynebacterium and many other species.

A number of fatty acids with a single methyl branch of this type have been isolated from specific bacteria. For example, 10-methylhexadecanoic and 11-methyloctadecanoic acids are relatively common. 12-Methylhexadecanoic acid and 14-methyloctadecanoic acid are major components of the halotolerant bacterium Rubrobacter radiotolerans. The latter occurs in the aquatic bacterium Rhodococcus equi also. 6- and 9-Methyltetradecanoic acids are found in lichenized fungi. Mycobacterium phlei contains a range of methyl-branched fatty acids, including 8- and 10-methylhexadecanoate, 9-methylheptadecanoate, 11-methylnonadecanoate, 12-methyleicosanoate, 14-methyldocosanoate and16-methyltetracosanoate. Very many different branched chain fatty acids of this type may be found in mixed microbial populations such as those isolated from soil or other environmental samples. Similarly sponges and some other marine organisms contain methyl-branched fatty acids derived from microorganisms in their diet or that live in symbiosis with them. For example, in addition to a number of iso- and anteiso-methyl-branched fatty acids, 10-methyl-16:0, 11-methyl-18:0, 14-methyl-20:0, 18-methyl-24:0 and 20-methyl-26:0 were found in the lipids of the sponge Verongia aerophoba.

Biosynthesis of branched chain fatty acids of this type involves methylation of oleic acid esterified as a component of a phospholipid, with S-adenosylmethionine as the methyl donor. The resulting 10-methyleneoctadecanoyl residue is reduced to the 10-methyl compound with NADPH as the cofactor. A related mechanism is in used for biosynthesis of cyclopropane fatty acids in bacteria. The intermediate 10-methyleneoctadecanoic acid has been isolated from a Corynebacterium.

A wide range of mono-methyl branched fatty acids occur in a few disparate mammalian tissues. Perhaps the best known of these is the uropygial (preen) gland of birds that secretes a waxy material that serves to waterproof the feathers. The precise composition of this varies from species to species, but all are characterized by high concentrations of branched-chain fatty acids (and alcohols). Usually the branch is a methyl group, but ethyl and propyl branches are also known. The positions of these and the chain-lengths of the various components cover a wide range, but for the monomethyl fatty acids, the branch-points are most often in positions 2 to 6. Di-, tri- and tetramethyl-branched fatty acids are also present. A common pattern is to find series such as 2,4-, 2,6-, 2,8- and 4,6-dimethyl, and so forth, with 2,4,6-, 2,4,8- and 2,6,8-trimethyl, and 2,4,6,8-tetramethyl fatty acids. In some species these can comprise 90% of the total fatty acids. However, the preen gland of the barn owl contains 3-methyl- and 3,5-, 3,7-, 3,9-, 3,11-, 3,13-, and 3,15-dimethyl-branched fatty acids As an example, the composition of the fatty acids in the uropygial gland of the fulmar is listed in Table 1. Much remains to be learned of the mechanism of biosynthesis of these fatty acids, but it appears that a high proportion at least is produced by a conventional type of fatty acid synthase that utilizes methylmalonyl-CoA to insert the methyl group as opposed to malonyl-CoA per se.

Ruminant fats also contain high proportions of branched-chain components, especially when they are fed carbohydrate-rich diets, when up to 9% of the subcutaneous fat can comprise such fatty acids. Relatively high proportions of propionic acid (as opposed to acetic and butyric) are produced by the rumen microorganisms, and this is in turn converted to methylmalonyl-CoA, which is incorporated into fatty acids by the fatty acid synthase. A consequence of this mechanism is that the methyl groups are all in the even-numbered positions, and are distributed randomly in fatty acids of varying chain-lengths. In fact more than 120 different mono-, di- and tri-methyl fatty acids (and some ethyl-branched components) have been identified in ruminant fats.

A further interesting example is Vernix caseosa, the waxy skin secretion that covers newborn babies. In addition to a high proportion of iso-/anteiso-methyl-branched fatty aids, this contains approximately 10% of components from C11 to C18 in chain-length with methyl groups in the even-numbered positions from 2 to 12, which are once more presumably synthesised using methylmalonate as a substrate.

Non-isoprenoid dimethyl-branched fatty acids are frequently reported from bacteria. For example, 4,9-dimethyl-10:0, 4,10- and 4,11-dimethyl-12:0, and 4,13-dimethyl-14:0 acids, with 2,13- and 2,12-dimethyl-14:0 acids were identified in a halophilic Bacillus sp. Multi-branched fatty acids with the methyl branches in positions 2-, 4-, 6- and 8- are present in certain Mycobacteria. Dimethyl fatty acids are occasionally reported from sponges, where they are presumed to be derived from bacteria in the food chain or that are symbiotic, e.g. 9,13- and 10,13-dimethyl-14:0, 8,10-dimethyl-16:0 and 3,13-dimethyl-14:0.

Dimethyl, dibasic acids such as 14,15-dimethyltriacontanedioic acid are sometimes found in plant waxes and in bacteria.

Isoprenoid Fatty Acids

A number of isoprenoid fatty acids occur naturally in animal tissues that are derived from the metabolism of phytol (3,7,11,15-tetramethylhexadec-trans-2-en-1-ol), the aliphatic alcohol moiety of chlorophyll. These range from 2,6-dimethylheptanoic to 5,9,13,17-tetramethyloctadecanoic acids, but those encountered most often are 3,7,11,15-tetramethylhexadecanoic (phytanic) and 2,6,10,14-tetramethylpentadecanoic (pristanic) acids. 4,8,12-Trimethyltridecanoic acid is especially common in fish and other marine organisms. Phytanic acid is formed in animal tissues by oxidation of phytol to phytenic acid (only encountered in tissues under artificial feeding conditions), followed by reduction. The shorter chain isoprenoid fatty acids are formed from this by sequential α- and/or β-oxidation reactions. In natural phytanic acid, each of the methyl groups would be expected to have the D-configuration, but in that prepared via chemical hydrogenation of phytol, the 3-methyl group is racemic (D,L).

Normally, these fatty acids occur at low levels only in tissues, with the concentrations being highest in herbivores. For example, phytanic acid is found at levels of up to 1% normally in milk fat and adipose tissue from cows. However, much higher concentrations can occur on occasion. For example, up to 20% of the fatty acids in the triacylglycerols of bovine plasma can consist of this acid, because the methyl-branch in position 3 of the chain inhibits the action of the enzyme lipoprotein lipase that clears triacylglycerols from plasma.

In humans, several inborn metabolic errors with one or more deficiencies in the degradation of phytanic and pristanic acids have been described that lead to an accumulation of these acids in tissues and body fluids. There are various clinical expressions of these disorders, some of which can be fatal, the best known of which is Refsum's disease. Because of the presence of the 3-methyl group, degradation of phytanic acid by β-oxidation is impossible. Rather, phytanic acid is oxidized by α-oxidation in peroxisomes, yielding pristanic acid, which can then be subjected to β-oxidation. Defects in one or other steps in the α-oxidation system lead to the clinical symptoms.

Isoprenoid branched-chain fatty acids, such as phytanic and pristanic acids, appear to be signalling molecules that function by regulating the expression of those genes that affect the catabolism of lipids in animal tissues. They are transported to the nucleus by binding to a liver-type fatty acid binding protein, and they exert their effects by binding to the α subtype of the peroxisome proliferator-activated receptors (PPAR), which induces the transcription of enzymes involved in fatty acid degradation by β- and ω-oxidation. In a sense, they are regulators of their own degradation. Phytanic acid is also a regulator of aspects of glucose and retinoic acid metabolism. Similarly, the retinoic acids, isoprenoid acids derived from vitamin A, are potent regulators of genes involved in cell growth and differentiation, via distinct transport proteins and nuclear receptors.

Unsaturated Methyl-Branched Fatty Acids

Monounsaturated methyl branched-chain fatty acids have been detected in bacteria and marine animals. Often, usually the branch is in the iso/anteiso-positions, but it can also be more central in the aliphatic chain. For example, one of the first acids of this type to be described was 7-methyl-7-hexadecenoic acid from lipids of the ocean sunfish (Mola mola), while 7-methyl-6- and 7-methyl-8-hexadecenoic acids were later found in a sponges. Similar fatty acids with iso-/anteiso-methyl groups found in related marine organisms include 13-methyltetradec-4-enoic, 14-methylhexadec-6-enoic, 14-methylpentadec-6-enoic and 17-methyloctadec-8-enoic acids, and many others. It is possible that the primary origin of these fatty acids is in bacteria, since many comparable fatty acids have been found in bacteria, for example in Bacillus cereus and Desulfovibrio desulfuricans.

Many different demospongic acids, i.e. with bis-methylene-interrupted double bonds (usually in the 5,9-positions), have been found with iso- and anteiso-methyl branches (see our web pages on sponge fatty acids). In addition, several related fatty acids have been described with the methyl group in more central positions, e.g. 17-methyl-5,9-24:2, 21-methyl-5,9-26:2 and 22-methyl-5,9-28:2. Unusual multibranched polyunsaturated and very-long-chain fatty acids have been located in slime moulds and freshwater sponges from Israel, including (2E,4E,7S,8E10E12E14S)-7,9,13,17-tetramethyl-7,14-dihydroxy-2,4,8,10,12,16-octadecahexaenoic acid from seven different species of myxomycetes.

Branched-chain fatty acids are uncommon in plants, but small amounts of 16-methyl-cis-9-octadecenoic and 16-methyl-cis-9,cis-12-octadecadienoic acids have been found in wood and seeds of certain Gymnosperm species.

Mycolic Acids

The mycolic acids, major components of the Mycobacteria and related species, are β-hydroxy-α-alkyl branched structures of high molecular weight (60 to 88 carbons or more). Depending on species, these can contain a variety of functional groups, including double bonds of both the cis- and trans-configuration (but when the latter they also possess an adjacent methyl branch) and cyclopropane rings, which can also be of the cis- and trans-configuration. In addition, they can contain, methoxy-, epoxy- and keto groups, which are also adjacent to a methyl branch normally. Some representative structures are illustrated below.

Structural analysis of such fatty acids is much more difficult than with conventional fatty acids. The first step usually involves pyrolysis to yield an alpha-branched fatty acid and a meroaldehyde, which can be analysed separately.

These two components are also synthesised separately by different enzyme systems in the microorganisms, before they are condensed to form a typical mycolic acid.

As mentioned above, mycobacteria also contain multimethyl-branched fatty acids (C₁₄ to C₃₂), often with the methyl branches in positions 2, 4, 6 and 8, and sometimes with hydroxyl groups in position 3.

The mycolic acids are key structural components of the membranes of mycobacteria, where they appear to confer distinctive properties, including a low permeability to hydrophobic compounds, resistance to dehydration, and the capacity to survive the hostile environment of the macrophage. The β-hydroxyl group is especially important in that it is believed to modulate both the phase transition temperature and the molecular packing within the membrane. The cell envelope of Mycobacterium tuberculosis, for example, has a distinctive lipid composition that is associated with its pathogenicity in tuberculosis infections. Thus, lipid mycolates are important as structural components of the cell wall. Also, there is a thick layer of lipid on the outer part of the cell, which protects the tubercle bacillus from the host’s immune system. Mycolic acids are the major constituents of this layer as components of distinctive lipids, including an arabinogalactanmycolate covalently linked to the cell wall peptidoglycan via a phosphodiester bond, phenolphthiocerol and phthiocerol dimycocerosates, and trehalose esters that include sulfatides and di- to polyacyltrehaloses. The cyclopropane rings in mycolic acids contribute to the structural integrity of the cell wall complex and are protective against oxidative stress. Experimentally induced changes to the structures of the mycolic acids lead to loss of virulence.

Many details of the biosynthesis of mycolic acids have yet to be determined experimentally. However, it is believed that in M. tuberculosis a fatty acid synthetase I provides C₂₀-S-coenzyme A to a fatty acid synthetase II system (see our web pages on the biosynthesis of saturated fatty acids). Cis double bonds are introduced at two locations on a growing meroacid chain to yield three different forms of cis,cis-diunsaturated fatty acyl intermediates, which can then be converted to methyl, cyclopropane, methoxy- and keto-meroacids. Finally, the mature meroacids and a C₂₆-S-acyl carrier protein enter into a Claisen-type condensation with a polyketide synthase to yield the mycolic acids.

Recommended Reading

• Adida, A. and Spener, F. Intracellular lipid binding proteins and nuclear receptors involved in branched-chain fatty acid signaling. Prostaglandins, Leukotrienes Essential Fatty Acids, 67, 91-98 (2002).
• Barry,C.E., Lee,R.E., Mdluli,K., Sampson,A.E., Schroeder,B.G., Slayden,R.A. and Yuan,Y. Mycolic acids: structure, biosynthesis and physiological functions. Prog. Lipid Res., 37, 143-179 (1998).
• Body, D.R. Branched-chain fatty acids. In: 'Handbook of Chromatography. Vol. I. Lipids', pp. 241-275 (edited by H.K. Mangold, CRC Press, Boca Raton) (1984).
• Dembitsky, V.M. Natural neo acids and neo alkanes: their analogs and derivatives. Lipids, 41, 309-340 (2006).
• Jacob, J. Bird waxes. In: 'Chemistry and Biochemistry of Natural Waxes', pp. 93-146 (ed. P.E. Kolattukudy, Elsevier, Amsterdam) (1976).
• Jones, L.N. and Rivett, D.E. The role of 18-methyleicosanoic acid in the structure and formation of mammalian hair fibres. Micron, 28, 469-485 (1997).
• Kaneda, T. iso-Fatty and anteiso-fatty acids in bacteria - biosynthesis, function, and taxonomic significance. Microbiol. Rev., 55, 288-302 (1991).
• Kniazeva, M., Crawford, Q.T., Seiber, M., Wang, C.Y. and Han, M. Monomethyl branched-chain fatty acids play an essential role in Caenorhabditis elegans development. PLOS Biol., 2, 1446-1459 (2004).
• Mukherji, M., Schofield, C.J., Wierzbicki, A.S., Jansen, G.A., Wanders, R.A.J. and Lloyd, M.D. The chemical biology of branched-chain lipid metabolism. Prog. Lipid Res., 42, 359-376 (2003).
• Nicolaides, N., Apon, J.M.B. and Wong, D.H. Further studies of the saturated methyl branched fatty acids of Vernix caseosa lipid. Lipids, 11, 781-790 (1976).
• Takayama, K., Wang, C. and Besra, G.S. Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis. Clin. Microbiol. Rev., 18, 81-101 (2005).
• van den Brink, D.M. and Wanders, R.J.A. Phytanic acid: production from phytol, its breakdown and role in human disease. Cell. Mol. Life Sci., 63, 1752-1765 (2006).

Updated: 25/2/2008 Credits/disclaimer	Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland