Nomenclature of Polyunsaturated Fatty Acids

The carbon atoms of fatty acids are numbered in two different ways. In the delta (Δ) numbering system, the carboxyl carbon is designated as carbon 1. The reverse occurs in the n− numbering system; the carbon at the methyl end of the hydrocarbon chain is designated as carbon 1. Another designation for the n− notation is ω, and both ω and n− notations are used interchangeably for numbering double bonds from the methyl end of a fatty acid. The n− notation is currently more popular and is used in this chapter.

When the methyl end notation is used, a number is usually placed after the n− or ω to indicate the location of the first double bond in relation to the methyl carbon. For example, ω3 indicates that the first double bond is the third carbon, counting from the methyl end of the fatty acid. The designation n−3 similarly indicates that the first double bond is the third carbon from the methyl carbon, although technically it indicates that the double bond begins at carbon number “n minus 3” counting from the carboxyl carbon. In every member of the n−3 class, the double bond closest to the methyl end is located 3 carbons from the methyl end.

In every member of the n−6 class, the double bond closest to the methyl end is located 6 carbons from the methyl end.

Fatty acids are often abbreviated as a ratio of the number of carbons to the number of double bonds (e.g., 18:0 for stearic acid). If the fatty acid is unsaturated, the location of the double bonds is also given. The location of the double bonds may be indicated by placing the location of each double bond before the number of carbons. For example, the notation for a PUFA that contains 18 carbons and two double bonds that are present at C9 and C12 is 9,12-18:2. Alternatively, the location of the double bonds for the commonly occurring PUFAs can be indicated by denoting the position of the first double bond counting from the methyl end (i.e., n−3, n−6, or n−9) because the double bonds are all methylene-interrupted. With this designation, for example, 18:3n−3 would be the same as 9,12,15-18:3. Thus, the location of a double bond in the Δ numbering system can be determined from the n− notation if the number of carbons that the fatty acid contains is known. For example, a double bond located in the n−3 position of an 18-carbon fatty acid is at C15 in the Δ nomenclature (i.e., n−3, or 18−3=15), and an n−6 double bond in an 18-carbon fatty acid is at C12 in the Δ nomenclature (i.e., 18−6=12). If the fatty acid is 18:3n−3, the double bonds will be between carbon atoms 15 and 16, 12 and 13, and 9 and 10, leaving a methylene carbon between each double bond.

The structure and nomenclature of fatty acids is described more fully in Chapter 6.

The n−6 Polyunsaturated Fatty Acids

Linoleic acid (18:2n−6), the first member of the n−6 class, is the main PUFA synthesized by terrestrial plants. It is the most abundant fatty acid contained in the triacylglycerols of corn oil, sunflower seed oil, and safflower oil, and linoleic acid accounts for most of the n−6 PUFAs obtained from the diet. Moreover, because there is much more n−6 than n−3 PUFA in most foods that we eat, linoleic acid usually is the most abundant PUFA in the diet.

The most prominent member of the n−6 class from a functional standpoint is arachidonic acid (20:4n−6; ARA). It is the main substrate used for the synthesis of the eicosanoid biomediators, such as the prostaglandins and leukotrienes, and it is also a major fatty acid component of the inositol glycerolphospholipids. Although a small amount of ARA is present in meat and other animal products in the diet, most of the ARA contained in the body is synthesized from linoleic acid. Adrenic acid (22:4), the elongation product of ARA, accumulates in tissues that have a high content of ARA. When necessary, adrenic acid can be converted back to ARA by removal of two carbons from its carboxyl end.

The n−3 Polyunsaturated Fatty Acids

The n−3 PUFAs are present in large amounts in the retina and certain areas of the brain. Like their n−6 counterparts, n−3 PUFAs can be structurally modified but cannot be synthesized completely in the body and ultimately must be obtained from the diet. The structures of the most important n−3 PUFAs are shown in Figure 18-1. α-Linolenic acid (18:3n−3), the 18-carbon member, is structurally similar to linoleic acid except for the presence of an additional double bond at C15. Some terrestrial plants synthesize small amounts of this fatty acid, and α-linolenic is present in soybean oil and canola oil. Larger amounts of α-linolenic acid are produced by vegetation that grows in cold water, and it is a prominent component in the food chain of fish and other marine animals. Although the intestinal mucosa can desaturate α-linolenic acid, most of the dietary intake is incorporated into the intestinal lipoproteins and absorbed by humans without structural modification.

Members of the n−3 fatty acid class that have five and six double bonds are present in fish, other marine animals, and foods that contain fish oils. The most abundant are eicosapentaenoic acid (20:5n−3; EPA) and docosahexaenoic acid (22:6n−3; DHA), which are often referred to as the fish oil fatty acids. Humans typically ingest a mixture of n−3 PUFAs, with the amount of α-linolenic acid compared to EPA and DHA depending on the relative amounts of plant products as compared with seafood and products containing fish oil in the diet. This differs from n−6 PUFA dietary intake, which is mostly in the form of linoleic acid.

Highly Unsaturated Fatty Acids, Long-Chain Pufas, and Very-Long-Chain Pufas

The terms highly unsaturated fatty acids, long-chain PUFAs, and very-long-chain PUFAs are sometimes used for PUFAs that contain four or more double bonds. These terms generally are applied to ARA (20:4n−6) and adrenic acid (22:4n−6) of the n−6 class and to EPA (20:5n−3) and DHA (22:6n−3) of the n−3 class (see Figure 18-1). The terms highly unsaturated fatty acids, long-chain PUFAs, and very-long-chain PUFAs were introduced to distinguish between the 20- and 22-carbon PUFAs, which produce most of the functional effects of essential fatty acids, and their 18-carbon precursors, which serve primarily as substrates for the synthesis of these more highly unsaturated derivatives. However, in chemistry the term long-chain fatty acid means any fatty acid greater than 12 carbons, thus leading to some confusion between the definitions of long-chain and very-long-chain fatty acids.

Synthesis of 20- And 22-Carbon Pufas

The synthesis of the longer, more highly unsaturated derivatives from the 18-carbon members of the n−3 and n−6 classes occurs through the pathway illustrated in Figure 18-2. Three types of reactions are involved: fatty acid chain elongation, desaturation, and β-oxidation (Sprecher, 2000). These reactions occur with both n−6 and n−3 PUFAs, but the two classes cannot be interconverted. Therefore an n−6 PUFA can be converted only to another n−6 PUFA, and likewise, an n−3 PUFA can be converted only to another n−3 PUFA. Therefore both classes of essential fatty acids are necessary in the diet. Of related interest, a gene from Caenorhabditis elegans encoding an n−3 desaturase, capable of converting n−6 PUFAs into n−3 PUFAs, has been isolated and transfected into mice and pigs, allowing them to synthesize n−3 PUFAs from n−6 PUFAs (Kang et al., 2004; Lai et al., 2006).

All the reactions in the PUFA metabolic pathway utilize fatty acids in the form of acyl-coenzyme A (CoA) derivatives. The complete pathway involves three elongation reactions, three desaturation reactions, and one retroconversion reaction. Fatty acids containing similar numbers of carbons and double bonds occur in the n−3 and n−6 classes (e.g., 18:3, 20:4, and 22:5). They are positional isomers, not identical compounds. Therefore the 18:3 in the n−3 pathway is α-linolenic acid (9,12,15-18:3, or 18:3n−3), whereas the 18:3 in the n−6 pathway is γ-linolenic acid (6,9,12-18:3, or 18:3n−6). Likewise, the 20:4 and 22:5 fatty acids that occur in both pathways are isomeric pairs. The 24-carbon fatty acids present in each class are metabolic intermediates that normally do not accumulate in either the plasma or the tissues.

Although each of the seven reactions in PUFA metabolism can utilize either n−3 or n−6 PUFAs, the pathway functions differently with the two classes of essential fatty acids under normal physiological conditions. The main n−6 PUFA product normally is ARA, and the last n−6 product normally formed is 22:4. The final three reactions in the n−6 PUFA metabolic pathway—(1) elongation to a 24-carbon intermediate, (2) Δ6-desaturation of this intermediate, and (3) retroconversion to the 22-carbon end-product—only become prominent when there is an n−3 PUFA deficiency. On the other hand, n−3 PUFA metabolism does lead to formation of the final 22:6n−3 product, DHA.

Fatty Acid Elongation

Fatty acids are elongated in the endoplasmic reticulum (ER) through the mechanism illustrated in Figure 18-3. The fatty acid must be in the form of an acyl-CoA, and malonyl-CoA is the elongating agent. In the condensation reaction, which is the rate-limiting step, the free carboxyl group of malonyl-CoA is released as CO₂ and the remaining 2-carbon fragment is attached to the fatty acid carbonyl group by displacement of CoA. Finally, the carbonyl group, which is C3 in the elongated product, is reduced in a three-step process that utilizes two NADPH molecules.

The position of the double bonds does not shift relative to the methyl end when a PUFA is elongated, and their numbering remains the same in the n− or omega nomenclature. However, the numbering of the double bonds changes in the Δ nomenclature because the 2-carbon fragment that adds becomes C1 and C2 of the lengthened product. Therefore when 6,9,12-18:3 undergoes one elongation, the resulting 20-carbon fatty acid is 8,11,14-20:3. A fatty acid can undergo more than one elongation. Each elongation sequence consists of the enzymatic reactions shown in Figure 18-3 and uses two NADPH, and the fatty acid is lengthened by the addition of two carbons to the carboxyl end.

All the elongation enzymes that have been studied effectively utilize both n−3 and n−6 PUFAs. However, there are at least five different human long-chain fatty acid elongase genes, denoted ELOVL1 to ELOVL5 (Jakobsson et al., 2006). The expression of these genes is tissue dependent. Furthermore, each ELOVL enzyme has different substrate specificity, although there is some overlap. For example, ELOVL5 acts on 18- and 20-carbon fatty acids, whereas ELOVL2 and ELOVL4 act on 20- and 22-carbon fatty acids. Consequently, at least two different fatty acid elongation enzymes operating in sequence are needed to convert an 18-carbon polyunsaturated fatty acid to the 24-carbon intermediate, and the enzymes that act in one tissue may be different from those that act in another tissue. These factors make elongation a complicated process that still is not fully understood.

Fatty Acid Desaturation

Double bonds are inserted into fatty acids by desaturation, a process that also occurs in the ER. The double bonds that are formed are always in the cis configuration. There are two classes of desaturase enzymes: (1) the stearoyl-CoA desaturases (SCDs) that act on saturated fatty acids, and (2) the fatty acyl-CoA desaturases (FADSs) that act on PUFAs.

Although several genes may encode the FADS enzymes, in terms of PUFA metabolism, FADS1 and FADS2 are the most studied. FADS1 is the fatty acid Δ5-desaturase, and FADS2 is the fatty acid Δ6-desaturase. The genes coding for FADS1 and FADS2 are located on human chromosome 11q12-q13.1 in reverse orientation, separated by about 10,000 bp (Marquardt et al., 2000). The expression of these two genes is coordinately regulated. In addition, a third desaturase gene, FADS3, is located in the 11q12-q13.1 region, but the function of its gene product is unknown (Lattka et al., 2010). Figure 18-2 illustrates where the fatty acid Δ5- and Δ6-desaturases act in essential fatty acid metabolism.

Both fatty acid desaturases can utilize either n−3 or n−6 polyunsaturated fatty acyl-CoA substrates, and they both require O₂, NADH, cytochrome b₅, and cytochrome b₅ reductase. Figure 18-4 illustrates the two reactions. The desaturases act on the segment of the acyl-CoA chain between the carboxyl group and the first existing double bond. The Δ5-desaturase acts on polyunsaturated acyl-CoAs that have the first double bond at C8, inserting the new double bond at C5. This enzyme acts at only one point in the metabolic pathway, converting 20:3n−6 to ARA in n−6 PUFA metabolism and 20:4n−3 to EPA in n−3 PUFA metabolism. The Δ6-desaturase acts on polyunsaturated fatty acyl-CoA substrates that have the first double bond at C9, and inserts the new double bond at C6. There is only one fatty acid Δ6-desaturase, and this enzyme functions twice in n−3 PUFA metabolism, converting α-linolenic acid to 18:4n−3 and 24:5n−3 to 24:6n−3 (Sprecher, 2000). The Δ6-desaturase ordinarily functions only once in n−6 PUFA metabolism, converting linoleic acid to 18:3n−6. It also is capable of converting 24:4n−6 to 24:5n−6, but this

CLINICAL CORRELATION

Fatty Acid Δ6-Desaturase Deficiency

A 4-year-old girl who had persistent health problems since birth was referred to a pediatric genetic disease specialist for evaluation because of poor growth, ulcerated cornea, severe photophobia, scaly skin lesions over her arms and legs, and cracking of the skin at the corners of her mouth. Analysis of her plasma revealed abnormally low levels of ARA and DHA. A supplement of fish oil and black currant seed oil, which contains γ-linolenic acid (18:3n−6), was prescribed. This treatment corrected the deficiencies of ARA and DHA in the plasma, and many of her symptoms gradually improved. A skin biopsy subsequently was obtained and fibroblasts were grown in culture. Biochemical studies revealed that the fatty acid Δ6-desaturase activity of the fibroblasts was very low as compared with normal human skin fibroblasts (Williard et al., 2001).

Thinking Critically

1. Why was black currant seed oil prescribed instead of corn oil as a source of n−6 PUFAs for this patient?

2. Could capsules containing purified EPA ethyl ester be used instead of fish oil to effectively treat the DHA deficiency in this patient?

3. Would you expect to find an elevation in 20:3n−9 in the patient’s plasma?

occurs to an appreciable extent only if there is a deficiency of n−3 PUFAs.

Retroconversion in the Peroxisomes

Conversion of the 24-carbon acyl-CoA intermediates to the 22-carbon end products is thought to occur through peroxisomal fatty acid oxidation, a β-oxidation system that shortens very-long-chain fatty acids. This process requires transport of the 24-carbon intermediate from the ER to the peroxisomes and, subsequently, transport of the 22-carbon product back to the ER where it is incorporated into tissue lipids. As shown in Figure 18-5, the retroconversion reaction requires O₂, FAD, NAD⁺, and CoA, and it removes two carbons in the form of acetyl-CoA from the carboxyl end of the fatty acyl-CoA. The peroxisomal enzymes that catalyze this β-oxidation process are straight-chain acyl-CoA oxidase, D-bifunctional protein, and either 3-ketoacyl-CoA thiolase or sterol carrier protein X (SCP-X) (Ferdinandusse et al., 2001).

In n−3 PUFA metabolism, this process converts 24:6n−3 to DHA. The numbering of the carbons in the Δ nomenclature changes when retroconversion occurs because the carbons that were numbered 1 and 2 in the original fatty acid are removed. Therefore the C6 double bond in the 24-carbon intermediate becomes the C4 double bond of DHA, the 22-carbon product. A similar process can occur with n−6 PUFAs to produce 22:5n−6 from 24:5n−6 (see Figure 18-2). Retroconversion also appears to be responsible for the increase in C20 PUFAs when C22 PUFAs are fed (e.g., increase in arachidonate when 22:4n−6 is fed, or of EPA when 22:5n−3 is fed). Thus elongation, desaturation, and retroconversion together may enable the body to utilize whichever n−3 and n−6 PUFAs are available in the diet to produce all of the necessary members of these essential fatty acid classes.

Peroxisomal fatty acid β-oxidation is deficient in cells of patients with Zellweger syndrome, which is caused by mutations in genes encoding proteins required for biogenesis of peroxisomes. Patients with Zellweger syndrome have elevated levels of very-long-chain fatty acids (e.g., C26:0 and C26:1), high ratios of C24/C22 and C26/C22 fatty acids, and low levels of DHA because they cannot produce DHA from the C24 n−3 PUFA precursor.