Chapter 18. Essential Fatty Acids. Arthur A. Spector, MD OUTLINE COMMON ABBREVIATIONS

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1 Chapter 18 Essential Fatty Acids Arthur A. Spector, MD OUTLINE HISTORICAL PERSPECTIVE STRUCTURE OF POLYUNSATURATED FATTY ACIDS ω6 Polyunsaturated Fatty Acids ω3 Polyunsaturated Fatty Acids Highly Unsaturated Fatty Acids ESSENTIAL FATTY ACID METABOLISM Fatty Acid Elongation Fatty Acid Desaturation Retroconversion Tissue Differences ESSENTIAL FATTY ACID COMPOSITION OF PLASMA AND TISSUE LIPIDS ESSENTIAL FATTY ACID FUNCTION ω6 Polyunsaturated Fatty Acids Arachidonic Acid and Eicosanoids Inositol Phosphoglycerides Linoleic Acid ω3 Polyunsaturated Fatty Acids Eicosapentaenoic Acid Docosahexaenoic Acid REGULATION OF GENE EXPRESSION BY ESSENTIAL FATTY ACIDS RECOMMENDATIONS FOR ESSENTIAL FATTY ACID INTAKE ESSENTIAL FATTY ACID DEFICIENCY PEROXIDATION OF POLYUNSATURATED FATTY ACIDS COMMON ABBREVIATIONS COX DHA ER EPA G-protein LDL cyclooxygenase docosahexaenoic acid endoplasmic reticulum eicosapentaenoic acid GTP-binding protein low density lipoprotein PG PPAR RXR SREBP prostaglandin peroxisome proliferator-activated receptor retinoid X receptor sterol regulatory element binding protein Essential fatty acids are polyunsaturated fatty acids that are necessary for growth and normal physiological function but cannot be completely synthesized in the body. There are two classes of essential fatty acids, omega (ω)6 and ω3. They cannot be interconverted. Therefore, the dietary fat intake must contain both of these classes in order to maintain good health and prevent an eventual deficiency. Plants have the ability to synthesize the first 18-carbon 518

2 Chapter 18 Essential Fatty Acids 519 member of each class, linoleic acid (ω6) and α-linolenic acid (ω3), and plant products are the ultimate sources of essential fatty acids in the human food chain. HISTORICAL PERSPECTIVE Early work demonstrated that a small amount of dietary fat was necessary for laboratory rats to grow normally, remain healthy, and reproduce. Two opposing views were put forward to explain this observation. Some thought that the protective action was due entirely to the vitamin E present in the dietary fat. Others thought that, in addition to vitamin E, some component of the fat itself was an essential nutrient. This controversy was resolved in 1929 when George O. Burr and Mildred M. Burr demonstrated that linoleic acid, the 18-carbon ω6 fatty acid that contains two double bonds, was an essential nutrient for the rat. The syndrome produced in rats by a lack of ω6 fatty acids, called essential fatty acid deficiency, was characterized by a cessation of growth, dermatitis, loss of water through the skin, loss of blood in the urine, fatty liver, and loss of reproductive capacity. Subsequent work showed that linoleic acid also is an essential nutrient for other mammals, including humans. No well-defined disease occurred when experimental animals were fed a diet deficient in α-linolenic acid, the corresponding 18-carbon member of the ω3 class. Therefore, it initially appeared that ω3 fatty acids were not essential nutrients and were present in the body simply because small amounts ordinarily are contained in the diet. This view gradually changed during the last 35 years because of increasing evidence that ω3 fatty acids are required for optimum vision and nervous system development (Lauritzen et al., 2001). A consensus now exists that, like the ω6 class, the ω3 fatty acids are essential nutrients for humans. STRUCTURE OF POLYUNSATURATED FATTY ACIDS Fatty acids contain a hydrocarbon chain and a carboxyl group. All fatty acids that have two or more double bonds in the hydrocarbon chain are classified as polyunsaturated. In humans and other mammals almost all of the polyunsaturated fatty acids present in the blood and tissues contain between 18 and 22 carbons and from two to six double bonds. The double bonds normally are three carbons apart; a carbon atom that is fully saturated (called a methylene carbon) separates them: CH CH CH 2 CH CH The presence of the methylene carbon between each pair of double bonds reduces the tendency of polyunsaturated fatty acids to undergo spontaneous oxidation in air, a process called lipid peroxidation. The double bonds in all unsaturated fatty acids synthesized by plants and animals are in the cis configuration. This introduces a rigid 45-degree bend at each double bond in the fatty acid chain. The bent conformation reduces the tightness with which adjacent fatty acid chains can pack, producing a more mobile physical state and thereby decreasing the melting point. The carbon atoms of fatty acids are numbered in two different ways. In the delta ( ) numbering system, the carboxyl carbon is designated as carbon 1. The reverse occurs in the ω numbering system; the carbon at the methyl end of the hydrocarbon chain is designated as carbon 1. Another designation for the ω notation is n, and both ω and n notations are used interchangeably for numbering double bonds from the methyl end of a fatty acid (see below). When the methyl end notations are used, a minus sign and number often are placed after them to indicate the location of the first double bond with respect to the methyl carbon; for CH 3 CH 2 CH 2 CH 2 CH 2 COOH Numbering system or n Numbering system

3 520 Unit IV Metabolism of the Macronutrients example, n-3 indicates that the first double bond is located 3-carbons from the methyl group. The location of double bonds in the numbering system can be determined from the ω or 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 C-15 in the nomenclature, and an n-6 double bond in an 18-carbon fatty acid is at C-12 in the nomenclature. Fatty acids are abbreviated as the number of carbons and a colon followed by the number of double bonds (e.g., 18:2 represents a fatty acid with 18 carbons and two double bonds). If the fatty acid is unsaturated, the location of the double bonds is placed before the number of carbons. Therefore, the notation for a polyunsaturated fatty acid that contains 18 carbons and two double bonds that are present at C-9 and C-12 is 9,12-18:2. However, the position of the double bonds often is omitted, and the commonly used notation for this fatty acid is 18:2. Although this shortened notation is more convenient, it is best to include the position of the double bonds to avoid confusion in situations where ambiguity might occur. In every member of the ω3 class, the double bond closest to the methyl end is located 3 carbons from the methyl end: 3 The double bond closest to the methyl end is located 6 carbons from the methyl end in every member of the ω6 class: 6 CH 3 CH 2 CH CH 3 CH 2 CH CH 2 Humans and other mammals do not have the enzymes necessary to form either the ω3 or ω6 double bonds that are present in essential fatty acids. However, plants have the capacity to synthesize 18-carbon polyunsaturated fatty acids containing these double bonds and are the ultimate sources of essential fatty acids in the human food chain. Figure 18-1 illustrates the chemical structures of the major ω6 and ω3 fatty acids present in 4 CH 2 CH 5 6 CH CH Omega-6 CH 3 Linoleic acid 9,12-18:2 COOH Arachidonic acid 5,8,11,14-20:4 Adrenic acid 7,10,13,16-22:4 COOH CH 3 COOH CH 3 Omega-3 COOH CH 3 Linolenic acid 9,12,15-18:3 COOH CH 3 Eicosapentaenoic acid 5,8,11,14,17-20:5 COOH CH 3 Docosahexanenoic acid 4,7,10,13,16,19-22:6 Figure 18-1 Structures of the most prominent ω6 and ω3 essential fatty acids. humans and animals. The ω6 fatty acids are shown on the left and the ω3 fatty acids on the right. Although each class contains eight fatty acids, the six fatty acids shown in this figure account for more than 90% of the polyunsaturated fatty acids present in the plasma and tissues under normal physiological conditions. ω6 POLYUNSATURATED FATTY ACIDS Linoleic acid (18:2), the first member of the ω6 series, is the main polyunsaturated fatty acid 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 ω6 fatty acid obtained from the diet. Moreover, because there is much more ω6 than ω3 fatty acid in most foods that we eat, linoleic acid usually is the most abundant polyunsaturated fatty acid in the diet. The most prominent member of the ω6 series from a functional standpoint is arachidonic acid (20:4). It is the main substrate utilized 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 phosphoglycerides.

4 Chapter 18 Essential Fatty Acids 521 Although a small amount of arachidonic acid is present in meat and other animal products in the diet, most of the arachidonic acid contained in the body is synthesized from dietary linoleic acid. Adrenic acid (22:4), the elongation product of arachidonic acid, accumulates in tissues that have a high content of arachidonic acid. When necessary, adrenic acid can be converted back to arachidonic acid by removal of two carbons from its carboxyl end. ω3 POLYUNSATURATED FATTY ACIDS The ω3 fatty acids are present in large amounts in the retina and certain areas of the brain. Like their ω6 counterparts, ω3 fatty acids 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 ω3 fatty acids are shown on the right side of Figure α-linolenic acid (18:3), the 18-carbon member, is structurally similar to linoleic acid except for the presence of an additional double bond at C-15. 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. α- Linolenic acid (18:3ω3) is commonly called linolenic acid, but α-linolenic is used here to avoid confusion with γ-linolenic acid (18:3ω6). Members of the ω3 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 (20:5; EPA) and docosahexaenoic acid (22:6; DHA), which are often referred to as the fish oil fatty acids. Fish and other marine animals feed on cold-water vegetation and convert the α-linolenic acid to EPA and DHA. Therefore, humans typically ingest a mixture of ω3 fatty acids, with the proportion of α-linolenic acid compared to EPA and DHA depending on the relative intake of plant products as compared with intake of seafood and products containing fish oil. This differs from the ω6 fatty acid dietary intake, which is mostly in the form of linoleic acid. HIGHLY UNSATURATED FATTY ACIDS The term highly unsaturated fatty acids is used for polyunsaturated fatty acids that contain four or more double bonds. It generally is applied to arachidonic acid (20:4) and adrenic acid (22:4) of the ω6 series and to EPA (20:5) and DHA (22:6) of the ω3 class (see Fig. 18-1). The term was introduced recently to distinguish between the 20- and 22-carbon polyunsaturated fatty acids, 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. ESSENTIAL FATTY ACID METABOLISM Humans cannot completely synthesize either ω3 or ω6 fatty acids. However, all humans, even infants, can convert the 18-carbon members of each class to the corresponding 20- and 22-carbon products (Salem et al., 1996). It is generally agreed that the human requirement for ω6 fatty acid can be fully satisfied by synthesis from dietary linoleic acid. However, there is ongoing debate as to whether humans, especially infants, can synthesize enough 20- and 22-carbon ω3 fatty acids from α-linolenic acid for optimum growth and development of the neural and visual systems. The synthesis of the longer, more highly unsaturated derivatives from the 18-carbon members of the ω3 and ω6 classes occurs through the pathway illustrated in Figure Three types of reactions are involved; fatty acid chain elongation, desaturation, and retroconversion (Sprecher, 2000). These reactions occur with both ω6 and ω3 fatty acids, but the two classes cannot be interconverted. Therefore, an ω6 fatty acid can be converted only to another ω6 fatty acid, and likewise, an ω3 fatty acid can be converted only to another ω3 fatty acid. Therefore, both classes of essential fatty acids are necessary in the diet. All the reactions in the polyunsaturated fatty acid metabolic pathway utilize fatty acids in

5 522 Unit IV Metabolism of the Macronutrients Omega-6 9,12 18:2 Omega-3 9,12,15 18:3 6 Desaturase 6,9,12 18:3 6,9,12,15 18:4 Elongase 8,11,14 20:3 8,11,14,17 20:4 5 Desaturase 5,8,11,14 20:4 5,8,11,14,17 20:5 Elongase 7,10,13,16 22:5 7,10,13,16,19 22:5 Elongase 9,12,15,18 24:4 9,12,15,18,21 24:5 6 Desaturase 6,9,12,15,18 24:5 6,9,12,15,18,21 24:6 Retroconversion 4,7,10,13,16 22:5 4,7,10,13,16,19 22:6 Figure 18-2 Pathway for the conversion of the 18-carbon ω6 and ω3 essential fatty acids to their elongated and more highly unsaturated products in mammalian tissues. The fatty acids are abbreviated as number of carbons and the number of double bonds, separated by a colon. This is preceded by the locations of the double bonds counting from the carboxyl end. Although not evident from the figure, this enzymatic pathway only utilizes fatty acids in the form of fatty acyl CoA. the form of their esters with coenzyme A (acyl CoAs). 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 ω3 and ω6 classes (e.g., 18:3, 20:4, and 22:5). They are positional isomers, not identical compounds. Therefore, the 18:3 in the ω3 pathway is α-linolenic acid (9,12,15-18:3), whereas the 18:3 in the ω6 pathway is γ-linolenic acid (6,9,12-18:3). 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 tissues. Although each of the seven reactions in polyunsaturated fatty acid metabolism can utilize either ω3 or ω6 fatty acids, the pathway functions differently with the two classes of essential fatty acids under normal physiological conditions. The ω3 fatty acids ordinarily pass through the entire pathway, and the most abundant product is DHA. By contrast, the main ω6 fatty acid product normally is arachidonic acid, and the last product normally formed is 22:4. The final three reactions in the ω6 fatty acid metabolic pathway (1) elongation to a 24-carbon intermediate, (2) 6-desaturation of this intermediate, and (3) retroconversion to the 22-carbon end-product only become prominent when there is an ω3 fatty acid deficiency. Figure 18-3 shows the fatty acid composition of normal human erythrocytes as determined by gas-liquid chromatography. Much more ω6 than ω3 fatty acids are contained in the erythrocyte lipids. The ω6 fatty acids present are 18:2, 20:3, 20:4, and 22:4, with linoleic acid (18:2) and arachidonic acid (20:4) accounting for about 80% of the total. The small amount of ω3 fatty acid is distributed almost equally between 22:5 and DHA (22:6). A similar distribution normally is present in human plasma and many other human tissues. This distribution reflects the large excess

6 Chapter 18 Essential Fatty Acids : :0 Amount :0 9 18:1 6 18:2 6 20:3 6 20:4 6 22:4 3 22:5 3 22: Time (min) Figure 18-3 Fatty acid composition of the human erythrocyte as determined by gas chromatography. The fatty acids are indicated as the number of carbons followed by a colon and then the number of double bonds. Margaric acid, 17:0, was added as an internal standard for the analysis and is not ordinarily present in erythrocyte lipids. The classes of the unsaturated fatty acids detected in the erythrocyte lipids are ω9, 18:1; ω6, 18:2, 20:3, 20:4, 22:4; ω3, 22:5, 22:6. of ω6 fatty acids typically present in the diet and the differences in the products that ordinarily accumulate in ω3 as compared with ω6 fatty acid metabolism. The most notable exceptions to this typical fatty acid distribution are the lipids of the retina and brain, which have a very high DHA content. FATTY ACID ELONGATION Fatty acids are elongated in the endoplasmic reticulum (ER) through the mechanism illustrated in Figure 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 CO 2 and the remaining 2-carbon fragment is attached to the fatty acid carbonyl group by displacement of CoA. Finally, the carbonyl group, which is C-3 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 polyunsaturated fatty acid is elongated, and their O O R C CoA O R C CH 2 C CoA R OH C H CH 2 O C CoA O R C H H C C CoA O R CH 2 CH 2 C CoA NADP Figure 18-4 Mechanism of fatty acid chain elongation. O C CO 2 CoA NADPH H NADP H 2 O CoA CH 2 COO NADPH H

7 524 Unit IV Metabolism of the Macronutrients numbering remains the same in the ω or n nomenclature. However, the numbering of the double bonds changes in the nomenclature because the 2-carbon fragment that adds becomes C-1 and C-2 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 18-4 and utilizes 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 ω3 and ω6 fatty acids. However, there are at least five different human long-chain fatty acid elongase genes, denoted ELOVL1-5 (Leonard et al., 2004). 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 ( 9) desaturases (SCDs) that act on saturated fatty acids and (2) the fatty acyl CoA desaturases (FADS) that act on polyunsaturated fatty acids. The FADS enzymes are encoded by two genes: (1) FADS1 and (2) FADS2. FADS1 encodes the fatty acid 5-desaturase, and FADS2 encodes the fatty acid 6-desaturase. The 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 Clinical Correlation Fatty Acid 6-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 arachidonic acid and DHA. Supplements of fish oil and of black currant seed oil, which contains γ-linolenic acid (18:3ω6), was prescribed. This treatment corrected the deficiencies of arachidonic acid 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 6-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 current seed oil prescribed instead of corn oil as a source of ω6 fatty acids 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 20:3ω9 in the patient s plasma? coordinately regulated (Cho et al., 1999). In addition, a third desaturase gene, FADS3, is located in the 11q12-q13.1 region, but its function is unknown. Figure 18-2 illustrates where the fatty acid 5- and 6-desaturases act in essential fatty acid metabolism. Both fatty acid desaturases can utilize either ω3 or ω6 polyunsaturated acyl CoA substrates, and they both require O 2, NADH, cytochrome b 5, and cytochrome b 5 reductase. Figure 18-5 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 5-desaturase acts on polyunsaturated acyl CoAs that have

8 Chapter 18 Essential Fatty Acids 525 R CH 2 CH CH CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CO CoA 9 8 O 2 5 Desaturase NADH H Cytochrome b 5 R CH 2 CH CH CH 2 CH CH CH 2 CH 2 CH 2 CO CoA R CH CH CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH O 2 CO CoA 6 Desaturase NADH H Cytochrome b 5 R CH CH CH 2 CH CH CH 2 CH 2 CH 2 CH 2 CO CoA Figure 18-5 Positional differences in the double bonds inserted by the fatty acid 5- and 6-desaturases. the first double bond at C-8, inserting the new double bond at C-5. This enzyme acts at only one point in the metabolic pathway, converting 20:3ω6 to arachidonic acid in ω6 fatty acid metabolism and 20:4ω3 to EPA in ω3 fatty acid metabolism. The 6-desaturase acts on polyunsaturated fatty acyl CoA substrates that have the first double bond at C-9, and inserts the new double bond at C-6. There is only one fatty acid 6-desaturase, and this enzyme functions twice in ω3 fatty acid metabolism, converting α-linolenic acid to 18:4ω3 and 24:5ω3 to 24:6ω3 (Sprecher, 2000). The 6-desaturase ordinarily functions only once in ω6 fatty acid metabolism, converting linoleic acid to 18:3ω6. It also is capable of converting 24:4ω6 to 24:5ω6, but this occurs to an appreciable extent only if there is a deficiency of ω3 fatty acids. RETROCONVERSION Conversion of the 24-carbon acyl CoA intermediates to the 22-carbon end products occurs through peroxisomal fatty acid oxidation, a β-oxidation system that functions to shorten 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. Recent evidence suggests that these transport processes may be mediated by cytosolic fatty acid binding proteins (Norris and Spector, 2002). As shown in Figure 18-6, the retroconversion reaction requires O 2, 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 (D-3- hydroxyacyl CoA dehydrogenase) and either 3- ketoacyl CoA thiolase or sterol carrier protein X (Ferdinandusse et al., 2001). In ω3 fatty acid metabolism, this process converts 24:6ω3 to DHA (22:6ω3). The numbering of the carbons in the nomenclature changes when retroconversion occurs because the carbons that were numbered 1 and 2 in the original fatty acid are removed. Therefore, the C-6 double bond in the 24-carbon intermediate becomes the C-4 double bond of DHA, the 22-carbon product.

9 526 Unit IV Metabolism of the Macronutrients R CH CH CH 2 CH CH CH 2 5 CH 2 Peroxisomes O 2 Straight-chain Acyl CoA oxidase 4 CH 2 3 CH CO CoA FAD NAD D-Bifunctional protein 3-Ketoacyl CoA thiolase/scp-x R CoA CH CH CH 2 CH CH CH Figure 18-6 Retroconversion reaction that occurs in essential fatty acid metabolism. SCP-X is the abbreviation for sterol carrier protein-x. CH 2 CO CoA CH 3 CO CoA Although a similar process can occur with ω6 fatty acids to produce 22:5ω6, the main function of retroconversion in ω6 fatty acid metabolism is to produce arachidonic acid from 22:4ω6. Likewise, in the ω3 pathway, DHA can be retroconverted to EPA. Therefore, just as linoleic and α-linolenic acids can be converted to their longer, more highly unsaturated derivatives, the 22-carbon members of the ω3 and ω6 classes can serve as a source of the corresponding shorter 20-carbon fatty acids. This enables the body to utilize whichever ω3 and ω6 fatty acids 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 s syndrome, a genetic defect in the biogenesis of peroxisomes. Patients who inherit this severe neurological disease have low levels of DHA because they cannot carry out the peroxisomal retroconversion step needed to produce DHA from ω3 fatty acid precursors. Dietary supplements containing DHA appear to improve the clinical condition in some of these patients (Martinez, 2001). TISSUE DIFFERENCES Differences in dietary intake and metabolism lead to the accumulation of different types of essential fatty acids in the body. Linoleic acid is the most abundant polyunsaturated fatty acid in the diet and, therefore, the ω6 fatty acids predominate in the plasma and most tissues. Many tissues are able to convert linoleic to arachidonic acid through the pathway illustrated in Figure 18-2, and linoleic and arachidonic acids are the main ω6 fatty acids that accumulate in the body. Very little α-linolenic acid ordinarily is present in the plasma or tissues, and unless the diet is supplemented with fish oil or ω3 fatty acid ethyl esters, there also is little EPA. Tissues like the retina and brain that have a high content of ω3 fatty acids contain mostly DHA. Some DHA is obtained directly from the diet, and dietary DHA is an important source of DHA for the brain (Su et al., 1999). The remainder is obtained by synthesis from α-linolenic acid and other ω3 fatty acids that may be present in the diet. Hepatocytes express the complete metabolic pathway shown in Figure 18-2, but many other cells normally do not convert α-linolenic acid to DHA and depend on the circulation for a supply of DHA synthesized elsewhere in the body. For example, the retina utilizes DHA that is formed in the liver from ω3 fatty acid precursors (Scott and Bazan, 1989). Similarly, neurons utilize DHA that is synthesized from α-linolenic acid by the combined actions of the astrocytes (neuroglial cells) and the microvascular endothelial cells that form the blood-brain barrier (Edmond, 2001; Moore, 2001). ESSENTIAL FATTY ACID COMPOSITION OF PLASMA AND TISSUE LIPIDS Human plasma contains a wide variety of essential fatty acids. The data in Table 18-1 were

10 Chapter 18 Essential Fatty Acids 527 Table 18-1 Essential Fatty Acid Composition of Normal Human Serum Lipids LIPOPROTEIN LIPIDS Fatty Acid* Free Fatty Acids Phospholipids Triacylglycerols Cholesteryl Esters (Fraction of Total Fatty Acids [% by Weight]) ω3 18: ± ± ± ± : ± ± ± ± 0.08 ω6 18: ± ± ± ± : ± ± ± ± : ± ± ± ± 0.39 Modified from data compiled by Edelstein C (1986) General properties of plasma lipoproteins and apoproteins. In: Scanu AM, Spector AA (eds) Biochemistry and Biology of the Plasma Lipoproteins. Marcel Dekker, New York, pp *Abbreviated as the number of carbons and number of double bonds. Phospholipids contain 0.65 ± 0.08% 20:5ω3 and 0.77 ± 0.03% 22:5ω3. The other lipid fractions contain only trace amounts (<0.3%) of these ω3 fatty acids. Cholesteryl esters contain 1.07 ± 0.07% 18:3ω6, but the other lipid fractions contain only trace amounts. The lipids contain only trace amounts (<0.5%) of 22:4ω6 and 22:5ω6. obtained from human subjects who consumed Western diets, which ordinarily contain about 10 times more ω6 than ω3 fatty acids. These data show that ω3 fatty acids comprise only 1% to 3% of the total fatty acids in any of the serum lipid fractions. By contrast, ω6 fatty acids account for 17% of the fatty acids in the plasma free fatty acid fraction, 37% in phospholipids, 22% in triacylglycerols, and 60% in cholesteryl esters. Linoleic and arachidonic acids comprise most of the ω6 fatty acids contained in these serum lipids. After an individual eats, the fatty acid composition of the triacylglycerols contained in chylomicrons, the lipoproteins produced by the small intestine, reflects that of the dietary fat. Therefore, in the immediate postprandial state, chylomicrons are a major source of essential fatty acids for the tissues. The other plasma lipoproteins and albumin continue to provide essential fatty acids to the tissues after the chylomicrons are removed from the circulation. Even though the concentrations of arachidonic acid and DHA are very low in the plasma free fatty acid pool, studies in the rat indicate that the rates of utilization of these unesterified fatty acids are sufficiently rapid to supply the needs of the brain (Rapoport et al., 2001). Essential fatty acids in tissues are contained primarily in phospholipids. They are located almost entirely in the phospholipid sn-2 position, the middle carbon of the glycerol moiety. Although each phospholipid class contains a mixture of essential fatty acids, one or two fatty acids usually predominate in each phospholipid class. Arachidonic acid is highly enriched in the inositol phosphoglycerides, whereas linoleic and arachidonic acids are contained in large amounts in the choline phosphoglycerides. The 22-carbon members, DHA and adrenic acid (22:4ω6), tend to accumulate in the ethanolamine and serine phosphoglycerides, and DHA is highly enriched in the ethanolamine plasmalogens. These differences in fatty acid distribution are due primarily to the substrate specificities of the acyltransferases that incorporate the acyl CoAs into the sn-2 position of the phospholipids. ESSENTIAL FATTY ACID FUNCTION The ω3 and ω6 classes of fatty acids are essential primarily because they are required for two important physiological processes, the synthesis of lipid biomediators and the production of membrane phospholipids that have optimal structural and signal transduction properties. Because these are fundamental processes, it is surprising that some animal cell lines can grow in culture for many passages in the absence of

11 528 Unit IV Metabolism of the Macronutrients Eicosanoids Phospholipids Signal transduction 6 PUFA PPARs Gene expression Sphingolipids Skin water barrier Figure 18-7 Physiological functions of the ω6 essential fatty acids. PUFA, Polyunsaturated fatty acid; PPARs, peroxisome proliferator-activated receptors. Nutrition Insight Dietary Effects on Membrane Fatty Acid Composition Modifying the dietary fat intake can alter the fatty acid composition of membrane phospholipids. Even the fatty acid composition of the heart, which one might think of as a very stable tissue, can be modified rapidly in experimental animals. The brain is the only organ that is resistant to such diet-induced changes. Except for those in the brain, the fatty acid composition of most membranes adapts to some extent to the type of fat available in the diet. This flexibility is surprising, considering the vital role that membranes play in so many cellular functions. Diet-induced changes in membrane lipid composition support the old saying that you are what you eat. However, there are limits to the extent of change that can take place in mammalian cells. Most of the variation occurs in the relative proportions of unsaturated fatty acids. For example, if the diet is enriched in sunflower seed oil, which contains 70% linoleic acid, the ω6 fatty acid content of the membrane phospholipids increases and is counterbalanced by a decrease in oleic acid. This reduction in monounsaturated fatty acid content is a compensation that protects against an excessive increase in membrane fluidity due to overabundance of unsaturated fatty acid in the membrane phospholipids. The relative amounts of ω3 and ω6 fatty acids in membrane phospholipids also is dependent on the dietary intake of these essential fatty acids. any detectable essential fatty acids. Many of these cell lines were derived from rodent malignant tumors that do not express the fatty acid 6-desaturase. Although a few biochemical functions are slightly compromised in these cells, they are viable and grow well. Therefore, essential fatty acids apparently are not required for the maintenance of basic life processes in a mammalian cell. The functions of essential fatty acids appear to become necessary when cells differentiate and form multicellular organisms, where intercellular communication, highly specialized membrane functions, and coordination of gene expression become vital. ω6 POLYUNSATURATED FATTY ACIDS Figure 18-7 illustrates the main functions of the ω6 fatty acids. These processes must operate properly for the body to function normally. Linoleic and arachidonic acid have membrane structural effects, and linoleic acid is especially required as a component of sphingolipids that prevent water loss from the skin. Arachidonic acid is the primary substrate for eicosanoid synthesis, and it is a major component of the inositol phosphoglycerides that are involved in membrane signal transduction. In addition, ω6 fatty acids and some of the eicosanoids are ligands for the peroxisome proliferator-activated receptors (PPARs), and thereby affect the expression of many genes involved in lipid metabolism.

12 Chapter 18 Essential Fatty Acids 529 Agonist Phospholipid-20:4 Phospholipase 20:4 COX, LOX, CYP Eicosanoids Autocrine response Signal transduction Signal transduction Paracrine response Figure 18-8 Synthesis and mechanism of action of eicosanoids. COX, Cyclooxygenase; CYP, cytochrome P450; LOX, lipoxygenase. Arachidonic Acid and Eicosanoids Eicosanoids synthesized from arachidonic acid are lipid biomediators that regulate many cellular functions. Figure 18-8 illustrates the production and action of these compounds. The binding of a cytokine or hormone to a plasma membrane receptor triggers eicosanoid production by the target cell via activation of a calcium-dependent cytoplasmic phospholipase A 2. This phospholipase hydrolyzes arachidonic acid from the sn-2 position of intracellular membrane phospholipids, primarily choline phosphoglycerides. The cyclooxygenase (COX), lipoxygenase, and/or cytochrome P450 pathways contained in the cell convert the arachidonic acid to one or more eicosanoid products. A major function of eicosanoids is cell-to-cell communication. These compounds are released into the extracellular fluid and function as autocrine and paracrine mediators by binding to plasma membrane GTP-binding protein (G-protein) coupled receptors that either activate or modulate the activity of intracellular signaling pathways. Some of the responses occur very rapidly, whereas others that involve transcriptional mechanisms occur more slowly. Figure 18-9 lists the types of eicosanoids formed by the COX, lipoxygenase, and cytochrome P450 pathways and illustrates the structures of several representative products. The cyclooxygenase pathway produces prostaglandins and thromboxanes. There are two cyclooxygenase isozymes, a constitutive form (COX-1) and an inducible form (COX-2). The structure of prostaglandin E 2 (PGE 2 ), one of the major prostaglandins, is shown. The subscript 2 denotes that the eicosanoid has two double bonds outside the ring structure, a characteristic of all COX products synthesized from arachidonic acid. Eicosanoids formed by the COX pathway have many physiological actions, including modulation of cardiovascular and renal function, blood coagulation, and inflammation. Lipoxygenases convert arachidonic acid to a hydroperoxyeicosatetraenoic acid (HPETE). There are three isozymes, 5-, 12-, and 15-lipoxygenase, which differ in their positional specificity for the arachidonic acid double bonds. The HPETE then is converted to a hydroxyeicosatetraenoic acid (HETE), leukotriene, or lipoxin. The structure of leukotriene B 4 (LTB 4 ), one of the main leukotrienes, is shown. Lipoxygenase products function primarily in the tissue response to inflammatory stimuli. Cytochrome P450 epoxygenases convert arachidonic acid to epoxyeicosatrienoic acids (EETs). The structure of 5,6-EET, one of the four EET positional isomers, is shown. Another class of cytochrome P450 enzymes, the ω-oxidases, insert a hydroxyl group at or

13 530 Unit IV Metabolism of the Macronutrients Arachidonic acid Cyclooxygenase Lipoxygenase Cytochrome P450 Prostaglandins Thromboxane HPETE HETE Leukotrienes Lipoxins EET DHET Omega-OH Omega-COOH COOH CH 3 OH OH COOH CH 3 COOH CH 3 OH OH Prostaglandin E 2 (PGE 2 ) Leukotriene B 4 (LTB 4 ) 5,6 Epoxyeicosatrienoic acid (5,6 EET) Figure 18-9 Pathways of eicosanoid synthesis and structures of some representative products. HPETE, Hydroperoxyeicosatetraenoic acid; HETE, hydroxyeicosatetraenoic acid; EET, epoxyeicosatrienoic acid; DHET, dihydroxyeicosatrienoic acid; omega-oh and omega-cooh refer to 20-hydroxy or 20-carboxy eicosatetraenoic products. near the methyl terminus of arachidonic acid; 20-HETE is the main product. Eicosanoids formed by the cytochrome P450 pathway act on small arteries and in the kidney. They modulate vascular resistance and blood pressure. Table 18-2 lists the prostaglandin receptors, the main prostanoids that they bind, the biochemical mechanism of action, and the physiological responses that are produced. Eight different types of prostaglandin receptors have been cloned; they are all G protein coupled receptors. Although there is some overlap in substrate specificity, each type is designated according to the main product that it binds. For example, IP is the PGI receptor, and EP is a PGE receptor. The four subtypes of EP receptors have different tissue distributions, are linked to different G-proteins, and produce different functional responses. Prostaglandin-mediated signaling is a very complicated process that can occur in different ways, depending on the type of prostaglandin and the receptor to which it binds. One mechanism is activation of adenylate cyclase. PGI 2 functions in this way. It binds to the IP receptor, which is coupled to a G s protein. This activates adenylate cyclase, causing a large increase in cyclic adenosine monophosphate (camp) within the target cell. PGE 2 produces a similar response when it binds to the EP 2 receptor. However, when PGE 2 binds to the EP 3 receptor, it activates a G i protein that reduces adenylate cyclase activity, thereby decreasing camp production. To further complicate matters, PGE 2 also can bind to EP 1 receptors, which are coupled to G-proteins that activate phospholipase C. This stimulates hydrolysis of phosphatidylinositol 4,5-bisphosphate, producing an increase in the cytosolic calcium concentration. Inositol Phosphoglycerides Another important function of arachidonic acid is phospholipid-mediated signal transduction. In particular, the inositol phosphoglycerides that activate the intracellular protein kinase C signaling pathway contain a relatively high percentage of arachidonic acid. When phospholipase C is activated and hydrolyzes

14 Chapter 18 Essential Fatty Acids 531 Table 18-2 Prostaglandin Receptors Receptor Main Ligand Signal Transduction Mechanism Major Physiological Functions DP PGD 2 Inositol phospholipids Platelet aggregation Increases intracellular Ca 2+ Smooth muscle contraction EP 1 PGE 2 Inositol phospholipids Smooth muscle contraction Increases intracellular Ca 2+ EP 2 PGE 2 Adenylate cyclase Smooth muscle relaxation Increases camp EP 3 PGE 2 Adenylate cyclase Decreases water reabsorption Decreases camp Inhibits gastric acid secretion Uterine contraction EP 4 PGE 2 Adenylate cyclase Smooth muscle relaxation Increases camp FP PGF 2α Inositol phospholipids Smooth muscle contraction Increases intracellular Ca 2+ IP PGI 2 Adenylate cyclase Arterial smooth muscle relaxation Increases camp Inhibits platelet aggregation TP TXA 2 Inositol phospholipids Platelet aggregation Increases intracellular Ca 2+ Vasoconstriction Bronchoconstriction PG, Prostaglandin; TX, thromboxane. phosphatidylinositol 4,5-bisphosphate, the resulting 1,2-diacylglycerol that is involved in activating protein kinase C contains the arachidonic acid. The function of the high arachidonic acid content is not known. The possibilities include targeting to specific membrane domains or imparting special binding properties to the diacylglycerol. Alternatively, the diacylglycerol may be hydrolyzed by a diacylglycerol lipase, releasing the arachidonic acid for eicosanoid production. Linoleic Acid The average intake of ω6 fatty acids in healthy adults, 11 to 17 g/day, greatly exceeds the amount of arachidonic acid needed for the synthesis of the eicosanoid biomediators. This suggests either that there are other actions of arachidonic acid or that other ω6 fatty acids have essential functions. Emphasis has focused on the skin because essential fatty acid deficiency leads to a breakdown of the epidermal barrier to water loss. Linoleic acid is strongly preferred for the synthesis of two sphingolipids, acylceramide, and acylglucosylceramide, which maintain the structure of the stratum corneum, the outer layer of the skin. This suggests that linoleic acid is required to impart an optimal barrier property to the skin surface sphingolipids, thereby preventing excessive water loss. Lipoxygenase enzymes can oxygenate linoleic acid, adding oxygen at one of its double bonds. 15-Lipoxygenase is the main lipoxygenase that acts on linoleic acid in human tissues, forming the 13-hydroperoxyoctadecadienoic acid. This product is reduced to the corresponding hydroxy-derivative, 13- hydroxyoctadecadienoic acid (13-HODE). No physiological requirement for this or other oxygenated linoleic acid derivatives has been established, and it is uncertain as to whether the lipoxygenase pathway of linoleic acid metabolism has any essential function. A decrease in the level of plasma low density lipoproteins (LDLs) occurs when fats enriched in linoleic acid, such as corn oil or safflower oil, are substituted for dietary saturated fats. LDLs contain the highest percentage of cholesterol in the plasma, and a decrease in

15 532 Unit IV Metabolism of the Macronutrients LDL level reduces the risk of developing coronary heart disease. The mechanism producing the decrease in LDL level is complex and may depend more on the reduction in saturated fat intake rather than a direct biochemical effect produced by linoleic acid or one of its ω6 fatty acid products. Although it is unlikely that reduction of the LDL level is an essential function of linoleic acid, it certainly is one that has health benefits. ω3 POLYUNSATURATED FATTY ACIDS Two approaches have been used to investigate the functions of ω3 fatty acids. One is to determine the effects of ω3 fatty acid deficient diets in experimental animals. The other is to study humans who consume a diet high in ω3 fatty acids or who are treated with dietary supplements containing either fish oil or ω3 fatty acid ethyl esters. Table 18-3 lists the biochemical and biophysical functions of ω3 fatty acids. EPA produces some of these effects, and DHA produces the others. No unique functional effects have been reported for α-linolenic acid in humans or animals, except to serve the substrate for EPA and DHA synthesis. Table 18-3 Biochemical and Biophysical Functions of ω3 Fatty Acids Fatty Acid EPA EPA EPA DHA DHA DHA DHA DHA Not specified Not specified Function Production of ω3 eicosanoids Inhibition of ω6 eicosanoid production Inhibition of hepatic triglyceride synthesis Enhanced coupling of rhodopsin to the retinal G protein Increased plasmalogen synthesis Ligand for brain RXR nuclear receptor Docosanoid synthesis Nonbilayer phospholipid configuration Modulation of sodium ion channel conductance Ligand for PPAR nuclear receptors DHA, Docosahexaenoic acid; EPA, eicosapentaenoic acid; PPAR, peroxisome proliferator-activated receptor; RXR, retinoid X receptor. Eicosapentaenoic Acid EPA is primarily responsible for the antithrombotic and antiinflammatory actions produced by ω3 fatty acid dietary supplements. This occurs through an effect on eicosanoid production. The suggested mechanism is illustrated schematically in Figure The COX and lipoxygenase pathways can convert EPA to eicosanoids. Although these EPA products have bioactivity, no unique or vital function has been demonstrated for them. Furthermore, EPA is a poor substrate for COX as compared with arachidonic acid, but it effectively inhibits the conversion of arachidonic acid to COX products, especially thromboxane A 2, which triggers platelet aggregation and thrombosis. EPA may produce its inhibitory effects by competing with arachidonic acid for incorporation into the tissue phospholipids that supply 3 Eicosanoids 20:5 3 20:4 6 20:5 Phospholipid 20:4 20:5 20:4 COX, LOX, CYP Competition Competition Decrease Decrease 6 Eicosanoids Figure Competition between EPA (20:5ω3) and arachidonic acid (20:4ω6) for eicosanoid synthesis. These essential fatty acids compete for incorporation into the membrane phospholipids that provide the fatty acid substrate for eicosanoid synthesis. Both fatty acids are released when phospholipase A 2 hydrolyzes the phospholipids. Although they compete effectively for access to the eicosanoid-producing enzymes, EPA is a less effective substrate. As a result there is a reduction in the quantity of eicosanoids synthesized from arachidonic acid without a compensatory replacement by products formed from EPA. In some cases, the products formed from EPA also have less bioactivity. The overall effect is a reduction in the eicosanoid response to cellular activation. The abbreviations are the same as in Figure 18-8.

16 Chapter 18 Essential Fatty Acids 533 substrate for eicosanoid synthesis. When cells are exposed to supplemental EPA, the arachidonic acid content of the phospholipids decreases and is replaced by EPA and its elongation product 22:5ω3. As a result, less arachidonic acid is released when phospholipases are activated, so that less is available for eicosanoid synthesis. The amount of COX products synthesized from EPA is either too small to compensate for the decrease in arachidonic acid products, or the eicosanoids produced from EPA have less bioactivity. This, combined with reduction in arachidonic acid availability, makes EPA an effective modulator of arachidonic acid function. EPA also produces the hypotriglyceridemic effect of fish oil, primarily by increasing mitochondrial β-oxidation (Fro/ yland et al., 1997). In addition, it probably contributes the antihypertensive effects of fish oil. These actions, together with its antiinflammatory and antithrombotic effects, can prevent or reduce the severity of life-threatening illnesses. However, none of these effects is likely to be essential for the maintenance of normal bodily function. Therefore, EPA probably is not the essential component of the ω3 fatty acids. Clinical Correlation Fish Oil for the Prevention of Coronary Thrombosis Daily fish oil supplements are being recommended by some physicians to reduce the risk of coronary thrombosis. Patients usually take between 1 and 3 g/day, administered either in the form of fish oil capsules or capsules containing purified EPA and DHA ethyl esters. Thinking Critically 1. How might ω3 fatty acids act to reduce the risk of coronary thrombosis? 2. Would the administration of canola oil, which contains α-linolenic acid as the only source of ω3 fatty acid, be as effective as fish oil for this purpose? Docosahexaenoic Acid DHA, which is highly enriched in retinal and brain phospholipids, is responsible for the visual and cognitive actions of the ω3 fatty acids. It is present in the phospholipids of cell membranes, primarily in the ethanolamine and serine phosphoglycerides and ethanolamine plasmalogens. These phospholipids are contained primarily in the inner (cytoplasmic) leaflet of the membrane lipid bilayer, so the functional effects produced by DHA most likely occur in this region of the membrane. The DHA chains contained in phospholipids are highly flexible and transition very rapidly between a large number of conformations, providing an optimum microenvironment for the function of certain proteins that are embedded in the membrane (Gawrisch et al., 2003). Phospholipids enriched in DHA augment the response of the retina to light and enhance the transmission of the visual signal. The retinal light receptor, rhodopsin, is a G protein coupled receptor. When rhodopsin is activated by light, the structure of the surrounding phospholipid DHA fatty acyl chains facilitates the conformational change of rhodopsin to the metarhodopsin II state, increasing the rate of coupling to the retinal G-protein. This enhances the amplification in the first stage of the visual pathway. The high DHA content of the retinal phospholipids also increases the activity of the phosphodiesterase, which is a measure of the integrated visual signal response (Mitchell et al., 2003). The need for DHA to produce optimum membrane lipid bilayer properties in the retina for the visual response is one reason why ω3 fatty acids are essential nutrients. If a similar DHAmediated mechanism applies to G protein coupled receptor signaling in the brain, it also may explain the cognitive benefits produced by ω3 fatty acids (Moriguchi et al., 2000). Biophysical studies indicate that some phospholipids have a tendency not to form a bilayer structure. One of the alternate arrangements is a hexagonal structure, an inverted configuration in which the polar head groups of the phospholipids cluster together on the inside and the hydrocarbon chains point outward. Phosphatidylethanolamine has an increased