5
Fats and Fatty Acids

Fats have the highest energy density among dietary components. Fatty acids are basic chemical units of fat, and the names and structural features of several are shown in Table 5-1. The fatty acids most commonly found in primates and in primate diets have 16 and 18 carbon atoms; those found less commonly have 12, 14, 20, and 22 carbon atoms. All are straight carbon chains that have zero to six double bonds in the cis conformation. Fatty acids with double bonds in the trans conformation are rare in nature and are unlikely to have an important presence in natural foods of primates. Multiples of double bonds typically occur in series, with a double bond beginning every fourth carbon. Essential fatty acids are those which cannot be made by the body; and for primates, these include the n-3 and n-6 fatty acids (Innis, 1991).

The designations n-3 and n-6 (sometimes written ω-3 and ω-6) refer to the number of carbons from the methyl end of the fatty acyl chain to the first double bond. The fatty acid that is the building block for the n-6 series is linoleic acid, an 18-carbon fatty acid containing two double bonds, the first between the sixth and seventh carbons. The building block for the n-3 fatty acids is α-linolenic acid, an 18-carbon fatty acid with three double bonds, the first between the third and fourth carbons. A short-form designation for fatty acids lists the number of carbons, a colon, the number of double bonds, and identity of the n series, for example, C18:2 n-6 for linoleic acid (Lin, et al., 1994; Buss and Cooper, 1970). The liver and, to a lesser extent, other tissues have enzymes needed to elongate and further desaturate linoleic and α-linolenic acid to make other fatty acids in these series. However, the primates that have been studied have no enzymes that can desaturate fatty acids at the third or sixth carbon. Thus, the basic fatty acids with these double bonds are termed essential and must be consumed in the diet.

Most dietary fats of animal or vegetable origin are triacylglycerols (TAGs; formerly called triglycerides); they have three fatty acids esterified to a glycerol molecule in one of three stereochemically distinct bonding positions: sn-1, sn-2, and sn-3. To a lesser extent, phospholipids also are parts of primate diets, typically with two fatty acids esterified to a glycerol phosphate molecule and an acidic or basic adduct attached to the phosphate residue.

The classification of a fatty acid as essential means that the fatty acid is not synthesized by the body. A recent article presents a proposal to reclassify essential fatty acids into categories of “conditionally indispensable” and “conditionally dispensable” (Cunnane, 2000). This proposal was made recognizing that the requirement for each of the n-3 and n-6 fatty acids is not the same throughout the life span of the animal. Adult animals do not need the same level of dietary intake as young, growing animals in part because they have well-developed body stores of each of the essential fatty acids. Further, the number of longer chain derivatives of either linoleate or α-linolenate with additional double bonds is recognized as large and each may sometimes be classified as essential by some individuals. However, the bulk of the evidence is that with the dietary precursor 16 or 18 carbon fatty acids with either the n-3 or n-6 double bond in place, the remainder of the n-3 or n-6 fatty acid series, respectively, can be generated in the body by the appropriate elongases and desaturases. While there is some evidence that not all of these enzymes for fatty acid modification are present in equivalent abundance, apparently all are available to the extent needed to provide for normal function. Some have suggested that the fatty acids in the n-3 and n-6 series will compete for access to one or more of the desaturases; however, the likelihood that such competition could lead to a deficiency has not been demonstrated. In addition, the efficiency of utilization of α-linolenate for making docosahexaenoic acid (22:6 n-3) for use in body functions is less than direct utilization of exogenous docosahexaenoic acid, due to the many energy requiring elongation and desaturation steps used in deriving the latter from the former. However, this does not change the fact that the body cannot generate either the n-3 or n-6 double bond in any fatty acid. We have used the term essential fatty acid to indicate this fact. We con-



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Nutrient Requirements of Nonhuman Primates: Second Revised Edition, 2003 5 Fats and Fatty Acids Fats have the highest energy density among dietary components. Fatty acids are basic chemical units of fat, and the names and structural features of several are shown in Table 5-1. The fatty acids most commonly found in primates and in primate diets have 16 and 18 carbon atoms; those found less commonly have 12, 14, 20, and 22 carbon atoms. All are straight carbon chains that have zero to six double bonds in the cis conformation. Fatty acids with double bonds in the trans conformation are rare in nature and are unlikely to have an important presence in natural foods of primates. Multiples of double bonds typically occur in series, with a double bond beginning every fourth carbon. Essential fatty acids are those which cannot be made by the body; and for primates, these include the n-3 and n-6 fatty acids (Innis, 1991). The designations n-3 and n-6 (sometimes written ω-3 and ω-6) refer to the number of carbons from the methyl end of the fatty acyl chain to the first double bond. The fatty acid that is the building block for the n-6 series is linoleic acid, an 18-carbon fatty acid containing two double bonds, the first between the sixth and seventh carbons. The building block for the n-3 fatty acids is α-linolenic acid, an 18-carbon fatty acid with three double bonds, the first between the third and fourth carbons. A short-form designation for fatty acids lists the number of carbons, a colon, the number of double bonds, and identity of the n series, for example, C18:2 n-6 for linoleic acid (Lin, et al., 1994; Buss and Cooper, 1970). The liver and, to a lesser extent, other tissues have enzymes needed to elongate and further desaturate linoleic and α-linolenic acid to make other fatty acids in these series. However, the primates that have been studied have no enzymes that can desaturate fatty acids at the third or sixth carbon. Thus, the basic fatty acids with these double bonds are termed essential and must be consumed in the diet. Most dietary fats of animal or vegetable origin are triacylglycerols (TAGs; formerly called triglycerides); they have three fatty acids esterified to a glycerol molecule in one of three stereochemically distinct bonding positions: sn-1, sn-2, and sn-3. To a lesser extent, phospholipids also are parts of primate diets, typically with two fatty acids esterified to a glycerol phosphate molecule and an acidic or basic adduct attached to the phosphate residue. The classification of a fatty acid as essential means that the fatty acid is not synthesized by the body. A recent article presents a proposal to reclassify essential fatty acids into categories of “conditionally indispensable” and “conditionally dispensable” (Cunnane, 2000). This proposal was made recognizing that the requirement for each of the n-3 and n-6 fatty acids is not the same throughout the life span of the animal. Adult animals do not need the same level of dietary intake as young, growing animals in part because they have well-developed body stores of each of the essential fatty acids. Further, the number of longer chain derivatives of either linoleate or α-linolenate with additional double bonds is recognized as large and each may sometimes be classified as essential by some individuals. However, the bulk of the evidence is that with the dietary precursor 16 or 18 carbon fatty acids with either the n-3 or n-6 double bond in place, the remainder of the n-3 or n-6 fatty acid series, respectively, can be generated in the body by the appropriate elongases and desaturases. While there is some evidence that not all of these enzymes for fatty acid modification are present in equivalent abundance, apparently all are available to the extent needed to provide for normal function. Some have suggested that the fatty acids in the n-3 and n-6 series will compete for access to one or more of the desaturases; however, the likelihood that such competition could lead to a deficiency has not been demonstrated. In addition, the efficiency of utilization of α-linolenate for making docosahexaenoic acid (22:6 n-3) for use in body functions is less than direct utilization of exogenous docosahexaenoic acid, due to the many energy requiring elongation and desaturation steps used in deriving the latter from the former. However, this does not change the fact that the body cannot generate either the n-3 or n-6 double bond in any fatty acid. We have used the term essential fatty acid to indicate this fact. We con-

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Nutrient Requirements of Nonhuman Primates: Second Revised Edition, 2003 TABLE 5-1 Common names, scientific names, and short-form designations of fatty acids Common Name Scientific Name Short-Form Designation Butyric acid butanoic acid C4:0 Caproic acid hexanoic acid C6:0 Caprylic acid octanoic acid C8:0 Capric acid decanoic acid C10:0 Lauric acid dodecanoic acid C12:0 Myristic acid tetradecanoic acid C14:0 Palmitic acid hexadecanoic acid C16:0 Stearic acid octadecanoic acid C18:0 Palmitoleic acid 9-hexadecaenoic acid C16:1 n-7 cis Oleic acid 9-octadecaenoic acid C18:1 n-9 cis Elaidic acid 9-octadecaenoic acid C18:1 n-9 trans Linoleic acid 9,12-octadecadienoic acid C18:2 n-6,9 all cis α-Linolenic acid 9,12,15-octadecatrienoic acid C18:3 n-3,6,9 all cis γ-Linolenic acid 6,9,12-octadecatrienoic acid C18:3 n-6,9,12 all cis Arachidic acid eicosanoic acid C20:0 Behenic acid docosanoic acid C22:0 Eicosenoic acid 11-eicosenoic acid C20:1 n-9 cis Erucic acid 13-docosaenoic acid C22:1 n-9 cis Brassidic acid 13-docosaenoic acid C22:1 n-9 trans Nervonic acid 15-tetracosaenoic acid C24:1 n-9 cis Dihomo-γ-linolenic acid 8,11,14-eicosatrienoic acid C20:3 n-6,9,12 all cis Arachidonic acid 5,8,11,14-eicosatetraenoic acid C20:4 n-6,9,12,15 all cis Timnodonic acid 5,8,11,14,17-eicosapentaenoic acid C20:5 n-3,6,9,12,15 all cis Clupanodonic acid 7,10,13,16,19-docosapentaenoic acid C22:5 n-3,6,9,12,15 all cis Docosahexaenoic acid 4,7,10,13,16,19-docosahexaenoic acid C22:6 n-3,6,9,12,15,18 all cis sider the most common dietary n-6 and n-3 fatty acids, linoleate and α-linolenate, as truly essential since these fatty acids must be ingested. Additional long chain polyunsaturated fatty acids can be constructed from these fatty acids, but ingestion of fatty acids with the n-3 and n-6 double bonds is a true requirement. FAT ABSORPTION In response to entry of fat into the intestine during digestion of a meal, the liver secretes bile into the gut; with the help of intestinal peristalsis, food fats are emulsified. Simultaneously, the pancreas secretes digestive enzymes, including lipases and esterases, into the small intestine. Fat digestion begins at the surface of emulsion particles with pancreatic lipase-catalyzed hydrolysis of triacylglycerol molecules into two fatty acids and a monoacylglyceride. These products of initial lipolytic activity and bile salts are active in further breakdown of emulsion particles and, with phospholipid and cholesterol bile micelles, form the micellar phase from which lipid absorption is maximal. In most laboratory studies of nonhuman primates, fat digestion and absorption were essentially quantitative, with less than 5% of dietary fat lost in the feces; this was true in a wide array of types and amounts (up to 40% of ME) of fat ingested (L. Rudel and P. Huth, unpublished). The processes of fat digestion and absorption also facilitate the absorption of cholesterol and fat-soluble vitamins from the intestine. These molecules are incorporated into emulsion particles and micelles, from which they pass into the enterocyte. However, only about 50% of cholesterol is absorbed from the intestine (Rudel et al., 1994; Wilson and Rudel, 1994), though some of the molecular processes involved are different for sterols and fatty acids. Once inside the intestinal enterocyte, fatty acids and glycerides are reassembled into triacylglycerol molecules and incorporated into newly forming chylomicrons that include a protein, apolipoprotein B48. Nonhuman primates and humans share the characteristic presence of only apolipoprotein B48 in the intestine for transport of TAGs in chylomicrons, in contrast with the liver, where only apolipoprotein B100 is used for TAG secretion in very-low-density lipoproteins (VLDLs) (Klein and Rudel, 1983). Newly absorbed cholesterol is also esterified and incorporated into chylomicrons, although it makes up only about one percent (by mass) of these particles. Capture of high-energy fatty acids from chylomicrons is efficient. The chylomicron particles are secreted by the enterocytes into basolateral spaces, where they cross into the lymphatic lacteals and enter the body via the thoracic lymph duct. This pathway of entry into the bloodstream directs fats first to the peripheral tissues, where interactions with lipoprotein lipase (LPL), attached to the endothelial cells of most tissues, can occur. Removal of TAG molecules from chylomicrons then proceeds with the LPL-catalyzed hydrolysis of TAGs into two fatty acids and a monoacylglyceride. In this form, the molecules pass across cell membranes and enter cells. In adipose tissue, TAG molecules are reassembled and stored for later use. In most other

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Nutrient Requirements of Nonhuman Primates: Second Revised Edition, 2003 tissues, the fatty acids are either oxidized for energy or used for assembly of the phospholipid molecules that are the primary building blocks of cell membranes. After the bulk of the TAGs are removed, the remainder of the chylomicron particle, termed a remnant lipoprotein and still containing absorbed cholesterol and fat-soluble vitamins, travels to the liver, where it is quantitatively removed from the bloodstream. MILK FATS The position of the three fatty acids on the three-carbon glycerol backbone of fats is not random, but appears to depend on fatty acyl specificities of enzymes involved in TAG synthesis and to some extent in TAG hydrolysis. These molecules are taken apart and resynthesized several times during their movement into the body, so it is important to recognize that fatty acids in the sn-1 and sn-3 positions are the most labile and most readily available for use in the tissues. The fat in milk gives perhaps the best indication of the relative importance of different fatty acids. The breast milk of most primates that have been studied contains a TAG concentration of about 4 g·dl-1, which represents about 50% of the GE provided by milk (Wolfe et al., 1993). Milk fats of several Old World nonhuman primates (including five different macaque species, African green monkeys, Talapoin monkeys, and the sooty mangabey) have been reported to have a fatty acid composition similar to that of the fat in human milk (Buss and Cooper, 1970; Jensen et al., 1980; Smith and Hardjo, 1974a; Smith and Hardjo, 1974b; Wolfe et al., 1993). In the work of Smith and Hardjo (1974b), caprylic (C8:0), stearic (C18:0), oleic (C18:1), and linoleic (C18:2) acid were found predominantly in the sn-1 and sn-3 positions, and lauric (C12:0), myristic (C14:0), palmitic (C16:0), and palmitoleic (C16:1) acids were found in the sn-2 position of the TAG molecule. Linoleic acid made up about 12-13% of the fatty acids, and 80% of this was in the sn-1 and sn-3 positions. The most abundant fatty acid was oleic acid (25-30%), and over 80% of it was found in the sn-1 and sn-3 positions. Palmitic acid was about 20% of total fatty acids and was the most abundant fatty acid in the sn-2 position, representing about 40% of total sn-2 fatty acids. Long-chain polyunsaturated fatty acids were not reported in this study. Buss and Cooper’s (1970) examination of the milk of Talapoin monkeys revealed a fatty acid composition that differed somewhat from that of milk of the other primate species. Linoleic acid made up about 40% and palmitic and oleic acids about 20% of total fatty acids. The Talapoin monkeys were fed commercial monkey biscuits, a diet low in fat (about 10% of ME) with about 44% of the fatty acids as linoleic acid. Talapoin milk fat contained about 5% more palmitic acid and 5% less oleic acid than the fat in the diet. a-Linolenic acid was found to be 2.5-5.5% of milk fatty acids compared with 7% of fatty acids in the diet. African green monkeys were fed two fat-enriched diets (40% of ME) containing isocaloric amounts of polyunsaturated or saturated fat (Wolfe et al., 1993). Milk analyses revealed that the dietary fat of the mothers was a major factor in determining the fatty acid composition of their milk, as previously shown in humans (Potter and Nestel, 1976). Linoleic acid was 14% of the total milk fatty acids when dietary fat was enriched in saturated fatty acids and 42% of total milk fatty acids when the diet was enriched in linoleic acid. The increase in linoleic acid in the milk of mothers fed polyunsaturated fat was at the expense of the other major fatty acids in the milk of the saturated fat group. Monounsaturated fatty acids (primarily oleic acid) were 45%, and saturated fatty acids (primarily palmitic acid) 40% of the fatty acids in the milk of the saturated-fat group. Concentrations of both monounsaturated and saturated fatty acids decreased to 28% of total fatty acids in the milk of the polyunsaturated fat group. Birth weight and growth and development of infants in both diet groups were comparable. Thus, the fatty acid shift in the mothers’ diet and in later milk fatty acid composition had no obvious detrimental effects on normal growth of the monkeys (Wolfe et al., 1993). From the perspective of milk fatty acid composition, the data suggest that the types of fatty acids acceptable for primate diets can vary widely, and they provide no support for the contention that linoleic acid levels above 20% of total dietary fatty acids might be harmful (discussed by Innis, 1991). ESSENTIAL n-3 FATTY ACIDS Primate diets should contain sufficient concentrations of both n-3 and n-6 fatty acids to support normal growth and development. In a study of African green monkeys (Wolfe et al., 1993), dietary ME was present as long-chain n-3 fatty acids (docosahexaenoic acid and eicosapentaenoic acid) at about 0.25%. Another 0.2% of dietary ME was present as a-linolenic acid. That was sufficient to provide the long-chain n-3 fatty acids needed for normal growth and development. Other studies have shown that if all of the n-3 fatty acids in the diet are to be provided in the form of a-linolenic acid, it can take as much as 1% of dietary ME to maintain normal brain and retinal development (Innis, 1991). Those findings are consistent with the observations of Greiner et al (1996) indicating that the efficiency with which docosahexaenoic acid is incorporated into brain and retinal lipids of fetal and infant rhesus monkeys is about 10 times higher than that of a-linolenic acid and the data from Su et al (1999b) showing a 7-fold higher efficiency in neonatal baboons. A large body of literature

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Nutrient Requirements of Nonhuman Primates: Second Revised Edition, 2003 indicates that the amount of long-chain n-3 polyunsaturated fatty acids required in the diet is less than the requirement for a-linolenic acid alone (reviewed by Innis, 1991; Greiner et al., 1997; Su et al. 1999a). The patterns of brain development in rhesus monkeys show a growth spurt in the last trimester of fetal development; the brain of a newborn weighs nearly 70% as much as that of an adult (Venkatraman et al., 1992). The presence of n-3 fatty acids, and particularly the long chain docosahexaenoic and eicosapentaenoic acids, in the diet of pregnant females (Greiner et al., 1996), is therefore critical for sustaining normal brain development. The work of Conner and associates (Connor et al., 1984; Neuringer et al., 1984; Neuringer et al., 1986; Lin et al., 1990; Reisbick et al., 1991; Lin et al., 1994; Reisbick et al., 1994) has demonstrated that a deficiency of n-3 fatty acids in diets of rhesus monkeys can result in demonstrable abnormalities in brain and retinal function. In a series of studies, two diets were fed to pregnant and lactating females, one with about 1% of dietary MEas a-linolenic acid (control) and one with less than 0.1% of dietary ME as a-linolenic acid (deficient). The infants raised on the n-3 fatty acid-deficient diet showed reduced visual acuity by the age of 4 weeks (Neuringer et al., 1984). Deficient monkeys also showed a tendency toward increased intake of water and other fluids (Reisbick et al., 1991) and more stereotypical behavior than the control monkeys (Reisbick et al., 1994). Observed biochemical changes included reduced docosahexaenoic acid levels in the phospholipids of brain and retina and replacement with long-chain n-6 fatty acids, principally 22:5 n-6 (Lin et al., 1990). Replenishment of the deficient diet with long-chain n-3 fatty acids from fish oil for 14 months resulted in complete reversal of the patterns of n-6 and n-3 fatty acids in brain phospholipids. The remodeling of brain phospholipids appeared to occur normally without significant loss of n-3 fatty acids (Innis, 1991). Furthermore, observations of Kanazawa et al. (1991) in cynomolgus monkeys and in Japanese macaques showed that the ability of the brain tissue to convert a-linolenic acid into docosahexaenoic acid is age-dependent, being essentially zero in newborn primates and increasing maximally in young adults. Nevertheless, apparently adequate amounts of docosahexaenoic acid are deposited in the brains of monkeys fed diets in which essentially the only n-3 fatty acid is a-linolenic acid (Lin et al. 1990). That indicates that other tissues, predominantly the liver, have the desaturases and elongases needed for conversion of a-linolenic acid to the docosahexaenoic acid required for lipid deposition in the gray matter of developing brain and in the retina. In the case of monkeys, in which much of brain development occurs in utero, the transfer of n-3 fatty acids across the placenta into the fetus supplies a major portion of the requirements for early life (Innis, 1991). The amounts of n-3 fatty acids that must be consumed for adequate deposition of docosahexaenoic acid in the developing nonhuman primate brain can be estimated by extrapolation (Kanazawa et al., 1995) from human data (Clandinin et al., 1980a, 1980b). However, it is difficult to define an exact dietary requirement because much of the needed n-3 fatty acid will be derived in utero from the mother, and the efficiency of this transfer process is unknown (Greiner et al., 1996). Subsequent studies by the same group (Greiner et al., 1997) gave the estimate of the requirement as 0.45% of ME as a-linolenic acid or 0.30% of ME as docosahexenoic acid (22:6 n-3) in fetal baboons for normal brain development. Other data show that diets of rhesus monkey mothers with 1% of ME as a-linolenic acid were adequate to maintain normal fetal brain development, as were infant diets that contained about 2% of ME as a-linolenic acid (Lin et al., 1990; Neuringer et al., 1984). Furthermore, the data derived from studies of African green monkeys showed that diets for mothers and infants containing about 0.25% of ME as long-chain n-3 fatty acids (eicosapentaenoic and docosahexaenoic acids), with another 0.2% of a-linolenic acid, resulted in normal development (Wolfe et al., 1993). In the mother’s milk, about 0.6% of ME was found as 22:5 n-3 and 22:6 n-3 fatty acids, and 0.2% as a-linolenic acid. Therefore, in the absence of dose-range studies, those data form the basis of the minimal amounts of n-3 fatty acids recommended for nonhuman primate diets. It is recommended that 0.5% (by weight) of dietary dry matter (about 1% of ME) be present as n-3 fatty acids to support normal development and maintenance of the brain and nervous system. ESSENTIAL n-6 FATTY ACIDS Research showing that n-6 fatty acids are dietary essentials for nonhuman primates was published by Greenberg and Moon (1961), who documented changes in blood fatty acids in rhesus monkeys fed a linoleic acid-deficient diet. Subsequently, Greenberg (1970) showed that diets containing corn oil at about 2% by weight (about 4% of ME) prevented the deficiency. Portman et al. (1959, 1961) demonstrated linoleic acid deficiency in cebus monkeys and described the changes in physical appearance of the animals and many biochemical changes in fatty acid composition and concentration in their tissues. Substantial pathophysiologic changes in cebus monkeys fed a fat-free diet for 19 months were limited to scaly skin, hyperplastic bone marrow, erythrophagocytosis by the reticuloendothelial system, and undersized gonads. The link between requirements for polyunsaturated fatty acids and vitamin E was studied in rhesus monkeys made vitamin E-deficient (Fitch et al., 1961, 1963). It was shown that vitamin E deficiency could be induced by using diets either unsupplemented

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Nutrient Requirements of Nonhuman Primates: Second Revised Edition, 2003 or supplemented with fat, but the level of vitamin E required for normalcy was higher in fat-supplemented diets, presumably because of the involvement of vitamin E in preventing lipid peroxidation. When dietary fats were more saturated, the requirements for vitamin E were lower. When monkeys are fed fat-deficient diets, the simultaneous occurrence of fatty acid deficiency and vitamin E deficiency is of concern because absorption of fat-soluble vitamins is limited in the absence of sufficient quantities of dietary fat, and some of the documented pathologic changes might have been due to vitamin E deficiency. The minimal amount of linoleic acid required in the diet of nonhuman primates is not known with certainty. The milk fat of many of the primate species described above contained about 10-15% linoleic acid, or a minimum of about 5% of GE. It should be noted that nonhuman-primate milk fat also typically contains 1-1.5% arachidonic acid, the bioactive metabolite of linoleic acid, which is the most prominent precusor of eicosanoids, bioactive molecules used in many signal transduction processes within and among cells. Linoleic acid and its metabolites (arachidonic acid (20:4 n-6), di-homo-γ-linolenic acid (20:3 n-6) and docosapentaenoic acid (22:5 n-6) are a significant part of the fatty acids in the developing brain, and to a lesser extent in membrane lipids in other tissues. Thus, for normal growth and development, it is essential that adequate linoleic acid or its products such as arachidonic acid be consumed in the diet. Su et al. (1999b) studied the kinetics of conversion of linoleic acid to its bioactive products in pregnant and fetal baboons fed a diet with 2% ME as linoleic acid and 0.2% α-linolenic acid, possibly a less than maximal dose of n-3 fatty acid as discussed above. The data suggest that the fetus derives about half of its arachidonate from conversion of linoleic acid and half from the diet, with the amount in the brain plateauing by 21 days after dosing. The ratio of n-6 to n-3 fatty acids in the diet was 10:1, a dose considered representative of the normal for human adults. The authors point out that this ratio might have affected the outcome, depending on the competition among n-3 and n-6 fatty acids for the desaturases and elongases required for conversion. From the available data, we can only infer that the minimal requirements of infant monkeys for linoleic acid and its metabolites may be in the range of 5% of dietary GE. Greenberg (1970) replaced about 2% of dietary ME with linoleic acid in young adult rhesus monkeys and appeared to reverse the signs of n-6 fatty acid deficiency, so the requirement in older animals may be somewhat less than in infant monkeys, perhaps 1-2% of dietary ME. Therefore, it is recommended that the dry matter in diets for nonhuman primates contain 2% of linoleic acid by weight to avoid a deficiency of n-6 polyunsaturated fatty acids. A separate issue is the upper limit of acceptable concentrations of n-6 polyunsaturated fatty acids in the diet. The value would appear to be high, on the basis of laboratory experiments with African green monkeys in which n-6 fatty acids in milk made up 42% of total fatty acids or about 20% of ME, and a n-6:n-3 ratio of >50 with no demonstrable ill effects on infant growth and development (Wolfe et al., 1993). Maintaining an optimal ratio of dietary n-3 to n-6 fatty acids has been proposed so that interactions among desaturase and elongase enzymes, involved in synthesis of long-chain polyunsaturated fatty acids, do not exacerbate any deficiency (Innis, 1991). However, data are insufficient to know where that is a concern. Normal development has been observed over a wide range of n-3:n-6 ratios. DETRIMENTAL FATTY ACIDS Fatty acids that could cause harmful effects in primates include the long-chain, monounsaturated docosaenoic acids—22 carbons with one n-9 or n-11 double bond (Loew et al., 1978; Schiefer et al., 1978). Diets studied contained very high concentrations (25% by weight or about 50% of ME) of rapeseed oil or partially hydrogenated herring oil. The Cebus monkeys in the studies had very high intakes of 22-carbon fatty acids with one double bond (constituting up to 25% of total fatty acids in rapeseed oil and 24% of the herring oil used in the experiments). Laboratory-reared animals were fed the diets for 120-170 days and were then killed and their hearts examined. A mild degeneration of cardiac and skeletal muscle with lipid infiltration was noted, although the pathophysiologic changes were mild compared to that seen in rats. It should be mentioned that the rapeseed now used for making canola oil has been genetically modified so that concentrations of docosaenoic acid are no longer increased. CHOLESTEROL A minimal dietary cholesterol concentration has not been established. Monkey milk has cholesterol at 10-20 mg·dl−1, which is equivalent to about 0.06 mg·GEkcal−1 (Wolfe et al., 1993). That would provide an infant with milligram quantities of cholesterol that could be used for brain development or incorporated into cell membranes of many tissues. However, commercial monkey biscuits that have essentially no cholesterol and have been shown to be hypocholesterolemic (Rudel, 1997) have been fed to many mothers during in utero development of the fetus and during nursing of infants with no obvious ill effects. Thus, a dietary requirement for cholesterol seems unlikely. If cholesterol is not available in the diet, it is synthesized in tissues that require it or is transported into those tissues from plasma via LDL receptor after synthesis in the liver or intestine (Brown and Goldstein, 1986). Dietschy and

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Nutrient Requirements of Nonhuman Primates: Second Revised Edition, 2003 Wilson (1968) showed essentially all tissues in the body of the squirrel monkey have the capacity to synthesize cholesterol. PRIMATES AS CARDIOVASCULAR DISEASE MODELS The typical diet of Western humans is rich in fat and cholesterol, and both constituents are believed to contribute to the coronary heart disease (CHD) epidemic in Western societies. Many studies have been conducted in nonhuman primates (reviewed in Strong, 1976) using diets imitating the Western diet to identify the nutritional factors important in development of atherosclerosis (hardening of the arteries), the disease process underlying CHD and the leading cause of death in Western societies (Marmot, 1992). When diets are fed containing 35-40% of ME as fat of different types, nonhuman primates do not develop significant atherosclerosis. However, when cholesterol is added to such diets, most species develop a degree of hypercholesterolemia that is species-specific (Rudel, 1997). Studies of the sensitivity of Macaca to dietary induction of atherosclerosis have included the rhesus monkey (Macaca mulatta), cynomolgus monkey (Macaca fascicularis), and pigtailed macaque (Macaca nemestrina) (Strong, 1976). Macaques, in general, are highly diet responsive, with cynomolgus monkeys and pigtailed macaques being particularly sensitive. Vervet monkeys (Cercopithecus aethiops) and patas monkey (Erythrocebus patas) are less so and require more dietary cholesterol to induce hypercholesterolemia and atherosclerosis (Rudel, 1997). The baboon (Papio spp.) has been extensively studied, is among the most diet-resistant primate species, and requires a dietary cholesterol concentration of 1.7 mg·MEkcal-1 for atherosclerosis to develop (McGill et al., 1981). If nonhuman primates are maintained on a hypercholesterolemic diet long enough, usually several years, coronary artery atherosclerosis will develop (Rudel et al., 1995a), and the coronary artery lesions will show essentially all of the characteristics seen in atherosclerosis in humans (Rudel et al., 1995b). Nonhuman-primate diets enriched in n-3 and n-6 polyunsaturated fatty acids appear to protect against coronary arterial atherosclerosis, whereas diets enriched in saturated and monounsaturated fatty acids appear to promote the disease, as demonstrated in several studies (Rudel et al., 1995a; Rudel et al., 1995b; Rudel et al., 1998; Wolfe et al., 1994). The phytoestrogen content of soy is protective, and the primate model has been useful in clarifying these effects (Anthony et al., 1997; Clarkson et al., 2001). 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