n-6 Fatty Acid Requirements
In studies on EFA, C18:2n-6 and C20:4n-6 have been emphasized because mammals have an absolute requirement for the n-6 family of FA. EFA are required for stimulation of growth, maintenance of skin and hair growth, regulation of CH metabolism, lipotropic activity, and maintenance of reproductive performance, among other physiologic effects. On a molecular level, EFA are components of specific lipids and maintain the integrity and optimal levels of unsaturation of tissue membranes. Because EFA are necessary for normal function of all tissues, the list of symptoms of EFAD is long (111). Detailed studies on the symptoms of EFAD have been done in young rats, in which EFAD was found to be avoided by providing 1 to 2% of calories as C18:2n-6 (112). In these rat studies, classic signs of EFAD included reduced growth rates, scaly dermatitis with increased loss of water by a change of skin permeability, male and female infertility, and depressed inflammatory responses. Also observed during EFAD are kidney abnormalities, abnormal liver mitochondria, decreased capillary resistance, increased fragility of erythrocytes, and reduced contraction of myocardial tissue (113).
C18:2n-6 is specifically required in the skin to maintain the integrity of the epidermal water barrier. In this regard, C18:2n-6 seems to be required as an integral component of acylglucoceramides. Animals with EFAD lose considerable amounts of water through the skin, which limits growth rates. Repletion of C18:2n-6 at 1% of calories corrects excessive transepidermal water loss, and growth is restored (114). Although transdermal water loss during EFAD symptoms may reflect the role of C18:2n-6 as a key component of skin acylglucoceramides, the major metabolic effects of C18:2n-6 derive from its further metabolism to C20:4n-6 and thence to eicosanoids. In EFAD, platelet adherence and aggregation are impaired because of limited thromboxane synthesis secondary to limiting supplies of C20:4n-6 and possible inhibition by accumulated eicosatrienoic acid C20:3n-9. The action of eicosanoids in modulating the release of hypothalamic and pituitary hormones has been indicated to be a major factor in the role of the n-3 and n-6 EFA in supporting growth and development (103). The skin is subject to rapid infection, and surgical wounds heal very slowly in humans who have EFAD. This probably reflects the lack of C20:4n-6, which is required for eicosanoid-mediated protective inflammatory and immune cell functions and for tissue proliferation (103). Monocyte and macrophage function is defective in EFAD because eicosanoid production is impaired. The scaliness of the skin of an EFA-deficient patient has been ascribed to insufficient synthesis of PG, and the efficacy of various EFA of the n-6 type against the scaly dermatitis has been demonstrated at low dose levels.
Columbinic acid (C18:3n-6, 9, 13 cis, cis, trans), found in the seed oil of the columbine, Aquilegia vulgaris, and dihomocolumbinic acid (C20:3n-6, 9, 13 cis, cis, trans) have been used to differentiate the roles of EFA as structural components in biomembranes versus their roles as eicosanoid precursors ( 115). Neither columbinic acid nor dihomocolumbinic acid can be converted to PG; however, columbinic acid can be incorporated into membrane PL in contrast to dihomocolumbinic acid. As EFAD results in decreased tissue concentrations of C20:4n-6, EFAD symptoms are worsened further by dietary addition of dihomocolumbinic acid. Columbinic acid given to EFA-deficient rats, either orally or by topical skin application, efficiently restores their growth rate and normal skin function ( 114). When EFA-deficient rats treated with columbinic acid became pregnant, however, they died of inadequate labor during parturition, since uterine labor depends on normal PG biosynthesis ( 116).
One of the most often used and sensitive diagnostic indicators of EFAD in all species tested, including humans, is the triene (n-9):tetraene (n-6) ratio ( HI); C20:3n-9 (triene) is the major product derived from nonessential FAs. C20:4n-6 with four double bonds (tetraene) is the major metabolite of C18:2n-6. The triene:tetraene ratio in plasma remains below 0.4 when dietary EFA are adequate and increases to above 0.4 with EFAD. Dietary intake of adequate amounts of EFA decreases formation of triene as a consequence of competitive inhibition among families of PUFA for desaturases and acyl transferases. If EFA are not available, the biosynthesis of PUFA with three double bonds derived from C18:1n-9 and C16:n-7 continues, leading to the accumulation of n-9 FA, specifically C20:3n-9, resulting in turn in an increased plasma triene:tetraene ratio. Feeding diets with 0.1 to 0.5% of C18:2n-6 normalizes an abnormally high triene/tetraene ratio in a few days ( 117). The optimum dietary C18:2n-6 intake required for a ratio less than 0.4 and prevention of EFAD symptoms is 1 to 2% of total calories. The triene:tetraene ratio, however, does not resolve if the EFAD is caused by a lack of either n-3 or n-6 EFA, since adequate intake of either C18:2n-6 or C18:3n-3 prevents synthesis of C20:3n-9 ( 118).
The exact requirement for EFA in humans is not clearly defined but is apparently very low. The first study of EFAD, in human adults maintained for 6 months on a diet extremely low in fat, did not produce dramatic symptoms (119). It has been suggested that because adults contain approximately a kilogram of C18:2n-6 in body stores, depletion of EFA stores to produce deficiency symptoms would require maintaining an EFAD diet for more than 6 months. Most diets contain enough EFA or their metabolic products to meet daily EFA requirements; thus EFAD is relatively rare in humans. When it does occur in humans, some of the symptoms characteristic in animals, such as abnormal skin conditions, increased susceptibility to infection, and an increase in triene:tetraene ratio, are observed.
An important role for C20:4n-6 in optimal fetal development has been suggested because C20:4n-6 exerts growth-promoting effects ( 120). Crawford et al. (121) demonstrated that mothers of low-birth-weight infants had lower intakes of C18:2n-6 than mothers of normal-birth-weight infants. However, lower C20:4n-6 concentrations in plasma and in plasma PC have been associated with depressed intrauterine and extrauterine growth, despite adequate dietary C18:2n-6 levels (122). In a doubly blinded, randomized, controlled trial, depressed plasma PC C20:4n-6 concentrations induced by supplementation of formulas with C20:5n-3-rich marine oils were associated with slower growth rates in preterm infants (123). Supplementation of formula with a low C20:5n-3-concentration marine oil caused relatively minor decreases in plasma PC C20:4n-6 concentration and in weight-to-length ratio in preterm infants (124).
Long-chain EFA of 20- and 22-carbon chain length are incorporated about 10 times more efficiently into the developing brain than are the parent EFA. However, whether term or preterm infants have sufficient enzymatic activity to synthesize their own long-chain PUFA from EFA to meet their requirement for brain growth and development is controversial. Despite knowledge that the developing and mature brain can desaturate and elongate C18:2n-6 and C18:3n-3 to their respective long-chain PUFA products and that brain and retina can incorporate C20:4n-6 and C22:6n-3 from plasma, the quantitative importance of these two pathways is uncertain. Lower levels of C20:4n-6 in the red blood cell PL of formula-fed infants than in PL from breast milk-fed infants has led to debate about whether C20:4n-6 is essential for optimal central nervous system development in infants (125). The lower erythrocyte C20:4n-6 levels of formula-fed babies (vs. breast-fed babies) can be normalized by inclusion of C20:4n-6 in formula. Stable isotope studies have indicated in vivo C20:4n-6 synthesis in term infants, but the rate of this synthesis is low and only about 6% of total plasma C20:4n-6 is renewed in this manner (126). However, postmortem studies of brain FA composition showed that brain C20:4n-6 is maintained in formula-fed infants (127).
The concept has emerged that an optimal ratio of n-3 and n-6 FA is required in the diet because n-3 and n-6 families compete for eicosanoid production. Various authorities have recommended that at least 3% of daily calories be provided as linoleate, to prevent EFAD; however, equal amounts of C18:2n-6 and various SAFA have been recommended to reduce serum CH for the prevention of atherosclerosis (117). Advocacy for increased intake of vegetable oils rich in C18:2n-6 has resulted in C18:2n-6 consumption of approximately 6 to 7% of calories in the United States, leading to a ratio of n-6:n-3 PUFA consumption above 10 ( 117). Although this amount of C18:2n-6 may be beneficial for reduction of elevated plasma CH in those on a high-fat diet, it has been argued that an n-6:n-3 PUFA ratio exceeding 10 is imbalanced compared with n-6:n-3 ratios of 2 to 4 found in food lipids of hunter/gatherer societies ( 103, 117). There is concern that a high intake of C18:2n-6 relative to n-3 PUFA may lead to excessive or imbalanced eicosanoid production conducive to various pathophysiologies. The optimal n-6:n-3 ratio in the diet is not yet clear and may vary with developmental stage, the presence of long-chain EFA, and other factors. Some authorities have suggested that the n-6:n-3 EFA ratio should be in the range of 4:1 to 10:1 (128); others believe optimal n-6:n-3 ratios to be 4:1 or lower (129, 130).
n-3 Fatty Acid Requirements
Requirements for n-3 FA have been less definitive because it has been difficult to demonstrate their essentiality in animal studies; n-3 FA levels in mammalian tissues are generally much lower than n-6 FA levels. Biochemical studies have indicated differences in the metabolism and tissue distribution of the two series of EFA. C20:4n-6 and C20:3n-6 tend to predominate in liver and platelets, while the main biologic activity of long-chain n-3 EFA appears to reside in retina, testes, and the central nervous system. C18:3n-3 is similar to C18:2n-6 with regard to growth rate, capillary resistance, erythrocyte fragility, and mitochondrial function. Dietary C18:3n-3 and C20:5n-3 are inferior to C18:2n-6 and the other n-6 PUFA in resolving skin lesions and preventing transepidermal water loss. Because of the inability of C18:3n-3 to normalize all physiologic functions during EFAD and because EFA activities attributed to C18:3n-3 were also expressed equally or more potently by C18:2n-6, n-3 FA were until recently designated nonessential or partially essential.
In the past 15 years, studies have suggested that n-3 FA may be essential in development of neural tissue and visual function, beyond the requirement for n-6 FA, for which they can partially substitute. Across mammalian species, levels of C22:6 n-3 in brain and retinal PL are extremely stable despite wide variations in diet ( 131). The strong affinity of brain lipids for C22:6n-3 suggest a requirement for n-3 EFA, but this requirement is difficult to study because n-3 EFAD develops only under extreme dietary conditions (125, 131). In particular, C22:6n-3 is selectively retained by the brain, and depletion of C22:6n-3 is difficult after weaning. Multigenerational studies in rats have been needed to produce drastic reductions in brain C22:6n-3 levels. For example, feeding rats fat-free diets from weaning reduced retinal C22:6n-3 concentrations in adults by only 10 to 20%. In the first generation, feeding diets containing 2.5% C18:2n-6 and free of n-3 PUFA decreased C22:6n-3 concentrations by 60% and in the second generation by more than 87% (132).
An essential role for C22:6n-3 in brain and retinal PL was described by Neuringer and Connor, who demonstrated C18:3n-3 deficiency in rhesus monkeys fed during gestation diets with safflower oil (n-6:n-3 ratio of 255:1) as the sole source of fat (133). Their offspring reared on the same diet developed abnormal electroretinograms compared with those of the control group of offspring fed soybean oil (n-6:n-3 ratio of 7). Decreased concentrations of C18:3n-3 and long-chain n-3 PUFA in plasma PL were observed in offspring who showed loss of visual activity. Learning capacity, as tested in a spatial-reversal learning task, was not affected, possibly because of the observed compensatory increase of n-6 PUFA, particularly C22:5n-6, in PL. Retinal n-3 PUFA deficiency was reversed at the ages of 10 and 24 months by feeding a fish oil diet rich in C20:5n-3 and C22:6n-3 (133). Although such extremely high n-6:n-3 ratios rarely occur in human nutrition because of the wide availability of n-3 PUFA in foods, these ratios have been induced by total parenteral nutrition. A 6-year-old child developed peripheral neuropathy and periods of blurred vision after receiving total parenteral nutrition whose sole source of lipid was a safflower oil emulsion ( 1). After 5 months, she experienced episodes of numbness, weakness, inability to walk, leg pain, and blurred vision. Very low serum concentrations of C18:3n-3 and other n-3 PUFA were detected. Replacement of the lipid source by a soybean oil emulsion containing C18:3n-3 caused all symptoms of deficiency to disappear, and serum concentrations of n-3 PUFA returned to normal (1). Recent reports on neurologic symptoms in an infant, associated with a parentally fed C18:3n-3-poor formula, and deficiency symptoms in adults that were corrected by C18:3n-3 support the essentiality of this FA in the diet (1, 134). In the above cases, however, the C18:3n-3 deficiency symptoms could arguably be attributed to low levels of vitamin E or total EFAD (127).
As human brain gray matter and retinal membranes contain significant amounts of C22:6n-3, the requirement for n-3 EFA may be more critical during the last trimester of gestation and first months of life, when rapid accretion of these FA occurs in the central nervous system (125, 131). Brain PL acquires only long-chain derivatives of EFA, not their 18-carbon precursors, and C22:6n-3 is the predominant PUFA in PL in synaptosomal membranes and photoreceptors (131). C22:6n-3 also accounts for approximately 50 to 60% of FA in the PL of the photoreceptor disks that contain rhodopsin and the G-protein. Much of the C22:6n-3 acquired by the brain is accrued during the suckling period, when the brain undergoes rapid development. A number of animal studies have demonstrated an impairment in the visual process, altered learning behavior, and low brain C22:6n-3 content because of a deficiency in C18:3n-3 and its metabolites C20:5n-3 and C22:6n-3 ( 125, 131). Permanent learning defects and alterations in synaptic function in the brain, observed in EFAD during pregnancy, can be prevented by feeding n-3 EFA ( 133, 135). In addition, a correlation has been noted between diet-induced changes in C22:6n-3 in the retina and a modification of electrical potentials induced in rod outer segments by light stimulation (136).
Although adequate dietary intake of n-3 EFA appears to be critical for central nervous system development, the optimum requirements for n-3 EFA for infants are not known. Human milk provides both C18:3n-3 and C22:6n-3 that are often absent from most infant formulas on the market. Formula-fed infants thus depend on endogenous synthesis of long-chain PUFA. Infant formulas provide nutrition that results in growth rates equal or superior to those of breast milk-fed infants. There is a suggestion, however, that long-chain n-3 PUFA may not be synthesized from their parent EFA at optimal rates for brain development during the first few weeks after birth, particularly in preterm infants. Clandinin et al. ( 137) have indicated that the infant's requirement for neural accumulation of long-chain PUFA can be met by intake of long-chain PUFA alone, without endogenous synthesis. Using the FA composition of red blood cell PL as an index of cerebral membrane composition, infants fed human milk had a significantly better C22:6n-3 status than formula-fed infants (138). The extent to which diet-induced changes in red blood cell membranes reflect changes in brain PL is not clear. However, recent postmortem studies indicate a lower C22:6n-3 brain content in formula-fed infants than in infants receiving breast milk (139). In a randomized trial of n-3 PUFA supplementation of formulas fed to term infants, C22:6n-3-treated infants had better visual acuities than infants fed standard formula (140). Other work, however, has shown no effect of long-chain PUFA formula supplementation on visual, psychomotor, or mental development (141).
Preterm infants may be especially susceptible to n-3 EFAD because of their relatively immature desaturase and elongase enzyme systems and their low fat stores. In two randomized clinical trials, intake of formula containing marine oil by preterm infants normalized blood levels of C22:6n-3 and improved certain aspects of visual function relative to breast-fed infants (142). In one of the randomized studies, however, marine oil supplementation was associated with decreases in linear growth, some measures of cognitive development, and blood C20:4n-6 content. These results are of concern in view of the important role of C20:4n-6 in growth and development (123). However, a more physiologic formulation containing pure C22:6n-3 resulted in better cognitive and visual performance and a less detrimental effect on growth than the mixture of C20:5n-3 and C22:6n-3 given over a shorter interval (124, 143).
Rapidly developing fetal organs, such as the liver and brain, incorporate large amounts of long-chain n-3 and n-6 EFA into membrane PL ( 144). The accumulation of EFA during human pregnancy has been approximated to be 620 g, which includes the demand for fetal, placental, mammary gland, and uterine growth and the increased maternal blood volume. On the basis of this estimate of expected EFA acquisition by maternal tissues and the conceptus, it is advised that maternal EFA consumption during pregnancy be increased from 3 to 4.5% of calories (145). In circumstances of relatively low dietary intake of n-6 EFA (i.e., 2 to 4% of calories), EFAD may be more likely to develop during periods of rapid cell division and growth.
In well-nourished mothers, approximately 4 to 5% of total calories in human milk is present as C18:2n-6 and C18:3n-3, and a further 1% as long-chain PUFA derived from these FA, amounting to about 6% of total energy as EFA and its metabolites. The efficiency of conversion of dietary EFA into milk FA is not clear; however, an additional 1 to 2% of calories in the form of EFA is recommended during the first 3 months of lactation. Another 2 to 4% of calories above the basic requirement is recommended thereafter (145).
The optimum requirements for EFA of the n-6 and n-3 families for infants are still not known, although normal growth of infants depends on an adequate supply of EFA. Growing individuals apparently require a minimum of 1 to 4.5% of total calories as C18:2n-6 to ensure an adequate supply of EFA for tissue proliferation, membrane integrity, and eicosanoid formation (128, 146). The need for n-3 EFA has been indicated to be higher during growth and development. Estimates based upon FA compositional data from autopsy tissue and breast milk n-3 EFA concentrations have ranged from 0.5 to 1.2% of calories (146). The Canadian Nutrition Recommendations suggest infant dietary intakes of C18:3n-3 of 1% of energy in the absence of intake of long-chain n-3 PUFA, compared with C18:3n-3 of 0.5% of energy when a supply of long-chain n-3 EFA is available in the diet (128). However, the bioequivalency of C18:3n-3 and its long-chain products, C20:5n-3 and C22:6n-3, has not yet been determined, although long-chain PUFA clearly contribute to the C20:5n-3 and C22:6n-3 content of plasma and erythrocyte PL ( 131). Another question that needs to be addressed is whether long-chain PUFA, especially C22:6n-3, are conditionally essential for optimal visual and neural development of preterm and term infants.
For adults, appropriate minimum amounts of n-6 EFA are in the range of 1 to 4% of energy to prevent signs of EFAD (131). The C18:3n-3 requirements for adults are suggested to range from 0.2 to 0.3% of energy to 1% of energy, although more studies are needed to define the minimal requirements in humans ( 3, 147).
Several dietary components are known to affect EFA requirements because of their interactions with EFA use or metabolism. Dietary SAFA slightly increase EFA requirements, as evaluated by growth and dermal symptoms of deficiency and the triene:tetraene ratio in plasma (148). This effect has been related to the action of SAFA in raising plasma levels of CH that forms esters with PUFA, thereby depleting the availability of the EFA pool for PL. In addition, in several animal species, induction of serum CH via a high-CH diet can aggravate EFAD. cis-MUFA (mainly C18:1n-9 and its product C20:3n-9) can replace EFA in the lipids of EFAD animals and humans. High dietary levels of C18:1n-9 suppressed desaturation of EFA such that if dietary concentrations of C18:1n-9 were 10 times higher than that of C18:2n-6, triene:tetraene ratios indicating EFAD were observed (149). Partial hydrogenation of vegetable oils in the production of margarines and shortenings forms SAFA and a variety of trans and positional isomers. The estimated average daily trans FA intake is 8 to 10 g, or 6 to 8% of the total dietary FA. Trans-MUFA increase the EFA requirement in animals when fed at moderate levels and can influence the desaturase reactions critical to the metabolism of PUFA ( 150). Trans-FA can also raise plasma levels of LDL and total CH, which could further increase EFA use.
Although development of human EFAD has traditionally been regarded as rare, use of the sensitive triene:tetraene ratio as a diagnostic index has recently indicated the existence of EFAD in a number of high-risk clinical conditions. EFAD appears to be exacerbated by increased metabolic demands associated with either growth or the hypermetabolism seen following stress, injury, or sepsis (151).
The supply of C18:2n-6 is of concern in premature infants because of their borderline stores of EFA and high caloric expenditure ( 151). Unless C18:2n-6 is supplied to premature infants in parenteral or enteral diets, early onset of EFAD may occur. Biochemical changes in the plasma and clinical signs indicating EFAD can develop rapidly within 5 to 10 days of life in premature infants (151, 152).
In patients receiving long-term parenteral nutrition without lipid, continuous glucose infusion results in high circulating levels of insulin that inhibit lipolysis and depress release of EFA from adipose fat stores (131). Development of EFAD in infants, children, and adults maintained on continuous fat-free or minimal-fat parenteral nutrition has been reversed by oral or intravenous administration of C18:2n-6 (151). Parenteral nutrition containing only amino acids and completely free of glucose does not produce evidence of EFAD (153). Clinical signs of EFAD include alopecia, scaly dermatitis, increased capillary fragility, poor wound healing, increased platelet aggregation, increased susceptibility to infection, fatty liver, and growth retardation in infants and children ( 153).
EFAD development has been described in several human diseases, including cystic fibrosis (154), acrodermatitis enteropathica (149), peripheral vascular disease (PVD) (155), and multiple sclerosis (156). Enteral supplementation of vegetable oils high in C18:2n-6 has been demonstrated to improve EFAD in patients with cystic fibrosis (154). Children with cystic fibrosis may require 7 to 10% of energy as C18:2n-6 to prevent reduced weight gain and growth, and infants with cystic fibrosis may require formula with a C18:2n-6 content above 12% of total calories (154, 1.57). Subjects with anorexia nervosa may have EFAD exhibited by plasma PL profiles showing lowered n-6 and n-3 PUFA concentrations (158). Low total plasma PUFA concentrations, particularly those of 20- and 22-carbon n-3 PUFA, have been noted in patients with acquired immune deficiency syndrome (AIDS) (159). Development of EFAD as measured by the triene:tetraene ratio has been demonstrated in elderly patients with PVD (160), in subjects with fat malabsorption after major intestinal resection, during low-fat, high-protein dietary supplementation for treatment of kwashiorkor (161), and after serious accidents and burns. Oral or intravenous feeding of C18:2n-6-containing TG corrects the biochemical and clinical abnormalities in these conditions.
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