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Home , Docosapentaenoic acid, Essential fatty acid, Triglyceride

European Journal of (1999) 53, Suppl 1, S66±S77 ß 1999 Stockton Press. All rights reserved 0954±3007/99 $12.00 http://www.stockton-press.co.uk/ejcn

Essential fatty acids as determinants of requirements in infants, children and adults

R Uauy1,2*, P Mena1 and A Valenzuela1

1Institute of Nutrition and Technology (INTA), University of Chile, Casilla 138-11, Santiago, Chile; and 2Retina Foundation of the Southwest, Dallas, Texas

Essential fatty acids (EFA) are the indispensable component of the lipid supply beyond the provision of energy as a fuel for oxidation. They serve as dietary precursors for the formation of and other thus are of great signi®cance in health and modulation of disease conditions. Eicosanoids are powerful autocrine and paracrine regulators of cell and tissue functions: thrombocyte aggregation, in¯ammatory reactions and leukocyte functions, vasoconstriction and vasodilatation, blood pressure, bronchial constriction, and uterine contraction. Recent attention has focused on the effect of n-3 and n-6 long chain EFAs in normal fetal development. Results from human infant studies suggest that n-3 fatty acids are needed for optimal development of visual and brain function. Human is the best and only time proven source of and dietary essential fatty acids for infant feeding. International recommendations for n-3 and n-6 EFA dietary intake are reviewed and suggested intakes for long chain EFAs are provided.

Basis for essentiality of EFAs, dietary de®cit, structural in the food chain; ®sh and other marine animals are able to and biochemical functional correlations and disease elongate and desaturate the parent EFAs forming the long prevention chain polyunsaturated fatty acids (LCPUFAs) (Sprecher, 1981; Willis, 1984; Simopoulos, 1991). Animal tissues, Dietary essential fatty acids (EFA) have long been con- especially the , are capable of further elongating and sidered part of the lipid supply necessary for energy, desaturating the parent EFAs generating families of com- growth, cellular , and muscle activity. The pounds as shown in Figure 1. As depicted in the ®gure, fact that some EFAs serve as indispensable dietary pre- [AA (20:4, n-6)] can be formed from cursors for formation has provided greater [LA (18:2, n-6)]; thus it becomes essential signi®cance to the study of their role in health and disease. only if the capacity for elongation and desaturation of During the past decade increasing attention has been placed linoleic acid is limited. This is the case in the cat and on the effect of n-3 and n-6 EFAs in normal fetal and infant other felines. Further details on EFA metabolism can be development (Sprecher, 1981; Bazan, 1989; Willis, 1984; found in recent reviews (Sprecher, 1981; Willis, 1984; Uauy & Hoffman, 1991; Simopoulos, 1991; Innis, 1991; Innis, 1991). The competitive desaturation of the n-3, n-6 Uauy et al, 1989). Dietary intake also affects and n-9 series by delta-6 desaturase is of major signi®cance metabolism throughout the life cycle and is since this step is controlled by the interaction of hormones associated with cardiovascular morbidity and mortality. and diet including the amount of LA in the diet. Sprecher's group has further demonstrated that the formation of Basis for nutritional essentiality of EFAs and derivatives [DHA (22:6, n-3)], initially thought The concept that speci®c components of fat may be to be catalyzed by a delta-4-desaturase, is indeed a 3-step necessary for the proper growth and development of process as depicted in Figure 1. The initial step is an animals and possibly humans was introduced in the 1930s elongation, followed by delta-6 desaturation, and then, (Burr & Burr, 1929). Yet EFAs were considered of mar- through a peroxisomal beta-oxidation, the chain is shor- ginal importance until the 1960s when signs of clinical tened to a 22 PUFA. This latter step has been de®ciency became apparent in infants fed skim-milk-based termed retroconversion (Voss et al, 1991). The Sprecher formula and in those given lipid-free parenteral nutrition. pathway has now been veri®ed for both DHA and docosa- The essentiality of n-6 and n-3 FAs for humans is best pentaenoic acid [DPA (22:5, n-6)] formation. The delta-6 explained by the inability of animal tissue to introduce desaturase in the Sprecher pathway is probably different double bonds in positions prior to carbon 9. from the responsible for the initial step of the Biochemical pathways for n-3 and n-6 FA desaturation parent EFA metabolism. If n-3 FAs are absent or de®cient are only present in chloroplasts. Terrestrial and marine in the diet, the elongation=desaturation of the n-6 com- plants and phytoplankton are the primary source of EFAs pounds generates a signi®cant elevation of DPA; if both EFAs are lacking eicosatrienoic acid [ETA (20:3, n-9)] *Correspondence: Dr R Uauy, Insitute of Nutrition and accumulates (Sprecher, 1981; Uauy & Hoffman, 1991; (INTA), University of Chile, Casilla 138-11, Santiago, Chile. Innis, 1991). The triene=tetraene ratio may be used as an Essential fatty acids R Uauy et al S67 delta-5 desaturase activity has been considered responsible for the lower AA observed when marine is consumed. Excess LA, as seen in enterally or parenterally-fed patients receiving corn oil or saf¯ower oil as the predominant FA supply, will inhibit the elongation=desaturation of the parent EFAs, thus lowering the long chain PUFA supply necessary for membrane synthesis. Marine PUFAs provide minimal preformed AA and substantial amounts of pre- formed n-3 long-chain PUFA such as EPA and DHA (Brockerhoff et al, 1963). Figure 2 summarizes trends in the consumption patterns of over the last two centuries. Saturated fats from animal , trans fats from hydrogenated vegetable and marine , and n-6 fatty acids from oil seeds have all Figure 1 Metabolic transformation of essential fatty acids (EFA) to form increased, while n-3 fatty acids derived from plant and long chain polyunsaturated fatty acids (LCPUFA). Parent EFAs are derived from dietary sources for both n-3 (18:3, a-linolenic acid) and n- marine foods have declined. The ratio of n-6 to n-3 fatty 6 series (18:2, linoleic acid). is able to produce only n-9 acids in the western diet has increased. LCPUFAs. Elongation occurs two at a time and delta desaturases The ratio of LA to LNA in the diet is an important factor (D-9,-6,-5) introduce double bonds at 9, 6, and 5 carbons from the in de®ning the capacity to biosynthesize AA and DHA. The carboxylic moiety. The ®nal step in the formation of n-3 and n-6 end- biological equivalence of LNA relative to DHA, that is, products is catalyzed by a peroxisomal partial beta-oxidation. PUFAs of interest include 18:3, n-6 gamma linolenic acid (GLA), arachidonic acid how much LNA needs to be given to have a similar effect 20:4, n-6 (AA), 22:5, n-6 (DPA), eicosatrienoic acid as compared to the direct provision of DHA, is variable 20:3, n-9 (ETA), 20:5, n-3 (EPA) and docosahexa- depending on the level of LA in the diet, total energy enoic acid 22:6, n-3 (DHA). EPA, AA and 20:3, n-6 are immediate supply and provision of other fatty acids. Studies from precursors of (PG) and other eicosanoids. India based on several biological responses suggest that when LA intake is around 5% of total energy, 10% of LNA index of de®ciency but is not valid as a is converted to n-3 LCPUFA (Indu & Ghafoorunisa, 1992). marker of isolated n-3 de®cit. In the latter case the ratio of Emken et al (1994) using stable-isotope-labeled LNA DPA to DHA is a good marker. demonstrated in human adults that when LA intake was The conversion of parent EFAs to long-chain PUFAs is increased from 4.7 ± 9.3% of energy, conversion of LNA to under active regulation, therefore, the effects of providing n-3 LCPUFAs was reduced by 50%. In human infant AA, EPA or DHA cannot be reproduced by providing the studies, the biological responses to DHA supplementation equivalent amount of LA or LNA. The uniqueness of the have not been fully reproduced by LNA supplementation in biological effects of feeding human milk or marine foods terms of concentrations of DHA in plasma and red cell on EFA metabolism is based on the direct supply of long- . In some cases equivalence in functional chain PUFAs bypassing the controlling step of the delta-6 responses has been achieved, while in other studies this desaturase. Excess dietary LA associated with some vege- has not been possible. No conclusive data on this issue is table oils, particularly saf¯ower, sun¯ower and corn oils, available from isotopic studies in infants. At very low consumed in most western countries, may decrease the LA=LNA ratios the supply of LA may in fact be limiting, formation of DHA from a-linolenic acid [LNA (18:3, n-3)] therefore restricting the use of this strategy to optimize since the delta-6 desaturase is inhibited by excess substrate. LNA conversion to long-chain n-3 PUFAs. Other dietary In addition AA formation is lower when excess LA is factors such as overall energy and supply, as well as provided (Sprecher, 1981; Willis, 1984; Simopoulos, 1991; zinc and copper de®ciency, affect EFA metabolism; for Innis, 1991; Brenner & Peluffo, 1969). The inhibitory example zinc de®cit is associated with decreased activity of effect of eicosapentaenoic acid [EPA (20:5, n-3)] on delta-6 desaturase, thus low AA and DHA. Hormones such as increase conversion of EFA to long-chain deri- vatives (Innis, 1991; Brenner & Peluffo, 1969). The fetus and the placenta are fully dependent on maternal EFA supply for normal growth and development. The diet before pregnancy plays an important role in determining maternal EFA status since these essential are stored in and can be mobilized over time. The major EFA deposition in the human fetus occurs during the third trimester; phospholipids in placental vessels and uterine vasculature are dependent on EFA supplied by the mother for eicosanoid formation. Maternal preconceptional nutrition determines, in part, which speci- ®c fats accumulate in the conceptus and placental tissues (Sprecher, 1981; Galli & Socini, 1983; Ongary et al, 1984). Placenta takes up preferencially LCPUFA by a plasma Figure 2 Trends in fatty acid consumption patterns over the last two membrane fatty acid-binding protein located in the micro- centuries. Saturated fats from animal foods, trans fats from hydrogenated villous membrane, so LCPUFA are in higher concentration vegetable and marine oils, and n-6 fatty acids from oil seeds have all in the fetus than in the mother (Dutta-Roy, 1997). increased while n-3 fatty acids derived from plant and marine foods have declined. The ratio of n-6 to n-3 fatty acids in the western diet has The dry weight of the human brain is predominantly increased. lipid. Studies in several animal species and recent evidence Essential fatty acids R Uauy et al S68 from humans have established that brain Structural role of EFAs and derived compounds DHA decreases while n-9 and n-7 mono- and polyunsatu- The implications of compositional changes in structural rated fatty acids increase when LA and LNA or only n-3 have been extensively studied (Wheeler et al, 1975; fatty acids are de®cient in the diet (Galli et al, 1971; Menon Bourre et al, 1989c; Stubbs & Smith, 1984). Membrane & Dhopeshwarkar, 1982; Bourre et al, 1989a). Typically, physiologists have suggested that the fatty acid composi- n-3 fatty acid de®cient cells have decreased DHA and tion of structural membrane lipids can affect membrane increased levels of the end products of n-6 metabolism, function by modifying overall membrane ¯uidity, by docosapentaenoic acid (DPA). Within the subcellular orga- affecting membrane thickness, by changing lipid phase nelles, synaptosomes and mitochondria seem to be more properties, by speci®c changes in the membrane microen- sensitive to a low dietary n-3 supply (Menon & Dhopesh- vironment, or by interaction of fatty acids with membrane warkar, 1982; Bourre et al, 1989b). (Wheeler et al, 1975; Stubbs & Smith, 1984; Lee The high concentrations of long-chain derivatives et al, 1986). The changes in neural membranes that are of of EFAs in cerebral cortex and retina suggest their greatest potential signi®cance to humans, are changes participation in neural and visual function. The n-3 end- affecting physical properties and membrane excitability product DHA reaches levels of up to 40% of total FAs in the (Wheeler et al, 1975; Bourre et al, 1989c; Stubbs & phospholipids of these tissues (Bazan, 1989; Widdowson & Smith, 1984; Lee et al, 1986). Dickerson, 1981; Clandinin et al, 1980). In the human, Multiple studies have shown that a de®ciency of essen- Clandinin et al (1980) have shown signi®cant increases in tial fatty acids can change physical properties of mem- the AA and DHA content of brain tissue during the last branes including membrane-bound , receptor trimester of gestation. More recently Martinez (1992), using activity, antigenic recognition, and signal transduction improved methodology, has provided complementary data (Stubbs & Smith, 1984; Lee et al, 1986; Salem et al, on retinal and brain accretion of LCEFA. 1988). Also, n-3 fatty acid de®ciency affects rotational The potential for dietary EFA de®ciency has become an mobility, as measured by ¯uorescent diphenylhexatriene important issue in the nutrition of preterm infants since probes, less than lateral mobility, as measured by pyrene these do not receive the third trimester intrauterine supply dimer formation within the lipid bilayer (Stubbs & Smith, of DHA and AA. Even full term infants are potentially at 1984; Lee et al, 1986; Salem et al, 1988; Treen et al, 1992). risk of DHA de®ciency since most formulas are devoid of We have found that excited dimer (eximer) pyrene forma- this critical EFA. Recent investigations have demonstrated tion in human retinal cells, which occurs when two pyrene that term infants who died suddenly, without a clear monomers collide, is increased by DHA supplementation of explanation, present a diet-dependent fatty acid composi- the culture media (Treen et al, 1992). Membrane composi- tion of their brain cortex. Infants who were breastfed had tion changes in human retinoblastoma and neuroblastoma higher DHA and lower DPA and AA in their cortex cell lines also affect membrane transport mechanisms phospholipids than infants having received cow's milk- (Treen et al, 1992; Yorek et al, 1984). A higher DHA based formulas (Farquharson et al, 1992; Makrides et al, content increases the af®nity and transport rates for 1994). DHA content increases with advancing age in and (Treen et al, 1992; Yorek et al, 1984). Further- breast-fed infants and is proportional to the duration of more, preliminary studies suggest that the dietary n-3 fatty human milk feeding, while AA content remains stable. acid supply may affect nucleotide cyclase activity and Furthermore the dietary ratio of n-6 to n-3 fatty acids rybosylation of guanine nucleotide binding proteins appears relevant as the lowest DHA and highest DPA (Ahmad et al, 1990). contents in brain cortex phospholipids were observed in Most dietary n-3 fatty acid-induced membrane changes preterm infants fed formulas with a high LA to LNA ratio are not re¯ected by an overall change in membrane ¯uidity (Makrides et al, 1994). but rather result in selective changes in membrane micro- Studies in rodents and non-human primates have char- environment affecting speci®c domains. The replacement acterized the biochemical and functional effects of dietary of DHA by DPA usually results in the same overall lipid n-3 de®ciency (Wheeler et al, 1975; Watanabe et al, 1987; unsaturation level. Thus ¯uidity on average would remain Neuringer et al, 1984, 1991). The animal models used unchanged. Furthermore, the main changes in the physical puri®ed LA as the only dietary fat or saf¯ower or sun¯ower state, induced by changes in the fatty acid composition of oil in which the n-6=n-3 FA ratio (approximately 250:1) is lipid bilayers, occur after the ®rst and second double bonds very high (Neuringer et al, 1984, 1991). High n-6=n-3 FA are introduced; namely when a saturated fatty acid such as ratios are found in powdered infant formulas currently in (18:0) is replaced by (18:1, n-9) or by use in some parts of the world (Koletzko & Bremer, 1989; linoleic acid (18:2, n-6) (Treen et al, 1992; Yorek et al, Hansen, 1993). If these diets are fed from conception 1984). Others have suggested that DHA supply modi®es and=or during early life, signi®cant declines in DHA the phospholipid molecular species present in neural tissues levels and elevations of DPA content of brain, retina and and also gene expression, therefore possibly affecting over- of other tissues occur in animals. Similar data have been all function (Lin et al, 1990; Clarke & Jump, 1993). obtained from plasma, red blood cell (RBC) lipids in Recently Litman & Mitchell (1996) have reported that preterm human infants fed corn-oil-based formula with a LCPUFAs present in membrane phospholipid molecular high LA=LNA ratio (Hoffman & Uauy, 1992). species have profound effects on G protein activation The measurement of the fatty acid composition of RBC and related structural modi®cations. The rhodopsin lipids as a potential index of EFA status in retinal mem- MI $ MII equilibrium constant is six times higher with branes is based on the observed correlations between fatty di DHA acylated phosphatidyl choline (PC) than with acid pro®les in RBCs and neural=retinal tissue from rats, di myristic PC, the di DHA PC having an equilibrium non-human primates, and human infants (Makrides et al, constant that is almost identical to that of native rod disks. 1994; Carlson et al, 1986; Connor et al, 1993). Yet these The effect is mostly explained by the increase in membrane correlations are not always signi®cant. free volume. This greater mobility within the rhodopsin Essential fatty acids R Uauy et al S69 microenvironment most likely explains the change in G consistently reduced duration of slow-wave sleep compared protein activation and the corresponding enhanced signal with children who received TPN plus essential lipids. The transduction to photon stimuli (Litman & Mitchell, 1996). main difference was a lower cumulative amount of slow- Diet-induced changes in structural lipids affect the wave sleep time from the second hour of night-sleep functional characteristics of excitable membranes in several onwards. These results suggest that early lipid nutrition animal species and in human neural cell lines (Wheeler et may modulate sleep regulation and may have implications al, 1975; Neuringer et al, 1984; Bourre et al, 1989b; Love for whole body energy expenditure during sleep, since et al, 1985; Holh & Rosen, 1987). Electrocardiographic slow-wave sleep is associated with a lower metabolic rate abnormalities, such as a notching in the QRS complex, than other stages of sleep (Stother & Warner, 1977). indicating impaired electrical conduction, occur in linoleic Interestingly, no signi®cant difference was found in and a-linolenic acid de®ciency before clinical signs appear either total sleep time or intra-sleep waking in the EFA- (Caster & Ahn, 1963). Either linoleic or a-linolenic acid de®cient group. Alterations in sleep-wake cycle develop- corrected these abnormalities. More recently, it has been ment may become evident only if the methods used include shown that dietary fat in¯uences the susceptibility to evaluations that are more subtle than total sleep or waking cardiac arrhythmias, their incidence and severity (Char- time measures. The possibility that fat-free TPN affected nock, 1991). Furthermore, studies with myocardial prepara- structural brain lipids is suggested by these results. In tions have indicated that the vulnerability to addition the excess LA relative to LNA in lipids used in catecholamine-induced arrhythmia is reduced in animals parenteral nutrition has also been reported to affect brain fed either n-6 or n-3 PUFA-enriched diets (Abeywardena et fatty acid composition of infants receiving intravenous lipid al, 1987). Feeding ®sh oil from blue®n tuna rather than emulsions in early life. Higher linoleic and lower DHA sun¯ower oil and resulted in a marked reduc- content in brain phospholipid fractions have been reported. tion in induced arrhythmias in two animal species and in The functional effect of this has not been studied (Martinez, isolated papillary muscle (Charnock, 1991). Changes in 1992). cardiac electrophysiologic responses to beta-mimetics and reduced excitability of cardiac myocytes and in the sus- Studies in preterm infants with controlled EFA intake ceptibility to arrhythmias have also been noted (Hallaq et Very-low-birth-weight (VLBW) infants are particularly al, 1992; Kang et al, 1995). Myocytes form minimal vulnerable to de®ciency given the virtual absence of amounts of cycloxygenase products and no lipoxygenase adipose tissue stores at birth. This de®ciency may be products, thus the changes in excitability and conduction attributable to a possible immaturity of the FA elonga- are probably related to structural lipid composition, and tion=desaturation pathways and the inadequate LNA and re¯ect changes in the function of ion channels (Holh & DHA intake provided by formula. We and others conducted Rosen, 1987). N-3 fatty acid supplementation ameliorates studies to evaluate the effect of n-3 FA in VLBW infants the ¯uidifying effect of ethanol on neural membranes while examining the effects of LNA or LNA plus DHA supple- linoleic and a-linolenic acid de®ciency potentiates volatile mentation on plasma and tissue lipid composition, retinal anesthetic action in rats; linoleic acid supplementation electrophysiologic function, maturation of the visual cortex speci®cally reverses this effect (Evers et al, 1986). and measures of infant growth and development (Koletzko The role of membrane lipid composition in determining et al, 1989; Uauy et al, 1990; Carlson et al, 1991; Hoffman the electrical properties of cultured neuronal cells exposed & Uauy, 1992; Carlson et al, 1993; Birch et al, 1992a, to exogenous fatty acids has been investigated (Lin et al, 1992b; Werkman & Carlson, 1996). 1990; Love et al, 1985). Both n-3 and n-6 fatty acids Infants fed the n-3 FA-de®cient formula, in all studies, induced slower rates of rise, and to a lesser extent a lower had signi®cant reductions in DHA of plasma and red cell amplitude, of Na‡ action potentials. The opposite effects lipids compared to the groups on other formula (Koletzko were observed when saturated or trans monoenoic fatty et al, 1989; Uauy et al, 1990; Carlson et al, 1991; Hoffman acids were added. It seems likely that these effects were & Uauy, 1992). DHA-supplemented infants presented mediated by a change in the number of active Na‡ higher DHA concentrations than those provided LNA but channels. A change in membrane composition or altered no DHA. In most studies where a human milk fed group has fatty acid availability to the cells could have caused this been used as a reference, the DHA ‡ AA-supplemented event (Love et al, 1985). Eicosanoid production is another group has matched the LCPUFA blood levels of human mechanism by which the effect of n-3 fatty acid supple- milk fed infants. Initial studies used marine oil, which mentation on neural function could be explained. Phos- contains high EPA, as a source of LCPUFA; this lead to pholipases liberate AA, EPA, and DHA from membrane drops in AA, which in one study was signi®cantly asso- lipids and make them available to cycloxygenase or ciated to poor growth (Carlson et al, 1993). lipoxygenase action for production of eicosanoids (Holh The functional measures of neurodevelopment have & Rosen, 1987). consistently shown delayed retinal maturation in the DHA-de®cient groups up to 40 weeks post-conceptional age; early differences in rod photoreceptor function dis- Models to study the effect of EFAs in `healthy' humans appeared after this time (Uauy et al, 1990; Birch et al, Fat-free parenteral nutrition as a model to study the effect 1992a). Retinal function responses demonstrated signi®- of EFA de®ciency cantly higher threshold values in rod photoreceptors in the The fact that the use of TPN initially did not include lipids, n-3 de®cient group indicating that the sensitivity and permitted to con®rm the characteristic acute effects on the maturity of the rod photoreceptors in n-3 FA-de®cient skin (Caldwell et al, 1972) and, of greater interest, the long- infants were reduced signi®cantly compared to infants fed term effects of EFA de®ciency on sleep organization DHA-supplemented formula or human milk. Small differ- (Fagioli et al, 1989). Studies in children maintained on ences in indices of inner retinal function (oscillatory fat-free TPN for 2 ± 6 months served to demonstrate potentials) persist for up to four months postnatal age Essential fatty acids R Uauy et al S70 Studies in term infants fed EFA-controlled diets We conducted a 3 y follow-up of healthy, full-term infants to test if human milk feeding, which contains signi®cant amounts of DHA, had an effect on the development of visual function (Birch et al, 1993). The cohorts were breast- fed from birth to at least 4 months or fed for 12 months formula containing ample LA and 0.5% of the total fat as LNA. The breast-fed group was weaned to an oleic acid (18:1)-predominant formula and received egg through 12 months of age. The breast-fed group maintained higher plasma and RBC membrane phospholipid DHA concentra- tions throughout the ®rst year of life. At 3 y of age, stereo acuity, as measured by operant preferential looking (OPL) techniques, was more mature in the breast-fed than in the formula-fed group; 92% of the breast-fed group had mature OPL stereo acuity whereas 35% of the infants in the formula-fed group met the maturity criteria. Visual recog- Figure 3 Effect of early diet on visual maturation responses. Rod ERG threshold at 36 weeks postconceptional age, visual evoked potential (VEP) nition in the breast-fed group was also better; only 61% of and forced-choice preferential looking (FCPL) visual acuities of preterm the formula fed infants had a perfect score, as compared to study infants at 57 weeks postconceptional age. Rod ERG threshold is the 93% in the breast-fed group (Birch et al, 1993). These and light intensity (scotopic troland-seconds) required to induce a b-wave other complementary data from comparisons of infants who amplitude of 2mV, higher thresholds are associated with lower photore- were breast fed with others who where given formula with ceptor sensitivity. DHA supplementation decreases threshold mimicking the human milk fed group. Larger log minimum angle of resolution ample LNA but devoid of LCPUFAs reported by Makrides (logMAR) visual acuity values are associated with poorer visual acuity. et al (1993) and by Jorgensen et al (1996) suggested a Bars within a functional measure having an asterisk (*) are signi®cantly , possibly DHA, effect on visual acuity matura- different from human milk fed group. LA, linoleic acid present in corn oil tion in term infants. One study showed a transient bene®t of is the precursor of the n-6 EFAs, LNA, a-linolenic acid present in soy oil is the precursor of the n-3 EFAs, marine oil containing formula provided breast milk: better visual acuity at two months (Carlson et LNA and DHA the long chain derivative of LNA. Data from Birch et al al, 1996). Some studies have attempted to optimize LNA to (1992a,b). DHA conversion by providing suf®cient LNA (> 0.7% of total energy) and a lower LA to LNA ratio in formula (< 10:1). Initial results by Ponder et al (1992) indicated that this alternate strategy could be simpler and less (Birch et al, 1992a). Visual acuity measurements using expensive than the addition of n-3 LCPUFA. Studies by forced choice preferential looking (a behavioral assess- Innis et al (1994, 1996, 1997) concluded that the need for ment) and visual evoked potentials (an electrophysiologic DHA supplementation should not be based on the alleged test) reveal that LPCUFA-supplemented infants are simi- improved visual acuity of breast fed infants since despite lar to human-milk-fed prematures in terms of acuity differences in DHA content in blood no differences in maturation, while the n-3 de®cient, even if provided preferential looking acuity between BF and unsupplemen- ample LNA, have delayed maturation over the ®rst four ted formula fed infants were found. The formula used in months of postnatal life (Birch et al, 1992b). Figure 3 this study contained 2.1% of total fat as LNA but no summarizes some of our results on the effect of dietary LCPUFAs. A controlled randomized study using a formula EFA supply on indices of visual maturation (Birch et al, supplemented with 0.36% DHA and compared with a 1992a, 1992b). formula providing ample LNA but no DHA and a breast Cognitive development measures using preferential fed reference group revealed delayed visual acuity at four looking novelty response and Bayley psychomotor devel- and six months of age in the group on formula lacking opment testing are not conclusive. The interaction with DHA (Makrides et al, 1995). On the other hand a recently enviromental and parental socioeconomic factors limits the published multicenter trial using formula supplemented capacity to detect potential diet-induced differences. In with 0.2% DHA and 0.4% AA did not show an effect of addition most studies have lacked the necessary sample this level of supplementation on visual acuity (Auestad et size to detect differences which may be of small magnitude. al, 1997). A bene®cial effect of DHA, AA and GLA Shorter look duration in the novelty response has been supplementation on psychomotor development at four and reported, but the interpretation of these ®ndings remains 12 months assessed by the Brunet-Lezine method has been controversial (Werkman & Carlson, 1996). reported (Agostoni et al, 1996), yet 1 y is too early to Some issues regarding LCPUFA provision to preterm predict future mental development in a consistent manner. infants that require further research include: (1) The need to Small effects of supplementation on weight-for-height have supplement both DHA and AA, or DHA alone, and whether been reported in some studies; the relevance of these other related fatty acids that are easier to obtain could ®ndings remains to be determined (Jensen et al, 1997). substitute them; (2) The optimal amount of LCPUFA to As discussed later, LCPUFA supplementation may have include in the formula given that human milk is variable in potential long-term effects on weight by regulating the DHA and AA content; (3) The duration of supplementation expression of genes responsible for adipocyte development. for optimal effects; (4) Possible effects of supplementation Recent evidence indicates n-3 supplementation in the form on prevalence of common neonatal morbidities, such as of perilla oil given to rats after weaning signi®cantly necrotizing enterocolitis and bronchopulmonary dysplasia; reduces the growth of visceral adipose tissue despite similar and (5) What sources of LCPUFAs should be used con- total food consumtion. The epididymal fat pad weight was sidering safety and costs. lower in the n-3 supplemented relative to or beef Essential fatty acids R Uauy et al S71 fed groups. The expression of genes responsible for evaluate the conversion of these into LCPUFA (Salem et late adipocyte differentiation, adipsin, adipocyte P2 and al, 1996). Concentration of deuterated precursors and peroxisome-activated receptor alpha where all down-regu- products were measured in plasma using negative ion lated in the perilla oil fed group (Masataka et al, 1997). mass spectrometry of penta¯uorobenzyl derivatives. Peak This summary of results must lead to a re¯ection on concentrations of labeled precursor EFAs were reached several issues: the biological signi®cance of the effects, the during the ®rst day after dosing, deuterated products selection of outcome measures, the duration and reversi- increased concentrations with time reaching peak levels bility of the change and ®nally the long-term consequences for the n-6 metabolites by 48 h in the term infants and of the effect. The presence of an effect does not in itself closer to 96 h in the preterm SGA or AGA infants. For the make it clinically signi®cant. For example, diet-induced n-3 series, concentrations of elongated=desaturated pro- changes in composition are of little conse- ducts increase with time reaching highest levels in the quence except to validate compliance with a particular term infants by 48 h; for preterm infants peak levels are diet. Tissue fatty acid pools, particularly those required reached by 96 h. The rise in labeled products for SGA for retinal and CNS neural membrane formation, may not infants is slower than that for AGA preterm infants. Other be re¯ected by plasma or even red cell fatty acid composi- studies using uniformly labeled 13C con®rm that preterm tion. Biochemical effects can not by themselves be used as and term neonates are able to elongate and desaturate surrogates for functional measures. parent EFAs (Sauerwald et al, 1996; Carnielli et al, Another relevant issue is the selected outcome measure. 1996). Estimation of pool size, relevant precursor pool Ideally it should be clinically relevant, but the sensitivity of enrichment, knowledge of homogeneity in distribution in clinical responses is usually low, therefore functional one or more pools and information on rates of oxidation of responses are considered valid to de®ne biologically sig- precursors and products are lacking in these studies. There- ni®cant outcomes. In the case of LCPUFA supplementa- fore, they have not been able to provide quantitative tion, growth is affected only in extreme n-6 fatty acid information on synthetic rates in order to orient dietary de®ciency therefore is not considered a sensitive measure recommendations. Earlier studies in adults have also docu- of n-6 suf®ciency. The effect of n-3 fatty acids on sensory mented n-3 LCPUFA formation from labeled LNA but no maturation and cognitive development are the outcomes of conversion of LA to arachidonic was documented (Emken greater interest in studies of n-3 supplementation. et al, 1992). El Boustani et al (1989) reported delta 5 The duration and reversibility of diet-induced effects is desaturation and arachidonic acid formation in four human another important issue. There may be transient effects that adults using deuterated 20:3, n-6. More recently Emken et re¯ect the acceleration or the slowing of a maturational al (1997) evaluated the in¯uence of AA on delta 5 process with a fully normal ®nal outcome. This is of special desaturation concluding that AA supplementation in relevance during the ®rst few months of life when visual adults increased Dihommo GLA (20:3, n-6) turnover and maturation is progressing rapidly. Several studies have desaturation into AA. demonstrated signi®cant effects of the dietary LCPUFAs on visual maturation in the ®rst four months of life, but in Studies in malnourished infants most cases the delayed response becomes normal at six Low-fat diets are a frequent cause of primary malnutrition months or at most by 1 y of age. Should we dismiss this in infants and children in developing countries. In addition phenomenon as transitory and of limited signi®cance or these diets are of low energy density and therefore limit the assume that we failed to detect a signi®cant change at 1 y total energy a child is able to consume in a single meal. because our tools were not sensitive enough, or that in fact There is very limited data on EFA composition of local other related function are indeed affected? Using this same diets used to feed infants as well as on EFA status of example, we failed to detect differences in visual acuity at malnourished infants. If children are not breast fed most six months but depth=spatial perception assessed by likely they will receive insuf®cient EFAs unless they are stereoacuity responses were different at 3 y of age, as given appropriate complementary foods. are low in shown by our studies comparing breast fed to routine fat and most oil seeds will provide only parent EFAs LA formula fed infants. These examples illustrate that unless rich and LNA poor, unless soy or rapeseed oil are available. sensitive outcome measures are used and suf®cient follow- Starchy roots, tubers and starchy fruits used as traditional up time is provided, it is impossible to fully discard the complementary foods are low in fat and low in EFAs. possible long-term consequences of early developmental Powdered cow milk used as a source of high quality protein effects. is also low in EFA and provides virtually no n-3 fatty acids; Evidence of potential bene®cial long-term effects of therefore malnourished children are at risk for EFA de®- LCPUFA supplementation on brain development of term ciency and specially n-3 de®cit. The risk for n-3 de®cit is infants is suggestive, proof is lacking. The resolution of this enhanced if during the recovery period infants are given issue should be forthcoming, the follow-up of term infants energy-dense diets high n-6 fatty acids. These diets are included in the controlled clinical trials of DHA ‡ AA usually formulated with vegetable oils that are high in n-6 supplementation into the school age period should help to and low in n-3 fatty acids (corn, sun¯ower, saf¯ower) address the question of long-term effects (Dobbing 1997). (Beare-Rogers et al, 1998). The breast fed infant will have suf®cient n-6 fatty acids Studies of essential using labeled up to 8 and probably up to 12 months of age. If we consider precursors that the n-3 fatty acid supply should be at least 0.5% of We and others have also conducted studies using labeled energy, the breast fed infant will have suf®cient n-3 EFA EFA precursors (Salem et al, 1996; Sauerwald et al, 1996). from human milk for the ®rst six months of life if the milk Our stable isotope studies have used as a label 5 deuterated volume is 800 ± 1000 mL=d and the fat content is 3 ± atoms distributed in the ®rst (n-1) and second (n-2) carbon 4g=dL. If the mother is malnourished, breast milk fat, atoms of the fatty acid chain (d5-LA and d5-LNA) to EFA content and total volume may be reduced; therefore Essential fatty acids R Uauy et al S72 of lipid mediators related to arachidonic acid, and that this situation leads to enhanced ischemic=in¯ammatory ten- dencies thereby increasing the incidence of chronic diseases ...'. It goes on to say: `We suggest that a relative n-3 de®ciency as evidenced by the very high n-6=n-3 ratios of plasma lipids might be affecting the behavioral patterns of a signi®cant part of the young generations in industria- lized countries.' (Okuyama et al, 1997). In the opposing camp, the FDA, after an extensive review of this subject as part of its effort to de®ne health claims for food labeling purposes, concluded that most of the documented bene®ts were attributable to the consumption of ®sh as a food and not of marine oil supplementation (FDA=HHS USA, 1991). The verdict is being contended by several groups, amongst them the International Society for Study of Fatty Acids and Lipids (ISSFAL) (Galli et al, 1994). The issue of whether the bene®cial effects are secondary to n-3 fatty acids or other phenomena associated with the consumption of ®sh Figure 4 Effect of n-3 polyunsaturated fatty acids (n-3 PUFA), inhibiting will only be settled by prospective randomized controlled key steps of the arachidonic acid (n-6) cascade. Phospholipases release arachidonate from membranes which serves to synthesize eicosanoids; n-6 clinical trials of speci®c fatty acids, alone or in combina- derived eicosanoids formed by cycloxigenase pathway (prostaglandins, tion. , ) or lipoxigenase pathway () are Omega-3 PUFA ingestion can have an effect on cardi- counterbalanced by n-3 PUFA derived eicosanoids. Biological effects are ovascular disease through a variety of mechanisms. The dependent on n-6=n-3 balance present in membrane phospholipids. experimental evidence does not permit deciding which of the many putative mechanisms are crucial in determining n-3 may be below accepted levels at an earlier age (Beare- the effects. Marine oils have antithrombotic effects via Rogers et al, 1998). modi®cation of platelet aggregation, decreasing vascular adhesiveness, enhancing small vessel relaxation mechan- Prevention of chronic disease in relation to EFAs supply isms, promoting endothelial repair and minimizing in¯am- Published experimental work indicates that consumption of matory responses in vessel walls (Simopoulos, 1991; Galli terrestial and marine foods with n-3 PUFA has a bene®cial et al, 1994; Shimokawa & Vanhoutte, 1988; Leaf & Weber, effect on primary and secondary prevention of athero- 1988). In addition, the risk of heart disease can be indir- sclerosis, thrombosis and embolism, , ectly modi®ed by effects on plasma , choles- , autoimmune disease and possibly allergic terol uptake by macrophages, foam cell formation and cell problems. Bleeding time in the human can be prolonged proliferation in vessel walls, blood pressure regulation, by 30 ± 50% by supplementing diets with marine oils; changes in cardiac electrophysiologic responses to beta- platelet aggregation can also be reduced. These effects mimetics, in the susceptibility to arrhythmias and reduced can be explained by changes in eicosanoid metabolism, excitability of cardiac myocytes (Charnock, 1991; Connor shifting it from the arachidonate-derived series2 metabo- & Connor, 1990; Dyerberg et al, 1978; Harris et al, 1988; lites to the EPA-derived series3 metabolites. Prostaglan- Knapp & FitzGerald, 1989; Kang et al, 1995). In a dins, thromboxanes, leukotrienes, hydroxy fatty acids and prospective randomized controlled trial of ®sh oil as an formed from EPA decrease blood viscosity, plate- antiarrythmic agent, patients with spontaneous ventricular let aggregation, increase bleeding time, promote vasodila- premature complexes (VPC) were given ®sh oil tion and insulin sensitivity; in addition they inhibit VLDL (0.9 g EPA ‡ 1.5 g DHA) or n-6-rich sun¯ower oil formation, cell proliferation, the in¯ammatory and allergic (5 g LA) for 16 weeks. VPCs were signi®cantly reduced response. Effects are evident with doses ranging from 5 ± in 44% of patients of the ®sh oil group and only in 15% of 30 g of marine oils or 2 ± 10 g of EPA per day (Uauy & the sun¯ower oil group (Sellmayer et al, 1995). The other Hoffman, 1991; Innis, 1991; Uauy et al, 1989; Glomset, intervention trial used the Mediterranean diet, rich in LNA 1985; Simopoulos, 1991; Galli et al, 1994; Katan, 1995; sources (2 g=d) as a model. Over 600 French patients who Bang & Dyerberg, 1972, 1986). Figure 4 summarizes the survived their ®rst infarcts where randomly assigned to this effects of n-3 polyunsaturated fatty acids inhibiting key or the control diet providing only a third of the LNA. The steps of the arachidonic acid cascade. Biological effects are group on the LNA-rich diet had 73% fewer cardiac deaths dependent on n-6=n-3 balance present in membrane phos- and non fatal heart attacks than controls (De Lorgeril & pholipids. Renaud, 1994). A recent report from the Chicago Western Recent reviews of the vast body of literature accumu- Electric Study to examine the possible bene®t of eating ®sh lated over the past decade are available. The conclusions on risk of dying from coronary heart disease showed in reached by the reviewers differ based on their biases and on over 1800 `healthy' middle aged men that consumption of the selection of evidence presented. Simopoulos concludes 35 g or more ®sh per day signi®cantly reduced the cumu- that `research reports make it increasingly evident that lative 30 y risk of death from coronary heart disease by eating o-3 fatty acids can have bene®cial effects on chronic 38% and that of by 44% (Daviglus et in¯ammatory and cardiovascular diseases' (Simopoulos, al, 1997). 1991; Galli et al, 1994). A more recent review by Okuyama The mechanism for the antiarrythmic effect has been et al, 1997 from Japan (491 refs) concludes: `It appears to studied by Leaf's group who has demonstrated that myo- us that an excess of linoleic acid and elevated n-6=n-3 cites become less excitable and more resistant to beta ratios in tissues result in over- and unbalanced production adrenergic stimuli due to a modulation of Na channels. Essential fatty acids R Uauy et al S73 The effect is observed with n-6 and n-3 PUFA, but is more oil has also been noted to signi®cantly reduce prominent in the latter (Kang & Leaf, 1995). systolic blood pressure in double-blind crossover controlled Initially it was thought that marine oils had an effect on studies. Increase in sodium excretion and a decrease in plasma cholesterol comparable to that of n-6 predominant metabolites derived from AA were also vegetable oils, however subsequent studies have demon- found. The lowering of diastolic blood pressure, of greater strated that only n-3 long-chain PUFA lower clinical signi®cance, has been the exception. The interac- in normal or hyperlipemic subjects (Harris, 1989). Patients tion of marine oil supplementation and need for antihyper- with type V have shown dramatic drops in tensive drugs has been less explored. A meta analysis VLDL and triglyceride levels in response to marine oil conducted by Morris et al (1993) involving a total of supplemented diets (Harris, 1989; Nestel et al, 1984). In over 1300 patients concluded that dietary n-3 fatty acids contrast, n-6 fatty acids from oil seeds caused a signi®cant induce a small but signi®cant reduction in blood pressure in rise in triglycerides in this group. The effect on triglycer- hypertensive but not in normal subjects. This area requires ides appears to be mediated by decreased production and further research before the use of marine oils can be increased fractional catabolic rate of the triglyceride-rich advocated for this purpose (Levinson et al, 1990). VLDL particle (Nestel et al, 1984). Small but In adult onset diabetes the effects are mixed since the signi®cant reductions in plasma cholesterol have also been potentially bene®cial effect on microvessel occlusion and noted after ®sh oil administration in patients with type II proliferation is offset by a potential increase in heterozygous , while in those with output and higher glucose levels observed in some type IV a rise has been noted (Simopoulos, 1991; Nestel studies. Additionally total cholesterol, LDL and apo B et al, 1984; Massarei et al, 1989). The evidence does not serum levels are increased by ®sh oil supplementation in support an LDL cholesterol lowering effect in response to diabetics. On the other hand recent evidence suggests that marine oil supplementation in patients with hypercholes- of adipose tissue may be ameliorated by terolemia. The response of HDL-cholesterol and apoprotein n-3 and by n-9 fatty acids (Ranaian et al, 1996). In addition A in supplementation trials has been variable but the triglyceride levels will commonly decrease with n-3 changes, if any, are of minor magnitude. The variability LCPUFA supplements. A major problem in the interpreta- in lipoprotein responses to supplementation in the various tion of metabolic effects of PUFAs is the genetic hetero- trials can be attributed to the differences in overall design, geneity of patients included in these studies; the response duration of study, patient populations, differences in base- may in fact be different within a group that appears to be of line diets tested, and different oils used. Some studies used a similar phenotype. This may be due to genetic factors ®sh or marine oils while others used concentrated which are only beginning to be unraveled. A study in EPA ‡ DHA (Simopoulos, 1991; FDA=HHS USA, 1991; patients with coronary heart disease and normal volunteers Leaf & Weber, 1988; Harris, 1989; Nestel et al, 1984). demonstrated that hyperinsulinemia, thus insulin resistance, Overall these studies suggest a bene®cial effect of ®sh and is inversely correlated with long-chain PUFA content of n-3 LCPUFAs on coronary disease mortality. muscle cell membrane phospholipid. The lower the AA and Results of studies on the use of marine oil in the DHA content of membranes the higher the insulin level. prevention of restenosis after coronary angioplasty or This association could be related to dietary EFA content but bypass surgery were suggestive of bene®cial effects of a also both conditions could be determined by a common combined use of ®sh oil and anticoagulant drugs in redu- cause since LCPUFA is affected by insulin cing thrombotic occlusion of coronary vessels. Furthermore activity (Storlien et al, 1996). No prospective controlled double-blind controlled studies using angiographic evi- study has addressed this point, thus for now we can only dence for presence of restenosis after coronary angioplasty speculate. In terms of mechanisms, it is well known that have failed to disclose the putative bene®ts (Leaf et al, PUFAs promote fatty acid oxidation and in the case of n-3 1994). A more recent randomized controlled study on graft LCPUFAs decrease VLDL secretion from the liver. The patency after bypass surgery using 4 g=d of ®sh oil in possibility that speci®c fatty acids promote or inhibit addition to anticoagulants demonstrated a clear bene®t in peroxisomal proliferation can be raised given the discovery terms of graft patency 1 y after surgery. The ®sh oil group of peroxisomal proliferation activator receptor (PPAR) and had only 27% graft occlusion vs 33% in the control group, the modulation of its level of expression by speci®c fatty an inverse relationship between increase in n-3 content of acids (Masataka et al, 1997; Schoonjans et al, 1996). plasma phospholipids and vein graft occlusion was noted Increase in saturated fat and=or trans fatty acids relative (Eritsland et al, 1996). to n-3 LCPUFA affect PPAR activity thus could potentially EPA and DHA prolong bleeding time and decrease adversely affect fatty acid oxidation and prevent the bio- A2 production. These effects seem to be synthesis of LCPUFAs by affecting peroxisomes, key site dose dependent but to date no major clinically signi®cant for LCPUFA synthesis from parent EFAs (Martinez, 1996). bleeding has been demonstrated in relation to the ingestion The main regulatory actions of PPAR on of ®sh oils (Lox, 1990), although one study showed are summarized in Table 1. The putative regulatory effects increased incidence of epistaxis when high dose ®sh oil was administered to hypercholesterolemic adolescents (Clarke et al, 1990). Platelet counts may be affected by Table 1 Regulatory actions of the peroxisome proliferator-activated large doses of marine oil and platelet half-life may be receptor (PPAR) in intracellular and extracellular lipid metabolism shortened. The effect on endothelial walls is receiving Activation of peroxisomal and mitochondrial b-oxidation increasing attention. The production of platelet-derived Regulatory action on transcription of HDL growth factor is decreased while the formation of endothe- Control of adipocyte differentiation and adipogenesis lium derived relaxing factor is increased (Fox & Dicor- Stimulation of genes that control lipoprotein activity Increased expression of fatty acid transport protein leto,1988). These effects may be important in mediating the Decreased production of VLDL and antiatherogenic effect of ®sh oil. Essential fatty acids R Uauy et al S74 DHA levels are below normal and that the biochemical response of these patients to ®sh oil test material is only partial. Sprecher (1981) has reported that the peroxisomal step necesary for C22 PUFA biosynthesis is restricted in Zellweger patients, thus they can not convert C 24:6, n-3 to C 22:6, n-3. Preliminary results on a limited number of patients studied by Martinez indicate that supplementation with pure DHA is capable of preventing and also of reverting the abnormal motor and mental function associated with these conditions. Concomitant ®broblast studies of these patients provide supportive evidence as to the relative importance of peroxisomal beta oxidation in determining the biochemical and func- tional consequences of this disease (Martinez, 1996).

Other potentially adverse health effects The vulnerability of lipid membranes to damage by free-radicals is directly related to their degree of unsaturation. The double bonds in the carbon chain of the fatty acids are the point of attack for oxygen free radicals. The number of pentadienic units present in a polyunsaturated fatty acid is Figure 5 Speculative mechanisms to explain the effect of dietary fatty acids on b-oxidation and LCPUFA biosynthesis and insulin resistance. directly related to its rate of oxidation (Willis, 1984). There- These effects could explain the effect of fat on diet related non commu- fore membranes enriched with AA, EPA and DHA are most nicable chronic diseases, obesity, hypertension, non insulin dependent susceptible to develop oxidative damage. The oxidation diabetes and hyperlipidemia. process is further catalyzed by the products of oxidation. This self-propagating process may severely affect membrane of dietary fatty acids on mitochondrial and peroxisomal structure and function unless quenched by proper antioxidants function has led to the proposed hypothesis summarized in in the membrane. The protection of LCPUFAs is dependent Figure 5. This could be central in explaining the effect of on enzymatic and non-enzymatic systems. Ingestion of high dietary fat on the epidemic of non communicable chronic doses of o-3 fatty acids in adults has raised the question of risk disease observed in industrialized countries and in some for tissue oxidative damage. This effect has been observed in segments of developing societies (Simopoulos, 1994). experimental animals by several authors. Moreover, aging The use of omega-3 marine oils as a natural immuno- appears to be an important factor in the development of this suppressant has been proposed based on their inhibitory susceptibility. Aged rats, fed ®sh oil show a higher suscept- role on arachidonic acid derived products such as prosta- ibility to develop tissue lipid peroxidation than young ones glandins and leukotrienes. Several studies on experimental (Garrido et al, 1989, 1993). animals have shown that ®sh oil increased survival of Evidence from human studies in young infants suggests animals genetically predisposed to lupus erythematosus that excess dietary PUFAs will increase red cell hemolysis and collagen-induced arthritis (Simopoulos, 1991; Galli et unless appropriate tocopherol supplementation is provided al, 1994). Controlled clinical studies in humans have (Oski & Barness, 1967). Experimental evidence indicates provided encouraging preliminary results; the effect of that hyperoxic-induced lung damage may be affected by ®sh oil are probably mediated by a suppression of LTB4 dietary fat, although the results from mice exposed to and increased LTB5 formation. IL-1 production is lowered oxygen immediately after birth indicate that PUFAs and by o-3 LCPUFAs while IL-2 is increased. The role of ®sh speci®cally ®sh oil feeding protect from oxygen-induced oil alone or in combination with antirheumatic medication damage. The proposed explanation for this apparent para- remains to be determined. Another potential use of marine dox is that PUFAs in plasma triglycerides may trap free PUFA being explored is the induction of an immunosup- radicals, thus protecting membrane phospholipid PUFAs pressed state to prevent the development or progression of (Sosenko et al, 1989). The role of o-3 LCPUFAs in type I insulin dependent diabetes in susceptible individuals. determining the increased susceptibility of the retina to Other uses documented by anecdotal evidence or uncon- light-induced oxidative damage has been recently studied trolled trials include bene®ts in the treatment of migraine, demonstrating that lower retinal DHA protects rod outer asthma and other allergic conditions and in the control of segments from light-induced damage. Tocopherol has been psoriasis (Simopoulos, 1991; Galli et al, 1994). shown to be protective (Bush et al, 1991). The role of PUFAs and immature antioxidant systems in determining oxygen-induced lung, eye and red cell damage in premature Studies of requirements under disease conditions infants is an area of great interest. Increased risk of Metabolic=genetic disease bronchopulmonary dysplasia and retinopathy of prematur- There is yet no conclusive evidence of an inherited meta- ity in preterm infants of Alaskan eskimo background have bolic defect of DHA and=or AA synthesis causing altered been observed (Arnold, 1992). The high o-3 diets and low brain or retinal function. However, there is strong evidence consumption of green vegetables or other sources of that Zellweger and other peroxisomal diseases may be tocopherol may play a role in this phenomenon (Wo & associated with defective formation of C 22 PUFAs. Draper, 1975). Martinez (1996) and others have provided data from Increased bleeding is a potentially adverse effect of several patients, indicating that in peroxisomal disorders, feeding o-3 fatty acids. The clinical implications of these brain DHA content is extremely low, that plasma and RBC have not been demonstrated except for anecdotal evidence Essential fatty acids R Uauy et al S75 of frequent bruising occurring among ®sh oil eating Green- Auestad N, Montalto MB, Hall RT, Fitzgerald KM, Wheeler RE, Connor land eskimos (Dyerberg & Bang, 1979). Clinical studies WE, Neuringer M, Connor SL, Taylor JA & Hartmann EE (1997): Visual acuity, erythrocytes fatty acid composition, and growth in term have demonstrated increased epistaxis in adolescents given infants fed formulas with and without long chain polyunsaturated fatty high doses of ®sh oil. On the other hand there is evidence acids for one year. Pediatr. Res. 41, 1 ± 10. for small non-signi®cant changes in bleeding time in infants Bang HO & Dyerberg J (1972): Plasma lipids and lipoproteins in Green- given a marine oil supplemented formula (Clarke et al, landic West-coast Eskimos. Acta Med. Scand. 192, 85 ± 94. Bang HO & Dyerberg J (1986): Lipid metabolism and ischemic heart 1990; Uauy et al, 1994). The use of marine oils has some disease in Greenland eskimos. Adv. Nutr. Res. 3, 1 ± 21. potential toxicological risks that can be circumvented by Bazan NG (1989): The metabolism of omega-3 polyunsaturated fatty acids in careful processing, storing and preservation of the unsatu- theeye:thepossibleroleofdocosahexaenoicacidanddocosanoidsinretinal rated fatty acids. physiology and ocular pathology. Prog. Clin. Biol. Res. 312, 95 ± 112. Beare-Rogers J, Ghafoorunissa, Korver O, Rocquelin G, Sundram K & Uauy R (1998): Dietary fats in developing countries. Food and Nutr. Bull. 19, 251 ± 267. Conclusions Birch D, Birch E, Hoffman D & Uauy R (1992a): Retinal development in very low birth weight infants fed diets differing in omega-3 fatty acids. Infants should be breast fed if at all possible. The fatty acid Invest. Ophtal. Vis. Sci. 33, 2365 ± 2376. Birch EE, Birch DG, Hoffman DR & Uauy R (1992b): Dietary essential composition of infant formula should correspond to the fatty acid supply and visual acuity development. Invest. Ophthal. amount and proportion of fatty acids contained in mature Vis.Sci. 33, 3242 ± 3253. breast milk from healthy omnivorous women. Birch E, Birch D, Hoffman D, Hale L & Uauy R (1993): Breast-feeding During weaning and at least until 2 y of age, a child's and optimal visual development. J. Pediatr. Ophtalmol. Strabismus 30, diet should contain at lest 30 ± 40% of energy from fat and 33 ± 38. Bourre JM, Durand G, Pascal G & Youyou A (1989a): Brain cell and tissue provide similar levels of essential fatty acids as are found in recovery in rats made de®cient in n-3 fatty acids by alteration of dietary human milk (FAO=WHO 1994). The n-6 and n-3 fatty acids fat. J. Nutr. 119, 15 ± 22. have critical roles in the membrane structure and as pre- Bourre JM, Francois M & Youyou A (1989b): The effects of dietary alpha- cursors of eicosanoids, which are potent and highly reactive linolenic acid on the composition of nerve membranes, enzymatic activity, amplitude of electrophysiological parameters, resistance to compounds. Since they compete for the same enzymes and poisons and performance of learning tasks in rats. J. Nutr. 119, have different biological roles, balance between n-6 and n-3 1880 ± 1892. fatty acids in the diet can be of considerable importance. Brenner RR & Peluffo RO (1969): Regulation of unsaturated fatty acid Desirable intakes of linoleic acid should provide between biosynthesis. Biochim. Biophys. Acta 176, 471 ± 479. 4 and 10% of energy (FAO=WHO 1994). Intakes at the Brockerhoff H, Ackman RG & Hoyle RJ (1963): Speci®c distribution of fatty acids in marine lipids. Arch. Biochem. Biophys. 100, 93 ± 100. upper end of this range are recommended when intakes of Burr GO & Burr MM (1929): A new de®ciency disease produced by rigid saturated fatty acids and cholesterol are relatively high. exclusion of fat from the diet. J. Biol. Chem. 82, 345 ± 367. Particular attention must be paid to promoting adequate Bush RA, Reme CE & Malnoe A (1991): Light damage in the rat retina: the maternal intakes of essential fatty acids throughout preg- effect of dietary deprivation of n-3 fatty acids on acute structural alterations. Exp. Eye Res. 53, 741 ± 752. nancy and lactation to meet the requirements of fetal and Caldwell MD, Johnson HT & Othersen HB (1972): Essential fatty acid infant development. de®ciency in an infant receiving prolonged parenteral alimentation. J. ISSFAL suggests an intake of 0.8 ± 1.1 g=d of LNA, Pediatr. 81, 894 ± 898. which is roughly 0.5% of energy; it also suggests 0.3 ± Carlson SE, Carver JD & House SG (1986): High fat diets varying in ratios 0.4 g=d of n-3 LCPUFAs. To prevent cardiovascular and of polyunsaturated to saturated fatty acid and linoleic to linolenic acid: a comparison of rat neural and red cell membrane phospholipids. J. Nutr. other non-communicable chronic diseases the recommenda- 116, 718 ± 726. tion is to eat ®sh 2 ± 3 times a week. Alternatively marine Carlson SE, Cooke RS & Rhodes PG (1991): Effect of vegetable and oil 3 ± 4 g per day or n-3 LCPUFAs (EPA ‡ DHA) 1.2 g per marine oils in preterm infant formulas on blood arachidonic and day or 0.5% of energy have been recommended by the docosahexaenoic acids. J. Pediatr. 120, S159 ± S167. Carlson SE, Ford AJ, Werkman SH, Peeples JM & Koo WW (1996): British Nutrition Foundation. Visual acuity and fatty acid status of term infants fed human milk and formulas with or without docosahexaenoate and arachidonate from egg yolk . Pediatr. Res. 39, 882 ± 888. Acknowledgements ÐWork by the authors of this publication is supported Carlson SE, Werkman SH, Peeples JM, Cooke RJ & Tolley EA (1993): in part by Fondecyt grant 1961001 and a CONICYT Presidential Award in Arachidonic acid status correlates with ®rst year growth in preterm infants. Proc. Natl. Acad. Sci. USA 90, 1073 ± 1077. Biological Sciences to RU. Carnielli V, Wattimena DJL, Luijendijk IHT, Boerlage A, Degenhart HJ & Sauer PJJ (1996): The very low birth weight premature infant is capable of synthesizing arachidonic and docosahexaenoic acids from linoleic and linolenic acids. Pediatr. Res. 40, 169 ± 174. References Caster WO & Ahn P (1963): Electrocardiographic notching in rats de®cient in EFA. Science 139, 1213. 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