ESSENTIAL FATTY ACID METABOLISM IN THE MICROPREMIE

ESSENTIAL FATTY ACID METABOLISM IN THE MICROPREMIE

NUTRITION AND METABOLISM OF THE MICROPREMIE 0095-5108/00 $15.00 + .OO ESSENTIAL FATTY ACID METABOLISM IN THE MICROPREMIE Ricardo Uauy, MD, PhD, Pat...

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NUTRITION AND METABOLISM OF THE MICROPREMIE

0095-5108/00 $15.00

+ .OO

ESSENTIAL FATTY ACID METABOLISM IN THE MICROPREMIE Ricardo Uauy, MD, PhD, Patricia Mena, MD, and Cecilia Rojas, PhD

Animal tissues, especially the liver, are capable of elongating and desaturating the parent essential fatty acids (EFA) generating a family of compounds for the respective families as shown in Figure 1. The essentiality of these fatty acids is determined by the inability of animal cells to introduce double bonds prior to carbon n-9. As shown in Figure 1, arachidonic acid (AA [20:4 n-61) can be formed from linoleic acid (LA [18:2 n-6]), and docosahexaenoic acid (DHA [22:6 n-31) from alpha-linolenic acid (LNA [18:3 n-31). In the case of EFA deficit, eicosatrienoic acid (ETA [20:3 n-91) is formed from oleic acid [18:1 n-9].Il2The competitive desaturation of the n-3, n-6, and n-9 series by delta-6 desiiturase is of major significance because tlus is considered to be the controlling step of the pathway. The synthesis of fatty acids longer than 20 carbon starts with one or more elongation steps, followed by a delta-6 desaturation and a partial peroxisoma1 beta-oxidation in a pathway termed retroconversion.'" The triene-tetraene (ETA-AA) ratio is used traditionally as an index of n-6 EFA deficiency. In n-3 fatty acid deficiency the n-6 long-chain polyunsaturated fatty acid (LCPUFA) docosapentaenoicacid (DPA [22:5 n-61) accumulates, whereas DHA decreases in plasma and tissue lipids.l12 The dry weight of the human brain is predominantly lipid; 22% of the cerebral cortex and 24% of white matter consist of phospholipids. Studies in

This work was supported by Fondecyt N01961001and Cdtedra Presidencial 1996.

From the Institute of Nutrition and Food Technology (INTA), University of Chile (RU, PM, CR); the Neonatal Unit, Hospital Dr. S6tero del Rio (PM), Santiago, Chile; the Retina Foundation of the Southwest, Dallas (RU); and the Division of Neonatology and Department of Pediatrics, University of Texas Health Science Center at Houston, Houston (RU), Texas

CLINICS IN PERINATOLOGY VOLUME 27 * NUMBER 1 * MARCH 2000

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OIeic acid ( 18:l n-9)

1.

( 18:2 n-9 )

Linoleic acid ( 18:2 n-6)

a-Linolenic acid ( 18:3 n-3 )

J.

+GzzGq( J. y Linolenic

Eicosatrienoic ( 20:3 n-9 )

( 18:4 n-3 )

( 20:4 n-3 )

( 20:2 n-9 )

I<

.1

I

Dihomo y -Linolenic ( 20:3 n-6 )

+G&Z+

J.

Eicosapentaenoic ( 20:5 n-3 )

Arachidonic ( 20:4 n-6 )

[-=I -I

( 22:4 n-6 )

( 22:5

n-3 )

( 24:4 n-6 )

( 24:5

n-3 1

A6 Desaturase

Docosapentaenoic ( 22:5 n-6 )

I

-

Docosahexaenoic ( 22:6 n-3 )

Figure 1. Metabolism of n-9, n-6, and n-3 fatty acids. Metabolic transformation of essential fatty acids (EFA) to form long chain polyunsaturated fatty acids (LCPUFA). Parent EFAs are derived from dietary sources for both n-3 (18:3, linolenic acid) and n-6 series (18:2, linoleic acid). De novo synthesis is able to produce only n-9 LCPUFAs. Elongation occurs two carbons at a time, and 6 desaturates (9, -6, - 5) introduce double bonds at 9, 6, and 5 carbons from the carboxylic moiety. The final step in the formation of n-3 and n-6 endproducts is catalyzed by a peroxisomal partial poxidation. Polyunsaturated fatty acids (PUFAs) of interest include 18:3 n-6 y linolenic acid (GLA), arachidonic acid 20:4 n-6 (AA), docosapentaenoic acid 2 2 5 n-6 (DPA), eicosatrienoic acid 20:3 n-9 (ETA), eicosapentaenoic acid 205 n-3 (EPA), and docosahexaenoic acid 22:6 n-3 (DHA). EPA, AA, and 20:3 n-6 are immediate precursors of prostaglandins and other eicosanoids.

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several animal species and recent evidence from humans have established that brain phospholipid AA and DHA decrease, whereas n-9 and n-7 monosaturated and polyunsaturated fatty acids increase when LA and LNA are deficient in the diet.15,16, 87 Typically, n-3 fatty acid-deficient cells have decreased DHA and increased levels of the end product of n-6 metabolism, DPA. Within the subcellular organelles, synaptosomes and mitochondria are more sensitive to a low dietary n-3 supply as evidenced by the relative abundance of DHA and the changes in composition of these organelles in response to dietary depri~ation.'~, 16,88 The animal data accumulated over the past decades strongly supports the essential nature of EFAs for humans and particularly a need for LCPUFAs in early life. Metabolic activity reflecting EFA elongation and desaturation is found mainly in the liver but the placenta, central nervous system (CNS), glial tissue, and choroid plexus vasculature also demonstrate this ~apacity.'~, 34 At the whole body level, it is also possible to measure elongase and desaturase activity in vito using deuterium or C13-labeled EFA by evaluating the product precursor ratios in plasma and tissue compartments. We have evaluated the biosynthesis of DHA and AA using LA and LNA labeled with five deuterium atoms positioned in carbons n-1 and n-2.lo3Concentrations of deuterated precursors and products were measured in plasma using negative ion mass spectrometry of pentafluorobenzyl derivatives. Peak concentrations of labeled precursor in plasma were reached during the first day after dosing; deuterated product concentrations increased over time peaking by 48 hours in the term infants and closer to 96 hours in the preterm infants. Figure 2 illustrates a representative response of a preterm compared with term infants; details have been pub1i~hed.l~~ The ratios of labeled final product, DHA or AA, respectively, relative to precursor are in the order of 1 to 100 for the n-6 series and 1 to 10 for the n-3 series. Unfortunately, neither these studies nor other published work permit a quantification of biosynthesis because no account of precursor pool size can be made. In addition, it is apparent from studies and nonhuman primates that uptake kinetics are quite different across tissues and that plasma may not fully reflect whole-body equilibrium.lo8Studies using uniformly labeled 13C precursors confirm that preterm and term neonates are able to synthesize DHA and AA by elongating and desaturating parent EFAs.~~, 40, Io4 There is also evidence for metabolic retroconversion in these studies. These studies appear to demonstrate greater biosynthesis LCPUFA at younger gestational and postnatal ages. Alternatively, the results indicate a reduced turnover rate of LCPUFA with advancing age.39

FETAL EFA SUPPLY

The fetus and the placenta are fully dependent on maternal EFA supply for their growth and development. The major fat deposition in the human fetus occurs during the third trimester, but key phospholipids in placental vessels and uterine vasculature are dependent on EFA supplied by the mother for eicosanoid 91 Crawford et a136have speculated formation from the moment of conception.61* that maternal and fetal vascular development are dependent on adequate EFA; this mechanism has been proposed to explain partly the significance of EFAs on fetal growth and cerebral vascular development. Maternal dietary LA and LNA supply serve as precursors for n-3 and n-6 LCPUFA synthesis by the maternal liver. The placental transfer of fatty acids is regulated in part by the transplacental fatty acid gradient. Serum albumin

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Figure 2. Plasma levels of d5-LNA, d5-DHA, d5-LA, and d5-AA after the administration of 50 mg-kg-’ body weight of d-5 LA and d-5 LNA, from a gestational age infant at 28 weeks (preterm) in comparison with a gestational age infant at 39 weeks (term).The disappear-

ance over time (24, 48, and 96 hours) of d-5 labeled precursors and appearance of d-5 labeled products demonstrates the capacity of micropremies to biosynthesize LCPUFAs. In each group: First bar = 24 hours; second bar = 48 hours; third bar = 96 hours. (Data from Salem N, Wegher B, Mena P, et al: Arachidonic and docosahexaenoic acids are biosynthesized from the 18-carbon precursors in human infants. Proc Natl Acad Sci USA 93:49-54, 1996.) concentration and alpha-fetoprotein have a high binding affinity for free fatty acids; this may be important for placental fatty acid transfer.4lr 75 Mammalian fetuin is a placental protein with a 50-fold greater efficiency in binding fatty acids relative to albumin. Lipoprotein lipase on the maternal surface of the sincytiotrophoblast hydrolyzes maternal triacylglicerol releasing free fatty acids. Fetal erythrocytes also appear to perform a significant role in the placental DHA There is a progressive enrichment in the concentration of AA and DHA in circulating lipids in the fetus during the h r d trimester, at a time when fetal demands for vascular and especially neural growth are greatest.63Significant increases in the AA and DHA content of fetal brain tissue during the last trimester of gestation and initial postnatal months have been observed.29A total of 600 g of EFA are transferred from mother to fetus during a full-term gestation; net uptake approximates 2.2 g per day. AA and DHA are supplied to the fetus from the maternal diet and by endogenous fetal biosynthesis (liver desaturation and elongation). Studies conducted in different populations using similar methodology have shown that the pattern of change in fatty acid composition during gestation and pregnancy is similar across different ethnic and diet groups?* Populations with higher maternal plasma concentrations of n-6 fatty acids have lower n-3 content and vice versa. DHA concentrations decrease during late gestation and n-6 cord levels in term infants are less dependent on maternal levels than cord DHA. Most n-3 fatty acids, which come into the fetal circulation, are accrued by the

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fetus despite low maternal n-3 concentrations. A need for LCPUFA supplementation during pregnancy is suggested by these results. This may be particularly important for populations with low n-3 EFA intake, for multiparous women, or during multiple pregnancies.5OS61 The changes in maternal fatty acid plasma and red cell content are paralleled by changes in fetal levels measured by cordocentesis. The latter are similar to values obtained at birth except for a significantly higher cord LA level measured during c e n t e ~ i sThe . ~ ~ results confirm that fetal DHA relative blood content and absolute concentration increase with gestational age. In the case of AA, levels increase or decrease with gestation depending on study, although there is a better positive correlation with birth weight than that observed with DHA." Differences in fatty acids profiles at 34 weeks' gestation in women delivering preterm and those delivering term babies have been noted. Red blood cell (RBC) and plasma AA are higher in mothers delivering preterm babies. The comparison suggests that maternal AA mobilization and availability may be altered in women delivering preterm. Indicators of n-3 EFA deficiency in preterm maternal RBCs and amniotic membranes are depressed. Preterm maternal n-3 and n-6 ratios suggest that perinatal n-3 EFA metabolism plays an important role in preterm birth.99This is of special significance because prostaglandins play a key role in the onset of labor; they also induce abortion and labor. AA in amniotic fluid increases at the initial process of human parturition. On the contrary, n-3 fatty acids may retard the onset of labor as shown by a randomized controlled trial of fish oil supplementation in humans that demonstrated prolonged gestation with higher birth weight in the n-3 supplemented.90 EFA AND GENE EXPRESSION, MOLECULAR MECHANISMS, AND POSSIBLE EFFECTS The interaction between nutrients, such as LCPUFAs, and genes occurs in several modes. Lipids as components of specialized cell membranes and organelles may affect membrane fluidity and protein-lipid interactions that result in changes in overall cell function. These effects may modulate receptor activity, transport in and out of cells, hormonal, and other signal transduction processes, including gene expression. In addition, eicosanoids derived from LCPUFAs are actively involved in the regulation of cell growth, differentiation, and multiple other functions that depend on gene expression. We focus our short discussion of this topic on the more specific aspects of fatty acids on gene expression. Fatty acids as oxidative substrates play an important role in modulating their own metabolism, synthesis, and oxidation. This is mediated, in part, by their effects on the expression of lipogenic enzymes, mitochondria1 oxidative enzymes, and gluconeogenic enzymes. In addition, LCPUFAs affect the expression of genes that regulate cell differentiation and growth. Specifically, for DHA a possible effect on retinal neuronal differentiation has been suggested.lolThis may have profound implications, because early diet may affect structural development of organs, and thus may have long-lasting consequences. We specifically address the effects of n-3 and n-6 LCPUFAs on these two aspects of nutrientgene interactions. Lipogenic enzymes n-3 and n-6 PUFAs reduce hepatic lipogenesis by decreasing the content of enzymes involved in lipid synthesis (fatty acid synthetase, acetyl-CoA carboxylase, stearoyl-CoA carboxylase, malic enzyme). This reduction is explained by regulation of gene transcription; that is, the level of the mRNAs for these enzymes is decreased.30, 31

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The development of white adipose tissue starts with the differentiation of fibroblasts into adipocytes. This process involves the induction of specific proteins, adipocyte fatty acid binding protein, phosphoenol-pyruvate creatine kinase (PEPCK), acyl-CoA synthetase, and lipoprotein lipase. Fatty acids or their derived compounds induce the expression of these adipocyte-specific gene products and stimulate adipocyte differentiation.6,7, 52 A specific role for AA in the expression of fibroblast transcription factors (c-fos and Erg-1) which modulate growth and differentiation, has been identified. T h s effect is mediated by forma*07 tion of prostaglandin E, (PGE,) and activation of protein kina~e.~*, The mechanism for the regulation of gene expression by LCPUFAs may include general transcription activation mediated by nuclear receptors or the specific activation of transcription factors belonging to the superfamily of nuclear receptors. In the first case, fatty acid acting as ligands may promote dimerization of nuclear receptors, specifying homodimer or heterodimer formation. In addition, fatty acids may promote the interaction of the receptor with coactivators; the binding site for the coactivator is formed upon fatty acid binding. In the second case, fatty acids exert transcriptional control through transcription factors that bind cis-regulatory elements found in target genes. Two types of transcription factors associated with the transcriptional effect of PUFA have been found: (1) the peroxisome proliferator-activated receptor (PPAR) and (2) the hepatic nuclear factor-4cr (HNF-4a). Both PPAR and HNF-4, belong to the superfamily of nuclear receptors that also includes steroid hormone receptors, the glucocorticoid receptor, vitamin D receptor, the tyroxine receptor, and the retinoic acid receptor (R X R)?O, 31 (Figure 3 provides a schematic representation of PPAR system regulation and Box 1 summarizes the main biochemical and cellular processes affected by PPAR expression.

Figure 3.The mechanism for transcriptional regulation of peroxisornal proliferator activated receptor (PPAR) system by fatty acids. PPRE is the peroxisornal proliferator responsive element and R x R is the retinoic acid receptor. In addition to fatty acids and drugs such as fibrates and Thiazolidinediones, eicosanoids can bind and activate PPAR. Other unknown potential activators or inhibitors are shown as a question mark. Box 1 provides a summary of processes regulated by PPAR modulation of gene transcription.

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Transcription-activation assays clearly demonstrate that PPAR transcription factors are activated by PUFA, apparently by direct binding of the free fatty acid. In addition, the PPARa null mice displays defective mitochondria1 fatty acid catabolism as compared with wild-type mice! It is not possible, however, to rule out that the observed transcriptional regulation is mediated by fatty acid metabolites or by indirect modification of PPAR activity. For example, in the case of the transcription factor HNF-4a, the identified ligand is the acyl-CoA hoester derivative of the long-chain fatty acid and not the fatty acid itself. Binding of acyl-CoA thioesters of long-chain fatty acids to the ligand binding domain of HNF-4a modulates its transcriptional activity. Agonistic ligands include saturated acyl-CoAs with 14 to 16 carbon chain length. Antagonistic ligands include n-3 and n-6 polyunsaturated fatty a c y l - C ~ A s . ~ ~ The PPAR family of nuclear receptors has received considerable recent attention due to its major role in the regulation of lipid and glucose metabolism and adipocyte differentiation. PPPAR isoforms a, p, and y are encoded by different genes; they can be distinguished based on their metabolic effects and display differential tissue specific expression. PPARa is involved predominantly in fatty acid metabolism in liver, but is also expressed in other tissues, such as kidney, heart, skeletal muscle, and brown adipose tissue. The expression of PPARy is predominantly observed in adipose tissue where it normally acts by suppressing adipocyte differentiation.There are two PPARy isoforms that derive from the same gene by alternative promoter usage and splicing. Specific mutations for PPARy, associated with enhanced adipocyte differentiation but with marginal effect on insulin sensitivity have been recently identified in humans.1oo A model depicting the regulation of PPAR activation is presented in Figure 3. The transcription activation process is mediated by a heterodimer formed by PPARs with R x R (Box 1.)Drugs such as fibrates and thiazolidinediones, also act by PPAR activation as shown in Figure 3. The net effects of these drugs on metabolism include enhanced peroxisomal proliferation, increased fatty acid oxidation, lower plasma triglyceride levels, and improved glucose t~lerance.~” 65, 116

Box 1. Biochemical and Cellular Processes Regulated by PPAR Modulation of Gene Transcription Mitochondria1 p-oxidation Microsomal oxidation Peroxisome proliferation Adipocyte differentiation

Lipogenesis Lipoprotein metabolism Gluconeogenesis Glycolisis

The regulation of the genes encoding enzymes responsible for fatty acid metabolism during the perinatal period represents an excellent model to evaluate nutrient gene interactions because these genes are affected by the changes in substrate availability, which occur after birth. The changes in perinatal fuel metabolism from glucose predominant to mixed fat-glucose oxidation and other nutritional changes, such as enteral nutrition, have profound effects on lipid m e t a b ~ l i s m The . ~ ~ roles of LCPUFAs in adipocyte differentiation and in CNS development add further interest to t h s emerging field.

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MECHANISMS FOR POTENTIAL EFFECTS OF EFA ON SIGNIFICANT NEONATAL MORBIDITIES Changes in Lipid Membrane Properties

Fatty acid composition of structural membrane lipids can affect membrane function by modifying overall membrane fluidity, by affecting membrane thickness, by changing lipid phase properties, by specific changes in the membrane microenvironment, or by interaction of fatty acids with membrane proteins.77, lz5 Most dietary n-3 fatty acid-induced membrane changes are not reflected by an overall change in membrane fluidity but rather result in selective changes in membrane microenvironment affecting specific domains. The replacement of DHA by DPA usually results in the same overall lipid unsaturation level. Thus, fluidity on average remains unchanged. Furthermore, the main changes in the physical state, induced by changes in the fatty acid composition of lipid bilayers, occur after the first and second double bonds are introduced; namely, when a saturated fatty acid, such as stearic acid (18:0), is replaced by oleic acid (18:l n9) or by linoleic acid (18:2 n-6).117,127 Others have suggested that DHA supply modifies the phospholipid molecular species present in neural tissues, thus possibly affecting overall function.78Recently, Litman and have reported that LCPUFAs present in membrane phospholipid molecular species have profound effects on G-protein activation and related structural modifications. The rhodopsin activation in response to light involves a transformation of the MI form to MII. This MI H MI1 equilibrium constant is six times higher with di-DHA acylated phosphatidylcholine (PC) than with dimyristic (saturated C14:O) PC. The di-DHA PC has an equilibrium constant that is almost identical to that of native rod disks. The effect is mostly explained by the increase in membrane-free volume. The greater mobility of rhodopsin withn the lipid microenvironment most likely explains the change to G-protein activation and the corresponding enhanced signal transduction to photon stimuli.79Figure 4 depicts the effect of lipid unsaturation on membrane microenvironment. Proteins, such as rhodopsin, have greater mobility in photoreceptor membranes if surrounded by DHA.

Membrane bilayer with SATURATED fatty acids is RIGID

Membrane bilayer with UNSATURATED fatty acids is FLUID

Figure 4. The effect of lipid unsaturation on membrane microenvironment and on lipid protein interaction. Solid elements represent protein within the lipid bilayer, mobility of proteins and membrane packing are clearly affected by fatty acid composition.

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Diet-induced changes in structural lipids affect the functional characteristics of excitable membranes in several animal species and in human neural cell lines.16,59* 89, 125 Electrocardiographic abnormalities, such as a notching in the QRS complex, indicating impaired electrical conduction, occur in LA and aLNA acid deficiency before clinical signs appeatZ6Either LA or a-LNA acid corrected these abnormalities. More recently, it has been shown that dietary fat influences the susceptibilityto cardiac arrhythmia, their incidence, and severity.= Furthermore, studies with myocardial preparations have indicated that the vulnerability to catecholamine-induced arrhythmia is reduced in animals fed either n-6 or n-3 PUFA-enriched diets.74Feeding fish oil from bluefin tuna rather than sunflower oil and saturated fat resulted in a marked reduction in induced arrhythmia in two animal species and in isolated papillary muscle.28Changes in cardiac electrophysiologic responses to beta-mimetics and reduced excitability of cardiac myocytes and in the susceptibility to arrhythmia have also been noted.54,74 Myocytes form minimal amounts of cycloxygenase products and no lipoxygenase products; thus, the changes in excitability and conduction are probably related to structural lipid composition, and reflect changes in the function of ion channels.59N-3 fatty acid supplementation ameliorates the fluidifying effect of ethanol on neural membranes, whereas LA linoleic and a-LNA acid deficiency enhanced volatile anesthetic action in rats; LA supplementation specifically reverses this effect.42 The role of membrane lipid composition in determining the electrical properties of cultured neuronal cells exposed to exogenous fatty acids has been i n v e ~ t i g a t e d .Both ~ ~ , ~n-3 ~ and n-6 fatty acids induced slower rates of rise, and to a lesser extent loser amplitude, of Na+ action potentials. The opposite effects were observed when saturated or transmonoenoic fatty acids were added. It seems likely that these effects were mediated by a change in the number of active Na+ channels. A change in membrane composition or altered.fatty acid availability to the cells could have caused this event.80 The clinical significance of these experimental findings is hard to determine, but clearly these relate to the function of critical organs, such as the lung, CNS, kidney, vascular responses, and the intestine. Disease conditions may affect membrane integrity, transport function, electrophysiologic responses, vasomotor tone, among others. Changes in Surfactant Composition and Properties

The reaction catalyzed by PC cytidiltransferase is the rate-limited step in the CDP-choline pathway for surfactant synthesis in the fetal lung. Cytidiltransferase is essentially inactive in the absence of lipids. Fatty acids induce the conversion of cytidiltransferase from a low to a high molecular weight form; t h s effect is potentiated by corticosteroids. Fatty acids also activate cytidiltransferase by increasing its translocation from cytosol to microsomes.'28Changes in phospholipid fatty acid composition may modify membrane structure affecting binding or activation of membrane-associated cytidyltransferase necessary for PC synthesis.'" Under conditions of low free fatty acid availability that exist in the fetal lung, type I1 cell glucocorticoid receptor binding may be inhibited and de novo fatty acid synthesis decreased. As a result, phospholipid synthesis in the fetal lung may be less dependent on an exogenous supply of fatty acids relative to mature lung.lZ2 Preliminary data in humans demonstrate the uptake of dietary fatty acids by the lung and the incorporation of these into surfactant obtained from tracheal

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lavage.32Thus, the supply of EFA and LCPUFA may affect surfactant composition with possible adverse consequences. Sosenko et aP1 have speculated that altered surfactant composition may explain the adverse effect of intravenous lipids administered during the first day of life. Parenteral n-3 PUFA have been demonstrated to be incorporated rapidly into lung tissue and reduced both vascular resistance and endothelial permeability in the pulmonary circulation, thus blunting edema formation in animal Effects Mediated by Eicosanoid Production Production of various eicosanoids is another mechanism by which the effect of LCPUFA supplementation on different physiologic functions may be explained. Phospholipases liberate AA and eicosapeutaenoic acid (EPA) from membrane lipids and through the action of cycloxygenase or lipoxygenase form eicosanoid productions. Prostaglandins, prostacyclins, thromboxanes, and leukotrienes derived from LCPUFA play a key role in modulating inflammation, cytokine release, immune response, platelet aggregation, vascular reactivity, thrombosis, and allergic phenomenon. The balance between AA(n-6) and EPA(n3) in biologic membranes is regulated based on dietary supply. The n-6:n-3 ratio in phospholipids modulates the balance between prostanoids of the 2 and 3 series derived from AA and EPA, respectively. Series 3 prostanoids are weak agonists or in some cases antagonize the activity of series 2 prostanoids. Eicosanoids of the 2 series promote inflammation, platelet aggregation, and activate the immune response. On the contrary, series 3 prostanoids tend to ameliorate these effects.I9,lz6 Figure 5 summarizes the role of n-6 and n-3 balance in regulating eicosanoid effects of potential interest to the micropremie. This balance could affect disease severity, progression, and recovery.

Linoleate n-3 PUFA

n-6 PUFA

Phospholipids Arachidonic acl Eicosapentaenoic ac

Inflammation Citokines

Inmune response Vascular reactivity

Thrombosis Bronchoconstriction

Bronchoconstriction Chemotaxis Inflammation

Figure 5. The role of n-6/n-3 fatty acid balance in determining membrane phospholipid composition and eicosanoid production. Excess n-6 favors arachidonate acid derived series 2 eicosanoids whereas eicosapentaenoic acid generates series 3 eicosanoids that antagonize the former.

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Pulmonary Disease

Hyaline Membrane Disease. This may be affected by n-6 and n-3 LCPUFA balance because surfactant function is dependent on its desaturated palmitic acid composition. If PUFA are incorporated a change in surface tension can be demonstrated. As discussed previously, a predominantly n-6 PUFA intravenous infusion affects survival of micropremies; for infants under 800 g receiving lipids in the first day of life overall survival was only 53% versus 75% in the controls."' The n-6 and n-3 balance may also affect changes in pulmonary vascular responses as has been demonstrated during acute intralipid infusion.76,97, 'lo Other mechanisms that come into play are changes in membrane permeability and edema formation, which may contribute to augment barotrauma and oxidative injury. Experimental data in animals indicate that dietary n-3 fatty acids may ameliorate endotoxin-induced acute lung injury by suppressing the levels of proinflammatory eicosanoids in bronchoalveolar lavage fluid and reducing pulmonary neutrophil accumulati0n.8~Further data in rodents demonstrate a decrease in mortality in rat pups of dams fed fish oil-supplemented diet throughout pregnancy and lactation; this was associated with lower PGE2 lung Bronchopulmonary Dysplasia. The main features of bronchopulmonary displasia (BPD) are related to alterations in the airway and loss of lung parenchyma. Airway obstruction owing to bronchoconstriction, increased mucous production, pulmonary edema, and often pulmonary hypertension accompany this disease. Virtually all these features correspond to known actions of AA metabolites; prostanoids; and leukotrienes C4, D4, and E4. Moreover, leukotrienes have been postulated to amplify oxygen radical-mediated lung injury by inducing chemotactic mediators, which attract polymorphonuclear cells and increase vascular permeability. In adult rats exposed to hyperoxia, lipooxygenase blockage results in decreased tissue damage. Lung lavage fluid in infants with BPD demonstrates significantly increased leukotriene levels as compared with age-matched infants with normal lungs or with hyaline membrane disease. These findings suggest a possible role for these inflammatory mediators in the pathophysiology of t h s disea~e.9~. 'lo During this decade, the use of intravenous lipids has been linked to BPD, although a causal relationship has not been clearly established. The earlier use of lipids in infants affected with severe lung disease may have contributed to making this association more evident. The effect of lipids in pulmonary hyper97 Thus, the tension has been demonstrated to be mediated by eico~anoids.~~, recommendation is to avoid the use of high-dose intravenous lipids in patients with instability of pulmonary vasculature or with high oxygen needs. The use of medium chain triglyceride (MCT) and a more balanced n-6:n-3 ratio in the PUFA supply could be justified on the basis of existing knowledge. The advent of a parenteral fat emulsion containing EPA and DHA to provide n3 LCPUFA in the management of critically ill patients is still under experimental 114 The specific fatty acid composition of this emulsion is designed evaluati~n.~~, to modulate cytokine production while preserving cell-mediated immunity.56A study by Carlson et alu demonstrated in a post hoc analysis a lower incidence of BPD in preterm infants fed formula supplemented with LCPUFA (AA+DHA). The control group (n=45) had a 40% prevalence, whereas the supplemented group (n = 49) had a 24% (P
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antioxidant status. The combined use of MCT and n-3 LCPUFA in emulsions as structured lipid has generated great interest and potentially could be used in parenteral nutrition in the next generation of lipid products. Infectious Morbidity

Necrotizing Enterocolitis. Present views on the pathogenesis of necrotizing enterocolitis (NEC) include a final common pathway of mucosal injury linked to feeding, bacterial proliferation, hypoxia, and ischemia. Mucosal injury is thought to be mediated by cytokine release activated by any of these factors. Recent controlled clinical observations using a randomized and masked design in premature infants suggest that a formula containing egg phospholipids as a source of LCPUFAs (DHA and AA) may reduce the incidence of NEC.'2 The control formula infants (n = 85) had a 17.6%prevalence of proved NEC, whereas the egg phospholipid (n = 34) formula-fed infants had only 2.9%. They speculate that one or more components present in the egg phospholipids enhanced gut maturation. LCPUFAs, phospholipids, or choline could potentially mediate this protective response. A larger-scale clinical trial is presently in progress in an attempt to validate this initial observation. The balance between AA and EPA could play a role in defining prostanoid synthesis and thus preventing intestinal mucosal injury. In a previous study this same investigator reports a nonsignificant association of LCPUFA supplementation and increased NEC incidence.21 Classic studies by Cerami's group in rodents have demonstrated a tumor necrosis factor (TNF)-induced gut injury that is nearly identical to NEC. These effects can be modulated by dietary n-6 and n-3. More recently, interleukin-6 has been demonstrated to play a key role in the activation sequence leading to tissue inj~ry.4~ Elevated interleukin-6 in amniotic fluid and in umbilical cord blood has been associated with NEC and other neonatal morbidity including sepsis and intraventricular hemorrhage. Whether diet modulation of cytokine release can prevent NEC deserves further study. Accumulation of AA and increased prostanoid production has been demonstrated during reperfusion of ischemic myocardium and ischemic gut in adults. This has been shown in newborn pigs with gut ischemia induced by occluding the superior mesenteric artery.93Efflux of 6-keto-PGF1a, an AA prostanoid derivative, represents a component of the response to mesenteric ischemia. In tlus study oxygen free radical scavengers did not alter the prostanoid increase after ischemia. The mechanisms by which dietary LCPUFA modulate cytokine production have not been fully elucidated. Changes in the production of the eicosanoids PGE, and leukotrienes B4 and a reduction in the intracellular signal transduction pathway involved in the synthesis of cytokines have been suggested as an explanation for the protective effects of n-3 PUFA.13,19 Others propose that n-3 fatty acids modulate protein kinase C activity, which may participate in transcriptional control of TNF gene expression via the activation of transcription factors NF-kappa B.56 Sepsis. Systemic infections resulting from bacterial, fungal, or viral agents are extremely common in the micropremie. As previously indicated, dietary lipid supply may affect inflammation and the immune resp0nse.5~Experimental studies suggest that n-3 LCPUFA may blunt the response to endotoxin and modulate undesirable sequelae secondary to sepsis by decreasing the production of inflammatory cyt~kines.'~ Interleukin-1 and TNF produced from stimulated mononuclear cells have a potent inflammatory and catabolic effect. The feeding of n-3 LCPUFA supplements to young piglets given endotoxin reduces the lactic acidosis and maintains or improves tissue perfusion of the intestine, heart, and

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lung. Studies in critical adult patients using an enteral nutrition product that contains n-3 LCPUFAs, arginine, and nucleic acids demonstrate a beneficial effect on clinical outcome possibly mediated by a modulation of the inflammatory and immune response. This study, however, does not permit the evaluation of isolo6 lated LPCUFA s~pplernentation.'~, Brain Injury (Ischemia and Hemorrhage).Hypoxic and hemorrhagic insults to the neonatal brain are frequent in the micropremie. Most ischemic injury occurs prior to or at birth; IVH occurs mostly in the first hours of life. Thus, it is difficult to propose a nutritional prevention of these conditions, unless the intervention is given to the mother. Whether maternal dietary LCPUFA supply plays a role in defining occurrence and severity of brain hemorrhagic injury is not known. Crawford et a136have speculated, based on limited data from animal observations, that poor maternal dietary LCPUFA supply could be responsible for the high prevalence of hemorrhagic injury observed in micropremies. In addition, the possibility of dietary modulation of cytokine release should be considered, because cytokines mediate much of the vascular and tissue damage observed during and after re~erfusi0n.l~ Evidence of cytokine role in periventricular leukomalasia has been reported.37,64 There is pharmacologic modulation of AA metabolism presently used in the form of indomethacin, a potent inhibitor of cyclooxygenase. The effect of early lipid supply on brain injury deserves further research. LIPID PEROXIDATION, OXIDANTS, AND ANTIOXIDANTS

The susceptibility of lipid membranes to oxidative damage is dependent on the degree of unsaturation. Feeding high PUFA diets is a potent oxidant stress, which if coupled to iron supplementation leads to the typical syodrome of vitamin E deficiency. In fact, the classic syndrome of hemolytic anemia was described in premature and malnourished infants given, h g h LA formula.53 Present formulas have compensated the addition of PUFA with the corresponding increase in tocopherol content. Other components of the antioxidant system include superoxide dismutase; catalase; glutathione; and other vitamins, such as alpha tocopherol, retinol, carotene, and ascorbic acid. Most antioxidant enzyme systems mature in the first weeks after birth. Several enzymes are dependent on micronutrients for their activity. Selenium and glutathione peroxidase, iron and catalase, copper, and zinc are necessary for super oxide dismutase activity. Feeding highly unsaturated fatty acids increases the demand for antioxidant and enhances the possible membrane damage induced by pro-oxidants. High plasma levels of malonyldialdehyde, or pentane plus ethane in expired air as an indication of oxidative damage in preterm babies has been correlated with adverse outcome: death or chronic pulmonary disease.66, 96 Peroxidation can augment damage in response to injury, namely cell membrane rupture, platelet aggregation, increase cell adhesion, and vasoconstriction. A significant inverse relationship between ethane and pentane in expired and gestational age has been noted; this may be explained by the decreased antioxidant capacity observed in preterm infants.lZ1 Lung damage induced by reactive oxygen species has previously been presented as an important factor in the pathogenesis of respiratory distress and BPD in preterm babies.=*'lo Hypoxia- and reoxygenation-induced free radical production may play an important role in the development of retinopathy of prematurity and intraventricular hemorrhage by damage to the microvasculature.

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Few clinical studies have addressed the possible adverse consequences of feeding LCPUFAs in terms of lipid peroxidation; most LCPUFA-supplemented formula have added antioxidants to compensate for the increase in oxidative potential. Now adverse clinical effects have been noted in terms of BPD or retinopathy of prematurity. In our published study on the safety of LCPUFA supplementation we evaluated red cell fragility in response to peroxide stress, malonyldialdehyde production, and tocopherol content. No adverse consequences were noted when testing intact red cell membrane. If we blocked antioxidant enzyme activity with sodium azide, however, increased malonyldialdehyde production was noted.'19,lZoFurther research is needed fully to resolve h s issue. EFFECTS OF EFA DEFICIT AND SUPPLEMENTATION IN PRETERM INFANTS

The short-term effects of combined n-6 and n-3 EFA deficiency have been well characterized in the past. Over the past decade the effect of isolated n-3 or n-6 deficiency has been described, principally affecting growth and CNS development during early life. The specific effects of LCPUFA deficit in the presence of adequate supply of LA and LNA have proved more difficult to elucidate, given that biosynthesis of AA and DHA from the respective precursors occurs even in premature infants. Whether endogenous biosynthesis from dietary precursors is sufficient to meet the needs for growth and development remains the object of present research efforts. Clinical signs of combined EFA deficiency became apparent in infants fed skim milk-based diets in the 1950s and in those given lipid-free parenteral nutrition in the late 1960s. These infants presented with dryness, desquamation and thickening of the skin, and growth faltering as manifestations of LA deficiencyz0,55 In LNA deficiency they include abnormal visual function and peripheral neuropathy.6°Subclinical deficiency of EFA has been studied in preterm and term infants. Most clinical alterations can be explained by changes in membrane-related function or eicosanoid-mediated changes. The subtle effects observed with LCPUFA supplementation are most likely related to discrete changes in membrane properties or possibly to changes in the developmental program related to the expression of specific genes. As previously noted, the lipid composition of the diet can affect the structure and function of membrane lipids modifying overall membrane properties, including fluidity and thickness. In addition, by specific changes in membrane microenvironment or by interaction of fatty acids with membrane proteins may affect specific membrane domains responsible for function. The changes in neural membranes of greatest potential significance to the human infant are those related to changes in physical properties and to changes that affect membrane excitability.102The functional response of the retina and the occipital cortex can be electrophysiologically measured: electroretinogram and pattern-reversal visual-evoked potential, or by behavioral methods (forced-choice preferential looking acuity). Additional studies have included measures of novelty-choice preferential looking43 and mental or motor development at 12 and 18 months. Studies of the effect of LCPUFA on sleep-wake cycle development, sympathetic tone, auditory evoked responses, and activity level have also been carried out in our laboratories. We have studied preterm infants receiving human milk or randomized to formulas with different EFA content. The functional impact of this fatty acid modification included enhanced maturation of the rod photoreceptor responses

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in LCPUFA supplemented babies, mimicking matched human milk-fed preterm infants of similar postconceptional age (36 weeks). By 57 weeks, a time when retinal development is nearly complete, the difference in photoreceptor function was not apparent, except for changes in oscillatory potentials, which reflect inner retinal signal processing. Visual acuity tests throughout the 6-month study were also less mature in infants receiving formula devoid of LCPUFA despite ample provision of LNA. The LCPUFA-supplemented group had significantly better visual acuity as measured by visual-evoked potential and forced-choicepreferential looking acuity than the control formula group. Highly significant correlations were found for both visual-evoked potential and forced-choice preferential looking acuity visual acuity when compared with the level of DHA in multiple lipid fractions from study infants.l1e 120 In our more recent studies conducted in Chle, no LCPUFA effect on auditory brain stem evoked responses were demonstrated; this coincides with FaldeIla et [email protected] was an increased maturation of the sleep-wake cycle in terms of less indeterminate sleep and more quiet (non-rapid eye movement) sleep in the human milk-fed compared with the randomized formula-fed groups. Bayley scale at 18 months demonstrated better developmental indices in the human milk-fed but no differences within formula-fed groups. The findings in developmental indices suggest that at least in our setting environmental influences have a greater effect than early diet effects. Carlson's et aP1, 24 randomized clinical study in preterm infants supplemented with LCPUFA demonstrated better visual acuity in infants up to 4 months of age. After this time, control infants caught-up in visual function measures. These investigators also report evidence of more rapid visual processing as measured by the Fagan test of visual recognition at 6 to 12 months of age in LCPUFA supplemented infants. The reduction in AA, when fish oil was provided as a source of n-3 fatty acids, was associated with reduced weight and length growth.1z4In a second preterm infant study using low EPA marine oil for up to 2 months corrected age, Werkman and C a r I ~ o n demonstrated '~~ improved visual development at the 2 months follow-up and a 10-point intelligence quotient difference favoring the DHA-supplemented group at 12 months. No significant drop in AA or deleterious effects on growth was observed when low EPA marine oil was used. The DHA-supplemented group had shorter look times in the novelty preference test at 9 months suggesting better visual processing. There are several recent studies in term infants; some, but not all, have shown transient effects of LCPUFA supplementation on visual function and mental 6b70, 72, 12, 21, 83 The difference in level of supplementation de~elopment.~, and the sensitivity of outcomes measured most likely explain the variability in results. Present efforts are centered on evaluating the duration of these effects in terms of CNS development and other relevant effects, such as body adiposity and risk for development of diet-related chronic disease. The role of DHA in myelin formation has been demonstrated in patients with generalized peroxisoma1 disorders who have greatly diminished brain DHA. This could be of potential importance in whte matter disease of micropremies.86Some indication of the long-term effects of LCPUFA supplementation can be drawn from studies of human milk-fed infants, because the presence of LCPUFA is one of the notable differences between these two feeding regiments. The data of Lucas et alsl suggest a remarkable benefit (intelligence quotient 5 points higher at 8 years) from even 28 days of human milk feeding in early life. It is virtually impossible, however, to draw conclusions for long-term effects of LCPUFAs from these type of studies, because despite the efforts to control for confounders there are

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differences in mothers and families who choose to provide breast milk to feed their preterm infants. The metabolic effect of LCPUFA supplementation can be explained by the combined effect of these fatty acids on genes related to lipid and glucose metabolism or by changes in signal transduction secondary to the higher DHA content in membrane phospholipids fatty acid composition. Membranes composition might directly influence the metabolic rate in mammals; a higher LCPUFA to saturated fatty acid ratio is associated with more permeable membranes requiring a higher Na-K pump activity, thereby raising the energy needs.35The study of LCPUFA supplementation of preterm infants has not clearly demonstrated an effect on growth, except for Carlson's et a1 study using formulas with high LA and high EPA marine oil, the lower levels of AA were associated with lower weight gains9 Recent studies of young infants indicate that having a higher LCPUFA content in membrane phospholipids from skeletal muscle phospholipids is associated with lower fasting plasma glucose. Changes in muscle membrane phospholipid fatty acid saturation may influence the subsequent development of insulin resistance.Io The prevalence of several diet-related chronic diseases, like type I1 diabetes, Alzheimer's dementia, age-related macular degeneration, cancer, and several autoimmune diseases, may be influenced by the balance of dietary n-6 and n-3 LCPUFA during the first two thirds of the life span as well as during the geriatric periodj7,48 Changes in membrane composition induced by dietary changes can be measured by gas cromatographic fatty acid analysis of plasma or red cell total lipids or subfractions, or from lipids extracted from scrapped buccal mucosal cells. The study of infants who died suddenly of an unexplained cause has served to document a strong correlation between the composition of the brain cortex and RBC total lipids, and that brain cortex composition is clearly affected 45, 46 by early dietary LCPUFA supply.18* Recently, in vivo MR spectroscopy was used to evaluate the fatty acid composition of adipose tissue noninvasively in 21 term and preterm infants. Differential spectral resonances arising from natural 13 carbon in different regions of the triglyceride acyl chain were discerned. The corresponding saturated, unsaturated, monounsaturated, and polyunsaturated fatty acids carbons were quantified on relative basis. Differences in the distribution of 13 carbon spectra for various moieties related to gestational age and mother-infant pairs were observed. The authors suggest that diet-induced differences could also be identified by this te~hnique."~ This and other noninvasive methods may expand the study of the effects of early dietary LCPUFA supply on health and disease in later life. ALTERNATIVES IN THE PROVISION OF EFA TO PRETERM INFANT Human Milk. A good starting point is to mimic the composition of human milk. Unfortunately, human milk lipid composition varies significantly depending on the mother's diet during pregnancy and lactation; postpartum age; preterm or term delivery, and maternal diseases affecting lipid metabolism (diabetes, cystic fibrosis, and abetalipoproteinemia). AA is the predominant n-6 and DHA is the most important of the n-3 LCPUFAs in human milk. The ratio of total n-6 to n3 is 5 : l to lO:l, ranging up to 18:l if corn, sunflower, or safflower oils are consumed. The ratio of AA to DHA is most commonly 1:1 to 2:l. EPA is found in minimal amounts except in populations consuming high

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fish intake." DHA levels range from close to 0.1% reported from Germany to 1.4% in Inuits of North America; however, typical values range from 0.3% to 0.4%.67,73 Makrides et a P reported a longitudinal reduction in breast milk DHA from Australian women on Western diets from 0.32% in 1981 to 0.21% in 1995. The definition of what to feed is not simply answered by deciding to follow the human milk model. Deciding how much to feed based on the upper range of LCPUFA content, the lower range, or the midpoint must be based on functional responses. If the effort is primarily focused on demonstrating efficacy, selecting a value in the upper range is preferable. On the other hand, if serious safety concerns were an issue, selecting a value in the lower range is more appropriate. Supplementation of formula-fed preterm with graded levels of AA (0% to 1.1%) and DHA (0% to 0.76% of total fat) compared with human milk-fed babies demonstrated that plasma and RBC LCPUFA levels can be mimicked by supplementing formula at similar levels. For omnivorous women this meant 0.54% AA and 0.3% DHA.51 Sources of LCPUFAs to Use in the Infant Formulas. The main source for the de novo synthesis of n-3 fatty acids in the aquatic environment are marine autotrophic bacteria, microalgae, and protozoa, whch constitute the zooplankton and phytoplankton. Fish higher in the food chain incorporate the omega-3 PUFA and further elongate them to form EPA and DHA. Thus, fish concentrate EPA and DHA as triglycerides, mainly in the adipose tissue and in the fat of muscle and visceral organs. The hgher the fat content of fish, the higher its content of n-3 fatty acids.' Another important source of LCPUFA is egg yolk phospholipids. The concentrations of PUFA are different depending on the feed given to animals. The ample use of fish meal in chicken feed has increased egg yolk DHA.105,109 LCPUFA products for blend in infants formulas can be produced successfully if chicken feed is carefully monitored and refined lipid extraction procedures are used. This is presently an important LCPUFA source used in some infant formulas. Bacterial strains and microalgae isolated from the intestinal content of some fish show a remarkably high content of EPA and DHA.J 33 Efforts to grow these microorganisms in natural or artificial sea water to obtain DHA for nutritional or pharmacologic use have been successful. In addition, selected fungal strains produce concentrated AA, which is suitable for human consumption. The industrial production of AA, EPA, and DHA from strains of single-cell organisms has lead to an expanded use of this source. Single-cell oils offer a promising new source of LCPUFAs provided mass production becomes commercially feasible.71Rigorous purity and toxicologic testing should be conducted on fatty acid sources intended for use in commercial infant formula. Initial studies used a mixture of vegetable oils to supply LA and LNA and marine oil as a source of n-3 LCPUFAS.~~, 118, 119 More recent studies, including our own, have used nearly pure DHA from marine oil fractions or DHA and AA from single-cell oils. Most published work to date is based on marine oil, marine oil fractions, and egg phospholipids as sources. Present clinical research efforts are concentrated in defining the implications of using different LCPUFA sources in infant formula. SUMMARY Lipids are structural components of all tissues and are indispensable for cell membrane synthesis. The brain, retina, and other. neural tissues are particularly rich in LCPUFAs, affecting neural structural development and function. LCPUFAs serve also as specific precursors for eicosanoid production (prostaglandins,

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prostacyclins, thromboxanes, and leukotrienes). These autocrine and paracrine mediators are powerful regulators of numerous cell and tissue functions (e.g., thrombocyte aggregation, inflammatory reactions, and leukocyte functions, vasoconstriction and vasodilatation, blood pressure, bronchial constriction, uterine contraction). Dietary lipid intake affects cholesterol metabolism at an early age and is associated with cardiovascular morbidity and mortality in later life. Over recent years, the role of fatty acids in modulating signal transduction and regulating gene expression have been described, emphasizing the complex of fatty acid effects. Dietary fatty acids, especially LCPUFA, can have significant effects in the modulation of developmental processes affecting the clinical outcomes of extremely premature infants. References 1. Abeywardena MY, McLennan PL, Charnock JS: Diet and cardiac arrhythmia: Involvement of eicosanoids. In Lands WEM (ed): Proceedings of AOCS Short Course of Polyunsaturated Fatty Acids and Eicosanoids. Champaign, IL, American Oil Chemist’s Society, 1987, p 62 2. Ackman RG: Structural homogeneity in unsaturated fatty acids of marine lipids: A review. J Fish Res Board Canada 21247-254, 1964 3. Akimoto MT, Ishii K, Yamagaki K, et al: Production of eicosapentaenoic acid by a bacterium isolated from mackerel intestines. J Am Oil Chem SOC67911-915, 1990 4. A1 MDM, Homstra G, Schouw YT, et al: Biochemical EFA status of mothers and their neonates after normal pregnancy. Early Hum Dev 24:239--248, 1990 5. A1 MDM, Van Houwelingen AC, Kester ADM, et al: Maternal essential fatty acid pattern during normal pregnancy and their relationship to the neonatal essential fatty acid status. BMJ 74:55-68, 1995 6. Amri E, Ailhaud G, Grimaldi P: Regulation of adipose cell differentiation: 11. Kinetics of induction of the aP2 gene by fatty acids and modulation by dexamethasone. J Lipid Res 321457-1463, 1991 7. Amri E, Bertrand B, Ailhaud G, et al: Regulation of adipose differentiation. I. Fatty acids are inducers of aP2 gene expression. J Lipid Res 32:1449-1456, 1991 8. Aoyama T, Peters JM, Iritani N, et al: Altered constitutive expression of fatty acidmetabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor a.J Biol Chem 273:5678-5684, 1998 9. Auestad N, Montaldo M, Hall R, et al: Visual acuity, erythrocyte fatty acid composition, and growth in term infants fed formulas with long chain polyunsaturated fatty acid for one year. Pediatr Res 4l:l-10, 1997 10. Baur L, OConnor J, Pan D, et al: The fatty acid composition of skeletal muscle membranes phopholipid: Its relationship with the type of feeding and plasma glucose levels in young children. Metabolism 47:106-112, 1998 11. Birch EE, Birch DG, Hoffman DR, Uauy RD: Retinal development in very low birth weight infants fed diets differing in omega-3 fatty acids. Invest Ophthalmol Vis Sci 33~236552376,1992 12. Birch EE, Hoffman DR, Uauy R, et al: Visual acuity and the essentiality of docosahexaenoic acid and arachidonic acid in the diet of term infants. Pediatr Res 441-9, 1998 13. Blok W, Katan M, van der Meer J: Modulation of inflammation and cytokine production by dietary (n-3) fatty acids. J Nutr 126:1515-1533, 1996 14. Bourre JM, Dihn L, Boithias C, et al: Possible role of the choroid plexus in the supply of brain tissue with polyunsaturated fatty acids. Neurosci Lett 224:14, 1997 15. Bourre JM, Durand G, Pascal G, et al: Brain cell and tissue recovery in rats made deficient in n-3 fatty acids by alteration of dietary fat. J Nutr 119:15-22, 1989 16. Bourre JM, Francois M, Youyou A The effects of dietary alpha-linolenic acid on the composition of nerve membranes, enzymatic activity, amplitude of electrophysiological parameters, resistance to poisons and performance of learning task in rats. J Nutr 119:1880-1892, 1989

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17. Bower RH, Cerra FB, Bershadsky 8, et al: Early enteral administration of a formula (impact) supplemented with arginine, nucleotides, and fish oil in intensive care unit patients: Results of a multicenter, prospective, randomized, clinical trial. Crit Care Med 23:436-449, 1995 18. Byard RW, Makrides M, Need M, et al: Sudden infant death syndrome: Effect of breast and formula feeding on frontal cortex and brainstem lipid composition. J Pediatr Child Health 31:14-16, 1995 19. Calder PC: N-3 polyunsaturated fatty acids and cytokine production in health and disease. Ann Nutr Metab 41:203-234, 1997 20. Caldwell MD, Johnson HT, Othersen HB: Essential fatty acids deficiency in an infant receiving prolonged parenteral alimentation. J Pediatr 81:894-898, 1972 21. Carlson SE, Ford AJ, Werkman SH, et al: Visual acuity and fatty acids status of term infants fed human milk and formulas with or without docosahexaenoate and arachinodate from egg yolk lecithin. Pediatr Res 39:882-888, 1996 22. Carlson SE, Montaldo MB, Ponder DL, et al: Lower incidence of necrotizing enterocolitis in infants fed a preterm formula with egg phospholipids. Pediatr Res M491498, 1998 23. Carlson SE, Werkman H, Tolley E: Effect of long-chain n-3 fatty acid supplementation on visual acuity and growth of preterm infants with and without bronchopulmonary dysplasia. Am J Clin Nutr 63:687-697, 1996 24. Carlson SE, Werkman SH, Peeples JM, et al: Arachidonic acid status correlates with first year growth in preterm infants. Proc Natl Acad Sci U S A 90:1073-1077, 1993 25. Carnielli VP, Wattimena DJL, Luijendijk I, et al: 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, 1996 26. Caster Wo, Ahn P: Electrocardiographic notching in rats deficient in EFA. Science 139:1213, 1963 27. Chan S, McCowen KC, Bistrian B: Medium-chain triglyceride and n-3 polyunsaturated fatty acid-containing emulsions in intravenous nutrition. Curr Opin Clin Nutr Metab Care 1:163-169, 1998 28. Charnock JS: Antiarrhythmic effects of fish oils. In Simopoulos AP, Kifer,RR, Martin RE, Barlow SM (eds): Health Effects of w-3 Polyunsaturated Fatty acids in Seafood. World Rev Nutr Diet 66:278-291, 1991 29. Clandinin MT, Chappell JE, Leong S, et al: Intrauterine fatty acid accretion rates in human brain: Implication for fatty acid requirements. Early Hum Dev 4121-130,1980 30. Clarke SD, Jump DD: Dietary polyunsaturated fatty acid regulation of gene transcription. Annu Rev Nutr 1483-98, 1994 31. Clarke SD, Jump DB: Fatty acid regulation of gene expression: A unique role for polyunsaturated fats. In Berdanier C, Hargrove JL (eds): Nutrition and Gene Expression. Boca Raton, FL, CRC Press, 1993, pp 227-246 32. Cog0 PE, Camielli VP, Bunt JE, et al: Endogenous surfactant turnover in critically ill human infants measured with stable isotopes. Pediatr Res 43:278A, 1998 33. Cohen Z , Norman HA, Heimer Y M Microalgae as a source of n-3 fatty acids. World Rev Nutr Diet 771-31, 1995 34. Cook Hw: In vitro formation of PUFA by desaturation in rat brain: Some properties of the enzymes in developing brain and comparison with liver. J Neurochem 30:13271334, 1978 35. Cortright R, Muoio D, Dohm L: Skeletal muscle lipid metabolism: A frontier for new insights into fuel homeostasis. Nutr Biochem 8:228-245 1997 36. Crawford M, Costeloe K, Ghebremeskel K, et al: Are deficits of arachidonic and docosahexaenoic acids responsible for the neural and vascular complications of preterm babies? Am J Clin Nutr 66:1032S-l041S, 1997 37. Dammann 0, Leviton A: Maternal intrauterine infection, cytokines, and brain damage in the preterm newborn. Pediatr Res 421-8, 1997 38. Danesch U, Weber PC, Sellmayer A Arachidonic acid increases c-fos and Erg-1 mRNA in 3T3 fibroblasts by formation of PGE2 and activation of protein C. J Biol Chem 269:27258-27263, 1994

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39. Demmelmair H, Sauerwald T, Koletzko B, et al: New insights into lipid and fatty acid metabolism via stable isotopes. Eur J Pediatr 156:S70-S74, 1997 40. Demmelmair H, Schenck W, Behrendt B, et al: Estimation of arachidonic acid synthesis in full term neonates, using natural variation of 13C content. J Pediatr Gastroenterol Nutr 21:31-36, 1995 41. Dutta-Roy AK Fatty acid transport and metabolism in the feto placental unit and the role of fatty acid binding protein. J Nutr Biochem 8:54&557, 1997 42. Evers AS, Elliot WJ, Lefkowith JB, et al: Manipulation of rat brain fatty acid composition alters volatile anesthetic potency. J Clin Invest 771028-1033, 1986 43. Fagan JF 111: The relationship of novelty preferences during infancy to later intelligence and later recognition memory. Intelligence 8:339-346, 1984 44. Faldella G, Govoni M, Alessandroni R, et al: Visual evoked potentials and dietary long chain polyunsaturated fatty acids in preterm infants. Arch Dis Child 75:F108F112, 1996 45. Farquharson J, Cockburn F, Ainslie P W Infant cerebral cortex phospholipid fattyacid composition and diet. Lancet 340:810-813, 1992 46. Farquharson J, Jamieson EC, Logan RW, et al: Age- and dietary related distributions of hepatic arachidonic and docosahexanoic acid in early infancy. Pediatr Res 38:361365, 1995 47. Fernandez G, Venkatraman J T Role of omega 3 fatty acids in health and disease. Nutr Res 13:S19-S45, 1993 48. Folsom AR, Ma J, Mc Govern P, et al: Relation between plasma phospholipid saturated fatty acids and hyperinsulinemia. Metabolism 45:223-228, 1996 49. Ford H, Watkins S, Reblock K, et al: the role of inflammatory cytokines and nitric oxide in the pathogenesis of necrotizing enterocolitis. J Pediatr Surg 32275-282, 1997 50. Foreman-van Drogelen M, A1 MDM, Van Houweligen A, et al: Comparison between the essential fatty acid status of preterm and full-term infants, measured in umbilical vessels. Early Hum Dev 42:241-251, 1995 51. Gibson R, Neumann M, Makrides M: Effect of increasing breast milk docosahexaenoic acid on plasma and erythrocyte phospholipid fatty acids and neural indices of exclusively breast fed infants. Eur J Clin Nutr 51:57&584, 1997 52. Grimaldi PA, Knobel SM, Whitesell R, et al: Induction of the aP2 gene by nonmetabolized long chain fatty acids. Proc Natl Acad Sci USA 89:10930-10934, 1992 53. Gross SJ: Vitamin E. Nutritional needs of the preterm infant. Scientific basis and practical guideline. In Tsang RC, Lucas A, Uauy R, et a1 (eds): New York, Caduceus Medical Publishers, 1993, p 103 54. Hallaq H, Smith T, Leaf A: Modulation of dihydropyridine-sensitive calcium channels in hearts cells by fish oil fatty acids. Proc Natl Acad Sci USA 89:1760-1764, 1992 55. Hansen AE, Wiese HF, Boelsche AN, et al: Role of linoleic acid in infant nutrition: Clinical and chemical study of 428 infants fed on milk mixtures varying in kind and amount of fat. Pediatrics 31:171-192, 1963 56. Hayashi N, Tashiro T, Yamamori H, et al: Effects of intravenous n-3 and n-6 fat emulsion on cytokine production and delayed type hypersensitivity in burned rats receiving total parenteral nutrition. J Parenteral Enteral Nutr 22363-367, 1998 57. Heller A, Koch T, Schmeck J, et al: Lipids mediators in inflammatory disorders. Drugs 55:487-496, 1998 58. Hertz R, Magenheim J, Berman I, et al: Fatty acyl-CoA thioesters are ligands of hepatic nuclear factor-4a. Nature 392:512-516, 1998 59. Holh CM, Rosen P: The role of arachidonic acid in rat heart cell metabolism. Biochem Biophys Acta 921:35&363, 1987 60. Holman RT, Johnson SB, Hatch TF: A case of human linolenic acid deficiency involving neurological abnormalities. Am J Clin Nutr 35:617423, 1982 61. Honstra G, A1 MDM, Van Houwelingen AC, et al: Essential fatty acids, pregnancy and pregnancy outcome. In Bindels JC, Goededhart AC, Visser HKA (eds): Recent Development in Infant Nutrition. London, Kluwer Academic Publishers, 1996, pp 5143 62. Horby Jorgensen M, Holmer G, Lund P, et al: Effect of formula supplemented with

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