Emerging Evidence for Long-chain Polyunsaturated Fatty Acids · derived n-3 fatty acids and an...

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Scientific Review:The Role of Nutrients in Immune Function of Infants and Young ChildrenEmerging Evidence for Long-chainPolyunsaturated Fatty Acids

PHILIP CALDER, PhD

SUSAN PRESCOTT, MD, PhD

MICHAEL CAPLAN, MD

Edited by

BMS5490_ImmunMonoBk_C21 6/12/07 11:23 AM

PrefaceMead Johnson & Company, the maker of Enfamil LIPIL® infant formulas, continues toinvestigate the effects of LCPUFA on health. This monograph reviews the role of nutrients inimmune function of infants and young children, including a more in-depth look at the roles ofLCPUFA.The intent of the monograph is to provide health care professionals a balanced overviewof research on this topic. It is to be used as an educational resource only and is not intended forproduct support or for the development of product claims.

We are pleased that you are interested in this emerging area of nutrition and we welcome thisopportunity to help you learn more about the roles of LCPUFA in nutrition.


Executive Summary 1

Introduction 5

LCPUFA in Health: An Overview 6

Immunology and the Immune System: An Overview 11

Effects of LCPUFA on Immune Function 16

LCPUFA and Healthy Immune System Development:Clinical Reports in Pregnancy, Lactation, and Early Childhood 20

Overall Summary and Conclusions 27

References 29

Table of Contents


Executive Summ

ary

Executive SummaryGrowing evidence suggests that long-chain polyunsaturated fatty acids (LCPUFA) havecritical roles in the growth and development of infants and children and may havebeneficial long-term effects on health throughout life. Studies have shown that twoLCPUFA in particular, docosahexaenoic acid (DHA; 22:6n-3) and arachidonic acid (ARA;20:4n-6), have important roles in infant cognitive development, visual acuity, andgrowth. These two LCPUFA are naturally present in human milk and are permitted assupplemental ingredients in infant formulas available in many countries. The purposeof this monograph is to review published studies evaluating the roles of dietary LCPUFAin supporting immune system development and function.

Widespread changes in dietary fat intake have occurred over the past 150 years in mostindustrialized nations, marked by a decrease in consumption of marine- and plant-derived n-3 fatty acids and an increase in consumption of n-6 fatty acids.1,2 As intake ofn-6 fatty acids has increased, the ratio of dietary n-6:n-3 fatty acids has shiftedsubstantially. Concomitant with changes in dietary LCPUFA intake have been significantincreases in the prevalence of atopic disease in a number of populations.3 Someresearchers have focused on dietary fatty acid intake as a potential factor in theincreased prevalence of atopy.

Because atopic sensitization tends to occur in early life, clinical studies evaluating therelationship between LCPUFA intake and incidence of atopic disease in infants andchildren have focused in two areas:

• The effects of maternal supplementation during pregnancy and lactation onbreast milk LCPUFA content and neonatal blood LCPUFA levels, and correlationswith atopic disease in infants, and

• The relationship between LCPUFA intake during infancy and childhood andsubsequent development of atopy.

Some epidemiological studies and preliminary clinical studies have suggested thatconsumption of n-3 LCPUFA in pregnancy, lactation, and early childhood may haveimplications for immune development and the subsequent risk of asthma andatopic disease.

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LCPUFA in Health: An OverviewThere is evidence from clinical studies that LCPUFA have key functions in maintaining healththrough their effects on the immune system and, in particular, through their modulation of theinflammatory response, which appear to be mediated through effects as precursors ofinflammatory mediators, as well as through other complex cellular mechanisms.4 Theseimmunomodulatory effects have generated obvious interest in the potential role of LCPUFA incommon inflammatory conditions in childhood, such as asthma, eczema, atopic dermatitis, andfood hypersensitivities. Epidemiological surveys support a protective role of oily fish, known tobe naturally rich in n-3 LCPUFA, against atopic disease and reactive airways.5,6 A number ofintervention studies using fish oil supplementation in early life are currently underway, asdiscussed later.

There is also accumulating evidence that supplementation of infant formula with preformedLCPUFA, particularly DHA, has positive effects on visual and mental development in preterminfants7-11 and term infants.12-16

Sources of LCPUFALCPUFA can be supplied in the diet, from tissue stores, or by de novo synthesis from precursorfatty acids. Infants who are breastfed obtain LCPUFA postnatally from human milk. LCPUFAconcentrations in human milk vary widely. Pregnant women who consume diets containinglittle fish or meats may have inadequate LCPUFA intake to meet demands for infant LCPUFAaccretion and replenishment of their own LCPUFA stores.17 Formula-fed infants who receiveformula without preformed LCPUFA must rely on endogenous synthesis from essential fattyacid precursors to meet their LCPUFA needs.18 Many experts now agree that rates of DHAsynthesis from alpha-linolenic acid typically provided in neonatal formula are variable frominfant to infant19 and may not provide adequate tissue DHA availability required for optimalneural development.20 These unpredictable systemic DHA levels may further contribute toaltered immune development at a critical timepoint during infancy and early childhood.

Immunology and the Immune System OverviewBreast milk intake is a key factor in the immune system development of newborns and infants,and breast milk contains numerous components that are thought to modulate immunologicalresponses, such as cytokines, growth factors, lactoferrin, oligosaccharides, leukocytes, IgA, andpolyunsaturated fatty acids. Clinical studies suggest that increased n-3 LCPUFA concentrationsin breast milk are associated with increased breast milk levels of IgA and some cytokines.21 Theeffects of such alterations in breast milk content on immune development have not yet beendetermined. However, it has been speculated that maternally derived immune factors couldhave direct immunomodulatory effects and/or promote infant IgA production.21

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Effects of nutrition on immune functionAppropriate functioning of the immune system is dependent on nutritional status.Malnutrition, including protein-energy malnutrition and micronutrient deficiencies, is animportant risk factor for illness and death, particularly among pregnant women, infants, andyoung children. Poor protein intake is associated with significantly impaired immunity.Inadequate levels of vitamins and minerals in the diet can have significant negativeconsequences for immune function. Key vitamins evaluated for their roles in immunity includethe fat-soluble vitamins A and E and the water-soluble vitamins C, B6, B12, and folate. Of themany minerals with functions in immune cell response, iron, zinc, and selenium have been theprimary focus of population-based supplementation studies.

Effects of LCPUFA on Immune FunctionLong-chain fatty acids, particularly DHA and ARA, are preferentially incorporated into cellmembrane phospholipids.22 In cell membranes, LCPUFA contribute to membrane fluidity, haveroles in signal transduction and gene expression, and provide substrate for production ofchemical mediators.22 All three functions contribute to immune cell responses.

Precursors of eicosanoids and docosanoidsChemical mediators derived from LCPUFA include eicosanoids, a family of mediators whichincludes prostaglandins, leukotrienes, and thromboxanes,22 as well as resolvins, docosatrienes,and neuroprotectins.23 ARA is a precursor of the 2-series prostaglandins (eg, PGE2) andthromboxanes and 4-series leukotrienes, all of which are predominantly pro-inflammatoryeicosanoids.22 DHA can be retroconverted to eicosapentaenoic acid (EPA; 20:5n-3), which ismetabolized to form the 3-series prostaglandins (eg, PGE3) and thromboxanes, as well as the 5-series leukotrienes. These EPA-derived chemical mediators tend to be less inflammatory thanthose produced from ARA.4,24 Both EPA and DHA give rise to resolvins and related mediatorsthat have potent anti-inflammatory activity.23

Early studies of LCPUFA effect on immune functionNumerous studies in adults and children suggest relationships between intake of specificLCPUFA and alterations in markers of immune function. Results have shown that:

• Persons with allergy may have altered levels and ratios of n-3 and n-6 fatty acids, includingDHA and ARA, compared with non-allergic persons.25-27

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• Supplementing the diet with preformed LCPUFA alters the levels of those LCPUFA inplasma phospholipids and immune cells.4,24,28,29

• Supplementing the diet with preformed LCPUFA results in discrete changes in immunecell and cytokine production; these changes differ with type of LCPUFA supplementationand are influenced by the ratio of n-6 to n-3 LCPUFA in the diet.30,31

LCPUFA and Healthy Immune System Development: ClinicalReports in Pregnancy, Lactation, and Early ChildhoodThere is a growing body of evidence suggesting that dietary intake of LCPUFA early in lifecould influence immune development and other health outcomes, including respiratoryhealth. Because maternal diet is the most important determinant of LCPUFA accretion forthe woman and her infant, dietary investigations have focused on the relationship betweenLCPUFA intake and status in pregnant and breastfeeding women and the incidence ofatopic diseases in their children. Numerous studies have demonstrated relationshipsbetween breast milk LCPUFA composition and incidence or markers of atopic disease ininfants and children. Several studies have shown relationships between maternal LCPUFAintake and development of atopic dermatitis in breastfed infants, although theserelationships are not consistent.

Emerging evidence in both animal and human studies suggests that n-6 LCPUFA, includingARA, have important roles in immune system development and regulation.29,32,33 Data fromanimal studies have demonstrated a substantial accretion of ARA in the thymus during earlygrowth and development,32 and ARA is consistently present in breast milk.

There are now a number of studies investigating the role of n-3 LCPUFA supplementation inearly life in the development and progression of atopy and childhood asthma. For example,studies have examined the relationships between maternal and infant intake of n-3 LCPUFA,immune development, and subsequent development of atopy. At this stage there is onlypreliminary evidence that exposure to adequate n-3 LCPUFA in utero and via breast milk isassociated with reduced development of atopic disease in infants and children.34 Furtherstudies are underway to confirm these associations. Several studies are also underway toevaluate the effects of early postnatal fish oil supplementation on asthma and allergicoutcomes. The only findings published so far indicate no clear benefits on these outcomes at5 years of age,35 although there were benefits at 1836 and 3637 months of age. A number ofother studies are on-going which will assess the effects of supplementation with higherdoses of n-3 PUFA from birth.

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Growing evidence in animal and human studies suggests that long-chain polyunsaturatedfatty acids (LCPUFA) have critical roles in the growth and development of infants andchildren and may have beneficial long-term effects on health throughout life. Studies haveshown that two LCPUFA in particular, docosahexaenoic acid (DHA; 22:6n-3) and arachidonicacid (ARA; 20:4n-6), have important roles in infant cognitive development, visual acuity, andgrowth. These two LCPUFA are naturally present in human milk and are provided assupplemental ingredients in many infant formulas available around the world. There isclinical evidence that infants may benefit from preformed dietary DHA and ARA either inbreast milk or in supplemented infant formulas. Furthermore, certain LCPUFA function asprecursors of compounds involved in modulation of the immune response. The purpose ofthis monograph is to review published studies evaluating the roles of dietary LCPUFA insupporting immune system development and function.

Widespread changes in dietary fat intake have occurred over the past 150 years in mostindustrialized nations, marked by a decrease in marine- and plant-derived n-3 fatty acids andan increase in consumption of n-6 fatty acids.1,2 As intake of n-6 fatty acids has increased, theratio of dietary n-6:n-3 fatty acids has shifted substantially. Whereas “traditional” diets ofearly humans provided a ratio of 1-2:1, contemporary diets provide a ratio up to 30:1.2

Population-based intake data used to establish Dietary Reference Intake (DRI) levels of n-6and n-3 fatty acids suggest that this ratio is approximately 10:1 in US diets.38

Concomitant with changes in dietary LCPUFA intake have been significant increases in theprevalence of atopic disease in a number of populations.3 The rapidity of this rise suggeststhat environmental factors, including alterations in dietary fat intake, may beresponsible.39,40 Because atopic sensitization tends to begin in early life,41,42 clinical studiesevaluating the relationship between LCPUFA intake and incidence of atopic disease in infantsand children have focused in two areas:

• The effects of maternal supplementation during pregnancy and lactation on breastmilk LCPUFA content and neonatal LCPUFA levels, and correlations with atopic diseasein infants, and

• The relationship between LCPUFA intake during infancy and childhood and subsequentdevelopment of atopy.

Current understanding of the effects of maternal LCPUFA supplementation in pregnancy isonly preliminary, but suggests early immune effects and reduced allergic outcomes.28,43

Further studies are underway. The reported effects of postnatal supplementation have beendisappointing to date, with no long-term benefits of fish oil supplementation in early infancyon asthma or allergy outcomes at 5 years of age.35 The results of other postnatal interventionstudies currently underway are awaited with interest.

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Introduction


There is evidence that LCPUFA have key functions in maintaining health. Clinical studies haveshown that dietary LCPUFA may have positive effects on visual and mental development,atopicdisease, and intestinal health, and may influence cardiovascular disease. LCPUFA are structuralcomponents of cell membranes and are precursors of other compounds which influence theinflammatory response. Inflammation is a key component of the normal immune response, yetinappropriate or uncontrolled responses can result in disease.4,24 LCPUFA and metabolites ofLCPUFA influence systemic immune responses to surgery, injury, and infection, as well aschronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease.4

Positive influence on visual and mental developmentIt has been well-established that supplementation of infant formula with preformed LCPUFA,particularly DHA, has positive effects on visual development in preterm infants7-9,11 and terminfants.12-16 DHA supplementation of infant formula has also been associated with improvedmeasures of mental development in preterm infants10 and term infants.12 Among breastfedinfants, increased blood DHA levels have been associated with improvements in neural andvisual development.44

Possible immune effectsConditions commonly associated with atopy, such as asthma, wheezing, eczema, and foodhypersensitivities, are common in children. These conditions have a strong inflammatorycomponent and could therefore be influenced by dietary LCPUFA. Epidemiological surveyssupport a protective role of oily fish, known to be naturally rich in long-chain n-3polyunsaturated fatty acids, against asthma.5,6,45 Data from the first US National Health andNutrition Survey (NHANES I) found that dietary fish intake was positively associated with lungfunction in adults.6 An Australian study noted that children who regularly consumed oily fishhad a significantly reduced risk of current asthma.5 Some recent prospective intervention andobservational studies have provided support for a role of LCPUFA in the development ofimmune function and atopic disease in young children, whereas others have not.

LCPUFA appear to modulate immune effects through a number of complex pathways, mostnotably as precursors of eicosanoids and through effects on intracellular signaling, whichaffects many aspects of cell function (including cytokine production and cell activationmolecules). LCPUFA may also have secondary effects by modifying oxidative stress.46 DietaryLCPUFA are preferentially incorporated into cellular membranes, where their highlyunsaturated chemical structure lends fluidity to the membranes, and where they provide areservoir for eicosanoid production.47 Thus, LCPUFA can affect broad-ranging aspects ofimmune cell function. Further studies are needed to determine the relevance of theseeffects during pregnancy and early childhood.

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Positive influence on intestinal healthIn vitro, animal, and preliminary clinical studies suggest that LCPUFA may have a role inpromoting intestinal health. LCPUFA have been shown to influence gut barrier function byaffecting intestinal secretions, mucus secretions, and density of surfactants in the mucuslayer.48 In vitro studies have shown that specific fatty acids, including LCPUFA, may influencegut microflora by inhibiting growth of pathogenic bacteria (anaerobes).49,50 It is well-established that the immature infant gut is susceptible to antigen sensitization, and thatearly feeding experiences can influence development of food allergies.51,52 Gut-associatedlymphoid tissue and numerous immune cells in the intestine provide a primary defenseagainst food-borne antigens and pathogens.52,53 Interventions which increase the strength ofthis defense may reduce infants’ risk of atopic sensitization.

Infants born prematurely are particularly at risk for necrotizing enterocolitis (NEC), aninflammatory disorder of the intestine which is associated with significant morbidity andmortality. In a double-blind, randomized, controlled study of LCPUFA supplementation inpreterm infants, it was found that infants fed preterm formula providing higher levels ofARA, DHA, and esterified choline developed significantly less NEC compared with infants feda control formula (2.9% vs 17.6%).54 A neonatal rat model also showed that feeding LCPUFAreduced the incidence of necrotizing enterocolitis (NEC) and intestinal inflammation.55

Additional clinical studies, however, showed no significant benefit of LCPUFA on theincidence of NEC in preterm infants,10,56 but differences in study design, dosing, andappropriate numbers of enrolled infants preclude a clear conclusion on this potentiallyinteresting relationship and further experimental investigation is warranted.

Potential influence on cardiovascular healthLCPUFA also are being investigated for their role in the prevention of endothelial activation,with implications for the initiation, progression, and clinical course of atherosclerosis.57,58

Preliminary clinical studies suggest that LCPUFA consumption early in life may have lastingeffects on cardiovascular risk; infants who received formula with added DHA and ARA forthe first 4 months of life had lower blood pressure at 6 years of age compared with infantswho received unsupplemented formula although both groups were within the normalrange.59

Sources of LCPUFALCPUFA can be supplied as a component of the diet, from tissue stores, or by de novosynthesis from precursor fatty acids. LCPUFA are naturally present in most animal tissues.n-3 LCPUFA, including DHA and eicosapentaenoic acid (EPA; 20:5n-3), tend to be higher in

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fish and shellfish than in other animal products, and are particularly high in fatty fish(Table 1). Although these foods are not recommended for young infants, they do providedietary sources of LCPUFA for pregnant and lactating women. Human milk containspreformed LCPUFA in varying amounts, which can be affected by the fat composition of thematernal diet.60-62 In a study of pregnant women in Canada, fish and shellfish contributedapproximately 80% of dietary DHA while meat and poultry contributed approximately 80%of dietary ARA.17 Eggs contributed smaller amounts of both DHA and ARA to subjects’ diets.

Table 1. Selected Food Sources of LCPUFA63

*Data for ARA are reported as “20:4 undifferentiated” by the USDA.

Prior to birth, infants can obtain LCPUFA directly from their mothers via placental transfer.LCPUFA, and DHA in particular, accrue in fetal adipose tissue with advancing gestational ageand may serve as a reservoir for LCPUFA after birth.18,47 Studies have consistently shown asignificant association between maternal and newborn infant DHA and ARA concentrations.64,65

Because much of the infant’s tissue LCPUFA accumulation occurs in the third trimester ofpregnancy, preterm infants are born with lower total LCPUFA reserves compared with terminfants.47,66,67

LCPUFA sources: breastfed infantsPostnatally, infants who are breastfed obtain LCPUFA from human milk. LCPUFA concentrationsin human milk vary widely. Numerous studies conducted among women in different


Food Source DHA (mg) EPA (mg) ARA (mg)*

Salmon filet, baked/broiled (3 oz) 638 456 85

Mackerel, baked/broiled (3 oz) 594 428 43

White tuna, canned in water (3 oz) 535 198 43

Crab, steamed (3 oz) 196 207 71

Chicken, roasted, dark meat (1 cup) 70 14 196

Chicken, roasted, light meat (1 cup) 42 14 98

Turkey, roasted, dark meat (1 cup) 84 0 364

Turkey, roasted, light meat (1 cup) 42 0 210

Egg, hard-boiled (1 large) 19 3 74


geographic regions have evaluated and quantified human milk levels of LCPUFA. A review ofstudies from European and African countries identified median human milk DHA levels of 0.3%of fatty acids and median ARA levels of 0.5% to 0.6% of fatty acids.62 Another review ofworldwide human milk LCPUFA levels reported mean DHA content of 0.45% to 0.88% of fattyacids and ARA content of 0.42% to 0.92% of fatty acids.61 Worldwide average concentrations ofDHA and ARA in human milk can be estimated at 17 mg DHA/100 Cal and 34 mg ARA/100 Cal(based on 0.3% of fatty acids as DHA and 0.6% as ARA, and 50% of calories from fat).60-62

Pregnant and lactating women who consume diets containing little fish or meat may haveinadequate LCPUFA intake to meet demands for infant LCPUFA accretion and for replenishmentof their own LCPUFA stores.17 Dietary supplements are available containing n-3 LCPUFA derivedfrom single-cell organisms or from fish oil which has been processed to remove most or allpotential contaminants.

LCPUFA sources: formula-fed infantsFormula-fed infants who receive formula without preformed LCPUFA must rely on tissue stores andendogenous synthesis from essential fatty acid precursors to meet their LCPUFA needs (Figure 1).18Through a series of desaturation and elongation reactions, enzymes present in the liver and othertissues can convert linoleic acid and alpha-linolenic acid into long-chain derivatives. Linoleic acid isconverted to ARA, which may be incorporated into membrane structures or metabolized furtherinto specific eicosanoids. Alpha-linolenic acid is converted to EPA and to DHA;EPA is used to producea different series of eicosanoids. DHA and EPA are also precursors of chemical mediators calledresolvins, docosatrienes, and neuroprotectins.23,68 Because n-3 and n-6 fatty acids compete forenzymes involved in LCPUFA metabolism and conversion to other compounds, an appropriatedietary balance of n-3 vs n-6 fatty acids is important. Animal studies have shown that the n-3 fattyacid alpha-linolenic acid is a strong suppressor of n-6 fatty acid conversion to LCPUFA; the n-6 fattyacid linoleic acid is less able to suppress n-3 fatty acid conversion to LCPUFA.47

Many experts now agree that rates of DHA synthesis from alpha-linolenic acid at levels typicallyprovided in neonatal formula are variable from infant to infant19 and may not provide adequatetissue DHA availability required for neural development.20 These unpredictable systemic DHA levelsmay further contribute to altered immune development at a critical timepoint during infancy andearly childhood. Infants appear to differ in the extent to which they can convert alpha-linolenic acidinto n-3 LCPUFA69 and in the rates at which newly formed n-3 LCPUFA are incorporated into plasmaphospholipid.70 Further,because alpha-linolenic acid may be preferentially oxidized for energy,71,72 itsavailability for conversion to n-3 LCPUFA may be limited in some infants despite apparentlyadequate intakes.69 Some expert organizations have called for the inclusion of preformed DHA andARA in term and preterm infant formulas.73-77 Commercial infant formulas containing DHA and ARAare available in many markets.

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Figure 1. Conversion of Essential Fatty Acids to LCPUFA

Adapted from Innis18 and Carlson.66



The immune system must achieve a fine balance between providing “protectivesurveillance” against threats, including infectious agents and malignancy, while at thesame time maintaining “tolerance” to harmless things, such as “self” and othercommonly encountered harmless environmental factors.

Allergy results when there is a breakdown in normal “tolerance” mechanisms, whichleads to inappropriate and detrimental immune responses to normally harmlesssubstances, including food allergens such as cow’s milk protein, eggs, nuts, or shellfish,and environmental allergens such as mold, dust mites, or pollen.30 Allergic reactionstypically result in one or more of a group of symptoms usually involving mucosal orcutaneous surfaces where environmental factors are first encountered, includingatopic dermatitis (eczema), asthma, wheezing, coughing, rhinitis, sneezing, nasalcongestion, vomiting, and diarrhea.78

The immune system is made up of an integrated network of organs, tissues, cells, andmolecules which work together to resist infection while maintaining tolerance toharmless factors such as “self” antigens and allergens.30 When a challenge is detected(ie, an allergen or pathogen), cell signaling within and between immune cell typesallows a coordinated immune response. At the cellular level, this involves the secretionof multiple cytokines and eicosanoids, which in normal situations enable cells tocommunicate with each other and to bind, neutralize, and eliminate the challenge.79,80

At birth, the immune system is immature, but it develops with age, antigenstimulation, and appropriate nutrition.81 In addition, bacterial colonization occursduring the first weeks of life, and interactions between intestinal flora and thedeveloping mucosa result in further development of immune responses and oraltolerance. Nutrition plays a key role in the development, maintenance, and optimalfunctioning of immune cells.30 Dietary nutrients such as DHA, EPA, and ARA influencethe nature of an immune response by modulating the types and amounts of cellularmessengers secreted22,82 and by reducing oxidative stress.46

Breast milk intake appears to be a key factor in immune system development amongneonates and infants. Clinical studies suggest that increased n-3 LCPUFAconcentrations in breast milk are associated with increased breast milk levels of IgAand some cytokines.21 The effects of these alterations in milk content on immunedevelopment have not yet been determined. However, it has been speculated thatmaternally derived immune factors could have direct immunomodulatory effectsand/or promote infant IgA production.21 Immune factors provided to the infant inbreast milk, including antigen-specific IgA, cytokines, and other immune cells, maycontribute to the preventive effects of breastfeeding on allergy,21 although this has notbeen confirmed.

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Imm

unology and the Imm

une System:An Overview


Effects of nutrition on immune functionOverall nutritional status is closely linked to appropriate functioning of the immune system.83

In general, deficiency of several nutrients will lead to impaired immune responses, andreplenishment of those specific components will typically restore the affected responses.82,83

Consuming excessive amounts of some nutrients can also be detrimental to immuneresponses.79 Worldwide, malnutrition is closely linked to infectious diseases that areresponsible for substantial morbidity and mortality among infants and children. Studies inboth developing and developed countries have focused on the effects of dietary protein, dietaryfat (including n-3 fatty acids), the fat-soluble vitamins A, D, and E, and minerals such as iron,selenium, and zinc. No single biomarker is available that accurately reflects the effect ofnutritional interventions on the immune response84; as a result, clinical outcomes are mostimportant in determining nutrient effect.

Malnutrition

Scientists have observed the close relationship between infectious disease and malnutritionfor more than 50 years.85 Malnutrition is an important risk factor for illness and death,particularly among pregnant women, infants, and young children. Being malnourishedincreases susceptibility to infection and severity of infection through multiple interactingpathways, which can lead to further deterioration of an individual’s nutritional status. Protein-energy malnutrition, including marasmus (starvation) and kwashiorkor (protein deficiency), isa major cause of immunodeficiency and is associated with significant morbidity and mortalityfrom infectious diseases.86 A 2005 review of literature estimated that malnutrition is indirectlyresponsible for about half of all deaths in young children worldwide and that the risk of deathis directly correlated with the degree of malnutrition.87 Deficiencies in iron, zinc, vitamin A, andiodine also impact immune function and susceptibility to infection among women andchildren in developing countries.87

Protein intake

Inadequate protein intake leads to suboptimal tissue repair and reduced resistance toinfection.86 Poor protein intake is associated with significantly impaired immunity, as

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DefinitionsCytokines: protein molecules that enable immune cells to communicate

with each other in generating an immune response.

Eicosanoids: biologically active substances derived from precursor LCPUFA with 20-carbon chain lengths (ie, ARA [an n-6 LCPUFA] and EPA [an n-3 LCPUFA]). Examples include prostaglandins, leukotrienes,and thromboxanes.


evidenced by altered immune responses and deficits in phagocyte function, complementcascade, antibody concentrations, and cytokine production.88 The amino acids arginine andglutamine have been studied extensively for their roles in promoting immune responsefollowing surgery, trauma, and sepsis; studies suggest that in certain populations these aminoacids may enhance wound healing, increase resistance to infection and tumorigenesis, andimprove immune function.86

Nucleotides

The effect of preformed nucleotides on immune function has been evaluated in a limitednumber of studies, but clinical data are inconclusive. Infants fed a nucleotide-supplementedformula had higher serum concentrations of IgA throughout a 48-week study compared toinfants fed a control formula.89 The supplemented infants had a lower risk of diarrhea between8 and 28 weeks, but this difference was not statistically significant over the period of 48 weeks.Supplemented infants had an increased risk of upper respiratory infections, similar incidence oflower respiratory infections, and similar antibody response to vaccination compared tounsupplemented infants. Another study suggested that nucleotide supplementation improvedHiB and diphtheria antibody responses following immunization.90 Nucleotides may becomeconditionally essential during periods of growth or immunological challenge, and there hasbeen a call for further research to define the importance of these nutrients, particularly infeeding situations involving single sources of nutrition.91

Vitamins and minerals

Inadequate levels of certain vitamins and minerals in the diet can have significant negativeconsequences for immune function.83 Much of what is known about the roles of vitamins andminerals in immune response has come from animal studies, where control of individualdietary components is easily accomplished.85 Population-based studies in countries wherenutrient deficiencies are endemic have substantially increased our understanding of theeffects of vitamins and minerals on the human immune response. Such studies have evaluatedthe impact of large-scale supplementation programs on morbidity and mortality in vulnerablepopulations—frequently infants and young children. While such studies do not directly showcausation, they provide compelling evidence of nutrient effects on immune function.

Key vitamins evaluated for their roles in immunity include the fat-soluble vitamins A and E andthe water-soluble vitamins C, B6, B12, and folate.79,83 Effects of these micronutrients on immunefunction are summarized briefly in Table 2. Of the many minerals with functions in immune cellresponse, population-based studies have focused primarily on iron, zinc, and selenium,85 whichare dietary deficiencies common among women, infants, and children in some developingcountries. Iron deficiency is the most common nutrient deficiency in both developed anddeveloping countries.92,93



Table 2. Effects of Selected Nutrients on Immune Response

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Nutrient Effects

Vitamin A • Improves gut barrier function• Maintains production of mucosal secretions• Protects against oxidative damage (in β-carotene form)• Improves immune response to antigens

Deficiency: increased morbidity and mortality, increased severity of infections,reduced number of lymphocytes, reduced lymphoid organ weight

Vitamin C • Protects against oxidative damage

Deficiency: decreased resistance to infection and cancer; decreased delayed-typehypersensitivity response; impaired wound healing

Vitamin B6 • Required for nucleic acid and protein synthesis (with implications for rapidimmune cell response to antigens)

Deficiency: lymphocytopenia; reduced lymphoid tissue weight; reduced responsesto mitogens; general deficiencies in cell-mediated immunity; lowered antibodyresponses

Vitamin B12* • Required for nucleic acid and protein synthesis• Mediates a variety of immune responses, including cell-mediated

and humoral immunity

Deficiency: depressed immune responses, including delayed-typehypersensitivity response and T-cell proliferation

Vitamin E • Acts as a strong antioxidant, reduces cell membrane peroxidation

Deficiency: rare in humans (except as secondary to fat malabsorption); reducedimmune response, anemia, fetal resorption in experimentally induced deficiency


Table 2. Effects of Selected Nutrients on Immune Response (continued)


Nutrient EffectsFolate* • Required for nucleic acid and protein synthesis

• Mediates a variety of immune responses, including cell-mediatedand humoral immunity

Deficiency: depressed immune responses, including delayed-typehypersensitivity response and T-cell proliferation

Iron • Is fundamental for normal immune system development• Allows proper functioning of enzymes involved in nucleic acid

synthesis and cell replication• Mediates components of inflammatory response

Deficiency: reduced capacity for an adequate immune response, as measured by:decreased delayed-type hypersensitivity response, mitogen responsiveness, andNK cell activity; decreased lymphocyte bactericidal activity; lower IL-6 levels

Selenium • Allows proper functioning of enzymes involved in drug/chemical metabolism and other processes

• Acts as an antioxidant; protects cells from oxidative damage

Deficiency: suppression of immune function; increased cancer incidence andcardiomyopathy in populations with chronic Se deficiency

Zinc • Allows proper functioning of enzymes involved in nucleic acidsynthesis and cell replication

• Improves intestinal barrier function• Mediates unspecific immunity, such as neutrophils and NK cells• Has a role in balance of T helper cell functions

Deficiency: increased susceptibility to infectious diarrhea, increaseddiarrheal and respiratory morbidity

*Note: Immune system effects of vitamin B12 deficiency and folate deficiency are clinically indistinguishable.86

References 86, 88, 94-96.


Long-chain fatty acids, particularly DHA and ARA, are preferentially incorporated intocell membrane phospholipids.22 LCPUFA in cell membranes contribute to variations inmembrane fluidity, influence on intracellular signal transduction and gene expression,and provide the substrate for eicosanoid production.22 Although all three effects couldcontribute to the immune response of many cell types, only effects related toeicosanoid production will be discussed herein.

Precursors of eicosanoids and docosanoidsChemical mediators derived from LCPUFA include eicosanoids (a family of mediatorswhich include prostaglandins (PG), leukotrienes, and thromboxanes), and docosanoids,as well as resolvins, docosatrienes, and neuroprotectins.22,23 These mediators act in amanner similar to hormones, but unlike hormones they have localized actions and arecleared rapidly from circulation.66 The production of LCPUFA-derived mediators isdirected by cytokines (and other stimuli), and in turn the production of cytokines isdirected by LCPUFA-derived mediators, forming complex feedback and feed-forwardinteractions. The overall physiological effects of eicosanoids depend on the organ ortissue of origin, the balance among individual eicosanoid types (eg, prostaglandins vsleukotrienes), and the balance among classes of eicosanoids (eg, 2-seriesprostaglandins derived from ARA vs 3-series prostaglandins derived from EPA).97,98

ARA is the precursor of the 2-series prostaglandins (eg, PGE2) and thromboxanes and 4-series leukotrienes (Figure 2). These compounds are predominantly pro-inflammatory,and they also influence T-cell, B-cell, and natural killer cell activities.4,24 The actions ofeicosanoids derived from dihomo-γ-linolenic acid (20:3n-6; the precursor of ARA) tendto oppose those of eicosanoids derived from ARA.22,66

Although ARA is associated with pro-inflammatory responses, in vitro and animalstudies suggest that ARA has an essential early role in immune cell growth in thethymus, and in differentiation, migration, and proliferation of immune cells. There is asubstantial accretion of ARA in the mouse thymus during early growth anddevelopment, which is consistent with other findings of placental ARA enrichment inthe fetus and the presence of ARA in breast milk.32 The pro-inflammatory effectsassociated with compounds derived from ARA have key roles in maintaining the healthof the individual.4,24

16 E f f e c t s o f L C P U F A o n I m m u n e F u n c t i o n

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f LCP

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Figure 2.22 Metabolic Derivatives of Linoleic Acid, Dihomo-γ-linolenic Acid, and ARA

DHA is readily incorporated into cell membrane phospholipids, where it exerts effects onfluidity and upon the function of membrane proteins such as receptors. DHA can also beretroconverted to eicosapentaenoic acid (EPA; 20:5n-3); EPA is a substrate for synthesis ofeicosanoids such as the 3-series prostaglandins (eg, PGE3) and thromboxanes, as well as the 5-series leukotrienes (Figure 3). These have a different structure from the eicosanoids producedfrom ARA, which affects their potency. In general, eicosanoids derived from EPA are lessinflammatory in nature than those produced from ARA.22

EPA also gives rise to resolvins of the E-series while DHA gives rise to D-series resolvins,docosatrienes, and neuroprotectins.23 These mediators have strong anti-inflammatory effects.23,68

In addition to giving rise to mediators of different activities and potencies, EPA and DHAcompete with ARA for metabolism. By doing so, n-3 LCPUFA slow the production of pro-inflammatory eicosanoids derived from ARA.4,22

17E f f e c t s o f L C P U F A o n I m m u n e F u n c t i o n


Figure 3.22,23 Metabolic Derivatives of Alpha-Linolenic Acid, EPA, and DHA

Early studies of LCPUFA effect on immune functionMuch of the evidence for the immunomodulatory role of LCPUFA has been derived from cellculture and animal studies. These studies have shown that LCPUFA influence several areas ofthe immune response, including T-cell activity and proliferation, as well as cytokine secretionand antigen presentation.99 These studies suggest that changes in the fatty acid compositionof immune cells and in the mediators they produce as a result of preformed LCPUFA intake(particularly n-3 LCPUFA intake) may result in altered immune responses and could potentiallylead to significant effects in promoting health.

Studies in adults and children suggest relationships between intake of specific LCPUFA andchanges in markers of immune function. Results have shown that:

• Persons with allergy may have altered levels and ratios of n-3 and n-6 fatty acids,including DHA and ARA, compared with non-allergic persons. For example, several studiesin infants and children have observed inverse relationships between risk or subsequentdevelopment of allergy and atopic disease and blood levels of PUFA, including ARA.25-27,100

• Supplementing the diet with preformed LCPUFA alters the levels of those LCPUFA inplasma phospholipids and immune cells.4,24 LCPUFA content in infant cell membranephospholipids is altered by dietary supplementation in both pregnancy28 and thepostnatal period.29

18 E f f e c t s o f L C P U F A o n I m m u n e F u n c t i o n


• Supplementing the diet with preformed LCPUFA results in discrete changes inimmune cell and cytokine production; these changes differ with DHA vs ARAsupplementation and are influenced by the ratio of n-6 to n-3 fatty acids in the diet.For example, Kelley et al. observed decreased production of PGE2 and LTB4 (aleukotriene), decreased natural killer (NK) cell activity, and decreased secretion of IL-1βand TNF-α cytokines in response to immunologic challenge to peripheral bloodmononuclear cells isolated from healthy men supplemented with 6 g DHA/day for 90days.31 B- and T-cell functions were not affected. In another study, men weresupplemented with 1.5 g ARA/day for 50 days and no changes were observed indelayed-type hypersensitivity reaction, NK cell activity, lymphocyte proliferation, orsecretion of IL-2 and TNF-α.101,102 However, significant increases were observed innumbers of circulating neutrophils and in production of PGE2 and LTB4.101,102 Thesediffering effects of DHA vs ARA suggest that dietary alterations to increase n-3 LCPUFAintake may have effects on markers of immune function. Furthermore, as evidenced bythe different responses observed with very large dosing in adults, additional studieswith varying doses in neonates and children may be necessary to fully appreciate thepotential benefit of these compounds.

19E f f e c t s o f L C P U F A o n I m m u n e F u n c t i o n


There has been a worldwide increase in allergic disease,103 with as many as 40% of childrenin industrialized countries developing allergic sensitization. Many of these children developassociated diseases. Recent Australian figures estimate that approximately 20% of school-aged children experienced wheezing in the previous 12 months, and that prevalence ofeczema and rhinitis were roughly 17% and 13%, respectively.104 This allergy “epidemic” haslead to intense interest in environmental factors that may either account for this change,and/or play a role in reversing this trend. There has been a good case for exploring the roleof n-3 PUFAs based on their declining levels of dietary intake and their anti-inflammatoryproperties. However, while a growing body of evidence suggests associations betweendietary LCPUFA intake early in life and immunological outcomes including atopy, resultshave not been conclusive, and new studies have challenged this notion.35

Development of atopic disease has a strong familial component; as a result, many researchstudies have focused on interventions in infants and children with an increased risk ofatopy. Children born into atopic families have a 50%–80% risk of developing allergicdiseases; children from families with no history of atopy have approximately a 20% risk.105

Risk of allergy appears to be higher if both parents are allergic (vs only one parent) and ifthe mother (vs the father) has allergic disease (reviewed by Prescott & Tang, 2004).105

Because maternal diet is the most important determinant of LCPUFA accretion for the womanand her infant, dietary investigations have focused on the relationship between LCPUFA intakeand status in pregnant and breastfeeding women and the incidence of atopic diseases in theirchildren. Studies have examined the effects of oily fish intake, fish oil supplementation, andsupplementation with oils high in n-3 LCPUFA. These studies have evaluated interventionsduring three different periods of development:

• The effects of maternal LCPUFA intake during pregnancy on neonatal indicators ofimmune response and atopic risk,

• The effects of maternal LCPUFA intake during lactation on breast milk LCPUFA contentand correlations with immune function and atopic disease in infants, and

• The relationship between LCPUFA intake during infancy and childhood and subsequentdevelopment or progression of atopic disease, particularly that affecting the respiratorysystem.

20 L C P U F A a n d H e a l t h y I m m u n e S y s t e m D e v e l o p m e n t :C l i n i c a l R e p o r t s i n P r e g n a n c y , L a c t a t i o n , a n d E a r l y C h i l d h o o d

LCPU

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Effects of LCPUFA intake in pregnancyThe effect of LCPUFA intake in pregnancy on infants’ risk of developing allergy is emerging asan area of clinical interest. As with all other systems, the immune system develops in utero, andthere is accumulating evidence that newborns who subsequently develop allergic disease havea number of alterations in immune function at birth (reviewed in Prescott, 2003).106 Togetherwith the rising rates of allergic disease, this association suggests that environmental factorsmay affect neonatal “allergy risk” during pregnancy. Dietary factors, with their recognizedpotential to influence immune functions, are among the environmental factors thought likelyto have such effect. Accordingly, several studies have noted differences in cord blood LCPUFAprofiles in neonates at high risk of allergic disease compared with low-risk newborns, althoughthe findings are not consistent.25,107,108 Others have noted that neonates at high risk of atopyalready had differences in in vitro T-cell responses at birth.109 Despite speculation aboutbiochemical alterations or metabolic abnormalities which could contribute to the developmentof atopy, specific relationships have not been confirmed.

More recently, Dunstan and Prescott proposed that supplementation of the maternal diet withn-3 LCPUFA during pregnancy may influence the development of allergic and other immune-mediated diseases.34 This proposal was based on their observations that fish oilsupplementation in pregnancy can modify immune responses.28,43 In a randomized, controlledtrial, allergic women (n=83) consumed 4 g fish oil/day (containing 3.7 g of n-3 LCPUFA; 56% asDHA and 27.7% as EPA) or placebo from 20 weeks gestation to delivery.43 Infants of LCPUFA-supplemented women had significantly higher levels of n-3 fatty acids in red blood cellmembranes than infants of unsupplemented women. All neonatal cytokine responses toallergens tended to be lower in infants of supplemented women, as evidenced by a trend tolower levels of IL-5, IL-13, IL-10, and IFN-γ produced compared to those for infants of women inthe control group. Cord blood IL-13 levels were significantly lower (by 65%) in the supplementedgroup compared to the control group; there was also a significant inverse relationship betweenn-3 PUFA levels in neonatal red blood cell membranes and plasma IL-13. Although the trial wasnot designed to evaluate clinical outcomes, infants of supplemented mothers were three timesless likely than control infants to develop a positive skin prick test to egg and had significantlyless severe atopic dermatitis at 1 year of age.43 Based on data from the same study, Barden et al.suggested that DHA may affect atopic disease by attenuating neonatal lipid peroxidation, ascord blood and urinary measures of lipid peroxidation were significantly decreased in theinfants of supplemented women.46 The authors suggest that the findings of these studiessupport a role of maternal dietary LCPUFA content in influencing the infant’s immune system,which in turn may affect development of atopy.21,28,43 Larger studies to assess the clinical effectsof early fish oil supplementation on allergy outcomes are currently underway.

21L C P U F A a n d H e a l t h y I m m u n e S y s t e m D e v e l o p m e n t :C l i n i c a l R e p o r t s i n P r e g n a n c y , L a c t a t i o n , a n d E a r l y C h i l d h o o d


Other groups have shown relationships between LCPUFA levels and neonatal cytokineresponses. In a US birth cohort study, higher cord blood levels of EPA and ARA were associatedwith reduced cord blood lymphocyte proliferation responses and reduced production of somecytokines.110

In contrast, Newson et al. found no association between fetal exposure to n-3 and n-6 fattyacids, measured in cord blood erythrocytes, and development of wheezing and atopic diseaseat 30 and 42 months of age.111 Positive associations were noted among cord blood ARA:EPA ratioand eczema as well as linoleic acid:alpha-linolenic acid ratio and later-onset wheeze, but theseassociations did not persist when data were adjusted for multiple comparisons. Womenenrolled in this longitudinal study were not supplemented with any particular form of fattyacids. The authors assert that, because of the interaction between maternal supply, fetaldemand, and transfer kinetics, cord blood fatty acid levels of LCPUFA are more likely to reflectfetal exposure in late gestation than maternal blood levels.111 As mentioned previously, dosingmay play a significant role in these apparently disparate effects.

Effects of maternal LCPUFA status during lactationAs a component of studies described previously, Dunstan and coworkers looked at the effectsof maternal fish oil supplementation on breast milk LCPUFA as well as markers of mucosalimmunity (IgA, sCD14) and cytokines (IL-5, IL-6, IL-10, TNF-α, and IFN-γ) in breast milk.21 At 3 dayspostpartum, breast milk DHA and EPA levels were significantly higher among supplementedwomen compared to control women, and milk ARA levels were significantly lower. Milk DHAlevels were positively correlated with milk IgA content. Furthermore, cytokines involved in IgAsynthesis (IL-10 and IL-6) were significantly correlated with milk levels of IgA and n-3 PUFA,although no differences were noted among study groups in breast milk levels of IgA, sCD14, orcytokines. These results suggest a potential effect of n-3 LCPUFA on mucosal immunedevelopment measured in the early postnatal period.

Numerous studies have provided evidence of relationships between breast milk LCPUFAcomposition and incidence or markers of atopic disease in infants and children.112-117 Inparticular, studies have shown that low levels of n-3 fatty acids in breast milk are associatedwith increased incidence of atopic disease and other allergic manifestations.118 Milk frommothers of allergic children was lower in total n-3 LCPUFA and had a higher n-6 to n-3 PUFAratio compared to milk from mothers of non-allergic children.112,113 Further, lowconcentrations of n-3 LCPUFA and a high ratio of ARA:EPA in breast milk were related tosymptoms of allergy at 18 months of age.

These effects are thought to be mediated by the influence of LCPUFA on maternal cytokines inbreast milk, although clinical studies have not yielded definitive conclusions. Hawkes et al.reported that supplementation of lactating women with 300 or 600 mg DHA for 4 weeksresulted in elevated DHA levels in breast milk, plasma, and selected immune cells, but did not



yield differences in cytokine levels.119 The authors suggested that large variability contributedto the inadequate power to detect differences, should they exist. In another study, in vitroproduction of IFN-γ in whole blood cultures from infants whose mothers received 1.5 g/day ofn-3 fatty acids from fish oil postnatally for 4 months had a median IFN-γ level four times higherthan that of control infants when measured at 2 1⁄2 years of age; IL-10 levels were similar.115 Theauthors speculated that fish oil supplementation during lactation may have caused a shift ininfants’ immune polarization and more rapid maturation of their immune systems, resulting ina long-term immunomodulating effect. A mechanism for such an effect was not defined.

Several studies have shown a relationship between maternal LCPUFA intake and developmentof atopic dermatitis in their breastfed infants and children.114,116,117 A study of women with atopicdisease (ie, asthma, allergic rhinitis, atopic dermatitis) and their breastfed infants found thatinfants who developed dermatitis (38% of the sample total) had consumed breast milk with ahigher ratio of saturated to polyunsaturated fatty acids.114 Notably, breast milk stearic acidlevels were increased and total n-3 fatty acids and EPA were decreased compared to milkconsumed by infants who did not develop atopic disease.

Oddy et al. examined the relationship between breast milk fatty acids and the laterdevelopment of atopy and eczema in high-risk children who were breastfed for at least 6months.116 They reported significant associations between non-atopic eczema at 6 months andan increased ratio of n-6 to n-3 fatty acids in breast milk samples collected at 6 weeks and 6months of lactation. The authors concluded that milk fatty acids significantly modulated non-atopic eczema in infants at 6 months.

Wijga et al. observed that risk of allergic symptoms in susceptible infants and children wasinversely associated with breast milk n-3 LCPUFA content.117 In a study of 265 mothers (158allergic and 107 nonallergic) and their children, inverse associations were observed amongchildren of mothers with allergy but not among children of mothers without allergy. Milk levelsof total n-3 LCPUFA and ratios of n-3 to n-6 LCPUFA were inversely associated with eczema at 1year among children of allergic mothers.

Kankaanpaa et al. found that the milk of atopic vs nonatopic women (n=20 each group)differed slightly in n-3 fatty acid content.120 Milk from atopic women also contained less γ-linolenic acid (an n-6 LCPUFA) than milk from nonatopic women. Similarly, atopic infants hadlower serum levels of γ-linolenic acid than healthy infants (n=10 each group). No differenceswere found in maternal PUFA intake. The authors suggested that risk of atopic disease may beincreased by high intake of dietary n-6 LCPUFA or low intake of γ-linolenic acid and n-3 LCPUFA.

These relationships are equivocal, however. Stoney et al. found a positive associationbetween breast milk (ie, colostrum) n-3 fatty acid levels and allergic sensitivity.121 Inparticular, they found that infants testing positive to skin prick tests of common allergenshad been exposed to colostrum containing higher levels of n-3 fatty acids, eg, DHA anddocosapentaenoic acid (DPA). The study evaluated 224 mother-infant pairs from families in



which at least one first-degree relative had an atopic disease. Stoney et al. concluded thathigher n-3 fatty acids in colostrum could be a risk factor for atopy in high-risk breastfedinfants. They suggested that the apparent contrast between their study findings and thoseof other investigators could be explained by differences in subjects’ risk of allergy, studyanalysis methods, age at which sensitization was measured, and definition of atopy used.

Associations were noted in another study evaluating the relationship between colostrumfatty acid levels and later development of allergy.122 At 1 year of age, high linoleic acid levelswere associated with high IgE response to cow’s milk protein, and low levels of DPA wereassociated with elevated total IgE, but no associations were observed between colostrumfatty acids and atopic eczema.

Although many questions remain to be answered, these studies and others providepreliminary evidence that exposure to n-3 LCPUFA in utero and via breast milk may beassociated with reduced development of atopic disease in infants and children.34 Thisassociation now needs to be confirmed. If atopic disease is in fact correlated with intake ofn-3 PUFA, then it is reasonable to consider that dietary interventions which result in alteredamounts and ratios of n-6 to n-3 fatty acids may be associated with modulation of atopicdisease. Such findings could have important implications for public health policy andnutrient intake recommendations, but more studies are needed.

Effects of infant formula supplementationWhether similar effects extend to infants fed LCPUFA-supplemented formula remains to bedetermined. In a study with preterm infants, Field et al. evaluated the effect on immunesystem measures of feeding supplemental DHA and ARA in preterm formula from aboutday 8 to day 42.29 Supplemented infants had lymphocyte populations, phospholipid LCPUFAcomposition, cytokine production, and antigen maturity more like that of a breastfedreference group and less like that of a non-supplemented control group. Similar resultswere observed in a study with term infants: infants fed supplemented formula hadlymphocyte populations and immune cell maturity more consistent with the breastfedgroup.123 Although results from these studies imply that providing supplementary DHA andARA in infant formula may enhance infants’ ability to respond to immune systemchallenges, the clinical significance is unknown.

A recent nonblinded, nonrandomized clinical survey (n=1342) reported that term infantswho were fed an LCPUFA-supplemented formula (17 mg DHA/100Cal and 34 mg ARA/100 Cal) for the first year of life had improved respiratory health compared to controlinfants.124 This survey was conducted with 357 pediatricians and was not blinded orrandomized. Infants in the control group were fed formulas commercially available inEurope, including unsupplemented formulas and a few formulas supplemented with DHAand ARA at levels that were lower than the LCPUFA-supplemented formula described



above. Whether formulas supplemented with lower levels or different sources of DHA orARA would show similar improvement was not tested. Nonetheless, because of the resultsobserved in this very large clinical survey, a large, randomized trial of LCPUFAsupplementation during infancy would be of interest.

Relationship between LCPUFA intake in infancy and childhood and subsequenthealth outcomesThe relationships between infant and childhood intake of LCPUFA and subsequent healthoutcomes have been of great interest for many years. Although interest was initially centeredaround effects on neurodevelopment, it has more recently expanded to include otheroutcomes, including potential benefits for immune development and allergy risk. A number ofstudies have explored the relationship between dietary LCPUFA in infancy and thedevelopment of allergic sensitization and associated conditions, including asthma and allergicairway inflammation and allergic rhinitis.

Asthma, a chronic inflammatory disease of the respiratory tract, is a common condition inchildhood and is commonly associated with atopy; its prevalence has been increasingworldwide in both children and adults for several decades.103 This rising prevalence has raisedsignificant health concern. Early population studies suggested that a decreased risk of asthmamay be associated with consumption of fish, a dietary source of n-3 fatty acids.5,125 Subsequentbirth cohort studies have also reported that the ratio of n-6 to n-3 LCPUFA may influence thedevelopment of asthma.126

To date, there has only been one published intervention study to examine the effects of fishoil supplementation in early childhood on the prevention of asthma and allergy.35-37 TheChildhood Asthma Prevention Study (CAPS) was a randomized, controlled trial whichseparately assessed the preventive effects of house dust mite avoidance and increasingconsumption of n-3 fatty acids. The study investigators recruited pregnant women (n=616)whose unborn children were deemed to be at risk of developing asthma. Women randomlyassigned to the dietary intervention group received a daily supplement of tuna fish oil (500mg oil/day) to give their children along with margarines and cooking oils rich in n-3 fattyacids to use in food preparation; women in the control group received a placebosupplement for their children and polyunsaturated margarines and cooking oils. Thechildren were assessed clinically at 18 months, 3 years, and 5 years of age.

Initial findings were encouraging, with reduced respiratory symptoms such as wheeze at 18months of age and reduced coughing (among atopic infants) in the “active” diet group at 3years of age.36,37 However, there was no reduction in the development of asthma. At 5 yearsof age, there was no reduction in the prevalence of asthma, wheezing, atopic dermatitis, orallergic sensitization in children who had received n-3 PUFA enriched diets35 despiteconfirmed effects on LCPUFA status during the intervention.36 A number of other studies



are on-going which will assess the effects of supplementation with higher doses of n-3PUFA from birth.

Omega-3 fatty acids have also been investigated in the management of established asthmain both children and adults. A recent Cochrane systematic review of nine randomized,controlled studies compared n-3 PUFA supplementation with placebo or with n-6 PUFAsupplementation and found no evidence to support the use of marine n-3 PUFA in themanagement of asthma.127 Seven of the studies were conducted in adults and two inchildren. Of the two studies conducted in children, one reported that fish oilsupplementation was associated with improved asthma symptom scores and reducedmedication usage.128 Hodge et al.129 observed no improvement in asthma severity inchildren supplemented with n-3 PUFA rich fish oil. However, a significant improvement inpeak expiratory flow and asthma medication use following fish oil supplementation wasdescribed when these results, as well as additional data provided by Hodge and colleagues,were evaluated in the Cochrane analyses.127 While inconclusive, these observationshighlight the need for further studies in this area, particularly in children with asthma.

Thus, it still may be too early to draw definitive conclusions about the influence of n-3 fattyacids on asthma, despite increasing positive evidence of their effect. At the request of theOffice of Dietary Supplements, US National Institutes of Health, Schachter et al. evaluatedthe published literature on the topic of n-3 fatty acid supplementation and asthma.130 Theyconcluded that there are not enough data from well-designed studies with which toevaluate the influence of n-3 fatty acids on mediators of inflammation thought to beinvolved in asthma, or the actual role of those mediators in asthma. Results from additionalcarefully designed studies will be useful.



It has been suggested that fatty acids act as “gatekeepers” of immune cell regulation,with direct effects on the activities of cells and mediators involved in immune systemresponse.131 Emerging data from clinical and animal studies suggest that dietary n-3fatty acids provided in appropriate ratios with n-6 LCPUFA and as part of a balanced dietin early life (beginning in utero) may influence immune system development, immunecell function, and incidence of atopic disease.3,81,112,113,115,132,133 Correlations betweenmaternal intake of n-3 LCPUFA and incidence of atopic disease in infants suggest thatthere may be a critical time period during which early exposure to n-3 and n-6 dietaryfatty acids may influence immune system development and function. Development ofatopy in the infant and child appears to be influenced by maternal intake and serumlevels of LCPUFA as well as the ratio of dietary n-6 to n-3 fatty acids during pregnancy.These findings suggest that prenatal fatty acid supply is more important to infantimmune development than previously understood.27,34,134

Because n-3 LCPUFA (eg, EPA and DHA) support anti-inflammatory and immuneresponses, dietary modification with these nutrients may have a potential role ininfluencing the development of allergic disease, including inflammation, in early life.4,46

Results of clinical studies in pregnant and lactating women, infants, and childrensuggest that interventions which increase intake of these fatty acids may havesignificant clinical effects on incidence and manifestations of atopy.34

Although some n-6 LCPUFA are associated with pro-inflammatory immune responses,emerging evidence in animal and human studies suggests that n-6 LCPUFA, includingARA, have important roles in immune system development and regulation.29,32,33

Providing preformed DHA and ARA in infant formula at levels close to those in humanmilk may help formula-fed infants compensate for a potential immunologicaldisadvantage compared to breastfed infants. Supplemented formulas have been shownto be safe for term and preterm infants when both DHA and ARA are added to theformula.10,135 The optimal levels of n-3 LCPUFA remain to be determined; it is prudent inthe meantime to follow worldwide average levels in human milk, although it isinteresting to consider the potential benefit of higher levels of supplementation.Nonetheless, despite evidence of benefit for some LCPUFA-supplemented formulas, theclinical effect of preformed LCPUFA in the infant diet remains controversial.

27O v e r a l l S u m m a r y a n d C o n c l u s i o n s

Overall Summ

ary and Conclusions


Although much insight has been gained in the past decade about how the immune systemdevelops and how dietary factors affect that development, numerous questions remain. Inparticular, additional research should consider:

• The effects of study design on trial outcome (such as sample size, immune parametersevaluated, methods of measurement, sources and levels of LCPUFA supplementation,duration of intervention, and age at initiation of supplementation);

• The effects of confounding factors on trial outcome and reproducibility (includinghereditary risk, sampling methods, regional dietary differences, and nutrient-nutrientand nutrient-gene interactions);

• The extent to which results from cell culture and animal studies can be replicated byclinical experiments;

• Potential mechanisms by which LCPUFA can modulate clinical outcomes; and

• The differing effects of ARA, EPA, and DHA on immune responses.

Despite unanswered questions, clinical evidence to date suggests that relatively simple dietaryinterventions may influence the incidence and severity of allergic diseases among infants andchildren worldwide. The particular characteristics of the most effective interventions remain tobe determined.

28 O v e r a l l S u m m a r y a n d C o n c l u s i o n s


1. Ailhaud G, Massiera F, Weill P, et al. Temporal changes in dietary fats: role of n-6 polyunsaturated fatty acids inexcessive adipose tissue development and relationship to obesity. Prog Lipid Res. 2006;45:203-236.

2. Simopoulos AP. Essential fatty acids in health and chronic disease. Am J Clin Nutr. 1999;70(suppl):560S-569S.

3. Calder PC. Polyunsaturated fatty acids and cytokine profiles: a clue to the changing prevalence of atopy?Clin Exp Allergy. 2003;33:412-415.

4. Calder PC. n-3 Polyunsaturated fatty acids and inflammation: from molecular biology to the clinic. Lipids.2003;38:343-352.

5. Hodge L, Salome CM, Peat JK, et al. Consumption of oily fish and childhood asthma risk. Med J Aust. 1996;164:137-140.

6. Schwartz J, Weiss ST. The relationship of dietary fish intake to level of pulmonary function in the first NationalHealth and Nutrition Survey (NHANES I). Eur Respir J. 1994;7:1821-1824.

7. Birch EE, Birch DG, Hoffman DR, Uauy RD. Dietary essential fatty acid supply and visual acuity development.Invest Ophthalmol Vis Sci. 1992;33:3242-3253.

8. Carlson SE, Werkman SH. A randomized trial of visual attention of preterm infants fed docosahexaenoic aciduntil two months. Lipids. 1996;31:85-90.

9. Carlson SE, Werkman SH, Rhodes PG, Tolley EA. Visual-acuity development in healthy preterm infants: effect ofmarine-oil supplementation. Am J Clin Nutr. 1993;58:35-42.

10. Clandinin MT, Van Aerde JE, Merkel KL, et al. Growth and development of preterm infants fed infant formulascontaining docosahexaenoic acid and arachidonic acid. J Pediatr. 2005;146:461-468.

11. Werkman SH, Carlson SE. A randomized trial of visual attention of preterm infants feddocosahexaenoic acid until nine months. Lipids. 1996;31:91-97.

12. Birch EE, Garfield S, Hoffman DR, et al. A randomized controlled trial of early dietary supplyof long-chain polyunsaturated fatty acids and mental development in term infants. Dev Med Child Neurol.2000;42:174-181.

13. Birch EE, Hoffman DR, Castañeda YS, et al. A randomized controlled trial of long-chain polyunsaturated fatty acidsupplementation of formula in term infants after weaning at 6 wk of age. Am J Clin Nutr. 2002;75:570-580.

14. Hoffman DR, Birch EE, Castañeda YS, et al. Visual function in breast-fed term infants weaned toformula with or without long-chain polyunsaturates at 4 to 6 months: a randomized clinical trial.J Pediatr. 2003;142:669-677.

15. Morale SE, Hoffman DR, Castañeda YS, et al. Duration of long-chain polyunsaturated fatty acids availability in thediet and visual acuity. Early Hum Dev. 2005;81:197-203.

16. Uauy RD, Hoffman DR, Mena P, et al. Term infant studies of DHA and ARA supplementation onneurodevelopment: results of randomized controlled trials. J Pediatr. 2003;143(suppl):S17-S25.

17. Innis SM, Elias SL. Intakes of essential n-6 and n-3 polyunsaturated fatty acids among pregnant Canadianwomen. Am J Clin Nutr. 2003;77:473-478.

18. Innis SM. Essential fatty acids in growth and development. Prog Lipid Res. 1991;30:39-103.

19. Brenna JT. Efficiency of conversion of α-linolenic acid to long chain n-3 fatty acids in man. Curr Opin Clin Nutr Metab Care. 2002;5:127-132.

20. Cunnane SC, Francescutti V, et al. Breast-fed infants achieve a higher rate of brain and whole bodydocosahexaenoate accumulation than formula-fed infants not consuming dietary docosahexaenoate. Lipids.2000;35:105-111.

29R e f e r e n c e s

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