Physiological aspects of human milk lipids

Early Human Development 65 Suppl. (2001) S3 – S18 www.elsevier.com/locate/earlhumdev

Review

Physiological aspects of human milk lipids Berthold Koletzkoa,* , Maria Rodriguez-Palmeroa, Hans Demmelmaira, Natasˇa Fidlera, Robert Jensenb, Thorsten Sauerwalda a

Department of Pediatrics, Division of Metabolic Disorders, Molecular Disease and Nutrition, Kinderklinik and Kinderpoliklinik, Dr. von Haunersches Kinderspitel, Ludwig-Maximilians-University of Munich, Lindwurmstr. 4, D-80337 Munich Germany b Department of Nutritional Science, University of Connecticut, Storrs, CT, USA

Abstract Human milk from healthy and well-nourished mothers is the preferred form of feeding for all healthy newborn infants. The nutrient supply with human milk supports normal growth and development of the infant. Here the general characteristics of human milk lipids and recent knowledge on lactational physiology, composition and functional aspects of human milk lipids are discussed. Lipids in human milk represent the main source of energy for the breastfed baby and supply essential nutrients such as fat-soluble vitamins and polyunsaturated fatty acids (PUFA). The essential fatty acids linoleic and a-linolenic acids (LA and ALA) are precursors of long-chain polyunsaturated fatty acids (LC-PUFA), including arachidonic (20:4n 6) and docosahexaenoic (22:6n 3) acids (AA and DHA). LC-PUFA serve as indispensable structural components of cellular membranes and are deposited to a considerable extent in the growing brain and the retina during perinatal development. The supply of preformed LC-PUFA with human milk lipids has been related to functional outcomes of the recipient infants such as visual acuity and development of cognitive functions during the first year of life. Recent stable isotope studies indicate that the major portion of milk PUFA is not derived directly from the maternal diet, but stems from endogenous body stores. Thus, not only the woman’s current but also her long-term dietary intake is of marked relevance for milk fat composition. D 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Human milk Arachidonic acid Docosahexaenoic acid Infant nutrition Infant development

Abbreviations: BSSL, bile-salt stimulated lipase; PUFA, polyunsaturated fatty acids; LC-PUFA, long-chain polyunsaturated fatty acids; VEP, visual evoked potentials; LA, linoleic acid (18:2n 6); ALA, a-linolenic acid (18:3n 3); AA, arachidonic acid (20:4n 6); EPA, eicosapentaenoic acid (22:6n 3); DHA, docosahexaenoic acid (22:6n 3). * Corresponding author. Fax: +49-89-5160-3336. E-mail address: [email protected] (B. Koletzko). 0378-3782/01/$ – see front matter D 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 3 7 8 2 ( 0 1 ) 0 0 2 0 4 - 3

S4

B. Koletzko et al. / Early Human Development 65 Suppl. (2001) S3–S18

1. Introduction Breast milk from healthy and well-nourished women is the preferred method of feeding for healthy infants for the first 6 months of life [1,2]. In the last two decades, special attention has been paid to the compositional and physiological aspects of the lipid fraction in human milk, which has recently been reviewed [3 – 5] and is summarised here. Human milk fat is the major source of energy for the breastfed infant, contributing some 40 –55% of the total energy intake. The lipids are part of the milk fat globules with an average diameter between 3 and 5 mm, which contain largely triacylglycerols in the core. The main lipid constituents of the amphipathic fat globule surface membrane, which is exposed to the watery milk fraction, are phospholipids and cholesterol. Human milk lipids provide essential lipid soluble vitamins and polyunsaturated fatty acids (PUFA), including linoleic acid of the n 6 series (LA, 18:2n 6) and alinolenic acid of the n 3 series (ALA, 18:3n 3). Different PUFA have specific biological functions; for example, LA is an indispensable structural component of certain dermal ceramides with importance for the maintenance of the epidermal water barrier. LA and ALA are precursors of long-chain PUFA with 20 and 22 carbon atoms of the n 6 and n 3 series, respectively. Metabolism of LA and ALA to long-chain PUFA (LC-PUFA) include consecutive desaturation and elongation steps (Fig. 1). Fatty acids of the n 6 and n 3 series compete for the enzyme systems. Among the biologically important LC-PUFA metabolites formed by these pathways are dihomo-g-linolenic acid (20:5n 3) and docosahexaenoic acid (DHA, 22:6n 3) of the n 3 series. LC-PUFA are important structural components of cell membranes and show a marked perinatal accumulation in membrane-rich tissues such as the brain and the retina [6]. The availability of LC-PUFA has been related to early human growth and development.

Fig. 1. Metabolism of essential fatty acids linoleic acid (18:2n polyunsaturated fatty acids (LC-PUFA).

6) and linolenic acid (18:3n

3) to long-chain

B. Koletzko et al. / Early Human Development 65 Suppl. (2001) S3–S18

S5

2. Lipids in human milk The average fat content of human milk is about 3.8 –3.9 g/100 ml, but it varies widely. A wide range of fat contents was found in the analysis of 2554 samples from 224 Danish mothers taken over 33 months of lactation [7] (Table 1), while the relative variation was smaller for protein and much less for lactose. The fat is present in milk in the form of milk fat globuli that are formed by the mammary alveolar cells. Milk fat globuli have a hydophobic core that is rich in triglycerides and also contains cholesteryl esters and retinyl esters. The surface of milk fat globules is comprised of amphipathic compounds such as phospholipids, proteins, cholesterol and enzymes in a loose network termed the milk fat globule membran. This amphipathic surface is required for the dispersion of milk fats in the watery environment of milk and for stability of the oil in water emulsion. Most of the membrane material is derived from the apical plasma and mature Golgi vesicle membranes, which envelop the globules as they are extruded from the secreting mammary cell. The diameter of the globules range from 1 to 10 mm, with most of the globules measuring 1 mm, but those of 4 mm account for most of the weight. The large surface area of the globules (4.5 m2/dl) can bind various lipase, and, thereby, contributes to effective triglyceride digestion. With the increase of the milk fat content milk during the first 4 weeks of lactation, the average size of the milk fat globules rises which is accompanied by a decreasing ratio of phospholipids and cholesterol (membrane lipids) to triglycerides (core lipids) [8,9]. Also during each nursing human milk fat content increases, and the ratio of phospholipids and cholesterol to triglycerides decreases. The formation of milk fat by mammary alveolar cells is stimulated by emptying of the breast through nursing and by prolactin secreted from the anterior lobe of the pituary gland. The major proportion of milk fat is formed from circulating lipids derived from the maternal diet, and from maternal body stores. In addition, part of the milk fat can be synthesized de novo in the mammary gland from glucose, which results primarily in the formation of saturated fatty acids with 10 – 14 carbon atoms [10]. The proportion of these endogenously synthesized fatty acids with medium and intermediate chain length increases when breastfeeding women consume diets with a low fat and a high carbohydrate content [11].

3. The composition of milk fat The major portion of milk fat is comprised by triglycerides accounting for  98% of the total fat. Phospholipids contribute about 0.7% and cholesterol 0.5%. Fresh human milk contains small amounts of lipolysis products, including free fatty acids, mono- and Table 1 Percentile distribution of fat content in 2554 human milk samples (adapted from Ref. [7]) Percentiles

Milk fat content (g/dl)

2.5

10

25

50

75

90

97.5

1.84

2.38

2.94

3.61

4.34

5.46

8.90

S6

B. Koletzko et al. / Early Human Development 65 Suppl. (2001) S3–S18

diacylglycerols. There are no major differences in lipid composition in milks from term and preterm mothers, although more medium and intermediate chain fatty acids (10:0 – 14:0) are found in preterm than term milks [12,13]. The composition of triglycerides (TG) is determined by the kinds and amounts of fatty acids present and their tertiary structure. A short formula is conventionally used to designate fatty acids, with the carbon number followed by the number of double and its location relative to the terminal (omega) carbon atom. For example, linoleic acid with 18 carbon atoms and two double bond with the final double bond in the omega-6 (n 6) position is 18:2n 6. The stereospecific numbering (sn) designates the location of fatty acids within the triglyceride molecule. If the glycerol is drawn with the first and third hydroxyl groups to the right and the second to the left, the first carbon is termed sn-1; the second, sn-2, and the third sn-3. In phosphatidyl choline, the choline moiety is located on the sn-3 carbon. Lipid structure is of importance for absorption. While saturated fatty acids in vegetable oils are more or less randomly positioned within the triglyceride molecules, the major portion of about 60% of the saturated palmitic acid (16:0) in human milk triglycerides is located in the sn-2-position. Since lipolytic enzymes will cleave the FA in sn-1 and sn-3-positions, human milk palmitic acid will appear primarily in the remaining monoglyceride which has a higher water-solubility than free palmitic acid. Thereby, absorption is facilitated because palmitic acid monoglyceride is more polar. Hence, most of the 16:0 is absorbed as the sn-2 MG and this structure is preserved through and beyond the intestinal wall. Clinical trials in formula-fed infants confirmed a better absorption of palmitic acid from triglycerides with preferential esterification in the sn-2 position compared to randomly esterified palmitic acid [14,15]. Milk phospholipids contribute about 25 mg/dl or 0.6%/100 g lipid [16]. The major classes of phospholipids are phosphatidyl choline (28.4%), phosphatidyl ethanolamine (27.7%), phosphatidyl serine (8.8%) phosphatidyl inositol (6.1%), and sphingomyelin (37.5%). Phospholipids have emulsifying properties and contribute to maintaining the globule emulsion. Among the several classes of sphingo- and glycolipids are gangliosides, some of which appear to contribute to the host defense by binding bacterial toxins [17]. Human milk has a high cholesterol content (10 –20 mg/dl or 250– 500 mg/100 g fat) [16]. Cholesterol is the major milk sterol contributing 90.1% to the total sterol content, followed by desmosterol (8.6% of total sterols). The quantities of phytosterols are negligible. Maternal diet has no appreciable effect on milk cholesterol. Breastfed infants have a relatively large cholesterol intake of about 25 mg/kg body weight relative to adults (about 4 mg/kg body weight). Plasma cholesterol values are higher in breastfed than in formula-fed infants [18], but no consistent effect on plasma lipids after weaning has been found in previously breast- or formula-fed infants [19].

4. Human milk fatty acids The characteristics of milk lipids are largely determined by their fatty acid composition. Data on the fatty acid composition of colostrum (1 – 5 days of lactation), transitional (6 –15 days of lactation) and mature milk (16 –35 days of lactation) are presented in Table 2. The

B. Koletzko et al. / Early Human Development 65 Suppl. (2001) S3–S18

S7

Table 2 Major fatty acids of human milk from omnivorous German women consuming self-selected foods (% wt/wt, median values and interquartile ranges) (adapted from Ref. [13])

Saturated fatty acids Total

Colostrum (5 days)

Transitional (10 days)

Mature (30 days)

43.65 (3.55)

43.05 (3.67)

44.30 (5.00)

32.10 (2.61)

31.50 (2.49)

10.30 0.15 0.42 0.43 0.59 0.15 1.59

(2.15) (0.08) (0.06) (0.11) (0.09) (0.03) (0.20)

11.33 0.18 0.30 0.38 0.45 0.08 1.28

(2.17) (0.10) (0.07) (0.08) (0.07) (0.02) (0.19)

0.81 0.05 0.00 0.18 0.39 0.66

(0.17) (0.03) (0.03) (0.04) (0.06) (0.11)

0.90 0.05 0.05 0.15 0.23 0.48

(0.20) (0.04) (0.07) (0.03) (0.06) (0.16)

Monounsaturated fatty acids C18:1n 9 32.1 (1.87) Polyunsaturated fatty acids (PUFA) n 6 PUFA C18:2n 6 8.86 (1.30) C18:3n 6 0.14 (0.31) C20:2n 6 0.57 (0.12) C20:3n 6 0.53 (0.13) C20:4n 6 0.72 (0.07) C22:4n 6 0.23 (6.00) n 6 LC-PUFA 2.15 (0.34) n 3 PUFA C18:3n 3 0.65 (0.07) C20:3n 3 0.09 (0.03) C20:5n 3 0.04 (0.08) C22:5n 3 0.22 (0.05) C22:6n 3 0.46 (0.08) n 3 LC-PUFA 0.80 (0.21)

predominant fraction are saturated fatty acids, followed by a relatively high proportion of monounsaturated fatty acids such as oleic acid (18:1n 9). The major milk PUFA is LA, but also ALA and some 10 different LC-PUFA of both n 6 and n 3 series are consistently found in human milk samples. In several recent studies analyzing fatty acid composition of mature milk from women living in industrialized countries, the percentage contribution of total n 6 LC-PUFA ranged from 0.83% to 1.40%, whereas total n 3 LC-PUFA ranged from 0.27% to 0.48% of total fatty acids.[13,20 – 22]. Data for human milk fatty acid composition from different studies are surprisingly consistent. When we compiled data on fatty acid composition in mature human milk from studies performed in Europe and Africa, we were surprised to find a relatively similar pattern of LC-PUFA contents in spite of marked differences in living conditions and dietary intakes (Table 3). The major LC-PUFA in human milk are 20:4n 6 (AA), 20:3n 6 and 20:2n 6 of the n 6 series, and 22:6n 3 (DHA) and 22:5n 3 of the n 3 series. The two LC-PUFA found in the highest proportions are 20:4n 6 (0.4% to 0.6% in most studies) and 22:6n 3 (0.2% to 0.4% in most studies). Median values and ranges reported for 18:2n 6 (LA) and 18:3n 3 (ALA) are also rather similar among studies in Europe and Africa [23]. The fatty acid composition of human milk varies with the duration of lactation. The contents of essential fatty acids LA and ALA increase with milk maturation [13,24], whereas the percentages of LC-PUFA of both the n 6 and n 3 series decrease markedly by [24,25] about 38% for arachidonic acid and about 50% for DHA during the first month

S8

B. Koletzko et al. / Early Human Development 65 Suppl. (2001) S3–S18

Table 3 Polyunsaturated fatty acids in mature human milk in 14 studies from Europe and 10 studies from and Africa (% wt/wt, medians and ranges) (adapted from Ref. [23]) Fatty acids

Europe (% wt/wt)

Africa (% wt/wt)

n 6 PUFA C18:2n 6 C20:2n 6 C20:3n 6 C20:4n 6 C22:4n 6 C22:5n 6 Total n 6 LC-PUFA

11.0 0.3 0.3 0.5 0.1 0.1 1.2

(6.9 – 16.4) (0.2 – 0.5) (0.2 – 0.7) (0.2 – 1.2) (0.1 – 0.2) (0.0 – 0.2) (0.4 – 2.2)

12.0 0.3 0.4 0.6 0.1 0.1 1.5

(5.7 – 17.2) (0.3 – 0.8) (0.2 – 0.5) (0.3 – 1.0) (0.0 – 0.1) (0.1 – 0.3) (0.9 – 2.0)

n 3 PUFA C18:3n 3 C20:5n 3 C22:5n 3 C22:6n 3 Total n 3 LC-PUFA

0.9 0.2 0.2 0.3 0.6

(0.7 – 1.3) (0.0 – 0.6) (0.1 – 0.5) (0.1 – 0.6) (0.3 – 1.8)

0.8 0.1 0.2 0.3 0.6

(0.1 – 0.4) (0.1 – 0.5) (0.1 – 0.4) (0.1 – 0.9) (0.3 – 2.9)

Ratios C18:2n n 6/n

6/C18:3n 3 3 LC-PUFA

12.1 (8.6 – 16.9) 2.7 (0.3 – 3.7)

14.2 (8.8 – 15.7) 2.4 (0.8 – 6)

after child birth [13] (Fig. 2). This profound decrease in the relative LC-PUFA content in human milk does not imply a marked reduction in the supply to the infant, since total fat content increases with the duration of lactation [12] and the amount of total LC-PUFA secreted with milk may remain relatively stable. It is tempting to speculate that a high percentage of LC-PUFA in colostrum might benefit the neonate, because the volume of milk consumed is still low but the infants’ requirements for PUFA are considered high due to the rapid growth rate [6,26,27].

Fig. 2. Changes in the percentage contents of arachidonic and docosahexaenoic (% wt/wt) in term and preterm milk during the first month of lactation. Drawn from data of Ref. [13].

B. Koletzko et al. / Early Human Development 65 Suppl. (2001) S3–S18

S9

After the first month of lactation, there are much less marked changes of fatty acid composition [28]. The percentage content of DHA was reported to decrease by about 20% from the 6th to the 16th week of lactation, while no further change was seen until the 30th week. Some n 6 PUFA also decrease with time, such as 18:3n 6, 20:3n 6, AA and 22:5n 6 [28]. One hypothesis to explain these changes is that long duration of lactation may drain maternal body stores of LC-PUFA, which can serve as a source of milk fatty acids. In studies comparing term and preterm human milk composition, Bitman et al. [12] reported higher LC-PUFA contents in the colostrum of mothers of preterm infants than those of term infants. In contrast, other investigators have consistently found that term and preterm milk do not differ in percentages of LA, ALA and LC-PUFA during the first month after delivery [13,29,30]. Furthermore, the decline in LC-PUFA during the first month of lactation was found similar in milk of mothers of both term and preterm infants [13,24] (Fig. 2). Although human milk feeding has great advantages both for term and preterm infants, the higher LC-PUFA requirements of preterm infants during the first weeks of life are not better met by the milk of their own mothers as compared to the milk of mothers of term babies [13]. After the first month of lactation, no further decrease was observed in LC-PUFA content in preterm milk but a continued decline in term milk [24]. Thus, during the further course of lactation, preterm human milk may provide higher proportions of LC-PUFA than term human milk, which could be of benefit for the breastfed preterm infant [24]. Mothers of term infants probably have a more marked AA and DHA depletion of their body stores after longer lactation, because they provide a greater placental LC-PUFA transfer during the longer duration of pregnancy, and also higher volumes of milk to their larger infants.

5. Effects of diet on milk fatty acids The diet of lactating women influences, to some extent, the fatty acid composition of human milk lipids. For example, the essential fatty acids LA and ALA must at some time be derived from the maternal diet since they cannot be synthesized de novo in the human organism. The milk contents of eicosapentaenoic acid (EPA, 20:5n 3) and DHA can be increased by consumption of fatty sea fish such as hering, mackerel and salmon, or by supplementation with fish oil. Also, the contents of trans fatty acids in milk reflect the maternal dietary intake of trans fatty acids and are higher in North America and Europe than in rural Africa, where little hydrogenated fats are consumed [11,31]. Trans isomeric fatty acids suchs as elaidic acid (18:1t) originate totally from the diet, mostly from partially hydrogenated food fats and oils, e.g. margarine and shortenings. A rapid increase of trans fatty acid content in human milk has been reported following maternal exposure [32]. Excessive contents of trans FA in the infant’s diet may have adverse effects on infants and children [33 – 35]. Another group of trans isomers called conjugated linoleic acid (CLA) has been considered as potential anticarcinogenic agents. CLA is found in beef and dairy products and, depending on maternal dietary habits, is also present in milk [4]. The active component is believed to be 9c, 11t-18:2, which may inhibit carcinogenesis in rats, but the effects in humans are largely unknown.

S10

B. Koletzko et al. / Early Human Development 65 Suppl. (2001) S3–S18

Maternal diet has not only short-term but also long-term effects on fatty acid contents of human milk. Francois et al. [36] supplied various dietary test fats to lactating women and found that marker fatty acids in breast milk increased within 6 h after ingestion, peaked between 10 and 24 h and remained elevated for 1– 3 days. Compared with other fatty acids, long-chain PUFA in milk showed later peak values (at 24 h after ingestion). The authors reported that the milk content of fatty acids with a large body pool size is less affected by dietary changes than of those with a small body pool size [36]. Studies in populations of vegan and vegetarian women show long-term dietary effects of a specific pattern of maternal fatty acid consumption. The diet of these populations tends to contain more LA than that of omnivores and is practically devoid of (in the case of vegans) or contains only small quantities of (in the case of vegetarians) preformed LC-PUFA such as AA, EPA and DHA. This difference in dietary intake is reflected in the fatty acid pattern of adipose tissue [37] as well as the fatty acid composition of breast milk. Human milk of vegans and vegetarians has considerably more LA and less DHA than that of omnivores [38]. Also in other populations the habitual consumption of vegetable oils with a high proportion of PUFA was associated with a higher milk content of LA and ALA [38,39], whereas the use of olive oil as the preferential source of dietary fat induces higher oleic acid (18:1n 9) and lower LA percentages in human milk lipids [40]. Similarly, populations consuming relatively large amounts of marine foods containing 20:5n 3 (EPA) and DHA show higher milk contents of n 3 LC-PUFA than Western women [23,41,42]. Supplementation of fish oil supplements during lactation induce a dosedependent increase in breast milk n 3 LC-PUFA [43 – 45].

6. Heat treatment and storage of human milk In hospitals expressed human milk is used particularly for the feeding of premature infants. Pasteurization (62.5 C for 30 min) or sterilization (120 C for 30 min) is often applied to inactive pathogenic bacteria such as Mycobacterium tuberculosis [46], Escherichia coli, Staphylococcus aureaus, and group B b-hemolytic streptococci [46,47]. Heat Table 4 Major polyunsaturated fatty acids in human milk samples analyzed fresh and after thermal pasteurization or sterilization (% wt/wt, means ± SD, identical superscripts indicate significant differences at p < 0.05) (adapted from Ref. [48]) Fresh

Pasteurized

Sterilized

n 6 18:2n 20:2n 20:3n 20:4n

6 6 6 6

9.86 ± 3.9a 0.47 ± 0.16 0.56 ± 0.18 0.77 ± 0.31a

9.83 ± 3.98 0.47 ± 0.16 0.56 ± 0.18 0.76 ± 0.30

9.79 ± 3.96a 0.47 ± 0.15 0.55 ± 0.17 0.75 ± 0.28a

n 3 18:3n 22:5n 22:6n

3 3 3

0.83 ± 0.58 0.29 ± 0.14 0.55 ± 0.26

0.83 ± 0.58 0.29 ± 0.14 0.55 ± 0.26

0.83 ± 0.57 0.28 ± 0.13 0.54 ± 0.24

B. Koletzko et al. / Early Human Development 65 Suppl. (2001) S3–S18

S11

treatment may induce undesirable changes, such as loss of several water soluble vitamins (C, folacin and B6) and adversely affect immune properties [48]. Polyunsaturated fatty acids are particularly susceptible to lipid peroxidation, which could have nutritional implications due to a decreased availability of essential fatty acids and might also cause oxidative damage to the newborn infant [49]. Therefore, we and others have studied the effects of heat treatment on human milk lipids. The results show that pasteurization does not alter milk fatty acid composition [48,50]. However, sterilization causes a marked decline in total available milk fat content by about 13% due to fat adhesion to the container surface, and a slight decrease in the percentage of LA and AA (Table 4) [48]. Moreover, heat treatment of human milk can inactivate human milk lipase (bile-salt stimulated lipase (BSSL)) and, thereby, further decrease the utilisable fat for the recipient infant [51,52]. In conclusion, if heat treatment of human milk is to be applied, one should only use pasterisation but not sterilisation.

7. Insights into the metabolism of polyunsaturated fatty acids during lactation The biologically important LC-PUFA in milk may originate from the maternal dietary intake (e.g. fatty fish, meat, liver and eggs), from maternal body stores or from endogenous synthesis from 18-carbon precursors in the liver, the mammary gland or other tissues. Cultured mammary epithelial cells contain the enzymes necessary for LCPUFA synthesis [53], but it is not known whether LC-PUFA are synthesized in the mammary gland in vivo and, if so, to which extent this pathway might contribute to the total milk LC-PUFA pool. Long-chain fatty acids from the diet are absorbed, reesterified in triacylglycerols, enter the circulation in the form of chylomicrons, and are quickly transferred into human milk. In fasting periods, triacylglycerols are transported from the liver as VLDL to the mammary gland and there are released by the action of lipoprotein lipase. During lactation, the activity of lipoprotein lipase decreases in adipose tissue and increases markedly in the mammary tissue [54,55], indicating an increased utilization of fatty acids in this tissue. Fatty acids from adipose tissue can be transported as unesterified fatty acids bound to albumin into the mammary alveolar cells [56]. Even though several studies indicated an effect of dietary composition on human milk fatty acid contents, there are indications that the amounts of LC-PUFA in human milk are to some extent metabolically regulated [23,25,41,43,44,57]. For example, the milk of women from rural areas in Africa have slightly higher n 6 LC-PUFA values than those found in European women [11,58] although these African women have only a very limited consumption of total and animal fats providing preformed n 6 LC-PUFA. Also, vegetarian women have similar or slightly higher milk levels of 20:3n 6 and AA than omnivorous women [37] although the dietary intake of preformed 20:3n 6 and AA is clearly lower in vegetarians [39]. It appears that n 6 LC-PUFA secretion in human milk does not only depend on their maternal dietary intake, but that they can be derived from maternal body stores or from endogenous synthesis. In contrast to the n 6 LC-PUFA, however, milk DHA levels are not maintained in strict vegans who consume high amounts of ALA but little amounts of preformed n 3 LC-PUFA [38]. Habitual intake of fish oil

S12

B. Koletzko et al. / Early Human Development 65 Suppl. (2001) S3–S18

results in higher n 3 LC-PUFA proportions in human milk [11], but although the major n 3 LC-PUFA in fish oil is 20:5n 3, DHA always remains the predominant n 3 LCPUFA in human milk lipids. No significant correlation has been found between the levels of the parent essential fatty acids LA and ALA and their respective long-chain metabolites AA and DHA, respectively, in mature human milk [57]. In contrast [21], we found a close correlation between the amounts of n 6 and n 3 LC-PUFA [57]. These data suggest that both series of fatty acids may share common pathways for synthesis and secretion into milk and may help to maintain a relatively constant n 6/n 3 LC-PUFA ratio in the dietary supply for the breastfed infant. Such a relatively constant ratio might be advantageous for the infant, not only because both fatty acid classes have a specific role in maintaining membrane integrity but also because LC-PUFA of the two series act as precursors in the synthesis of different eicosanoids with very different biological effects [26,59]. Further insights into the mechanisms of PUFA metabolism in human lactation can be derived by stable isotope studies. Since the use of uniformly 13C-labeled, highly enriched fatty acids is safe and without adverse effects even in newborn infants [60], such tracers can be used to study the physiology of substrate flux in lactation. We studied six breastfeeding women and gave them an oral dose of 1 mg/kg body weight of U – 13Clabeled LA at three times in the 2nd, 6th and 12th week of lactation, respectively [61]. The proportion of the ingested, 13C-labeled LA that was oxidized to carbon dioxide was estimated from analysis of breath gas enrichment. The fatty acid transfer into milk was determined by analysis of milk samples collected before and at several time points during a 5-day study period after the tracer intake, while the volume of daily milk production was recorded.

Fig. 3. Schematic depiction of linoleic acid flux in healthy breastfeeding women, based on results of a stable isotope study with oral application of 1 mg/kg bodyweight of U – 13C-labeled linoleic acid and measurements of its oxidation from breath gas analyses and its transfer into milk by analysis of milk samples collected over 5 days. Modified after Ref. [4] based on data of Ref. [61].

B. Koletzko et al. / Early Human Development 65 Suppl. (2001) S3–S18

S13

In these studies we found no significant differences in fatty acid oxidation or transfer into milk between the three time points of lactation in the range from the 2nd to the 12th week. Some 30% of the total LA in milk was directly transferred from the diet, whereas only about 11% of 20:3n 6 and no more than 1.2% of milk AA were derived from endogenous conversion of dietary LA (Fig. 3). Thus, maternal body stores with a relatively slow turnover contribute greatly to the formation of human milk lipids. Hence, short-term variations of dietary fat composition are to some extent metabolically buffered, resulting in a relatively constant PUFA and particularly LC-PUFA content in human milk which could be of biological benefit for the breastfed baby.

8. Do polyunsaturated fatty acids from human milk benefit the recipient infant? The mean supply of LC-PUFA in fully breastfed infants, based on the average composition of human milk, is about 100 mg/kg body weight/day [23,26]. In contrast to breastfed infants, who receive considerable amounts of LC-PUFA with human milk, infants fed traditional vegetable oil based infant formulas without LC-PUFA receive no appreciable dietary supply of AA and DHA [3,62,63] and thus depend on endogenous synthesis to meet LC-PUFA requirements. However, the activity of endogenous enzymatic LC-PUFA synthesis from the precursor fatty acids appears to be limited in both term and preterm infants. Stable isotope techniques have allowed us to study the essential fatty acid turnover in infants in vivo. When we gave full term infants during the first month of life a diet providing LA with a naturally increased content of the stable isotope 13C, we found a rise of 13C enrichment not only in plasma LA, but also in the LC-PUFA metabolites 20:3n 6 and AA [64]. These results demonstrated for the first time active endogenous synthesis of LC-PUFA in human infants, however, the rate of conversion appears to be relatively low. Based on a simplified isotope balance equation, we estimate that only some 6% of the plasma AA pool is renewed per day by endogenous synthesis. In further studies supplying uniformly 13C-labeled LA and ALA to term infants during the first week of life [65] and to preterm infants [66], we confirmed endogenous LC-PUFA synthesis but again found only relatively small changes of enrichment in the major LC-PUFA metabolites, i.e. AA and DHA. Thus, the rate of conversion of the parent essential fatty acids into LCPUFA in both term and preterm infants seems low. This also explains why the dietary supply of linoleic and a-linolenic acids with vegetable oil based infant formula LC-PUFA does not prevent the decline of blood LC-PUFA concentrations relative to levels found in breastfed infants [67]. Therefore, enrichment of infant formulas with LC-PUFA approximating the typical levels of human milk lipids (n 6 LC-PUFA, 1%; n 3 LC-PUFA, 0.5% of total fatty acids) has been considered to improve substrate supply to formula-fed babies [27,68 –70]. Some studies in preterm infants fed human milk or formulas supplemented with DHA have indicated functional effects of the DHA supply on electroretinogram recordings, development of visual acuity assessed by visual evoked potentials (VEP), and performance in psychometric tests relative to infants fed formulas which contain LA and ALA but without preformed LC-PUFA [66,71 – 75]. These functional data are associated with higher percentages of the long-chain fatty acids AA and DHA in plasma

S14

B. Koletzko et al. / Early Human Development 65 Suppl. (2001) S3–S18

and erythrocyte lipids of infants fed human milk or a formula supplemented with LCPUFA [68,69,71,76,77]. In recent studies, we found a close dose –response relationship between the DHA supply and visual acuity in preterm infants [66]. Term infants fed formulas with precursor essential fatty acids but low in LC-PUFA also show a pronounced AA and DHA depletion after birth as compared to infants fed human milk or formulas supplemented with LC-PUFA, with differences persisting to the age of 7 –8 months for erythrocyte DHA concentrations [78] and up to the age of 4 months for plasma and erythrocyte arachidonic acid concentrations [78,79]. Analyses of brain tissue obtained post mortem from the bodies of infants who had died from sudden infant death syndrome documented that not only blood lipids but also cerebral lipid concentrations of DHA are higher in infants who had been previously breastfed than in infants fed nonsupplemented formulas [80]. Nonetheless, the question whether or not dietary LC-PUFA are beneficial for term infants is still a matter of debate. Some recent studies have assessed development in term infants fed human milk or formulas without and with LCPUFA. Innis et al. [81] compared visual acuity of infants fed human milk or a formula with a higher content of ALA than breast milk (2.1%, wt/wt). At 3 months, visual acuity was assessed with a behavioural method, the Teller card forced choice preferential looking test. The authors found no group differences in this measure of visual function. In a multicenter study using a somewhat different methodology at the various study centers, the authors also found no effect of LC-PUFA supply on growth or visual function [82]. In contrast, other recent studies using precise electrophysiological methods documented better visual acuity in infants fed breast milk or formulas providing DHA than in infants receiving formula without preformed DHA. In a randomized study, Makrides et al. [78] detected improved visual acuities measured by VEP at both 16 and 30 weeks of age in breastfed infants and those fed a fish oil supplemented formula than in infants fed non-supplemented formula. DHA was the predictor of the differences in visual acuity, since erythrocyte DHA was the only fatty acid that correlated with VEP acuity in all infants at any age tested [78,83]. Other studies have found that breastfeeding is associated with higher erythrocyte DHA concentrations and improved visual acuity up to the age of 12 months [84,85]. Willatts et al. [86] reported in a randomised study higher problem-solving scores in term infants previously fed formula supplemented with LC-PUFA as compared to a group on nonsupplemented formula. Thus, there are data indicating that dietary LC-PUFA may well have beneficial effects on neurodevelopmental maturation in term infants. This findings should encourage further detailed investigation on the metabolism of polyunsaturated fatty acids in pregnant and lactating women as well as their infants and on the biological effects of these substrates. This research should aim at providing a basis for an optimal substrate supply during the critical time period of early growth and development.

Acknowledgements The work of the authors is financially supported in part by Deutsche Forschungsgemeinschaft, Bonn, Germany (Ko 912/5-2). Dr. M. Rodriguez-Palmero was the recipient of a scholarship awarded by the Alexander von Humboldt-Foundation, Bonn, Germany.

B. Koletzko et al. / Early Human Development 65 Suppl. (2001) S3–S18

S15

References [1] ESPGAN Committee on Nutrition. Guidelines on infant nutrition: I. Recommendations for the composition of an adapted formula. Acta Paediatr Scand 1977;262:1 – 20 (Suppl). [2] ESPGAN Committee on Nutrition. Guidelines on infant nutrition: III. Recommendations for infant feeding. Acta Paediatr Scand 1982;302:1 – 27 (Suppl). [3] Koletzko B, Tsang R, Zlotkin SH, Nichols B, Hansen JW, editors. Nutrition during infancy. Principles and practice. Importance of dietary lipids. Cincinatti: Digital Educational Publishing; 1997. p. 123. [4] Koletzko B, Rodriguez-Palmero M. Polyunsaturated fatty acids in human milk and their role in early infant development. J Mammary Gland Biol Lactation 1999;4(3):269 – 84. [5] Rodriguez-Palmero M, Koletzko B, Kunz C, Jensen R. Nutritional and biochemical properties of human milk. Nutritional and biochemical properties of human milk: II. Lipids, micronutrients and bioactive factors. Clin Perinatol 1999;26(2):335 – 59. [6] Martinez M. Tissue levels of polyunsaturated fatty acids during early human development. J Pediatr 1992;120(4 Pt 2):129 – 38. [7] Michaelsen KF, Skafte L, Badsberg JH, Jorgensen M. Variation in macronutrients in human bank milk: influencing factors and implications for human milk banking. J Pediatr Gastroenterol Nutr 1990;11(2): 229 – 39. [8] Harzer G, Haug M, Dieterich I, Gentner PR. Changing patterns of human milk lipids in the course of the lactation and during the day. Am J Clin Nutr 1983;37(4):612 – 21. [9] Jensen RG. Handbook of milk composition. San Diego: Academic Press; 1995. [10] Thompson BJ, Smith S. Biosynthesis of fatty acids by lactating human breast epithelial cells: an evaluation of the contribution to the overall composition of human milk fat. Pediatr Res 1985;19(1):139 – 43. [11] Koletzko B, Thiel I, Abiodun PO. Fatty acid composition of mature human milk in Nigeria. Z Ernahrungswiss 1991;30(4):289 – 97. [12] Bitman J, Wood L, Hamosh M, Hamosh P, Mehta NR. Comparison of the lipid composition of breast milk from mothers of term and preterm infants. Am J Clin Nutr 1983;38(2):300 – 12. [13] Genzel-Boroviczeny O, Wahle J, Koletzko B. Fatty acid composition of human milk during the 1st month after term and preterm delivery. Eur J Pediatr 1997;156(2):142 – 7. [14] Carnielli VP, Luijendijk IH, van Beek RH, Boerma GJ, Degenhart HJ, Sauer PJ. Effect of dietary triacylglycerol fatty acid positional distribution on plasma lipid classes and their fatty acid composition in preterm infants. Am J Clin Nutr 1995;62(4):776 – 81. [15] Carnielli VP, Luijendijk IH, Van GJ, Sulkers EJ, Boerlage AA, Degenhart HJ, et al. Structural position and amount of palmitic acid in infant formulas: effects on fat, fatty acid, and mineral balance. J Pediatr Gastroenterol Nutr 1996;23(5):553 – 60. [16] Jensen RG. The lipids of human milk. Prog Lipid Res 1996;35:53 – 92. [17] Laegreid A, Kolsto OA. Trace amounts of ganglioside GM1 in human milk inhibit enterotoxins from Vibrio cholerae and Escherichia coli. Life Sci 1987;40(1):55 – 62. [18] Decsi T, Fekete M, Koletzko B. Plasma lipid and apolipoprotein concentrations in full term infants fed formula supplemented with long-chain polyunsaturated fatty acids and cholesterol. Eur J Pediatr 1997; 156(5):397 – 400. [19] Koletzko B. Beeinflußt die Fru¨hkindliche Substratzufuhr die Spa¨tere Gesundheit und Entwicklung? Monatsschr Kinderheilkd 1998;146:S99 – 110. [20] Jackson MB, Lammi-Keefe CJ, Jensen RG, Couch SC, Ferris AM. Total lipid and fatty acid composition of milk from women with and without insulin-dependent diabetes mellitus. Am J Clin Nutr 1994;60(3):353 – 61. [21] Ratnayake WM, Chen ZY. Trans, n 3, and n 6 fatty acids in Canadian human milk. Lipids 1996; 31:279 – 82 (Suppl). [22] Serra G, Marletta A, Bonacci W, Campone F, Bertini I, Lantieri PB, et al. Fatty acid composition of human milk in Italy. Biol Neonate 1997;72(1):1 – 8. [23] Koletzko B, Thiel I, Abiodun PO. The fatty acid composition of human milk in Europe and Africa [see comments]. J Pediatr 1992;120(4 Pt 2):62 – 70. [24] Luukkainen P, Salo MK, Nikkari T. Changes in the fatty acid composition of preterm and term human milk from 1 week to 6 months of lactation. J Pediatr Gastroenterol Nutr 1994;18(3):355 – 60.

S16

B. Koletzko et al. / Early Human Development 65 Suppl. (2001) S3–S18

[25] Chen ZY, Kwan KY, Tong KK, Ratnayake WM, Li HQ, Leung SS. Breast milk fatty acid composition: a comparative study between Hong Kong and Chongqing Chinese. Lipids 1997;32(10):1061 – 7. [26] Koletzko B. Fats for brains. Eur J Clin Nutr 1992;46(Suppl 1):S51 – 62. [27] Aggett PJ, Haschke F, Heine W, Hernell O, Koletzko B, Launiala K, et al. Comment on the content and composition of lipids in infant formulas. ESPGAN Committee on Nutrition. Acta Paediatr Scand 1991; 80(8 – 9):887 – 96. [28] Makrides M, Simmer K, Neumann M, Gibson R. Changes in the polyunsaturated fatty acids of breast milk from mothers of full-term infants over 30 wk of lactation. Am J Clin Nutr 1995;61(6):1231 – 3. [29] Lemons JA, Moye L, Hall D, Simmons M. Differences in composition of preterm and term human milk during lactation. Pediatr Res 1982;16:113 – 7. [30] Rueda R, Ramirez M, Garcia-Salmeron JL, Maldonado J, Gil A. Gestational age and origin of human milk influence total lipid and fatty acid contents. Ann Nutr Metab 1998;42(1):12 – 22. [31] Koletzko B. Zufuhr, Stoffwechsel und biologische Wirkungen trans-isomerer Fettsa¨uren bei Sa¨uglingen. Die Nahrung 1991;35:229 – 83. [32] Chappell JE, Clandinin MT, Kearney-Volpe C. Trans fatty acids in human milk lipids: influence of maternal diet and weight loss. Am J Clin Nutr 1985;42:49 – 56. [33] Koletzko B. Trans fatty acids may impair biosynthesis of long-chain polyunsaturates and growth in man. Acta Paediatr Scand 1992;81:302 – 6. [34] Danish Nutrition Council. The influence of trans fatty acids on health. Clin Sci 1995;88:375 – 92. [35] Koletzko B, Galli C, Simopoulos AP, Tremoli E, editors. Fatty acids and lipids: biological aspects. Trans fatty acids and the human infant. Basel: Karger; 1994. p. 82 – 5. [36] Francois CA, Connor SL, Wander RC, Connor WE. Acute effects of dietary fatty acids on the fatty acids of human milk. Am J Clin Nutr 1998;67:301 – 8. [37] Sanders TAB, Ellis FR, Dickerson JWT. Studies of vegans: the fatty acid composition of plasma choline phosphoglycerides, erythrocytes, adipose tissue, and breast milk and some indicators of susceptibility to ischemic heart disease in vegans and omnivore controls. Am J Clin Nutr 1978;31:805 – 13. [38] Sanders TAB, Reddy S. The influence of a vegetarian diet on the fatty acid composition of human milk and the essential fatty acid status of the infant. J Pediatr 1992;120:S71 – 7. [39] Finley DA, Lonnerdal B, Dewey KG, Grivetti LE. Breast milk composition: fat content and fatty acid composition in vegetarians and non-vegetarians. Am J Clin Nutr 1985;41(4):787 – 800. [40] de la Presa-Owens S, Lopez-Sabater MC, Rivero-Urgell M. Fatty acid composition of human milk in Spain. J Pediatr Gastroenterol Nutr 1996;22(2):180 – 5. [41] Innis SM, Kuhnlein HV. Long-chain n 3 fatty acids in breast milk of Inuit women consuming traditional foods. Early Hum Dev 1988;18(2 – 3):185 – 9. [42] Rocquelin G, Tapsoba S, Dop MC, Mbemba F, Traissac P, Martin-Prevel Y. Lipid content and essential fatty acid (EFA) composition of mature Congolese breast milk are influenced by mothers’ nutritional status: impact on infants’ EFA supply. Eur J Clin Nutr 1998;52(3):164 – 71. [43] Henderson RA, Jensen RG, Lammi-Keefe CJ, Ferris AM, Dardick KR. Effect of fish oil on the fatty acid composition of human milk and maternal and infant erythrocytes. Lipids 1992;27:863 – 9. [44] Cherian G, Sim JS. Changes in the breast milk fatty acids and plasma lipids of nursing mothers following consumption of n 3 polyunsaturated fatty acid enriched eggs. Nutrition 1996;12(1):8 – 12. [45] Harris WS, Connor WE, Lindsey S. Will dietary omega-3 fatty acids change the composition of human milk? Am J Clin Nutr 1984;40(4):780 – 5. [46] Walstra P, Jenness R. Dairy chemistry and physics. Heating. New York: Wiley-Interscience; 1984. p. 162 – 85. [47] Wardel JM, Hill CM, D’Souza SW. Effect of pasteurisation and of freezing and thawing human milk on its triglyceride content. Acta Paediatr Scand 1981;70:467 – 71. [48] Fidler N, Sauerwald T, Koletzko B, Demmelmair H. Effects of human milk pasteurization and sterilization on available fat content and fatty acid composition. J Pediatr Gastroenterol Nutr 1998;27:317 – 22. [49] Hurrell RF, Nielsen HK, Horisberger M, Bracco U, editors. Lipids in modern nutrition. Nutritional aspects of oxidized lipids and their interaction with other dietary components. New York: Raven Press; 1987. p. 223 – 37. [50] Henderson TR, Fay TN, Hamosh M. Effect of pasteurization on long chain polyunsaturated fatty acid levels and enzyme activities of human milk. J Pediatr 1998;132(5):876 – 8.

B. Koletzko et al. / Early Human Development 65 Suppl. (2001) S3–S18

S17

[51] Williamson S, Finucane E, Ellis H, Gamsu HR. Effect of heat treatment of human milk on absorption of nitrogen, fat, sodium, calcium and phosphorus by preterm infants. Arch Dis Child 1978;53:555 – 63. [52] Hamosh M, Henderson TR, Ellis LA, Mao JI, Hamosh P. Digestive enzymes in human milk: stability at suboptimal storage temperatures. J Pediatr Gastroenterol Nutr 1997;24:38 – 43. [53] Grammatikos SI, Subbaiah PV, Victor TA, Miller WM. n 3 and n 6 fatty acid processing and growth effects in neoplastic and non-cancerous human mammary epithelial cell lines. Br J Cancer 1994;70:219 – 27. [54] Rogers MP, Zhao X. Lipoprotein lipase mRNA levels in adipose tissue in lactation. Biochem Soc Trans 1996;24:169S. [55] Rebuffe´-Scrive M, Enk L, Crona N, et al. Fat cell metabolism in different regions in women. Effect of menstrual cycle, pregnancy and lactation. J Clin Invest 1985;75:1973 – 6. [56] Hachey DL, Thomas MR, Emken EA, et al. Human lactation: maternal transfer of dietary triglycerides labeled with stable isotopes. J Lipid Res 1987;28:1185 – 92. [57] Koletzko B, Mrotzek M, Bremer HJ. Fatty acid composition of mature human milk in Germany. Am J Clin Nutr 1988;47(6):954 – 9. [58] van der Westhuyzen J, Chetty N, Atkinson PM. Fatty acid composition of human milk from South African black mothers consuming a traditional maize diet. Eur J Clin Nutr 1988;42(3):213 – 20. [59] Simopoulos AP. Omega-3 fatty acids in health and disease and in growth and development [see comments]. Am J Clin Nutr 1991;54(3):438 – 63. [60] Koletzko B, Sauerwald T, Demmelmair H. Safety of stable isotope use. Eur J Pediatr 1997;156(Suppl 1): S12 – 7. [61] Demmelmair H, Baumheuer M, Koletzko B, Dokoupil K, Kratl G. Metabolism of U13C-labeled linoleic acid in lactating women. J Lipid Res 1998;39(7):1389 – 96. [62] Decsi T, Koletzko B. Polyunsaturated fatty acids in infant nutrition. Acta Paediatr 1994;83(395):31 – 7 (Suppl). [63] Koletzko B, Bremer HJ. Fat content and fatty acid composition of infant formulae. Acta Paediatr Scand 1989;78:513 – 21. [64] Demmelmair H, von SU, Behrendt E, Sauerwald T, Koletzko B. Estimation of arachidonic acid synthesis in full term neonates using natural variation of 13C content. J Pediatr Gastroenterol Nutr 1995;21(1):31 – 6. [65] Szitanyi P, Koletzko B, Mydlova A, Demmelmair H. Metabolism of 13C-labeled linoleic acid in newborn infants during the first week of life. Pediatr Res 1999;45(5 Pt 1):669 – 73. [66] Koletzko B, Diener U, Fink M, et al. In: Ziegler E, Lucas A, Moro G, editors. Nutrition of the extremely low birthweight infant. Supply and biological effects of long-chain polyunsaturated fatty acids (LC-PUFA) in premature infants. Philadelphia: Lippincott-Raven; 1999. p. 33 – 52. [67] Decsi T, Thiel I, Koletzko B. Essential fatty acids in full term infants fed breast milk or formula. Arch Dis Child Fetal Neonatal Ed 1995;72(1):23 – 8. [68] Koletzko B, Schmidt E, Bremer HJ, Haug M, Harzer G. Effects of dietary long-chain polyunsaturated fatty acids on the essential fatty acid status of premature infants. Eur J Pediatr 1989;148(7):669 – 75. [69] Koletzko B, Edenhofer S, Lipowsky G, Reinhardt D. Effects of a low birthweight infant formula containing human milk levels of docosahexaenoic and arachidonic acids. J Pediatr Gastroenterol Nutr 1995;21(2): 200 – 8. [70] Decsi T, Koletzko B. Growth, fatty acid composition of plasma lipid classes, and plasma retinol and alphatocopherol concentrations in full-term infants fed formula enriched with omega-6 and omega-3 long-chain polyunsaturated fatty acids. Acta Paediatr 1995;84(7):725 – 32. [71] Uauy RD, Birch DG, Birch EE, Tyson JE, Hoffman DR. Effect of dietary omega-3 fatty acids on retinal function of very-low-birth-weight neonates. Pediatr Res 1990;28(5):485 – 92. [72] Birch EE, Birch DG, Hoffman DR, Uauy R. Dietary essential fatty acid supply and visual acuity development. Invest Ophthalmol Visual Sci 1992;33(11):3242 – 53. [73] Lucas A, Morley R, Cole TJ, et al. Breast milk and subsequent intelligent quotient in children bron preterm. Lancet 1992;339:261 – 4. [74] Carlson SE, Werkman SH, Rhodes PG, Tolley EA. Visual acuity development in healthy preterm infants: effects of marine oil supplementation. Am J Clin Nutr 1993;58:35 – 42. [75] Carlson SE, Werkman SH. A randomized trial of visual attention of preterm infants fed docosahexaenoic acid until two months. Lipids 1996;31:85 – 90.

S18

B. Koletzko et al. / Early Human Development 65 Suppl. (2001) S3–S18

[76] Carlson SE, Cooke J, Rhodes PG, Peeples JM, Werkman SH, Tolley EA. Long-term feeding of formulas high in linoleic acid and marine oil to very low birth weight infants: phospholipid fatty acids. Pediatr Res 1991;30:404 – 12. [77] Innis SM, Foote KD, MacKinnon MJ, King DJ. Plasma and red cell fatty acids of low birth weight infants fed their mothers expressed breast milk or preterm infant formula. Am J Clin Nutr 1990;51:994 – 1000. [78] Makrides M, Neumann M, Simmer K, Pater J, Gibson R. Are long chain polyunsaturated fatty acids essential nutrients in infancy? Lancet 1995;345:1463 – 8. [79] Koletzko B, Decsi T, Demmelmair H. Arachidonic acid supply and metabolism in human infants born at full term. Lipids 1996;31(1):79 – 83. [80] Farquharson J, Cockburn F, Patrick WA, Jamieson EC, Logan RW. Infant cerebral cortex phospholipid fattyacid composition and diet [see comments]. Lancet 1992;340(8823):810 – 3. [81] Innis SM, Nelson CM, Rioux MF, King DJ. Development of visual acuity in relation to plasma and erythrocyte omega-6 and omega-3 fatty acids in healthy term gestation infants. Am J Clin Nutr 1994;60: 347 – 52. [82] Auestad N, Montalto MB, Hall RT, Fitzgerald KM, Wheeler RE, Connor WE, et al. Visual acuity, erythrocyte fatty acid composition, and growth in term infants fed formulas with long chain polyunsaturated fatty acids for one year. Ross pediatric lipid study. Pediatr Res 1997;41(1):1 – 10. [83] Makrides M, Neumann M, Byard J, Simmer K, Gibson R. Fatty acid composition of brain, retina and ertyhrocytes in breast- and formula-fed infants. Am J Clin Nutr 1994;60:189 – 94. [84] Birch EE, Birch DG, Hoffman DR, Hale L, Everett M, Uauy R. Breast-feeding and optimal visual development. J Pediatr Ophthalmol Strab 1993;30:33 – 8. [85] Birch EE, Hoffman DR, Uauy R, Birch DG, Prestidge C. Visual acuity and the essentiality of docosahexaenoic acid and arachidonic acid in the diet of term infants. Pediatr Res 1998;44:201 – 9. [86] Willatts P, Forsyth JS, DiModugno MK, Varma S, Colvin M. Effect of long-chain polyunsaturated fatty acids in infant formula on problem solving at 10 months of age. Lancet 1998;352(9129):688 – 91.