Human milk and formula fatty acids Sheila M. Innis, PhD From the Department of Paediatrics, University of British Columbia, Vancouver, British Columbia
Recent years have seen a growing debate over the amounts of various fatty acids required in infant diets. Particular emphasis has been given to long-chain polyenoie fatty acids, such as arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3). These fatty acids are found in high proportions in the structural lipids of cell membranes, particularly those of the central nervous system, and are present in human milk but not in the vegetable oils used to prepare infant formulas. This article provides a brief introduction to fatty acid metabolism and the fatty acid composition of mature human milk and the proprietary infant formulas available in North America. FATTY ACID METABOLISM Mammalian cells can synthesize saturated fatty acids de novo from acetylcoenzyme A by sequential addition of 2 carbon units, usually culminating at palmitic acid (16:0). Palmitic acid can be elongated to stearic acid (18:0) and desaturated by the delta-9 desaturase enzyme to oleic acid (18:1n-9). Mammalian cells do not have desaturase enzymes capable of introducing an unsaturated bond at the n-6 or n-3 position of a fatty acid carbon chain. Fatty acids of the n-6 and n-3 series, however, provide the unsaturated "fluid" core of cell membranes and are the precursors for eicosanoid biosynthesis. Consequently, a dietary source of n-6 and n-3 fatty acids is essential for all animals. This must be derived either directly by ingestion of plants and phytoplankton or from the tissues of other animals obtained as a result of transfer up the food chain. The n-6 and n-3 fatty acids recognized as essential in human nutrition are the 18 carbon chain n-6 and n-3 fatty acids, linoleic acid (18:2n-6) and linolenic acid (18:3n-3). 1, 2 These fatty acids can be metabolized further by alternating desaturation and elongation to give a series of carbon chain
Reprint requests: Sheila M. Innis, PhD, Department of Paediatrics, University of British Columbia, The Research Centre, Room 179, 950 W. 28th Ave., Vancouver, BC, Canada, V5Z 4H4.
9/0/36792
$56
20 and 22 n-6 and n-3 fatty acids with between 3 and 6 carbon-carbon double bonds. Of these, the best known are arachidonic acid (20:4n-6), docosatetraenoic acid (22:4n6), eicosapentaenoic acid (20:5n-3), and docosahexaenoic acid (22:6n-3). These highly unsaturated fatty acids are found almost exclusively in the phospholipids that make up the structural matrix of all cell membranes. Thus although linoleic acid and linolenic acid are recognized as the essential dietary nutrients, normal cell function requires their more' highly unsaturated derivatives. The synthesis of arachidonic acid and docosahexaenoic acid requires not only adequate amounts of the substrates, linoleic acid and linolenic acid, respectively, but also adequate fatty acid desaturase enzyme activity. There is no direct information on the development of these enzymes in human tissues. Circumstantial and direct experimental information for a variety of other species shows that these enzymes are fully active in the appropriately growing term newborn.3The biochemistry of fatty acid desaturation is complex, and many aspects are still not well understood. It is known that the desaturation of n-9, n-6, and n-3 fatty acids relies on the same enzymes and there is competition among potential substrates for a given desaturase enzyme. Preferential desaturation occurs in the order 18:3n3 > 18:2n-6 > 18:1n-9.4' 5 This suggests that the balance, as well as the absolute amounts, of linoleic acid and linolenic acid in the milk or formula diet may impact on the ability of the infant to synthesize arachidonic acid or docosahexaenoic acid. The desaturase enzymes are also inhibited by their reaction products, such as arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. 4, 5 Presumably, this inhibition provides a mechanism to ensure that the rate of synthesis of these fatty acids is balanced with the needs of new membrane lipid growth. However, disproportionate amounts of the desaturation products of either linoleic or linolenic acid in the diet may interfere with synthesis of fatty acids from the alternate pathway. Decreased tissue levels of arachidonic acid in adult and infant humans or animals fed eicosapentaenoic and docosahexaenoic acids from
Volume 120 Number 4, Part 2
fish oils have been described extensively69; this reduction is explained in part by inhibition of 18:2n-6 desaturation by the n-3 fatty acid desaturation products. Although texts on essential fatty acid metabolism frequently depict the pathways of desaturation and elongation, it must also be remembered that linoleic acid and linolenic acid are readily oxidized for energy. Linoteie acid, and to a much lesser extent linolenic acid, is also found in stored adipose tissue triglycerides and cholesteryl esters. The levels of linoleic acid are also high in many tissue phospholipids. The partitioning of 18:2n-6 and 18:3n-3 among potential pathways of oxidation, direct aeylation into tissue lipids, or desaturation is not yet understood. However, it is reasonable to expect that energy balance may be important in determining the need to oxidize essential fatty acids to provide the energy for basic metabolic and physiologic functions. The positive energy balance associated with normal infant nutrition would presumably facilitate the use of fatty acids for biosynthetic purposes, whereas a negative energy balance may theoretically be expected to direct fatty acids toward oxidation to meet maintenance energy needs. In contrast to linoleic acid and linolenic acid, arachidonic acid and docosahexaenoic acid are rapidly incorporated into tissue phospholipids a n d undergo mitochondrial/~-oxidation to a much lesser extent. This provides a reasonable biochemical explanation for repeated demonstrations that dietary linoleic and linolenic acids are not equivalent to their longer-chain, more unsaturated products. This aspect of fatty acid metabolism is important because it indicates that not all dietary n-6 and n-3 fatty acids are equivalent in either their dietary or metabolic effects. In summary, the assimilation of appropriate amounts of n-6 and n-3 fatty acids in growing infant tissues will depend on the dietary fatty acid supply, tissue desaturase enzyme activity, and energy intake. Human milk provides the infant with linoleic acid and linolenic acid and a preformed source of arachidonie acid and docosahexaenoic acid.l~The arachidonic acid and docosahexaenoic acid provided in human milk is likely to contribute directly to the infant's structural lipid pools of these fatty acids. Infants who receive formulas without these fatty acids must fulfill their tissue requirements exclusively from desaturation of linoleic acid or linolenic acid. Attempts to estimate the requirements for, or efficacy of, linoleic acid and linolenic acid for synthesis of longer-chain metabolites for membrane growth must consider the amounts of these .fatty acids lost to oxidation, energy intake, tissue desaturase enzyme activity, and the dietary fatty acid balance. A small amount of information indicates that dietary saturated and monounsaturated fatty acids also influence the metabolism and tissue levels of n-6 and n-3 fatty acids. 11 Although these studies do not yet al-
Human milk and formula fatty acids
$57
Table. Average levels of the major saturated and monounsaturated fatty acids and linoleic and linolenic acid in mature human milk in the United States* Range of means (% total)
Fatty acids
Pooled mean (% total)
Minimum
Maximum
Median (% total)
8:0 10:0 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3
0.32 1.21 4.79 6.55 21.58 3.23 7.97 35.18 16.15 1.07
0.17 0.89 1.40 3.80 19.80 2.50 7.10 30.70 14.47 0.30
0.60 1.60 6.50 10.20 23.96 3.83 9.00 38.20 18.80 1.85
0.26 1.01 5.08 6.20 21.80 3.29 8.14 35.51 15.80 1.03
*Meanvaluesof individualfatty acid componentsreported in references12 through 20.
low definitive conclusions, it would be unwise to assume that the composition of nonessential fat in infant diets is of no consequence. H U M A N M I L K AND F O R M U L A F A T Fat represents the major portion of energy in most human milk and infant formulas, usually about 45% to 50% of kcal. About 98% of the fat is present as triglycerides, which contain one molecule of glycerol to which three fatty acids are esterified. Human milk triglycerides are present in the core of the milk fat globules, which are held in solution in the aqueous milk environment by a surface layer of phospholipids and proteins, l~ Dispersion of triglycerides in infant formulas is maintained with plant lecithins, sometimes with the aid of monoglyeerides and diglycerides. Human milk contains a large number of fatty acids, including saturated and monounsaturated fatty acids and n-6 and n-3 series polyunsaturated fatty acids. The latter include both the dietary essential fatty acids linoleic acid and linolenie acid, as well as small amounts of longer-chain, more unsaturated n-6 and n-3 fatty acids such as arachidonic acid and doeosahexaenoic acid. These longer-chain, more unsaturated n-6 and n-3 fatty acids usually represent a total of about 1% to 1.5% of the milk fatty acids, roughly equivalent to about 0.5% to 0.75% of kcal. Although there is considerable information on the major fatty acid components of human milk, information on the long-chain polyunsaturated fatty acids is more limited. Average levels of the major saturated and monounsaturated fatty acids and linoleic and linolenic acid in mature human milk in the United States are given in the Table. The data given represent mean values for nine studies of women fol-
S 58
Innis
The Journal of Pediatrics April 1992
50-
40
?
~/).
o o
50
20 ~d 4~
%
10
i
_
MCSFA ICSFA LCSFA
_
MFA
_
--~]
_
18:2
18:5
[:i9. t. Saturated and monounsaturated fatty acid and linoleic and linolenic acid profile of mature human milk in the United States: pooled means and minimum and maximum means calculated from data in references 12 through 20. MCSFA, Medium-chain saturated fatty acids, 8:0 + 10:0, 1CSFA, intermediate-chain saturated fatty acids, 12:0 + 14:0; LCSFA, long-chain saturated fatty acids, 16:0 + 18:0;MFA, monounsaturated fatty acids 16:1 + 18:1; 18:2, linoleic acid (18:2n-6); 18.'3, linolenic acid (18:3n-3).
lowing unrestricted diets that were published within the past 10 years. ~22~The levels of medium-chain fatty acids were low (1% to 2%), whereas palmitic acid (16:0) and oleic acid (18:1) were major components, representing about 20% to 24% and 30% to 38% of milk fatty acids, respectively. The mean levels of linoleic acid (18:2n-6) and linolenic acid (18:3n-3) were about 16% and 1%, respectively (Table). The fat in North American infant formulas is a blend of one or more vegetable oils, with the addition of oleo oils in the products of some manufacturers. Corn oil and soybean oil contain about 50% to 60% linoleic acid and are well absorbed; soybean oil contains about 6% to 9% linolenic acid and is the usual oil of choice to provide this fatty acid. These vegetable oils contain low levels of oleic acid (20% to 25%) and palmitic acid (10% to 12%), and they must be blended with other oils if a saturated/m~176176 urated fat profile resembling human milk is desired (Fig. 1). In Fig. 1 the saturated fatty acids have been grouped as the medium-chain fatty acids 8:0 and 10:0, the intermediatechain saturated fatty acids 12:0 and 14:0 (which are synthesized by the mammary glandl9), the long-chain saturated fatty acids 16:0 and 18:0, and the monounsaturated fatty acids 16:1 and 18:1. Oleo oils, derived from beef fat, and vegetable oils, such as palm oil, contain palmitic acid. The palmitie acid in these oils, however, is esterified predominantly to the sn-l,3 car-
bon atoms of the 3 carbon glycerol backbone of the triglyceride. I~ In contrast, more than 70% of the palmitic acid in human milk is esterified to the center, sn-2 glycerol carbon. 1~ This is important because the products of intestinal hydrolysis of fat are sn-2 monoglycerides and two free fatty acids released from the sn-1 and sn-3 positions.21 The coefficiency of absorption of free palmitic acid is low in very small preterm infants.22, 23 Consequently, some manufacturers prefer to use coconut oils or medium-chain triglycerides as a source of well-absorbed saturated fatty acids for infant formulas. The constitutive milk lipase, known as bile salt-stimulated lipase, is believed to hydrolyze the sn-2 monoglycerides left after triglyceride hydrolysis by gastric and intestinal lipases. 24 The action of this enzyme should release 16:0 from the sn-2 position of human milk. The coefficiency of absorption of human milk fat is high and apparently not limited in any way by free 16:0. Human milk contains a variety of n-6 and n-3 fatty acids with 20 or 22 carbon atoms. Detailed information on the levels of these fatty acids can be found in subsequent articles in this supplement. Usually arachidonic acid is the major long-chain n-6 fatty acid and represents about 0.5% to 0.7% of total milk fatty acids (Fig. 2, B). Docosahexaenoic acid is the major longer-chain n-3 fatty acid in human milk (Fig. 2, D), even when the mother's dietary intake of eicosapentaenoic acid is higher than that of docosahexaenoic ac-
Volume 120 Number 4, Part 2
Human milk and formula fatty acids
$5 9
A: Linoleie Acid (18:2n-6)
24
18
l
12
l I g 1.4
B: Araehidonie Add (20:4n-6)
1.2 1.0 0.8
0.2 0.0
Fig. 2. Percent of total human milk fatty acids. A, Linoleic acid (18:2n-6); B, arachidonic acid (20:4n-6). Values modified from references given in parentheses represent mean values _+SD, with the exception of reference 31, for which the median value was reported and is reproduced here. In some cases, estimates of variance were not published. id. 25 Commercially available infant formula prepared from vegetable oils with or without oleo oils do not contain significant amounts of these fatty acids. The appropriateness of adding these longer-chain polyunsaturated fatty acids to infant formulas is the subject of subsequent articles in this supplement. Fat is one of the most variable nutrients in human milk, varying with the stage of lactation, among different women, with the time of day, and in numerous other factors. ~ The variability in the fatty acids of mature human milk poses
considerable problems in attempting to establish optimal patterns of fatty acid intake from the infant fed human milk. Fig. 2 shows a comparison of the major n-6 and n-3 fatty acids, linoleic acid (18:2n-6), arachidonic acid (20:4n6), linolenic acid (18:3n-3), and docosahexaenoic acid (22:6n-3), reported in studies of mature human milk from women following unrestricted diets in different regions of the world or from groups with diverse cultural backgrounds.12, zs, 25-33 Some of the articles in this supplement provide evidence that these wide variations in human milk
S 60
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The Journal of Pediatrics April 1992
C: Linolenie Acid (18:3n-3)
2.5
2.0
1.5
1.0
0.0
g
-
g
g
D: Docosahex~enoie ANd (22:6n-3)
2.5
2.0-
1.5 b4X~
•215
1.0
xxx • N'x)< •215 XNX X'>oo< XNX NMN X )~ .x, xxx X• X~'NX
0.0
xz,x
g
g
o=
~
,
tn
~2
Fi9. 2, Cont. Percent of total human milk fatty acids. C, Linolenic acid (18:3n-3); D, docosahexaenoic acid (22:6n-3). Values modified from references given in parentheses represent mean values _+ SD, with the exception of reference 31, for which the median value was reported and is reproduced here. In some cases, estimates of variance were not published.
fatty acids are at least partly the result of differences in the mother's dietary fat intake. H u m a n milk fatty acids in North America may well reflect changes in dietary fat toward increased use of corn, soybean, and other vegetable oils 34 that provide h i g h amounts of linoleic acid and linolenic acid, in response to recommendations from various agencies concerned with morbidity and death caused by atherosclerotic diseases. Extrapolation of infant fatty acid requirements from in-
formation on the amounts received by infants fed human milk is limited by several factors other than the variability in human milk. Subsequent articles in this supplement debate which fatty acids are essential nutrients for the newborn infant, the importance of fatty acid balance, and the possible influence of saturated or monounsaturated fatty acids on lipid metabolism. Recommended intakes calculated as the mean intake from human milk plus an estimate of variance (usually 2 SDs, or about 25% to 30% mean
Volume 120 Number 4, Part 2
value) to cover the estimated requirement of 97.5% of the population will also, by definition, define 97.5% of all h u m a n milk as deficient. Finally, if the total fat intake is maintained, high amounts of one fatty acid must be accompanied by low a m o u n t s of a n o t h e r fatty acid. In the absence of scientific reasons to consider otherwise, the average or median levels of fatty acids in h u m a n milk, with regard to all fatty acids, provide an estimate most likely to cover the lipid nutrient requirements of infants.
REFERENCES 1. Tinoco J. Dietary requirements and function of c~-linolenic acid in animals. Prog Lipid Res 1982;21:1-45. 2. u WK, Clemans GW, Hutchinson ML. Essential fatty acid deficiency in humans. Prog Lipid Res 1981;19:187215. 3. Innis SM. Essential fatty acids in growth and development. Prog Lipid Res 1991;30:39-103. 4. Brenner RR. The oxidative desaturation of unsaturated fatty acids in animals. Moll Cell Biochem 1974;3:41-52. 5. Brenner RR. Nutritional and hormonal factors influencing desaturation of essential fatty acids. Prog Lipid Res 1981; 20:41-7. 6. Hwang DH, Boudreau M, Chanmugam P. Dietary linoleic acid and longer-chain n-3 fatty acids.: comparison of effects on arachidonic acid metabolism in rats. J Nutr 1988;118:42737. 7. Arbuckle LD, Rioux FM, Mackinnon M J, Hrboticky N, Innis SM. Response of n-3 and n-6 fatty acids in brain, liver and plasma of piglets fed formula to increasing, but low, levels of fish oil supplementation. J Nutr 1991;121:1536-47. 8. Carlson S, Cooke RJ, Rhodes PG, Peeples JM, Werkman SH, Tolley EA. Long-term feeding of formulas high in linolenic acid and marine oil to very low birth-weight infant: phospholipid fatty acids. Pediatr Res 1991;30:404-12. 9. Popp-Snijders C, Schouten JA, van Blitterswijk W J, van der Veen EA. Changes in membrane lipid composition of human erythrocytes after dietary supplementation of (n-3) polyunsaturated fatty acids: maintenance of membrane fluidity. Biochim Biophys Acta 1986;854:31-7. 10. Jensen RG. Lipids in human milk: composition and fat-soluble vitamins. In: Lebenthal E, ed. Textbook of gastroenterology and nutrition in infancy. 2rid ed. New York: Raven Press, 1989:157-208. 11. Garg ML, Wierzbicki AA, Thompson ABR, Clandinin MT. Dietary saturated fat level alters the competition between ~-linolenic acid and linoleic acid. Lipids 1989;24:334-9. 12. Bitman J, Wood L, Hamosh M, Mehta NR. Comparison of the lipid composition of breast milk from mothers of term and preterm infants. Am J Clin Nutr 1983;38:300-12. 13. Borschel MW, Elkin RG, Kirkse A, et al. Fatty acid composition of mature human milk of Egyptian and American women. Am J Clin Nutr 1986;44:330-5. 14. Clark RM, Ferris AM, Fey M, Brown PB, Hundrieser KE, Jensen RG. Changes in the lipids of human milk from 2 to 16 weeks postpartum. J Pediatr Gastroenterol Nutr 1982; 1:311-5. 15. 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;41:787800.
Human milk and formula fatty acids
S61
16. Harris WS, Connor WE, Lindsey S. Will dietary ~o-3fatty acids change the composition of human milk. Am J Clin Nutr 1984;40:780-5. 17. Putnam JC, Carlson SE, Devoe PW, Barness LA. The effect of variations in dietary fatty acids on the fatty acid composition of erythrocyte phosphatidylcholine and phosphatidylethanolamine in human infants. Am J Clin Nutr 1982;36:106-14. 18. Specker BL, Wey HE, Miller D. Differences in fatty acid composition of human milk in vegetarian and nonvegetarian women: long-term effect of diet. J Pediatr Gastroenterol Nutr 1987;6:764-8. 19. Thompson BJ, Smith S. Biosynthesis of fatty acids by lactating human breast milk epithelial cells: an evaluation of the contribution to the overall composition of human milk fat. Pediatr Res 1985;19:139-43. 20. Wang C-S, Illingsworth DR. Lipid composition and lipolytic activities in milk from a patient with homozygous familial hypobetalipoproteinemia. Am J Clin Nutr 1987;45:730-6. 21. Carey MC, Small DM, Bliss CM. Lipid digestion and absorption. Ann Rev Physiol 1983;45:651-77. 22. Chappell JE, Clandinin MT, Kearney-Volpe C, Reichman B, Swyer PW. Fatty acid balance studies in premature infants fed human milk or formula: effect of calcium supplementation. J Pediatr 1986;108:439-47. 23. Jensen C, Buist NRM, Wilson T. Absorption of individual fatty acids from long-chain or medium-chain triglycerides in very small infants. Am J Clin Nutr 1988;43:745-51. 24. Hernell O, Blackberg L, Bernback S. Digestion of human milk fat in early infancy. Acta Paediatr Scand Suppl 1989;351:5762. 25. Innis SM, Kuhnlein HV. Long-chain n-3 fatty acids in breast milk of Inuit women consuming traditional foods. Early Hum Dev 1988;18:185-9. 26. Boersma ER, Offringa P J, Muskiet FAJ, Chase WM, Simmons IJ. Vitamin E, lipid fractions, and fatty acid composition of colostrum, transitional milk, and mature milk: an international comparative study. Am J Clin Nutr 1991 ;53:1197-204. 27. Gibson RA, Kneebone GM. Fatty acid composition of human colostrum and mature breast milk. Am J Clin Nutr 1981; 34:252-7. 28. Harzer G, Haug M, Dieterich I, Gentner PR. Changing patterns of human milk lipids in the course of lactation and during the day. Am J Clin Nutr 1983;37:612-21. 29. Jansson L, Akesson B, Holmberg L. Vitamin E and fatty acid composition of human milk. Am J Clin Nutr 1981;34:8-13. 30. Kneebone GM, Kneebone R, Gibson RA. Fatty acid composition of breast milk from three racial groups from Penang, Malaysia. Am J Clin Nutr 1985;41:765-9. 31. Koletzko B, Mrotzek M, Eng B, Bremer HJ. Fatty acid composition of mature human milk in Germany. Am J Clin Nutr 1988;47:954-9. 32. Muskiet FA, Hutter NH, Martini IA, Jonxis JH, Offringa P J, Boersma ER. Comparison of the fatty acid composition of human milk from mothers in Tanzania, Curacao and Surinam. Hum Nutr Clin Nutr 1987;41:149-59. 33. van Beusekom CM, Martini IA, Rutgers HM, Boersma ER, Muskiet FAJ. A carbohydrate-rich diet not only leads to incorporation of medium-chain fatty acids (6:0-14:0) in milk triglycerides but also in each milk phospholipid subclass. Am J Clin Nutr 1990;52:326-34. 34. Stephen AM, Wald NJ. Trends in individual consumption of dietary fat in the United States, 1920-1984. Am J Clin Nutr 1990;52:457-69.
Recent years have seen a growing debate over the amounts of various fatty acids required in infant diets. Particular emphasis has been given to long-chain polyenoie fatty acids, such as arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3). These fatty acids are found in high proportions in the structural lipids of cell membranes, particularly those of the central nervous system, and are present in human milk but not in the vegetable oils used to prepare infant formulas. This article provides a brief introduction to fatty acid metabolism and the fatty acid composition of mature human milk and the proprietary infant formulas available in North America. FATTY ACID METABOLISM Mammalian cells can synthesize saturated fatty acids de novo from acetylcoenzyme A by sequential addition of 2 carbon units, usually culminating at palmitic acid (16:0). Palmitic acid can be elongated to stearic acid (18:0) and desaturated by the delta-9 desaturase enzyme to oleic acid (18:1n-9). Mammalian cells do not have desaturase enzymes capable of introducing an unsaturated bond at the n-6 or n-3 position of a fatty acid carbon chain. Fatty acids of the n-6 and n-3 series, however, provide the unsaturated "fluid" core of cell membranes and are the precursors for eicosanoid biosynthesis. Consequently, a dietary source of n-6 and n-3 fatty acids is essential for all animals. This must be derived either directly by ingestion of plants and phytoplankton or from the tissues of other animals obtained as a result of transfer up the food chain. The n-6 and n-3 fatty acids recognized as essential in human nutrition are the 18 carbon chain n-6 and n-3 fatty acids, linoleic acid (18:2n-6) and linolenic acid (18:3n-3). 1, 2 These fatty acids can be metabolized further by alternating desaturation and elongation to give a series of carbon chain
Reprint requests: Sheila M. Innis, PhD, Department of Paediatrics, University of British Columbia, The Research Centre, Room 179, 950 W. 28th Ave., Vancouver, BC, Canada, V5Z 4H4.
9/0/36792
$56
20 and 22 n-6 and n-3 fatty acids with between 3 and 6 carbon-carbon double bonds. Of these, the best known are arachidonic acid (20:4n-6), docosatetraenoic acid (22:4n6), eicosapentaenoic acid (20:5n-3), and docosahexaenoic acid (22:6n-3). These highly unsaturated fatty acids are found almost exclusively in the phospholipids that make up the structural matrix of all cell membranes. Thus although linoleic acid and linolenic acid are recognized as the essential dietary nutrients, normal cell function requires their more' highly unsaturated derivatives. The synthesis of arachidonic acid and docosahexaenoic acid requires not only adequate amounts of the substrates, linoleic acid and linolenic acid, respectively, but also adequate fatty acid desaturase enzyme activity. There is no direct information on the development of these enzymes in human tissues. Circumstantial and direct experimental information for a variety of other species shows that these enzymes are fully active in the appropriately growing term newborn.3The biochemistry of fatty acid desaturation is complex, and many aspects are still not well understood. It is known that the desaturation of n-9, n-6, and n-3 fatty acids relies on the same enzymes and there is competition among potential substrates for a given desaturase enzyme. Preferential desaturation occurs in the order 18:3n3 > 18:2n-6 > 18:1n-9.4' 5 This suggests that the balance, as well as the absolute amounts, of linoleic acid and linolenic acid in the milk or formula diet may impact on the ability of the infant to synthesize arachidonic acid or docosahexaenoic acid. The desaturase enzymes are also inhibited by their reaction products, such as arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. 4, 5 Presumably, this inhibition provides a mechanism to ensure that the rate of synthesis of these fatty acids is balanced with the needs of new membrane lipid growth. However, disproportionate amounts of the desaturation products of either linoleic or linolenic acid in the diet may interfere with synthesis of fatty acids from the alternate pathway. Decreased tissue levels of arachidonic acid in adult and infant humans or animals fed eicosapentaenoic and docosahexaenoic acids from
Volume 120 Number 4, Part 2
fish oils have been described extensively69; this reduction is explained in part by inhibition of 18:2n-6 desaturation by the n-3 fatty acid desaturation products. Although texts on essential fatty acid metabolism frequently depict the pathways of desaturation and elongation, it must also be remembered that linoleic acid and linolenic acid are readily oxidized for energy. Linoteie acid, and to a much lesser extent linolenic acid, is also found in stored adipose tissue triglycerides and cholesteryl esters. The levels of linoleic acid are also high in many tissue phospholipids. The partitioning of 18:2n-6 and 18:3n-3 among potential pathways of oxidation, direct aeylation into tissue lipids, or desaturation is not yet understood. However, it is reasonable to expect that energy balance may be important in determining the need to oxidize essential fatty acids to provide the energy for basic metabolic and physiologic functions. The positive energy balance associated with normal infant nutrition would presumably facilitate the use of fatty acids for biosynthetic purposes, whereas a negative energy balance may theoretically be expected to direct fatty acids toward oxidation to meet maintenance energy needs. In contrast to linoleic acid and linolenic acid, arachidonic acid and docosahexaenoic acid are rapidly incorporated into tissue phospholipids a n d undergo mitochondrial/~-oxidation to a much lesser extent. This provides a reasonable biochemical explanation for repeated demonstrations that dietary linoleic and linolenic acids are not equivalent to their longer-chain, more unsaturated products. This aspect of fatty acid metabolism is important because it indicates that not all dietary n-6 and n-3 fatty acids are equivalent in either their dietary or metabolic effects. In summary, the assimilation of appropriate amounts of n-6 and n-3 fatty acids in growing infant tissues will depend on the dietary fatty acid supply, tissue desaturase enzyme activity, and energy intake. Human milk provides the infant with linoleic acid and linolenic acid and a preformed source of arachidonie acid and docosahexaenoic acid.l~The arachidonic acid and docosahexaenoic acid provided in human milk is likely to contribute directly to the infant's structural lipid pools of these fatty acids. Infants who receive formulas without these fatty acids must fulfill their tissue requirements exclusively from desaturation of linoleic acid or linolenic acid. Attempts to estimate the requirements for, or efficacy of, linoleic acid and linolenic acid for synthesis of longer-chain metabolites for membrane growth must consider the amounts of these .fatty acids lost to oxidation, energy intake, tissue desaturase enzyme activity, and the dietary fatty acid balance. A small amount of information indicates that dietary saturated and monounsaturated fatty acids also influence the metabolism and tissue levels of n-6 and n-3 fatty acids. 11 Although these studies do not yet al-
Human milk and formula fatty acids
$57
Table. Average levels of the major saturated and monounsaturated fatty acids and linoleic and linolenic acid in mature human milk in the United States* Range of means (% total)
Fatty acids
Pooled mean (% total)
Minimum
Maximum
Median (% total)
8:0 10:0 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3
0.32 1.21 4.79 6.55 21.58 3.23 7.97 35.18 16.15 1.07
0.17 0.89 1.40 3.80 19.80 2.50 7.10 30.70 14.47 0.30
0.60 1.60 6.50 10.20 23.96 3.83 9.00 38.20 18.80 1.85
0.26 1.01 5.08 6.20 21.80 3.29 8.14 35.51 15.80 1.03
*Meanvaluesof individualfatty acid componentsreported in references12 through 20.
low definitive conclusions, it would be unwise to assume that the composition of nonessential fat in infant diets is of no consequence. H U M A N M I L K AND F O R M U L A F A T Fat represents the major portion of energy in most human milk and infant formulas, usually about 45% to 50% of kcal. About 98% of the fat is present as triglycerides, which contain one molecule of glycerol to which three fatty acids are esterified. Human milk triglycerides are present in the core of the milk fat globules, which are held in solution in the aqueous milk environment by a surface layer of phospholipids and proteins, l~ Dispersion of triglycerides in infant formulas is maintained with plant lecithins, sometimes with the aid of monoglyeerides and diglycerides. Human milk contains a large number of fatty acids, including saturated and monounsaturated fatty acids and n-6 and n-3 series polyunsaturated fatty acids. The latter include both the dietary essential fatty acids linoleic acid and linolenie acid, as well as small amounts of longer-chain, more unsaturated n-6 and n-3 fatty acids such as arachidonic acid and doeosahexaenoic acid. These longer-chain, more unsaturated n-6 and n-3 fatty acids usually represent a total of about 1% to 1.5% of the milk fatty acids, roughly equivalent to about 0.5% to 0.75% of kcal. Although there is considerable information on the major fatty acid components of human milk, information on the long-chain polyunsaturated fatty acids is more limited. Average levels of the major saturated and monounsaturated fatty acids and linoleic and linolenic acid in mature human milk in the United States are given in the Table. The data given represent mean values for nine studies of women fol-
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The Journal of Pediatrics April 1992
50-
40
?
~/).
o o
50
20 ~d 4~
%
10
i
_
MCSFA ICSFA LCSFA
_
MFA
_
--~]
_
18:2
18:5
[:i9. t. Saturated and monounsaturated fatty acid and linoleic and linolenic acid profile of mature human milk in the United States: pooled means and minimum and maximum means calculated from data in references 12 through 20. MCSFA, Medium-chain saturated fatty acids, 8:0 + 10:0, 1CSFA, intermediate-chain saturated fatty acids, 12:0 + 14:0; LCSFA, long-chain saturated fatty acids, 16:0 + 18:0;MFA, monounsaturated fatty acids 16:1 + 18:1; 18:2, linoleic acid (18:2n-6); 18.'3, linolenic acid (18:3n-3).
lowing unrestricted diets that were published within the past 10 years. ~22~The levels of medium-chain fatty acids were low (1% to 2%), whereas palmitic acid (16:0) and oleic acid (18:1) were major components, representing about 20% to 24% and 30% to 38% of milk fatty acids, respectively. The mean levels of linoleic acid (18:2n-6) and linolenic acid (18:3n-3) were about 16% and 1%, respectively (Table). The fat in North American infant formulas is a blend of one or more vegetable oils, with the addition of oleo oils in the products of some manufacturers. Corn oil and soybean oil contain about 50% to 60% linoleic acid and are well absorbed; soybean oil contains about 6% to 9% linolenic acid and is the usual oil of choice to provide this fatty acid. These vegetable oils contain low levels of oleic acid (20% to 25%) and palmitic acid (10% to 12%), and they must be blended with other oils if a saturated/m~176176 urated fat profile resembling human milk is desired (Fig. 1). In Fig. 1 the saturated fatty acids have been grouped as the medium-chain fatty acids 8:0 and 10:0, the intermediatechain saturated fatty acids 12:0 and 14:0 (which are synthesized by the mammary glandl9), the long-chain saturated fatty acids 16:0 and 18:0, and the monounsaturated fatty acids 16:1 and 18:1. Oleo oils, derived from beef fat, and vegetable oils, such as palm oil, contain palmitic acid. The palmitie acid in these oils, however, is esterified predominantly to the sn-l,3 car-
bon atoms of the 3 carbon glycerol backbone of the triglyceride. I~ In contrast, more than 70% of the palmitic acid in human milk is esterified to the center, sn-2 glycerol carbon. 1~ This is important because the products of intestinal hydrolysis of fat are sn-2 monoglycerides and two free fatty acids released from the sn-1 and sn-3 positions.21 The coefficiency of absorption of free palmitic acid is low in very small preterm infants.22, 23 Consequently, some manufacturers prefer to use coconut oils or medium-chain triglycerides as a source of well-absorbed saturated fatty acids for infant formulas. The constitutive milk lipase, known as bile salt-stimulated lipase, is believed to hydrolyze the sn-2 monoglycerides left after triglyceride hydrolysis by gastric and intestinal lipases. 24 The action of this enzyme should release 16:0 from the sn-2 position of human milk. The coefficiency of absorption of human milk fat is high and apparently not limited in any way by free 16:0. Human milk contains a variety of n-6 and n-3 fatty acids with 20 or 22 carbon atoms. Detailed information on the levels of these fatty acids can be found in subsequent articles in this supplement. Usually arachidonic acid is the major long-chain n-6 fatty acid and represents about 0.5% to 0.7% of total milk fatty acids (Fig. 2, B). Docosahexaenoic acid is the major longer-chain n-3 fatty acid in human milk (Fig. 2, D), even when the mother's dietary intake of eicosapentaenoic acid is higher than that of docosahexaenoic ac-
Volume 120 Number 4, Part 2
Human milk and formula fatty acids
$5 9
A: Linoleie Acid (18:2n-6)
24
18
l
12
l I g 1.4
B: Araehidonie Add (20:4n-6)
1.2 1.0 0.8
0.2 0.0
Fig. 2. Percent of total human milk fatty acids. A, Linoleic acid (18:2n-6); B, arachidonic acid (20:4n-6). Values modified from references given in parentheses represent mean values _+SD, with the exception of reference 31, for which the median value was reported and is reproduced here. In some cases, estimates of variance were not published. id. 25 Commercially available infant formula prepared from vegetable oils with or without oleo oils do not contain significant amounts of these fatty acids. The appropriateness of adding these longer-chain polyunsaturated fatty acids to infant formulas is the subject of subsequent articles in this supplement. Fat is one of the most variable nutrients in human milk, varying with the stage of lactation, among different women, with the time of day, and in numerous other factors. ~ The variability in the fatty acids of mature human milk poses
considerable problems in attempting to establish optimal patterns of fatty acid intake from the infant fed human milk. Fig. 2 shows a comparison of the major n-6 and n-3 fatty acids, linoleic acid (18:2n-6), arachidonic acid (20:4n6), linolenic acid (18:3n-3), and docosahexaenoic acid (22:6n-3), reported in studies of mature human milk from women following unrestricted diets in different regions of the world or from groups with diverse cultural backgrounds.12, zs, 25-33 Some of the articles in this supplement provide evidence that these wide variations in human milk
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The Journal of Pediatrics April 1992
C: Linolenie Acid (18:3n-3)
2.5
2.0
1.5
1.0
0.0
g
-
g
g
D: Docosahex~enoie ANd (22:6n-3)
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2.0-
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xxx • N'x)< •215 XNX X'>oo< XNX NMN X )~ .x, xxx X• X~'NX
0.0
xz,x
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g
o=
~
,
tn
~2
Fi9. 2, Cont. Percent of total human milk fatty acids. C, Linolenic acid (18:3n-3); D, docosahexaenoic acid (22:6n-3). Values modified from references given in parentheses represent mean values _+ SD, with the exception of reference 31, for which the median value was reported and is reproduced here. In some cases, estimates of variance were not published.
fatty acids are at least partly the result of differences in the mother's dietary fat intake. H u m a n milk fatty acids in North America may well reflect changes in dietary fat toward increased use of corn, soybean, and other vegetable oils 34 that provide h i g h amounts of linoleic acid and linolenic acid, in response to recommendations from various agencies concerned with morbidity and death caused by atherosclerotic diseases. Extrapolation of infant fatty acid requirements from in-
formation on the amounts received by infants fed human milk is limited by several factors other than the variability in human milk. Subsequent articles in this supplement debate which fatty acids are essential nutrients for the newborn infant, the importance of fatty acid balance, and the possible influence of saturated or monounsaturated fatty acids on lipid metabolism. Recommended intakes calculated as the mean intake from human milk plus an estimate of variance (usually 2 SDs, or about 25% to 30% mean
Volume 120 Number 4, Part 2
value) to cover the estimated requirement of 97.5% of the population will also, by definition, define 97.5% of all h u m a n milk as deficient. Finally, if the total fat intake is maintained, high amounts of one fatty acid must be accompanied by low a m o u n t s of a n o t h e r fatty acid. In the absence of scientific reasons to consider otherwise, the average or median levels of fatty acids in h u m a n milk, with regard to all fatty acids, provide an estimate most likely to cover the lipid nutrient requirements of infants.
REFERENCES 1. Tinoco J. Dietary requirements and function of c~-linolenic acid in animals. Prog Lipid Res 1982;21:1-45. 2. u WK, Clemans GW, Hutchinson ML. Essential fatty acid deficiency in humans. Prog Lipid Res 1981;19:187215. 3. Innis SM. Essential fatty acids in growth and development. Prog Lipid Res 1991;30:39-103. 4. Brenner RR. The oxidative desaturation of unsaturated fatty acids in animals. Moll Cell Biochem 1974;3:41-52. 5. Brenner RR. Nutritional and hormonal factors influencing desaturation of essential fatty acids. Prog Lipid Res 1981; 20:41-7. 6. Hwang DH, Boudreau M, Chanmugam P. Dietary linoleic acid and longer-chain n-3 fatty acids.: comparison of effects on arachidonic acid metabolism in rats. J Nutr 1988;118:42737. 7. Arbuckle LD, Rioux FM, Mackinnon M J, Hrboticky N, Innis SM. Response of n-3 and n-6 fatty acids in brain, liver and plasma of piglets fed formula to increasing, but low, levels of fish oil supplementation. J Nutr 1991;121:1536-47. 8. Carlson S, Cooke RJ, Rhodes PG, Peeples JM, Werkman SH, Tolley EA. Long-term feeding of formulas high in linolenic acid and marine oil to very low birth-weight infant: phospholipid fatty acids. Pediatr Res 1991;30:404-12. 9. Popp-Snijders C, Schouten JA, van Blitterswijk W J, van der Veen EA. Changes in membrane lipid composition of human erythrocytes after dietary supplementation of (n-3) polyunsaturated fatty acids: maintenance of membrane fluidity. Biochim Biophys Acta 1986;854:31-7. 10. Jensen RG. Lipids in human milk: composition and fat-soluble vitamins. In: Lebenthal E, ed. Textbook of gastroenterology and nutrition in infancy. 2rid ed. New York: Raven Press, 1989:157-208. 11. Garg ML, Wierzbicki AA, Thompson ABR, Clandinin MT. Dietary saturated fat level alters the competition between ~-linolenic acid and linoleic acid. Lipids 1989;24:334-9. 12. Bitman J, Wood L, Hamosh M, Mehta NR. Comparison of the lipid composition of breast milk from mothers of term and preterm infants. Am J Clin Nutr 1983;38:300-12. 13. Borschel MW, Elkin RG, Kirkse A, et al. Fatty acid composition of mature human milk of Egyptian and American women. Am J Clin Nutr 1986;44:330-5. 14. Clark RM, Ferris AM, Fey M, Brown PB, Hundrieser KE, Jensen RG. Changes in the lipids of human milk from 2 to 16 weeks postpartum. J Pediatr Gastroenterol Nutr 1982; 1:311-5. 15. 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;41:787800.
Human milk and formula fatty acids
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16. Harris WS, Connor WE, Lindsey S. Will dietary ~o-3fatty acids change the composition of human milk. Am J Clin Nutr 1984;40:780-5. 17. Putnam JC, Carlson SE, Devoe PW, Barness LA. The effect of variations in dietary fatty acids on the fatty acid composition of erythrocyte phosphatidylcholine and phosphatidylethanolamine in human infants. Am J Clin Nutr 1982;36:106-14. 18. Specker BL, Wey HE, Miller D. Differences in fatty acid composition of human milk in vegetarian and nonvegetarian women: long-term effect of diet. J Pediatr Gastroenterol Nutr 1987;6:764-8. 19. Thompson BJ, Smith S. Biosynthesis of fatty acids by lactating human breast milk epithelial cells: an evaluation of the contribution to the overall composition of human milk fat. Pediatr Res 1985;19:139-43. 20. Wang C-S, Illingsworth DR. Lipid composition and lipolytic activities in milk from a patient with homozygous familial hypobetalipoproteinemia. Am J Clin Nutr 1987;45:730-6. 21. Carey MC, Small DM, Bliss CM. Lipid digestion and absorption. Ann Rev Physiol 1983;45:651-77. 22. Chappell JE, Clandinin MT, Kearney-Volpe C, Reichman B, Swyer PW. Fatty acid balance studies in premature infants fed human milk or formula: effect of calcium supplementation. J Pediatr 1986;108:439-47. 23. Jensen C, Buist NRM, Wilson T. Absorption of individual fatty acids from long-chain or medium-chain triglycerides in very small infants. Am J Clin Nutr 1988;43:745-51. 24. Hernell O, Blackberg L, Bernback S. Digestion of human milk fat in early infancy. Acta Paediatr Scand Suppl 1989;351:5762. 25. Innis SM, Kuhnlein HV. Long-chain n-3 fatty acids in breast milk of Inuit women consuming traditional foods. Early Hum Dev 1988;18:185-9. 26. Boersma ER, Offringa P J, Muskiet FAJ, Chase WM, Simmons IJ. Vitamin E, lipid fractions, and fatty acid composition of colostrum, transitional milk, and mature milk: an international comparative study. Am J Clin Nutr 1991 ;53:1197-204. 27. Gibson RA, Kneebone GM. Fatty acid composition of human colostrum and mature breast milk. Am J Clin Nutr 1981; 34:252-7. 28. Harzer G, Haug M, Dieterich I, Gentner PR. Changing patterns of human milk lipids in the course of lactation and during the day. Am J Clin Nutr 1983;37:612-21. 29. Jansson L, Akesson B, Holmberg L. Vitamin E and fatty acid composition of human milk. Am J Clin Nutr 1981;34:8-13. 30. Kneebone GM, Kneebone R, Gibson RA. Fatty acid composition of breast milk from three racial groups from Penang, Malaysia. Am J Clin Nutr 1985;41:765-9. 31. Koletzko B, Mrotzek M, Eng B, Bremer HJ. Fatty acid composition of mature human milk in Germany. Am J Clin Nutr 1988;47:954-9. 32. Muskiet FA, Hutter NH, Martini IA, Jonxis JH, Offringa P J, Boersma ER. Comparison of the fatty acid composition of human milk from mothers in Tanzania, Curacao and Surinam. Hum Nutr Clin Nutr 1987;41:149-59. 33. van Beusekom CM, Martini IA, Rutgers HM, Boersma ER, Muskiet FAJ. A carbohydrate-rich diet not only leads to incorporation of medium-chain fatty acids (6:0-14:0) in milk triglycerides but also in each milk phospholipid subclass. Am J Clin Nutr 1990;52:326-34. 34. Stephen AM, Wald NJ. Trends in individual consumption of dietary fat in the United States, 1920-1984. Am J Clin Nutr 1990;52:457-69.
