The nutritional implications of replacing bovine milk fat with vegetable oils in infant formulas are evaluated in this review. Milk fat contains higher levels of trans fatty acids (FAs; a negative health factor), higher levels of conjugated linoleic acid (a positive health factor), higher levels of short-chain FAs (neutral effect), and higher levels of phospholipids and sphingolipids (possibly positive, but further investigation is necessary). Palmitate of vegetable oil is less well absorbed than palmitate of milk fat because of its different location on the glycerol backbone. Nonabsorbed palmitate can be excreted as a calcium soap, and there are suggestions that this may compromise overall calcium balance. Vegetable oils contain higher levels of plant sterols, and these have a neutral effect on cholesterol levels in infants. Infants fed vegetable oil–based formulas do not receive dietary cholesterol and must synthesize it. Studies examining the long-term effects of neonatal dietary cholesterol level on cholesterol metabolism in adult life have yielded mixed results. In some studies, infants fed higher levels of cholesterol had better ability to regulate plasma cholesterol levels in later life, but in others this was not the case. In conclusion, there are presently no contraindications for replacing milk fat with vegetable oils in infant formulas. Because there is a plethora of different bioactive compounds in bovine milk fat and vegetable oil whose biologic roles are still under investigation (several of which are described herein), this issue should continue to be closely monitored.
FATS IN INFANT FORMULAS
Infant formulas generally contain approximately 50% energy as fat. This lipid is of animal or vegetable origin and is generally a combination of different fats, mixed in proportion to mimic the FA composition of human breast milk. Traditionally, formulas have contained substantial quantities of milk fat, but more recently they have tended to be richer in vegetable oils. The main vegetable oils used are coconut oil, corn oil, soybean oil, palm olein, palm kernel oil, palm oil, high oleic safflower oil, peanut oil, and, in Europe, low–erucic acid rapeseed oil. Vegetable oil–based formulas can contain up to 4% residual milk fat, even if milk powder from skimmed milk is used as a protein source.
When milk fat is replaced with vegetable oils in formulas, certain biologically active dietary substances are different. These include minerals and ions (1), antioxidants, water-and fat-soluble vitamins (2), contaminants (3), flavonoids, hormones (4), growth factors, nucleotides (5), carnitine (6), enzymes (7), defense agents (8,9), oligosaccharides (10), ether lipids (11), and phytosterols (12).
In this article, the advantages and drawbacks of milk fat (9, 11–14) versus vegetable oil use in infant formula are evaluated regarding their effects on nutritional quality. The main discussion focuses on differences in FA composition, triacylglycerol (TAG) structure, phospholipid (PL) and sphingolipid content, and cholesterol content. Comparisons to breast milk-feeding are included, but it should be emphasized that breast milk-feeding provides the infant with a plethora of additional nutritional and psychological support. Although not the subject of this review, for the interested reader, there are numerous reviews on the potential importance of including long-chain FAs such as arachidonic acid (AA) and docosahexaenoic acid (DHA) in the diets of pregnant mothers and preterm and term infants (15–18).
Fat mixes used for infant formulas are designed to yield an FA profile as close as possible to that of human milk, including the two essential FAs, linoleic acid (C18:2 n-6) and α-linolenic acid (C18:3 n-3). Nevertheless, between the two main types of fat mixes, (i.e., vegetable fat– and milk fat–based) there are still some potentially important differences. Some FAs such as short-chain FAs (SCFAs) are present in cow's milk fat at much higher levels than in human milk and vegetable oils. Other FAs are present in bovine and human milk but are absent from vegetable oils—for example, conjugated linoleic acid (CLA). The currently available vegetable oil mixes also contain more C12:0, more C18:1 n-9, slightly more C18:2 n-6 and C18:3 n-3, and less C14:0 and C18:0 than the typical milk fat–based formula (19). Bovine fat also contains odd-chain FAs such as C11:0, C13:0, C15:0, C17:0, C19:0, C21:0, C23:0, and C25:0 (20), the physiological importance of which is unknown. In addition to FA compositional differences between milk and vegetable sources, there are differences in positional distribution on the glycerol backbone that can affect FA absorbability (see section on Triacylglycerol Structure). Between milk fat and vegetable oil mixes, there may also be differences in the types and quantity of bioactive N-acyl ethanolamines and related lipids that bind to CB1/2 receptors, or potentiate the activity of CB-receptor–binding lipids (21,22).
Short-Chain Fatty Acids
Short-chain FAs are consumed in milk (23). Milk fat contains more SCFAs than vegetable oils, including up to 10% butyrate (C4:0). The C4:0 is present in 30% of the TAG in milk, typically in the sn- 3 position, associated with two long-chain FAs (24). The content of the final formula depends on the amount of milk fat used, but typical values may be 3.2% C4:0 and 1.7% C6:0, in comparison with vegetable oil–based formulas at the much lower levels of 0.04% C4:0 and 0.2% C6:0 (Table 1).
The SCFAs in milk fat may be an easily metabolizable energy source for the infant. However, no other claims regarding health benefits of dietary SCFA can be made at this time because of the lack of data from controlled studies. Butyrate can be produced by colonic bacteria and is known to have important biologic effects in the colon (25). However dietary preformed butyrate is absorbed in the stomach and upper intestine of mammals, and little if any can be expected to reach the colon, based on rapid release of SCFAs from TAG by lingual and gastric lipases, followed by pancreatic lipase in the upper small intestine (26–29). It is assumed that humans, similar to other mammals, metabolize C4:0 as described, but this has not been shown definitively. Also, it is not known whether dietary butyrate could have local effects on the gastric and upper intestinal wall before absorption. Care must be taken in interpreting results of butyrate studies: Most in vitro and some in vivo studies have used chemical derivatives of butyrate, butyrate salts, and tributyrate.
Medium-Chain Fatty Acids
As shown in Table 1, vegetable oil mixes prepared from coconut oil used in infant formula can have higher levels of C12:0 (approximately 11%) than is found in bovine milk formulations. C12:0 and monoacylglycerols (MAGs) containing C12:0 (monolaurin; formed from TAG containing C12:0 during digestion) are known to have antimicrobial actions against Gram-negative bacteria (30). In vitro, C12:0 and monolaurin (alone or mixed into infant formulas) were found to have bacteriocidal properties against Helicobacter pylori, the bacterium associated with chronic superficial gastritis and peptic ulcer disease (31). Monoacylglycerols rich in monolaurin synthesized from coconut oil have also been shown to have antimicrobial activity against the dairy pathogen, Listeria monocytogenes, in vitro (32). Coconut oil–derived MAGs have been found to be more listericidal than monolaurin itself, and more listericidal than bovine milk fat–derived MAGs. The aspirated stomach contents from infants fed formulas containing mixtures of bovine milk fat, medium-chain TAGs, corn oil, and coconut oil have been found to reduce titers of enveloped virus and to kill Staphylococcus epidermidis and Escherichia coli(33,34). There were not sufficient numbers of infants in these studies to test whether the type and quantity of fats present in the infant formula affect killing activity.
Polyunsaturated Fatty Acids
Vegetable oils are rich sources of the nutritionally essential polyunsaturated FAs (PUFAs), 18:2 n-6 and 18:3 n-3. Because of the microbial hydrogenation occurring in the rumen, fats of bovine origin have a notoriously poor content of polyunsaturates. For this reason, infant formulas based on bovine fats must include sufficient vegetable oils to provide adequate essential FA intake (Table 1). The long-chain PUFAs, AA and DHA, are not found in commonly used vegetable oils, nor are they found in bovine fat, although they are present in human milk. This issue is the subject of extensive reports in the literature, and the reader is directed to recent relevant reviews (15–18).
Branched-Chain Fatty Acids
Milk fat contains approximately 3.1% branched-chain FAs (BCFAs), whereas there is only 1.5% in human milk and only traces in vegetable oils (20,35). Typical BCFAs in bovine milk fat are isobutyrate, isovalerate, isocaproate, and isocaprylate (35). These FAs have been found in both the small and large intestine of adult humans (36). They may be better absorbed when present on the sn-2 position of the glycerol backbone (37) and are then metabolized by hepatic α or ω oxidation in peroxisomes, followed by mitochondrial β oxidation. The benefits or lack thereof of BCFAs in cow's milk are not known in healthy infants.
Conjugated Linoleic Acid
Conjugated linoleic acids (9 cis, 11 trans 18:2, and other isomers) have been the focus of intense interest in the scientific community (38). They are present in bovine and human milks, but the levels in vegetable oils are very low (39–44). Human milk is reported to contain 3.8 ± 0.3 mg CLA/g fat, originating mainly from the dairy products consumed by the mother. In cow's milk, these FAs are products of partial biohydrogenation of linoleic acid in the rumen, and their content has been found to be 3.38 to 6.39 mg/g fat (41). In infant formulas containing animal fat, the level is 2.28 ± 0.04 mg/g fat, and in infant formulas containing vegetable oil the level is 0.51 ± 0.08 mg/g fat (44).
Conjugated linoleic acids have been shown to reduce very effectively the incidence of chemically induced skin and forestomach cancers in mice, mammary tumors in rats, and numerous other cancers (43). They have similar effects as a free FA and as a TAG in cancer models. They have also been found to reduce low-density lipoprotein (LDL) cholesterol and aortic atherosclerosis in hamsters. Some researchers, but not others, also have found CLAs to have antioxidant properties. Recently, it was shown that CLAs can be elongated to 20 carbons and desaturated to derivatives with three double bonds in rats and three to four double bonds in lambs and incorporated into liver PLs. Work is in progress to determine whether novel eicosanoid products may be formed from the conjugated diene 20:4 (43).
Trans Fatty Acids
Hydrogenated vegetable oils are not used in infant formulations, but trans fatty acids (tFAs) have been found in products sold in the United States (45), Canada (46), and France (47) (Table 2). In milk fat–based formulas, the trans isomer of 18:1 n-9 originates from biohydrogenation of lipids incorporated into milk fat. Summer milk is richer in t18:1 than winter milk. Because of this natural occurrence, milk fat–based formulas can contain substantially higher levels of total tFAs (mainly t18:1) than vegetable oil–based formulas.
In vegetable oils, low amounts of trans isomers of PUFA can be produced through high-temperature deodorization procedures (48–50). Deodorization results in organoleptically acceptable oils by eliminating peroxides and undesirable odors. If mild deodorization conditions are used, isomerization is minimized.
Infants also receive tFAs in breast milk. Mothers obtain their tFAs through consumption of dairy products and partially hydrogenated oil products such as margarine (51). The level of tFAs in human milk varies with the amounts obtained by the mother through diet. In Canada, human milk contains an average of 7.2% total tFAs—up to a 17% maximum (51). Koletzko et al. (52) found an average of 4.15% in German mothers' milk. Thus infants receiving breast milk may have an intake of tFAs equal to or even greater than that ingested by milk-fat-based formula-fed infants.
The significance of this requires further study. High plasma tFA content (derived from breast milk) has been negatively correlated with levels of AA and other long-chain n-6 FAs (excluding linoleic acid) in the plasma PL, TAG, and cholesterol esters of premature infants (53). Similarly, in children 1 to 15 years of age, there is a negative correlation between dietary tFA content and plasma PL AA levels (54). Arachidonic acid is thought to be important for infant tissue growth and development, and it would be prudent to limit tFA in infant diets. In newborn mice and rats, tFA consumption has been associated with impaired postnatal weight gain (54).
When milk fat is replaced with the major currently available vegetable oils, formulas contain less SCFAs and C18:0, and higher C12:0 and C18:1. A product containing only vegetable oil can be adjusted to produce an FA profile similar to that found in bovine milk fat–based products, except that levels of SCFAs (C4:0 and C6:0) are lower, and levels of C12:0 are higher. There is no obvious need to be concerned about the lower levels of SCFAs in vegetable oil, because human breast milk also contains very low levels of SCFAs (12). Functional benefits of higher amounts of SCFAs in bovine milk are not known. The higher levels of C12:0 in vegetable oil could be an advantage because of increased bacteriocidal activity, but this premise has not been adequately evaluated. If vegetable oils replace milk fat, there would be a slight reduction in the levels of tFAs. We would not expect that this small reduction in tFAs would have a significant health benefit for the infant. Formulas containing bovine milk fat contain approximately five times more CLAs than vegetable oil–based infant formulas. Based on extrapolations from rodent models, it is possible that the amount of CLA in milk fat (∼1%) may be sufficient to suppress mammary cancer and to lower cholesterol, although this has never been tested in humans. Health benefits have been attributed to butter in comparison with vegetable oils (55–59), but because vegetable oils and milk fat have many differences other than CLA, we cannot unequivocally conclude that CLA is the active ingredient in these studies.
Differences Between Milk Fat and Vegetable Oils
In milk fat TAG, saturated FAs (SFAs) and linoleic acid are located on the sn-2 position of the glycerol backbone, and SCFAs are preferentially located on the sn-1 (3) positions. Oleic acid is equally distributed among all three positions (60, 61).
In corn, palm, and soy oils, the main oils used in infant formulas, SFAs are located mainly in sn-1 (3) positions of the TAG and linoleic is in sn-2. In vegetable oils, only 5% to 20% of palmitate is in the sn-2 position (62–65), whereas in human milk fat, approximately 70% of palmitate is in the sn-2 position (66) and in bovine milk fat, 40% is in sn-2 (67). Oleic acid may be equally distributed on all three positions (corn, soy oils) or preferentially located in sn-2 (palm oil, palm olein). Linolenic acid is equally distributed among the three carbons (soy oil).
Thus, the main difference between milk fat and vegetable oils commonly used in infant formula is that long-chain SFAs such as palmitate are located in the sn-2 position of the glycerol in milk fat and in the external positions (1 and 3) in vegetable oils. It is possible to rearrange or randomize the fatty acyl positional distribution by esterification, chemical means, and genetic manipulation, if so desired (68).
Consequences of These Differences
Most clinical studies have established that fats are better absorbed from human milk than from formulas (63, 69). Many factors affect fat absorption in infants, such as types of FA, types of proteins, mineral content, presence or absence of bile salt stimulated lipase, and milk fat globule membrane (MFGM) components (61,70). Another important factor known to affect absorbability is FAs' positional distribution on glycerol (71). In human milk and in infant formulas, palmitate (16:0) is the major SFA representing 22% to 26% of total FA. As mentioned, palmitate is predominantly located on the sn-2 position in human and bovine milk fat but is predominantly located on the sn-1 (3) positions in palm oil. Palmitate on the sn-1 (3) is more poorly absorbed than palmitate on the sn-2. During normal digestion, TAG hydrolysis by sn-1 (3) specific pancreatic lipase, liberates free FA in the gut. Free palmitate is not well absorbed, because it has a high melting temperature (63°C) and is rapidly converted into insoluble hydrated acid soaps at the pH of the intestine (5.8–6.5). If sufficient calcium is present in the intestinal lumen, calcium soaps can also be formed. These soaps have very high melting points that limit their absorption and result in their recovery in feces. Lower absorption of palmitate from TAG positions 1 and 3 has been shown in rats (63,72,73), term babies (74–76), and preterm neonates (69,77).
In a crossover study by Carnielli et al. (69) two formulas with sn-2 palmitate of either 13% or 70% were fed to preterm infants. Although total fat absorption was not significantly different, palmitate absorption increased respectively from 51% with the lower levels of sn-2 palmitate to 73% with the predominantly sn-2 palmitate formula. A similar result was found in another study with preterm infants (78). In a rat study, cow's milk with 40% palmitate in the sn-2 position was shown to result in an intermediate absorption of palmitate, compared with 5%sn-2 palmitate and 79%sn-2 palmitate diets (73). Nelson et al. (76) calculated that the loss of palmitate in feces from formulas containing palm olein (9%sn-2 position) would be equivalent to approximately 2.0 kcal/kg body weight per day. This amount could be easily made up in a 5-kg infant by feeding the infant an extra 10 to 15 ml formula per day.
In a recent study in piglets, it was shown that ingestion of a formula having 32%sn-2 palmitate leads to lower levels of chylomicron TAG AA and DHA compared with ingestion of a formula having 4.2%sn-2 palmitate (palm olein), suggesting that positional distribution of palmitate affects not only absorption of palmitate but also absorption of long-chain n-6 and n-3 PUFAs (79).
The type and positional specificity of fat present in formula affects not only fat absorption but also calcium absorption and retention in infants. Thus, fecal fat excretion has been closely correlated with fecal calcium excretion (69,75,76,80). Williams et al. (81) demonstrated that calcium absorption and retention were lower in infants fed formulas containing proportionately more SFA (palmitate and stearate) and less oleate. Similarly, Nelson et al. (76,80) found that infants fed formulas containing more palmitate from palm olein had less calcium absorption and retention and thus more insoluble and nonabsorbable calcium soaps in the gut. In two rather elegant studies, infants fed formulas with palmitate primarily in the sn-2 position had decreased fecal calcium excretion, but increased urinary calcium excretion (69,75). The increase in urinary calcium showed a trend in term infants (75) and was statistically significant in preterm infants (69). In both these studies, the quantity of palmitate on the sn-2 position did not significantly affect overall calcium retention, but there was a trend toward increased calcium retention in both term and preterm infants.
Infants fed formulas have harder stools than infants fed human milk, and stool hardness has been positively associated with the quantity of calcium soaps of FA excreted in the feces (82). Feeding human milk or formula enriched with palmitate in the sn-2 position to term infants results in softer stools by reducing the amount of FA soaps. This is an important issue, because constipation is not just uncomfortable but can lead to life-threatening complications in preterm infants.
Triacylglycerol structure can influence cholesterol metabolism in various species. Compared with the original fats, randomly interesterified lard, tallow, peanut oil, and butter had much reduced atherogenicity in rabbits and humans (83,84). However, in a recent study in adult humans fed palm oil (18% palmitic in sn-2) or enzymatically modified palm oil (65% palmitic in sn-2), only very minor effects were seen on total and LDL cholesterol (65). In two other studies, there was also little effect of positional distribution of palmitate and stearate on fasting cholesterol levels (85,86). It is possible that TAG structure has a greater effect on cholesterol levels in neonates than adults, but data in humans are lacking so far. Larger effects of palmitate position on cholesterol level have been previously noted in piglets (87) (see later discussion), but it is difficult to extrapolate across species.
Overall, we expect palmitate in vegetable oil fat–based formula to be less well absorbed than palmitate in bovine milk–based formula because of the lesser proportion located on the sn-2 position. The loss of energy from this decreased absorption is minor and can be expected to be easily compensated by the child with the consumption of a few more milliliters of formula per day. Whether palmitate malabsorption has significant repercussions on calcium balance in premature and term infants needs confirmation. For premature infants, the tendency to enhanced calcium retention and reduced stool hardness after consumption of formulas having more palmitate on the sn-2 position (as in milk fat or modified vegetable oils) could have important clinical advantages. Some vegetable oil-based formulas for premature infants (having less palmitate on the sn-2 position) are thus supplemented with more calcium than formulas for term infants. The positional distribution of palmitate on the TAG could affect plasma cholesterol levels and subsequent palmitate metabolism, but data on infants are not available.
PHOSPHOLIPIDS AND SPHINGOLIPIDS
Milk fat is complex in its composition and properties. Bovine milk contains approximately 4% fat in the form of globules. The core of these globules contains nonpolar lipids such as TAG, cholesteryl esters, and esters of vitamins. This core is surrounded by a loose membrane layer (the MFGM), which comprises 1% to 2% of the total milk lipids and also contains proteins, enzymes, and other components. The lipids that make up the MFGM (Table 3) include phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phosphatidylinositol, phosphatidylserine, and other sphingolipid or PL components in trace amounts, such as gangliosides, sphingosine, and ceramides (9). The functionality and possible nutritional roles of PLs and sphingolipids have been reviewed previously (9,88).
Milk-based formulas are expected to have higher levels of these compounds than vegetable oil–based formulas which would only contain trace amounts coming from residual milk fat inclusion from skimmed milk powder.
Specific functionality may exist for the PLs in milk. In a recent study, rats were fed diets containing either 10.4 or 1.7 μmol PL per gram of diet. The animals receiving the higher levels of PL demonstrated improved host defense against subsequent infection with L. monocytogenes 4B (89). In a recent double-blind clinical study, preterm infants fed formula supplemented with egg PL (providing 0.4% AA, 0.13% DHA, and seven times more esterified choline than the control) had less stage II and III necrotizing enterocolitis (90). It is possible that the choline component influenced intestinal surfactant production and levels of the vasodilatory neurotransmitter acetylcholine, and enhanced immature intestinal functions. However, the added AA and DHA could also have affected intestinal functioning by modulation of mucosal membrane FAs and eicosanoids. Whether the same benefit of adding egg PL would be seen in mature infants is also not known. Dipalmitoyl phosphatidylcholine, present in bovine milk, is reported to protect the gastric mucosa against acidic secretions in rats (91). Phosphatidylinositol is well established as a source of the two second messengers, diacylglycerol and inositoltriphosphate, when acted on by phospholipase C. Whether such hydrolysis occurs in the gut or milk is uncertain. Ether PLs, which have numerous biologic properties (tumor cytotoxic effects) are present in bovine milk fat-neutral lipids at the 0.01% level and in human milk at the 0.2% level (92). It is not known whether the low levels of ether lipids in milk fat have biologic properties (9).
Sphingomyelin consists of a sphingosine backbone (16–19 carbons), an N-acyl chain (16–24 carbons with 0–1 double bonds), and a 1-phosphorylcholine head group (Fig. 1) (93,94). It represents 0.2% of total milk fat. In cells, it is associated with cholesterol in membranes and imparts a structural rigidity to membranes. The nutritional and anticancer properties of dietary sphingomyelin have been studied by Dillehay et al. (95), Schmelz et al. (96,97), and Merrill and coworkers (98,99). In one study, CF1 mice received the inducing agent 1,2-dimethylhydrazine followed by diets supplemented with 0% to 0.1% (wt/wt) milk sphingomyelin (95). The number of aberrant colonic crypts per foci (early indications of colon carcinogenesis), and the proportion of colonic malignant adenocarcinomas (larger tumors that invade through the muscularis mucosa) relative to adenomas (noninvasive) was significantly lower in mice fed 0.05% to 0.1% sphingomyelin compared with control subjects. In longer term studies, dietary milk-derived sphingomyelin had no effect on colon tumor incidence or multiplicity; however, up to 31% of the tumors in mice fed sphingomyelin were adenomas, whereas all the tumors in mice fed the diet without sphingomyelin were adenocarcinomas (96). These findings demonstrate that milk sphingomyelin suppresses the appearance of more advanced malignant tumors as well as early markers of colon carcinogenesis. Sphingomyelin is consumed in dairy products, meat, fish, eggs, and vegetables and consumption has been estimated to be 300–400 mg sphingomyelin consumed per day (98). Thus, consumption of sphingomyelin affects the behavior of colonic cells, and sphingomyelin may be another important class of nutritional modulators of carcinogenesis and may contribute to the cancer-preventive effects of some foods. Clearly, more work is needed to demonstrate a nutritional benefit of sphingomyelin in the diet.
Sphingolipid derivatives (sphingosine, ceramide, gangliosides, cerebrosides, sulfatides) are known to be present in human milk and cow's milk in trace amounts. Intracellularly, they are formed in response to growth factors that stimulate the activity of enzymes involved in sphingolipid catabolism. They have a plethora of bioactive properties that have been intensively studied in recent years, predominantly for their roles in normal and malignant cell growth regulation.
Cleavage of the 1-phosphorylcholine group of sphingomyelin by acidic, neutral, and alkaline sphingomyelinases in the gut or intracellularly results in the formation of ceramides (N-acyl sphingosine) (100,101). There is also the possibility that the bioactive molecule sphingosylphosphorylcholine (deacylated sphingomyelin) (102,103) is formed during digestion (100,104). In in vitro studies, ceramides have numerous bioactive properties including induction of apoptosis (programmed cell death). Butter contains higher levels of ceramides than vegetable oils.
Intracellularly, sphingosine can be formed by deacylation of ceramide. Both ceramide and sphingosine can also be formed during the de novo synthesis of sphingomyelin. The phosphorylated derivative of sphingosine (sphingosine-1-phosphate) is a signaling molecule that releases calcium intracellularly, inhibits protein kinase C in some cell types, and either inhibits or stimulates cell proliferation, depending on the cell type. The nutritional role of dietary ceramide, sphingosine, and related compounds ingested intact or formed in the gut is not known.
N-acyl sphingosines with a polar carbohydrate head group (attached by a glycoside bond) are called neutral glycosphingolipids. Gangliosides are acidic glycosphingolipids, which in addition to the neutral sugar, also contain one or more sialic acid residues (105). In human colostrum, the major ganglioside is GD3; in mature human milk, GM3 predominates. In bovine milk, GD3 is the major ganglioside, and GM3 is the next most abundant. Human and bovine milk gangliosides have been shown to inhibit enterotoxigenic–enteropathogenic E. coli from adhering to intestinal cells in vitro (106) and to bind to cholera toxin, H. pylori, Campylobacter pylori, rotaviruses, and human immunodeficiency virus-1 surface envelope glycoprotein gp120 (107,108). The ingestion of bovine milk infant formulas supplemented with porcine brain–derived gangliosides was reported to reduce fecal levels of E. coli in premature infants at 7 days postnatal age and to increase fecal counts of bifidobacteria at 30 days after birth (109). The speculation was that ganglioside GD3 could affect proliferation, activation, and differentiation of intestinal immune cells, because GD3 has been shown to be involved in T-lymphocyte activation.
Choline has recently been officially recognized to be an essential nutrient by the Food and Nutrition Board of the U.S. National Academy of Sciences. Dietary daily reference intakes are 550 mg for adult men, 425 mg for adult women, 450 mg for pregnant women, and 550 mg for lactating women. Lower amounts for infant and children have been recommended. Tolerable upper daily limits are 1 g for children and 3.5 g for adults (110).
Although formulas containing bovine milk and formulas containing exclusively vegetable oils are supplemented with additional choline, bovine milk–containing formulas may contain forms of choline (free choline, phosphocholine, glycerophosphocholine, and phosphatidylcholine) that are more, or less, bioavailable (111–113). These different forms of choline may be delivered to different target tissues and used for different purposes. For example, choline can be used as a methyl donor, through betaine; as a precursor to the neurotransmitter acetylcholine; as a PL precursor, through phosphocholine and phosphatidylcholine; and possibly as a delivery vehicle for FAs such as AA and DHA to the brain, through lysophosphatidylcholine (114–117). Thus, in the future it may be desirable to design formulas with the same choline moieties as are found in human milk.
Phospholipids and sphingolipids together can have antimicrobial, toxin-binding, and anticancer properties; and can possibly affect cell signaling in the gastrointestinal tract and intracellularly. Phospholipids can also be sources of important dietary FA. Levels of PLs in refined vegetable oils are very low, less than in bovine milk.
There is no direct, strong scientific evidence showing a beneficial effect for the PLs and sphingolipids in milk nor a disadvantage in excluding them. If it is determined that they impart a health benefit to the infant, they could be added separately to vegetable oil and bovine milk fat–based formulas. Clearly, more research is needed in this important area.
Cholesterol Levels in Milk and Formulas
Human milk is known to be a rich source of cholesterol for the developing infant. It contains approximately 150 mg/l of cholesterol (2.59–3.88 mM) (118,119). Cow's milk contains 0.28 to 0.85 mM cholesterol—3 to 14 times less than human milk. Small amounts of cholesterol precursors are also present in milks (120). Exclusively breast-fed infants receive 15 to 20 mg cholesterol per kilogram body weight daily, whereas formula-fed infants receive 2 to 5 mg/kg (121). Formulas produced with vegetable oils such as corn, coconut, palm olein, and soybean oil contain low levels of cholesterol, originating mainly from residual milk fat of the protein source. Cholesterol oxides are known to be present in infant formulas; however, the levels (using newer methods that avoid cholesterol autoxidation artifacts) are very low (0.15–1.2 μg/g powder), and are probably not a health risk for infants (122), although this issue should be carefully monitored (123). The infant needs cholesterol for membrane synthesis, as a hormone precursor, for cholesterylation of signaling molecules (124), and for proper brain development and myelin formation. Most infants have a clearly demonstrated ability for endogenous cholesterol synthesis. It is important to note that those infants with metabolic abnormalities of cholesterol synthesis such as in Smith–Lemli–Opitz syndrome have retarded mental development, illustrating the importance of cholesterol for normal development (125,126). The issue of whether to add cholesterol to infant formulas has been under review for at least 30 years (127), but no definite conclusion has yet been reached.
Most studies on neonatal cholesterol metabolism have focused on comparing human milk with formulas but have the limitation that cholesterol metabolism can be affected by many factors other than dietary cholesterol (hormones, growth factors, proteins, nucleotides, antibodies). Studies comparing formulas containing graded amounts of cholesterol are most relevant to this question. The following discussion focuses on experiments with pigs, baboons, and human neonates because these species have cholesterol metabolism that is similar to humans (128). However, the baboon model has been criticized, because serum LDL cholesterol levels are much lower in baboons than in humans (119).
Cholesterol Levels and Synthesis in Sow-and Formula-Fed Piglets
In piglets fed milk or formula from day 0 to 18, formula-fed animals had lower plasma cholesterol than sow's milk-fed animals, even when the formula was supplemented with three times the amount of cholesterol (1.09 mM) found in sow's milk (129) ! The feeding of identical formulas containing 0.05 or 1.09 mM cholesterol resulted in similar levels of plasma cholesterol, plasma cholesteryl esters, and high-density lipoprotein (HDL) and very low-density lipoprotein (VLDL) cholesterol. However, feeding the highest cholesterol-containing formula led to the highest levels of hepatic cholesterol, cholesteryl ester, PLs, and bile acids, and the lowest levels of liver hydroxymethylglutarylCoA reductase. Thus, some of the exogenous cholesterol was absorbed, entered the liver, suppressed de novo synthesis, and was esterified by acyl cholesterol acyl transferase (ACAT) to form cholesteryl esters. The number of LDL receptors was the same in all three treatments.
In a recent work (130), piglets received formulas with and without added cholesterol and containing a similar FA composition, except for the percentage of palmitate on the sn-2 position, which was varied. After 18 days' feeding, sow's milk–fed piglets had higher plasma cholesterol than all formula-fed groups. The addition of cholesterol to formulas increased plasma cholesterol, although the increase was not statistically significant. That there was no significant increase is most likely because dietary cholesterol downregulates cholesterol synthesis in the pig (131).
Positional distribution of palmitate on the glycerol backbone affected cholesterol metabolism: total cholesterol, apolipoprotein (apo) B–containing lipoprotein (chylomicrons + VLDL + LDL) cholesterol, and TAG were lower when most palmitate was esterified to the sn-2 position. In contrast to this study, in piglets fed an infant formula enriched with 70% palmitate in the sn-2 position for 18 days, there was an increase in plasma total and HDL cholesterol compared with those fed a formula of similar FA composition but in which palmitate was in the sn-1 (3) position (87).
Cholesterol Levels and Synthesis in Breast-and Formula-Fed Infants
Formula-fed infants have been shown to have lower levels of serum cholesterol than breast-fed infants (121,127,132–139). The effect of adding cholesterol to milk-and vegetable oil–based infant formulas on fractional synthesis rates (FSRs) and cholesterol levels is not as clear.
In term (138–141) and preterm (142) infants, the addition of cholesterol to formula did not increase serum cholesterol levels to the same extent as breast milk. Increasing the cholesterol content from 5 to 10 mg/dl in formulas did not increase serum total cholesterol at days 0, 7, or 30 after birth, although serum free cholesterol was increased (138).
Apolipoprotein E is an arginine-rich glycoprotein, which, similar to apo B, serves as a ligand for the LDL receptor and the LDL receptor-related protein. The apo E phenotype influences binding affinity of apo E containing lipoproteins to the LDL receptor. The E4 phenotype (4/4 and 4/3), compared with E2 (4/2, 3/2, 2/2), has a higher absorption efficiency of cholesterol, and persons of apo E phenotype are usually responders to dietary cholesterol and fat content. In a recent study with 151 infants, serum total cholesterol at ages 4 and 12 months and 5 years, and apo E phenotype in breast-fed infants were found to be important factors in predicting serum total cholesterol and LDL cholesterol, but not HDL cholesterol at 11 years of age (143). The greatest difference between breast-fed and formula-fed infants was seen in the E4 group during the first year of life (121). Therefore, discrepancies between studies looking at cholesterol levels in breast-fed and formula-fed infants could be due to genetic factors such as apo E, previously not examined.
In 1994, Cruz et al. (139) found that term infants at 4 months of age have the ability to adapt cholesterol synthesis rate to cholesterol intake. Cholesterol FSRs were lowest in infants with the highest cholesterol intake (human milk), intermediate in infants with an intermediate intake (cow's milk formula), and highest in infants with no cholesterol intake (soy formulas). Serum cholesterol levels were significantly higher in human milk-fed infants than in formula-fed infants. There were no significant differences in serum total cholesterol and LDL cholesterol among formula-fed infants. This study shows that serum cholesterol levels in infants fed lower cholesterol formulas are not significantly lower than in infants fed higher cholesterol formulas. The infant has the adaptive ability to increase cholesterol synthesis.
In a follow-up study, the FSR for cholesterol at 4 months of age was four times lower in breast-fed infants, but not significantly different when infants were provided with either 33 mg/l (cow's milk formula) or 133 mg/l cholesterol (cow's milk formula with added cholesterol in the diet, i.e., approximately the cholesterol level in breast milk) for the first 4 months of life (141).
Taken together with results from the earlier 1994 study (139), we could conclude that infants have the adaptive ability to synthesize more cholesterol when less cholesterol is provided in the diet but that the relationship is not a straightforward dose–response relationship. When very low levels are provided in the diet, cholesterol synthesis is upregulated (soy formula versus cow's milk formula), but when some cholesterol is already provided in the diet (cow's milk formula versus cow's milk formula with added cholesterol), additional dietary cholesterol does not result in a decreased FSR. As in the previous study (139), increasing the concentration of cholesterol in formulas from 33 mg/l to 133 mg/l had no statistically significant effect on plasma cholesterol or LDL cholesterol levels at 4 months of age. However, the sample size was only 9 to 10 infants per group (of mixed sex) in the 1998 study (141), and there were quantitative (statistically nonsignificant) differences in total cholesterol and LDL cholesterol. Thus, with a larger sample size and fewer covariates, statistically significant differences might have been achieved.
From the results in these studies, it is also apparent that simply mimicking the cholesterol content of human milk in formulas does not lead to equivalent plasma cholesterol levels. Factors other than cholesterol itself in human milk are responsible for the higher levels of plasma cholesterol in human milk-fed babies than in formula-fed babies. These factors may include SFAs, MFGM components, thyroid hormones, growth hormones, growth factors (130), and nucleotides (144). Nucleotide addition to formula has been shown to enhance total lipoprotein concentrations without affecting total cholesterol levels. This appears to relate to enhanced lipoprotein synthesis and lecithin cholesterol acyltransferase activity (144). The enhancement of lipoprotein concentrations was more pronounced in preterm than in term infants. Although palmitate position on the glycerol backbone yielded mixed results in piglet studies, there is the possibility that palmitate positional distribution could affect cholesterol metabolism in humans. With respect to FAs, SFAs such as those found in cow's milk fat can increase the absorption of cholesterol in rats and African green monkeys (145,146) and may also decrease the number of LDL receptors in adult animal models (147).
Neonatal Cholesterol Intake and Adult Cholesterol Metabolism
An important question is whether intake of high levels of cholesterol during early infancy (as found in human milk) affects adult cholesterol metabolism, leading to either cardioprotective effects (the Reiser hypothesis (148)), or conversely, leading to initiation of atherosclerosis and premature artery disease. There is no definite answer to this question, because of discrepancies among results in different studies, interspecies differences in cholesterol metabolism, and the lack of human studies. Most studies with rats, rabbits, and guinea pigs have failed to demonstrate long-term effects of neonatal cholesterol intake (118,119,127,149).
In rats, as reviewed by Wong (119), supplementing formulas with cholesterol or ingesting breast milk had a protective effect against hypercholesterolemia in later life in some studies, but not in others. Rats that had been fed beef tallow plus 1% cholesterol for the first 2 postweaning weeks of life had lower levels of phosphatidylcholine in ileal brush border membranes than rats receiving beef tallow without cholesterol during those first 2 weeks, when measured at 11 weeks' postweaning age (150). Both groups received beef tallow alone during postweaning weeks 2 through 9, and beef tallow plus cholesterol (the cholesterol challenge) during postweaning weeks 9 through 11. Conversely, rats that had been fed fish oil plus 1% cholesterol for the first 2 postweaning weeks of life had higher levels of sphingomyelin in ileal brush border membranes than rats receiving fish oil without cholesterol during these first 2 weeks, when measured at 11 weeks' postweaning age. Both groups received fish oil alone during postweaning weeks 2 through 9, and fish oil plus cholesterol during postweaning weeks 9 through 11 (150). These experiments demonstrate that early cholesterol induction can have late effects on the specific ileal PL content, which could affect intestinal nutrient absorption.
In pigs, a high intake of cholesterol during weaning induced resistance to dietary cholesterol in later life (148,151). In other studies, however, the addition of 0.5% cholesterol to the diet of genetically bred pigs during days 1 to 28 of age led to elevated cerebrum cholesterol levels at 6 months of age (152). In pigs bred to have low plasma cholesterol levels, the addition of cholesterol also increased some aspects of exploratory behavior at day 28 (reviewed in reference 153). Also, in DBA/2 mice (which have impaired learning and memory), subcutaneous implantation of cholesterol pellets improved learning performance (154). In a recent study, pigs received either no cholesterol or 0.5% cholesterol from days 1 through 28. From days 29 through 56 they received no cholesterol, and from day 57 through 6 months (or day 57 through 5 months) they received 0.5% cholesterol (the cholesterol challenge) (155). Early cholesterol feeding did not lead to lower cholesterol levels in later life after the cholesterol challenge period or to protection against development of early aortic atherogenic lesions.
Studies on neonatal metabolism of cholesterol in baboons have been reviewed (156,157). Baboons were fed breast milk or formulas containing 2, 30 (comparable to baboon's milk), or 60 mg/dl cholesterol for 16 weeks. They then received either high-or low-cholesterol diets until young adulthood (7–8 years). At 6 to 8 years of age, the cholesterol content of the initial formulas did not influence serum cholesterol levels. Breast-feeding led to lower HDL cholesterol, higher LDL cholesterol, lower cholesterol production rates, and steroid excretion rates in adults. The differing effects of baboon's milk and formulas (even if providing the same level of cholesterol, 30 mg/dl) on adult cholesterol metabolism confirms that breast-feeding can affect adult cholesterol metabolism through various complex mechanisms.
The effects of neonatal feeding on adult cholesterol metabolism in humans have been reviewed (119,127). Fall et al. (158) examined the medical records of 5718 men in Hertfordshire, United Kingdom, born from 1911 through 1930. They found that men who were breast-fed or fed both human milk and formula during infancy had lower serum total cholesterol, LDL cholesterol, apo B, and lower mortality from ischemic heart disease than men who were bottle-fed. Because the formulas of 80 years ago differ markedly from those of today (formulas had more saturated fats and no mineral and vitamin fortification), the relevance of these findings is questionable.
Marmot et al. (159) showed that plasma cholesterol was lower in women, but not in men, who were breast-fed as infants compared with those bottle-fed. Although differences were not statistically significant, findings in the Bogalusa Heart Study indicated that children fed cow's milk as infants had lower cholesterol and TAG levels at 7 years of age than children fed low-cholesterol formulas (160). In recent work, it has been shown that there is a significant correlation between total and LDL cholesterol levels in children at 4 and 12 months of age and cholesterol at 11 years of age, and that subclassifying the infants by apo E phenotype enhanced the correlation. Cholesterol in children beyond 11 years has not been examined (143). In this study, paired comparisons for each infant were not shown, and breast-feeding and formula-feeding and the duration of breast-feeding and formula-feeding were not considered as covariates. It is not possible to conclude from this study whether the level of cholesterol in infant formula affects cholesterol levels in later life, because dietary cholesterol intake was not recorded. In at least four other studies, method of feeding in infancy was not found to affect adult serum cholesterol levels (132,161–163).
Cholesteryl Esters as a Source of Liver Fatty Acids
An unanswered question is the extent to which cholesteryl ester is important as a carrier to bring FAs such as linoleic or AA to the liver. Once transported to the liver, linoleic acid could be desaturated and elongated to AA, an important precursor of eicosanoid products.
In pigs, increasing the cholesterol content of the diet from 0.05 to 1.09 mM in formula during days 0 through 18 did not increase the amount of 18:2 n-6 or AA in liver cholesteryl esters, suggesting that dietary cholesterol conversion to cholesteryl ester is not quantitatively important for bringing FAs to the liver (129).
Similarly, in newborn infants, the addition of cholesterol to formula (5–10 mg/dl) did not increase the amount of serum cholesteryl esters (palmitoyl, oleoyl, linoleoyl, arachidonyl) at days 7 and 30 after birth (138).
In addition to cholesterol, cow's milk and human milk fats contain numerous other sterols (12,164). Vegetable oils and soy milk also contain noncholesterol plant sterols and phytoestrogens with bioactive properties (165). Plant sterols can impair the absorption of cholesterol, but are themselves poorly absorbed (approximately 5% for sitosterol, and higher for campesterol). An area of intense discussion in plant sterol research is the potential bioactive properties of the plant sterols that are absorbed (166–168).
In one early study, sterol excretion was higher and plasma cholesterol levels were lower in term infants fed soy milk than in those fed cow's milk, even if the former was supplemented with cholesterol, and it was postulated that compounds in soy milk may impair cholesterol absorption (169). More recently (139), serum cholesterol levels in 4-month-old infants were found not to differ significantly among infants fed cow's milk (0.28–0.85 mM cholesterol), soy milk (no cholesterol, phytoestrogens), or modified soy milk (0.28 mM cholesterol). Thus soy phytoestrogens did not significantly lower serum cholesterol levels.
Breast Milk– Versus Formula-Feeding and Plasma Cholesterol
In several studies, breast-fed infants and sow-fed piglets have been found to have higher levels of plasma cholesterol than formula-fed neonates, even if these formulas were supplemented with the same or higher levels of cholesterol as found in breast milk. This suggests that unidentified factors contained in mothers' milk other than cholesterol itself may be important in elevating plasma cholesterol.
Supplementation of Formulas With Cholesterol and Levels of Plasma Cholesterol
In several studies, cholesterol supplementation of formulas did not lead to an elevation of plasma cholesterol in infants. This may be because those infants fed less cholesterol have the adaptive ability to increase cholesterol synthesis. Thus, it is unlikely that adding cholesterol to pure vegetable oil formulas, either as a purified ingredient or as a component of milk fat, significantly elevates infant plasma cholesterol levels. Levels of cholesterol are less than in breast-fed infants whether the formulas are pure vegetable oil–based or partly milk fat–based.
Neonatal Cholesterol Intake and Adult Cholesterol Metabolism
Studies examining the effects of neonatal dietary cholesterol level on cholesterol metabolism in adult life have yielded mixed results. In the future, apo E phenotype (and possibly other genetic factors) should be examined, because this can affect the responsiveness to dietary fat and cholesterol. In some studies, infants fed higher levels of cholesterol had better ability to regulate cholesterol in later life, and in others this was not the case. Thus, there is no contraindication for excluding cholesterol from the infant diet.
Infant formulas contain 50% energy as fat, composed of mixes of cow's milk fat and vegetable oils or mixes of vegetable oils without milk fat. The proportions of the different fats are calculated to be as close as possible to human milk FA composition. However, because of the presence or the absence of certain FAs in the different fats, there are some differences in FA composition between milk fat and vegetable oil–based formulas. We would not expect a significant health effect due to the small differences in SCFAs and tFAs. The presence of CLA in milk fat–based formulas could have a beneficial effect, although clinical studies are still needed.
The differences in TAG structure between cow's milk fat and vegetable oils have no major effect on the quantity of energy absorbed from fat and an unknown effect on calcium absorption. Stool hardness may be greater with vegetable oils. These effects on calcium absorption and stool hardness could be important for preterm infants, and further study is needed.
There is presently no evidence showing that the lower levels of PLs and sphingolipids in vegetable oils compared with milk fat would be a health disadvantage to infants. The absence of cholesterol in vegetable oils and its presence in human and bovine milk suggest that it may be prudent to supplement vegetable oil–based infant formulas with cholesterol. However, studies have shown that such supplementation has little effect on plasma cholesterol levels and that levels of plasma cholesterol are less in formula-fed than in breast-fed infants, whether the formula is pure vegetable oil–based or partly milk fat–based. Studies on the effect of neonatal dietary cholesterol level on cholesterol metabolism in adulthood have produced contradictory results. Thus, there is no contraindication for excluding cholesterol from the infant diet, but clearly this issue must continue to be studied, with focus on infant apo E phenotype and other genetic parameters that can affect cholesterol metabolism. Overall, there are presently no strong contraindications for replacing milk fat with vegetable oils in infant formulas, but a number of lipid components in vegetable oil and milk fat, as described in this review, have potential for important physiological effects and should be the focus of future studies.
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