To meet the relatively high energy requirements of infancy, human milk contains approximately 50% of its calories as fat. Classes of saturated and monounsaturated fatty acids are dominant in human milk (each constituting approximately 40% of fat), with palmitic acid being the most abundant saturated fatty acid (20-25% of total fatty acids), and oleic acid being the predominant monounsaturated fatty acid (30-38%). The polyunsaturated essential fatty acids generally comprise less than 20% of milk fatty acids (1). Infant formulas are similar to human milk in their total fat content and supply sufficient linoleic acid, an essential fatty acid, to meet the needs of the infant as mandated by numerous regulations (2,3). However, the long- chain saturated fatty acid (LCSFA, C14:0, C16:0, C18:0) content of formulas varies widely. Although it is possible to produce fat blends in infant formula with fatty acid profiles similar to those of human milk, the absorption of fat and calcium in young infants consuming these blends may not be optimal (4). The fatty acid profile in human milk is characterized by a high proportion of the generally less well-absorbed LCSFAs, essentially palmitic acid (C16:0). Evidence, primarily from animal studies, indicates that the superior absorption of human milk fat relates to a difference in the positional distribution of palmitic acid in the triglycerides of human milk compared with that of the fat and oil blends of infant formulas selected to mimic the fatty acid profile of human milk (5).
The triglyceride digestive process of the neonate is complex. It is initiated by a gastric phase catalyzed by gastric or lingual lipase (6). This initial lipolysis allows maximal activity of pancreatic colipase-dependent lipase during the intestinal phase of digestion. The pancreatic lipase system attacks the triglyceride with a high degree of positional specificity. Lipolysis occurs predominantly at the sn-1 and sn-3 positions, yielding two free fatty acids and a 2-monoglyceride (7). Monoglycerides are well absorbed independent of their constituent fatty acid. In contrast, the absorption of free fatty acids varies greatly, depending on chemical structure. Mono- and polyunsaturated fatty acids are well absorbed, as are saturated fatty acids of 12 carbons or less in chain length. Free LCSFAs form insoluble soaps with calcium and are more poorly absorbed, with a negative correlation between chain length and absorption. The fat of human milk is well absorbed because palmitic acid is predominately in the sn-2 (beta) position of the triglycerides (70%) (5,8,9) and, after lipolysis by pancreatic lipase, is found in the intestine as the readily absorbed 2-monopalmitin. In the common fats and oils of infant formula blends, palmitic acid is predominantly in the sn-1 and sn-3 positions of the triglyceride molecule. The percentage of palmitic acid in the sn-2 position of cow's milk triglycerides is 40% (5,8) and in the range of 5% to 20% in vegetable oils (5,10,11).
Several possible means exist for elevating the proportion of 2-palmitic acid in infant formula. In contrast to most animal depot fats, lard contains an elevated proportion of sn-2 palmitic acid (80%). In a study in rats, a fat blend containing lard was as well absorbed as human milk fat (5). Fat absorption in infants fed formula containing lard was reduced when the high proportion of sn-2 palmitin in lard was reduced to 33.3% by chemical randomization (12). However, numerous religious and cultural prohibitions mitigate against the widespread use of this fat in infant formula. The process of interesterification (randomization) elevates the proportion of LCSFAs at the sn-2 position to approximately 33% in vegetable oils, and we have previously reported improved LCSFA absorption with these types of preparations (11). The proportion of 2-palmitic acid is still substantially lower than the level found in human milk.
The development of an enzymatic interesterification process that yields triglycerides rich in sn-2 palmitic acid has lead to the formulation of a commerical product, Betapol, which mimics human milk fat in fatty acid profile and in sn-2 palmitic acid content. This product was used in examining the absorption of total fat, individual fatty acids, and the distribution of fatty acids in the fecal fat of rats fed diets of nearly identical fatty acid content but varying in the proportion of palmitic acid in the sn-2 position of the triglycerides.
MATERIALS AND METHODS
The proportion of fatty acid in the sn-2 position was determined by a modification (13) of the Brockerhoff procedure (14). Lipid samples were digested with pancreatic lipase to hydrolyze the ester bonds selectively at the 1 and 3 positions of the triglycerides. The 2-monoglycerides were isolated through a liquid-liquid extraction followed by thin layer chromatography. The purified 2-monoglyceride fraction was transesterified and the fatty acid methyl esters extracted and quantitated by capillary gas chromatography.
Total Fatty Acid Analysis
Dietary and fecal fatty acids were determined titrimetrically on extracts from acidified saponified samples (15).
The fatty acid methyl esters were prepared and analyzed by gas chromatography using the method of Morrison and Smith (16).
Neutral fat (including free fatty acids) and soap fatty acids were determined gravimetrically. For the determination of neutral fecal fat, a weighed sample of feces was reflux extracted with petroleum ether, the extract evaporated, and the residue weighed. The sample remaining in the extraction thimble was reextracted with petroleum ether-acetic acid 20:1, the solvent evaporated, and the residue of soap fatty acids weighed. Each fraction was dissolved in chloroform, an internal standard, LCSFA C17:0 added and the fatty acids derivatized to the methyl esters for gas chromatographic analysis.
Calcium was determined by atomic absorption spectroscopy (17).
Fat Absorption Assay
The absorption of fat and individual fatty acids was determined by the procedure published previously (5). In brief, young male Sprague-Dawley rats (Harlan; Indianapolis, IN), 90 to 110 g in body weight, were fed a fat-free diet for 10 days, with 0.1 ml of corn oil administered daily until 3 days before the start of the assay. The composition of the fat-free diet was as follows: casein, 220 g; dextrose, 719 g; cellulose, 10 g; mineral mix, 40 g; AIN-76 vitamin mix, 10 g; choline chloride, 1 g. Groups of 10 rats were fed diets containing the test fat (150 g/kg), replacing an equal weight of dextrose in the fat-free diet) for 3 days followed by the fat-free diet for the next 3 days. Feces were collected throughout the 6-day assay. Consumption of the fat-containing food was measured for calculation of fatty acid intake. A control group was fed the fat-free diet for the 6-day period to permit a correction for endogenous fatty acid excretion. Procedures were approved by the Wyeth-Ayerst Institutional Animal Care and Use Committee.
Statistical significance of differences was determined by a one-way ANOVA with Student-Newman-Keuls comparisons (18). Differences were considered significant at p < 0.05.
The fatty acid composition and the positional distribution of the fatty acids of Betapol and human milk fat are presented in Table 1.
Table 2 presents the absorption of Betapol compared with that of the fat blend of a current proprietary infant formula. Both blends were very well absorbed but the excretion of fatty acids from Betapol was significantly less, despite nearly twice as much palmitic acid in the product. As shown in Fig. 1, the excretion of each of the saturated fatty acids, C12:0-C18:0, was significantly lower in the Betapol group. The excretion of palmitic acid (C16:0) in the Betapol group was one fifth that of the group fed the infant formula fat. Because the unsaturated fatty acids are almost completely absorbed, excretion values are not presented in the figures. The percentage of palmitic acid in the sn-2 position of the formula blend was calculated to be approximately 8%.
The data of Table 3 and Fig. 2 demonstrate the poor fat absorption that results when fat blends are formulated to simulate the fatty acid composition of human milk without regard for positional distribution of LCSFAs. Blends A and B contain similar total amounts of myristic, palmitic, and stearic acids; the primary source of LCSFA in blend A is palm olein and in blend B is oleo. Excretion of total fatty acids from the two simulated blends A and B was more than ten times greater than from Betapol (Table 3). As shown in Fig. 2, palmitic acid excretion from Betapol was approximately 1% compared with approximately 18% from the two simulated blends. The calculated amounts of palmitic acid in the sn-2 position of blends A and B were 8% and 9%, respectively.
This experiment was designed to determine the relationship between fat absorption and the percentage of palmitic acid in the sn-2 position of a fat blend. Blend C, a mixture of vegetable oils formulated to have the same fatty acid composition as Betapol, was mixed in varying proportions with Betapol to yield fat blends with sn-2 palmitate content from 5% to 79% of the total palmitic acid.
As the percentage of palmitic acid in the sn-2 position decreased, the excretion of total fatty acids increased (Table 4). The differences in the excretion of the individual saturated fatty acids (Fig. 3) increased progressively from the 100% Betapol group to the native oils group, with most differences between groups being statistically significant. The plotted excretion values of Fig. 4 reveal that the excretion of total fatty acids and also of palmitic acid is a logarithmic function of the proportion of palmitic acid in the sn-2 position of the triglycerides.
Rats were fed diets containing Betapol, or the oil blend C, whose fatty acid profile duplicated that of Betapol, or a 50:50 mixture of the two. The feces collected throughout the 6-day experiment (3 days of fat-containing diets followed by 3 days of fat-free diets) were dried and analyzed for calcium, soap fatty acids, and neutral fat (essentially free fatty acids and nonsaponifiables, in that only traces of glycerides were detected by thin layer chromatography).
There was no difference of statistical significance in the calcium content of feces during the collection period: 0.49 ± 0.26 mg, 0.47 ± 0.22 mg, 0.47 ± 0.26 mg for blend C, the 50:50 mixture, and Betapol, respectively.
The results of fecal fat analysis are presented in Table 5. Total fat is expressed as the sum of the weights of neutral fat and soap fatty acids. The individuals fatty acids in each fraction were determined; only the major identifiable fatty acids are presented. As the percentage of palmitic acid in the sn-2 position increased from 5%, to 42%, to 79%, the total fat in the feces decreased from 350, to 160, to 70 mg. The major component in the decrease was in the soap fatty acid fraction, a more than tenfold decrease from 267 mg to 23 mg. Neutral fat also decreased but at lesser degrees so that the soap fatty acids decreased from 76%, to 55%, to 33% of the total fat.
As shown in Table 5, almost all of the major dietary fatty acids in the feces were in the fecal soap fraction-99%, 96%, and 92%, in the three groups. As the percentage of palmitic acid in the sn-2 position increased in the dietary fat, reduced amounts of every fatty acid were found in the fecal soaps. Soap palmitic (C16:0) and stearic (C18:0) in the feces of the Betapol group were 191 mg less than in the blend C group and constituted 83% of the 229-mg reduction of the total determined fatty acids.
Triglyceride digestion in the bottle-fed infant relies on two enzyme systems. This process is initiated by a gastric phase (6) employing either lingual lipase (derived from the serous glands on the dorsal posterior of the tongue) or gastric lipase (from glands within the gastric mucosa). This phase of digestion yields almost exclusively diglycerides and free fatty acids because of the high specificity of gastric lipase and lingual lipase for the sn-3 position. In vitro studies have suggested that this phase of digestion may be relatively limited because of feedback inhibition from free fatty acids and enzymatic inactivation by pancreatic proteases when bile salts are present (19); however, data from in vivo studies in dogs indicate that this phase of digestion may provide substantial triglyceride hydrolysis (20). The intestinal phase of lipolysis is dominated by the colipase-dependent pancreatic lipase system, which is highly triglyceride position-specific (7). The resulting products consist of sn-1 and sn-3 fatty acids and sn-2 monoglycerides.
The greater absorption of fat and calcium in breast-fed infants compared with that in those fed formula has been ascribed to two factors: the presence in breast milk of a lipolytic enzyme (the bile salt-stimulated lipase) and the relatively high proportion of palmitic acid in the sn-2 position of the triglycerides, resulting in the release of the palmitic acid in the intestine as the readily absorbable 2-monopalmitin rather than as the free acid, which could react with calcium to form an insoluble soap. The importance of triglyceride structure to the absorption of human milk fat has been questioned, in that bile salt-stimulated lipase has been shown to hydrolyze the fatty acids at all three positions of the triglycerides (19,21). Although complete hydrolysis can be demonstrated, the sn-2 position is more resistant to lipolysis, as demonstrated in in vitro studies (21) and it is likely that monopalmitin would be absorbed before being exposed to hydrolysis by bile salt-stimulated lipase. This assumption is supported by the findings of Innis et al. of elevated amounts of sn-2 palmitate in the plasma triglycerides of breast-fed infants (22).
In the present study, we have investigated the effect of positional distribution on fat absorption employing the rat as a model for formula-fed infants. The weanling rat absorbs fat more efficiently than the neonatal human infant, but in the few cases where the same fat blend was fed to rats and infants, the rank order of absorption was similar, suggesting similar absorptive mechanisms (11).
The availability of the Betapol product, with its similarity to human milk fat in fatty acid profile and in the positional distribution of palmitic acid, permitted the detailed studies reported here on the importance of triglyceride structure to fat absorption. Several blends of vegetable and animal fat with fatty acid profiles similar to Betapol, but with a low proportion of sn-2 palmitic acid, were shown to be poorly absorbed compared with Betapol. Widdowson et al. (4) almost 30 years ago, in a study with neonatal infants, demonstrated that the formulation of an fat blend in infant formula with a fatty acid profile simulating that of human milk would not necessarily ensure equal absorbability. The commercial fats and oils acceptable for infant formula manufacture are low in sn-2 palmitate and cannot be formulated to approach the total palmitic acid content of human milk without compromising absorption. Appropriate mixtures of fats and oils will achieve the equal parts of saturated and unsaturated fatty acids of human milk, and whereas levels of oleic, linoleic and α-linolenic acids may be similar to those of human milk, the saturated fatty acid proportion is maintained by reducing the palmitic acid concentration and by substituting the better absorbed, shorter chain, lauric acid (C12:0). The proprietary infant formula blend of Table 2 is an example of this approach to achieve acceptable absorption. This fat blend, studied in experiment 1, (Table 2 and Fig. 1) contains 13.2% of the total fatty acids as palmitic acid (1.88 g/100 g of the rat diet). Five percent was excreted or a loss of approximately 94 mg/100 g of diet. In contrast, the formula blend with palmitic acid content of human milk (simulated blend A of experiment 2; Table 3 and Fig. 2) contains 29% of the total fatty acids as palmitic acid (4.13 g/100 g diet); with 20% excreted, 827 mg lost in the feces for every 100 g of diet consumed. Similar results were observed in human neonates when the fat blend of experiment 1 was compared with blends similar to the blends of experiment 2 (23).
The results of experiment 3 show that the absorption of total fatty acids is a logarithmic function of the percentage of palmitic acid in the sn-2 position of the dietary fat (Fig. 3). Because palmitic acid is the major dietary LCSFA, this relationship also applies to the absorption of this fatty acid. As noted in earlier studies (5,11) whenever total absorption of fatty acid is improved as a result of triglyceride restructuring, the absorption of all the saturated fatty acids is improved, regardless of their positional distribution (Figs. 1, 2, and 4).
It is well known from studies in animals and human infants that fat malabsorption may be accompanied by a reduction in the absorption of calcium. It has been concluded that the reaction between LCSFAs and calcium results in insoluble soaps that are not absorbed and are passed into the feces. Of the many studies correlating fat and calcium absorption in human infants, one of the earliest was that of Holt et al. (24) who, in addition to demonstrating that unsaturated fatty acids are better absorbed than saturated fatty acids, and that short chain saturated fatty acids are better absorbed than LCSFAs, also demonstrated that malabsorption of calcium paralleled that of fat. The studies of Widdowson (4) alerted the infant formula industry that a formula fat with a human milk level of saturated fatty acids, although adequately absorbed by infants 3 or 4 months old, was not well absorbed during the first week of life, and that the loss of fat was accompanied by a loss of calcium. Hanna et al. (25) in a study with infants fed either human milk or one of two infant formulas showed a linear relationship between the excretion of calcium and that of palmitic or stearic acids. More recently, Chappellet al. (26) reported a decrease in fat absorption when preterm infants fed either preterm human milk or an infant formula were given oral calcium supplements.
We found no differences in the fecal calcium content of the group fed Betapol or the oil blend C with the same fatty acid profile, but differing in sn-2 palmitate. This result is not unexpected because on a stoichiometric basis the difference in the sum of fecal myristic, palmitic and stearic acids between the two groups is 0.767 mEq (0.802-0.035 mEq), whereas the millieqivalent of calcium in the fecal samples was 24.45 and 23.30 mEq for the groups blend C and Betapol, respectively. Experimental error would prevent a difference of less than one millieqivalent from being statistically significant. However, the predominance of LCSFA content over calcium in an infant formula would explain the correlation found in clinical studies between fat and calcium absorption. The rat diets containing 15% Betapol or blend C and 0.9% calcium would supply 17.9 mEq of myristic, palmitic, and stearic acids/100 g and 45 mEq calcium/100 g diet; the calcium/LCSFA ratio would be 2.5. In contrast, an infant formula (27% fat and 0.33% calcium) supplying 27 mEq of myristic, palmitic, and stearic acids and 16.5 mEq of calcium/100 g would have a calcium/LCSFA ratio of 0.6.
The relationship of dietary calcium and magnesium to LCSFA absorption was examined by Mattson et al. (27) in a study in which rats were fed a number of synthetic triglycerides of oleic and stearic acids in which the fatty acids were esterified to specific positions on the glycerol molecules. Rats were fed a diet with either sufficient calcium and magnesium content or one that was deficient. Stearic acid was almost completely absorbed from a diolein stearate when the stearic acid was in the sn-2 position when fed with either the mineral-sufficient or -deficient diet. With stearic acid in the sn-1, absorption was also nearly complete when fed with the mineral deficient diet, but dropped to 55% with the diet containing sufficient calcium and magnesium.
In conclusion, the current study demonstrated the preferential absorption of LCSFAs when present at the triglyceride sn-2 position. Current fat blends in infant formula are well absorbed, but this absorption is accomplished by deviating from the fatty acid profile of human milk by substituting lauric acid for palmitic acid and, in some cases, by increasing the polyunsaturated fatty acid content. It is known that the fatty acid composition of the diet influences the fatty acid composition of developing infant tissue (28,29), and the lipoprotein and lipid metabolism differ between breast-fed and formula-fed infants (30,31). Adherence to the fatty acid profile of human milk may have physiological consequences beyond that of absorption.
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