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Department of Pediatrics, University of British Columbia, Vancouver, BC V5Z 4H4 Canada and * Wyeth Nutrition International, Nutritional Research Department, Philadelphia, PA 19101
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
LITERATURE CITED
ABSTRACT 
Human and pig milk fat contains a high proportion of palmitic acid (16:0) which is largely esterified to the 2-position of the triglycerides. In contrast, the 16:0 in most nonmilk fats and in infant formulas is mainly esterified at the triglyceride 1,3 positions. Gastric and pancreatic lipases hydrolyze fatty acids from the dietary triglyceride 1- and 3-positions to produce unesterified fatty acids and 2-monoglycerides which are absorbed and re-esterified. In this study, we determined whether formula with chemically randomized oils, which equally distributes 16:0 among all the positions of triglycerides, influences growth or the distribution of fatty acids in plasma and liver lipid of formula-fed piglets compared with piglets fed formula with native oils or sow's milk. After feeding from birth to 18 d, piglets fed formula with palm olein randomized with canola oil (co-randomized) had higher weight gain per liter of formula intake and higher 16:0 in the chylomicron triglyceride 2-position than piglets fed formula with randomized or native palm olein oil blended with canola oil. The fatty acid distribution of formula triglycerides is an important determinant of pathways of 16:0 absorption, and consequently of plasma lipid fatty acids in formula-fed piglets.
KEY WORDS: co-randomized oils · infant formula · milk · triglycerides · palmitic acid · pigletsINTRODUCTION
The positional distribution, as well as the composition, of fatty acids in dietary triglycerides is an important determinant of fat digestion and absorption (Small 1991
). Gastric and pancreatic lipases hydrolyze fatty acids from the 1,3 positions of dietary triglycerides to produce free fatty acids and 2-monoglycerides (Carriere et al. 1993, Small 1991). Human milk triglycerides contain about 20-25% palmitic acid (16:0), with over 70% of the 16:0 esterified to the sn-2 position of the milk triglyceride (Martin et al. 1993). In contrast, the 16:0 in vegetable and most nonmilk fats is predominately esterified at the 1,3 positions, and mono- and polyunsaturated fatty acids are usually esterified at the 2-position of the triglyceride (Small 1991). As a result, the intraluminal hydrolysis of human milk by gastric and pancreatic lipases should result in formation of 2-monopalmitin, whereas hydrolysis of other dietary fats should produce unesterified 16:0.
Unesterified 16:0 is less well absorbed from the lumen than shorter chain saturated, or carbon chain 18 unsaturated fatty acids such as oleic acid (18:1) or linoleic acid [18:2(n-6)] (de Fouw et al. 1994, Filer et al. 1969, Jensen et al. 1986). This may be due at least in part to a melting point above body temperatures (>60°C) and a strong tendency of unesterified 16:0 to form insoluble soaps with divalent cations such as calcium and magnesium at the pH of the small intestine (Chappell et al. 1986, de Fouw et al. 1994, Filer et al. 1969, Jensen et al. 1986, Mattson and Volpenheim 1964, Tantibhedhyangkul and Hashim 1979). To avoid this, some infant formulas contain 8:0-14:0 (caprylic, caproic, lauric and myristic acids) from medium-chain triglycerides and/or coconut oil rather than 16:0 as the major source of saturated fatty acids. The specific positioning of 16:0 at the 2-position of human milk triglycerides has been suggested as one of the reasons for the high efficiency of absorption of fat from human milk (Filer et al. 1969, Tomarelli et al. 1968). Support for this has been provided by studies showing better absorption of total dietary fat or 16:0 by infants fed formula with 16:0 esterified to the 2 rather than the 1,3 positions of the dietary triglycerides (Carnielli et al. 1995b, Filer et al. 1969). This is still controversial, however, because of the postulated role of the milk enzyme bile salt-stimulated lipase in completing the hydrolysis of the milk triglycerides to glycerol and unesterified fatty acids (Bernbäck et al. 1990).
The 2-monoglyceride pathway is believed to account for about 80% of the triglycerides synthesized and secreted in chylomicrons following the absorption of dietary fat (Small 1991). Triglyceride synthesis via this pathway, rather than the 3-glycerophosphate pathway, results in formation of triglycerides with the same fatty acid in the 2-position as in the dietary fat (Myher et al. 1985). Thus, in the absence of complete hydrolysis by bile salt-stimulated lipase, the digestion and absorption of human milk fat should result in higher proportions of 16:0 in the 2-position of plasma triglycerides than should the digestion and absorption of infant formula with vegetable fats. Recent studies have provided evidence for this through the demonstration of higher proportions of 16:0 in the 2-position of plasma triglycerides in breast-fed infants than in infants fed formula containing similar amounts of 16:0, but predominately esterified to the triglyceride 1,3 positions (Innis et al. 1994). Similar differences occur between piglets fed sow's milk, which also has a high proportion of 16:0 in the 2-position of the milk triglyceride (Innis 1993, Parodi 1982), and piglets fed formula containing vegetable oils (Innis et al. 1995).
The physiological importance of dietary 16:0 or of its position in the plasma triglycerides of young infants is not well known. The well-known preferential portal-venous transport and rapid hepatic 
-oxidation of 8:0-12:0, compared with chylomicron triglyceride transport of 16:0 (Bach and Babayan 1982, Senior 1968), however, can reasonably be expected to result in differences in fatty acid metabolism between infants fed formula with 8:0-12:0 and breast-fed infants (Innis 1992). Further, the distribution of fatty acids in plasma triglycerides is known to influence the rate of hydrolysis by lipoprotein lipase and the composition of partial glycerides cleared by the liver (Mortimer et al. 1992, Nilsson et al. 1992, Small 1991). However, no information is available as yet to indicate whether the plasma triglyceride fatty acid distributions of young infants are important to fatty acid turnover or delivery to specific tissues.
The proportion of 16:0 esterified to the 2-position of dietary triglycerides can be increased by chemical randomization. This process involves release of fatty acids from their natural position followed by equal re-esterification among all three positions of the glycerol molecule. Whether formulas containing randomized fats can be used to support a distribution of fatty acids in plasma lipids similar to that found during milk feeding is not known. This study, therefore, determined if the feeding of formula with randomized fats can increase the proportion of 16:0 in the 2-position of chylomicron triglycerides and if increased absorption of saturated 2-monoglycerides is accompanied by changes in the composition of chylomicron or liver lipid fatty acids in formula-fed piglets. The small blood volumes available in studies with infants often limit analyses to measures of total plasma phospholipid, triglyceride and cholesteryl ester fatty acids. However, it is possible that chylomicron fatty acids, which should reflect the absorbed lipids, will differ from those of other plasma lipoproteins, e.g., VLDL or LDL. This study, therefore, also compared the fatty acid composition of triglycerides, phospholipids and cholesteryl esters in plasma with that in chylomicron.
MATERIALS AND METHODS
containing (% energy) about 45.5% fat, 14.1% protein and 40.4% carbohydrate. The composition of the formula and the sow's milk total fatty acids, and the percentage of each fatty acid in the 2-position, determined as described elsewhere (Innis 1993, Lien et al. 1993), are shown in Table 1.
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Table 1. Composition and positional distribution of fatty acids in formula and sow's milk1 |
). Passive immunity was provided by inclusion of colostrum-derived immunoglobulins (La Belle, Bellingham, WA) in the formulas for the first 72 h after birth. Piglets fed sow's milk (n = 6) were fed by their natural mothers and studied at a similar age and time after the last feed. Littermates were not assigned to the same diet group. All of the procedures involving the piglets were approved by the Animal Care Committee of the University of British Columbia and conformed with the guidelines of the Canadian Council on Animal Care.
80°C. The liver was immediately removed, weighed, homogenized in ice-cold saline and stored at 80°C. Chylomicron were isolated at d < 1.006 kg/L by ultracentrifugation, 60 min at 140,000 × g using a Beckman model L5 ultracentrifuge and SW-28 rotor (Beckman Instruments, Palo Alto, CA).
Biochemical analyses.
Plasma and liver total and free cholesterol, and triglycerides were determined with enzymatic kits (no. 225-26, 210-75, respectively, from Diagnostic Chemicals Charlottetown, PE, Canada). Esterified cholesterol was calculated as the difference between free and total cholesterol. HDL cholesterol was determined following precipitation of the apolipoprotein (apo) B-containing lipoproteins with heparin-manganese chloride (Gidez et al. 1982) within 6 h of blood collection. The amount of cholesterol associated with the apo B-containing lipoproteins was calculated as the difference between the total and HDL cholesterol.
). The separated lipid fractions were recovered, and an aliquot of the triglyceride and phospholipid fractions was taken for analysis of the 2-position fatty acids; the fatty acid components in the remaining samples were converted to their respective methyl esters using methanoic HCl (1:5 v/v) 100°C × 5 min for phospholipids, and 14% boron trifloride in methanol/benzene/methanol (25:20:25, v/v/v) 100°C × 30 min or 45 min for triglyceride and cholesteryl esters, respectively. Enzymatic hydrolysis to determine the composition of the chylomicron triglyceride and phospholipid 2-position fatty acids used pig pancreatic lipase (EC 3.1.1.4) and phospholipase A2 (EC 3.1.1.3 Type 11) (both from Sigma Chemical, St. Louis, MO), respectively, as described by Kuksis (1984). Mono-heptadecanoic or heptadecanoic acid was added after the pancreatic lipase or phospholipase A2 enzyme reactions, respectively, as internal standards. The monoglyceride and free fatty acid and lysophospholipid and free fatty products were then separated by TLC using hexane/diethyl ether/acetic acid, 60:40:1.5 (v/v/v/) and chloroform/acetic acid/methanol/water, 75:25:5:2 (v/v/v/v), respectively. Parallel reactions without triglyceride or phospholipid substrate were always done to correct for any fatty acid components in the enzyme preparations. The completeness of the reactions was established in preliminary experiments and monitored throughout these studies from the amount of the substrate (triglyceride and diglyceride, or phospholipid) present after TLC separation of the reaction products. Fatty acid methyl esters were separated and quantified using a Varian 3400 gas liquid chromatograph equipped for analysis with capillary columns and a Varian Star data system (Varian Canada, Georgetown, ON, Canada) (Innis et al. 1994 and 1995). Milk fatty acids were determined following direct methylation of fatty acids (Lepage and Roy 1987) as in previous studies (Innis et al. 1994).
RESULTS
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Table 2. Composition of the major total and position 2 fatty acids in chylomicron triglycerides of piglets fed formula with native or randomized oils, or sow's milk1 |
), is esterified mainly at the 1-position; 18:1, 18:2(n-6) and 18:3(n-3) are predominantly esterified at the 1- and 3-positions; and 20:4(n-6) and 22:6(n-3) (which was absent in the formula) are equally distributed between the 2- and 3-positions of milk triglycerides (Martin et al. 1993).
Table 3.
Composition of the major total and position 2 fatty acids in chylomicron phospholipids of piglets fed formula with native or randomized oils, or sow's milk1
Table 4.
Composition and distribution of major fatty acids in chylomicron cholesteryl esters of piglets fed formula with native or randomized oils, or sow's milk1
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Table 5. Composition of major fatty acids in liver lipid classes of piglets fed formula with native or randomized oils, or sow's milk1 |
0.05, of the difference between the plasma and chylomicrons is indicated above the bars.
DISCUSSION 
Fig. 2.
Comparison of the major fatty acids in the plasma and chylomicron phospholipids of piglets fed the formula with native palm olein and canola oil, randomized palm olein and native canola oil, or co-randomized palm olein and canola oil, or sow's milk. The results shown are means ± SEM, n = 6/group. The statistical significance, where P 0.05, of the difference between the plasma and chylomicrons is indicated above the bars.
[View Larger Version of this Image (38K GIF file)]
Fig. 3.
Comparison of the major fatty acids in the plasma and chylomicron cholesteryl esters of piglets fed the formula with native palm olein and canola oil, randomized palm olein and native canola oil, or co-randomized palm olein and canola oil, or sow milk. The results shown are means ± SEM, n = 6/group. The statistical significance, where P 0.05, of the difference between the plasma and chylomicrons is indicated above the bars.
[View Larger Version of this Image (38K GIF file)]
, Tomarelli et al. 1968). This hypothesis is based on the relatively poor absorption of unesterified 16:0 and better absorption of 16:0 when present at the 2-position of dietary triglycerides. The milk enzyme, bile salt-stimulated lipase, however, can complete the hydrolysis of milk triglycerides in vitro to glycerol and unesterified fatty acids (Bernbäck et al. 1990). The extent of hydrolysis of 16:0 from milk triglycerides by this enzyme in infants in vivo, however, is uncertain. Recent studies have reported a high proportion (about 27% fatty acids) of 16:0 esterified at the 2-position of the plasma triglycerides in breast-fed infants (Innis et al. 1994). This suggests that a considerable proportion of human milk 16:0 is absorbed as 2-monopalmitin. For this reason, studies on the effects of dietary oils that also result in absorption of 2-monopalmitin are clearly of interest.
, Yang and Kuksis 1991), 16:0 should represent 39-46% of fatty acids in the 2-position of the chylomicron triglycerides of piglets fed sow's milk. This theoretical value agrees closely with the results of this study to show 39.5 ± 2.09% 16:0 in fatty acids at the 2-position of chylomicron triglycerides from piglets fed sow's milk. This result offers strong evidence that, at least in piglets, milk lipases do not hydrolyze substantial amounts of 16:0 from the 2-position of the milk triglycerides. The high absorption of fat from human milk (Barnes et al. 1974) and relatively high amounts of 16:0 esterified to the 2-position of plasma triglycerides in breast-fed infants (Innis et al. 1994) suggest that 16:0 is also at least partly absorbed as 2-monopalmitin in human infants.
, Innis et al. 1994 and 1996) rather than on separated lipoproteins. These studies with piglets show that the presence and direction (higher or lower) of difference between the plasma and chylomicron fatty acid levels depends on whether milk or formula is fed. Differences in the fatty acid composition of piglet plasma and chylomicron phospholipids, which have been reported in several studies on (n-6) and (n-3) fatty acid requirements, however, were relatively small and inconsistent, at least in these milk- and formula-fed piglets. This suggests that although studies pertaining to the transport of absorbed fatty acids should consider separation and analysis of chylomicron lipids, the incorporation of fatty acids into phospholipids might be adequately addressed through analysis of total plasma phospholipids.
). Further, studies conducted several decades ago found that triglyceride configuration, specifically the position of 16:0, is an important determinant of fat absorption in formula-fed infants (Filer et al. 1969). More recent studies have confirmed this and have shown that fecal 16:0 excretion is lower in premature infants fed formula with 16:0 predominantly esterified at the triglyceride 2-position rather than formula containing the usual oil sources of 16:0 (Carnielli et al. 1995b).
). The improved absorption of 16:0 conferred by co-randomization has been suggested to result from differences in the amount of totally saturated triglycerides in the diets. In the rat studies, the amounts of tripalmitin and dipalmitoylstearoyl glycerol were about 44 and 40% lower, respectively, when palm olein was co-randomized with coconut oil rather than blended with coconut oil (Lien et al. 1993).
). In contrast, in the study reported here, no differences were found in plasma total or HDL cholesterol among piglets fed the formula with co-randomized, randomized or native palm olein oil. The randomized and co-randomized palm olein in the study here, however, had less (about 30%) 16:0 in the triglyceride 2-position fatty acids (Table 1). Further, the enzymatic catalysis used to prepare the synthesized triglycerides resulted in relatively little 16:0 in the triglyceride 1,3 positions (Innis et al. 1993). In contrast, the chemical randomization used to prepare the randomized and co-randomized oils resulted in equal redistribution of 16:0 among all three positions in the triglyceride. Differences in the effects of dietary triglyceride 16:0 distribution on plasma cholesterol in this compared with previous (Innis et al. 1995) studies could, therefore, be explained by the difference in enrichment of 16:0 at the triglyceride 2-position or in the amount of 16:0 remaining at the 1,3 positions. Recent studies with adult humans, however, found no effect on fasting serum lipoprotein cholesterol levels after feeding diets with 28% energy from fat with 66.9% 16:0 compared with 6.4% 16:0 in the triglyceride 2-position fatty acids (Zock et al. 1995). Human and pig milk and infant formula typically contain about 50% dietary energy as fat (Innis 1992). Possibly, both the dietary triglyceride quantity and distribution of 16:0 in dietary triglycerides are important in determining any effects of 16:0 on plasma cholesterol metabolism.
) and piglets fed sow's milk (Innis et al. 1993, Table 4 of the present study). The origin of these saturated cholesteryl esters is not known. Previous studies found higher 16:0 in plasma cholesteryl ester fatty acids of piglets fed formula containing about 70% 16:0 in the triglyceride 2-position fatty acids than in piglets fed formula with 16:0 from the usual vegetable oils (Innis et al. 1993 and 1995). A recent study with premature infants (Carnielli et al. 1995a) also found higher plasma cholesteryl ester 16:0 in infants fed formula containing triglycerides with 58% rather than 4% 16:0 in the triglyceride 2-position fatty acids. In contrast, no differences in plasma or chylomicron cholesteryl ester 16:0 were found among piglets fed the randomized, co-randomized or native palm olein oils in the studies reported here. Whether the absence of any effect on cholesteryl ester 16:0 in this study is explained by the lower proportion of 16:0 in the 2-position and/or the relatively high proportion of 16:0 remaining in the 1,3 positions of the randomized and co-randomized oil triglycerides is not known.
, Yang and Kuksis 1991). Synthesis of triglycerides via the 3-glycerol phosphate pathway, on the other hand, incurs the usual preferential positioning of unsaturated fatty acids at the 2-position of the glycerol. The results of the studies reported here provide the first evidence to show that as the percentage of 16:0 in the 2-position of the plasma chylomicron triglycerides increases, presumably due to increased absorption of 2-monopalmitin, the proportion of unsaturated fatty acids decreases. Of note, the higher absorption of 2-monopalmitin in piglets fed the formula with co-randomized palm olein and canola oils was accompanied by lower 20:4(n-6) and 22:6(n-3), and higher 18:2(n-6), but not 16:0, in the chylomicron phospholipid total and 2-position fatty acids. These results suggest that the effect of dietary triglyceride structure on the chylomicron phospholipid 20:4(n-6) and 22:6(n-3) is explained by differences in the pathway of 18:2(n-6) absorption, i.e., as a monoglyceride or unesterified fatty acid, rather than a specific effect of 16:0. Little information is available on the origin of 20:4(n-6) and 22:6(n-3) incorporated into chylomicron phospholipids during the feeding of diets without 20:4(n-6) and 22:6(n-3). Potential sources include 20:4(n-6) and 22:6(n-3) secreted in bile phospholipids, uptake of systemic fatty acids, or desaturation-elongation of dietary 18:2(n-6) and 18:3(n-3) by the intestinal cells. Potentially, a decrease in chylomicron phospholipid 20:4(n-6) and 22:6(n-3) could involve inhibition of 20:4(n-6) and 22:6(n-3) synthesis, or competition by 18:2(n-6) for acylation.
FOOTNOTES
Manuscript received 13 September 1996. Initial reviews completed 19 November 1996. Revision accepted 7 March 1997.
LITERATURE CITED
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