The distribution and composition of fatty acids in dietary triacylglycerols are important determinants of fat digestion and absorption. Human milk fatty acids contain about 20-30% palmitic acid (16:0), with over 70% of the 16:0 esterified in the sn-2 position of the milk triacylglycerols (Breckenridge et al. 1969, Christie and Clapperton 1982, Martin et al. 1993). In contrast, the 16:0 in vegetable oils and non-milk fats is predominately esterified at the sn-1 and 3 positions, and the unsaturated fatty acids oleic acid (18:1) and linoleic acid [18:2(n-6)] are esterified at the sn-2 position of the glycerol molecule (Mattson and Lutton 1958). In milk, on the other hand, a substantial proportion of the 18:1, 18:2(n-6) and about half of the arachidonic acid [20:4(n-6)] and docosahexaenoic acid [22:6(n-3)] are esterified in the sn-1,3 positions of the triacylglycerols (Martin et al. 1993).

Intraluminal hydrolysis of dietary triacylglycerols by endogenous lipases releases sn-2 monoacylglycerols and unesterified fatty acids from the 1 and 3 positions (Small 1991). The 2-monoacylglycerol and unesterified fatty acid products are then absorbed and reassembled in the enterocytes to triacylglycerols for secretion in chylomicrons. The positional distribution of fatty acids in dietary triacylglycerols thus determines whether fatty acids are absorbed as 2-monoacylglycerols or unesterified fatty acids. In the fed state, the 2-monoacylglycerol pathway, which involves re-esterification of the glycerol 1 and 3 positions by monoacylglycerol transferase, then diacylglycerol acyl transferase, predominates in the synthesis of triacylglycerols (Small 1991). This pathway results in formation of triacylglycerols in which the fatty acid in the sn-2 position is the same as in the dietary fat, and little specificity is shown toward the unesterified fatty acids that are re-esterified to the sn-1 or 3 positions (Myher et al. 1985, Small 1991). Estimates from studies in rodents have suggested that, under normal circumstances, the monoacylglycerol and 3-glycerophosphate pathways contribute about 80 and 20%, respectively, to chylomicron triacylglycerol formation in the enterocytes (Yang and Kuksis 1991).

Long-chain unesterified saturated fatty acids such as 16:0 are not well absorbed from the lumen, in part because of melting points substantially above body temperatures and a strong tendency to form insoluble soaps with divalent cations, such as calcium and magnesium, at the alkaline pH of the small intestine (Barnes et al. 1974, Filer et al. 1968, Innis et al. 1993, Jensen et al. 1986, Tomarelli et al. 1968). The specific positioning of most of the 16:0 at the sn-2 position of milk triacylglycerols has been suggested to be one of the reasons for the high efficiency of absorption of fat from human milk (Filer et al. 1968, Tomarelli et al. 1968). However, others have hypothesized that the milk enzyme, bile salt-stimulated lipase completes the hydrolysis of milk fat to glycerol and free fatty acids (Bernback et al. 1990), inferring that the 16:0 provided by milk is absorbed as an unesterified fatty acid. The recent demonstration of a high proportion of 16:0 in the plasma triacylglycerol sn-2 position of breast-fed infants, in contrast, suggests that bile salt-stimulated lipase does not quantitatively hydrolyze 2-monopalmitin in vivo (Innis et al. 1994a). Similar to human infants, piglets fed sow's milk have higher proportions of 16:0 in the sn-2 position of plasma triacylglycerols than piglets fed formula containing vegetable oil sources of 16:0 (Innis et al. 1995). Sow's milk, like that of the human and many other mammalian species, contains most of the 16:0 esterified at the sn-2, rather than sn-1, 3 positions of the triacylglycerols (Parodi 1982).

Only limited information is available on the physiological importance of the composition of sn-2 monoacylglycerols absorbed by the intestine. Intravascular clearance of triacylglycerols involves hydrolysis by lipoprotein lipase and/or hepatic lipase to form 2-monoacylglycerols and unesterified fatty acids (Small 1991). Plasma triacylglycerol hydrolysis and uptake of remnants by the liver is slower for triacylglycerols with 16:0 rather than an unsaturated fatty acid at the sn-2 position (Mortimer et al. 1992, Redgrave et al. 1988). In previous studies, piglets fed formula with about 70% 16:0 in fatty acids at the triacyglycerol sn-2 position had higher fasting plasma total and HDL cholesterol and higher cholesteryl 16:0 than piglets fed formula with similar amounts of 16:0, but with <5% 16:0 in the milk triacylglycerol 2-position fatty acids (Innis et al. 1993). Whether the positioning of 16:0 at the sn-2 position influences cholesterol metabolism in breast-fed infants is not clear. Similarly, studies have not yet considered if differences in pathways of absorption of dietary fatty acids, as monoacylglycerols or unesterified fatty acids, contribute to the differences in blood lipid levels of 18:1, or (n-6) and (n-3) fatty acids in formula-fed compared with breast-fed infants (Innis et al. 1994b, Makrides et al. 1993, Ponder et al. 1992) and piglets (Hrboticky et al. 1990). These studies, therefore, undertook to determine if the distribution of fatty acids in formula triacylglycerols influences the composition and distribution of fatty acids in serum chylomicron, LDL and HDL lipids of formula-fed piglets, with comparison to a reference group of piglets fed milk. Because of the possibility that dietary cholesterol might influence the response of plasma lipoprotein fatty acids to triacylglycerol fatty acid distribution, each formula was studied with and without added cholesterol. The study included analyses of chylomicron, LDL and HDL triacylglycerols, phospholipids and cholesteryl ester fatty acids, and of fatty acids in the 2-position of chylomicron triacylglycerols and phospholipids in postprandial plasma. Most plasma lipoprotein cholesteryl esters are considered to result from intravascular synthesis via the lecithin cholesterol acyl transferase (LCAT) reaction, which in humans and pigs, involves esterification of free cholesterol using the fatty acid from the 2-position of HDL phospholipid (Gloset 1979, Grove and Parnall 1991, Lui et al. 1995). The composition of HDL phospholipid 2-position fatty acids was thus also analyzed to provide information on possible reasons for the differences in cholesteryl ester fatty acids that accompany formula and milk feeding (Innis et al. 1993, 1994a and 1994b).

MATERIALS AND METHODS

Table 1. Fatty acid composition of formulas with different saturated fatty acids and triacylglycerol (TG) structures 1 [View Table]

Newborn male piglets of birth weight >1 kg were obtained from Peter Hill Holdings, Langley, BC, Canada within 12 h of birth. The piglets were randomly assigned to one of the four formulas, 6 piglets each, and bottle-fed (Hrboticky et al. 1990, Innis et al. 1995) until 0600 h on d 18 after birth. The piglets were bottle-fed by hand to appetite every 1.5-2 h for the first 5 d, then every 3 h from 0600-2400 h. Passive immunity was provided by addition of colostrum-derived immunoglobulins (La Belle Associates, Bellingham, WA) to the formula fed during the first 72 h after birth. Six piglets were fed by their natural mothers and were studied at a similar age and time after the last feed. The amount of milk received by the milk-fed piglets was not controlled or recorded. Littermates were not assigned to the same diet group.

At 18 d of age and 4 h after consuming 60 mL formula, the piglets were anesthetized with ketamine/rompun (37.5 and 3.75 mg/kg, MTC Pharmaceuticals, Cambridge, ON, Canada and Chemagro, ON, Canada, respectively), by intramuscular injection. Blood samples were drawn by cardiac puncture into tubes containing EDTA (150 mg EDTA/L in 9 g NaCl/L) as the anticoagulant. Plasma was prepared by low speed centrifugation at 2000 × g for 15 min and, with the exception of aliquots for HDL cholesterol, the samples were frozen at 80°C. All of the procedures involving the piglets were approved by the Animal Care Committee of the University of British Columbia and conformed to the guidelines of the Canadian Council on Animal Care.

The triglyceride-rich chylomicrons were isolated from plasma at d = 1.006 kg/L after ultracentrifugation at 141,000 × g for 60 min. LDL and HDL (d > 1.063-1.21 g/L) were then separated and recovered (Redgrave et al. 1975) by further ultracentrifugation at 141,000 × g in a SW28 rotor, 66 h at 15°C. The small band corresponding to VLDL in the plasma collected 4 h after feeding was not analyzed. The absence of apo A and apo B in the LDL and HDL fractions, respectively, was confirmed by SDS-polyacrylamide gel electrophoresis (Maguire et al. 1989).

Lipoprotein total lipids were extracted, and the phospholipids, cholesteryl esters and triacylglycerols separated on TLC plates (Hrboticky et al. 1990). Fatty acid components were converted to their respective methyl esters with 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 triacylglycerols and cholesteryl esters, respectively (Innis et al. 1993). Analysis of the composition of fatty acids at the sn-2 position of separated triacylglycerols and phospholipids used porcine pancreatic lipase and phospholipase A₂, respectively (EC 3.1.1.3 Type 11, crude, and EC 3.1.1.4, respectively, Sigma Chemical, St. Louis, MO) as previously described (Innis et al. 1994a and 1995). After the enzyme reactions, the samples were extracted, the monoacylglycerols separated by TLC and recovered, and the fatty acids converted to their respective methyl esters. Fatty acid methyl esters were separated and quantified using capillary GLC (Innis et al. 1994a). Milk and formula fatty acids were analyzed without solvent extraction (Lepage and Roy 1987) as in previous studies (Innis et al. 1994a).

RESULTS

The addition of ~0.6mmol/L cholesterol to the formula resulted in significantly higher 18:2(n-6), but had no significant effect on the level or distribution of other fatty acids in the chylomicron triacylglycerols. The formula triacylglycerol fatty acid distribution, however, influenced both the composition and distribution of chylomicron triacylglycerol saturated, mono-, (n-6) and (n-3) unsaturated fatty acids in the formula-fed piglets. Of note, although the distribution of 16:0 in the formula triacylglycerols had no significant effect on the percentage of 16:0 in the chylomicron triacylglycerol total fatty acids (range of means, 21.7-22.9% 16:0), the enrichment of 16:0 in the 2 position was significantly and substantially higher in piglets fed the formula with synthesized triacylglycerols (range of means 17.4-19.3% 16:0) than in piglets fed the palm-olein formulas (4.0-5.6% 16:0, Fig. 1). As a group, piglets fed the synthesized triacylglycerol formulas had significantly higher 18:1 and lower 18:3(n-3), 20:4(n-6) and 22:6(n-3) in their chylomicron triacylglycerol total fatty acids, and higher 18:1 but lower 18:2(n-6) and 20:4(n-6) in the 2-position fatty acids than piglets fed the palm-olein formulas. Analyses of the interaction between the dietary triacylglycerol source and cholesterol content, however, showed that synthesized triacylglycerols resulted in significantly lower 20:4(n-6) and 22:6(n-3) only in the absence of added cholesterol (Fig. 1).

As for the triacylglycerols, the chylomicron phospholipids of the piglets fed formula had significantly lower 16:0 and higher 18:0 than the piglets fed sow's milk (Fig. 2), with the exception of 16:0 in the group fed the synthesized triacylglycerol formula without cholesterol (Fig. 2). Piglets fed the synthesized triacylglycerol formula without cholesterol, but not piglets fed the same formula with cholesterol or piglets fed the formulas with palm-olein oil, also had significantly lower chylomicron phospholipid 20:4(n-6) than piglets fed sow's milk. As noted previously, piglets fed the synthesized triacylglycerol formula without cholesterol also had significantly lower 20:4(n-6) in chylomicron triacylglycerol fatty acids than piglets fed the other formulas. The percentage of 20:4(n-6) in the chylomicron phospholipid fatty acids was significantly lower in all of the formula-fed piglets than in those fed sow's milk; the reduction in 20:4(n-6) was particularly pronounced in the phospholipid 2-position (Fig. 2).

Piglets fed the formula with added cholesterol had significantly lower 16:0 and higher 20:4(n-6) in chylomicron phospholipid total and sn-2 position fatty acids than piglets fed the same formula without added cholesterol. Piglets fed the formulas with synthesized triacylglycerols, on the other hand, had significantly higher 16:0 in phospholipid total fatty acids, and higher 16:0 and 18:0 and lower 18:1 and 18:2(n-6) in sn-2 position fatty acids than piglets fed the palm-olein formula (Fig. 2).

Table 2. Plasma LDL triacylglycerol (TG) and phospholipid (PL) fatty acids of piglets fed formula with palm olein or synthesized TG without () or with (+) cholesterol or sow's milk1 [View Table]

The addition of cholesterol to the formula was associated with significantly higher 20:4(n-6) in the LDL phospholipid fatty acids, but had no other significant effect on the LDL triacylglycerol or phospholipid fatty acids. The LDL phospholipid 16:0 was significantly higher, but 20:4(n-6) and 22:6(n-3) were lower in piglets fed the synthesized triacylglycerol formulas than in piglets fed the palm-olein formula (Table 2).

Table 3. Plasma HDL triacylglycerol (TG) and phospholipid (PL) fatty acids of piglets fed formula with palm olein or synthesized TG without () or with (+) cholesterol or sow's milk1 [View Table]

The composition of fatty acids esterified to the HDL phospholipid 2-position was analyzed to gain further understanding of the possible reason for differences in plasma cholesteryl ester fatty acids in infants (Innis et al. 1994a) and piglets (Innis et al. 1993 and 1995) fed milk and formula. The piglets fed formula all had significantly lower 16:0 and higher 18:2(n-6) in HDL phospholipid sn-2 position fatty acids than piglets fed sow's milk (Fig. 3). There were no differences, however, in 16:0 or 18:2(n-6) among piglets fed the palm-olein and synthesized triacylglycerol formulas. Levels of 18:1, in contrast, were significantly higher in piglets fed the formulas with palm olein than in piglets fed sow's milk, or the formulas with synthesized triacylglycerols. The addition of cholesterol to the formulas had no effect on the composition of the HDL phospholipid 2-position fatty acids in the formula-fed piglets.

As found previously (Innis et al. 1993), the cholesteryl ester fatty acid composition of the formula-fed piglets was influenced by the formula triacylglycerol fatty acid distribution. In this study, piglets fed the formulas with synthesized triacylglycerols had significantly higher 16:0 in the chylomicron, LDL and HDL cholesteryl esters than piglets fed the formulas with palm olein (Fig. 4). The significantly higher chylomicron and HDL cholesteryl ester 16:0 of piglets fed the formulas with synthesized triacylglycerols was accompanied by lower 18:1, but in the LDL cholesteryl esters, the higher 16:0 was accompanied by lower 18:3(n-3), 20:4(n-6) and 22:6(n-3). The inclusion of cholesterol in the formulas was associated with significantly higher 18:1 and lower 22:6(n-3) in the chylomicron, but not LDL or HDL cholesteryl esters of the formula-fed piglets.

DISCUSSION

The physiological significance of the unusual positioning of 16:0 at the sn-2 position of milk triacylglycerols has been the subject of some uncertainty. Early studies suggested that the absorption of 16:0 in 2-monoacylglycerols, following gastric and pancreatic lipase hydrolysis of fatty acids from the triacylglycerol sn-1 and 3 positions (Small 1991), may be one of the reasons for the high absorption of fat from milk (Filer et al. 1969, Tomarelli et al. 1969). Subsequent in vitro studies showing that milk bile salt-stimulated lipase can complete the hydrolysis of milk triacylglycerols to glycerol and free fatty acids (Bernback et al. 1990) suggested that 16:0 may be released in the intestinal lumen as an unesterified fatty acid. However, the absorption of 16:0 is relatively low compared with shorter-chain saturated, or monounsaturated and polyunsaturated fatty acids (Jensen et al. 1986). This laboratory recently provided circumstantial evidence that 16:0 is absorbed as a 2-monoacylglycerol in milk-fed piglets (Innis et al. 1995) and human infants (Innis et al. 1994) through demonstration of relatively high amounts of 16:0 in the plasma triacylglycerol 2-position after feeding. The study reported here shows that ~80% of the 16:0 in the milk triacylglycerol 2-position (~55% 16:0 in milk triacylglycerol sn-2 fatty acids, Table 1) was recovered in the piglet chylomicron triacylglycerol 2-position (44.9 ± 4.9% 16:0, Fig. 1). This is surprisingly consistent with estimates that re-esterification of 2-monoacylglycerols via the monoacylglycerol pathway accounts for ~80% of triacylglycerol synthesis in intestinal cells in the fed state, with the remaining 20% of triacylglycerol synthesis proceeding via the -glycerophosphate pathway (Small 1991).

The positional distribution of fatty acids in dietary triacylglycerols clearly involves not only potential effects with regard to the fatty acids absorbed as 2-monoacylglycerols, but also has implications for re-esterification, transport and tissue delivery of fatty acids absorbed as unesterified fatty acids. The enrichment of 16:0 in the 2-position of milk and formula triacylglycerols involves redistribution of 18:1, 18:2(n-6) and 18:3(n-3) to the 1,3-positions. Feeding the formulas with synthesized triacylglycerols, which had a similar composition but different triacylglycerol arrangement of fatty acids than that of the palm-olein formulas, resulted in lower 20:4(n-6) and 22:6(n-3) in chylomicron triacylglycerols and LDL phospholipids and cholesteryl esters, and lower 20:4(n-6) in chylomicron phospholipids. Possibly, the decrease in 20:4(n-6) and 22:6(n-3) in chylomicron triacylglycerols and cholesteryl esters, and particularly of 20:4(n-6) in chylomicron phospholipids of piglets fed the synthesized triacylglycerols, was due to competition by 18:1, 18:2(n-6) and/or 18:3(n-3) absorbed as unesterified fatty acids with 20:4(n-6) and 22:6(n-3) for acylation to the 2-position of triacylglycerols or phospholipids formed via the -glycerophosphate pathway. Reduced synthesis of 20:4(n-6) and/or 22:6(n-3) via desaturation/elongation of 18:2(n-6) and 18:3(n-3) may be an alternate explanation. Possibly, this effect of dietary triacylglycerol fatty acid distribution was not seen in piglets fed sow's milk, nor is it seen in breast-fed infants, because milk provides 20:4(n-6) and 22:6(n-3) in both the phospholipids and in the sn-2 and sn-3 positions of the milk triacylglycerols (Martin et al. 1993).

Previous studies with piglets fed formula containing ~70% 16:0 in the triacylglycerol sn-2 position fatty acids found a small, but significantly higher (~0.34 mmol/L) fasting plasma HDL cholesterol, and twofold higher plasma cholesteryl ester 16:0 than in piglets fed a formula with <5% 16:0 in the triacylglycerol sn-2 position fatty acids. No differences were found in postprandial plasma cholesterol between piglets fed formula with ~32% 16:0 compared with <5% 16:0 in the triacylglycerol 2-position in the studies reported here. Similarly, fasting serum lipoprotein concentrations were not different in adult men and women consuming diets with 28% daily energy from fat with 6.4% compared with 66.9% 16:0 in the triacylglycerol sn-2 position fatty acids (Zock et al. 1995). The formula fed to the piglets in these (Table 1) and previous (Innis et al. 1993) studies had 50-55% dietary energy as fat. Possibly, the potential effects of dietary triacylglycerol fatty acid distribution on plasma cholesterol depend on both the quantity (percentage of energy) and enrichment of 16:0 in the 2-position of the dietary triacylglycerols.

The studies reported here confirm earlier work (Innis et al. 1993) showing that dietary triacylglycerol fatty acid distribution is an important determinant of plasma cholesterol ester fatty acids, at least in piglets. Cholesteryl palmitate was increased in all of the major serum lipoprotein fractions of piglets fed the formulas with synthesized triacylglycerols, compared with piglets fed formulas containing similar amounts of 16:0 but from palm olein. In these studies, piglets fed sow's milk with ~55% 16:0 in the milk triacylglycerol 2-position fatty acids had higher lipoprotein cholesteryl 16:0 than piglets fed the formula with synthesized triacylglycerols containing ~32% 16:0 in the 2-position fatty acids. Palmitic acid (16:0) represented about 24, 16 and 13% of the chylomicron cholesteryl ester fatty acids in piglets fed sow's milk, or the formulas with synthesized triacylglycerols and palm olein, respectively. In previous studies, the plasma cholesteryl esters of piglets fed sow's milk, synthesized triacylglycerols with 70% 16:0 in the 2-position, or formula with palm-olein oil had (mean ± SEM) 20.5 ± 0.5, 21.6 ± 0.5 and 12.2 ± 0.2% 16:0, respectively. These results suggest a direct relationship between the amount of 16:0 in the 2-position of dietary triacylglycerols and the proportion of saturated cholesteryl esters in plasma lipoproteins in piglets. Similarly, recent studies with premature infants and with adult men and women have found higher plasma cholesteryl 16:0 levels with diets containing synthesized triacylglycerols than with diets containing 16:0 from the usual vegetable oils (Carnielli et al. 1995, Zock et al. 1996). The effect of dietary synthesized triacylglycerols on cholesteryl ester fatty acids in humans, however, seems to be of smaller magnitude than in piglets; but the specific effects of dietary fatty acid distribution on the composition of cholesteryl esters in individual lipoproteins in humans are not known.

The pathway(s) by which the dietary triacylglycerol fatty acid distribution influences the composition of plasma cholesteryl esters is not known. Fatty acids esterified to the 2-position of HDL phospholipids are considered a major source of fatty acids for cholesteryl esters formed by LCAT (EC 2.3.1.43) (Glomset 1979). It has been suggested that in native plasma, LCAT prefers 18:2(n-6) > 18:1 > 22:6(n-3) > 20:4(n-6) at the 2-position, and 16:0 > 18:1 > 18:0 at the 1-position of plasma phospholipid (Subbaiah and Monshizadegan 1988). In the studies reported here, the HDL cholesteryl esters had a pattern of 16:0 enrichment similar to that of the dietary triacylglycerol 2-position, i.e., piglets fed sow's milk > synthesized triacylglycerol formulas > palm-olein formulas. The composition of HDL phospholipid 2-position fatty acids did not correspond with the lipoprotein cholesteryl ester fatty acids. Of particular relevance, 16:0 represented only 4.0-6.6% of the HDL phospholipid 2-position fatty acids in piglets fed the milk and formula diets. Further, 20:4(n-6) represented about 20-22% and 22:6(n-3) represented about 4.9-5.5% of the HDL phospholipid 2-position fatty acids, whereas the HDL cholesterol esters had only 3.1-4.1% 20:4(n-6) and 0.2% 22:6(n-3). A similar discrepancy, with lower 16:0 and higher 20:4(n-6) in the sn-2 acyl groups of plasma phospholipids than in cholesteryl esters has been noted in studies with human plasma (Subbaiah et al. 1992). Results from in vitro studies have indicated that a large proportion of cholesteryl esters formed from phospholipids with 20:4(n-6) or 22:6(n-3) in the 2-position derive their fatty acyl moiety from the sn-1 acyl group, possibly as a result of steric restrictions, favoring against formation of cholesteryl 20:4(n-6) or 22:6(n-3) (Subbaiah and Monshizadegan 1992). It has also been suggested that the increased 16:0 in cholesteryl esters after fish oil feeding (Holub et al. 1987, Illingsworth et al. 1984, Parks et al. 1989) is derived from the sn-1 position as a result of increased 22:6(n-3) at the sn-2 position of plasma phospholipids (Subbaiah and Monshizadegan 1988). Indeed, in vitro, 22:6(n-3) seems to be a poor substrate for LCAT (Subbaiah et al. 1993). Differences in enrichment of 20:4(n-6) or 22:6(n-3) in the HDL phospholipid sn-2 position fatty acids among piglets fed the different formulas or sow's milk in the studies reported here, however, do not correspond with the consistently higher 16:0 in the lipoprotein cholesteryl esters in the order sow milk > formulas with synthesized triacylglycerols > formulas with palm olein. In these studies, the plasma lysophospholipid fatty acids of piglets fed sow's milk, the palm-olein formula without and with cholesterol, or the synthesized triacylglycerol formula without and with cholesterol had (mean) 43.2, 32.7, 32.9, 40.4 and 40.9% 16:0 (pooled SEM = 1.8), and 1.8, 1.7, 2.5, 0.8 and 0.9% 20:4(n-6) (pooled SEM = 0.2), respectively. The high 16:0 and very low 20:4(n-6) in the plasma lysophospholipid compared with 20-25% 16:0 and 10.0-11.6% 20:4(n-6) in the HDL phospholipid fatty acids are also not readily compatible with a hypothesis that cholesteryl 16:0 was derived utilizing 16:0 from the HDL phospholipid 1-position.

Although cholesterol is taken up into the enterocyte unesterified, it is secreted largely in the esterified form. The relationship between the enrichment of 16:0 in the 2-position of the milk and formula triacylglycerols and the enrichment in the chylomicron cholesteryl esters suggests the possibility that the absorbed 2-monoacylglycerols might be used as a substrate for esterification of cholesterol in piglet enterocytes. No experimental evidence to support this suggestion is available. In this regard, it seems possible that differences in pathways of cholesteryl esterification, or in the contribution of cholesteryl esters from intracellular compared with intravascular synthesis to the plasma pool, may explain the higher plasma cholesteryl 16:0 in response to feeding milk or triacylglycerols enriched in 16:0 in the sn-2 position in piglets (Fig. 4, Innis et al. 1993) than in infants (Carnelli et al. 1995) or adults (Zock et al. 1996).

In summary, these studies provide substantial evidence that the positional distribution of fatty acids in milk and formula triacylglycerols determines the route of fatty acid absorption, as 2-monoacylglycerols or unesterified fatty acids, and the distribution of plasma chylomicron triacylglycerol fatty acids. Of note, differences in lipoprotein triacylglycerol fatty acid distributions can be present despite the absence of differences in composition of triacylglycerol total fatty acids. These studies have also indicated that, at least for piglets, the digestion of milk triacylglycerols involves hydrolysis of fatty acids esterified in the 1 and 3 positions, with little if any hydrolysis of 16:0 from the triacylglycerol 2-position. Further, in the absence of a dietary intake of 20:4(n-6) and 22:6(n-3), redistribution of 18:1, 18:2(n-6) and 18:3(n-3) to the 1,3-positions of dietary triacylglycerols is associated with decreased chylomicron triglyceride 20:4(n-6) and 22:6(n-3). The sn-2 positioning of 16:0 in milk and in synthesized triacylglycerols also seems to be related to plasma lipoprotein levels of saturated (16:0) cholesteryl esters, and these seem most likely to be derived from intracellular rather than intravascular sources. The physiological significance of these findings, for example, involving differences in rates of triacylglycerol turnover (Mortimer et al. 1992, Redgrave et al. 1988), or fatty acid delivery to specific tissues or their relevance to infant nutrition has yet to be determined.