Most dietary triacylglycerols are digested into fatty acids and 2-monoacylglycerol. They are absorbed into intestinal mucosal cells and resynthesized into triacylglycerols, which are assembled into chylomicrons and secreted into lymph. It is believed that the stereospecific structure of dietary triacylglycerols may affect their absorption from the gut, their metabolism in enterocytes and their distribution into tissues (Hoagland and Snider 1943, Mattil and Higgins 1945, Small 1991). Fatty acids at the sn-2 position of glycerol might play an important role in any effect of triacylglycerol composition on metabolism (Mortimer et al. 1988).

Human milk fat contains approximately 20% palmitic acid, with 75-80% of the palmitic acid esterified to the sn-2 position of the triacylglycerols (Breckenridge et al. 1969, Nakano et al. 1995). Earlier studies showed that, in infants fed triacylglycerols, the absorption of saturated fatty acids was higher when palmitic acid was esterified to the sn-2 position than when palmitic acid was esterified to the sn-1,3 positions (Filer et al. 1969, Fomon et al. 1970, Tomarelli et al. 1968). Recent studies also showed that palmitic acid was absorbed as sn-2 monopalmitin from milk, and overall absorption of palmitic acid was higher in rats fed palmitic acid-containing triacylglycerols resembling human milk fat (De Fouw et al. 1994, Innis et al. 1994). These reports support the hypothesis that palmitic acid in the sn-2 position can be metabolized in a different way than palmitic acid in the sn-1,3 positions. However, more data are needed to determine the metabolic importance of sn-2 monopalmitin.

Studies of the stereospecific positions of triacylglycerol fatty acids after a more complicated fat diet have been even more difficult to interpret (Small 1991). To further study the differences in fatty acid metabolism, a simplification of the system was needed. Human milk fat is also composed of many different complex triacylglycerols (Breckenridge et al. 1969, Brühl et al. 1994), but one of the major triacylglycerols in human milk fat is 1,3-dioleoyl-2-palmitoyl glycerol (OPO). Therefore our study used this simple stereospecific triacylglycerol to study the importance of sn-2 monopalmitin. The purpose of this study was to determine the metabolic characteristics of OPO. The influences of OPO and 1,2-dioleoyl-3-palmitoyl glycerol (OOP) on lymph chylomicron transport, composition and size in rats were investigated and compared.

MATERIALS AND METHODS

Table 1. Molecular species of the triacylglycerols and composition of the fatty acids in 1,3-dioleoyl-2-palmitoyl glycerol (OPO) and 1,2-dioleoyl-3-palmitoyl glycerol (OOP) preparations [View Table]

Lymph chylomicrons collected at 2-3 h after infusion were used for the measurement of apolipoprotein A-I. Apolipoprotein A-I was determined by a modification of the method of Oda and Yoshida (1994). Total protein concentrations of chylomicrons were measured by Bio-Rad Protein Assay (Bio-Rad Lab., Richmond, CA) with bovine albumin (fraction V, Sigma Chemical) as standard. Proteins in chylomicron fractions were concentrated with 150 g/L trichloroacetic acid (Wako Pure Chemicals), centrifuged at 18,000 × g (30 min, 15,000 rpm, 4°C), and washed twice with cold acetone. Precipitated proteins were dissolved in 25 µL of SDS-PAGE sample buffer containing 50 mmol/L Tris-HCl (pH 6.8), 40 g/L SDS, 120 g/L glycerol, 22.4 nmol/L bromophenol blue, and 20 mL/L 2-mercaptoethanol, heated at 90°C for 5 min, and analyzed with Tris-Tricine SDS-PAGE system (100 g/L acrylamide, Schagger and Von Jagow 1987). The gels were loaded with 100 µg of protein. All reagents for electrophoresis were purchased from Wako Pure Chemicals. The gels were stained with Coomassie brilliant blue (Wako Pure Chemicals). The proportions of apolipoprotein A-I were determined by the density of bands correspond to apolipoprotein A-I (28 kDa) using a densitometer at 595 nm (CS-930, Shimadzu, Kyoto, Japan). Apolipoprotein A-I concentrations were calculated from the total protein concentrations and the proportions of apolipoprotein A-I.

RESULTS

Lymph flow rates were similar for each group (2-3 mL/h). The time-dependent change in the transport rates was similar for each group, but the maximum transport rates (2-3 h after injection) in the OPO group were significantly higher than those in the OOP group (Fig. 1). The absorption rates of the triacylglycerols were calculated from the infused dose (200 mg) and transported triacylglycerols. The time-dependent change in the cumulative absorption rates and overall absorption rates (12 h) were significantly different between the two groups (Fig. 2); OPO was absorbed faster than OOP, and the overall absorption rates in the OPO group (80%) were higher than those in the OOP group (67%). The absorption rates of palmitic acid in the OPO and OOP groups after 12 h were 80% and 63%, respectively (P < 0.01, Fig. 3). Absorption rates of oleic acid also differed significantly. The compositions of the fatty acids in the sn-2 position of the chylomicron triacylglycerols are shown in Table 2. The OPO preparation contained triacylglycerols with 76.6% of the palmitic acid in the sn-2 position, whereas 100% of the oleic acid was esterified to the sn-2 position in the OOP preparation. Most (60-70%) of these fatty acids were transported at the original position of the triacylglycerols. The major molecular species were dioleoyl-palmitoyl-glycerol, dipalmitoyl-oleoyl-glycerol and triolein (Table 3). These molecular species were directly influenced by the molecular species of the infused triacylglycerols. However, the level of dioleoyl-palmitoyl-glycerol in the OPO group was significantly higher than that in the OOP group. In contrast, the level of triolein in the OOP group was significantly higher than that in the OPO group.

Table 2. Composition of the fatty acids in the sn-2 position of the chylomicron triacylglycerols collected at 2-3 h after infusion of the experimental emulsions containing 1,3-dioleoyl-2-palmitoyl glycerol (OPO) or 1,2-dioleoyl-3-palmitoyl glycerol (OOP)1 [View Table]

Table 3. Molecular species of the triacylglycerols in the chylomicrons collected at 2-3 h after infusion of the experimental emulsions containing 1,3-dioleoyl-2-palmitoyl glycerol (OPO) or 1,2-dioleoyl-3-palmitoyl glycerol (OOP)1 [View Table]

The transport rates for triacylglycerols at 2-3 h after infusion in the OPO group were higher than those in the OOP group (Table 4). The transport rates for apolipoprotein A-I did not differ between groups, whereas the mean diameter of the chylomicrons in the OPO group was significantly larger than that for the OOP group.

Table 4. Transport rates of the triacylglycerols and apolipoprotein A-I, and particle sizes of the chylomicrons collected at 2-3 h after infusion into rats of the experimental emulsions containing 1,3-dioleoyl-2-palmitoyl glycerol (OPO) or 1,2-dioleoyl-3-palmitoyl glycerol (OOP)1 [View Table]

DISCUSSION

We also showed that OPO is transported faster than OOP through the secretion of larger chylomicrons into lymph. The influence of the distribution of saturated fatty acids in human milk triacylglycerols on chylomicron triacylglycerol composition or turnover in infants is still unknown (Innis et al. 1995). Our results show that the particle sizes of the chylomicrons and transport rates of the chylomicron triacylglycerols in rats infused OPO and OOP are different. These facts suggest a specific effect on chylomicron metabolism. It has been reported that chylomicrons of different size were metabolized differently (Quardfort and Goodman 1966). Recent studies also showed that the differences in particle size may have contributed to the metabolic effects (Redgrave et al. 1988) and that larger chylomicrons were metabolized faster than smaller chylomicrons (Levy et al. 1991). The positional distribution of palmitic acid may affect the subsequent metabolism of lymph chylomicrons, but further infusion studies will be needed to show this. Levy et al. (1991) concluded that differences in chylomicron removal are not related to apolipoprotein composition. Our results show that the transport rates of apolipoprotein A-I in the chylomicrons of OPO and OOP are similar. It has been reported that apolipoprotein A-I is a major protein component of chylomicrons in rat mesenteric lymph (Fainaru et al. 1976, Imaizumi et al. 1978). Our experiments demonstrated that the number of the chylomicron particles is unaffected by the positional distribution of the dioleoyl-palmitoyl glycerols.

In our study, a bile salt-phospholipid emulsion was used for intragastric infusion to prepare the stable emulsion. An infusion of a bile salt into the stomach may affect stomach tissues. However, we considered that this potential injury might not be serious because our study did not involve continuous infusion. It was conducted to compare the transport rates of the OPO and OOP emulsions containing a bile salt at a same concentration.

The purity of the OPO and OOP preparations was 65.7 g/100 g. We considered that the comparison of OPO and OOP at this level of purity would be possible, because the concentrations of OOO in both preparations were comparable.

Kalogeris and Story (1992) reported that at least 4 h of continuous infusion is required to establish steady-state triacylglycerol transport. A steady-state condition would be one of the best models to study differences in the efficiency of absorption between saturated and unsaturated fatty acids in normal intestinal triacylglycerol transport (Bergstedt et al. 1990, Ockner et al. 1972, Renner et al. 1986). However, we used a non-steady-state model (Feldman et al. 1983) to measure the transport rates of triacylglycerols containing similar fatty acid compositions but having different positional distribution. Human infants are usually fed human milk or infant formula for short periods at intervals of several hours. Therefore, the model of a short-term infusion to rats may better explain the metabolic importance of OPO in human milk, and the results might be more applicable to human infants than those of the continuous infusion model.

Puppione et al. (1982) mentioned that triacylglycerol-rich particles, isolated from bovine lymph, contains a high proportion of saturated fatty acid triacylglycerols that were dense and irregularly shaped and that scattered light anomalously. It was demonstrated that these particles were crystalline triacylglycerols (Small et al. 1980). Clark et al. (1982) suggested that cooling triacylglycerol-rich particles to 16°C could initiate crystallization of the triacylglycerols and alter the shape and density of the particles. Florén and Nilsson (1977) also noted that chylomicrons from butter-fed rats, isolated at low temperatures, behaved differently from chylomicrons isolated from corn oil-fed animals. To avoid the alterations associated with the partial crystallization of saturated core lipids, the chylomicrons in our study were prepared at 25-27°C. Observed chylomicrons were regularly shaped and scattered light normally (electronmicrographs not shown).

In conclusion, these results indicate that OPO is absorbed and transported more effectively than OOP. Further studies of the effect of long-term consumption and of the metabolic difference between steady-state and non-steady-state infusion are needed to confirm our conclusion.