Dietary Supplementation with Arachidonic and Docosahexaenoic Acids Has No Effect on Pulmonary Surfactant in Artificially Reared Infant Rats Yu-Yan Yeha,*, Kerry Anne Whitelocka, Shaw-Mei Yeha, and Eric L. Lienb a
Department of Nutrition, The Pennsylvania State University, University Park, Pennsylvania 16802, and b Wyeth Nutritionals International, Philadelphia, Pennsylvania 19101
ABSTRACT: Despite the potential use of long-chain polyunsaturated fatty acid (LCPUFA) supplementation to promote growth and neural development of the infant, little is known about potential harmful effects of the supplementation. The present study determined whether supplementation with arachidonic acid (AA) and/or docosahexaenoic acid (DHA) in rat milk formula (RMF) affects saturation of pulmonary surfactant phospholipids (PL). Beginning at 7 d of age, infant rats were artificially fed for 10 d with RMF supplemented with AA at 0, 0.5, and 1.0% of total fatty acid, or supplemented with DHA at 0, 0.5, and 1.0%, or cosupplemented with AA and DHA at levels of 0:0, 0.5:0.3, and 1.0:0.6% of the fat blend. Lung tissue PL contained 43 weight percent palmitate (16:0) of total fatty acids in infant rats fed the unsupplemented RMF. The supplementation with AA at both 0.5 and 1.0% decreased the weight percentage of 16:0 and stearate (18:0), indicating a decrease in saturation of PL. The observed decreases were accompanied by increases in AA and linoleic acid (18:2n-6). Surfactant phosphatidylcholine (PC) consisted of 71 weight percent 16:0 in the unsupplemented group, and this highly saturated PC was not altered by the cosupplementation with AA and DHA although there was a slight increase in DHA. Similarly, the cosupplementation did not change fatty acid composition of surfactant PL when compared with the unsupplemented group. The cosupplementation slightly decreased the weight percentage of 16:0 with a proportional increase in 18:0 leading to an unchanged weight percentage of total saturated fatty acids. These results suggest that, unlike lung tissue PL, the composition of saturated fatty acids in surfactant PL, particularly PC, is resistant to change by dietary AA and DHA supplementation. This, together with the unchanged concentration of total fatty acids in surfactant PC, indicates that LCPUFA cosupplementation causes no effect on pulmonary surfactant. Paper no. L7980 in Lipids 34, 483–488 (May 1999).
Pulmonary surfactant is composed of lipoprotein complexes rich in phospholipids (PL), especially phosphatidylcholine (PC) (1). Among lipids, disaturated phosphatidylcholine (DSPC) accounts for approximately two-thirds of the total PC and is the major metabolically active component of lung surfactant (2). Surfactant lipids stabilize pulmonary alveoli and prevent alveolar collapse by reducing surface tension at the air-liquid interface (3). An insufficient amount of surfactant causes respiratory distress syndrome in both newborns and adults (4,5). Surfactant content in alveoli is regulated by its synthesis and secretion by alveolar type II cells (6,7). The synthesis of PL, on the other hand, is dependent on the availability of fatty acids (8,9). Although fatty acid synthesized de novo contributes about 40% of DSPC production (8), dietary fatty acids presented to tissue via the circulation play an important role in modulating the quantity and composition of surfactant lipids (10). Similar to tissues such as liver, heart, kidney and brain, lung tissue fatty acid content is responsive to dietary lipids (11–16). Thus, it is not surprising that supplementation with long-chain polyunsaturated fatty acids (LCPUFA) of the n-6 and n-3 families—including arachidonic acid (AA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3), and docosahexaenoic acid (DHA, 22:6n-3)—increased the corresponding fatty acids and increased the level of total unsaturated fatty acids of lung tissue PL (11–14). Despite the extensive studies with lung tissue little is known about the impact of dietary LCPUFA on surfactant PL. DHA and AA are integral components of retina and neural tissue and are considered to be essential for growth and development in humans and animals (17,18). Accordingly, supplementation of LCPUFA has been studied to determine if dietary DHA and AA benefit neurodevelopment of human infants (19–24). In these studies, such supplementation increased LCPUFA concentrations in plasma and erythrocytes of human infants (19–24). Animal studies, on the other hand, showed that aside from an increase in LCPUFA in blood, the supplementation enhanced accretion of the fatty acids in the brain and liver leading to increased unsaturation of PL (16,25,26). Since increased unsaturation of surfactant PC could potentially perturb the respiratory function of pul-
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monary surfactant (1), the present experiments were undertaken to determine whether LCPUFA supplementation in milk formula alters fatty acid composition and the degree of saturation of surfactant PL. To this end infant rats were artificially fed milk formula via an intragastric tube as described previously (16). The results showed that, unlike lung tissue PL, the fatty acid composition of surfactant PC and other PL is resistant to change by dietary AA and DHA. MATERIALS AND METHODS Animals. Pregnant Sprague-Dawley rats obtained from Harlan Sprague-Dawley (Indianapolis, IN) were housed in individual plastic containers. Water and food (Purina Rat Chow, Ralston Purina, St. Louis, MO) were available to the dams at all times. For each experiment, newborns from four pregnant rats were delivered naturally, culled to 10 pups per dam within 24 h postpartum, and were nourished by their dams until the beginning of artificial rearing (AR). At 7 d of age, one or two weight-matched rat pups from each litter were assigned to one of three AR groups. Thus, each group consisted of rat pups derived from four dams. In the first series of experiments, three groups of infant rats were fed rat milk formulas (RMF) which contained 0, 0.5, and 1.0% AA as a percentage of the total fatty acids. The second experiment featured three RMF that contained 0, 0.5, and 1.0% DHA. In the third series of experiments infant rats were fed RMF cosupplemented with AA and DHA at concentrations of 0:0, 0.5:0.3, or 1.0:0.6% for AA and DHA, respectively. AR system. Artificially reared infant rats were used throughout the study. The detailed account of AR has been previously described (16). Briefly, at the age of 7 d infant rats were permanently implanted with intragastric cannulae, connected to a semiautomatic feeding pump, and fed intragastrically for 15 min each hour. The intragastric cannulation on day 7 after birth was essential to ensure the survival of the pups during the course of AR. The pups were weighed daily between 0930 and 1100 in the animal care room. All animal care and surgical procedures were in accordance with protocols established by the Institutional Animal Care and Use Committee of The Pennsylvania State University. The RMF were adopted from that of Auestad et al. (27). As described in the previous study (16), all formulas contained 11.8% fat (w/w) consisting of fat from milk formula base (3.4%), medium-chain triglycerides (2.8%), and corn oil plus microbial oil (6.4%). A fungal source of AA (i.e., Microbial oil A) and an algal source of DHA (i.e., Microbial oil B) were employed (Martek Bioscience, Columbia, MD). Microbial oil A consisted of 23.4% 18:1n-9, 17.7% AA, 16.3% 16:0, 15.1% 18:0, 11.5% 18:2n-6, but no DHA and EPA. Microbial oil B contained 41.9% DHA, 19.1% 16:0, 15.5% 14:0, 13.8% 18:1n-9, but no AA and EPA. All RMF contained micro- and macronutrients comparable to those found in rat milk (27). Upon the completion of the preparation, RMF were degassed with nitrogen and allocated into 100-mL plastic botLipids, Vol. 34, no. 5 (1999)
tles for storage at −20°C. The formulas were rehomogenized daily immediately prior to feeding. Tissue preparation and lipid analysis. The AR system provides a useful model to investigate the effects of nutrients on postnatal development of the rat. In a series of studies we evaluated the effects of exogenous LCPUFA on brain lipid metabolism (16). In addition, lung tissues were obtained from the same study to assess whether highly unsaturated fatty acids provided in the diet alter the degree of saturation of lung tissue and surfactant PL. Artificial feeding was terminated at the age of 17 d (i.e., 10 d after AR began) when the rat is known to have reached the peak of brain myelination (28). On the day of termination the brain, liver, and lung were excised from the rat pups under anesthetic state after intraperitoneal injection of pentobarbital (5 mg/100 g body weight). Immediately after the removal, tissues were rinsed in 0.9% saline, blot-dried, frozen in liquid nitrogen, and then stored at −80°C until subsequent analyses. The brain and liver were analyzed for fatty acids as reported elsewhere (16). In the present study, lipids were extracted from lung tissues with chloroform/methanol (2:1, vol/vol) by the procedure modified from that of Folch et al. (29). In a series of experiments, surfactant isolated from lung tissue was used for lipid extraction. Lung tissues were homogenized using a Ten-Broek homogenizer, and surfactant was isolated by the method of Frosolono et al. (30) as modified by Sanders and Longmore (31). The extracted lipids were separated into lipid classes by thin-layer chromatography using Silica Gel H plates and a solvent system consisting of hexane/ethyl ether/glacial acetic acid (80:20:1; by vol) (32). After the plates had dried, they were exposed to iodine vapor to identify total PL with standards. For analysis of PL species, the lipid extracts were separated into PC and phosphatidylethanolamine (PE) as described elsewhere (16). The isolated PL and PL species were scraped into ampules and immediately transmethylated with 12% boron trifluoride in methanol (wt/vol). Heptadecanoate (100 µg) was added to the transmethylation ampule as an internal standard. The sealed ampules were heated at 100°C in a heating block for 30 min. The methylated fatty acids were analyzed by using a gas chromatograph (Model 5890 Series II; Hewlett-Packard, Palo Alto, CA) equipped with a fused-silica capillary column (SP-2330, 30 m × 0.25 mm, 20 µm film; Supelco, Inc., Bellefonte, PA). The gas chromatographic analyses were performed under the following conditions: column temperature, 150°C; injector temperature, 220°C; detector temperature, 250°C; flow rate of the carrier gas (helium), 21.4 mL/min; and a split ratio of 76:1. Appropriate methyl ester standards (Supelco, Inc., Bellefonte, PA) were used to identify fatty acids. Fatty acid composition was expressed as weight percentage (wt%) of total fatty acids or µg fatty acid/g tissue. Data and statistical analysis. All statistical analyses were performed on an IBM PS/2 computer using Minitab 10.1 (Minitab, Inc., State College, PA). Data were expressed as mean ± SD, and significance was assigned at P < 0.05. Difference in values among experimental groups was analyzed by one-way analysis of variance, and Tukey’s pairwise com-
n-6 AND n-3 FATTY ACIDS IN SURFACTANT
parisons were used to identify where the difference existed (33). RESULTS Rat milk formulas were analyzed for fatty acids. As expected, the formulas supplemented with AA, or DHA, or AA plus DHA were enriched with the respective fatty acids (Table 1). The actual wt% of AA (i.e., 0.4 and 1.1%) and of DHA (i.e., 0.5 and 0.9%) were close to the targeted 0.5 and 1.0%, respectively (Table 1). Similarly, the AA to DHA ratios of 0.5:0.3 and 0.9:0.5 in AA- and DHA-supplemented formula were the same as or close to the expected target ratio of 0.5:0.3 and 1.0:0.6. There was no significant difference in other fatty acids except a decrease in 18:2n-6 in AA-supplemented formula, and a slight reduction in 18:1n-9 in 1.0% DHA-supplemented formula. The supplementation did not affect the sum of saturated fatty acids and of polyunsaturated fatty acids. At 7 d of age infant rats were weight-matched and randomly assigned to three groups for AR. After 10 d of feeding of RMF supplemented with or without AA plus DHA, the body weights ranging from 32.6 to 34.6 g, and lung weights ranging from 0.37 to 0.38 g were comparable among the three groups (Table 2). These values resulted in unaltered lung to body weight ratios. Comparable body weights were also noted in the experiments in which infant rats were fed diets supplemented with or without AA, or DHA alone (data not shown). Total PL of lung tissue contained 43% palmitate (16:0) as a portion of the total fatty acids in infant rats fed the unsupplemented formula (Table 3). The supplementation with AA at 0.5 and 1.0% equally lowered the weight percentage of 16:0 and stearate (18:0) by 12–16% and 14–16%, respectively. Conversely, the AA supplementation resulted in higher weight percentages of AA and linoleate (18:2n-6) by 38–72% and 25–33%, respectively. These changes led to decreased total saturated fatty acids and increased total polyunsaturated fatty
acids. The supplementation with DHA, on the other hand, did not alter fatty acids composition of total PL except that the weight percentage of DHA was higher in the supplemented groups than in the unsupplemented group (Table 3). Despite the small net change in weight percentage of DHA, total saturated and polyunsaturated fatty acids remained unchanged. Table 4 shows the fatty acid composition of total PL and PL species isolated from pulmonary surfactant. The weight percentage of 16:0 was the highest in PC (71%), and the lowest in total PL (49%) in rat pups fed the unsupplemented RMF (Table 4). Conversely, the highest weight percentage of 18:0 was found in PE (24%) and the lowest in PC (5.5%). The cosupplementation with AA and DHA at either the 0.5:0.3 or the 1.0:0.6 ratio did not alter fatty acid composition of total PL. Similarly the cosupplementation did not change the composition of PC despite a slight increase of AA in the group supplemented with a high ratio of AA to DHA. The weight percentage of 16:0 in PE was lower in the cosupplemented groups by 11–12% than that of the unsupplemented counterparts. Also, the weight percentage of 20:2n-6 in PE was lower in the cosupplemented group than in the unsupplemented group. The observed reduction in 16:0 and 20:2n-6 was accompanied by an increase in 18:0. The sum of all saturated fatty acids was unchanged by dietary AA and DHA supplementation. The determination of the fatty acid concentration in surfactant isolated from 1 g of lung tissue disclosed that total fatty acids in PC were comparable in all three groups of infant rats (i.e., 400 ± 66 vs. 403 ± 12 vs. 415 ± 32 µg/g tissues). Similarly, fatty acid concentrations in PE and total PL remained unchanged by the AA and DHA cosupplementation. DISCUSSION Dietary fat is the most important determinant of tissue lipid both quantitatively and qualitatively. For example, a number of studies have shown that lipid composition of brain tissue
TABLE 1 Fatty Acid Composition of Rat Milk Formulas Supplemented with Arachidonate and/or Docosahexaenoatea AA (%) Fatty acid 8:0 10:0 12:0 14:0 16:0 18:0 18:1n-9 18:2n-6 18:3n-6 20:4n-6 22:6n-3 Sum SFA Sum PUFA
Values expressed as weight percentage of total fatty acids are means ± SD for four samples. Fatty acids included only major saturated and unsaturated (n-9, n-6, and n-3) species, and hence did not add up to 100%. Values for each supplement with different superscript roman letter in the same row are significantly different at P < 0.05. AA, arachidonic acid; DHA, docosahexaenoic acid; SFA, saturated fatty acids; PUFA, polyunsaturated fatty acids.
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TABLE 2 Body and Lung Weights of Infant Rats Fed Milk Formulas Containing Arachidonate and Docosahexaenoatea AA/DHA (%/%)
Body wt (g) Lung wt (g) Lung/body (%)
32.8 ± 1.9 0.37 ± 0.05 1.20 ± 0.11
34.6 ± 0.1 0.38 ± .01 1.10 ± 0.01
32.6 ± 1.1 0.38 ± 0.02 1.15 ± 0.07
Values are means ± SD for four rats. Lung/body (%) = lung (g)/body weight (g) × 100. For abbreviations see Table 1.
(15,34,35), cells (36), and subcellular organelles (25,36) of infant rats reflected that of maternal diets. Specifically, the tissue and cellular levels of n-6 and n-3 LCPUFA were effectively enriched by the corresponding fatty acids presented in maternal diets (15,25,26,34–36). These fatty acids supplemented in rat milk formulas also were rapidly incorporated into various tissues (e.g., liver and brain) in artificially reared infant rats (16,25). The present study further determined the role of dietary fat in lung tissue and surfactant during postnatal development in AR model. Aside from many advantages of AR described elsewhere (16), the model permits evalua-
tion of the direct impact of dietary nutrient on neonates without interference of maternal involvement. Consistent with earlier studies by other investigators (11–14), the results of the present study showed that dietary AA increased the accretion of AA with a proportional decrease in saturated fatty acids, i.e., 16:0 and 18:0 in PL isolated from lung tissue. These changes led to a reduction in PL saturation as indicated by the decreased weight percentage of total saturated fatty acids and increased weight percentage of total polyunsaturated fatty acids. However, the supplementation of DHA had no such effect on saturation of lung tissue PL despite a slight increase in DHA accretion. Although the effect of cosupplementation with AA and DHA on lung tissue PL was not determined, the fatty acid composition and the level of saturation of surfactant PL was not altered by simultaneous supplementation with the two LCPUFA. An analysis of individual PL species of surfactant revealed that the cosupplementation with AA and DHA did not reduce the degree of saturation of PE or PC, which is the major component of surfactant lipids. This finding was consistent with our previous study which showed that fish oil rich in DHA and EPA did not decrease the proportion of saturated fatty acids
TABLE 3 Fatty Acids of Lung Phospholipids in Infant Rats Fed Formulas Supplemented with Arachidonate or Docosahexaenoatea AA (%)
16:0 18:0 18:1n-9 18:2n-6 20:2n-6 20:4n-6 22:6n-3 Sum SFA Sum PUFA
Values expressed as weight percentage of total fatty acids are means ± SD for four samples. Fatty acids included only major saturated and unsaturated (n-9, n-6, and n-3) species, and hence did not add up to 100%. Values for each supplement with different superscript roman letters in the same row are significantly different at P < 0.05. For abbreviations see Table 1.
TABLE 4 Fatty Acids of Pulmonary Surfactant Phospholipids and Phospholipid Species in Infant Rats Fed Formulas Supplemented with Arachidonate and Docosahexaenoatea Phospholipids [AA/DHA (%/%)] Fatty acid 16:0 18:0 18:1n-9 18:2n-6 20:2n-6 20:4n-6 22:6n-3 Sum SFA Sum PUFA
a Values expressed as weight percentage of fatty acids are means ± SD for four samples. Fatty acids included only major saturated and unsaturated (n-9, n-6, and n-3) species, and hence did not add up to 100%. Values for each phospholipid with different superscript roman letters in the same row are significantly different at P < 0.05. For abbreviations see Table 1.
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in PL of alveolar type II cells (37). Clearly, unlike lung tissue PL, surfactant PL, especially PC, is resistant to modification by dietary AA and DHA. The reason for the differential effects on lung tissue and surfactant is not completely understood but may be attributed to the complex cellular organization of the lung, which is known to consist of more than 40 cell types (38). Surfactant is synthesized by alveolar type II cells which constitute only 14% of the total cell mass in the lung (38). It is reasonable to speculate that the lack of modification of surfactant PC by dietary LCPUFA may stem in part from preferential incorporation of saturated fatty acids into PC by alveolar type II cells. In support of such speculation, we showed that PL of alveolar type II cells were able to maintain the level of saturated fatty acids without increasing the sum of n-3 and n-6 LCPUFA by fish oil supplementation (37). Alternatively, the maintenance of PC saturation under the present experimental conditions may be achieved by the deacylation-reacylation mechanism by remodeling of de novo synthesized unsaturated PC (1). The function of alveoli depends not only on the composition of surfactant PL but also on the amount of surfactant secreted onto alveolar space (39). It is therefore important to determine whether dietary AA and DHA influence the amount of surfactant present in the lung. Although no attempt was made in the present study to quantify surfactant, the constant concentration of total fatty acids measured in surfactant PC among three groups of infant rats strongly suggests that dietary LCPUFA did not reduce the quantity of lung surfactant. However, it is not known whether the findings of lung surfactant reflect that of alveolar surfactant. In an earlier study we demonstrated that fish oil fed to adult rats had no effect on mass of DSPC and unsaturated PC in alveolar surfactant isolated from lung lavage (37). This, together with the fact that tissue surfactant isolated in the present study represents extracellular and intracellular materials (31), led us to speculate that saturation of surfactant PC on the alveolar space remains unchanged by AA and DHA supplementation. In summary, the neutral effect of the LCPUFA supplementation on lung surfactant observed in the present study lend support for n-6 and n-3 fatty acid supplementation to promote growth and development of infants (17,18).
6. 7. 8. 9.
ACKNOWLEDGMENTS This work was supported in part by Wyeth Nutritionals International and by Elmore Funds.
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[Received July 13, 1998, and in final revised form and accepted March 23, 1999]