The Journal of Nutrition Vol. 128 No. 12 December 1998, pp. 2473-2487

Long-Chain Polyunsaturated Fatty Acid Levels in Formulae Influence Deposition of Docosahexaenoic Acid and Arachidonic Acid in Brain and Red Blood Cells of Artificially Reared Neonatal Rats^1,2,3

G. R. Ward^{*, 4}, Y. S. Huang, E. Bobik, H-C. Xing^*, L. Mutsaers^*, N. Auestad, M. Montalto, and P. Wainwright^*

^* Department of Health Studies and Gerontology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1 and  Ross Laboratories, Columbus, Ohio, USA 43215-1724.

	ABSTRACT

Abstract Introduction Methods Results Discussion References

We studied the effects of dietary long-chain polyunsaturated fatty acids (PUFA) on the fatty acid composition of the brain and red blood cells in gastrostomized rat pups reared artificially from postnatal Days 5-18. These pups were fed rat milk substitutes in which the fat comprised 10% linoleic acid and 1% -linolenic acid and, using a 3 × 3 factorial design, one of three levels of both arachidonic acid (AA) and docosahexaenoic acid (DHA) supplied as single cell microbial oils (0.0, 0.4 and 2.4% fatty acids). A tenth group was reared by nursing dams. The fatty acid composition of the phosphatidylethanolamine (PE) and phosphatidylserine/phosphatidylinositol (PS/PI) phospholipids in the brain and red blood cells on Day 18 reflected the dietary composition in that pups receiving long-chain supplementation of each had higher levels of the supplemented PUFA, but lower levels of the other, relative to unsupplemented groups. In contrast to these results, there were few changes in the brain in phosphatidylcholine (PC) phospholipids whereas, in the red blood cells, changes in PC were similar to those in PE and PS/PI. Regression analyses showed that DHA levels in the brain correlated more closely with those of the red blood cells than did AA levels. The results of this study indicate that, although supplementation of formula with AA or DHA during the period of rapid brain development in rats increases deposition of the long-chain PUFA in the developing tissues, each also affects the levels of the other.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

The mammalian brain is particularly rich in long-chain polyunsaturated fatty acids (PUFA)⁵, especially docosahexaenoic acid (DHA) [22:6(n-3)] and arachidonic acid (AA) [20:4(n-6)] (see review by Innis 1991). These fats accrue mainly during the period of rapid brain development, which in humans begins in utero during the last trimester of pregnancy and continues through the early postnatal period. The accretion of lipid at this time supports the formation of cell membranes that, in addition to their role in the structural support of cells, also play a dynamic role in cellular physiology. AA and DHA are component fatty acids of the phospholipid bilayer of these membranes, and are particularly concentrated in synaptic nerve endings whereas, in the retina, the rod outer segment membranes of the photoreceptors are highly enriched with DHA.

Unlike cholesterol and saturated fatty acids, which are formed de novo in the developing brain (Sastry 1985), DHA and AA must be obtained either directly from dietary sources or derived from the 18-C precursor essential fatty acids linolenic acid (LN) [18:3(n-3)] and linoleic acid (LA) [18:2(n-6)], respectively, through a series of metabolic steps involving chain elongation and desaturation. Whereas the liver appears to play a major role in these processes (Anderson and Connor 1994, Bazan et al. 1994), evidence indicates that the capacity for the formation of DHA from its precursor also exists at the blood-brain interface (Moore et al. 1991). Therefore, most adults are able to meet brain requirements for long-chain PUFA by consuming adequate amounts of the 18-C precursors (Innis 1991). On the other hand, although there is evidence that infants are able to synthesize AA and DHA from LA and LN, (e.g. Carnielli et al. 1996), the increased requirements for the longer chain metabolites during development has raised the concern that infants may also require some preformed long-chain PUFA. During pregnancy, DHA and AA are provided to the developing brain by the maternal blood supply whereas, during the early postnatal period, they are provided by breast milk (Jensen 1978). In contrast to breast milk, however, most commercial infant formulae available in North America do not contain oils and lipids that provide long-chain PUFA. Infants fed standard, unsupplemented formulae have lower plasma and red blood cell (RBC) levels of these fatty acids than do infants fed breastmilk (Jorgensen et al. 1996) or supplemented formulae (Carlson et al. 1987). Whereas the extent to which plasma or red blood cell levels provide information about long-chain PUFA levels in the brain and retina is unclear, data from a small number of studies on formula-fed infants who died in infancy indicate that, although brain levels of AA were similar to those of breast-fed infants, levels of DHA were lower (Farquharson et al. 1992, Makrides et al. 1994).

View larger version (27K): [in this window] [in a new window]   Fig 1. Concentrations of selected polyunsaturated fatty acids in phosphatidylethanolamine membrane fraction from forebrain, cerebellum and red blood cells of 18 d-old rats fed experimental formulae from Day 5. Values are means ± SEM, n = 7-9. Filled symbols indicate that group is significantly different from suckled control group. (a) denotes main effect of DHA, (b) denotes main effect of AA, (c) denotes interaction, P < 0.05.

View larger version (13K): [in this window] [in a new window]   Fig 2. Linear relationships between phosphatidylethanolamine levels of arachidonic acid in cerebellum and red blood cells (A), and between docosahexaenoic acid in cerebellum and red blood cells (B), and sigmoidal exponential relationship between docosahexaenoic acid in cerebellum and red blood cells (C). Note that identical data are presented in (B) and (C).

Although little is known about neurochemical processes that may be affected by differences in brain levels of PUFA, marked reductions in DHA levels in brain phospholipid membranes in experimental animals have been associated with alterations in electrophysiological (Bourre et al. 1989. Neuringer et al. 1986, Weisinger et al. 1996) or behavioral outcomes (Lamptey and Walker 1976, Neuringer et al. 1984, Wainwright 1992). Furthermore, studies in which animals were supplemented with pharmacologically high concentrations of DHA have sometimes reported an association between increased DHA levels in brain or retina and deficient electrophysiological (Weisinger et al. 1996) and behavioral (Wainwright et al. 1997) responses and, in the case of the latter study, the behavioral impairment could be prevented by substituting AA for some of the LA. It is important to keep in mind that in all these studies the dietary manipulations were extreme, and could not be considered physiological. However, they do suggest that supplementation of DHA to infant formula requires knowledge of the relationship between intake of DHA and subsequent effects on tissue AA concentrations, with or without concomitant supplementation of AA.

The relationship between dietary PUFA, brain DHA and AA levels, and functional development of the brain is of particular interest in the case of formula-fed premature infants, who are removed from the transplacental supply of long-chain PUFA early in the brain growth spurt. For example, studies with preterm infants fed unsupplemented formula compared to those fed formula containing DHA report lower visual acuity (Carlson et al. 1993 and 1996) or lower electroretinogram responses (Birch et al. 1992) up to 4 mo corrected age. Differences in responding on tests of looking behaviors were reported in studies in which preterm infants were supplemented with DHA (Carlson and Werkman 1996). Studies examining visual and developmental outcomes in preterm infants fed formula containing both AA and DHA are not yet available. Additionally, when term infants have been fed formula with or without added DHA, or both AA and DHA, effects on visual or cognitive development were inconsistent (Makrides et al. 1996), perhaps due to inconsistencies across studies in sources, amounts and ratios of various 18-carbon and long-chain (n-6) and (n-3) fatty acids and differences in the populations studied and in the methods used to test visual and cognitive outcomes (Lucas 1997, Wainwright and Ward 1997).

Animal studies in which the amounts of both AA and DHA could be varied during the period of rapid brain development would help establish the potential roles of at least some of these variables. In particular, given that members of the (n-3) and (n-6) families compete for the same desaturase enzymes (Innis 1991), the effects of the supplementation of PUFA from one family on tissue deposition of the PUFA from the other family may be an important factor to consider when determining which dietary PUFA composition is optimal for neural development. Therefore in the present study, we chose to use the artificial rearing model to directly feed infant rats experimental formulae during the period of rapid brain development. In contrast to other rodent models in which dietary fatty acid manipulations are delivered to the developing pup via the maternal blood supply, and later milk, this model allows for the precise control of both the relative and absolute amounts of specific dietary PUFA. Furthermore, in contrast to the piglet (Innis 1993) and primate (Reisbick et al. 1997) models, which allow for formula feeding of the subject during a period of brain development corresponding to that of the term and older infant, respectively, our method allows for the direct delivery of the diet to the pups during a period of brain development that roughly parallels the third trimester and early postnatal period in humans (Dobbing and Sands 1979) or early infancy in the preterm infant. Recent studies using this model have shown that rat pups reared on (n-3)-deficient formula exhibit large reductions in DHA concentrations in the brain (Ward et al. 1996), and that LN, even when provided in amounts much greater than that normally found in rat breast milk, does not lead to levels of DHA in the plasma and erythrocytes (Winters et al. 1994) or brain (Woods et al. 1996) equal to those of breast-fed rat pups.

We hypothesize that supplementing formula with DHA or AA will lead to increased deposition of PUFA of the (n-3) and (n-6) series, respectively, in developing tissue but will decrease deposition of the other series. Similarly, we hypothesize that the addition of both DHA and AA will be necessary to attain the desired overall brain fatty acid composition. Because any model of the relationship between two PUFA should account for a wide range of possible concentrations, we used a 3 × 3 design to supplement formula with concentrations of both AA and DHA, ranging from 0% fatty acids, through what could be considered a physiological level of 0.4%, to a very high level of 2.4%. Rat milk substitutes containing 10% LA and 1% LN, with or without DHA and /or AA, were fed from Days 5-18 of life. Furthermore, we assessed fatty acid composition in both the blood and the brain to study the relationship between brain and RBC composition. We also subdivided the brain into forebrain (FB) and cerebellum (CB) because, in rats, developmental events that occur prenatally in the FB occur to a large extent postnatally in the CB (Altman 1982). Therefore, dietary effects that have specific effects on rapidly developing tissue should, during the early postnatal period in rats, produce greater effects on the CB than on the FB.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Subjects.  The subjects in this study were offspring of 17 timed-pregnant Long-Evans rats (Harlan Sprague Dawley, Indianapolis, IN). The pregnant dams were obtained at 10-12 days gestation and housed individually with free access to AIN-93M laboratory diet (Reeves et al. 1993) (Dyets, Inc., Bethlehem, PA) and tap water. They were maintained under a reversed dark:light cycle (lights off at 0600 h) at 22 ± 1° C. Offspring were culled, when necessary, to 12 pups per litter within 24 h of birth, and approximately equal numbers of male and female offspring were selected as subjects. Up to 10 pups from each litter were assigned randomly to groups such that no more than one pup from any litter was assigned to any one group.

Diets.  The composition of the rat milk-substitute was a modification of the formula developed previously (Auestad et al. 1989) and consisted of a base solution of bovine whey and casein in distilled water to which were added the other dietary components (Table 1). The base solution was prepared by heating the water to 50°C before adding the whey and casein and then refrigerating the dissolved solution for approximately 20 h before adding the rest of the components. The resulting formula was polytroned at high speed for 10 min, stored under nitrogen in aliquots of 50 mL, and frozen at 20°C until use.

Basal dietary oils comprised mixtures of medium-chain triglyceride oil (MCT oil), coconut, soy, and high-oleic safflower oils, supplied by Ross Laboratories (Columbus, OH). Long-chain PUFA were provided by the single-cell microbial oils ARAsco and DHAsco (Martek Biosciences Corp., Columbia, MD), which were added in such a way that the overall amounts and ratios of LN and LA remained approximately the same in each formula (Table 2).

Experimental design.  We used a 3 × 3 factorial design, with three levels of AA [0.0, 0.4 and 2.4% of fatty acids (NoAA, LowAAand HighAA, respectively)] and three levels of DHA [0.0, 0.4 and 2.4% (NoDHA, LowDHAand HighDHA, respectively)]. The group receiving neither AA nor DHA was designated the baseline control group. A tenth group of pups was fostered to nursing dams and reared in litters of 11-12 pups. This group served as a suckled control (SC) group.

Artificial rearing procedure.  All procedures used in this study were reviewed and approved by the Animal Care Committee at the University of Waterloo, in compliance with the Animals for Research Act of Ontario (Revised Statutes of Ontario) and the Guide for the Care and Use of Experimental Animals from the Canadian Council on Animal Care. The artificial rearing procedure was modified from a procedure first described by Messer et al. (1969) and Hall (1975) and a detailed description has been published elsewhere (Woods et al. 1996). Briefly, the gastrostomy tube was a length (~15 cm) of Intramedic tubing (PE 10, Clay Adams, Parsippany, NJ) with a small plastic flange at one end. On Day 27 postconception (approximately Day 5 after birth), Long-Evans rat pups were anesthetized with methoxyfluorane inhalant (Metofane), and the gastrostomy tube was attached to a short wire contained within silastic tubing and lubricated with MCT oil, inserted into the mouth, down the esophagus, and out through the stomach wall. Pups were housed individually in plastic cups floating in a water bath maintained at 36 ± 1°C and were fed one of the experimental diets via PE tubing attached at one end to their gastrostomy tubes and at the other end to syringes attached to an infusion pump (Model #55-4143, Harvard Apparatus, South Natick, MA). The pumps were programmed to deliver the formulae for 10 min every hour. Artificially reared pups were fed an amount of diet representing 29% of their body weight (adjusted daily) at the start of the procedure and increasing to 36% of body weight after approximately 1 wk. Each day, all pups were weighed, their gastrostomy tubes were flushed with 0.1 mL of water, and their anogenital region was gently rubbed with a wet tissue to stimulate urination and defecation. Suckled control pups were not gastrostomized, but were fostered to nursing dams on the day of gastrostomy. On Day 40 postconception (approximately Day 18 postbirth), all pups were deeply anesthetized by Halothane and, when completely unconscious, blood was removed by cardiac puncture, and brains were removed and divided into FB and CB.

Milk samples were obtained from six nursing dams, selected from those which had provided pups to the various groups but were not used to raise SC pups. These dams were fed and housed similarly to the SC dams until Days 12-14 when they were injected with oxytocin (0.4 IU/kg body weight) and lightly anesthetized with Halothane. Breast milk was extracted as described previously (Mills et al. 1990).

Biochemical analysis.  The lipids in red blood cells were extracted by the method of Dodge and Phillips (1967), milk samples by the method of Bligh and Dyer (1959) and forebrain and cerebellum by the method of Folch et al. (1957). Aliquots of total tissue lipid extracts were separated into different phospholipid fractions by thin layer chromatography using chloroform/methanol/water/triethlamine (4:5:1:4, v/v) as the developing system. The fatty acids in the phosphatidylcholine, phosphatidylserine and -inositol, and phosphatidylethanolamine fractions were methylated under nitrogen according to the method of Morrison and Smith (1964). Heptadecaenoic acid was added as the internal standard. Fatty acid methyl esters were analyzed by capillary gas chromatography (Hewlett-Packard 5890 II Plus, Hewlett-Packard, Palo Alto, CA) equipped with a 30 m (0.32 mm id) capillary column (Omegawax, 0.25 µm film thickness, Supelco, Bellefonte, PA) and integrated by a Hewlett-Packard Chem Station. The oven temperature was programmed to increase from 120 to 205°C at 4°C/min and to hold for a final 15 min. The identification of each fatty acid was made with authentic standard mixtures (Nu-Chek Prep, Elysian, MN).

Statistical analysis.  All data were analyzed using SAS v.6.0.9 (SAS Institute, Cary, NC). Main and interactive effects of dietary treatments were analyzed using the general linear models approach for ANOVA, and significant effects were further analyzed by preplanned comparisons (Cody and Smith, 1991). Main effects of AA were analyzed by comparing the levels of AA collapsed across the levels of DHA and, similarly, main effects of DHA were explained by comparing the levels of DHA collapsed across the levels of AA. Significant interactions were interpreted by conducting comparisons of the pattern of simple effects (for example, the main effects of AA within each level of DHA, and those of DHA within each level of AA). Effects of formula feeding were assessed by comparison of each group with the SC group by Tukey's test. The level was set at 0.05. The relationships between the fatty acid composition of the FB and CB and of each with the RBC, were analyzed between pups across all nine formula-fed groups using multiple regression, and this was done for both the PE and PC fractions.

	RESULTS

Abstract Introduction Methods Results Discussion References

Morphological development.  There were no significant differences in body growth on Day 18 or wet weight of the FB, CB or liver among any of the artificially reared groups, nor between the artificially reared groups and the suckled control group (data not shown).

Fatty acid composition of the brain. 

Effects on long-chain PUFA in PE and PS/PI.  The effects of the dietary treatments on selected PUFA are shown in Table 3 and the effects on PE are shown in Figure 1. Generally, results were similar between the PE (Tables 4 and 5) and PS/PI (Tables 6 and 7) fractions in that PUFA composition reflected the dietary treatments. Groups receiving additional AA in formula, in most cases, had higher levels of AA and 22:4(n-6) but lower levels of DHA. Conversely, the groups receiving additional DHA in formula had higher levels of DHA but lower levels of the long-chain (n-6) PUFA. Also, effects of diet on tissue DHA were generally larger than those on AA. The effects seen in the CB and FB were similar in direction but, when expressed as proportion of the baseline control group, the effects of long-chain supplementation in CB were more pronounced, i.e., mean AA and DHA levels in the FB ranged from 86 to 110%, and 82 to 142%, respectively, whereas, in the CB, they ranged from 75 to 112% for AA and from 77 to 153% for DHA.

The effects of low levels of DHA supplementation were more consistent than were those of low levels of AA. For example, groups supplemented with low levels of DHA had higher tissue DHA and lower levels of AA in most cases, with the exceptions being that only high levels of DHA were associated with lower levels of AA and 22:4(n-6) in PS/PI in FB. On the other hand, supplementation with low levels of AA did not lead to higher levels of AA in PS/PI in FB and CB and did not lead to lower levels of DHA in cerebellum PS/PI. In most instances, whereas there were main effects for AA and DHA on tissue concentrations of AA, 22:4(n-6) and DHA, the effects on 22:5(n-6) levels were due to an interaction between dietary AA and DHA in both FB and CB. Individual group comparisons showed that groups receiving both low and high levels of DHA supplementation had lower levels of 22:5(n-6), regardless of the level of arachidonic acid supplementation in PE and PS/PI in both forebrain and cerebellum. However, high levels of AA supplementation were associated with high levels of 22:5(n-6) only in the absence of DHA supplementation or with low levels of DHA. The only other interaction effect on PUFA composition in the brain was for forebrain DHA in PS/PI and, in this case, the pattern was somewhat similar but in the opposite direction (i.e., groups receiving low or high levels of DHA supplementation had higher DHA levels regardless of the level of AA supplementation, whereas those receiving high levels of AA supplementation had lower levels of DHA only in the absence of DHA supplementation or with high levels of DHA).

Effects on long-chain PUFA in PC.  In contrast to the effects on PE and PS/PI, the effects of dietary supplementation on PC were inconsistent (Tables 8 and 9). There were no effects of dietary supplementation on PUFA composition in the PC fraction in the FB and effects of AA supplementation only in the CB. The effects in the CB were not dose-dependent (i.e., the effects of high doses of AA were not greater than those of low doses), perhaps because of the low levels of AA and DHA in brain PC, accompanied by the large relative variability.

Effects on LA in all fractions.  The effects of the dietary supplementation on levels of the 18-C (n-6) fatty acid LA were in the opposite direction of those on the longer-chain (n-6) PUFA: whereas groups receiving AA supplementation had lower tissue levels of LA, DHA supplementation increased LA levels. Furthermore, whereas the effects of AA were dose-dependent for both PE and PS/PI fractions in both forebrain and cerebellum, DHA supplementation did not affect LA concentrations in cerebellum PS/PI, with only high doses of DHA associated with higher levels of LA in forebrain PE and PS/PI and in cerebellum PE. In the PC fraction of both CB and FB the effects were interactive, with groups supplemented with AA having lower levels of LA in a dose-dependent manner regardless of the level of DHA supplementation. But only high levels of DHA increased the accrual of LA, and only in the NoAA condition in the FB, and in both the NoAA and LowAA conditions in the CB.

Effects on nonessential fatty acids  There were also differences among the groups in the concentrations of several of the nonessential fatty acids (FA), particularly in PE. In forebrain PE, for example, AA supplementation led to reductions in the deposition of both monounsaturated FA 18:1(n-9) and 18:1(n-7), [main effects for AA, F(2,63) = 7.52, 0.0012 and F (2,63) = 10.86, P < 0.0001, respectively], a main effect of DHA reducting deposition of 18:0, F(2,63) = 8.73, P <0.004) and 18:1(n-7), F(2,63) = 3.98, P < 0.0235. In cerebellum PE, there were main effects of DHA on 18:0, F(2,63) = 3.84, P < 0.0268; 18:1(n-9), F(2,63) = 4.09, P < 0.0213; and 18:1(n-7), F(2,63) = 6.74, P < 0.0022, and a main effect of AA on 18:1(n-9), F(2,63) = 11.63, P < 0.0001. There were few effects of either AA or DHA on nonessential fatty acids in PC or PS/PI in the brain. These results are shown in Tables 4-9.

Effects of formula feeding.  The SC group, suckled to lactating dams that were fed purified diet (AIN-93 M), provides a normative group for comparing the effects of formula feeding with the effects of breast-feeding. There were relatively consistent relationships in fatty acid composition between the SC and specific formula-fed groups. For brevity we will expand upon only selected PUFA. Comparisons of the SC group with each of the formula-fed groups are indicated in Tables 4-9 for all major fatty acids. As seen in Table 2, the LA/LN ratio of rat breast milk resembled that of the artificial formulae, and AA was present at a level (1.1%) intermediate between the LowAA and HighAA formulae. However, the milk contained no detectable levels of DHA. Despite this fact, in both the FB and CB the only group that did not differ from the SC pups in terms of its overall phosphatidylethanolamine PUFA was the group supplemented with low levels of both AA and DHA. Similarly, in PS/PI in FB, none of the LowDHA groups differed from the SC pups in fatty acid composition, with the sole exception of the HighAA group, which had higher 22:4(n-6) levels. Generally, supplementation with high levels of DHA increased deposition of DHA in PE relative to suckled control pups, and decreased accrual of AA, unless AA was also supplemented at high levels: supplementation with low and high levels of AA (PE) and high levels of AA (PS/PI) in the NoDHA condition reduced DHA deposition below that of the SC group.

Fatty acid composition of the red blood cells. 

Effects on long-chain PUFA in PE and PS/PI.  The effects of the dietary treatments on selected PUFA are shown in Table 3, and the effects on PE are shown in Figure 1. The effects of dietary supplementation on red blood cell PUFA in PE (Table 10) and PS/PI (Table 11) fractions, whereas in the same direction as those in the brain, differed in both pattern and magnitude. With the exception of RBC AA, for which low levels of DHA supplementation had no effect, groups receiving both low and high levels of DHA had lower levels of 22:4(n-6) and 22:5(n-6) and higher levels of DHA, these effects were dose-dependent (i.e., the effects of high doses of DHA were greater than those of low doses). The effects of AA supplementation, on the other hand, were specific to the PUFA under study. For example, while groups receiving either low or high doses of AA had higher RBC AA levels; there was no significant differential effect of high rather than low levels. For 22:4(n-6) levels, however, the effects were dose-dependent, with groups receiving high AA doses exhibiting higher levels of 22:4(n-6) than those receiving low doses. For both 22:5(n-6) and DHA, there were no effects of low doses of AA, and the effects of high doses were in opposite directions, lowering deposition of DHA levels but raising that of 22:5(n-6).

Effects on Long-chain PUFA in PC.  There were main effects of both AA and DHA on concentrations of AA, 22:4(n-6) and DHA, and an interactive effect on 22:5(n-6) (Table 12). In the case of AA and 22:4(n-6), the effect of AA supplementation was dose-dependent with only high doses reducing deposition of DHA. Conversely, the effects of DHA supplementation on RBC PC 22:4(n-6) and DHA were also dose-dependent, with only high doses of DHA reducing accumulation of AA. The interactive effect on 22:5(n-6) was complex, with groups receiving low levels of AA supplementation exhibiting higher 22:5(n-6) concentrations only when low levels of DHA were added to formula, whereas groups receiving DHA supplementation had lower levels of 22:5(n-6) only in the presence of AA supplementation.

Effects on LA and on nonessential fatty acids in all fractions.  High levels of AA supplementation reduced deposition of LA in the PC fraction, whereas there were interactive effects in both PE and PS/PI (Tables 10-12). In both cases, whereas AA supplementation reduced LA deposition regardless of the level of DHA supplementation, only high levels of DHA supplementation increased deposition, and only in the absence of AA supplementation.

There were few effects of either AA or DHA on nonessential fatty acids in PC or PS/PI in either brain or RBC or on RBC nonessential fatty acids in general.

Effects of formula feeding.  In red blood cell PE and PC, the suckled control group again showed DHA deposition similar to that of the groups receiving low levels of DHA, higher than those of the NoDHA groups, and lower than those of the HighDHA groups. Similarly in PS/PI, the SC group had lower accrual of DHA than did the HighDHA groups, but they also had less than the LowDHA/NoAA group and more than the NoDHA/HighAA group. With respect to arachidonic acid in PE and PS/PI, the only group to differ from the SC was the HighDHA/NoAA group, which had the lowest deposition of AA of all groups. In PC, on the other hand, the pattern was somewhat more complicated, with accrual of AA in the SC pups being greater than those of the NoAA and LowAA groups (with the exception of that group receiving no DHA) and less than those of the HighAA groups. These results are also shown in Tables 10-12.

Regression analyses. 

Brain vs. red blood cell.  Results of the regression analyses of the relationship between PE DHA in forebrain and cerebellum and in RBC are shown in Figure 2 and Table 13. DHA exhibited a stronger linear correlation than did AA for both FB (r² = 0.794 vs. 0.379) and CB (r² = 0.819 vs. 0.331). In both brain regions, the best fit for DHA was obtained for the exponential model (FB, r² = 0.861, CB, r² = 0.919) (Fig. 2). Among the (n-6) PUFA, both 22:4(n-6) and 22:5(n-6) exhibited stronger correlations than did AA between either FB and RBC [(r² = 0.799 and r² = 0.835, for 22:4(n-6) and 22:5(n-6), respectively)], or CB and RBC [(r² = 0.864 and r² = 0.853, for 22:4(n-6) and 22:5(n-6), respectively)].

Cerebellum vs. forebrain.  (Data not shown.) There were significant correlations between forbrain and cerebellum for both AA and DHA, and as expected from the lack of a dietary effect on PC composition, these were greater in PE than in PC (data not shown). In PE, AA levels in the FB were strongly correlated with those in the CB, r² = 0.869, P < 0.0000, as were the DHA levels r² = 0.891, P < 0.000, and these correlations were greater than those seen in PC (AA, r² = 0.056, P = 0.036; DHA, r² = 0.200, P = <0.0001).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

In this study of infant rats fed formula containing different amounts and ratios of AA and DHA from postnatal Days 5 to 18 (the period of rapid brain development), long-chain PUFA levels in the brain and in RBC reflected the relative amounts fed. Generally, the addition of AA to formula increased deposition of AA and 22:4(n-6) in brain and RBC. It also increased 22:5(n-6), but generally only when AA was high and in the presence of no or low DHA. Similarly, the addition of DHA led to increased deposition of DHA in these tissues. Furthermore each long-chain PUFA, when added to formula, led to a concurrent decrease in the deposition of the long-chain PUFA of the other series [i.e., AA supplementation reduced deposition of DHA, whereas DHA supplementation reduced deposition of all long-chain (n-6) PUFA]. On the other hand, the effects of LA on tissue deposition were in the opposite direction of those seen in the longer-chain PUFA in that AA supplementation decreased LA deposition whereas DHA supplementation increased it. These changes occurred in both brain regions and in RBC, but to varying degrees. For example, whereas the effects of AA and DHA intake on PUFA composition were seen in both the FB and CB, the changes in CB relative to the baseline control were of a greater magnitude, and the changes in RBC were of a much greater magnitude, than in either brain region.

The fact that tissue PUFA levels reflected levels of dietary PUFA is in contrast to a recent study (Jimenez et al. 1997) that reported no differences in plasma, retina, or brain levels of DHA between piglets fed formulae with or without long-chain PUFA (1.1% AA and 0.3% DHA) from Day 1. Another piglet formula feeding study (Craig-Schmidt et al. 1996) reported that dietary levels of ~1% AA and 0.8% DHA increased deposition of AA and DHA, respectively, in retinal PE, and that there was a trend toward lower levels of the long-chain PUFA of the other series. This trend was not significant, however, possibly due to either the small sample size (n = 5) or, given the relatively advanced state of development of the piglet brain at birth relative to that of the rat, to the possibility that the piglet brain is less sensitive to dietary changes at that point of development. Bourre et al. (1993), who employed a maternal feeding rat model, reported a relationship between maternal intake of long-chain (n-3) fatty acids through pregnancy and lactation and DHA, but not AA, levels in the brain of offspring at 21 d of age. Maternal intake of (n-3) fatty acids, of which DHA comprised 89%, varied from 0 to 100 mg (n-3) fatty acids/100 g of diet and the ratios of AA:DHA (0.77:1), LA:AA (~15:1) and LN:DHA (84:1) remained relatively constant. Wainwright et al. (1997) also used a maternal feeding model to vary dietary levels of very long-chain PUFA, using single cell microbial oils, across a wide range in mice [(n-6):(n-3) ratios of 49, 4 and 0.32, respectively]. In this study the (n-6) FA were provided either entirely as LA or as LA in combination with AA, and the (n-6):(n-3) ratio was varied by partial replacement of the (n-6) FA with DHA. The levels of DHA were very high and the results showed that addition of AA to the diet increased AA accrual in the brain and, at high levels only, decreased accrual of DHA. Increasing levels of DHA in the diet were associated with higher levels of DHA and lower levels of AA, and there was also evidence of retroconversion of DHA to 20:5(n-)3 in the mice fed high levels of DHA. In contrast, the 3 × 3 design of the present study, together with the direct feeding method employed, provided an opportunity to evaluate intakes of AA and DHA (from single cell oils) independently and in combination, across a range of dietary levels, including physiological levels, during the brain growth spurt.

It should be noted that, in using the present study design, where LN:LA was kept constant at 1:10, when DHA was added to formula, the total (n-3) complement of the formula increased proportionately, from 1 to ~3.5% of fatty acids. However, it is unlikely that the effects of this supplementation were due solely to a general increase in available (n-3) fatty acids. In a previous study (Woods et al. 1996), when artificially reared infant rats were fed formula in which the ratio of LN to LA was raised from 1:10 to 1:1, DHA levels in the brains at weaning were only slightly higher in the latter group (i.e., 12 vs. 13% fatty acids), even though the total amount of LN in the formula was raised from 2.8 to 14%.

There were notable differences in the degree to which the dietary treatments affected the various phospholipid fractions. Specifically, whereas AA and DHA levels were affected similarly in PE, PC and PS/PI fractions in the RBC, the effects in the brain were restricted primarily to the PE and PS/PI fractions: PUFA levels in brain PC were rarely influenced in rat pups at this age. At the same time, levels of all PUFA in PC were much lower, and levels of saturated and monounsaturated fatty acids much higher, than those found in the other fractions. This is consistent with previous findings, including two recently published studies in which the PUFA composition of each phospholipid fraction was measured in isolated neuronal cells of rats of various ages (Jumpsen et al. 1997a and b). The results obtained from rats at 3 wk of age (i.e., 3 d older than the rats in the present study) were similar to our results. That is, the levels of DHA in PE and PS/PI were much higher than those seen in PC, which ranged from less than 1 mol/100 mol to no more than 5 mol/100 molt. AA levels in PC were also lower in that study, although slightly higher than those reported here. Future research should focus on the relationship between fluctuations over time of fatty acid content in specific fractions of the developing brain and particular developmental processes.

The fact that the changes in the CB were greater than those in the FB is expected due to the differential timing of development of the two regions. By the time the dietary manipulations began in this study, the FB was in a relatively more mature stage of development than was the CB. Still, it should be noted that only the magnitude of the group differences differs between regions: the overall pattern of changes is similar between the two major divisions of the brain.

There were also significant relationships in the PUFA levels between various tissues. Long-chain PUFA levels in both FB and CB at 18 d of age were associated with long-chain PUFA levels in RBC, and the levels in RBC reflected the amounts in formula. The correlations between the AA levels in RBC and those in the FB or CB were smaller than those for the other (n-6) long-chain PUFA or for DHA. The associations between the DHA levels in RBC and those in the FB and CB were slightly greater when described using an exponential model than with linear correlations. It is also important to note that the changes in RBC levels due to the dietary treatments were much greater than were those in the brain. Few other studies in the rat have used maternal dietary manipulations to study the relationship between red blood cell PUFA status and that of the brain. In one such study (Carlson et al. 1986), pregnant and lactating rats were fed diets with varying LA/LN ratios but no long-chain PUFA and, although there were significant differences in the AA and DHA levels in PE for red blood cells and brain, the design of that study did not allow for the quantitative assessment of those relationships. Thus, the data presented here, showing a strong relationship between the levels of DHA in the brain and those in the RBC, at least for PE, have important implications for human clinical research, in which brain PUFA composition cannot be assessed directly. The strongest relationship in the present data was that described by an exponential model and, as clearly seen in the data, a linear relationship was only evident at low levels of DHA. These data suggest therefore that changes in RBC DHA levels may not reflect similar changes in the brain of human infants, particularly at high levels of DHA.

That DHA supplementation reduced tissue AA levels is interesting in light of the fact that, as reported previously (for discussion, see Wainwright et al. 1997), the effect of fish oil supplementation on AA levels may be due to the inhibitory effects of 20:5(n-3), commonly found in fish oil, on AA production and/or deposition. Moreover, in that previous study, when pregnant and lactating dams were fed diets containing high levels of DHA but no 20:5(n-3), the brains of offspring at weaning exhibited high levels of DHA and low but measurable amounts of 20:5(n-3), accompanied by reductions in AA levels. In that study, therefore, 20:5(n-3) may have been produced by retroconversion of DHA, by the lactating mother, in the nursing pup or both. In the present study, 20:5(n-3) was not present in any of the formulae (although it was present in small amounts in rat breast-milk), and we did not find measurable quantities of 20:5(n-3) in the rat brains or RBC. Because the offspring in the previous study that exhibited higher levels of 20:5(n-3) in the brain were assessed in adulthood, this suggests that preweanling rodents may be unable to synthesize and/or deposit more than very small quantities of 20:5(n-3) in the brain.

In this study, the breast-milk of the nursing dams did not contain detectable levels of DHA. Although the presence of DHA in rodent breast-milk has been reported previously, this may reflect the amount of long-chain (n-3) PUFA in the food supply. For example, in the study of different rat strains by Mills et al. (1990), where DHA levels in the milk ranged from 0.4 to 0.7% of fatty acids, the rats were fed a nonpurified diet containing 5 % total fatty acids as long-chain (n-3) PUFA [20:5(n-3) and DHA]. Similarly, in a study by Woods et al. (1996), where the maternal diet contained fish meal again with a long-chain (n-3) PUFA complement of 5% [20:5(n-3) and DHA], rat breast-milk (as measured by pup stomach contents) had 1.1% DHA. Finally, in a study conducted on mice in our laboratory (Wainwright et al 1992), when (n-3) levels were varied using fish oil, at the lowest (n-3) level (~7% of total fatty acids) the levels of DHA in breast-milk were 0.5%. Only at the much higher (n-3) levels were there appreciable amounts of DHA in the milk, ranging from 1 to 1.5%. Interestingly, the 20:5(n-3) levels varied across a much wider range (0.7-5.6%). Although the semipurified AIN-93M laboratory chow used in the present study supplied both (n-6) and (n-3) FA through a mixture of corn and soybean oil, these were in the form of LA and LN. Therefore, the fact that the nursing dams did not have DHA in their milk is likely a reflection of its absence in their diet. Thus it is interesting to note that, despite the lack of a direct dietary source, the milk did contain detectable amounts of AA and 20:5(n-3) (~1 and 0.7%, respectively), suggesting that dietary LA and LN are adequate sources for AA and 20:5(n-3), but not of DHA, in milk. Despite the absence of DHA in the milk, the PUFA composition in the FB and CB of the suckle control pups generally corresponded to that of the artificially reared groups receiving 0.4% DHA (and AA). It would seem, therefore, that rat breast-milk, even when it contained no detectable levels of DHA, still supported DHA deposition in the brain as well as did the artificial formula containing physiological amounts of DHA and AA and almost similar amounts of LA. This suggests that there may be factors other than dietary PUFA levels in the milk that also contribute to the deposition of long-chain PUFA in the developing brain.

For many researchers studying the effects of early nutrition on behavioral development, the rat is the species of choice. Much is known of its behavior and of the influence of early developmental events on that behavior and so, given the proposed behavioral effects of dietary (n-3) fatty acids, the rat model has the potential to make an important contribution in this regard. Moreover, its size and ease of care and maintenance mean that it requires relatively fewer resources than does a model such as the primate or piglet, an important consideration when designing large experiments, such as the present study. However, the fact that nutritional manipulations are usually delivered to the young rat via the maternal system limits its usefulness when studying manipulations of specific long-chain PUFA in the diet. The artificial rearing model described here overcomes this limitation by providing the nutritional manipulation directly to the pup during the critical period of brain development, and the present results support the utility of this model. What remains to be seen, however, is whether any behavioral correlates are associated with the resulting changes in tissue PUFA composition.

In general, these results suggest that the levels of both AA and DHA in infant formula and/or breast-milk should be considered carefully when determining the optimal PUFA composition of infant formula. In this study, whereas the effects on brain composition of supplementing small amounts (0.4%) of AA and DHA appeared to be quite small, subsequent effects on brain function cannot yet be discounted. Further work is currently underway to establish the possible effects of these dietary manipulations on behavioral outcomes related to specific aspects of brain function.

	FOOTNOTES

Manuscript received 4 March 1998. Initial reviews completed 27 May 1998. Revision accepted 24 August 1998.

	ACKNOWLEDGMENTS

The authors thank Dawn McCutcheon for her dedicated technical assistance and Martek Biosciences (Columbia, MD) for generously providing the dietary oil mixtures.

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