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Journal of Nutrition, doi:10.3945/jn.108.094136
Vol. 138, No. 11, 2164-2171, November 2008
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© 2008 American Society for Nutrition J. Nutr. 138:2164-2171, November 2008

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Enrichment of Intestinal Mucosal Phospholipids with Arachidonic and Eicosapentaenoic Acids Fed to Suckling Piglets Is Dose and Time Dependent1–3,

Holly A. Hess4, Benjamin A. Corl4, Xi Lin4, Sheila K. Jacobi4, Robert J. Harrell4, Anthony T. Blikslager5 and Jack Odle4,*

4 Department of Animal Science and 5 Department of Clinical Sciences, North Carolina State University, Raleigh, NC 27695

* To whom correspondence should be addressed. E-mail: jack_odle{at}ncsu.edu.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Infant formula companies began fortifying formulas with long-chain PUFA in 2002, including arachidonic acid (ARA) at ~0.5% of total fatty acids. The primary objective of this study was to determine the time-specific effects of feeding formula enriched with supra-physiologic ARA on fatty acid composition of intestinal mucosal phospholipids. One-day-old pigs (n = 96) were fed a milk-based formula for 4, 8, or 16 d. Diets contained either no PUFA (0% ARA, negative control), 0.5% ARA, 2.5% ARA, 5% ARA, or 5% eicosapentaenoic acid (EPA) of total fatty acids (wt:wt). Growth (299 ± 21 g/d) and clinical hematology were unaffected by treatment (P > 0.6). Although minimal on d 4, concentrations of ARA in jejunal mucosa were enriched 47, 272 and 428% by d 8 and 144, 356, and 415% by d 16 in pigs fed the 0.5% ARA, 2.5% ARA, and 5% ARA diets, respectively, compared with the 0% ARA control pigs (P < 0.01). On d 16, ARA enrichment increased progressively with increasing dietary ARA supplementation from 0 to 2.5% but plateaued as dietary ARA rose to 5%. A similar pattern of ARA enrichment was observed in ileal mucosal phospholipids, but maximal enrichment in the ileum exceed that in the jejunum by >50%. As ARA increased, linoleic acid content decreased reciprocally. Although maximal enterocyte enrichment with EPA approached 20-fold by d 8, concentrations were only ~50% of those attained for ARA. Negligible effects on gross villus/crypt morphology were observed. These data demonstrate a dose-dependent response of intestinal mucosal phospholipid ARA concentration to dietary ARA with nearly full enrichment attained within 8 d of feeding formula containing ARA at 2.5% of total fatty acids and that supra-physiologic supplementation of ARA is not detrimental to growth.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Provision of optimal nutritional support for the preterm infant poses a major challenge to neonatologists. Among the complicating factors is the underdeveloped state of the digestive system, making gastrointestinal disturbances among the most common health challenges faced by the suckling neonate. Whereas total parenteral nutrition can be used in the short term, it is increasingly clear that some intestinal stimulation via enteral nutrition is needed to promote health and development of the intestine, and yet enteral nutrition (1) is itself a risk factor for the development of necrotizing enterocolitis.

Although many nutritional variables may modulate the severity of intestinal inflammation and the rate of recovery, our laboratory has focused on the role of long-chain PUFA. We have previously shown that arachidonic acid (ARA)6-derived prostaglandins stimulate rapid recovery of gut barrier function after injury (2). Specifically, we demonstrated that prostaglandins orchestrate recovery of paracellular resistance within restituting epithelium (3). Because these reparative prostaglandins are produced from ARA via the cyclooxygenase enzymes (4), and because the status of ARA may be impaired (5) in preterm infants (6,7), we speculate that dietary supplementation with supra-physiological concentrations of ARA might enrich enterocyte membranes and facilitate repair following injury. Accordingly, in this study, we employed a suckling piglet model to elaborate the time course of ARA enrichment of intestinal mucosa following dietary supplementation and to determine the dose-response relationship between intestinal mucosal phospholipid ARA enrichment and dietary ARA concentration. We also examined the effects of supra-physiological ARA supplementation on growth and clinical health status of the neonates.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
Animal care and study design

    Piglets and experimental diets. All animals were cared for in accordance with the Institutional Animal Care and Use Committee of North Carolina State University. Colostrum-fed piglets (n = 96 across 3 replicates) were acquired within 12–24 h of birth and housed individually in a room under ambient temperature control (30°C). Piglets weighing <1 kg were not selected. All pigs were injected with 200 mg of iron dextran on d 1 and were given 5 mg of gentamycin orally on d 1 and 3. Piglets were randomly allocated to 1 of 5 dietary treatments differing in fatty acid composition and fed by a gravity feeding system (8) that allowed for accurate measurement of formula consumption. The dry matter content of the basal liquid diet (Table 1) was 15% and the chemical composition (dry matter basis) included: crude protein, 31%; lactose, 36%; ether extract, 26%; and total energy, 19.3 MJ/kg (2.91 MJ/L). Diets contained either no (n-6) PUFA (0% ARA, negative control), 0.5% ARA, 2.5% ARA, 5% ARA, or 5% eicosapentaenoic acid (EPA) of total fatty acids (Table 2). We blended tallow, coconut, and medium-chain triglycerides, supplying predominantly SFA, and soy oil, supplying unsaturated fatty acids. This yielded a final linoleic acid concentration of ~20%. Incremental ARA was achieved using ARASO oil (Martek Biosciences). We also formulated an extreme PUFA-deficient negative control blend, lacking both ARA as well as linoleic precursor and a diet enriched in EPA, matching the highest level of ARA (5%). This resulted in a range of diets varying markedly in long-chain PUFA with little confounding variation in other PUFA. The 0.5% ARA diet was designed to approximate the ARA concentration prescribed for preterm infant formulas (9) and ARA included in the 2.5% ARA and the 5% ARA diets was supplied at elevated (~5x and 10x) concentrations to prophylactically enrich enterocyte membranes with ARA.


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  		TABLE 1 Composition of basal formula fed to suckling piglets

 

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  		TABLE 2 Formulated dietary oil blends and analyzed fatty acid compositions

 
    Piglet feeding and management. Fresh formula was prepared daily, refrigerated, and fed 3 times/d (0800, 1600, and 2300). Diets were reconstituted at 180 g/L of water [(133.2 g dry diet + 46.8 g oil)/L water]. Piglets were fed formula at ~60% of ad libitum intake to match typical growth rates of sow-reared piglets. The feeding rate increased progressively from 100 mL/kg0.75 on d 1 to 600 mL/kg0.75 on d 7 and thereafter. To feed pigs according to the prescribed plan, body weights and milk intakes were recorded daily. Pigs (n = 6 per treatment x day), including age-matched, sow-reared controls, were killed on d 0, 4, 8, and 16 of treatment by AVMA-approved electrocution and then exsanguinated. Tissue collection occurred immediately thereafter.

Sample collection and analytical procedures

    Tissue collection. Blood was collected postmortem during exsanguination and serum was prepared and stored at –80°C for subsequent clinical analyses of 25 enzymes and metabolites via a commercial autoanalyzer (Vetscreen, Antech Diagnostics). The abdominal cavity was opened and the intestinal tract was removed. The duodenum, jejunum, and ileum were separated and flushed with 0.9% NaCl. Segments (~3 cm long) from each region were placed in formalin fixative for histological analysis. Mucosal samples were obtained by cutting segments lengthwise and scraping mucosa from the connective tissue. Samples from the jejunal and ileal regions were snap-frozen in liquid nitrogen for phospholipid fatty acid analysis and a composite of duodenum, jejunum, and ileum mucosa was frozen for measurement of lactase-specific activity.

    Mucosal histology and enzymology. Intestinal sections were assessed histologically for gross morphology, villus height, and crypt depth measurements as previously described (10). Formalin-fixed segments were embedded in paraffin and 5-µm-thick sections were cut and stained with hematoxylin and eosin for examination by light microscopy. Computer-assisted morphometric measurements were conducted with a video-imaging system (Nikon- FXA). All samples were measured by an observer unaware of the treatment groups. Six well-oriented villi were measured to determine mean villus height and width. Crypt depths were measured on 6 sites from the same sections. Villus surface areas for representative villi were calculated as reported previously (11).

For lactase determination, mucosal scrapings were homogenized in iced buffer (0.2 mol/L Tris, 0.15 mol/L KCI, 2.5 mmol/L EDTA, pH 7.4) and immediately frozen in aliquots at –20°C until analyzed. Specific activity was measured as previously described (8) and expressed as µmol glucose released/(min·g protein).

    Fatty acid analysis. Total fatty acids in phospholipids were determined in the mucosa collected from the jejunum and ileum. Our sampling technique (mucosal scraping) did not allow discrimination between phospholipids from enterocyte membranes vs. other supporting tissues and immune cells inhabiting the lamina propria. Lipids were extracted from the scraped mucosa (12) and phospholipids were separated from other lipid classes using silica (normal phase) solid-phase extraction columns (Sigma-Aldrich) (13). Fatty acids were methylated (14) and analyzed by a 5890 Hewlett Packard gas chromatograph equipped with 100-m capillary column (SP-2380, Supelco), a flame ionization detector, and a 6890 autosampler. Peaks were identified by comparison of retention times with authentic FAME (Sigma-Aldrich). Fatty acid concentrations were calculated using C17:0 as an internal standard and concentrations were expressed as g/100 g fatty acid. Dietary fatty acid analysis employed identical methods except that the phospholipid fraction was not segregated.

    Statistical analysis. Daily gain, feed intake, serum metabolite and enzyme concentrations, and intestinal morphology data were analyzed by 1-way ANOVA using the general linear models procedure of SAS (SAS Institute), and data from lactase and fatty acid analyses were subjected to 2-way ANOVA (dietary treatments x day of study). Means were separated using a least significant difference post hoc test (SAS Institute). Values were expressed as least square means ± SEM and differences were considered significant when P < 0.05.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Growth and food intake. Piglets on all treatments grew rapidly (299 ± 21 g/d) and daily gains were unaffected by dietary content of long-chain PUFA (P > 0.1; data not shown). Notably, the formula-fed pigs gained at rates similar to sow-reared controls. Correspondingly, mean daily formula intakes were 591 g/d over the first 4 d and 1434 g/d over the 16-d trial, being similar among treatments (data not shown).

    Serum metabolite and enzyme concentrations. Serum concentrations of creatinine, bilirubin, alkaline phosphatase, cholesterol, and triglycerides were lower in the formula-fed pigs than in the sow-fed control pigs (P < 0.05) but did not differ among formula-fed pigs (Table 3). The serum ratios of urea-N:creatinine were higher in most formula-fed pigs than in the sow-fed controls. Other blood metabolites and enzymes, including total protein, albumin, globulin, lipase, amylase, alanine aminotransferase, aspartate aminotransferase, creatine phosphokinase, {gamma}-glutamyl transpeptidase, Ca, Mg, Na, K, P, and Cl concentrations, did not differ among the groups (P > 0.05).


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  		TABLE 3 Serum metabolite and enzyme concentrations in piglets after 16 d of consuming sow milk or formula enriched with 0–5% ARA or 5% EPA12

 
    Small intestine morphology. Ileal villus height and area did not differ among any of the tested groups (Table 4; P > 0.05). Ileal villus width was slightly less (P < 0.05) for pigs fed 5% ARA and in those nursing the sow than in those fed 0.5 or 2.5% AA. Ileal crypt depths were greater in the formula-fed pigs than in the d 0 and sow-fed pigs but did not differ between day 0 and sow-fed pigs. The villus:crypt ratio was greater in d 0 pigs than in all of the formula-fed pigs (P < 0.05) sampled 16 d later. The villus:crypt ratios did not differ among the formula-fed pigs.


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  		TABLE 4 Ileal morphology of piglets after 16 d of consuming sow milk or formula enriched with 0–5% ARA or 5% EPA12

 
    Lactase-specific activity. Of the 4 time points sampled, lactase-specific activity was highest on d 4 (Fig. 1, day effect; P < 0.05). Furthermore, over all sampling times, the activity in sow-fed pigs exceeded that of formula-fed pigs (P < 0.05). All formula-fed pigs had similar lactase-specific activity levels at each time point that were similar in magnitude to levels measured in pigs sampled on d 0.


Figure 1
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  		FIGURE 1  Lactase-specific activity in the small intestinal mucosa of suckling pigs consuming formula with 0–5% ARA or 5% EPA and in sow-fed controls after 0, 4, 8, or 16 d. Values are means ± SEM, n = 6 composite (duodenum, jejunum plus ileum) mucosal samples. Time points without a common letter differ, P < 0.05. *Formula-fed pigs < sow milk-fed pigs, P < 0.05.

 
    Intestinal fatty acid profiles. Jejunal mucosal phospholipid ARA [20:4(n-6)] concentration increased (P < 0.05) with increasing dietary ARA in a dose-response fashion that strengthened with time (Fig. 2A; day x treatment interaction; P < 0.01). Although minimal on d 4, the concentrations of ARA were enriched by 47, 272, and 428% by d 8 and 144, 356, and 415% by d 16 in pigs fed the 0.5% ARA, 2.5% ARA, and 5% ARA diets, respectively, compared with the 0% ARA control pigs. On d 16, ARA enrichment increased progressively with increasing dietary ARA supplementation from 0 to 2.5% but plateaued as the dietary ARA rose to 5%. A similar pattern of ARA enrichment was observed in ileal mucosal phospholipids (Fig. 2B), but the maximal degree of enrichment in the ileum (26.5 g/100 g fatty acid) exceeded that in the jejunum (17.5 g/100 g fatty acid) by >50%. Mucosal ARA concentration of the 5% EPA-fed pigs was similar to the 0% ARA-fed pigs at all time points in both the ileum and the jejunum. Congruent with concentrations of ARA measured in sow milk (Table 2; 0.55% of fatty acids), ARA enrichment of mucosal phospholipids from sow-milk-fed pigs did not differ from 0.5% ARA formula-fed pigs, but interestingly, ileal enrichment was most similar to the 2.5% ARA-fed pigs by d 16.


Figure 2
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  		FIGURE 2  ARA [20:4(n-6)] enrichment of isolated jejunal (A) and ileal (B) mucosal phospholipids of suckling pigs consuming formula with 0–5% ARA or 5% EPA and in sow-fed controls after 0, 4, 8, or 16 d. Values are means ± SEM, n = 6. Labeled means without a common letter differ, P < 0.05. Data for initial and sow-reared pigs are presented for reference only and were not analyzed statistically.

 
As ARA increased in jejunal and ileal phospholipids, the enrichment of linoleic acid [18:2(n-6)] reciprocally decreased (Fig. 3) despite similar concentrations of dietary linoleic acid (e.g. 20% of dietary fatty acid; Table 2).


Figure 3
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  		FIGURE 3  Linoleic acid [18:2(n-6)] enrichment of isolated jejunal (A) and ileal (B) mucosal phospholipids of pigs consuming formula supplemented with 0–5% ARA or EPA and in sow-fed controls after 0, 4, 8, or 16 d. Values are means ± SEM, n = 6. Labeled means without a common letter differ, P < 0.05. Data for initial and sow-reared pigs are presented as reference only and were not analyzed statistically.

 
Jejunal and ileal mucosal phospholipid concentrations of EPA [20:5(n-3)] increased from negligible levels on d 0 and 4 to maximal concentrations by d 8 in piglets fed the EPA-enriched formula (Fig. 4; P < 0.05). However, the maximal EPA enrichment attained in 5% EPA-fed pigs was <50% of the ARA enrichment attained in the 5% ARA-fed pigs (Fig. 2). Consistent with dietary concentrations of EPA and 18:3(n-3) precursor (Table 2), intestinal mucosal EPA concentration in all other treatment groups was negligible (Supplemental Tables 1 and 2). Docosahexaenoic acid [DHA; C22:6(n-3)] enrichment also reflected dietary concentrations, being higher in the EPA-supplemented group than in all other groups and increasing progressively with time (Fig. 4; P < 0.05). Regardless of dietary treatment, intestinal mucosal phospholipids from younger pigs (d 4) contained greater proportions of SFA (day effect, P < 0.05) compared with older pigs (d 8 and 16) (Supplemental Tables 1 and 2).


Figure 4
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  		FIGURE 4  EPA [20:5(n-3)] and DHA [22:6(n-3)] enrichment of isolated jejunal (A) and ileal (B) mucosal phospholipids of pigs consuming formula supplemented with 5% EPA and 1.3% DHA for 0, 4, 8, or 16 d. Values are means ± SEM, n = 6. For each fatty acid, means without a common letter differ, P < 0.05.

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
Proper development of the brain, retina, and other body tissues in infants is dependent on provision of ARA and DHA either directly in the diet or through synthesis from linoleic and linolenic acids (15). Human breast milk naturally contains linoleic, linolenic, arachidonic, and docosahexaenoic acids (16,17). The concentration of DHA in milk of women consuming a typical North American diet is generally in the range of 0.1–0.3% of total fatty acids and the level of ARA ranges from 0.4–0.6% of total fatty acids (16). In 2002, the FDA approved of the supplementation of ARA and DHA into U.S. infant formula to help provide the appropriate amounts of ARA and DHA for normal growth and development. The recommended concentration of ARA (maximum of 0.6% of total FA) and DHA (maximum of 0.35% of total FA) included in infant formula is similar to that observed in breast milk (9). The goal of our research was to incrementally increase the dietary ARA up to ~10 times the currently prescribed concentration and to determine the time course for maximal enrichment of ARA in phospholipids of the intestinal mucosa. This goal was predicated on previous research in our laboratory studying the role of prostanoids in the recovery of intestinal barrier function and tight-junction architecture following ischemic injury (18). Prostanoids produced from arachidonate (via cyclooxygenase enzymes) have been implicated in accelerating recovery of gut barrier function following ischemic injury (2), which is thought to play a role in the pathogenesis of necrotizing enterocolitis (19,20).

Supplementation of ARA or EPA up to 5% of dietary fatty acids for 16 d had no detrimental effects on piglet growth, intake, blood metabolites (Table 3), or intestinal morphology (Table 4). Similar to previous studies in which formula containing both ARA and DHA was fed to infants (21–23), there were no differences in growth of ARA-supplemented piglets compared with unsupplemented piglets. Huang et al. (24) confirmed the apparent safety of elevated dietary concentrations in piglets fed ARA and DHA up to 5 times the currently recommended levels. We expected growth depression in pigs fed the deficient diet (8), but others (25) have suggested that 5–10 wk may be required for full manifestation of deficiency symptoms when diets provide <2% of energy as linoleic acid. We did note lower concentrations of circulating lipids and higher concentrations of urea-N in formula-fed pigs compared with sow-suckled controls, as we have reported previously (8). These differences are not surprising, because they reflect the lower lipid and higher protein content of formulas compared with sow milk.

Intestinal lactase activity is higher in the newborn pig than in the mature pig and decreases over the first 2 mo of life with little subsequent change in the enzyme (26). Manners et al. (26) observed that the decline in intestinal lactase activity from birth to 2 wk of age occurred rapidly compared with the decline occurring from 2–8 wk of age. Lactase activity in the present study was lower at d 8 and 16 than at d 4 but did not differ between d 8 and 16.

Prostaglandin E2 exerts trophic effects on the small bowel of suckling rats (27) and when present in high doses, has been shown to increase brush border enzyme activities, possibly indicating accelerated mucosal maturation (28). This might explain in part why our 0% ARA-supplemented pigs showed lower levels of lactase activity compared with the sow-fed pigs. It also is possible that other milk-born growth factors present in sow milk contributed to this difference (29). Gross intestinal health status often is inferred from villus/crypt architecture. The formula-fed piglets in this study on average had greater crypt depths and lower villus:crypt ratios than either the initial (d 0) piglets or the sow-fed controls, but crypt depth and villus:crypt ratios did not differ among the formula-fed pigs. Overall, morphology measurements showed no deleterious effects of feeding elevated levels of ARA or EPA.

Previous research has demonstrated that PUFA content of tissue lipids is strongly modulated by dietary intake (30–33), noting that when dietary linoleic acid was increased in the diet, its metabolite, ARA, increased in tissue lipids. Further research indicated that specific fatty acids may be targeted for synthesis of membrane phospholipids in specific tissues (34). Most recently, a piglet dose-response study illustrated differential enrichment rates of ARA and DHA in various tissues (24). Interestingly, ARA in blood, adipose, and liver responded to increased diet concentration, whereas brain and retina did not. In the present study, elevated levels of dietary ARA were reflected in intestinal mucosal phospholipids. Both jejunal and ileal mucosal phospholipid concentrations of ARA increased with dietary increases in ARA concentration at all time points in the study. Enrichment of jejunal and ileal intestinal mucosa phospholipids plateaued at 8 d of feeding and no further enrichment occurred between d 8 and 16 in either the jejunum or the ileum. We anticipated a greater enrichment by d 4 based upon a 3- to 4-d lifespan of enterocytes as they migrate from the crypt and slough from the villus tip. However, a slower enterocyte turnover rate in suckling neonates (35) may account in part for our observed delay in enrichment.

Unexpectedly, the ARA concentration within the mucosal phospholipids of the 5% EPA-fed group did not differ from the 0% ARA-fed pigs at any of the time points. This came as a surprise due to the competition between parent (n-6) and (n-3) fatty acids for metabolism to their long-chain PUFA metabolites, such as ARA (n-6) and EPA (n-3) (36,37). Perhaps EPA must be fed at a concentration higher than 5% of total fatty acids to competitively inhibit the desaturation and elongation of linoleic acid to ARA.

Because (n-6) and (n-3) fatty acids also compete for esterification, their relative composition in the diet can directly influence their incorporation into phospholipids (38,39). The importance of altering the (n-6):(n-3) fatty acid ratio is that changing the ratio may modulate physiological processes under the influence or regulation of eicosanoids synthesized there from (33,40). Accordingly, we observed that the ratio of intestinal mucosal (n-6):(n-3) fatty acids in the ARA-supplemented pigs was reflective of the PUFA content of the diets. However, EPA did not enrich the intestinal mucosa to the extent that ARA did when fed at equal (5%) concentrations. There was greater and more efficient enrichment of ARA. We speculate that the basis for this observation could relate to differential fatty acid specificity of the multiple isoforms of glycerol-phosphate-acyltransferase and acylglycerolphosphateacyltransferase enzymes responsible for enterocyte phospholipid synthesis (41,42). Our results are consistent, in general, with previous studies (33,43) demonstrating that in vitro and in vivo supplementation of (n-3) PUFA is associated with an increase in rat intestinal cell and mouse hepatic and peritoneal tissue (n-3), respectively, and a decrease in (n-6) PUFA. Additionally, Campbell et al. (44) demonstrated that porcine colonic phospholipids could be enriched in vivo with long-chain PUFA and that enrichment was maintained when supplementation was continued.

In conclusion, this study demonstrated that supra-physiologic supplementation of ARA may be safe for neonates, because it was not detrimental to piglet growth, clinical blood parameters, or morphologic and functional development of the small intestine. Fatty acid analysis of intestinal mucosa showed a dose-response relationship between dietary ARA concentration and ARA content of mucosal phospholipids, with an enrichment plateau at 2.5% ARA and 8 d of feeding. The goal of our continuing research is to investigate the effects of prophylactically altering intestinal fatty acid composition, through dietary manipulation, on enhancing acute recovery from ischemic injury and to confirm the role of prostanoids in the mechanism of gut recovery from ischemic injury induced when intestinal fatty acid composition is altered by dietary means.


   ACKNOWLEDGMENTS
 
We thank Dr. Lori Gatlin, Oulayvanh Phillips, and Ryan Odle for their assistance with animal care and laboratory analyses and Martek Biosciences and Pronova Biocare for donation of the long-chain PUFA oils.


   FOOTNOTES
 
1 Supported in part by Cooperative State Research, Education and Extension Service, USDA-National Research Initiative grant no. 2005-35200-16174 and by the North Carolina Agricultural Research Service. Back

2 Author disclosures: H. A. Hess, B. A. Corl, X. Lin, S. K. Jacobi, R. J. Harrell, A. T. Blikslager, and J. Odle, no conflicts of interest. Back

3 Supplemental Tables 1 and 2 containing complete intestinal fatty acid profiles are available with the online posting of this article at jn.nutrition.org. Back

6 Abbreviations used: ARA, arachidonic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid. Back

Manuscript received 12 June 2008. Initial review completed 29 July 2008. Revision accepted 10 August 2008.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 

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