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Human Nutrition and Metabolism

The Source of Long-Chain PUFA in Formula Supplements Does Not Affect the Fatty Acid Composition of Plasma Lipids in Full-Term Infants¹

Aleix Sala-Vila, Ana I. Castellote, Cristina Campoy^*, Montserrat Rivero, María Rodriguez-Palmero and M. Carmen López-Sabater²

Department Nutrició i Bromatologia, Centre de Referència en Tecnologia dels Aliments (CeRTA), Facultat de Farmàcia, Universitat de Barcelona, 08028 Barcelona, Spain; ^* Department Neonatology, Hospital Universitario de Granada, Granada, Spain; and Scientific Department, ORDESA Lab. SL, Barcelona, Spain

²To whom correspondence should be addressed. E-mail: mclopez{at}ub.edu.

	ABSTRACT

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Supplementation of formulas for full-term infants with long-chain (LC) PUFA [arachidonic acid (AA) and docosahexaenoic acid (DHA)] at levels resembling human milk is recommended because they provide biochemical and functional benefits to the neonate. The objective of this work was to determine whether the source of dietary LC-PUFA affects the bioavailability in full-term infants. Treatment groups were as follows: full-term infants were fed from birth to 3 mo breast-milk (n = 11, 0.4 and 0.3 g/100 g total fatty acids as AA and DHA, respectively), formula containing LC-PUFA in the form of egg phospholipids (n = 12), or a formula supplemented with LC-PUFA in the form of triglycerides synthesized by single cells of algal and fungal microorganisms (n = 12). Both formulas provided 0.4 and 0.1 g/100 g total fatty acids as AA and DHA, respectively. We compared the fatty acid compositions of the main plasma lipid fractions (phospholipids, triglycerides, and cholesteryl esters) at birth and 3 mo. At 3 mo, lower levels of nervonic acid (NA), docosapentaenoic (DPA) acid, and DHA were found in all plasma lipid fractions from infants fed formula compared with those in the human milk–fed infants, irrespective of the source of the formula supplement (P < 0.02). These data demonstrate that the form of dietary LC-PUFA (triglycerides or phospholipids) does not influence their bioavailability. Similarly, absorption of LC-PUFA depends mainly on the lipid composition of the diet fed. These results suggest that the levels of NA, DPA, and DHA in formulas for full-term infants should be increased.

KEY WORDS: • human milk • infant formula • LC-PUFA • phospholipids • plasma

The quality and composition of dietary fat, especially long-chain PUFA (LC-PUFA),³ supplied to infants have important structural and functional effects on their development (1–4). The most important LC-PUFA are arachidonic acid [AA; 20:4(n-6)] and DHA [DHA; 22:6(n-3)]. AA and DHA are incorporated into the phospholipid membranes of the retina and brain during intrauterine development, and continue to accumulate during the first 2 y of life (5–7). Thus, maintenance of neural AA and DHA levels is necessary to achieve optimal development and function of the newborn (8).

Full-term infants can synthesize LC-PUFA from their dietary 18-carbon precursors {linoleic acid [LA; 18:2(n-6)] and -linolenic acid [ALA; 18:3(n-3)]} via the desaturation-elongation pathway (9–11). Nevertheless, the addition of preformed LC-PUFA to formulas for full-term infants may enhance DHA and AA status in blood, brain, and retina lipids, resembling the levels of breast-fed infants (12–14), suggesting that endogenous synthesis of LC-PUFA in full-term infants is insufficient (15,16).

In recognition of the need to match the LC-PUFA composition of human breast milk, the European Society of Pediatric Gastroenterology and Nutrition (ESPGHAN) Committee on Nutrition (17) and the FAO/WHO Expert Committee on Fats and Oils in Human Nutrition (18) recommended that both AA and DHA should be included in infant formulas at levels equivalent to those in human milk because they are the sole source of nutrition for infants (17). A number of highly unsaturated dietary lipid sources are currently available for supplementing infant formulas with LC-PUFA, such as egg yolk lipids, low-eicosapentaenoic acid (EPA) [EPA; 20:5(n-3)] fish oils and oils synthesized from Mortierella alpina and Crypthenocodinium cohnii, and fungal and algal organisms that synthesize oils rich in AA and DHA, respectively. It was shown that these oils had no toxic effects (19–21).

Nevertheless, the sources of LC-PUFA differ in their lipid structure. In egg yolk lipids the LC-PUFA are in the form of phospholipids (PLs), whereas in fungal and algal oils, the LC-PUFA are in the form of triglycerides (TGs). In human milk, 85% of LC-PUFA are in form of TGs (98% of total fat), whereas only 15% of LC-PUFA are in form of PLs (0.8% of total fat) (22). Due to their different chemical structures, the two sources have different metabolic pathways, mainly in the processes of enzymatic hydrolysis, absorption and incorporation into lipoproteins. This involves differences in plasma accretion and, therefore, uptake of LC-PUFA for developing tissues (23). The choice of the dietary source of LC-PUFA is important not only nutritionally but also in terms of economic and industrial criteria.

Although several randomized controlled trials were conducted to investigate the need to add LC-PUFA in formula destined for full-term infants (24–31), few compared the two LC-PUFA sources (PLs and TGs) (32–38). In addition, the results obtained are controversial. The majority of these comparative studies used animals, such as piglets (32,34,35,37), rats (36), or baboons (38), which are appropriate models for human infants. To our knowledge, only one study was performed in preterm infants (33). In that study, LC-PUFA bioavailability was measured as apparent fat absorption via analysis of feces.

Here we report a trial investigating the incorporation of LC-PUFA in full-term infants fed formula enriched in different sources of LC-PUFA, and comparing the fatty acid profile of plasma lipid fractions with that of infants fed human milk (HM). It was demonstrated that a correlation exists between DHA levels in plasma phospholipids and retinal phospholipids (32) and cerebral and hepatic phosphatidylethanolamine (34).

We hypothesized that delivery of LC-PUFA in the same chemical form found in human breast milk (TGs), as in all mammals, would enhance their absorption in full-term infants compared with the earlier used sources of LC-PUFA (egg PLs). The initial fatty acid composition of all plasma fractions served as a reference point for comparison with all treatments. The infants included in the HM group, which received the ideal source of nutrients for the neonate, were used as a comparison for the formula-fed groups. An unsupplemented formula-feeding group was not included here because, in our opinion, it has been widely demonstrated that infants who receive formulas supplemented with LC-PUFA have higher plasma levels of LC-PUFA than infants fed LC-PUFA–free formulas.

	SUBJECTS AND METHODS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Subjects. Neonates (n = 35) born in the Hospital Universitario de Granada (Spain) were enrolled in the study at birth. Eligible subjects were full-term infants (37–42 wk gestation), of appropriate weight-for-gestation-age. None of the infants had delivery complications, evidence of intrauterine malnutrition, congenital malformations, or metabolic abnormalities, and their mothers had experienced a normal pregnancy. All mothers were apparently healthy and nonvegetarian. Their overall mean age was 28.3 y. The Hospital Ethical Committee approved the study protocol, which agreed with the principles in the Declaration of Helsinki. Participation in the study was voluntary; the study was explained to one or both parents and written consent obtained before enrolment. Anthropometric measurements (Table 1) were performed at birth and at 3 mo. Allocation to human milk (HM) or formula groups was based on maternal preference. Infants were assigned to formula groups (group PL or TG) using a computer-generated randomization table (IDU Rancode). Analysts were unaware of the origin of the samples.

Sample collection and analytical procedures

    Blood sampling. Blood samples were collected by venipuncture from each infant at birth and at 3 mo. They were put in brown polypropylene tubes containing EDTA-K as an anticoagulant. Blood samples were centrifuged (3000 x g, 5 min) and the plasma was coded and immediately frozen at -80°C. Samples were preserved in liquid nitrogen and flown from Granada to Barcelona, where they were stored at -80°C until analysis.

Analytical methods

n-Hexane (100 µL) solution containing 2 g/L BHT was added to 150 µL of plasma to prevent lipid oxidation. Plasma lipids were extracted by the method of Kolarovic and Fournier (41). Separation of lipid fractions [PLs, TGs, and cholesteryl esters (CEs)] from plasma extract was accomplished by the method of Skipski and Barclay (42). Lipid fractions were scraped off and placed in test tubes capped with teflon-lined caps. The FAMEs of each fraction were obtained as follows: anhydrous methanolic 3 mol/L HCl (2 mL) was added to the tubes and subjected to methanolysis at 90°C for 1 h. The tubes were then cooled in water and a mixture of Na₂CO₃:Na₂SO₄:NaHCO₃ (2:2:1, by vol) solution was added until the contents of the tubes were neutralized. Then, 1.2 mL of n-hexane was added to the tubes. The tubes were mixed on a vortex and centrifuged (2200 x g, 3 min), and the clear n-hexane top layer, containing the FAMEs, was transferred with a micropipette into an Eppendorf tube. After evaporation of hexane under a stream of nitrogen, FAMEs were redissolved in 80 µL of n-hexane containing 1 g/L BHT and transferred into a GC autosampler vial. After encapsulation, vials were stored at -20°C until injection into the gas chromatograph.

FAMEs were analyzed using a Hewlett-Packard 6890 GC equipped with a flame ionization detector (FID) and HP-6890 Series Injector. Separation of FAME was carried out on a fused silica column (30 m x 0.25 mm i.d.), coated with SP-2330 stationary phase [poly (80% byscyanopropyl-20% cyanopropylphenyl) siloxane, 0.20-µm film thickness] from Supelco.

The split-splitless injector was used in split mode with a split ratio of 1:30. The injection volume of the sample was 3 µL. The injector and detector temperatures were kept at 250 and 270°C, respectively. The oven temperature was programmed as follows: initial temperature 130°C for 3 min, then the temperature was increased by 7°C/min to 170°C and held at this temperature for 2 min. The temperature was increased again at 6°C/min to 240°C, and then left to stand for 10 min at 240°C. Helium was used as the carrier gas, with a linear velocity of 22.5 cm/s. The recording of chromatograms and the quantitative measurement of peak areas were performed with a HP-Chemstation from GC systems. Chromatographic peaks were identified by comparing the retention times with those of known standards.

The fatty acid compositions of the infant formulas (n = 2 for each formula) and mature human milk samples (n = 6) were also determined by GC, using the method described by Lopez (43).

    Statistical analysis. All determinations were made in duplicate. The results are reported as means ± SEM. Analyses were made using the statistical package SPSS 10.0. To test differences among groups and within-group changes, repeated-measures ANOVA was performed. When a significant difference was observed (P < 0.05), pairwise multiple comparisons were performed using Tukey’s test.

	RESULTS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Anthropometric measures. The overall mean body weight at the beginning of the study was 3192.38 g. Body weight, cranial perimeter, and thoracic perimeter increased exponentially. The anthropometric variables did not differ among the 3 groups at birth or after the 3-mo feeding period (Table 1).

Fatty acid composition of plasma lipid fractions

    Plasma PL. In all 3 feeding groups, there was a fall in the levels of 16:0, 16:1(n-7), and 20:3(n-6) (P < 0.005) in plasma PL during the first 3 mo of life, whereas levels of LA and EPA increased (P < 0.001) (Table 3). Moreover, breast-fed infants had higher levels of DHA (P < 0.001), docosapentaenoic acid [DPA; 22:5(n-3)] (P < 0.02) and nervonic acid [NA; 24:1(n-9)] (P < 0.02) at 3 mo than formula-fed infants. Other fatty acids did not differ among the 3 feeding groups.

    Plasma CE. Levels of 16:1(n-7), 18:1(n-9), 20:3(n-6), 20:4(n-6), and 20:5(n-3) decreased during the feeding period, whereas the level of LA in plasma CE at 3 mo was significantly higher than at birth. Infants fed PL had higher concentrations of 16:0 than infants in the other groups at the end of the feeding period. As with the other plasma lipid fractions, formula-fed infants had lower levels of DHA, DPA, and NA at 3 mo than breast-fed infants.

	DISCUSSION

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
In the present work, we compared two widely used dietary LC-PUFA sources. Egg PLs and TGs of single-cell oils differ not only in their chemical form, but also in their fatty acid composition, intramolecular distribution of fatty acids, and presence of components such as cholesterol or phosphate groups that may influence digestion and absorption of LC-PUFA. The diets used in this study were designed to be as similar as possible in fatty acid composition, except for the origin and chemical form of LC-PUFA. The level of DHA supplementation of the formulas used in our trial was chosen according to the content of the fatty acid in LC-PUFA–supplemented formulas commercially available in Spain. Nevertheless, the DHA concentrations of commercial formulas are lower than those in the breast milk of Spanish women (44). This is of little relevance here because we were not seeking to determine the optimal PUFA concentration for formulas but rather which PUFA source was better absorbed.

At the beginning of the study, blood samples were collected. Full-term infants were fed different diets for 13 wk after birth because the mean duration of breast-feeding in Spain is 3.2 mo (45). After this feeding period, blood samples were taken again to determine the effect of the diet on the fatty acid composition of the plasma fractions, mainly the PL composition. The fatty acid profile of plasma PL, together with the fatty acid composition of buccal check cell PL (46), is used to determine the metabolism and uptake of dietary LC-PUFA in newborns.

Profiles of plasma lipid fractions did not differ between the two groups of formula-fed infants, except for 16:0; its incorporation into the CEs of E-PL formula-fed infants was higher than that in infants fed SC-TG formulas. Our results for AA and DHA agree with those obtained by Goustard-Langelier et al. (34) and Amate et al. (35) in piglets, who observed that in equal concentrations, incorporation of LC-PUFA into plasma PLs did not differ. Nevertheless, our results do not agree with those obtained by Mathews et al. (37) in piglets and Wijendran et al. (38) in baboons because they found that LC-PUFA absorption was higher when they were delivered in the form of TGs (37) or PLs (38), respectively. Nevertheless, in these trials, LC-PUFA absorption was not determined by the fatty acid composition of plasma PLs but by measurements of total plasma fatty acids (37) and radioactivity in tissues after feeding baboons with radiolabeled fatty acids (38). We suggest that our results are not comparable to those obtained by Alessandri et al. (32) in piglets or Carnielli et al. (33) because in those trials, the quantities of LC-PUFA were not balanced in the LC-PUFA–supplemented diets.

DHA concentrations in all plasma lipid fractions of formula-fed infants (irrespective of source) were significantly lower than those of breast-fed infants. In contrast, the 3 feeding groups did not differ in AA content of PLs, TGs, or CEs, suggesting that the DHA content in plasma lipid fractions reflects the diet fed. This agrees with two trials performed previously in piglets (32,34) comparing the incorporation of LC-PUFA into plasma PLs of formula-fed piglets with those fed sow’s milk; feeding sow’s milk resulted in much lower DHA plasma phospholipid concentrations compared with the formulas. These results can be explained by the composition of the diets administered because the DHA concentration of sow’s milk was significantly lower than those in the formulas used.

On other hand, breast-fed infants also had higher levels of NA in plasma PLs. Because NA is not a PUFA, we did not consider its concentration relevant to the formula design used in this trial. Nevertheless, we have observed a growing interest in NA in pediatric nutrition because it is incorporated into the developing central nervous system, mainly from mid-gestation until the end of the second postnatal year (47,48). Several studies performed in mammals demonstrated that NA does not cross the placental barrier in rats, but it can cross the mammary epithelial and intestinal barriers (49). The NA content in cerebellar white matter increases with age in human newborns (50), and decreases in lactating rat dams (49). Despite the low concentration of NA in human milk, levels were still higher than those in the formulas used in the present trial because they were not supplemented with sources containing NA, such as canola oil. Therefore, differences in the NA content of plasma PLs can be explained by the difference in the NA concentration in diets administered to neonates. Moreover, this fact confirms that even though newborns are able to synthesize NA from 18:1(n-9) by desaturation-elongation pathways (51), supplying preformed NA (via breast-milk or supplemented formulas) is a more efficient way to enhance NA levels in newborns.

In conclusion, incorporation of LC-PUFA into plasma PLs (and therefore into neural and retinal tissues) depends more on the fatty acid composition of the diet fed than on the source (TG or PL) of the dietary LC-PUFA.

	ACKNOWLEDGMENTS

 
We thank all parents and their infants. Robin Rycroft revised the English manuscript.

	FOOTNOTES

 
¹ Supported by a fellowship from Fundació Bosch i Gimpera, project 3427 and CeRTA (Centre de Referència en Tecnologia dels Aliments, Catalunya, Spain).

³ Abbreviations used: AA, arachidonic acid; ALA, -linolenic acid; CE, cholesteryl ester; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; E-PL, formula containing LC-PUFA as egg phospholipids; HM, human breast milk; LA, linoleic acid; LC-PUFA, long-chain PUFA; NA, nervonic acid; PL, phospholipid; SC-TG, formula containing LC-PUFA as single-cell triglycerides.; TG, triglyceride.

Manuscript received 29 October 2003. Initial review completed 2 January 2004. Revision accepted 14 January 2004.

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TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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