The influence of a formula supplemented with dairy lipids and plant oils on the erythrocyte membrane omega-3 fatty acid profile in healthy full-term infants: a double-blind randomized controlled trial

Human milk is recognized as the gold standard for infant nutrition [1‐3]. Lipids are a major component of human milk and provide 45% of total energy intake. The main constituents are triacylglycerols, phospholipids and their components fatty acids and sterols. Lipids contribute to several biological functions with regard to growth and development. In particular, human milk provides essential fatty acids (EFA) and long chain polyunsaturated fatty acids (LC-PUFA) of omega-3 and omega-6 families, such as docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (AA, 20:4n-6) [4]. These fatty acids constitute the main components of brain tissue and have an important impact on neuronal and visual functions [5]. LC-PUFA accretion in human brain is a slow gradual process [6]. The last trimester of pregnancy represents a critical period for LC-PUFA deposition whose accretion in the nervous system endures after birth [7‐9]. The deposit of AA during the last trimester of pregnancy differs from that of DHA so that at term human brain contains relatively more AA than DHA. However, after term, DHA brain content increases, resulting in DHA being the main LC-PUFA in adult brain. It has been demonstrated that lipids represent 50 to 60% of dry adult brain weight [10]. In addition, cerebral LC-PUFA concentration seems to be higher in the cortical gray matter and lower in the white matter [11, 12]. In baboon neonates, DHA and AA have been found to be present at highest concentrations in the precentral, postcentral, prefrontal and occipital cortices, and also in basal ganglia, hippocampus, thalamus and cerebellum [12]. As these areas are mainly involved in the development of sensor-motor integration, attention and memory functions, these data suggest the critical role of LC-PUFA in promoting these skills.

Experimental studies confirmed that a deficient dietary intake of DHA can lead to a reduced neuronal cell size, a decreased visual function and a compromised learning behavior [13, 14].

DHA and AA can be directly provided from the diet or be synthesized from endogenous conversion of the precursors alpha-linolenic acid (ALA; 18:3ω3) and linoleic acid (LA; 18:2ω6) by enzymatic processes, which are present also in fetuses and infants. However, these enzymatic systems seem to be unable to satisfy LC-PUFA requirements in infants until 16 weeks after birth [15, 16]. Accordingly, LC-PUFA during pregnancy are mainly supplied by the placental transfer, whereas during the first months of life the infant is dependent on LC-PUFA supply through breast milk or formula [17].

Fatty acid status can be evaluated by measuring the fatty acid composition of functional tissue such as brain or retina. However, in clinical studies, substitute parameters can be used. It has been demonstrated that erythrocyte membrane fatty acids are representative of brain cell membranes composition, whereas serum lipid levels are influenced by other transient factors, such as contingent diet [18, 19]. Therefore, DHA levels in erythrocyte membrane phospholipids are commonly used as an indicator of brain DHA status.

When breastfeeding is not possible, milk substitutes represent the nutritional alternative. It has been demonstrated that formula fed infants have lower levels of LC-PUFA in their cerebral cortex than breastfed infants. This relative deficit of LC-PUFA may partially explain the lower Intelligence Quotient scores reported in formula fed infants in comparison with breastfed infants [20, 21]. Makrides M. et al. [22] reported a positive correlation between the erythrocyte DHA levels and the visual-evoked potential acuity. In addition, the authors demonstrated that full-term formula fed infants had erythrocyte DHA levels lower than breast fed infants [22]. Farquharson J. et al. [23] have found lower levels of LC-PUFA in the cerebral cortex of infants fed a formula enriched only with EFA than in breastfed infants. These results suggest that formulas containing only ALA and LA may not be adequate to satisfy the actual requirements of infants in terms of LC-PUFA. Furthermore, it has been reported that infants fed a formula supplemented with DHA and AA have higher erythrocyte membrane omega-3 concentrations at 9 months of age as compared to infants fed an unsupplemented formula [24]. Indeed, supplementation of infant formula with LC-PUFA appears to be associated with a beneficial effect on short-term neurodevelopmental outcome and visual function [25‐27]. Although evidence concerning the persistence of the beneficial effect beyond the fourth month of age is lacking, it cannot be excluded that the positive effect of LC-PUFA supplementation may become again evident at school age when infants are required to perform tasks that necessitate more complex neural functions [15].

Besides, DHA consumption appears to influence infants’ body composition by promoting the development of fat free mass without any detrimental effect on growth [27]. Courville AB. et al. [28] have recently demonstrated that infants of mothers consuming food supplemented with DHA during the last half of pregnancy have lower ponderal indices and umbilical cord blood insulin concentrations than infants of mothers consuming the placebo.

Most of infant formulas available nowadays on the market contain plant oils as the only source of fat [29]. Indeed, infant formulas have been enriched in EFA-rich plant oils as cow milk fat does not contain enough EFA to meet infant’s needs [30]. The main plant oils used are coconut oil, corn oil, soybean oil, palm olein, palm kernel oil, palm oil, high oleic safflower oil, peanut oil, and, in Europe, low-erucic acid rapeseed oil. Vegetable oil-based formulas can contain up to 4% residual milk fat [31]. However, plant oils do not contain specific fatty acids, particularly short chain fatty acids, that are present in human and cow milk [32, 33] and constitute a pertinent energy source for infants [31]. In addition, plant oils do not encounter milk fat triglyceride structure [30].

The supplementation of infant formulas with dairy lipids could provide a fat composition and structure closer to human milk, thus improving the quality of formula fat composition. Dabadie et al. [34] demonstrated that dairy lipids associated with rapeseed oil significantly increased erythrocyte DHA levels adults. Recently, it has been reported that rodents consuming a diet with a mix of dairy lipids and plant oils showed levels of brain DHA higher than rodents consuming a diet containing only plant oils or a DHA-enriched diet containing plant oils, with the same ALA contents [35]. These data suggest that a mix of dairy lipids and ALA-rich plant oils could potentiate endogenous n-3 LC-PUFA synthesis.

The study is designed as a mono-centric, double-blind, randomized controlled trial. The Ethical Committee of the Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, University of Milan, Italy, approves the study protocol before the start of the study. Written informed consent is obtained from both parents.

Human milk represents the optimal nutrition for infants and ensures optimal growth and development. LC-PUFA, which are provided through breast milk, play a critical role in the development of neuronal and visual functions. Hence, the achievement of an appropriate neonatal LC-PUFA status represents an important goal in neonatal nutrition, especially when breastfeeding is not possible [5, 7, 8]. Formula fed infants have been demonstrated to exhibit lower cerebral and erythrocyte LC-PUFA levels as compared to breast fed infants when consuming an unsupplemented formula or a formula supplemented only with EFA [22, 23]. On the contrary, infants receiving a formula supplemented with DHA and AA show higher erythrocyte membrane omega-3 concentrations than infants fed an unsupplemented formula [24]. Recent experimental data indicate that a dairy fat blend providing as little as 1.5% ALA is superior to the plant oil blend for increasing brain DHA, even when the recommended DHA and AA levels are exogenously provided, suggesting that the endogenous synthesis of n-3 LC-PUFA synthesis can be enhanced [35]. Formula fed infants could then benefit from the supplementation of formula with dairy lipids and plant oils by attaining higher erythrocyte membrane omega-3 FA content than infants receiving a formula supplemented with plant oils or plants oils enriched with LC-PUFA. The achievement of optimal DHA levels in formula fed infants could also be associated with improved neurocognitive and visual functions, as previously underlined by other studies [25‐27]. Not only the erythrocyte membrane omega-3 FA levels will be investigated but also the other erythrocyte membrane FA levels, the plasma lipid profile, the insulin growth factor 1, the formula tolerance, the growth and body composition. Gaining insight in the effects of the supplementation of a formula with dairy lipids and plant oils in healthy full-term infants could improve the fat content, composition and structure of infant formulas, making them closer to human milk.