The results showed that, although formulas containing a mixture of palm olein, palm kernel oil, and canola oil (PALM) provide proportions of palmitic acid similar to those of human milk fat, they result in significantly lower absorption of fat and retention of calcium by infants compared with a blend of sunflower, coconut, and soy oils (NoPALM) (Tables 1 and 4). Furthermore, we observed different absorption percentages for fatty acids in the formulas examined in this study (Table 4). The results are concordant with those of other studies [4, 9, 12, 13] comparing calcium and fat absorption from palm olein–predominant milk-based formulas versus formulas without palm olein.
Compared to calcium absorption, calcium retention is more accurate markers of functional outcomes for the impact of dietary calcium on calcium homeostasis [9, 20]. Borschel et al. [21] demonstrated a significantly (p = 0.041) lower bone mineral content in term infants fed a palm olein containing partially hydrolyzed whey protein-based formula compared to a similar formula containing no palm olein. In another clinical trial, quantitative balance studies were performed to compare calcium absorption in healthy, full term infants fed casein hydrolysate-based and soy protein-based infant formulas with or without palm olein.
In another clinical trial, quantitative balance studies were performed to compare calcium absorption in healthy, full term infants fed casein hydrolysate-based and soy protein-based infant formulas with or without palm olein. Calcium intake did not differ between the groups. However, infant’s calcium absorption was less when fed with casein hydrolysate-based and soy protein-based with palm olein compared to when fed without olein [13].
It is important to highlight that the fat absorptions for both formulas assessed in this study (PALM, 95.50%; NoPALM, 96.55%) were comparable to that of human milk (90.5–97.10%) [5, 22]. However, only the NoPALM formula offered calcium absorption and retention (58.00 and 55.10%, respectively) similar to the reported values of breast milk (58.70 and 52.40%, respectively) [23]. An important physiological consequence of reduced calcium bioavailability is the negative effect on bone mass accretion. Moreover, good fat absorption is important for infants because of the high calorie content of fat and its role in brain development [24].
A systematic review of human intervention studies on the effects of infant formulas with the addition of palm olein on bone mineral content and bone mineral density concluded that healthy infants fed a formula containing palm olein as the predominant oil had significantly lower values for both parameters than those fed a formula without olein. The inclusion of this oil in infant formula to provide a fatty acid profile at required levels leads to lower bone mineralization [6].
Infants fed NoPALM in the current study had significantly higher fecal concentrations of oleic, palmitic, and stearic acids versus higher fecal concentrations of palmitic, oleic, and linoleic acid after PALM formula feeding. Palmitic acid accounted for a large proportion of unabsorbed fatty acids in the PALM formula (38.96%). Studies have reported that infants fed a milk-based formula containing palm olein as a predominant fat have a higher fecal excretion and lower absorption of palmitic acid [4, 11, 12]. In palm olein, the palmitic acid is preferentially esterified at positions sn-1 and sn-3 of the triglyceride molecule. Thus, it is absorbed as a free fatty acid that can bind with calcium in the intestine, forming fatty acid soaps that are excreted fecally, resulting in the low absorption of both nutrients. Moreover, in the intestine, the fatty acid soaps solidify because of their high melting temperature causing hard stools and constipation in the infant. [6, 8, 9, 13].
As previously demonstrated [4, 12], the percentage of palmitic acid absorption was similar for the PALM (97.56%) and NoPALM (95.77%) (p = 0.094) formulas (Table 4). Nelson et al. [4] compared the absorption of fatty acids in a group of term infants fed a milk-based formula containing palm olein (45%), soy (20%), coconut (20%), and sunflower (15%) oils with a group fed a formula that contained a blend of safflower, coconut, and soy oils, concluding that the absorption of palmitic acid (91.70%) was better in the formula without palm olein. However, unlike the study of Nelson et al. [4], the PALM formula in the current investigation contained palm kernel oil in addition to palm olein. While palm olein is extracted from the mesocarp of the fruit Elaeis guineans, the palm kernel oil is derived from the seed of this fruit and the two have different fatty acid compositions. Palm olein contains 40–42.5% palmitic acid, 9% of which is esterified in the sn-2 position, 9.4–13.52% is already in kernel oil, and 6% is in the sn-2 position [22, 25]. This change in fat composition may influence the fatty acid absorption by infants.
The lauric (C12:0) and myristic (C14:0) saturated fatty acids were significantly (p < 0.05) better absorbed by infants fed the NoPALM (99.70 and 98.54%, respectively) formula compared to those fed the PALM (98.89 and 97.65%, respectively) formula. Raiten [1], as in the Assessment of Nutrient Requirements for Infant Formulas report, did not recommend adding myristic or lauric acids to infant formulas since there are no data to indicate their specific roles as dietary nutrients. However, these fatty acids are components of some oils used in infant formulas, and the author does not proscribe the use of such oils [1]. Since no data are available on which to base a recommendation, the Codex Alimentarius [15] recommends that the maximum levels of lauric and myristic acid in infant formulas not exceed 20% of the total fatty acids. The evaluated formulas had concentrations within these values (PALM, 12.26%; NoPALM, 18.99%) (Table 1). Furthermore, the infants fed both formulas demonstrated absorption percentages of lauric and myristic acid similar to those of infants fed breast milk [5].
The absorption of essential fatty acids (18:2n6 and 18:3n3) were similar for both formulas. However, the absorption of LCPUFA (ARA and DHA) were significantly (p < 0.05) greater for the NoPALM formula even when the intake was used as a covariate (Tables 3 and 4). The values found in this study were superior to those found by Moya et al. [26] and Canielli et al. [27]. However, both of those studies measured the absorption of fatty acids in premature infants, which may explain the lower values. To date, our current study is the first and only study to report the impact of dietary palm olein on the absorption of DHA and ARA in infants. Previous studies on palm olein evaluated infant formulas which were not supplemented with DHA and ARA.
The importance of essential fatty acids, as dietary precursors for eicosanoid and docosanoid formation, has been widely reported. The LCPUFA DHA and ARA are derived from their precursors ALA and LA, respectively. However, ALA and LA cannot be synthesized owing to the lack of the required dietary enzymatic desaturases [28]. DHA and ARA are found in high proportions in the structural lipids of cell membranes, particularly those of the retina and central nervous system, and their accretion primarily occurs during the last trimester of pregnancy and the first year of life [28, 29].
It had previously been assumed that infants could synthesize LCPUFA from essential fatty acids (ALA and LA) through the elongase and desaturase systems. However, evidence that infants fed formula deficient in LCPUFA have a significantly lower plasma or red blood cell levels of DHA and ARA compared with those who were breastfed or fed formula supplemented with LCPUFA suggests that the enzyme systems in infants may be inefficient during the first months of life [30].
In the neonatal period, dietary n-6 and n-3 fatty acid balance is necessary to provide essential polyunsaturated fatty acids for normal growth and development, particularly that of the brain. This ratio is important because both essential fatty acids (ALA and LA) compete for the same enzyme during the synthesis of LCPUFA (DHA and ARA). In this study, both formulas were within the margin of 5:1 and 15:1 suggested by the Codex Alimentarius [15] (PALM, 8:1; NoPALM, 12:1) and the ratio reported in breast milk (10:1) [5, 27].
Supplementation of infant formulas with DHA and ARA for term infants remains controversial. A meta-analysis by Qawasmi et al. [31] concluded that the supplementation of infant formulas with LCPUFA failed to show any significant effect on improving early infant cognition; however, opposite results were reported by Jiao et al. [32]. Another meta-analysis showed that LCPUFA supplementation of infant formulas improves infant visual acuity up to 12 months of age [33]. The European Food Safety Authority concluded from a review of the literature that, although DHA is required for infant formula, ARA is not [34]. However, Crawford et al. [35] did not agree with this opinion and have commented on the recommendations around the need for ARA in infant formulas.
The data presented in this study show that the absorptions of the fatty acids DHA and ARA were as efficient as those from breast milk [27] for the two evaluated formulas. However, absorption percentages were significantly higher for the NoPALM formula.
Fish and algal oils are the main sources of DHA added to infant formulas. However, unlike in breast milk triacylglycerols, in which DHA is preferentially esterified in the sn-2 position, algal and fish oils do not have a strong positional specificity; rather, there are similar proportions at the sn-1, sn-2, and sn-3 positions [36]. Differences in the molecular structure of the triacylglycerols in these oils may contribute to the differences in digestibility and absorption of these two products [37].
Our results showed that DHA was better absorbed by infants fed the NoPALM formula than those fed the PALM formula. The source of DHA may partly explain this difference (NoPALM, algal oil; PALM, fish oil) since the intake of this fatty acid was significantly higher with the PALM formula. Clandinin et al. [38] evaluated the benefits of feeding preterm infants formula supplemented with fish and algal oils as a source of DHA. The authors observed an increase in weight and length of infants fed DHA from algal oil but not from fish oil; however, the mechanism for this increase was unclear. Tou et al. [39] also observed the influence of DHA source in digestibility and tissue incorporation of rats fed diets containing different oils. Unlike the above studies in preterm infants and rats, the source of DHA is less likely to be impactful on DHA absorption in comparison to the impact of palm olein in our current study because we evaluated term human infants and there were no differences noted in weight or growth. Nonetheless, the influence of DHA sources on DHA absorption human term infants remains untested.
An association between the fecal excretions of calcium and fatty acids, especially palmitic and stearic acids, was demonstrated for the PALM formula. The increase in calcium excretion was significantly (p < 0.01) and directly proportional to the excretions of palmitic and stearic acid in the PALM formula (r
s
= 0.71 and r
s
= 0.69, respectively). However, these correlations were inversely proportional but not significant (p > 0.05) in the NoPALM formula (r
s
= −0.10 and r
s
= −0.40, respectively) (Fig. 2). These data reinforce the hypothesis that the excretion and consequent absorption of calcium are closely related to the palmitic acid source in infant formula. The palmitic acid from palm olein is not absorbed efficiently; rather, it forms insoluble calcium soaps in the intestinal tract, rendering a portion of dietary calcium unavailable for absorption. The observation of a high correlation between calcium and palmitic acid excretion in infants fed formula containing palm olein resulting in low calcium absorption and retention is also supported by other authors [4, 12].
Whether the reduced absorption percentage of fat, fatty acids, and calcium retention caused by the inclusion of palm olein in infant formula is clinically relevant is a matter of perspective. Fecal loss of 0.22 g fat/kg (PALM) and 0.14 g/fat/kg (NoPALM) represents a loss of 9.95 kJ/kg (2.4 kcal) and 6.30 kJ/kg (1.50 kcal), respectively, each day. Normal infants are certainly capable of increasing energy intake proportionately to make up for an energy loss of this magnitude, but preterm infants may have difficulty because of intestinal immaturity. The fecal loss per day can be considered low, but should be taken into consideration during the first year of life when infants are almost exclusively fed formula. As fat provides up to 50% of the total calorie content of most infant formulas, it is important to make allowance for the variations in absorption with different sources of fats. Estimation of caloric intake on the basis of milk composition alone is likely to be a confounding factor when fats of different origins are considered. Similarly, decreased retention of calcium suggests decreased bone mineral deposition. Koo et al. [40] demonstrated that differences in calcium absorption in infants fed formulas with and without palm olein, led to significant differences in bone mineral content at three and 6 months of age. However, additional long-term studies are necessary to evaluate this influence.
A limitation of our current concerns the carryover effect, observed for some variables, between the analyzed periods, wihich can distort the results obtained after the second period. The crossover design is used in clinical trials to provide an unbiased estimate of the difference between the treatment effects. In the presence of a differential carryover effect, such an estimate can only be obtained by: using data from the first treatment period only or assuming that there is no differential carryover [41]. According to William and Pater [42] in many situations the carryover effect is unlikely to exist. However, these authors and others advise that if crossover design has been used, unless carryover effects are negligible, the analysis is based on only the first-period data. But, evaluating only the data of the first period, is a limiting factor, because it increases the variance by not eliminating the variability between subjects. In other studies, the use of washout periods between administrations of interventions can be used to combat carryover effects.