Infant formula containing large, milk phospholipid-coated lipid droplets and dairy lipids affects cognitive performance at school age.

Background: Breastfeeding has been positively associated with infant and child neurocognitive development and function. Contributing to this effect may be differences between human milk and infant formula in the milk lipid composition and milk fat globule structure. Objective: To evaluate the effects of an infant formula mimicking human milk lipid composition and milk fat globule structure on childhood cognitive performance. Methods: In a randomized, controlled trial, healthy term infants received until 4 months of age either a Standard infant formula (n = 108) or a Concept infant formula (n = 115) with large, milk phospholipid coated lipid droplets and containing dairy lipids. A breastfed reference group (n = 88) was included. Erythrocyte fatty acid composition was determined at 3 months of age. Neurocognitive function was assessed as exploratory follow-up outcome at 3, 4, and 5 years of age using the Flanker test, Dimensional Change Card Sort (DCCS) test and Picture Sequence Memory test from the National Institutes of Health Toolbox Cognition Battery. Mann-Whitney U test and Fisher exact test were used to compare groups. Results: Erythrocyte omega-6 to -3 long-chain polyunsaturated fatty acid ratio appeared to be lower in the Concept compared to the Standard group (P = 0.025). At age 5, only the Concept group was comparable to the Breastfed group in the highest reached levels on the Flanker test, and the DCCS computed score was higher in the Concept compared to the Standard group (P = 0.021). Conclusion: These outcomes suggest that exposure to an infant formula mimicking human milk lipid composition and milk fat globule structure positively affects child neurocognitive development. Underlying mechanisms may include a different omega-3 fatty acid status during the first months of life. Clinical trial registration: https://onderzoekmetmensen.nl/en/trial/28614, identifier NTR3683 and NTR5538.

Levels of n-3 and n-6 Fatty Acids in Maternal Erythrocytes during Pregnancy and in Human Milk and Its Association with Perinatal Mental Health.

Omega-3 long-chain polyunsaturated fatty acid (n-3 FA) status may be associated with mood disorders. Here, we evaluated the potential association between antenatal depression/anxiety and n-3/n-6 FA in (a) maternal erythrocytes and (b) human milk. In addition, we explored associations between n-3/n-6 FA in erythrocytes and in human milk and postpartum depression, while controlling for antenatal depression. Twenty-seven pregnant women diagnosed with a current major depressive disorder (MDD; n = 9), anxiety disorder (AD; n = 10) or a mixed anxiety-depression disorder (MADD; n = 8), and 40 healthy controls were included. n-3/n-6 FA were determined in maternal erythrocytes in gestational week 32 and in human milk in postpartum week 1. In the first week postpartum, the Edinburgh-Postnatal-Depression-Questionnaire was used to assess postpartum depression. Results show that women with M(A)DD had significantly lower erythrocyte levels of total n-3 FA, EPA, DHA and DGLA, and significantly higher n-6 DPA, and n-6:n-3, AA:EPA and n-6 DPA:DHA ratios compared to healthy controls. No significant associations between antenatal depression or anxiety and n-3/n-6 FA in human milk were found. After controlling for antenatal mental health, n-3/n-6 FA in maternal erythrocytes or in human milk were not significantly associated with postpartum depression. In conclusion, antenatal depression, alone or with an anxiety disorder, was associated with lower n-3 FA levels and higher n-6:n-3 FA ratios in maternal erythrocytes during gestation. This study provides some insights into the associations between n-3/n-6 FA levels during pregnancy and lactation and perinatal mental health.

Structure of human nSMase2 reveals an interdomain allosteric activation mechanism for ceramide generation.

Neutral sphingomyelinase 2 (nSMase2, product of the SMPD3 gene) is a key enzyme for ceramide generation that is involved in regulating cellular stress responses and exosome-mediated intercellular communication. nSMase2 is activated by diverse stimuli, including the anionic phospholipid phosphatidylserine. Phosphatidylserine binds to an integral-membrane N-terminal domain (NTD); however, how the NTD activates the C-terminal catalytic domain is unclear. Here, we identify the complete catalytic domain of nSMase2, which was misannotated because of a large insertion. We find the soluble catalytic domain interacts directly with the membrane-associated NTD, which serves as both a membrane anchor and an allosteric activator. The juxtamembrane region, which links the NTD and the catalytic domain, is necessary and sufficient for activation. Furthermore, we provide a mechanistic basis for this phenomenon using the crystal structure of the human nSMase2 catalytic domain determined at 1.85-Å resolution. The structure reveals a DNase-I-type fold with a hydrophobic track leading to the active site that is blocked by an evolutionarily conserved motif which we term the "DK switch." Structural analysis of nSMase2 and the extended N-SMase family shows that the DK switch can adopt different conformations to reposition a universally conserved Asp (D) residue involved in catalysis. Mutation of this Asp residue in nSMase2 disrupts catalysis, allosteric activation, stimulation by phosphatidylserine, and pharmacological inhibition by the lipid-competitive inhibitor GW4869. Taken together, these results demonstrate that the DK switch regulates ceramide generation by nSMase2 and is governed by an allosteric interdomain interaction at the membrane interface.

A Postnatal Diet Containing Phospholipids, Processed to Yield Large, Phospholipid-Coated Lipid Droplets, Affects Specific Cognitive Behaviors in Healthy Male Mice.

BACKGROUND: Infant cognitive development can be positively influenced by breastfeeding rather than formula feeding. The composition of breast milk, especially lipid quality, and the duration of breastfeeding have been linked to this effect. OBJECTIVE: We investigated whether the physical properties and composition of lipid droplets in milk may contribute to cognitive development. METHODS: From postnatal day (P) 16 to P44, healthy male C57BL/6JOlaHsd mice were fed either a control or a concept rodent diet, in which the dietary lipid droplets were large and coated with milk phospholipids, resembling more closely the physical properties and composition of breast milk lipids. Thereafter, all mice were fed an AIN-93M semisynthetic rodent diet. The mice were subjected to various cognitive tests during adolescence (P35-P44) and adulthood (P70-P101). On P102, mice were killed and brain phospholipids were analyzed. RESULTS: The concept diet improved performance in short-term memory tasks that rely on novelty exploration during adolescence (T-maze; spontaneous alternation 87% in concept-fed mice compared with 74% in mice fed control diet; P < 0.05) and adulthood (novel object recognition; preference index 0.48 in concept-fed mice compared with 0.05 in control-fed mice; P < 0.05). Cognitive performance in long-term memory tasks, however, was unaffected by diet. Brain phospholipid composition at P102 was not different between diet groups. CONCLUSIONS: Exposure to a diet with lipids mimicking more closely the structure and composition of lipids in breast milk improved specific cognitive behaviors in mice. These data suggest that lipid structure should be considered as a relevant target to improve dietary lipid quality in infant milk formulas.

Pectin-Derived Acidic Oligosaccharides Improve the Outcome of Pseudomonas aeruginosa Lung Infection in C57BL/6 Mice.

The administration of prebiotics as oligosaccharides (OS), by acting on intestinal microbiota, could modulate the immune and inflammatory response and represent a new strategy to improve the outcome of bacterial infection. The aim of this study was to determine whether pectin-derived acidic oligosaccharides (pAOS) could modulate the outcome of pulmonary P. aeruginosa (PA) infection in C57BL/6 mice, which develop a Th1 response to PA lung infection. Mice were randomized for 5 weeks to consume a control or a 5% pAOS diet and chronically infected by PA. Resistance to a second PA infection was also analyzed by reinfecting the surviving mice 2 weeks after the first infection. Compared with control mice, mice fed pAOS had reduced mortality (P<0.05). This improvement correlated with a better control of the inflammatory response with a lower neutrophil count on day 1 (P<0.05), a sustained neutrophil and macrophage recruitment on days 2 and 3 (P<0.01) a greater and sustained IL-10 release in lung (P<0.05) and a reduction of the Th1 response and M1 activation with a lower IFN-γ/IL-4 (P<0.01) and nos2/arg1 (P<0.05) ratios. These results coincided with a modulation of the intestinal microbiota as shown by an increased butyric acid concentration in feces (P<0.05). Moreover, pAOS decreased the bacterial load (P<0.01) in mice reinfected 2 weeks after the first infection, suggesting that pAOS could reduce pulmonary exacerbations. In conclusion, pAOS improved the outcome of PA infection in C57BL/6 mice by modulating the intestinal microbiota and the inflammatory and immune responses.

Impact of fish oils on the outcomes of a mouse model of acute Pseudomonas aeruginosa pulmonary infection.

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that causes pneumonia in immunocompromised humans and severe pulmonary damage in patients with cystic fibrosis. Imbalanced fatty acid incorporation in membranes, including increased arachidonic acid and decreased DHA concentrations, is known to play a critical role in chronic inflammation associated with bacterial infection. Other lipids, such as EPA and alkylglycerols, are also known to play a role in inflammation, particularly by stimulating the immune system, decreasing inflammation and inhibiting bacterial growth. In this context, the goal of the present study was to assess the effect of dietary DHA/EPA, in a 2:1 ratio, and alkylglycerols, as natural compounds extracted from oils of rays and chimeras, respectively, on the inflammatory reaction induced by P. aeruginosa pulmonary infection in mice. To this end, mice were fed with a control diet or isolipidic, isoenergetic diets prepared with oils enriched in DHA/EPA (2:1) or alkylglycerols for 5 weeks before the induction of acute P. aeruginosa lung infection by endotracheal instillation. In our model, DHA/EPA (2:1) significantly improved the survival of mice after infection, which was associated with the acceleration of bacterial clearance and the resolution of inflammation leading to the improvement of pulmonary injuries. By contrast, alkylglycerols did not affect the outcomes of P. aeruginosa infection. Our findings suggest that supplementation with ray oil enriched in DHA/EPA (2:1) can be considered as a preventive treatment for patients at risk for P. aeruginosa infection.

Dietary pectin-derived acidic oligosaccharides improve the pulmonary bacterial clearance of Pseudomonas aeruginosa lung infection in mice by modulating intestinal microbiota and immunity.

BACKGROUND: A predominantly T-helper type 2 (Th2) immune response is critical in the prognosis of pulmonary Pseudomonas aeruginosa infection. But the mucosal and systemic immune responses can be influenced by the intestinal microbiota. METHODS: We assessed the effect of microbiota compositional changes induced by a diet enriched in 5% acidic oligosaccharides derived from pectin (pAOS) on the immune response and outcome of chronic pulmonary P. aeruginosa infection in mice. RESULTS: pAOS promoted Th1 polarization by increasing interferon γ release, upregulating t-bet gene expression, decreasing interleukin 4 secretion, and downregulating gata3 gene expression. pAOS also sustained the release of keratinocyte chemoattractant, recruited polynuclear leukocytes and macrophages, stimulated M1 macrophage activation and interleukin 10 release, and decreased tumor necrosis factor α release in the lung. These effects led to increased bacterial clearance after the first and second P. aeruginosa infections. pAOS modified the intestinal microbiota by stimulating the growth of species involved in immunity development, such as Bifidobacterium species, Sutturella wadsworthia, and Clostridium cluster XIVa organisms, and at the same time increased the production of butyrate and propionate. CONCLUSION: These results suggest that pAOS may have beneficial effects by limiting the number and severity of pulmonary exacerbations in patients chronically infected with P. aeruginosa, such as individuals with cystic fibrosis.

Higher efficacy of dietary DHA provided as a phospholipid than as a triglyceride for brain DHA accretion in neonatal piglets.

Long-chain PUFAs (LCPUFAs) occur in foods primarily in the natural lipid classes, triacylglycerols (TAGs) or phospholipids (PLs). We studied the relative efficacy of the neural omega-3 DHA provided in formula to growing piglets as a dose of (13)C-DHA bound to either TAG or phosphatidylcholine (PC). Piglets were assigned to identical formula-based diets from early life and provided with TAG-(13)C-DHA or PC-(13)C-DHA orally at 16 days. Days later, piglet organs were analyzed for (13)C-DHA and other FA metabolites. PC-(13)C-DHA was 1.9-fold more efficacious for brain gray matter DHA accretion than TAG-(13)C-DHA, and was similarly more efficacious in gray matter synaptosomes, retina, liver, and red blood cells (RBCs). Liver labeling was greatest, implying initial processing in that organ followed by export to other organs, and suggesting that transfer from gut to bloodstream to liver in part drove the difference in relative efficacy for tissue accretion. Apparent retroconversion to 22:5n-3 was more than double for PC-(13)C-DHA and was more prominent in neural tissue than in liver or RBCs. These data directly support greater efficacy for PC as a carrier for LCPUFAs compared with TAG, consistent with previous studies of arachidonic acid and DHA measured in other species.

Functional glycans and glycoconjugates in human milk.

Human milk contains complex carbohydrates that are important dietary factors with multiple functions during early life. Several aspects of these glycostructures are human specific; some aspects vary between lactating women, and some change during the course of lactation. This review outlines how variability of complex glycostructures present in human milk is linked to changing infants' needs.

Breast milk fatty acid profile in relation to infant growth and body composition: results from the INFAT study.

BACKGROUND: There is some evidence that the n-6/n-3 long-chain polyunsaturated fatty acids (LCPUFAs) ratio in early nutrition, and thus in breast milk, could influence infant body composition. METHODS: In an open-label randomized controlled trial (RCT), 208 healthy pregnant women were allocated to a dietary intervention (supplementation with 1,200 mg n-3 LCPUFAs per day and instructions to reduce arachidonic acid (AA) intake) from the 15th wk of gestation until 4 mo of lactation or to follow their habitual diet. Breast milk LCPUFAs at 6 wk and 4 mo postpartum were related to infant body composition assessed by skinfold thickness (SFT) measurements and ultrasonography during the first year of life. RESULTS: Dietary intervention significantly reduced breast milk n-6/n-3 LCPUFAs ratio. In the whole sample, early breast milk docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and n-3 LCPUFAs at 6 wk postpartum were positively related to the sum of four SFT measurements at age 1. Breast milk AA and n-6 LCPUFAs at 6 wk postpartum were negatively associated with weight, BMI, and lean body mass (LBM) up to 4 mo postpartum. CONCLUSION: Breast milk n-3 LCPUFAs appear to stimulate fat mass growth over the first year of life, whereas AA seems to be involved in the regulation of overall growth, especially in the early postpartum period.

Development of the Digestive System-Experimental Challenges and Approaches of Infant Lipid Digestion.

At least during the first 6 months after birth, the nutrition of infants should ideally consist of human milk which provides 40-60 % of energy from lipids. Beyond energy, human milk also delivers lipids with a specific functionality, such as essential fatty acids (FA), phospholipids, and cholesterol. Healthy development, especially of the nervous and digestive systems, depends fundamentally on these. Epidemiological data suggest that human milk provides unique health benefits during early infancy that extend to long-lasting benefits. Preclinical findings show that qualitative changes in dietary lipids, i.e., lipid structure and FA composition, during early life may contribute to the reported long-term effects. Little is known in this respect about the development of digestive function and the digestion and absorption of lipids by the newborn. This review gives a detailed overview of the distinct functionalities that dietary lipids from human milk and infant formula provide and the profound differences in the physiology and biochemistry of lipid digestion between infants and adults. Fundamental mechanisms of infant lipid digestion can, however, almost exclusively be elucidated in vitro. Experimental approaches and their challenges are reviewed in depth.

Structural profiling and quantification of sphingomyelin in human breast milk by HPLC-MS/MS.

The sphingolipid composition of food as well as of physiological samples has received considerable interest due to their positive biological activities. This study quantified the total amount of sphingomyelin (SM) in 20 human breast milk samples from healthy volunteers and determined the structures of SM by detailed mass spectrometric studies in combination with enzymatic cleavage. The quantification of SM was performed by hydrophilic interaction liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (HILIC-HPLC-ESI-MS/MS) measuring the characteristic fragment ion of the phosphorylcholine group at m/z 184.2 and by using hexanoylsphingomyelin (C6-SM) and heptadecanoylsphingomyelin (C17-SM) as internal standards. The structures of SM species were identified after enzymatic cleavage with alkaline sphingomyelinase (SMase) to the corresponding ceramides. Structure elucidation of the sphingoid base and fatty acid backbone was performed by reversed-phase HPLC-ESI-MS/MS. The method includes the sphingoid bases dihydrosphingosine (d18:0), sphingosine (d18:1(Δ4)), 4,8-sphingadienine (d18:2(Δ4,8)), 4-hydroxysphinganine (phytosphingosine (t18:0)), and 4-hydroxy-8-sphingenine (t18:1(Δ8)) and fatty acids with even-numbered carbon atoms (C12-C26) as well as their (poly)unsaturated and monohydroxylated analogues. The total amount of SM in human breast milk varied from 3.87 to 9.07 mg/100 g fresh weight. Sphingosine (d18:1) was the predominant sphingoid base, with 83.6 ± 3.5% in human breast milk, followed by 4,8-sphingadienine (d18:2) (7.2 ± 1.9%) and 4-hydroxysphinganine (t18:0) (5.7 ± 0.7%). The main SM species contained sphingosine and palmitic acid (14.9 ± 2.2%), stearic acid (12.7 ± 1.5%), docosanoic acid (16.2 ± 3.6%), and tetracosenoic acid (15.0 ± 3.1%). Interestingly, the fatty acid composition of SM species in this study differs from the total fatty acids in human breast milk, and the fatty acids are not consistently distributed among the different sphingoid bases.

Identification of novel anionic phospholipid binding domains in neutral sphingomyelinase 2 with selective binding preference.

Sphingolipids such as ceramide are recognized as vital regulators of many biological processes. Neutral sphingomyelinase 2 (nSMase2) is one of the key enzymes regulating ceramide production. It was previously shown that the enzymatic activity of nSMase2 was dependent on anionic phospholipids (APLs). In this study, the structural requirements for APL-selective binding of nSMase2 were determined and characterized. Using lipid-protein overlay assays, nSMase2 interacted specifically and directly with several APLs, including phosphatidylserine and phosphatidic acid. Lipid-protein binding studies of deletion mutants identified two discrete APL binding domains in the N terminus of nSMase2. Further, mutagenesis experiments pinpointed the core sequences and major cationic amino acids in the domains that are necessary for the cooperative activation of nSMase2 by APLs. The first domain included the first amino-terminal hydrophobic segment and Arg-33, which were essential for nSMase2 to interact with APLs. The second binding domain was comprised of the second hydrophobic segment and Arg-92 and Arg-93. Moreover, mutation of one or both domains decreased APL binding and APL-dependent catalytic activity of nSMase2. Further, mutation of both domains in nSMase2 reduced its plasma membrane localization. Finally, these binding domains are also important for the capability of nSMase2 to rescue the defects of yeast lacking the nSMase homologue, ISC1. In conclusion, these data have identified the APL binding domains of nSMase2 for the first time. The analysis of interactions between nSMase2 and APLs will contribute to our understanding of signaling pathways mediated by sphingolipid metabolites.

(n-3) long-chain PUFA differentially affect resistance to Pseudomonas aeruginosa infection of male and female cftr-/- mice.

The aim of this study was to determine whether oral supplementation with EPA/DHA (10.5 and 5.1% of fat, respectively) could improve the outcome of pulmonary P. aeruginosa infection in cftr(-/-) mice compared with wild-type (Wt) mice similarly treated. Because gender could influence the susceptibility of cftr-deficient mice, results were analyzed by gender. Wt and (-/-) mice were randomized for 6 wk to consume a control or EPA/DHA diet, infected with endotracheal injection of 5 × 10(7) CFU/mouse of P. aeruginosa, and killed 24 h later. Cftr(-/-) mice were more susceptible to infection than were Wt mice; (-/-) males had more neutrophils (P < 0.01) and a higher keratinocyte-derived chemokine (KC) level (P < 0.05), and (-/-) females had greater lung injury and mortality (P < 0.05). Female (-/-) mice were more susceptible than (-/-) males with a higher mortality and lung injury (P < 0.05). The EPA/DHA diet reduced neutrophil numbers and KC and IL-6 levels (P < 0.05) in (-/-) males and reduced mortality rate (P < 0.001), lung permeability, and IL-6 level (P < 0.05) in (-/-) females compared with (-/-) mice fed the control diet. These results were associated with a reduction in the pulmonary bacterial load (P < 0.05), an increase in the EPA/DHA concentration in cell membranes of (-/-) males and females (P < 0.01), and an increased weight gain only in males compared with (-/-) mice fed the control diet (P < 0.01). In conclusion, EPA/DHA improves the host resistance of (-/-) mice, although the beneficial effect differed in males and females.

Bioactive sphingolipids: metabolism and function.

Sphingolipids (SLs) are essential constituents of eukaryotic cells. Besides playing structural roles in cellular membranes, some metabolites, including ceramide, sphingosine, and sphingosine-1-phosphate, have drawn attention as bioactive signaling molecules involved in the regulation of cell growth, differentiation, senescence, and apoptosis. Understanding the many cell regulatory functions of SL metabolites requires an advanced knowledge of how and where in the cell they are generated, converted, or degraded. This review will provide a short overview of the metabolism, localization, and compartmentalization of SLs. Also, a discussion on bioactive members of the SL family and inducers of SL enzymes that lead to ceramide generation will be presented.

Analysis of sphingolipids in potatoes (Solanum tuberosum L.) and sweet potatoes (Ipomoea batatas (L.) Lam.) by reversed phase high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS).

Ceramides and glucocerebrosides of potatoes (Solanum tuberosum L.) and sweet potatoes (Ipomoea batatas (L.) Lam.) were analyzed using RP-HPLC-ESI-MS/MS. Ceramides and glucocerebrosides containing the three different long-chain bases 4,8-sphingadienine (d18:2(delta4,delta8)), 4-hydroxy-8-sphingenine (t18:1(delta8)), and 8-sphingenine (d18:1(delta8)) acylated to saturated and unsaturated hydroxy- and nonhydroxy fatty acids with 16-26 carbon atoms were detected. For ceramides and glucocerebrosides 4,8-sphingadienine (d18:2(delta4,delta8)) was found as the major long-chain base, with lesser amounts of 4-hydroxy-8-sphingenine (t18:1(delta8)) and 8-sphingenine (d18:1(delta8)). 2-(Alpha-)hydroxypalmitic acid (C16:0h) was the major fatty acid, which was found to be acylated to the long-chain bases. For quantification of these compounds, an RP-HPLC-ESI-MS/MS method with an "echo-peak"-technique simulating internal standard injection was developed. The analyzed samples of potatoes and sweet potatoes showed amounts of approximately 0.1-8 microg/kg single ceramides and amounts up to 500 microg/kg glucocerebrosides, with C16:0h-glucosyl-4,8-sphingadienine as the major component.