Dietary Polar Lipids and Cognitive Development: A Narrative Review

ABSTRACT

Polar lipids are amphiphilic lipids with a hydrophilic head and a hydrophobic tail. Polar lipids mainly include phospholipids and sphingolipids. They are structural components of neural tissues, with the peak rate of accretion overlapping with neurodevelopmental milestones. The critical role of polar lipids in cognitive development is thought to be mediated through the regulation of signal transduction, myelination, and synaptic plasticity. Animal products (egg, meat, and dairy) are the major dietary sources of polar lipids for children and adults, whereas human milk and infant formula provide polar lipids to infants. Due to the differences observed in both concentration and proportion of polar lipids in human milk, the estimated daily intake in infants encompasses a wide range. In addition, health authorities define neither intake recommendations nor guidelines for polar lipid intake. However, adequate intake is defined for 2 nutrients that are elements of these polar lipids, namely choline and DHA. To date, limited studies exist on the brain bioavailability of dietary polar lipids via either placental transfer or the blood–brain barrier. Nevertheless, due to their role in pre- and postnatal development of the brain, there is a growing interest for the use of gangliosides, which are sphingolipids, as a dietary supplement for pregnant/lactating mothers or infants. In line with this, supplementing gangliosides and phospholipids in wild-type animals and healthy infants does suggest some positive effects on cognitive performance. Whether there is indeed added benefit of supplementing polar lipids in pregnant/lactating mothers or infants requires more clinical research. In this article, we report findings of a review of the state-of-the-art evidence on polar lipid supplementation and cognitive development. Dietary sources, recommended intake, and brain bioavailability of polar lipids are also discussed.

Introduction

The human brain grows rapidly during early life, with a growth spurt starting midgestation and continuing postnatally, increasing its weight from ∼27% of its adult weight at birth to ∼80% by age 2 y (1). This process is accompanied by notable lipid deposition in the brain, of which complex lipids account for the most significant proportion.

Complex lipids are amphiphilic polar lipids with a hydrophilic head and a hydrophobic tail, and they comprise mainly phospholipids and sphingolipids. They are major constituents of cell membranes of all living organisms but are also found in circulating fluids. In addition, they play an important role in membrane functions through interacting with membrane proteins (2). As structural components of neural tissues, they are critical for neurodevelopment and cognition, as indicated by the overlap of the peak rate in the accretion of certain polar lipids with neurodevelopmental milestones (3–5). The polar lipids reviewed in this article include phospholipids (glycerophospholipids and sphingomyelin) and sphingolipids (ceramides, cerebrosides, and gangliosides).

Glycerophospholipids (GPLs) are key components of cell membranes. They are the major source of long-chain PUFAs and the reservoir of signaling molecules. Membrane GPLs mainly include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) (6). They can be derived from de novo synthesis or from resynthesis—for example, in enterocytes after hydrolysis of dietary GPL to free fatty acids and lysophospholipids (7–9). PC and PS have been investigated for their roles in brain development, with some positive effects in cognitive enhancement in preclinical models (10, 11) and mixed results in clinical studies (12, 13).

Sphingomyelin (SM) is a sphingophospholipid and thus classified as either a phospholipid or a sphingolipid. In this review, we generally refer to it as a phospholipid. SM is particularly rich in the myelin sheath of the central nervous system (14, 15), and due to its role in myelin integrity (16) and axonal maturation (17), SM has been implicated in a number of different cognitive disorders (18).

Sphingolipids are particularly abundant in the brain. Three types of sphingolipids—ceramides, cerebrosides, and gangliosides—have been reported to be present in higher concentrations in the brain than in many organs and play important roles in brain development during the early years (19). A large part of brain sphingolipids originate from endogenous synthesis; however, dietary supplementation influences sphingolipid composition in rodent brain (20, 21) and has positive effects on infant cognitive and neurobehavioral development (22). These findings highlight the potential importance of dietary sphingolipids during early development.

In this review, we summarize and discuss the available scientific evidence supporting the role of polar lipids in brain and cognitive development, with a focus on the nutritional impact during pregnancy, lactation, and infancy. We review the food sources of polar lipids, their bioavailability, and the effects on cognitive measures via dietary supplementation. Although recent publications of preclinical findings indicate a role of milk phospholipids in cognitive impairment and aging (23, 24), this review aims at identifying gaps and potential future direction of research for dietary milk polar lipids in early life.

Methodology

Literature searches in PubMed and Google Scholar were conducted up to January 2018, with no time restriction for the publication date. Several references were recovered during writing of the manuscript, up to August 2018.

Although the primary focus was on results from human randomized controlled trials, animal and in vitro studies were also included. Different combinations of the following search terms were used: ganglioside (including subspecies such as GM3, GD3, and GM1), ceramide, cerebroside (including interchangeably used terms such as glucosylceramide and galactosylceramide), sialic acid, phospholipid, phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, plasmalogens, sphingomyelin, sphingolipid, choline, fatty acid, milk, MFGM (milk fat globule membrane), diet, food, nutrition, infant formula, supplementation, maternal, pregnant, lactating, administration, prenatal, antenatal, perinatal, postnatal, fetal, infant, baby, child, children, brain, neuron, cognitive, neurological, neurodevelopment, gut, intestine, stomach, enzyme, hydrolysis, digestion, ileum, jejunum, stomach, bioavailability, biosynthesis, de novo synthesis, absorption, uptake, chylomicron, LDL, VLDL, HDL, apolipoprotein, distribution, metabolism, tissue accretion, accumulation, blood–brain barrier, brain (neuron, oligodendrocyte, astrocyte, microglia, etc.), cognition, and development.

Inclusion limits were set to literature with abstracts and available in English. After assessing abstracts of the search results, full texts of relevant articles were reviewed and their reference lists were examined for further source material, if relevant.

Roles of Polar Lipids in Molecular, Cellular, and Physiological Processes

GPLs are composed of a glycerol backbone esterified with 2 hydrophobic fatty acid “tail” groups and a hydrophilic (=polar) “head” made of a phosphate group plus a hydrophilic residue (Figure 1). They are ubiquitous molecules present in all cell membranes, as well as membranes of subcellular organelles such as mitochondria. In addition to their function as membrane building blocks, they modulate membrane properties such as fluidity, permeability, integrity, phase transition temperature, bilayer thickness, and lateral domains, allowing them to influence crucial intracellular and intercellular signaling (25). GPLs also stabilize proteins within membrane structures, and they act as cofactors in enzymatic reactions (2). In addition, GPLs, particularly PC, are secreted in bile and together with bile salts and bile bicarbonate facilitate the absorption of dietary fat.

FIGURE 1.

Representative structures of glycerophospholipids: (A) phosphatidylcholine; (B) phosphatidylethanolamine; (C) phosphatidylinositol; (D) phosphatidylserine; (E) sphingomyelin: example with choline as a base (CerPCho); (F) ceramide: example with d18:0/18:1 (sphingosine and oleic acid); (G) cerebroside: example with monosaccharide; and (H) ganglioside: example with galactose and glucose and 2 sialic acids. Adapted from www.lipidmaps.org, a free resource sponsored by the Wellcome Trust.

SM, consisting of ceramide and a phosphocholine or phosphoethanolamine head group, varies in length as well as saturation level in its fatty acid chains (26, 27). Figure 1 includes a typical example of SM, which has an 18-carbon sphingosine and a phosphocholine head. The specific roles of the diverse SM species in plasma membranes are yet to be clearly elucidated (18), although there is evidence that SM with differing fatty acids modifies membrane fluidity (28, 29). SM is critical in the formation of lipid rafts, which are rich in cholesterol as well as SM (30). Furthermore, SM interacts with neighboring plasma membrane components such as membrane proteins and receptors, modulating the activities of the plasma membrane (18, 28).

Sphingolipids consist of a sphingoid (long-chain amino alcohol; e.g., sphingosine with 18 carbon) backbone attached to varying fatty acids and head groups (Figure 1). This is a complex family made of many molecules varying in their fatty acid moiety, carbohydrate moiety, and sialic acid molecule numbers. They include several subclasses, namely ceramides, cerebrosides, and gangliosides.

Ceramides are the simplest sphingolipids. They are composed of a sphingoid base backbone and a fatty acid (Figure 1). Ceramide is a lipid mediator regulating cell death and survival, differentiation, senescence, autophagy, and migration (31–33). Increased ceramide has been linked to cell death (34, 35), possibly through the interaction with latent binding sites on the outer or inner mitochondrial membranes, followed by an increase in membrane permeability (36). In addition, being the precursor for biosynthesis of all sphingolipids in the brain, ceramides are pivotal during early development, in particular, when SM and glycosphingolipids are required for myelin formation and signal transfer in the infant brain (37).

Cerebrosides are sphingolipids with a single-unit sugar residue, either glucose or galactose, attached to a ceramide through a glycosidic bond. Galactocerebrosides are rich in neural tissue, particularly myelin, whereas glucocerebrosides are typically found in other tissues (38–41).

Gangliosides are glycosphingolipids with sialic acid residues (42, 43). The structures of gangliosides are highly diverse due to the large variations in the carbohydrate chain and the lipophilic components, as well as the number, position, and structure of sialic acid residues (44). Gangliosides are primarily positioned in lipid rafts (45) in plasma membranes and other intracellular membranes. The ceramide part is anchored in the membrane with the glycan chains exposed on cell surfaces. Gangliosides have a role in the regulation of signal transduction pathways in cell recognition, adhesion, proliferation, and neuronal protection (46, 47). They also function as anchors or entry points for various toxins, bacteria, viruses, and autoantibodies. Their bioactive roles include gut maturation (48), protection from inflammation (49–51), and neurodegeneration (21, 52–54).

The Role of Polar Lipids in Neuronal Function

The rapid accretion of some polar lipids in the nervous system during critical cognitive development stages has been demonstrated in various studies (3–5), with a growing body of evidence supporting the role of these lipids in cognition.

GPLs act as a source of PUFAs, which are present in large quantities in the brain and central nervous system. It has been hypothesized that the degree of unsaturation in neuronal phospholipid fatty acids as well as their compositions can influence neurological functions as they modulate membrane activities, such as those of bound enzymes (55). GPLs are also crucial in signal transduction as a reservoir for second messengers such as arachidonic acid, eicosanoids, platelet-activating factor, and diacylglycerol. Also, PC, the most abundant phospholipid in the plasma membrane accounting for ∼50% of total plasma membrane phospholipids, is a precursor of the neurotransmitter acetylcholine (56). Choline, 1 of PC's components, is thought to be critical during fetal brain development, as demonstrated in animal models. Specifically, in rodents, choline has reported effects on stem cell proliferation and apoptosis, and subsequent change in neuron morphology and function (57, 58). In addition, a choline-deficient diet given to sows and piglets induced delayed brain development in the piglets (59). PE is the second most abundant phospholipid in plasma membranes, and it accounts for 20–30% of total membrane phospholipids. It is used for the production of glycosylphosphatidylinositol, which facilitates the anchoring of proteins to the membrane for a wide range of biological processes (60). With a notable presence in the membrane of mitochondria, PE is essential for the growth and stability of these energy-generating organelles, which are particularly important for the brain (61). PS accounts for 5–10% of membrane phospholipids and is particularly enriched in myelin (62, 63). It is involved in cell-to-cell communication, cell growth regulation, secretory vesicle release, and signal transduction (64). In addition, the incorporation of PS into neuron cell membranes influences the metabolism of the neurotransmitters acetylcholine, norepinephrine, serotonin, and dopamine (65–67). PI is particularly abundant in inner membrane and accounts for 10% of total inner leaflet phospholipids. The metabolism of PI gives rise to 7 known polyphosphoinositides (also known as phosphoinositides or phosphatidylinositol phosphates), which have prominent roles in signal transduction events in the central nervous system (68–70). They are precursors of inositol phosphates (IP₁, IP₂, IP₃, IP₄, etc.), intracellular second messenger molecules modulating the homeostasis and mobilization of Ca²⁺ in both neural and nonneural tissues (68, 70, 71).

SM plays a pivotal role in the formation of the myelin sheath that surrounds and insulates the axons. Increased level of SM in young rat neurons was reported to induce the maturation of the axonal plasma membrane (17). In addition to its role in brain structure, SM is also a source of bioactive sphingolipids. Whereas SM is generated de novo from ceramide and PC in the Golgi apparatus (71), its degradation by hydrolysis releases ceramide, which is at the center of sphingolipid metabolism and is a critical second messenger (72). Although SM does not have a recognized role in regulating cell function such as cell proliferation, survival, and migration, the homeostasis of SM and ceramide influences these processes (15, 18, 72). Indeed, alterations in SM metabolism can lead to conditions with significant neuronal symptoms, compromised function, and/or death (e.g., Niemann–Pick disease) (73).

Regarding sphingolipids, findings of a study using Wistar rat embryos, which inhibited the ceramide synthesis in cerebellar Purkinje neurons, suggest that ceramide plays a role in neuron survival and dendritic differentiation—potentially mediated via ISP-1, a specific inhibitor of serine palmitoyltransferase (74). The accretion of cerebroside, another sphingolipid and 1 of the major myelin lipids in the brain, is proportional to myelin formation (75, 76). The different species of cerebrosides are reported to have distinct roles in neurodevelopment: Whereas galactocerebroside is a critical constituent of the myelin sheath, glucosylcerebroside plays an important role in regulating axonal growth rate (39–41). By applying inhibitors of glycosylceramide synthesis to cultured hippocampal neurons, axonal morphology was significantly altered toward shorter axon plexus (as axonal branches retracted) and fewer axonal branches (77, 78). In contrast when lysosomal degradation of glucosylceramide to ceramide was inhibited, the number of axonal branches and the length of the axon plexus were increased (77, 78).

Gangliosides are involved in synaptic networking, dendritic branching, and cell multiplication and migration during neonatal development (79). More recent studies using glycosyltransferase gene knockout mice demonstrate the role of gangliosides in neurodevelopment and the integrity of the nervous system (80). Mice lacking all gangliosides of the ganglio-series were reported to develop severe degeneration of the central nervous system and died soon after weaning. These mice also had lower brain weights, and they experienced axon degeneration in white matter and abnormal axon–glia interactions (21). In addition, the mutation of GM3 synthase, with an absence of GM3 and key complex gangliosides in infant brain, leads to neurological decline, seizures, and epilepsy (81), further indicating the potentially important role of gangliosides in early brain development.

Food Sources of Polar Lipids

Food sources for the general population

Foods that are good sources of phospholipids (GPL + SM) include eggs, liver, lean meats, fish, shellfish, cereal grains, and oilseeds (82). Some of the highest phospholipid-containing foods are egg yolk (10.3 g PL/100 g), beef brain (5.4 g/100 g), pig or chicken liver (2.9–2.5 g/100 g), herring dark muscle (2.6 g/100 g), soybeans (2.0 g/100 g), dehulled oat (1.4 g/100 g), and rapeseed (1.5 g/100 g) (79). The richest organ is brain, followed by liver, kidney, lung, spleen, and muscle. Most leafy vegetables, fruits, and tubers, on the other hand, are poor dietary sources for phospholipids (82). The most common PL in food is PC, followed by PE. The other phospholipids, PS and PI, occur at much lower concentrations, and SM is only present in products of animal origin (82). The average daily intake of phospholipids has been estimated to be ∼2–8 g (1–10% of total lipid intake) (83). Ceramide from the diet is primarily derived from the hydrolysis of complex sphingolipids, predominantly SM. Data on free ceramide contents of foods are scarce. Rice bran has 5.6 mg/100 g (84) and soybean has 11.5 mg/100 g (85). Plants such as soybean, maize, rice bran, and wheat contain cerebrosides, with more diverse sphingosine base structures than those of mammalian sources (86). Gangliosides are found in animal products such as meat, especially organ meat and fish (87, 88). Gangliosides in beef, chicken, and pork have both N-acetylneuraminic and N-glycolylneuraminic acids (2 forms of sialic acids), whereas fish-based gangliosides contain only N-acetylneuraminic acid (88). Direct measurements of gangliosides in meat and fish have quantified the total gangliosides to be 0.95–1.44 mg/100 g of chicken, 0.48–0.95 mg/100 g of beef, 0.49 mg/100 g of pork, and 0.76–6.48 mg/100 g of fish (88). GM3 is by far the most abundant ganglioside species in these food sources, whereas GD3, GD1a, and GD1b are present in minor quantities (88). Egg yolk is another source of gangliosides (87, 89), and 1 egg yolk reportedly has 0.71 mg of GM3, 0.21 mg of GM4, and 0.13 mg of GD3 (89). The total daily ganglioside intake has been estimated in Canada in a small group of healthy adults to be <200 μg/d; however, only lipid-bound N-acetylneuraminic acid was determined, and not N-glycolylneuraminic (87, 88). Based on dietary records, Vesper et al. (90) estimated that US adults consume daily 300–400 mg of sphingolipids, including SM, whereas in Japan, using chemical analysis of the 3 daily meals during 2 d, sphingolipid intake was estimated to be between 45 and 292 mg/d and consisted mainly of cerebrosides and SM (91).

Bovine milk

In bovine milk, polar lipids range from 9.4 to 40 mg/100 g raw milk and are mainly located in the MFGM (92, 93). In liquid milk, they typically range from 0.25% to 1% of total fat (93). Some aqueous milk coproducts obtained during butter production, such as buttermilk and butter serum, are enriched in MFGM and thus in polar lipids (19.4 and 43.9 g/100 g fat, respectively) (94). Milk and dairy products have been of increasing interest as an alternative to soybean or egg as a commercial source of polar lipids because they also are a source of SM and PS (95). As percentages of total phospholipid content, the most abundant phospholipids in milk fat are PE (26.4–72.3%), PC (8.0–45.5%), and SM (4.1–29.2%), followed by PS (2.0–16.1%) and PI (1.4–14.1%) (93). Bovine milk is also a source of glycosphingolipids, with neutral glycosphingolipids ranging from 1.8 to 2.7 mg/100 g raw milk and accounting for 6–9% of milk polar lipids (92). These are mainly nonhydroxylated fatty acid lactosylceramide (0.67–1.94 mg/100 g; 63.8% of the neutral glycosphingolipids) and glucosylceramide (a cerebroside; 0.78–1.15 mg/100 g) and lower quantities of globotriaosylceramide (Gb3) and globoside (Gb4) (96). Gangliosides (acidic glycosphingolipids) account for 0.14 mg/100 g in mature milk (97, 98), with >90% of the contained gangliosides associated with MFGM (99). GD3 alone represents 60–70% of the total gangliosides in bovine milk, with the rest likely to be GM3, GT3, GD1a, or GD1b (97, 100–102). Similar to the other polar lipids, gangliosides are more concentrated in cultured dairy foods such as yogurt or in some aqueous milk coproducts such as buttermilk than in whole milk (103). Skim milk, on the other hand, has only 11.2% of the ganglioside level compared to whole milk (103). Large variations in the contents or distribution of polar lipids are observed according to lactation stage, with, for example, higher levels of gangliosides in colostrum and transitional milk than in mature milk (97, 98). Bovine feeding, treatment of the milk, or the analytical method used also impact on polar lipid concentration or subclass distribution (92).

Food sources for infants

Breastfeeding provides the ideal form of nutrition for infants, and human milk is the only source of nutrients for exclusively breastfed infants. The average content of phospholipids in human milk ranges from 9.8 to 47.4 mg/100 mL (104), and it is estimated that a 4-wk-old breastfed infant has a daily intake of 140 mg phospholipids/d (105), calculated based on a polar lipid concentration of 23.8 mg phospholipids/100 mL milk [mature milk from women in Singapore (106)] and consumption of 600 mL human milk at age 4 wk (107). Table 1 summarizes the range of polar lipid content in human milk and reports the most abundant phospholipids in human milk fat are usually SM and PC (expressed as percentages of total phospholipid content) (104, 106, 108–116). Glycosphingolipids are present in low amounts, with cerebrosides the most abundant (0.28 mg/100 mL; 74% of the neutral glycosphingolipids), followed by lactosyl, Gb3 and Gb4, and lower amounts of gangliosides, mostly GM3 and GD3 (96, 100, 106, 110, 117–126). Polar lipid concentrations and proportions vary greatly, depending on the method of milk expression or analysis and the lactation period, with increased GM3 and decreased GD3 concentrations over lactation time (122). Maternal diet during lactation plays a major role in human milk composition; for example, human milk fatty acid profile and content vary over lactation depending on geographical regions due to different dietary habits and genetic polymorphisms influencing metabolism of fatty acids (127–129). Indeed, human milk fatty acids are either endogenously synthesized by the mammary gland or taken up from the maternal plasma, and both of these fatty acid sources are influenced by maternal diet (130). In addition, nutritional studies in cows show that phospholipid and sphingolipid compositions of the bovine MFGM are modified by diet (131).

TABLE 1.

Polar lipid content in mature human milk and starter infant formulas¹

						Ganglioside subspecies
	SM	PC	PE	PI	PS	GD3	GM3
Human milk, mg/100 mL2	3–14	2–11	0.2–9	0.2–3.3	0.8–4.5	0.01–2.0	0.06–2.1
Milk-based infant formula (n = 4), mg/100 mL3,4	1.6–3.4	4.3–15.9	2.6–7.2	1.8–12.7	NA	NA	NA
MFGM-enriched infant formula (n = 3), mg/100 mL3,5	7–14.2	20.1–46.6	8.5–29.6	8.8–22.0	NA	1.3–3.4	<0.005–0.11

¹MFGM, milk fat globule membrane; NA, not analyzed; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin.

²Range for polar lipid content in mature human milk from different countries in Europe, Asia, and North America (references cited in article text).

³Range for polar lipids measured in infant formulas. The results on powder product were converted in 100 mL of formula as consumed using the formulas’ minimum and maximum amount according to instructions on packaging.

⁴Four bovine milk-based formulas from different brands manufactured in Asia (India, Singapore, and Thailand) (133).

⁵Five MFGM-enriched formulas from 2 brands manufactured in Asia (Hong Kong and Thailand) (internal data; each formula analyzed minimum in duplicate).

Mature bovine and human milk display comparable amounts and distribution of phospholipids (112, 126, 132). Neutral glycosphingolipid concentrations are higher in bovine milk (96), whereas ganglioside concentrations appear higher in human milk than in bovine milk (1.2 μg/100 mL compared with 0.1 μg/100 mL, respectively) (126). In mature milk, GD3 is the major ganglioside in bovine milk, whereas GM3 is the most abundant in human milk (124).

When breastfeeding is not possible, infant formula can be provided as an alternative feeding solution. Formula provides infants with polar lipids, with variable content and distribution depending on the ingredients used and their concentrations (104, 112). PC is usually the most abundant phospholipid, especially in soy-based formula, and both milk- and soy-based formulas show lower concentrations of SM than human milk (104). Table 1 gives phospholipid and ganglioside content of different bovine-based (133) and MFGM-enriched infant formulas (internal data from in-house analyses). The addition of MFGM does increase the concentration of all polar lipids, allowing the formula to reach the SM and GD3 concentration of human milk while the other phospholipids are higher and GM3 remains lower.

Recommended Intake

Currently, health authorities do not define intake recommendations for either phospholipids or sphingolipids. However, they do provide recommendations for 2 nutrients that are elements of these polar lipids, namely choline (present in PC and SM) and PUFAs, particularly DHA (present in GPL) (134–136). Indeed, choline and DHA cannot be synthesized by the body in sufficient amounts during periods of increased need, such as during pregnancy, lactation, and infancy (57, 134). Choline is ingested mainly as phosphocholine and glycerophosphocholine in humans, but polar lipids contribute to this intake. For example, breastfed infants consume ∼15% of choline as PC and SM (137). In 2016, the European Food Safety Authority (EFSA) proposed a daily Adequate Intake (AI) for choline of 400 mg for the general adult population (135). A higher AI was proposed for pregnant women (480 mg/d), extrapolated from the AI for nonpregnant women and allowing for weight increase during pregnancy. An adequate choline intake was set at 520 mg/d for lactating women, based on an estimated daily amount of choline secreted in human milk (120 mg/d) on top of the AI for nonlactating women (138–141). An estimated 20% of pregnant women may not meet the minimum recommended intake of choline (138). Infants aged 0–6 mo should consume 120 mg/d (the intake estimated from human milk), and infants aged 7–11 mo should consume 160 mg/d because of the high demand for phospholipid synthesis by the developing human brain. DHA is a PUFA that is more concentrated in the brain GPL than in GPL of other tissues, and due to the high need of the developing fetus and infant, especially for brain accretion, higher values for adequate intake are recommended during infancy, pregnancy, and lactation. EFSA set the value at 100 mg/d for 6–12 mo and an additional 100–200 mg DHA/d for pregnant and lactating women (134).

As is the case for phospholipids and sphingolipids, there are currently no guidelines or recommendations from authorities on the intake of gangliosides for any population. However, in light of their crucial role in pre- and postnatal development of the brain, which coincides with the critical window of rapid brain growth around birth, there has been growing interest and encouragement to use gangliosides as a dietary component or supplement for either the infant or the mother (142). Beneficial effects of gangliosides on gut health and intestinal immunity have also been suggested (98, 124). Following these observations, it has been proposed to include polar lipids from dairy sources to enrich infant formulas by using concentrations in human milk as a reference model (22).

Bioavailability of Polar Lipids in Perinatal Developing Brain

Maternal placental transfer

Experiments carried out in a rabbit model exploring the placental transfer of phospholipids showed inconsistent results. One study reported that exogenous phospholipids dramatically alter the phospholipid composition of the maternal plasma, although they do not cross the placenta intact or induce changes in umbilical vein–artery phospholipid concentrations (143). Another study suggested a small direct phospholipid transfer in the earliest period of gestation (144). Further research across different preclinical models and in clinical settings is required to understand the placental transfer of phospholipids during pregnancy.

There is limited research on the maternal transfer of gangliosides to the fetus. The most abundant form of ganglioside in human serum is GM3, followed by GD3 and other minor forms (145). An ex vivo model of dually perfused isolated human placenta reported increases in both GM3 and GD3 on the maternal side of the perfused placenta after exposure, although only GM3 has been shown to increase on the fetal side (146). This is consistent with a previous rat model that demonstrated an uptake of GM1, first by maternal organs and then transfer across the placenta before being taken up by fetal tissues after an injection (147). In another study which examined the composition of gangliosides in the placental tissue from early to term pregnancy, it was found that ganglioside content in trophoblast plasma membrane samples declined with gestational age. Although the overall ganglioside distribution showed qualitative and quantitative differences between the first- and third-trimester samples, the most striking difference was observed in GM3, which increased from being almost absent in the first-trimester sample to high concentrations in term placentae (148). Overall, the results suggest that gangliosides are transferred from the maternal circulation to the fetal circulation and made available to the fetus for its development.

Bioavailability of polar lipids in the developing brain through dietary intake

Polar lipid digestion occurs mainly in the small intestine. In adults, pancreatic lipase A2 is largely responsible for PC hydrolysis, whereas in infants pancreatic lipase-related protein-2 and carboxyl ester lipase [also called bile salt-stimulated lipase (BSSL)] are the main enzymes that hydrolyze PC into lyso-PC and free fatty acids. After entry into the enterocyte, some absorbed lyso-PC is reacylated to PC and incorporated into lipoproteins, enabling distribution throughout the body. Passage of lipoproteins into the brain is limited by the blood–brain barrier, which allows passage of only small HDL (149). Digestion of SM is slow, extended along the whole length of the small intestine, and depends on enzymes of the enterocyte brush border. Dietary SM is hydrolyzed to ceramides in the gut lumen by alkaline sphingomyelinase located in the brush border of the enterocyte (150); this enzyme has also been found in human bile (151). Ceramides are then hydrolyzed by neutral ceramidase to sphingosine and free fatty acids, with this hydrolysis taking place at the level of the middle and lower part of the small intestine (jejunum and ileum). These enzymes are expressed in young infants. Human milk also contains enzymes that are able to perform the first 2 steps of SM hydrolysis, namely acidic sphingomyelinase yielding ceramides and BSSL yielding free fatty acids and sphingosine, respectively (152). The extent of SM hydrolysis can be dose dependent. For example, a rat study showed a higher proportion of SM in feces with a dose of 22 mg compared with 0.1 mg (153), and a human study found undigested SM in ileostomized patients after a dose of 250 mg milk SM. Sphingosine enters the enterocyte, where it is either catabolized in palmitic acid or transformed in ceramides. Free fatty acids originating from ceramide cleavage and absorbed by the enterocytes, as well as palmitic acid from sphingosine, are re-esterified into triglycerides, which enter chylomicrons that are transported from the enterocyte to the lymph and then the blood. Studies investigating the transport of dietary SM to the brain in conditions of normal endogenous SM synthesis are sparse. A study using intravenous injection to adult rats of radiolabeled (³H and ¹⁴C) SM in HDL, with HDL being the major carrier of SM in blood, suggests some low but direct uptake of SM to the brain 24 h after injection; however, the majority of SM was resynthesized in the brain (154, 155).

Digestion of dietary gangliosides has not been well characterized. Kawakami et al. (156), who incubated GD3 and GM3 gangliosides in gastric juices (pH 1.3) and acid solutions (pH l.3, 2.2, 3.1, and 4.0) in vitro, postulated that the digestion of gangliosides begins in the infant's stomach because ∼20% of gangliosides are hydrolyzed under the same acidic conditions found in the infant's stomach. Then the remaining gangliosides reach the small intestine, where they are taken up intact by the enterocyte via passive uptake or receptor-mediated endocytosis, and they insert into the brush border membrane of the cell. Some can then be mobilized and transported to lysozymes for catabolism to ceramide and other metabolites; some can reach the Golgi apparatus to produce different gangliosides; and some can be transferred to the blood and distributed to various tissues throughout the body, including the brain (157–160). Limited studies have examined the bioavailability of dietary gangliosides for the brain (161). Despite poor bioavailability, some studies have suggested that dietary gangliosides have an impact on early brain development, demonstrating that they are able to cross the blood–brain barrier (159–162).

Evidence of Cognitive Benefits through Dietary Supplementation of Polar Lipids in Preclinical Models

Regarding the impact of phospholipids on cognitive development, phosphatidylcholine (with a focus on its choline component) and phosphatidylserine are most widely studied. It was shown that the offspring of rats supplemented with 4.6 mmol/kg body weight of choline chloride per day from day 11 to day 17 of pregnancy showed improved ability to navigate water maze tasks compared with those fed a standard diet (1.3 mmol choline/kg/d) (163). Another rat study suggested that prenatal exposure to choline supplements during pregnancy may modify discriminative abilities engaged in memory tasks (11). Alternatively, postnatal choline supplements were also shown to have long-term cognitive enhancement in spatial memory (164). One mechanism proposed to explain this association is the morphological change in basal forebrain neurons that innervate the hippocampus (165). Regarding PS, an early experiment on adult rats indicated enhanced behavior in active avoidance acquisition and passive avoidance retention through maternal PS supplementation (166). Park et al. (167) administered 50 or 100 mg/kg krill-PS daily for 30 d to young rats (140–160 g) and found that the group fed the higher dose performed better on a Morris water maze test as well as a retention test. In addition, although not via maternal supplementation, postnatal DHA and/or PS supplements showed improved oxidative parameters in the brain and improved learning and memory abilities (10). Interestingly, PS from different sources (bovine cortex, soybean, and egg) did not exhibit the same cognition-enhancing properties, which the authors speculate could be due to a loss of bioactivity due to issues with processing and/or storage (168).

Supplementation of gangliosides and phospholipids has been investigated in a number of studies carried out in both rat and piglet models. Vickers et al. (169) conducted behavioral tests on young postpubertal rats supplemented with complex milk lipid (CML) derived from the MFGM rich in gangliosides and phospholipids from 10 d post birth. A significant improvement in learning and memory, as measured by Morris water maze and novelty recognition tests, was observed in the supplemented group compared with the control group (169). CML supplementation also increased the striatal dopamine output as well as the average area of synaptophysin staining in the hippocampal CA3 region, which might indicate enhanced neuroplasticity (170).

As a gyrencephalic species with brain growth and development similar to those of humans, the piglet is an appropriate model for studying neurodevelopment (171). Liu et al. (172) examined the impact of a diet supplemented with phospholipids and gangliosides (Lacprodan PL-20; Arla Foods) from postnatal day 2 to postnatal day 28 on cognitive development using a spatial T-maze task. Fewer errors were made by the supplemented piglets compared with nonsupplemented control piglets, and choices were made more quickly. An increased brain weight (5%) was also observed in supplemented piglets, along with increases in PC-derived metabolites in hippocampus. However, a limitation of this work is that both gangliosides and phospholipids were supplemented simultaneously, so it cannot be determined whether the improvements in cognition were due to the individual components or a combined effect.

Clinical Evidence for Polar Lipid Supplementation and Benefits in Cognitive Development

Maternal dietary supplementation

Given the ethical considerations of manipulating maternal diet, there are limited studies examining maternal dietary supplementation of gangliosides and its impact on infant development. However, to our knowledge, the first such trial is underway assessing the effect of supplementation of CML during pregnancy on maternal ganglioside status and subsequent cognitive outcomes (for a comparison of this and other relevant clinical studies, see Table 2) (173). The treatment group is administered a daily amount of 8 mg gangliosides, an increase of 4 mg from the control group. This clinical trial is the first large human study on CML supplementation during pregnancy. The results of this trial are awaited to aid understanding of maternal dietary supplementation of gangliosides and its impact on infant development.

TABLE 2.

Summary of clinical studies (in chronological order) examining polar lipid supplementation and associated changes in cognitive development¹

Reference	Intervention	Methodology	Outcome
Gurnida et al. 2012 (22)	CML (gangliosides)	Double-blind, randomized, controlled, parallel group trial; term infants from ages 2–8 to 24 wk. The treatment group (n = 29) was fed ganglioside-supplemented formula (to increase GD3 content by 2–3 mg/100 g), the control group (n = 30) was fed standard formula, and the reference group (n = 32) was breastfed. All infants were exclusively breastfed up to the point of intervention. Cognitive development (using the Griffiths scales) was tested before (ages 2–8 wk) and after intervention (age 24 wk).	Ganglioside supplementation resulted in improved scores for Hand and Eye coordination IQ, Performance IQ, and General IQ on the Griffiths scales. Scores for the treatment group and breastfed reference group did not differ significantly.
Cheatham et al. 2012 (174)	Phosphatidylcholine (750 mg/day)—maternal supplementation	Double-blind, randomized controlled trial (n = 140 pregnant women) supplemented from 18 weeks of gestation through 90 d postpartum. Phosphatidylcholine supplementation (750 mg) vs. placebo (corn oil). Infants’ (n = 99) cognitive development (Mullen Scales of Early Learning, language development, and visual spatial and episodic memory) tested at ages 10 and 12 mo.	No difference in infant cognitive development at either time point.
Tanaka et al. 2013 (177)	SM-fortified milk	Double-blind, randomized controlled trial, low-birth-weight infants (<1.5 kg; n = 24) supplemented with fortified milk [SM = 20% of all phospholipids (n = 12) vs. 13% in the control group (n = 12)] for first 12 mo of life. Assessed plasma phospholipids, VEPs, Fagan, BSID-II, attention and memory tests at 3, 6, 9, 12, and 18 mo of corrected age.	SM-fortified group reported an increased percentage of SM in total phospholipids at 4, 6, and 8 wk. In addition, the SM-fortified group had better Behaviour Rating Scale of the BSID-II, Fagan test, VEP latency, and sustained attention scores at 18 mo compared with control.
Ross et al. 2013 (175)	Phosphatidylcholine (6300 mg/d)— maternal supplementation and 100 mg/d for infants	Double-blind, randomized, placebo-controlled trial (n = 100). Phosphatidylcholine supplementation (6300 mg/d for mothers) vs. placebo (corn oil) from second trimester of pregnancy until third postnatal month; 100 mg/d for infants from birth to 52 wk. Infants’ cerebral evoked responses to auditory stimuli were assessed at ages 5 and 13 wk.	No adverse effects of phosphatidylcholine supplementation were observed on maternal health and delivery, birth, or infant development. At postnatal week 5, P50 response was suppressed in phosphatidylcholine-treated infants; there was no difference at 13 wk.
Timby et al. 2014 (176)	Bovine MFGM [4% of total (wt:wt) protein content] supplemented, low-energy, low-protein experimental formula.	Prospective, double-blind, randomized controlled trial (n = 160). MFGM supplemented, low-energy, low-protein experimental formula vs. standard formula from 2 mo to 6 mo of age. Infant cognitive development was assessed using BSID-III.	At age 12 mo, cognitive score for the experimental group was higher than that for the standard group. There were no significant differences in other measures (linear growth, weight gain, body mass index, etc.).
Ross et al. 2015 (13)	Phosphatidylcholine (6300 mg/d), maternal supplementation	Double-blind, randomized, placebo-controlled trial (n = 49). As for Ross et al. 2013 (175). Infant behavior was measured at 40 mo using the Child Behavior Checklist (n = 23 treatment group; n = 26 placebo group).	At 40 mo, parents rated children receiving treatment as having fewer attention problems and less social withdrawal compared to placebo.
Huang et al. 2017 (173)	CML: standard CML formula and a ganglioside-enhanced CML formula (4 mg/serving; 8 mg/d)	Multicenter, 3-group, parallel randomized controlled trial—the CLIMB (Complex Lipids In Mothers and Babies) study. Three groups—standard maternal milk formulation, CML-enhanced maternal milk formulation, and no maternal milk formulation but provided standard pregnancy advice. Postnatal follow-up at 6 wk and 12 mo: cognitive development assessed with BSID-I.	Trial is ongoing.

¹BSID-I, -II, and -III, Bayley Scales of Infant Development, First Edition, Second Edition, and Third Edition, respectively; CML, complex milk lipids; IQ, intelligence quotient; MFGM, milk fat globule membrane; P50, P50 auditory evoked potential; SM, sphingomyelin; VEP, visual evoked potential.

Although PC has been investigated to the greatest extent among phospholipids, results on PC supplementation are limited and mixed. A clinical trial on maternal PC supplementation for pregnant women consuming diets containing moderate amounts of choline showed no enhancement of infant brain function when assessed at ages 10 and 12 mo. However, the investigators presented the possibility that benefits could emerge if a longer follow-up period was used or for pregnant women with lower dietary intake of choline (174). Perinatal PC supplementation given to pregnant women in an attempt to lower childhood behavioral issues associated with mental illness later in life, such as schizophrenia, autism, and attention-deficit/hyperactivity disorder, has also been investigated. The results suggest that perinatal PC supplementation initiates timely progression in cerebral inhibition and encourages normal brain development, and these may reduce the risk of future mental illness for infants (13, 175).

Dietary supplementation in infants

A randomized controlled trial investigating the potential benefits of infant formula supplemented with CML, containing gangliosides (with the majority being GD3) and phospholipids, on cognitive function of healthy infants aged 0–6 mo has been conducted (22). CML was added to the treatment group with the intention to increase the GD3 content in the supplemented formula by ∼2–3 mg/100 g of formula product (from 6 mg/100 g to 9 mg/100 g). Results showed that the group given the CML supplement achieved higher scores compared with the control group for the categories of hand and eye coordination, performance, and general IQ using the Griffiths scales at 6 mo of age. However, their scores did not differ from those of a reference group of breastfed infants. This suggests that the supplementation of CML, which contains gangliosides and phospholipids, compared with no supplementation may benefit infants in terms of cognitive development and bring them closer to the development of breastfed infants.

In another randomized controlled trial, infants aged <2 mo were fed MFGM-supplemented, low-protein, low-energy formula until 6 mo of age. Compared with the control group, which received standard formula, the experiment group showed significantly higher cognitive scores, as measured by the Bayley scale, at age 12 mo; however, the experiment group's scores were no different from those of the breastfed group (176). The effects of MFGM have been at least partially attributed to the enriched polar lipids (e.g., gangliosides and phospholipids). However, in this study, confounding factors are present (low energy and low protein) in the MFGM-supplemented group; as such, the improvement in cognitive performance may be a consequence of multiple factors apart from the presence of polar lipids.

In terms of dietary supplementation of a single polar lipid, studies have been mainly performed in infants with compromised health or developmental status. A randomized controlled trial on the direct administration of SM-fortified formula to very low-weight infants reported a positive association with neurobehavioral development, as measured by the Behavior Rating Scale of the Bayley Scales of Infant Development II, the Fagan test of infant intelligence, the latency of visual evoked potentials, and a sustained attention test (177). Because the follow-up assessments were carried out up to 18 mo, more detailed studies over time periods of >1 y may be required to establish any long-term benefits of SM fortification and their significance. In addition, studies on normal-weight term-born infants would clarify whether the benefits extend beyond low-birth-weight infants.

Conclusion

The role of polar lipids in supporting brain function and development has been widely investigated, with convincing evidence in cell and animal models. Specifically, phospholipids, ceramides, and glycosphingolipids show promise. However, whether there are added benefits of supplementing these polar lipids in pregnant and/or lactating women or healthy infants for infant cognitive development requires more clinical research. Dairy products are good sources of polar lipids; however, their brain bioavailability through dietary intake remains to be verified. Currently, no specific intake recommendations can be provided for pregnant or lactating women. Regarding infants, although the literature continues to evolve, referring to the concentrations and species of these polar lipids in human milk would be prudent to guide recommended intake levels.

Notes

This work was solely sponsored by Nestec Ltd. No additional external support was used.

Author disclosures: LZ, MF, FG, BVO, and NS, no conflicts of interest.

Abbreviations used: AI, adequate intake; BSSL, bile salt stimulated lipase; CML, complex milk lipid; EFSA, the European Food Safety Authority; GPL, glycerophospholipid; IP, inositol phosphates; MFGM, milk fat globule membrane; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin.