**Maternal DHA Impact on Child Neurodevelopment**

Editor

**Asim K. Duttaroy**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editor* Asim K. Duttaroy University of Oslo Norway

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This is a reprint of articles from the Special Issue published online in the open access journal *Nutrients* (ISSN 2072-6643) (available at: www.mdpi.com/journal/nutrients/special issues/DHA cognition).

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## **Contents**

## **About the Editor**

#### **Asim K. Duttaroy**

Dr. Asim K. Duttaroy is a Professor and Group Leader at the Clinical Nutrition at the Faculty of Medicine, University of Oslo, Norway. His main research area is feto-placental growth and development, and the cardioprotective function of naturally derived molecules. His scientific work has been published in over 270 original contributions, reviews, books, book chapters, editorials, and 9 books. He has successfully supervised 35 PhD/MD students so far. He has examined more than 270 PhD theses submitted from different countries, including India, the USA, the UK, South Africa, and Australia. He has several international patents that led to the establishment of two multinational companies, Provexis Ltd., UK, and IDIA AS, Norway. His patented work on the anti-platelet regime (Fruitflow®) isolated from tomatoes, an alternative to aspirin, was the EU's first approved EFSA product. He received numerous international awards for his contribution to clinical research.

## **Preface to "Maternal DHA Impact on Child Neurodevelopment"**

This preprint book focuses on maternal docosahexaenoic acid, 22:6n-3 (DHA), and arachidonic acid, 20:4n-6 (ARA), on children's neurodevelopment.

I express my gratitude to all authors for their excellent contributions from mechanical aspects to interventional studies.

I am also grateful to Ms. Stella Duo, the managing editor, the journal *Nutrients*, MDPI, and all of the people involved in making this a successful Special Issue on maternal DHA impact on child neurodevelopment.

I hope the readers find the papers in this preprint book very helpful as they contain new information.

> **Asim K. Duttaroy** *Editor*

## *Review* **Maternal Docosahexaenoic Acid Status during Pregnancy and Its Impact on Infant Neurodevelopment**

#### **Sanjay Basak, Rahul Mallick and Asim K. Duttaroy**

Received: 19 October 2020; Accepted: 23 November 2020; Published: 25 November 2020

**Abstract:** Dietary components are essential for the structural and functional development of the brain. Among these, docosahexaenoic acid, 22:6n-3 (DHA), is critically necessary for the structure and development of the growing fetal brain *in utero*. DHA is the major n-3 long-chain polyunsaturated fatty acid in brain gray matter representing about 15% of all fatty acids in the human frontal cortex. DHA affects neurogenesis, neurotransmitter, synaptic plasticity and transmission, and signal transduction in the brain. Data from human and animal studies suggest that adequate levels of DHA in neural membranes are required for maturation of cortical astrocyte, neurovascular coupling, and glucose uptake and metabolism. Besides, some metabolites of DHA protect from oxidative tissue injury and stress in the brain. A low DHA level in the brain results in behavioral changes and is associated with learning difficulties and dementia. In humans, the third trimester-placental supply of maternal DHA to the growing fetus is critically important as the growing brain obligatory requires DHA during this window period. Besides, DHA is also involved in the early placentation process, essential for placental development. This underscores the importance of maternal intake of DHA for the structural and functional development of the brain. This review describes DHA's multiple roles during gestation, lactation, and the consequences of its lower intake during pregnancy and postnatally on the 2019 brain development and function.

**Keywords:** DHA; brain; MFSD2a; SPM; fetus; placenta; infant; neurogenesis; pregnancy; pre-term

#### **1. Introduction**

Docosahexaenoic acid, 22:6n-3(DHA), an n-3 (omega-3) long-chain polyunsaturated fatty acid (LCPUFA), plays various essential roles in human health [1–4]. DHA takes part in several biological processes, such as angiogenesis, immune modulation, inflammatory response, signal transduction, apoptosis, cell proliferation, and a host of the membrane, cellular and molecular functions that affect health and diseases [3,5,6]. The DHA metabolites also mediate their roles in cellular signaling processes [7]. Inadequate dietary consumption of DHA and eicosapentaenoic acid, 22:5n-3 (EPA) impairs the optimal growth of the feto-placental unit and imposes plausible risks of cognitive decline, inflammatory disease, cardiovascular disease, inflammatory disorders, behavioral changes, and mental stress in later life [5,8,9]. While the metabolites of EPA and DHA, such as eicosanoids and docosanoids, are involved in cell signaling, DHA is primarily used for membrane structure and function [3,4]. Modern refined diets are mostly deficient in n-3 PUFAs, leading to sub-optimal organ function that may predispose individuals to an increased risk of diseases [9–11].

The recommended intake of DHA and the EPA is based on the quantum of data supporting beneficial outcomes in protecting cognitive development and cardiovascular diseases. Experts are agreed upon to enhance n-3 LCPUFA intake via increased seafood or supplementary approaches. An n-3 LCPUFA intake of no less than 250–500 mg/d should be made available to maintain healthy adults' physiological needs in their daily routine. Even though DHA consumption during pregnancy is an essential consideration as it is required for fetal brain development and growth, a limited number of countries have adopted and implemented appropriate guidelines for the n-3 LCPUFA consumption during pregnancy. DHA's requirement during placental growth and early development has been highlighted recently. Thus, low maternal DHA status may lead to the placenta's functional inadequacy and accordingly alter fetoplacental growth and development. Therefore, DHA supplementation well before gestation can be considered to prevent feto-placental associated developmental disorders.

Several comprehensive articles have focused on DHA and its impacts on human health [3,4,12–14]. However, this review has primarily concentrated on maternal DHA status during pregnancy on neurocognitive development in infants.

#### **2. DHA and Its Metabolites: E**ff**ects on the Structure and Function of the Human Brain**

N-3 PUFAs have essential functions on human health, and various studies have explained the molecular mechanisms underlying the effects of DHA in various tissues, including the brain. DHA is the predominant n-3 LCPUFA within the brain. Several neurophysiological functions are attributed to DHA, including the cell-survival, neuroinflammation, neurogenesis cellular signaling, and its protective function in maintaining blood-brain barrier integrity. Because of these vital roles of DHA in the brain, any alteration of DHA metabolism in the brain, as a cause or consequence, affects several neurological and psychiatric conditions.

DHA derivatives include the families of specialized pro-resolving mediators (SPMs), such as lipoxins, resolvins (resolvin D (RvD), and resolvin E (RvE)), protectins, and maresins, are known to have diverse biological activities [15,16]. SPMs actively thwarted the inflammatory response by stimulating specific G-protein-coupled receptors (GPR) expressed on immune cells that propagate both the anti-inflammatory and pro-resolving processes [15,16]. The anti-inflammatory activities of SPMs are mediated by inflammatory scavenging interleukins (IL) such as IL-10, IL-1 decoy receptors, and IL-1 receptor antagonists and anti-inflammatory cytokines [15,16]. The SPMs activate their actions by several mechanisms that include the anti-inflammatory resolution, blocking the intracellular pathways leading to inflammation, downregulation of pro-inflammatory cytokines, and clearance of inflammatory cell debris macrophages, as well as stabilization of immune cells counts to basal levels [15,16]. The importance of SPMs in the inflammatory resolution is widely reported in chronic pathologies, including brain inflammation when their production remained insufficient, and the administration of SPMs exogenously reduces the inflammatory process and protects inflamed tissue(s) [15,17]. Dietary supplementation with metabolic precursors of SPMs can increase their availability and resolve the inflammatory process following neurological injury.

In the brain, DHA and arachidonic acid, 20:4n-6 (ARA) are incorporated into cell membrane phospholipids and thus influence their metabolism. ARA represents approximately 20% of the total amount of fatty acids in neurons. ARA, primarily esterified in phospholipids of the membranes, is released by the phospholipases A2. Several enzymes convert free ARA into several eicosanoids, and these metabolites may be involved in neuroinflammation [18]. Unesterified ARA can also directly modify synaptic functions [19]. Therefore, the level of intracellular free ARA plays a critical role in both neuroinflammation and synaptic functions. Inflammation may prolong due to the presence of ARA-derived eicosanoids, such as prostaglandin E2 and leukotriene B4, whereas n-3-derived eicosanoids decrease the levels of these inflammatory compounds and their activity. Thus, supplementation of n-3 fatty acids can restore or reverse the balance of lipid mediator precursors. Metabolic precursors of the inflammation-resolving SPMs, including DHA itself, having vital inflammation resolution effects in brain injury [20]. Intravenously administered low-dose DHA in rats showed significant tissue-sparing

effects in the peri-infarct region of the middle cerebral artery occlusion compared to those who received high or medium DHA doses [21]. DHA-treated rats improved significantly in neurological performance up to 7 days following middle cerebral artery occlusion. Following experimental traumatic brain injury in rats, a DHA-enriched diet for 12 days preserved brain-derived neurotrophic factor (BDNF) concomitant improved learning capability [20]. Although DHA's mechanism in rescuing neurological injury is not known, its potential therapeutic warrants further investigations.

Resolvins, protectins, and maresins have SPM functions. SPMs are produced from DHA either spontaneously or by aspirin, involving enzymes, such as lipoxygenase (LOX) and acetylated cyclooxygenase-2 (COX-2) enzymatic pathway. Two distinct classes of the resolvins (Rvs) are synthesized from DHA by different biosynthetic paths, denoted as 17S- and 17RD- series resolvins during inflammation of the resolution [22,23]. The strong anti-inflammatory and pro-resolving activities of Rvs were demonstrated in animal models [7]. Rv of E and D series are endogenously produced from EPA and DHA, respectively. Both RvD1 and RvE1 may potentially lower inflammation. Conversion of DHA to 17(S)-hydroxy-containing RvD1–D4 and conjugation to triene-containing docosanoid structures are carried out by LOX [24]. Rvs can reduce the augmented pain by regulating the mediators of inflammation, ion channels with transient receptor potential, and transmission via the spinal cord. RvD1 (17(S)-trihydroxy-docosahexaenoic acid) is structurally different from aspirin-triggered RvD1. COX-2 promotes the synthesis of 17(R)-trihydroxy-docosahexaenoic acid, followed by the LOX on 13-hydroxyDHA [25]. The transcription of IL-1β is prevented by RvD1 that reduces the infiltration of neutrophils into the brain [23]. Since all Rvs are potent anti-inflammatory agents, dietary supplementation of n-3LCPUFAs is beneficial in the inflammatory situation.

The anti-inflammatory protectin D1 (PD1) is also synthesized endogenously from DHA in neuronal tissues and, therefore, termed neuroprotectin (NPD1). The internally formed NPD1,17(S)-trihydroxy DHA varies from aspirin-triggered NPD1; 17(R)-trihydroxy DHA structurally. Protectins are anti-microbial agents. PD1 was shown to prevent infiltration PMN both in vivo and in vitro. NPD1 effectively prevents damage to several mice tissues, such as the retina, brain, liver, kidney, and fibrosis [26]. The PD1/NPD1 isomer of PDX can prevent both inflammation and atherogenesis [27,28]. Besides its insulin-sensitizing and glucose regulatory actions, PDX can inhibit inflammation via IL-6 synthesis [29,30]. Maresins, MaR (macrophage mediators in resolving inflammation) are endogenously synthesized from DHA by macrophages through 12-LOX, followed by epoxy hydrolase activity. Maresins have both anti-inflammatory and pro-resolving actions [31]. The two structural variants of MaR are MaR 1 and MaR 2. MaR 1 (7*R*,14*S*-dihydroxy-docosa-4*Z*,8*E*,10*E*,12*Z*,16*Z*,19*Z*-hexaenoic acid) is a family of structurally distinct autacoids [32]. Studies on MaR 1 has potent anti-inflammatory activity even at the nano molar range [33].

Resolvins and protectins are produced from EPA and DHA. Both resolvins and protectins have an anti-inflammatory function. While 18R E-series resolvins (RvE1 and RvE2) are generated from EPA, DHA-derived 17S D-series resolvins (RvD1 to D6), PD1, and MaRs are synthesized during the inflammatory resolution. These inflammation preventive mediators are critically involved in resolving inflammation for the healing process. These EPA- and DHA-derived anti-inflammatory compounds inhibit trans-endothelial migration of PMN, suppress dendritic cell migration, reduce leukocyte infiltration [34]. PD1 blocks the synthesis of T cells-derived tumor necrosis factor alpha TNF-α and interferon gamma IFN-γ and induces apoptosis of these cells, implying its role in the down-regulation of Th1-mediated responses. LOX-dependent Th2-skewed human peripheral blood mononuclear cells-derived PD1 may favor the Th2 phenotype [35]. Several studies suggest that both n-3LCPUFAs, DHA, EPA, and their inflammation preventive mediators, such as MaRs, PDs, and Rvs, play vital role in mitigating the inflammatory process and support wound healing. Therefore, these bioactive lipids could be developed to manage and prevent various inflammatory diseases [34].

#### *2.1. DHA Accretion, Supplementation, and Fetal Brain Development*

DHA is vital for the development of a healthy brain [12,36,37]. Quantitatively in the brain, DHA is considered as the vital fatty acid [38]. In the human brain, the DHA level is 250–300 times higher than EPA. DHA is predominantly found in the phospholipid fraction of the brain grey matter [39]. The phospholipid distribution of n-3 LCPUFA in the brain showed a striking difference between EPA and DHA. DHA is primarily present in phosphatidylethanolamine and phosphatidylserine fractions, whereas EPA is mostly distributed in the phosphatidylinositol fraction of the membrane phospholipids. DHA is the most abundant n-3 fatty acids in the entire nervous system. DHA is critically required in the neuron regeneration and formation of synapse during the fetus's development and the first two years following birth [40].

Accumulation of DHA in the fetal brain occurs continuously throughout the gestation but is most active during week 29 to week 40. Supplementation of DHA (200 mg DHA/day) during the third trimester of pregnancy prevented the decrease of maternal DHA status [41]. Due to continuum fetal DHA accretion, the nutritional status of maternal DHA during pre-conception, pregnancy, and lactation demands DHA requirements for the brain and retinal development of babies [42,43]. Neonates with higher DHA concentrations in umbilical plasma phospholipids showed a longer gestational length than neonates with a lower concentration of DHA [44]. The pregnant women, supplemented with 600 mg DHA/day before 20 weeks of gestation until delivery, significantly reduced the preterm delivery and low-birth-weight babies [45]. DHA supplementation improves the DHA status both in the mother and her child because of its efficient transfer through the placenta [46], and breast milk [47,48]. DHA supplementation was beneficial for mothers having low consumption of seafoods [49]. DHA supplementation during pregnancy increases the placental transport of n-3 LCPUFAs via increased expression of fatty acid-binding/ transport proteins [50,51]. Consuming DHA (2.2 g DHA/day) and EPA (1.1 g EPA/day), from the 20th week of pregnancy until the partum showed an improved visual and coordination capacity of the children [52]. Similar beneficial effects were obtained after the supplementation of mothers with 500 mg DHA/day during pregnancy, which correlated the high blood DHA levels with the improved cognitive development of 5.5-year-old children [53]. Similarly, daily supplements of fish oil (500 mg DHA + 150 mg EPA), along with 5-methyltetrahydrofolate (400 µg/day), showed improved cognitive development until 6.5 years of age [54]. Higher DHA levels in plasma and breast milk positively correlate the brain's growth, development, and visual acuity in the neonate [55,56]. These studies demonstrated that DHA supplementing mothers during pregnancy and lactation or baby food fortified with DHA enhanced DHA levels in the infant tissues with improved neurological and visual development [57]. Conversely, a diet low in n-3 LCPUFA during pregnancy and/or lactation may negatively impact the child's visual and neurological development [58,59].

Infants fed with breast milk (<0.17% of total fatty acid in milk in contrast to optimal 0.3–0.4% DHA) showed a lower amount of DHA in erythrocytes, reduced visual acuity, and delayed language development as compared to infant fed by breast milk containing 0.36% DHA [43,60]. Women who received DHA (600 mg/day) supplementation during pregnancy from <20 weeks until delivery showed a substantial increase in visual acuity, particularly in male newborns. DHA supplementation may be the best predictor for nervous system development [61]. A direct relationship between higher DHA levels in erythrocytes (in mother and children) and the optimal visual and neuronal development of children was reported [62–65]. Perinatal DHA supplementation was also reported to reduce the risk of lower intelligence quotient (IQ) scores in children from very low-income families [66,67]. Newborn, when supplemented with DHA enriched formulas, improved cortical maturation and visual function [68]. Six-month-old babies who did not receive maternal milk and fed a formula containing egg yolk enriched with DHA (115 mg DHA/100 g food) showed a significant increase in erythrocyte DHA phospholipids and a better visual development measured at 1 year [69].

 In addition to DHA's dietary intake, ARA is obligatory for optimal brain and eye development in

infants [71]. The child who did not receive maternal milk must be fed with a formula containing DHA and ARA for optimum brain growth and development until 39 months after birth [72] and even 4 years after birth [71]. A study in newborns (*n* = 343) of 1–9-day-olds fed with formula with varying levels of DHA ((control (DHA 0%); 0.32%; 0.64%; 0.96%, with ARA 0.64% in all formulas) until the 12th month showed that babies fed with formulas containing 0.32% DHA showed a significant improvement in cognitive development as compared to control group [73]. An improved problem-solving skills and memory were noticed when human milk supplemented in DHA (32 mg/day) and ARA (31 mg/day) were fed to the preterm infant (birth weight < 1500 g) until 9 weeks after birth [74]. DHA (0.05 g/100 g) and ARA (0.1 g/100 g) fortified feeding to the preterm infant (birth weight > 2000 g) performed a higher index in mental development scores [75]. The term newborn (*n* = 420) supplemented with n-3 LCPUFA (60 mg of EPA and 250 mg of DHA daily) for six months following birth showed a significant increase of DHA deposition in the erythrocyte phospholipids and language and communication skills development [76]. A follow-up study with a formula (0.5% DHA) feeding preterm children (*n* = 107) from their birth until nine months showed a significant improvement in the verbal and total intellectual coefficient, language capacity, and memory in girl children as compared to control (0.0% DHA) [77]. A comparative study among the infants fed on breast milk, a preterm formula supplemented with LCPUFAs, or a traditional preterm formula without LCPUFAs, showed that preterm formulas with LCPUFAs improve the visual understanding and development of infants like those fed with maternal milk [78]. A large study involving 28 countries demonstrated that better mathematics test scores in children from low-income families are proportionate to the DHA levels in breastfed children's performance was found superior to non-breastfed children from high-income families and/or increased spending on education [79].

DHA level in the blood is positively correlated to cognitive abilities and inversely associated with cognitive decline. Activation of membrane GPR40 receptor by LCPUFA is responsible for driving neurogenesis by modulating synaptic plasticity in the adult brain. Dysfunction of GPR40 receptors can produce lipotoxicity in brain endothelial cells and neurons [80]. LCPUFA mediate GPR40 signaling modulates neurogenesis's functional aspects, anti-nociceptive effects, anti-apoptotic effect, and Ca2+ homeostasis in Alzheimer's disease (AD), and the nigrostriatal pathways. GPR40 is chosen as a potential drug target in treating several neuropathological disorders, including AD. During brain development, the DHA availability is also influenced by genetic polymorphisms of enzymes responsible for the endogenous conversion to LCPUFAs from their precursors.

Linoleic acid, 18:2n-6 (LA), and alpha-linolenic acid,18:3n-3 (ALA), are the two essential fatty acids. Imbalanced dietary intake of n-3 and n-6 fatty acids may lead to several neurological conditions. Brain concentrations of n-3 and n-6 PUFA derivatives are modulated by their dietary level and play a central role in regulating mood and cognition [81]. Endogenous abilities in the conversion of n-3 LCPUFAs from its precursor is affected by altered gene polymorphisms. The presence of gene-specific polymorphisms encode∆-5 and ∆-6 desaturases, enzymes involved in the synthesis of n-3 LCPUFAs from the precursor ALA, altered these fatty acid levels in particular DHA [82]. The f gene polymorphism (rs 174575) of ∆-6 desaturase enzyme in children allows higher DHA levels and better IQ test scores [83]. This indicates the importance of gene variations in the metabolism of n-3 LCPUFA and a consequential beneficial effect on brain development. Nine-month-old children supplemented with high DHA content showed significant cognitive performance and better arterial pressure at the end of infancy. N-3 LCPUFA supplementation, especially DHA, may favor optimum brain development that also has a protective effect against adult life's cognitive and cardiovascular diseases [84]. Supplementation of DHA (400 mg/ daily) for 4 months significantly raised blood DHA levels, positively correlated with an increment of punctuation score obtained for vocabulary and comprehension tests in 4-year-old children [85]. Supplementation of children (*n* = 409, 3 to 13-year-old) in Australia, with 750 mg DHA and 60 mg EPA per day for 20 weeks, demonstrated a significant increment in children's academic performance between 7 and 12 years old [86]. The supplementation of fish oil rich in DHA to 7–12-year-old ADHD children in Australia showed improved word reading, spelling capacity, and parents' conduct. These changes were positively related to an increase in the DHA level of erythrocyte phospholipids [87].

Despite this evidence, absolute compliance with DHA supplementation of baby food has not been established yet. The Scientific Opinion of the European Food Safety Authority (EFSA) Panel (2014) expressed that *"* . . . *there is no convincing evidence that the addition of DHA to infant and follow-on formulae has benefits beyond infancy on any functional outcomes"*. The panel's proposal about DHA supplementation of infant formula is based on DHA's structural and functional roles in nervous tissue and retina and its exclusive presence in the brain and retina's normal development.

#### *2.2. Maternal DHA and Its E*ff*ects on the Placental Structure and Functional Development*

Inappropriate structure and functional property of the placenta can impair the adequate exchange of nutrient and waste products between materno-fetal compartments during gestation [88]. The placenta's transport efficiency critically determines the fetus's optimal development and the well-being of the mother. Majorities of complicated pregnancies in humans are linked with the altered structure and function of the placenta. Inadequate supply of maternal blood to the fetus via the placenta is correlated adversity with risks of preeclampsia (PE), and intrauterine growth restriction (IUGR), preterm delivery (PT), etc. Shallow invasion of uterine spiral arteries by extravillous trophoblasts (EVTs) is a characteristics phenotype in PE, significant complications of the human pregnancy [89]. The EVTs are engaged in remodeling the uterine spiral artery to improve blood flow from the maternal circulation to the fetoplacental unit. Typically, humans' trophoblast invasion is confined to the endometrium and the first third of the myometrium and restricted within the first trimester of pregnancy. The pathogenesis of PE is primarily caused due to inadequate angiogenesis and placental dysfunction, leading to adversity in pregnancy outcomes for both mother and babies. Angiogenesis forms a new blood vessel from pre-existing ones, critically important for placental development and vasculature [90]. Several growth factors are known to influence the angiogenesis processes that include vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), angiopoietin-like protein 4 (ANGPTL4), platelet-activating factor (PAF), matrix metalloproteinases (MMP), etc. [91].

Recent data highlights about DHA are that in addition to its essential requirement in the third trimester of pregnancy, it is also involved in the first trimester's placentation processes. DHA stimulated tube-like formation in vitro to mimic angiogenesis during the early trimester of pregnancy in first-trimester human placental trophoblast cells, HTR8/SVneo cells [92,93]. Among the long-chain fatty acids tested in vitro, DHA was found the most potent stimulator of the in vitro angiogenesis in placental cells [92,94]. Moreover, DHA stimulated tube formation was partly mediated by increased expression and secretion of VEGFA synthesis, whereas oleic acid OA, ARA, EPA mediated their effects via increased synthesis of ANGPTL4 in these cells. The finding of n-3LCPUFAs on VEGFA synthesis and angiogenesis in placental cells opposed their other cell types' actions [95,96]. The induction in the expression of VEGFA by DHA was found unique in many ways as its expression was induced by several growth factors and cytokines, including EGF, PDGF, TGFβ1, TNFα, and IL-1β, but not by any fatty acids. The mechanism of DHA's action is still to be defined. However, DHA metabolites are unlikely to be involved as DHA induced VEGFA expression was not accompanied by COX-2 expression, a predominant mediator of DHA metabolites. Since peroxisome proliferator-activated receptor (PPAR,) agonist, and ligands failed to stimulate VEGFA expression, the involvement of PPARγ in DHA-stimulated VEGFA expression was unlikely. Both DHA and the EPA showed anti-angiogenic effects in cancer cells by inhibiting angiogenic mediators' production, VEGF, nitric oxide (NO,) PDGF, COX-2, PGE2 [95,97]. EPA enhanced VEGF synthesis by activating GPR120 and PPARγ pathway in 3T3-L1 cells [98]. Although DHA metabolite, such as 4-hydroxy-docosahexaenoic acid (4-HDHA), inhibited PPARγ-mediated angiogenesis in endothelial cells [99], however, DHA metabolites are not involved in the tube formation and/ VEGF synthesis of the placental trophoblast cells as fatty acid receptor genes fatty acid-binding protein-4 (FABP4, GPR120, GPR40) and lipid metabolic genes (COX-2 and CAV-1) is not upregulated by DHA [94]. The precise mechanism of enhanced tube formation by

EPA and DHA in placental trophoblast cells still to be ascertained. These LCPUFAs increased COX-2 messenger RNA mRNA expression, which may play a role in angiogenesis [94]. The mechanism that distinguishes DHA from other LCPUFA may be due to structural differences of the fatty acids. Further data postulates that DHA stimulates the expression of FABP4, an upstream target of VEGF in other cell systems. However, detailed work is essential to underscore DHA's differential effects over other fatty acids in endothelial, tumor, and placental trophoblast cells.

DHA delivery is essential for fetal brain and eye development during the third trimester of pregnancy [93,100]. Due to the limited synthesis and endogenous DHA conversion, the growing fetus relies mostly on the maternal DHA's placental supply [100]. Maternal plasma free fatty acids are taken up by the placental trophoblast via several membranes spanning proteins, such as fatty acid transport proteins (FAT/ cluster of differentiation 36 CD36, FATPs, and plasma membrane fatty acid-binding protein FABPpm) and intracellular fatty acid-binding proteins (FABPs) [101]. The mechanism of DHA uptake and transport in the placenta is not fully understood yet. However, a preferential uptake of DHA transport over other fatty acids via placental plasma membrane fatty acid-binding protein (p-FABPpm) was demonstrated [51,102,103]. Recently, a transporter major facilitator superfamily domain-containing 2A (MFSD2a) is present in the human placenta. MFSD2a) is located in the blood-brain barrier vessel and is involved in transporting the lysophosphatidylcholine form of DHA from the circulation into the brain. The role of MFSD2a in the placenta is not clear at this moment. Expression of the MFSD2a gene during pregnancy is used as a biomarker in predicting fetal neurodevelopment [104]. Maternal MFSD2a levels in blood were positively associated with the Z-score of head circumference at multiple time points at the neonate's first year. Placental MFSD2a transporter expression decreased and correlated to decreased DHA in cord blood of women with gestational diabetes, indicating its role in contributing to materno-fetal DHA transport [51,105,106]. Not much is known about the cellular localization of MFSD2a in the human placenta yet, although its expression is altered in the placenta of gestational diabetes placenta and PE. It is still not known whether the expression of MFSD2a in normal pregnancy may be used as an indicator of DHA deficiency in the fetus's brain.

Several researchers reported an increased incidence of preeclampsia in women with low n-3 fatty acids content in their red blood cells [107]. Increased n-3 LCPUFAs intake during pregnancy was beneficial for overall fetal growth and lowered the risk of early delivery and preeclampsia incidence [2,108]. In preterm birth, a positive association between placental DHA contents with placental weight was reported [109]. Maternal DHA content is, therefore, not only crucial for fetal growth and development but may also contribute a role in determining the placental size by stimulating first trimester trophoblasts mediated early placentation processes. International scientific experts recommend maternal consumption of pre-formed DHA for the prevention of premature birth [110], based on prevailing data from meta-analyses and extensive randomized controlled trial (RCT) studies [17,111]. The majority of the n-3LCPUFA supplementation trials were conducted during 16–20 weeks of gestation, well before the development of first-trimester pregnancy. Therefore, studies are required to evaluate the effects of n-3LCPUFA supplementation during pre-conception or before placentation prior to the 14th week of pregnancy to ensure optimal placentation and protect placental developmental-related abnormalities.

DHA incorporates a considerable amount of the fetal brain and retina during the third trimester of pregnancy [112,113], and this correlates with the development of normal eyesight and cognitive function [112]. DHA deficient condition can exist for an extended period after birth of very low birth weight (VLBW) babies since neonates miss the placenta's DHA supply window due to shortened gestation time. Various studies demonstrated the beneficial effects of maternal intakes of n-3 fats during pregnancy in optimum growth and development of the brain and retina [114]. Maternal supplementation of n-3 LCPUFAs during pregnancy increased gestational duration with a slight increase in birth weight [115,116]. The improvement in birth weight and prolong gestation was confirmed by meta-analyses of several n-3 LCPUFAs supplementation studies during pregnancy [115]. Despite these, the DHA supplementation had mixed impacts on gestation length in the general

population of pregnant women [74,117]. Low consumption of marine fish is found a decisive risk factor for preterm delivery [118]. Daily intake of n-3PCPUFA significantly lowered the rate of recurrent preterm delivery as concluded from a large multicentric clinical trial involving subjects with a history of preterm delivery [115]. The effects of n-3LCPUFA supplementation in high-risk pregnancy showed an inconsistent outcome [117]. Regular intake of n-3PCPUFA or placebo failed to prevent the recurrence of high-risk IUGR or pregnancy-induced hypertension [119]. The power and sample size can determine the measurable differences in the n-3LCPUFA supplementation trial's birth outcome during pregnancy [17,113]. The improved birth weight due to n-3LCPUFA supplementation often arises due to the longer gestational length of these pregnancies [120]. The adequate intake of n-3 LCPUFA by the mother ensures increased availability of these fatty acids in the fetus [121]. Besides, post-natal supplementation of LCPUFAs also improved the subsequent development of vision and brain in VLBW infants [122].

#### *2.3. Maternal Intakes of DHA and Its Impact on Fetal Brain Development*

Despite studies supported the nutritional and metabolic requirement of n-3 PUFAs, but functional implications of the n-3 PUFA, like ALA, were not demonstrated. The first report on ALA deficiency was reported in 1982 when the parenteral formula containing a higher ALA (42.4% LA and 6.9% ALA) corrected the neurological disorders of a 6-years old girl [123]. Holman and coworkers then proposed that ALA was an essential fatty acid. The minimum requirement to prevent symptoms caused by ALA deficiency was in the range of 0.5–0.6% of total energy intake [123]. After consuming an ALA deficit diet, elderly patients developed dermatological disorders, particularly dermatitis, and flaky skin, together with deficient circulating EPA and DHA levels. The adverse skin symptoms were resolved, and plasma levels of EPA and DHA regained normal value once ALA was added to the formula [124]. Based on available evidence, a minimum intake of ALA and EPA plus DHA for an adult was fixed at 0.2–0.3 en% per day and 0.1–0.2 en% per day, respectively, indicating that in the absence of EPA and DHA, the endogenous biosynthesis of these fatty acids from ALA is significantly increased [125]. Considering data available to date and the substantial presence of DHA in the human nervous system, including in the brain and retina, it is agreed that humans can transform 1% of total ingested ALA into DHA [126]. The breast milk remained the only food supply to the neonate containing all the essential nutrients, including the necessary and obligatory n-3 and n-6 LCPUFAs, to ensure optimal brain development [127–129].

 In contrast, LA levels in colostrum are negatively associated with child cognitive scores at ages 2 and 3 years, independently of breastfeeding duration [131]. DHA's relative content is ranged from 0.1% to 1% of the total fatty acids in human breast milk. The DHA content is vastly varied among populations globally, depending on their eating habits of fish or seafood intake or food cycle with land animals that feed on fish-meal or fish oils [43,132]. Over time, the level of DHA in breast milk is significantly decreased in the Western population, primarily due to lower intake of DHA containing foods [133]. According to the Food and Agriculture Organization (FAO), minimum dietary intake of 200 mg/day is suggested for pregnant women [134]; however, there is no universal consensus on the recommendation. However, the compliance was much better when pregnant women were advised or received dietary counseling about DHA's physiological importance in pregnancy, as reflected in their increased consumption of n-3 LCPUFAs containing foods and/or supplements [135]. Dietary counseling about the benefits of marine fish or fish product consumption during pregnancy significantly boosted n-3 LCPUFAs [136]. FAO and many studies have established that benefit of consuming marine fish rich in n-3 LCPUFA quash the possible adverse effects of any heavy metal or other organic contaminants present [137]. Consumption of two portions of fish rich in DHA, such as salmon, tuna, anchovy, or mackerel, in a week may adequately comply with the minimum dietary requirements during pregnancy [138,139]. Low intake of marine foods that provide DHA during pregnancy is critical to obtain enough of these fatty acid levels, reflected in low DHA levels in umbilical blood [140]. Intake of

*trans* fatty acid ingestion may also decrease the availability of n-3 LCPUFA to the mother and her child [141]. All these data suggest that the public health policies must be developed using DHA consumption for the population, especially pregnant and nursing women.

#### **3. The DHA Uptake System in the Human Brain**

Usually, non-esterified DHA is the major plasma pool for its supply to the brain [142,143]. DHA is also present as an esterified to lysophosphatidylcholine (LPC-DHA) pools in similar amounts. The brain maintains its fatty acid level via fatty acid uptake from circulation [144]. Astrocytes and endothelial cells of the blood-brain barrier (BBB) are significantly involved in the brain's uptake of plasma fatty acids. The brain's fatty acid uptake may occur via different mechanisms, such as passive diffusion and a saturable protein-mediated transport system.

In circulation, free fatty acids (FFAs) are mainly bound by albumin. At the inner surface of endothelial cell membranes, a small fraction of FFAs is delivered into the subcellular compartments for further metabolism. At the same time, most FFAs diffuse into the cytosol with or without the help of the battery of cell membrane- and cytoplasmic fatty acid-binding proteins. Figure 1 shows the putative fatty acid transport system of the brain. There are four classes of fatty acid transport proteins involved in transportation in the adult brain, including fatty acid translocase (FAT/CD36), plasma membrane-fatty acid-binding proteins (FABPpm), fatty acid transport proteins (FATPs), and cytoplasmic FABPs. Additionally, MFSD2a is newly identified as a DHA transporter in the brain. Even though there is a preferential uptake by the brain of esterified DHA (LPC-DHA), this is not the major pathway by which DHA is transported into the brain. Despite being an abundant fatty acid in brain phospholipids, DHA cannot be synthesized de novo in the brain and must be imported across the blood-brain barrier, but its transportation pathways are unknown yet. MFSD2a is the major DHA transporter, is expressed exclusively in the endothelium of the BBB of micro-vessels. The MFSD2a-deficient mice brain had significantly reduced DHA concentrations with neuronal cell loss in the hippocampus and cerebellum. These mice also exhibited had microcephaly with severe cognitive deficits and anxiety. MFSD2a transports LPC-DHA, but not unesterified DHA, in a sodium-dependent manner. Notably, MFSD2a transports plasma pool LPCs carrying long-chain fatty acids (C>14). Long-chain acyl-CoA synthetases (ACSLs) are also involved in brain DHA uptake [51,102]. Brain development requires an incredible increase in the de novo synthesis and accretion of DHA, mediated by several factors, including sterol regulatory element-binding protein SREBP. In normal physiology, the activity of MFSD2a is regulated by SREBP to maintain a balance between de novo lipogenesis and exogenous uptake of LPC-DHA [145]. *Nutrients* **2020**, *12*, x FOR PEER REVIEW 9 of 24 plasma fatty acids. The brain's fatty acid uptake may occur via different mechanisms, such as passive diffusion and a saturable protein-mediated transport system. In circulation, free fatty acids (FFAs) are mainly bound by albumin. At the inner surface of endothelial cell membranes, a small fraction of FFAs is delivered into the subcellular compartments for further metabolism. At the same time, most FFAs diffuse into the cytosol with or without the help of the battery of cell membrane- and cytoplasmic fatty acid-binding proteins. Figure 1 shows the putative fatty acid transport system of the brain. There are four classes of fatty acid transport proteins involved in transportation in the adult brain, including fatty acid translocase (FAT/CD36), plasma membrane-fatty acid-binding proteins (FABPpm), fatty acid transport proteins (FATPs), and cytoplasmic FABPs. Additionally, MFSD2a is newly identified as a DHA transporter in the brain. Even though there is a preferential uptake by the brain of esterified DHA (LPC-DHA), this is not the major pathway by which DHA is transported into the brain. Despite being an abundant fatty acid in brain phospholipids, DHA cannot be synthesized de novo in the brain and must be imported across the blood-brain barrier, but its transportation pathways are unknown yet. MFSD2a is the major DHA transporter, is expressed exclusively in the endothelium of the BBB of micro-vessels. The MFSD2adeficient mice brain had significantly reduced DHA concentrations with neuronal cell loss in the hippocampus and cerebellum. These mice also exhibited had microcephaly with severe cognitive deficits and anxiety. MFSD2a transports LPC-DHA, but not unesterified DHA, in a sodiumdependent manner. Notably, MFSD2a transports plasma pool LPCs carrying long-chain fatty acids (C>14). Long-chain acyl-CoA synthetases (ACSLs) are also involved in brain DHA uptake [51,102]. Brain development requires an incredible increase in the de novo synthesis and accretion of DHA, mediated by several factors, including sterol regulatory element-binding protein SREBP. In normal physiology, the activity of MFSD2a is regulated by SREBP to maintain a balance between de novo lipogenesis and exogenous uptake of LPC-DHA [145].

**Figure 1.** Docosahexaenoic acid (DHA) uptake system of the brain.  **Figure 1.** Docosahexaenoic acid (DHA) uptake system of the brain.

LPC-DHA, but not other DHA forms, across the blood-brain barrier to the neuron.

*DHA Deficiency during Fetal Brain Development and Its Impact on Cognitive Functions*

DHA is carried through the plasma by albumin and circulating lipoproteins. There are four

DHA is carried through the plasma by albumin and circulating lipoproteins. There are four classes of lipid transport proteins involved in DHA uptake of the brain that includes fatty acid translocase (FAT/CD36), plasma membrane fatty acid-transport proteins (FABPpm), and fatty acid transport proteins (FATPs) and cytoplasmic FABPs. MFSD2a, a specific protein, can transport plasma LPC-DHA, but not other DHA forms, across the blood-brain barrier to the neuron.

#### *DHA Deficiency during Fetal Brain Development and Its Impact on Cognitive Functions*

During pregnancy and lactation, maternal intake of DHA ensures adequate maternal reserve deposited during pregnancy to support six-month breast-fed post-natal life. Maternal adipose resources during pregnancy cover the increasing demand for DHA in the early post-natal stage. Although it is difficult to estimate the quantity of DHA require in the diet for optimum brain development, the study from Kuiper et al. [146] first estimated the absolute requirement of DHA, ARA, and LA at 25 (conceptual age), 35 (preterm), and 45 (term) weeks. Based on the mother's data fed on the western diet, the DHA accretion rate was found 42 mg/day in the last five weeks of pregnancy. The accretion of DHA was found double in the last five weeks of pregnancy compared with the first thirty-five weeks together. The DHA accretion rate largely determines the DHA deficiency during brain development in the brain during a term and preterm conditions. Thus, it is important to devise the DHA requirement for preterm babies based on the mother's data from a different ethnic and dietary background. The majority of the available data suggests DHA's optimal requirement during brain development in infants is fed on the Western diet. A scenario with high LA intake in the diet will further enhance the need for pre-formed DHA in infant formula. To fulfill enough maternal reserve of DHA, intake may be required well before conception.

In developing populations, where pregnant women's usual diet is low in fats in which n-6 PUFAs are predominantly present with little intake of ALA or DHA as n-3PUFAs, most of the women start pregnancy with inadequate or insufficient n-3 PUFA status in their reserve. Under such a scenario, where a maternal reserve of DHA is low, endogenous synthesis of DHA from precursors is inadequate, resulting in an insufficient supply of DHA to the fetus that may affect DHA accretion in the brain during the brain development phase of the neonate. DHA's placental deficiency can be correlated with the lower DHA accretion in the brain of the babies born preterm in the developing population. There is no clinical data available about DHA accretion in the brain and neonatal's cognitive performance from the mother fed on the n-3 deficient diet in such a population. Therefore, optimal maternal intake of n-3 LCPUFAs is essential to fulfilling DHA's neonatal requirement for the first six months. Maternal intake of DHA is correlated with the problem-solving skills of the children in some aspects. It was argued that inadequate intake of DHA could affect rapid accretion of DHA in the human brain, mainly when brain growth is maximal as with the third trimester or first six months of life. Adequate DHA during this period ensures the maturation of the prefrontal cortex's specific brain domain that may support the problem-solving skills.

The impact of DHA deficiencies on cognitive functions is extensively studied using animal models in vivo. DHA deficiency can influence DHA's endogenous synthesis in the brain and its impact on synaptic plasticity. A recent study with DHA deficient mice by silencing ELOVL2, an enzyme that is responsible for endogenous synthesis of DHA from its precursors, showed downregulation in the expression of neural plasticity factors (BDNF, Arc-1) with a concomitant increase in the pro-inflammatory markers (TNFα, IL-1β, inducible nitric oxide synthase iNOS) in the cerebral cortex of the DHA deficient mice [147]. The altered expression of these factors is also associated with the learning and memory function in the brain. The endogenous DHA deficient mice showed an alteration in microglial architecture and cytokine factors without involving astrocytes indicates that resident immune cells are affected in the brain by endogenous DHA deficiency. The replenishment with DHA restored the physiological expression of neuroinflammatory and neuroplasticity factors in the cerebral cortex. These data indicate that DHA plays a critical role in neuroimmune communication in brain function and synaptic plasticity.

The damaging effects of n-3 PUFA deficiency on brain lipid composition and memory performance were evidenced in lipopolysaccharides (LPS)-induced rat models, suggesting that in utero n-3 PUFAs deficiency could be a potential risk factor for neurodevelopmental disorders [148]. The n-3 PUFA deficiency in rats shows downregulated glutamate receptors and upregulated pro-inflammatory TNFα gene expression in the central nervous tissue independent of their effects on membrane composition [149]. Chronic deficiency of DHA over multiple generations during brain spurt affects the process of neurodevelopment by modulating the neuronal cell growth and differentiation, as well as neuronal signaling. The deficiency may cause a functional deficit in the offspring's learning and cognitive efficiency by reducing intellectual potential and enhancing the risks of neurological diseases in adult life [150]. The n-3 fatty acid deficiency disrupts the peripheral balance of pro-and anti-inflammatory states in the brain due to altered systemic ARA: DHA ratio [151]. The excess ARA generates high prostaglandin concentrations, leukotriene, and thromboxane that lead to a pro-inflammatory state in the brain that disrupts the balance of anti- (n-3) and pro-inflammatory (n-6) eicosanoids due to alteration in GPR receptors' signaling [152]. In the animal model, DHA's maternal deficiency revealed reduced telencephalon structure in the hippocampus region [153]. Such a deficiency state affects region-specific brain development areas where the cerebral frontal cortex region is affected mostly, leading to hyper motor activity, reduced learning ability, and altered monoamine transmission [154]. The maternal DHA deficiency affects the offspring's brain development in a gender-specific manner due to differential efficiency of endogenous DHA converting enzyme in males and females.

As the endogenous conversion of DHA from its precursor is more efficient in female newborns due to estrogen presence, the infant male is more susceptible to the risk of brain disorders, such as ADHD, Autism, etc., in their later life [155]. The maternal DHA deficiency state profoundly affects behavioral change in feed intake, anxiety, and stress response in the offspring [156]. The n-3 deficiency state triggers sucrose-motivated food intake preference due to alteration in the brain-rewarding pathway that may prompt children to consume calorie-dense foods [157]. Chronic deficiency of n-3 fatty acids for multiple generations induces anxiety-related stress behavior in the offspring due to altered expression of neuropeptide Y-1 receptor and glucocorticoid receptor in the pre-frontal and hippocampus of the rat brain [150,158]. Data from several in vivo studies suggest that DHA promotes neurogenesis by improving the membrane fluidity in the structural domain of the hippocampus, prefrontal cortex, and hypothalamus region to stabilize the neurodevelopment circuitry network required for learning and memory recognition processes [159]. N-3 fatty acid deficiency during utero development and in the postnatal state negatively impacts cognitive abilities [74,160]. DHA's dietary deficiency increases the risk for neurocognitive disorders, whereas a diet enriched with DHA increases learning and memory and protects against cognitive decline during aging. However, whether increased intake of DHA can prevent the risk of brain disorders requires further investigation.

#### **4. Maternal DHA Supplementation and Brain Development**

During pregnancy, DHA's importance for fetal brain development has been shown in a large observational study (*N* = 11,875). The study found that children born to mothers with a higher intake of seafood during pregnancy improved fine motor skills, more significant pro-social behavior, higher verbal intelligence, and higher social development scores at eight years of age [66]. A recent longitudinal cohort concludes that higher DHA status during pregnancy and lactation is associated with an infant's problem-solving skills at 12 months [161]. But Crozier did not find any relationship between DHA level during pregnancy and cognitive performance of 4- or 6-year-old children [161].

A randomized controlled trial (RCT) found that taking 200 mg DHA orally daily for 4 months after delivery caused children's higher cognitive abilities at 5 years of age [162]. Ogundipe et al. showed that 300 mg/day DHA supplementation on the last trimester of pregnancy correlates positively with an infant's brain volumes (on MRI scan) [163]. DHA (600 mg/day) supplementation from 14.5 weeks of pregnancy until the delivery on KUDOS study found improved visual attention in infancy but

no consistent long-term benefit in childhood [164]. DHA (120 mg/day) supplementation, along with EPA (180 mg/day), from the 20th week of pregnancy until the 30th day of the postpartum period among 18–35 years older women in Iran found primary neurodevelopment improvement among 4 to 6 months old children [165]. However, another RCT from Australia did not find any effect among the toddlers at 18 months of age after an 800 mg DHA intake in 2399 pregnant women during <21st weeks of gestation to delivery [17]. Maternal supplementation of DHA (400 mg/day) during pregnancy positively reflected the child's (5–6 years) performance on language skills and short-term memory [166]. The positive effect of maternal DHA intake during gestation was observed in 18-month-old infants but not when they were 5 years old. DHA's long-term effects may be too small to detect, or it is possible that the DHA intake was not good enough during gestation to have lasting effects.

A considerable DHA accretion occurs during the brain growth spurt beginning in the third trimester. However, there is no adequate information on whether DHA deficiency during early gestation can impact fetal brain development in the third trimester. Most DHA intervention studies on neurodevelopment were carried out from the second trimester of pregnancy. Therefore, the impacts of DHA intervention at 16 weeks of gestation may be too late as DHA is required during the first trimester for early placental growth and development, the critical step for future placenta's adequate roles for the maternal supply of DHA for the fetal brain development. The inverse relationship of child Beery scores with maternal erythrocytes 22:4*n*-6 and 22: 5*n*-6 suggests that visual-motor integration development is compromised at low prenatal DHA. This is consistent with the time course of brain maturation; maturation occurs in the visual cortex before the prefrontal cortex.

Consuming DHA-rich eggs (135 mg DHA/egg) during pregnancy showed higher erythrocyte and umbilical cord DHA levels as compared with those pregnant women who consumed non-enriched-eggs (18 mg DHA/egg) [167]. Babies born to mothers who consumed DHA-enriched cereal-bars (300 mg DHA/bar) had increased visual acuity until four months of postpartum [168], and a better capacity to resolve problems with an improved organization of their dream [169]. Despite the beneficial effects of fish oil n-3 fatty acids in fetal development, recommendations to increase fish consumption for pregnant women are often met by the fear of heavy metal contaminations in these foods. However, many health professionals recommend avoiding or reducing fish consumption, especially for pregnant women. Despite some organoleptic problems, because the reconstituted product developed some uncomfortable smell that is not accepted for some mothers, the formula has proved to increase the DHA content of breast milk [170]. In Chile, a newly developed inexpensive formula overcame this organoleptic limitation and increased DHA availability for pregnant and nursing population [171].

Available data based on randomized controlled trials suggest beneficial effects of maternal supplementation of DHA on neonatal growth and cognitive development (Table 1). Several RCTs found that DHA supplementation on term and pre-term infants found a significant outcome on visual development during infancy [172]. Table 2 presents RCTs from the postnatal supplementation of DHA on the neonatal visual, verbal, and cognitive development. A meta-analysis of RCTs on routinely supplemented infant formula milk with DHA has found no beneficial role in neurodevelopmental outcomes [173]. A large trial (DHANI) was carried out in India, where prenatal and 6 months of post-partum 400 mg/day maternal DHA supplementation effects measure to evaluate the neurodevelopment outcome [174,175]. The study reported that the mean development quotient (DQ) scores in the DHA and placebo groups were not statistically significant after 12 months of mothers' supplementation through pregnancy and lactation with 400 mg/d DHA [175].

**Table 1.** Supplementation of DHA on the neonatal growth and cognitive development: a consolidated Randomized Controlled Trial (RCT).

Abbreviations: DHA: Docosahexaenoic acid; ARA: Arachidonic acid; EPA: Eicosapentaenoic acid; Mo: Month; Yr: Year; Wk: Week; MRI: Magnetic resonance imaging. ↑ denotes "increased".

**Table 2.** Supplementation of DHA on the neonatal visual, verbal, and cognitive development: a collection of Randomized Controlled Trials (RCTs).

↑ denotes "increased".

Figure 2 shows the accretion of DHA at different development stages that affect fetoplacental and fetal brain development. DHA accumulates substantially in the retina and cerebral cortex during the last trimester and the second year of life. Intervention studies have shown that improving maternal DHA nutrition reduces the risk of visual and neural development in infants and children. Several pieces of evidence supports the notion that maternal transfer of DHA to the infant before and after birth, with short and long-term, modulates neural functions. However, genetic variation responsible for endogenous conversion of DHA by fatty acid desaturases also influences essential fatty acid metabolism and may affect individuals' optimal DHA requirements. Consideration of adequate DHA intake includes

Pre-term infant, *n* = 361, USA

Pre-term 23–24 wk infant, *n* = 90, USA

ADHD 7–12 yr children, *n* = 90, Australia

Larger pre-term 30– 37 wk, BW > 2 kg, *n* = 27, Taiwan

Breastfed 6 mo infant, *n* = 25–26, USA

brain development, a balanced intake of n-3, and n-6 PUFAs in gestation, and lactation, and optimal fatty acid nutrition during pregnancy is required for infant neurodevelopment. Premature infants have a deficit in DHA shortly after delivery for several reasons, including missing the third trimester DHA accretion. More studies are required to assess DHA's optimal dosage, delivery method, and duration of supplementation to evaluate DHA intake in premature infants. adequate DHA intake includes brain development, a balanced intake of n-3, and n-6 PUFAs in gestation, and lactation, and optimal fatty acid nutrition during pregnancy is required for infant neurodevelopment. Premature infants have a deficit in DHA shortly after delivery for several reasons, including missing the third trimester DHA accretion. More studies are required to assess DHA's optimal dosage, delivery method, and duration of supplementation to evaluate DHA intake in premature infants.

fatty acid metabolism and may affect individuals' optimal DHA requirements. Consideration of

↑ denotes "increased".

Figure 2 shows the accretion of DHA at different development stages that affect fetoplacental and fetal brain development. DHA accumulates substantially in the retina and cerebral cortex during the last trimester and the second year of life. Intervention studies have shown that improving maternal DHA nutrition reduces the risk of visual and neural development in infants and children. Several pieces of evidence supports the notion that maternal transfer of DHA to the infant before and after birth, with short and long-term, modulates neural functions. However, genetic variation

*Nutrients* **2020**, *12*, x FOR PEER REVIEW 14 of 24

Growth and development of pre-term infant till 118 wk ↑

DHA levels of pre-term infant comparable to term placebo ↑

Reading and spelling correlated DHA levels in the blood ↑

Mental development index at 6–12 mo ↑

Maturation of retina and visual cortex at 12 mo ↑

Clandinin et al., 2005[176]

Baack et Al, 2016 [177]

Milte et Al, 2012 [87]

Fang et Al, 2005 [75]

Hoffman et al., 204 [69]

Algal DHA oil (17 mg/100 kcal), fungal ARA oil (34 mg/100 mL) from birth to 4 mo

Algal DHA 50 mg per day from 1 to 6–7 wk at discharge

DHA 1032 mg or 108 mg, EPA 264 mg or 1109 mg for 4 mo

DHA (0.05%), ARA(0.1%) infant formula for 6 mo after birth

DHA 115 mg/100 g baby food from 6 mo to 12 mo of the breastfed infant

**Figure 2.** Maternal source and delivery of DHA at different stages of development affect fetoplacental and fetal brain development. **Figure 2.** Maternal source and delivery of DHA at different stages of development affect fetoplacental and fetal brain development.

DHA plays different roles in the gestation specific development of the feto-placental unit. During the first trimester, DHA possibly involves in decidual remodeling to establish early placentation by promoting the expression and secretion of angiogenic growth factors, such as VEGFA, FABP4, ANGPTL4, leptin, etc. DHA and its metabolites improve maternal-fetal immunocompetence by maintaining the oxidative stress, production of reactive oxygen species (ROS), and pro-anti-inflammatory balance maternal-fetal interface. DHA is delivered to the fetal brain during the third trimester via the placenta and subsequently via breast milk. In addition to DHA's presence in the neonatal brain matter's structural skeleton, mounting evidence suggests DHA helps several brain development processes, including neurogenesis, synaptogenesis, brain plasticity, inflammatory signaling, neuroprotection, etc.

#### **5. Conclusions**

The relationships between DHA on the brain development and function have been extensively studied. Under its ability to control membrane fluidity, the DHA also modulates neuronal density, neurotransmitter concentration, and synaptic activity by regulating the brain's neuroinflammatory state. Increasing evidence suggests that DHA, the foremost important n-3 long-chain fatty acid in the brain, has neurotrophic and neuroprotective properties. Dietary n-3 PUFA deficiency during early development decreased the brain n-3 PUFA levels. Together, this body of evidence supports the proposition that DHA deficiency in utero increases the vulnerability of brain development. Most evidence indicates that the DHA accumulation is mainly influenced by dietary intake, specifically of preformed DHA. Insufficient intake of n-3 PUFA may lead to DHA deficiency states that could affect the offspring's metabolic phenotypes by altering placental structure and function, fetal adiposity, body fat distribution, energy utilization, musculoskeletal growth, signaling between brain-adipose, epigenome stability, and inflammation [10].

**Author Contributions:** Conceptualization, A.K.D.; Methodology, A.K.D. and S.B.; Investigation, A.K.D. and S.B. Writing—Original Draft Preparation A.K.D.; Writing—Review & Editing, A.K.D., S.B., and R.M.; Visualization, R.M., S.B., and A.K.D.; Supervision, A.K.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** This work is supported in part by the grants from Throne Holst Foundation, and the Faculty of Medicine, University Oslo, Norway.

**Conflicts of Interest:** Authors express no conflict of interest.

#### **References**

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### *Review* **Maternal Supply of Both Arachidonic and Docosahexaenoic Acids Is Required for Optimal Neurodevelopment**

**Sanjay Basak, Rahul Mallick, Antara Banerjee, Surajit Pathak and Asim K. Duttaroy**

**Abstract:** During the last trimester of gestation and for the first 18 months after birth, both docosahexaenoic acid,22:6n-3 (DHA) and arachidonic acid,20:4n-6 (ARA) are preferentially deposited within the cerebral cortex at a rapid rate. Although the structural and functional roles of DHA in brain development are well investigated, similar roles of ARA are not well documented. The mode of action of these two fatty acids and their derivatives at different structural–functional roles and their levels in the gene expression and signaling pathways of the brain have been continuously emanating. In addition to DHA, the importance of ARA has been much discussed in recent years for fetal and postnatal brain development and the maternal supply of ARA and DHA. These fatty acids are also involved in various brain developmental processes; however, their mechanistic cross talks are not clearly known yet. This review describes the importance of ARA, in addition to DHA, in supporting the optimal brain development and growth and functional roles in the brain.

**Keywords:** arachidonic acid,20:4n-6; brain; docosahexaenoic acid,22:6n-3; fetus; maternal diet; cognitive; infants; neurodevelopment; neurogenesis

#### **1. Introduction**

The neurodevelopmental process involves a complex interplay between nutrients, genes, and environmental factors that result in the optimal growth, development, and maturation of the brain. The development of the brain in utero critically depends on the maternal supply of several components for its well-regulated structural–developmental process, characterized by specifically defined developmental periods, growth, a cellular signaling system, and maturation. During the brain growth spurt, neurodevelopment is particularly vulnerable to nutritional deficiencies [1].

The long-chain polyunsaturated fatty acids (LCPUFAs), docosahexaenoic acid,22:6n-3 (DHA), and arachidonic acid,20:4n-6 (ARA) are important nutrients required for fetal brain growth and development. The accumulation of DHA and ARA in the fetal brain predominantly occurs in the third trimester of a human pregnancy. The de novo synthesis of these LCPUFAs seems low in a growing fetus and placenta [2]; the maternal intake of these fatty acids contributes a significant share for brain development. Maternal DHA and ARA are accumulated rapidly within the cerebral cortex during the last trimester of pregnancy and postnatal 18 months [2–4]. The dietary balance of DHA and ARA intake and their interactions are thought to be important for the development and function of the brain. Several experimental studies suggested a crucial involvement of these two fatty acids in neural membrane formation and various roles of their metabolites, production of

**Citation:** Basak, S.; Mallick, R.; Banerjee, A.; Pathak, S.; Duttaroy, A.K. Maternal Supply of Both Arachidonic and Docosahexaenoic Acids Is Required for Optimal Neurodevelopment. *Nutrients* **2021**, *13*, 2061. https://doi.org/ 10.3390/nu13062061

Academic Editor: Andrew J. Sinclair

Received: 25 April 2021 Accepted: 14 June 2021 Published: 16 June 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

eicosanoids, and their influence on depression- and anxiety-related behaviors. Moreover, multiple trials have found that higher plasma or erythrocyte DHA levels positively correlate with infant neurocognitive outcomes [5–10]. late with infant neurocognitive outcomes [5–10]. High levels of DHA in the brain are achieved during early life and are maintained throughout life. DHA accretion to the brain continues into childhood, and the incorpora-

acids in neural membrane formation and various roles of their metabolites, production of eicosanoids, and their influence on depression- and anxiety-related behaviors. Moreover, multiple trials have found that higher plasma or erythrocyte DHA levels positively corre-

*Nutrients* **2021**, *13*, x FOR PEER REVIEW 2 of 29

High levels of DHA in the brain are achieved during early life and are maintained throughout life. DHA accretion to the brain continues into childhood, and the incorporation of DHA is still high despite its reduced accumulation rate. The preferential transfer of maternal DHA and ARA by the placenta to fetal circulation and its mechanisms are reviewed extensively [11,12]. LCPUFA content in human milk also regulate the amount of DHA and ARA transferred to the infant during breastfeeding [13]. However, this phenomenon may depend on an optimal maternal dietary intake of DHA from the supplement or marine fish [14], whereas ARA levels are usually maintained due to high n-6/n-3 ratios in the diet. tion of DHA is still high despite its reduced accumulation rate. The preferential transfer of maternal DHA and ARA by the placenta to fetal circulation and its mechanisms are reviewed extensively [11,12]. LCPUFA content in human milk also regulate the amount of DHA and ARA transferred to the infant during breastfeeding [13]. However, this phenomenon may depend on an optimal maternal dietary intake of DHA from the supplement or marine fish [14], whereas ARA levels are usually maintained due to high n-6/n-3 ratios in the diet. In the central nervous system (CNS), the proportion of DHA with other membrane

In the central nervous system (CNS), the proportion of DHA with other membrane fatty acids increases as the brain size increases. The increase in the proportion of these fatty acids continues for the second year of life. DHA has significant neurobiochemical roles in ion channel and receptor functions, the release of neurotransmitters, synaptic plasticity, and gene expression in the neurons. Both DHA and ARA of synaptic membrane phospholipids are released as free fatty acids (FFAs) by activated phospholipase A2 (PLA2) and are converted to different bioactive metabolites. These liberated fatty acids and the metabolites play critical roles during ischemia seizure activity, inflammation, and other types of brain disorders. Figure 1 describes the putative roles of DHA in neural membranes. fatty acids increases as the brain size increases. The increase in the proportion of these fatty acids continues for the second year of life. DHA has significant neurobiochemical roles in ion channel and receptor functions, the release of neurotransmitters, synaptic plasticity, and gene expression in the neurons. Both DHA and ARA of synaptic membrane phospholipids are released as free fatty acids (FFAs) by activated phospholipase A2 (PLA2) and are converted to different bioactive metabolites. These liberated fatty acids and the metabolites play critical roles during ischemia seizure activity, inflammation, and other types of brain disorders. Figure 1 describes the putative roles of DHA in neural membranes.

**Figure 1.** DHA modulates several aspects of structural and functional activities of neuronal mem-**Figure 1.** DHA modulates several aspects of structural and functional activities of neuronal membrane.

Maternal n-3 PUFA deficiency during pregnancy was associated with impaired brain development in offspring [15] and defective neuroblast migration [16]. The deficiency of n-3 LCPUFAs during development causes hypomyelination in the brain, resulting in mood and anxiety disorders [17,18]. Consuming a diet containing a high amount of n-3 Maternal n-3 PUFA deficiency during pregnancy was associated with impaired brain development in offspring [15] and defective neuroblast migration [16]. The deficiency of n-3 LCPUFAs during development causes hypomyelination in the brain, resulting in mood and anxiety disorders [17,18]. Consuming a diet containing a high amount of n-3 fatty acids during pregnancy protected infants against the detrimental effects of maternal stress [19].

fatty acids during pregnancy protected infants against the detrimental effects of maternal stress [19]. The essentiality of DHA is well recognized in childhood and adult life, as its deficiency causes cognitive decline and other psychiatric disorders [20]. Plasma DHA levels are inversely correlated with depressive symptoms in infants and adolescents with bipolar disorder [21]. Human breast milk containing higher n-3 and n-6 LCPUFAs was associated with decreased infant despair and distress [22]. The impact of n-3 PUFAs in human milk in influencing infant mood or anxiety is still not clearly established, since cortisol levels in milk are also associated with infant temperament [23]. Moreover, the variation The essentiality of DHA is well recognized in childhood and adult life, as its deficiency causes cognitive decline and other psychiatric disorders [20]. Plasma DHA levels are inversely correlated with depressive symptoms in infants and adolescents with bipolar disorder [21]. Human breast milk containing higher n-3 and n-6 LCPUFAs was associated with decreased infant despair and distress [22]. The impact of n-3 PUFAs in human milk in influencing infant mood or anxiety is still not clearly established, since cortisol levels in milk are also associated with infant temperament [23]. Moreover, the variation in the fatty acid composition of mothers' milk may play an essential role in the outcome of offspring's mental status and overall health.

Although n-3 LCPUFAs supplementation is beneficial in preventing and treating major depression, bipolar disorder, and anxiety disorders in adults [24,25], much less is known about how the imbalance of these LCPUFA levels impact the mood and behavior of

brane.

infants. Studies in experimental models suggested that early exposure to n-3 fatty acids had a lasting effect on temperament and behavioral phenotypes of offspring [26]. Interventional studies in adults also showed an association between the n-3 PUFA status with improved mood and mental health [17,26].

Though precise molecular mechanisms are not well defined, DHA and its bioactive derivatives play various essential structural and functional roles in the brain [12,27,28]. High DHA levels in the phospholipids of synaptic membranes provide membrane flexibility and improve the efficiency of protein–protein interaction necessary for signal transduction [27]. Different aspects of metabolism, and the structure–function of the brain depend on optimum ARA and DHA levels and interactions between their metabolites [29]. Cognitive benefits from supplementation of combined ARA and DHA in early life have also been observed through early and middle childhood [30,31]. Furthermore, several human brain diseases, such as Alzheimer's and bipolar disorders, involve disturbed n-3 and n-6 LCPUFA uptake and metabolism. Therefore, understanding the dynamics of the maternal supply of DHA and ARA to the developing brain may be important for managing brain disorders. An additional neuroprotective mechanism may involve bioactive molecules derived from DHA and ARA, which are also involved in several cellular neuronal biochemical processes. These bioactive derivatives modify the functions of several genes in the brain by acting as ligands for transcription factors involved in critical brain functions, including signal transduction and synaptic plasticity.

A literature search was performed on the PubMed database by using search terms such as DHA, ARA, brain development, infant, fetal, breastfeeding nutrients deficiency, DHA and ARA supplementation. All types of articles related to human and mechanistic studies on models were included for evaluation. The articles for which full text was not available or not reported in English were excluded. The articles retrieved in the first round of searches identified additional references by a manual search among the cited references. This review describes the latest development of the interplay of DHA and ARA transfer and their impacts on brain development, their complementarity, the structure–function relationship, and their mechanisms of action in the brain. Moreover, the evidence of the essentiality of ARA in brain development is summarized.

#### **2. Maternal Delivery of DHA and ARA to the Developing Brain**

Both DHA and ARA are major components of the brain. DHA comprises 10–20% of the total fatty acid composition in the brain, whereas 9% is present in the form of ARA [32]. During the third trimester of pregnancy, these LCPUFAs preferentially accumulate in the fetus and reach higher fetal/neonatal blood levels than those in the mother [33]. Both DHA and ARA are deposited in large amounts relative to the accretion rates of other fatty acids in the fetal brain during a maximum brain growth spurt, which occurs from the last trimester in utero and continues through the breastfed postnatal life [4,34–36]. The development of the brain is critically dependent on the adequate maternal supply of LCPUFAs in this period, since their synthesis from the parent EFAs is insufficient to meet the high requirement [4,37]. The lower fetal status of both DHA and ARA is associated with neurological development [38]. Consequently, ample fetal and neonatal LCPUFA supply via transplacental transport and human milk is critically important, implying that maternal LCPUFA status should be adequate.

Maternal dietary intakes of fatty acids influence the fatty acid composition of breast milk and plasma levels of lactating women and their infants [39]. DHA incorporation in the neuronal membrane in early fetal life solely depends on placental transfer [11], breastfeeding, and the endogenous synthesis of DHA [40–42]. However, DHA accretion in the CNS depends on the dietary provision of DHA, i.e., on the duration and concentration of DHA supplementation. A similar nutritional dependency is absent for ARA accretion in the brain, i.e., the dosage and duration of postnatal ARA supplementation do not affect ARA accretion in the CNS [43].

A robust linear relationship between maternal DHA level and umbilical cord blood phospholipid was reported [44]. Both ARA and DHA also have a role in the early placentation process, in addition to their roles in fetal neurodevelopment and in the postnatal lactation period [45–47]. The high-affinity placental plasma membrane, fatty acid-binding protein (p-FABPpm), is involved in the preferential supply of both maternal DHA and ARA to the fetus [12,41,48]. Since the rapid deposition of these LCPUFAs into the brain occurs during the last trimester of pregnancy and subsequently in lactation, the maternal status must be maintained well during the critical time of brain development. The dietary intake and maternal stores of DHA are the determinants of infant blood DHA concentrations at birth [49]. Blood LCPUFAs in breast-fed infants remain higher than those in maternal circulation postnatally [50,51]. The blood levels of PUFAs of infants are higher than those in the maternal circulation postnatally during the breastfeeding period. The preferential postnatal deposition of LCPUFAs in the infant's brain is mediated via breastmilk. The dietary supplementation of DHA to pregnant and nursing mothers dose-dependently increases the DHA level in breast milk, which causes higher tissue accretion of DHA in breastfed infants with improved outcomes of mental performance [52–54]. The erythrocyte DHA status of breastfed infants is correlated with the maternal DHA status of erythrocytes during lactation; however, no such association was observed for ARA [13]. Human breast milk levels of DHA and ARA are relatively stable throughout the lactation [55]. The worldwide mean concentration of DHA is 0.3 ± 0.2%, and that of ARA is 0.5 ± 0.1% in breast milk [56]. Typically, human breast milk has 1.5- to 2-fold more ARA than DHA, though the breast milk's DHA content can be higher than ARA in populations with high marine fish consumption. It has been shown that the DHA level in breast milk is directly related to the DHA content of the maternal diet [57]. Still, it is unknown whether metabolic or dietary mechanisms explain the lower variability in breast milk ARA. Breast milk ARA was not affected in lactating mothers allocated to consume increasing doses of DHA [57]. Two recent studies confirmed the different regulation of ARA and DHA in breast milk, indicating that ARA is affected by the genetic pattern in the *FADS*-gene cluster [58] and is less sensitive than DHA to dietary supplementation [59]. The essentiality of ARA in infant nutrition is supported by the observation of the potentially adverse effects in preterm infants of consuming a marine-oil-containing formula [60]. Dietary ARA has roles in growth, neuronal development, and cognitive function in infants. Both ARA and DHA are necessary for fetal development, and a deficiency in one may compromise growth [61]. It has been shown that growth deficiency induced by fish oil supplementation is related to a reduced ARA availability due to the excess of DHA [62]. Western diets usually have a much higher ratio of n-6/n-3 fatty acids [63]. Therefore, it has been proposed that supplementation during gestation should be based on intake of n-3 fatty acids.

Children's intelligence quotient (IQ) was increased by 0.8 to 1.8 points when their mothers consumed DHA from pregnancy to the lactation period and beyond [64,65]. The mean levels of DHA and ARA in breast milk are found at 0.37% and 0.55% of total fatty acids across the globe, respectively. Prospective observational studies suggested that breastfed infants had a significant neurocognitive advantage compared with formula-fed infants [5,66,67], possibly due to the higher incorporation of DHA and ARA in breast milk relative to formula milk. The association between breastfeeding and child IQ concerning FADS2 genetic profile, specifically in SNP rs174575b, was observed [68]. Breastfed infants with rs174575 C-dominant carriers achieved higher scores on standardized IQ tests than non-breastfed C-carrier infants. However, observational data are confounded by the heterogeneous composition of breast milk, and environmental factors that influence the infant's mental development.

Based on established guidelines, it is emphasized that maternal dietary DHA requirements should be increased during pregnancy and lactation. Precisely, a minimum of 200 mg of DHA per day is recommended during these periods [69]. In both full-term and preterm populations, the evidence is compelling that breastfeeding is vital for an infant's neurodevelopment.

#### **3. The Fatty Acid Uptake System of the Brain**

The de novo synthesis of DHA in the brain is almost non-existent, and therefore, it must be imported from the circulation across the blood–brain barrier (BBB) [70]. The BBB comprises endothelial cells in the capillary connected with tight junctions, astrocytic end-foot processes, pericytes, and neurons [71]. The endothelial cells offer highly selective permeability in the BBB by their specialized tight junction function. Thus, the BBB is provided with a neuronal system immune-privileged environment permitting only small molecules into the brain.

Free fatty acids (FFAs) are transported into the cytosol via cell membrane- and cytoplasmic fatty acid-binding proteins (FABPs) [12]. There are four classes of membrane fatty acid transport proteins present in the brain, such as fatty acid translocase (FAT/CD36), plasma membrane fatty acid-binding proteins (FABPpm), fatty acid transport proteins (FATPs), and several cytosolic fatty acid-binding proteins (FABPs) [41,72,73]. Additionally, another transporter, Mfsd2a (major facilitator superfamily domain-containing protein 2A), is present in the BBB [12]. Mfsd2a transports lysophosphatidylcholine (LPC)-DHA, but not unesterified DHA [12,74–76]. The DHA-LPC produced from DHA-containing phosphatidylcholine when acted upon by PLA1. A sterol regulatory element-binding protein regulates the activity of Mfsd2a to maintain a balance between de novo lipogenesis and exogenous uptake of LPC-DHA. The brain of the Mfsd2a-deficient mice had significantly reduced DHA levels and experienced loss of neurons in the hippocampus and cerebellum. The mice later developed microcephaly with severe cognitive deficits and anxiety. Altered plasma levels of Mfsd2a during pregnancy influence the placental transport of DHA and neurodevelopment in utero [77]. The maternal blood levels of Mfsd2a in the third trimester were inversely correlated to DHA-LPC in maternal plasma [77]. DHA or DHA-LPC is taken up by the endothelial cells via FAT/CD36, FATPs, or Mfsd2a. Plasma albumin binds both unesterified DHA and DHA-LPC.

Like LPC-DHA, the accretion of ARA to the brain from ARA-containing lysophospholipids is six-fold more efficient than for unesterified ARA [78]. The brain relies on different transport systems for shuttling the varied forms of the metabolized DHA across the BBB. Active pathways of DHA and ARA incorporation into the brain include members of the FABP family. In human brain microvessel endothelial cells, FAT/CD36 and FATP-1, FATP4 also transport fatty acids across the monolayer [73]. However, DHA incorporation into brain phospholipids was not affected in CD36−/− mice, indicating this transporter may not be involved in the transport of DHA.

FABPs involve in the FFA uptake and transport to various intracellular compartments of a cell [73,79]. The expression of various FABP genes occurs at different developmental stages of the brain, as shown in animal studies. FABP3 is expressed in the brain after birth, and its expression levels increases until adulthood [80].  FABP7 is expressed mainly in radial glial cells at the early stages of brain development [80,82]. The level of FABP7 expression decreases starkly in the neonate and adult brain [80]. FABP7 plays a role in the establishment of the radial glial fiber system [82]. Moreover, FABP7 has been suggested to be associated with the decreased survival of glioblastoma patients [83–85].

FABPs have broad binding specificity, including the binding affinity for long-chain fatty acids (≥c16), eicosanoids, bile salts, and PPARs [41,86]. Both FABP3 and FABP4 bind ARA with high affinity [87,88], while FABP5 preferentially binds long-chain saturated fatty acids (c ≥ 16) [89]. FABP7 binds both DHA and ARA but with four-fold more affinity for DHA [90], indicating that DHA may be a preferred ligand for FABP7. Although FABP7, FABP5, and FABP3 can also bind different types of fatty acids [89,91].

FABP3, FABP5, and FABP7 are involved in both developing and mature adult brains [86,92]. Functional studies have demonstrated a variety of roles for FABPs in brain development, including the generation of neuronal and/or glial cells, differentiation, neuronal cell

migration, and axis patterning. Like DHA, FABP7 increases the proliferation of neural stem cells and neural progenitor cells and differentiates into mature neurons both in vitro and in vivo [93,94].

FABPs are multi-functional proteins, and complex signaling networks and transcription factors regulate their expression. FABPs are major downstream effectors of the Reelin-Dab1/Notch pathway that involves neuron–glia crosstalk during brain development. Since LCPUFAs and several FABPs are involved in brain development and function, it is important to further elucidate their roles in brain disorders' pathogenesis.

As FABPs are involved in developing, establishing, and maintaining the central nervous system, FABPs are implicated in the pathogenesis of Down syndrome. FABP7 is overexpressed in Down syndrome adult [95] and fetal brains [96], whereas FABP3 is significantly decreased in Down syndrome adult brains [95]. Furthermore, FABP7 upregulation correlates with PKNOX1 gene-dosage imbalance in the brains of Down syndrome patients. PKNOX1 is a POU domain protein that may directly control FABP7 expression by interacting with the Pbx/POU binding element of the FABP7 promoter [96]. Human FABP7 mRNA levels were significantly upregulated in the postmortem brains of schizophrenia patients. A correlation between an SNP variant within the second exon of human FABP7 and schizophrenia pathology was observed [97]. The impairment of prepulse inhibition (PPI) occurs in many brain disorders such as Alzheimer's disease, autism, bipolar disorders, Tourette syndrome, and schizophrenia [98]. The association between FABP7 and PPI status suggests roles of the FABP7 gene in the pathology of PPI-mediated neuropsychiatric and/or neurodegenerative diseases. The association of FABP3 and FABP5 with several neurodegenerative diseases is also reported [99,100]. DHA influences brain functions and protects from different brain tumors such as astrocytoma and glioma by binding to the FABP, resulting in the activation of transcription factor PPARγ to the nucleus-reduced cell migration, growth arrest, and apoptosis of tumor cells [101]. However, the roles of these proteins in human brain development are not well known.

#### **4. Structural and Functional Roles of DHA in the Human Brain**

The brain is known as the body's fattiest organ, containing phospholipids around 2/3 of its weight. The brain harness 20% of its total energy from β-oxidation of fatty acids in the mitochondria of astrocytes. The presence of DHA on the membrane influences neuronal information transmission, signal transduction velocity, and interaction with ion channels or receptor proteins and their activity [102]. DHA is also predominantly present in cortical gray matter, representing approximately 15% of total fatty acids in the adult human prefrontal cortex. As a crucial structural ingredient of the brain, DHA comprises the regions of the cerebral cortex and synaptic membrane. Neuronal membranes have approximately 50% DHA [44]. DHA is also vital for hippocampal and cortical neurogenesis, neuronal migration, and outgrowth [93,103,104].

The brain fatty acid levels, mostly LCPUFAs, are maintained via different mechanisms, as described above. The circulating plasma levels of DHA is positively related to cognitive abilities during aging and is inversely associated with decline in cognitive function. DHA, being a part of the cell membrane phospholipid, contributes to maintaining optimal fluidity and lipid raft assembly in the membranes, membrane electrical and antigenic signals of the cells. DHA also halts cell death by stimulating cell-cycle exit in neuro-progenitor cells [93,105]. DHA is involved in monoaminergic and cholinergic systems during brain development processes [52,106,107]. DHA has a long-term effect on serotonergic and dopaminergic systems during the fetal brain development in utero [35,107,108]. Data emphasized the importance of examining the long-term critical impact on brain development due to inadequate DHA supplies to the fetus during pregnancy. DHA stimulates neurite outgrowth in cell culture systems. Neurite outgrowth is an important process in the developing nervous system and also in the regeneration of nerves. The alpha linoleneic acid, 18:3n-3 (the precursor of DHA)-restricted diet decreased neurogenesis in rat dams' fetal brains, possibly due to the deficiency of DHA [103]. DHA

influences gene expression, neurotransmission and protects the brain from oxidative stress during development [109]. DHA is an essential factor for neurogenesis, phospholipid synthesis, and turnover [93,110,111].

Again, DHA can act as a ligand for peroxisome proliferator-activated receptor-gamma (PPARγ) and retinoid X receptor (RXR). RXR plays a vital role in embryonic neurogenesis, neuronal plasticity, and catecholaminergic neuron differentiation along with retinoic acid receptors. RXR is highly expressed in the hippocampus [112,113]. The PPARγ-RXR heterodimer modulates early brain development by regulating transcription genes [112,114].

DHA also protects the developing brain from peroxidative damage of lipids and proteins [115–117]. DHA and eicosapentaenoic acid,20:5n-3 (EPA) were reported as suppressors of angiogenesis in cancer cells, but they stimulate angiogenesis in the placenta [45]. However, DHA and DHA-LPC may act as pro-angiogenic and anti-angiogenic depending on the concentration and microenvironments [118].

Several epidemiological data show an inverse association of low habitual dietary intake of DHA and a higher risk of brain diseases [2,119,120]. A diet containing high amounts of n-3 fats and/or a lower amount of n-6 fats was strongly associated with the lower incidence of Alzheimer's disease and other brain diseases [121–124]. Intake of DHA improves attention deficit hyperactivity disorder (ADHD), bipolar disorder, schizophrenia, impulsive behavior, and other brain disorders [20,123,125].

Nevertheless, the data of intervention studies with DHA supplements are conflicting, despite the fact that many such studies demonstrated an apparent benefit of DHA intake in brain function. Several studies failed to reproducibly show that the absence of DHA and its metabolites are involved in various adult brain diseases. More well-designed clinical trials considering background diets and genetic makeup are needed for definitive conclusions.

#### **5. Roles of DHA and Its Metabolites in the Brain**

DHA and its metabolites play vital roles in the functional brain development of the fetus in utero and infants and healthy brain function in adults. DHA and its metabolites play significant roles in cellular and biological functions. The oxidation of DHA by lipoxygenases produces several types of metabolites such as oxylipins that regulate various biochemical processes of the brain [126].

DHA stimulates membrane-associated G-protein-coupled receptor (GPR) 120 mediated gene activation to promote anti-inflammatory activities [127,128]. DHA also activates PPARs and upregulates the expression of genes responsible for increasing insulin sensitivity and reducing plasma triglyceride level and inflammation. [44]. DHA and its metabolites' signaling pathways are involved in neurogenesis, anti-nociceptive effects, anti-apoptotic effects, the plasticity of the synapse, Ca2+ homeostasis in the brain, and nigrostriatal activities [129]. DHA itself and its metabolites have a broad spectrum of actions at different levels and sites in the brain [129,130]. Figure 2 shows the DHA and EPA metabolites and their function in the brain.

DHA is converted to maresin 1(MaR1), neuroprotectin D1(NPD1), and resolvins by human 12-LOX, 15-LOX, and CYP or enzymatically by aspirin-treated COX-2. They play various functions in the brain. DHA is metabolized by the P450 system, cyclooxygenase, and lipoxygenase enzymes under different metabolic conditions. 14-HDHA: 14-hydroxydocosahexaenoic acid; 17-HDHA: 17-hydroxy-docosahexaenoic acid; 18-HEPE: 18-hydroxyeicosapentaenoic acid; LG R6: G protein-coupled receptor 6; ALX/Fpr2: N-formyl peptide receptor 2; BLT1: leukotriene B4 receptor; COX-2: cyclooxygenases; DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; GPR32/37: G protein-coupled receptor 32/37; LC-PUFAs: long-chain polyunsaturated fatty acids; LOX: lipoxygenases.

**Figure 2.** Metabolites of EPA and DHA, and their membrane receptors. **Figure 2.** Metabolites of EPA and DHA, and their membrane receptors.

DHA is converted to maresin 1(MaR1), neuroprotectin D1(NPD1), and resolvins by human 12-LOX, 15-LOX, and CYP or enzymatically by aspirin-treated COX-2. They play various functions in the brain. DHA is metabolized by the P450 system, cyclooxygenase, and lipoxygenase enzymes under different metabolic conditions. 14-HDHA: 14-hydroxydocosahexaenoic acid; 17-HDHA: 17-hydroxy-docosahexaenoic acid; 18-HEPE: 18-hydroxy-eicosapentaenoic acid; LG R6: G protein-coupled receptor 6; ALX/Fpr2: N-formyl peptide receptor 2; BLT1: leukotriene B4 receptor; COX-2: cyclooxygenases; DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; GPR32/37: G protein-coupled receptor 32/37; LC-PUFAs: long-chain polyunsaturated fatty acids; LOX: lipoxygenases. DHA-derived specialized pro-resolving mediators (SPMs) such as DHA epoxides, oxo-derivatives (EFOX) of DHA, neuroprostanes, ethanolamines, acylglycerols, docosahexaenoyl amides of amino acids, and branched DHA esters of hydroxy fatty acids play important roles in brain functions [131,132]. Additionally, epoxydocosapentaenoic acids (EDPs) and 22-hydroxydocosahexaenoic acids (22-HDoHEs) are produced from DHA [133,134]. DHA is mainly metabolized by enzymes such as 5-, 12- and 15-lipoxygenases (LOX), COX-2, and cytochrome P450 (CYP). As the most demanding by-products of DHA, resolvins are formed by either LOX15 or CYP or aspirin-treated COX-2 activity [28]. The LOX15-derived resolvins are homologous to CYP-, or aspirin-treated COX-2-derived resolvins [28]. As the inflammation resolution mediators, resolvins act via different Gprotein coupled receptors (GPRs) [28,135]. Both resolvin D1 and aspirin-triggered resolvin D1 improve brain functions and impede neuronal death by down-regulating several factors such as NFkB, TLR4, CD200, and IL6R [136,137]. They even induce remote functional recovery after brain damage [136]. Both resolvin D2 and aspirin-triggered resolvin D2 protect from cerebral ischemic injury via phosphorylation ERK1/2. Subsequently, this pathway stimulates nNOS or eNOS to inhibit neuronal cell death and maintain BBB integrity by increasing zonula occludens-1 [138]. Resolvin D3, resolvin D5, aspirin-triggered COX-2 -derived resolvin D3, and aspirin-triggered resolvin D5 halt the neuroinflamma-DHA-derived specialized pro-resolving mediators (SPMs) such as DHA epoxides, oxo-derivatives (EFOX) of DHA, neuroprostanes, ethanolamines, acylglycerols, docosahexaenoyl amides of amino acids, and branched DHA esters of hydroxy fatty acids play important roles in brain functions [131,132]. Additionally, epoxydocosapentaenoic acids (EDPs) and 22-hydroxydocosahexaenoic acids (22-HDoHEs) are produced from DHA [133,134]. DHA is mainly metabolized by enzymes such as 5-, 12- and 15-lipoxygenases (LOX), COX-2, and cytochrome P450 (CYP). As the most demanding by-products of DHA, resolvins are formed by either LOX15 or CYP or aspirin-treated COX-2 activity [28]. The LOX15-derived resolvins are homologous to CYP-, or aspirin-treated COX-2-derived resolvins [28]. As the inflammation resolution mediators, resolvins act via different G-protein coupled receptors (GPRs) [28,135]. Both resolvin D1 and aspirin-triggered resolvin D1 improve brain functions and impede neuronal death by down-regulating several factors such as NFkB, TLR4, CD200, and IL6R [136,137]. They even induce remote functional recovery after brain damage [136]. Both resolvin D2 and aspirin-triggered resolvin D2 protect from cerebral ischemic injury via phosphorylation ERK1/2. Subsequently, this pathway stimulates nNOS or eNOS to inhibit neuronal cell death and maintain BBB integrity by increasing zonula occludens-1 [138]. Resolvin D3, resolvin D5, aspirin-triggered COX-2 -derived resolvin D3, and aspirin-triggered resolvin D5 halt the neuroinflammation [139,140]. However, the functions of other resolvins are still a mystery. Maresin (MaR), the anti-inflammatory pro-resolving mediator, is produced from DHA in macrophages during the inflammation, healing, and regeneration process [141–143]. MaR1 is predominant among other maresins. MaR1 decreases LTB4 synthesis and stimulates phagocytosis at the site of inflammation [144–146]. MaR1 enhances tissue repair by stimulating stem cell differentiation and plays an analgesic role through TRPV1-mediated response blockage [145,147]. MaR1 involves neurocognitive functions by regulating the infiltration of macrophages, regulating NF-κB signaling, oxidative stress, and cytokine release. Maresin 1 reduces neuroinflammation perioperative neurodegenerative disorders in an animal model [148]. MaR1 significantly affects the post-spinal cord injury model [149,150].

tion [139,140]. However, the functions of other resolvins are still a mystery. Maresin (MaR), the anti-inflammatory pro-resolving mediator, is produced from DHA in macrophages during the inflammation, healing, and regeneration process [141–143]. MaR1 is predominant among other maresins. MaR1 decreases LTB4 synthesis and stimulates phagocytosis at the site of inflammation [144–146]. MaR1 enhances tissue repair by stimulating Another SPM, neuroprotectin D1(NPD1), derived from DHA, improves cell survival and cell repair in brain disorders [151]. Like MaR1, NPD1 also possesses anti-inflammatory and neuroprotective activities [152]. In response to neuroinflammation, NPD1 is produced from endogenous DHA in the retina and brain [153,154]. Besides antiviral protection, NPD1 helps in neurocognitive functions [155–157]. NDP1 blocks the progression of Alzheimer's

stem cell differentiation and plays an analgesic role through TRPV1-mediated response

disease by stimulating the expression of PPARγ, amyloid precursor protein-α, and reducing the β-amyloid precursor protein [156]. Figure 3 describes DHA metabolites and their global effects on gene expression and second messenger systems affecting multiple cellular functions in the brain. Alzheimer's disease by stimulating the expression of PPARγ, amyloid precursor proteinα, and reducing the β-amyloid precursor protein [156]. Figure 3 describes DHA metabolites and their global effects on gene expression and second messenger systems affecting multiple cellular functions in the brain.

blockage [145,147]. MaR1 involves neurocognitive functions by regulating the infiltration of macrophages, regulating NF-κB signaling, oxidative stress, and cytokine release. Maresin 1 reduces neuroinflammation perioperative neurodegenerative disorders in an animal model [148]. MaR1 significantly affects the post-spinal cord injury model [149,150]. Another SPM, neuroprotectin D1(NPD1), derived from DHA, improves cell survival and cell repair in brain disorders [151]. Like MaR1, NPD1 also possesses anti-inflammatory and neuroprotective activities [152]. In response to neuroinflammation, NPD1 is produced from endogenous DHA in the retina and brain [153,154]. Besides antiviral protection, NPD1 helps in neurocognitive functions [155–157]. NDP1 blocks the progression of

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**Figure 3.** DHA metabolites formation and function in the brain. **Figure 3.** DHA metabolites formation and function in the brain.

Maresin 1(MaR1), neuroprotectin D1(NPD1), and resolvins are produced from DHA by human 12-LOX, 15-LOX, and CYP or aspirin-treated COX-2 enzymatically. These metabolites have multiple functions in the brain, which have been mentioned in the boxes. DHA is metabolized by the P450 system, cyclooxygenase, and lipoxygenase enzymes under different metabolic conditions. Maresin 1(MaR1), neuroprotectin D1(NPD1), and resolvins are produced from DHA by human 12-LOX, 15-LOX, and CYP or aspirin-treated COX-2 enzymatically. These metabolites have multiple functions in the brain, which have been mentioned in the boxes. DHA is metabolized by the P450 system, cyclooxygenase, and lipoxygenase enzymes under different metabolic conditions.

Anti-inflammatory activities of EFOX and neuroprostanes protect neuroinflammation in various diseases, such as Parkinson's disease and Alzheimer's disease [158–160]. Another DHA derivative, docosahexaenoyl ethanolamide, improves mood, pain, inflammation status, hunger, and glucose uptake by the brain endocannabinoid system [161– 166]. The function of DHA metabolites is summarized in Table 1. Anti-inflammatory activities of EFOX and neuroprostanes protect neuroinflammation in various diseases, such as Parkinson's disease and Alzheimer's disease [158–160]. Another DHA derivative, docosahexaenoyl ethanolamide, improves mood, pain, inflammation status, hunger, and glucose uptake by the brain endocannabinoid system [161–166]. The function of DHA metabolites is summarized in Table 1.

**Table 1.** Functions of DHA and ARA metabolites. **Metabolites Name Biological Effects** Maresins Resolution of inflammation, wound healing, anal-DHA glyceryl ester regulates the intake of food and neuroinflammation, similarly to the way docosahexaenoyl ethanolamide uses the endocannabinoid system [167,168]. The endocannabinoid system plays an integral part in memory, cognition, and pain perception [169,170]. DHA conjugates via cannabinoid receptors reduce neuroinflammation and improve neurogenesis [171,172].

DHA Metabolites gesic effects Protectins Resolution of inflammation, neuroprotection Resolvins Resolution of inflammation and wound healing DHA is involved in alleviating short-term stress, preventing anxiety and stress in later life [132,173]. DHA is reported to improve various psychiatric disorders such as schizophrenia, mood and anxiety disorders, obsessive-compulsive disorder, ADHD, autism, aggression, hostility and impulsivity, borderline personality disorder, substance abuse, and anorexia nervosa [174]. There is strong evidence that the consumption of marine fish reduces depression [175].

The mild symptoms of ADHD are corrected with DHA supplementation [176]. DHA was also shown to improve depressive symptoms of bipolar disorder by increasing Nacetyl-aspartate brain levels without affecting mania [174]. Even IQ outcomes in children and cognitive function in the aging brain are improved by DHA supplementation [2,24]. DHA-derived anti-inflammatory eicosanoids' neuroprotection prevent Alzheimer's disease pathogenesis [177]. DHA may modulate the metabolism of cholesterol and apolipoprotein E, lipid raft assembly, and the cell signaling system in Alzheimer's disease [178,179]. DHA protects neuronal brain function by reducing NO production, calcium influx, and apoptosis

while activating antioxidant enzymes such as glutathione peroxidase and glutathione reductase [180]. The second most prevalent neurodegenerative disease, Parkinson's disease, can be halted by DHA's neuroprotective role [180].

**Table 1.** Functions of DHA and ARA metabolites.

#### **6. DHA Deficiency in Utero and Human Brain Function**

DHA deficiency is linked with different brain disorders such as major depressive and bipolar disorder [181,182]. DHA levels are positively correlated with improved learning and memory and reduced neuronal loss [24]. DHA deficiency affects epigenetic development in the feto-placental unit [183,184]. The improvement in attention scores, adaptability to new surroundings, mental development, memory performance, and hand–eye coordination are associated with higher maternal DHA delivery to the fetal brain [7,185].

DHA deficiency during pregnancy suggests the lower development of language learning skills in children [186]. Even autistic spectrum disorder or ADHD among teenagers is associated with DHA deficiency [187,188]. Neurocognitive functional insufficiency in young adults or loneliness-related memory problems in middle age have been associated with DHA deficiency [189,190]. DHA deficiency in the third trimester significantly causes preterm brain development due to the insufficient maternal consumption of n-3 fatty acids. Even following delivery, infants are entirely dependent on breast milk or formula milk for DHA and ARA. Reduced DHA consumption during this critical brain development period may influence brain functionalities in adult life [191].

Dementia has shown an inverse relationship with regular marine fish consumption in different continents [120,192] and higher blood DHA levels inversely related to dementia [193,194].  The most prevalent dementia is Alzheimer's disease, which is inversely related to brain DHA level. Serum DHA level reduces significantly, and its addition positively correlates with memory scores in elderly Alzheimer patients [119,197,198]. The plasma DHA level is significantly associated with the risk of Alzheimer's disease [199].

Intake of 200 mg of DHA-containing fish per week reduces the risk of AD by 60% [200]. However, different randomized controlled trials (RCTs) found mixed results with DHA supplementation. DHA alone or combined with ARA or the EPA found no significant neuropsychiatric status changes in Alzheimer's disease patients [123,124,201]. The neurodegenerative disorder, Parkinson's disease's etiology is unknown. However, its primary palliative treatment is dopamine-based therapy. Various animal studies found the neuroprotective effect of DHA in the Parkinson's disease model. DHA has been shown to improve L-DOPA-induced dyskinesia [202] and reduce dopaminergic neuron apoptosis in mouse models [121]. DHA supplements offer a beneficial neuroprotective effect for Parkinson's disease management [203].

Multiple factors influence serotonin biosynthesis and function. The brain's serotonin level correlates with various behavioral consequences, e.g., control of executive function, sensory gating, social behavior, and impulsivity [204]. Serotonin-related gene polymorphism is associated with mental illnesses, e.g., autism spectrum disorders, ADHD, bipolar disorder, schizophrenia, etc. DHA modulates the activity of serotonin in the brain. DHA increases serotonin receptor accessibility by increasing membrane fluidity in postsynaptic neurons [204]. Concentric serotonin and a low DHA level in the orbitofrontal cortex are correlated with schizophrenia [205].

Observational studies showed that ADHD also has a relationship with DHA levels. RCTs of DHA with EPA supplementation and the addition of medications have demonstrated significant improvement in ADHD symptoms [122,187,206–216]. However, DHA supplementation with methylphenidate did not improve ADHD symptoms [217].

There are mixed results in RCTs that have been observed in early psychosis symptoms improvement with DHA supplementation. When DHA is supplemented with EPA for at least 12 weeks, the functional improvement and reduction in psychiatric symptoms are visible in different studies [218–220]. DHA deficiency elicits the chances of schizophrenia by promoter hypermethylation of nuclear receptor genes RxR and PPAR, which results in the downregulation of the gamma-aminobutyric acid-ergic system and the prefrontal cortex involved in oligodendrocyte integrity [221]. Lower erythrocyte DHA status is associated with the development of bipolar disorder [222]. However, combined DHA and EPA supplementation for 6 weeks improved mania and depression among juvenile patients [223–225]. However, recent RCTs found DHA has no role in improving the symptoms of bipolar disorder [20].

#### **7. Can DHA Supplementation Improve Brain Function of Infants: Results of Clinical Trials**

Various RCTs proved significant effects of DHA supplementation on infant brain development in pregnancy. The meta-analytic study in 2007 found a static co-relation between visual growth and DHA supplementation in first year of life [125]. An RCT in 2011 showed DHA-supplemented 18-month-old children had higher index scores in mental development [8]. Other RCTs showed DHA-enriched fish oil supplemented children had significantly higher percentile ranks of the total number of gestures at 1 to 1.5 years of age [226]. DHA-formula-fed infants scored equal visual equity scores with breast-fed infants at the age of four [227]. The recent meta-analytic review also found the positive effects of seafood consumption in pregnancies in developing childhood neurocognitive function [53]. The supplementation of DHA to pregnant and nursing mothers and the first year of infant life have developed better cognitive ability. During the last trimester of pregnancy, fetal brain development demands a higher amount of DHA, which can be interrupted in the case of a preterm born baby and can result in mental growth retardation. Different RCTs showed that higher DHA-enriched formula (around 1% of total fatty acids) is essential for the preterm baby for mental growth and development [24].

DHA supplemented along with EPA enhance the outcome for cognitive and mood disorders. However, conflicting data exist about the effect of DHA supplementation on cognition during childhood. The earlier RCT conducted in Australia and Indonesia did not show any improvement in general intelligence or attention among 6–10-year-old children following 88 mg/d DHA supplementation [228]. The DHA Oxford Learning and Behavior (DOLAB) study showed a significant improvement in brain function among aged 7–9-year-olds [229]. Another RCT showed improved memory and learning ability among 7–9-year-old children [230]. A few years ago, another RCT from Australia found no significant difference in academic performance between the DHA-supplemented and control groups [231]. Although the authors have mentioned their study's limitation that the final assessment was finished by less than half of the study population, DHA supplementation was low. The study was conducted with slightly older children, and in some cases, mothers did not give consent or provide data. The Third National Health and Nutrition Examination Survey (NHANES III) found a higher DHA supplementation effect in girls than boys, despite both sexes' receiving cognitive benefit [232]. The pregnant women need higher DHA supplementation for their own growth and for the growth and development of the newborn. Even the source of DHA and the ratio of EPA and DHA may influence the bioavailability of DHA.

A summary of a few recent clinical trials has been shown in the following Table 2. Although a slight improvement, these studies did not show any significant positive outcome from DHA supplementation effects on young children. However, a meta-analysis of DHA supplementation with the EPA improved childhood visual and psychomotor development without significant global IQ effects later in childhood [233]. However, there are no clinical data available where DHA supplementation was conducted before 14.5 weeks of the gestational period.

**Table 2.** Various clinical studies of DHA supplementation in mothers and infants about brain function.

#### **8. Roles of Arachidonic acid20:4n-6 (ARA) in Brain Development and Function**

In addition to DHA, the mother preferentially supplies ARA to the growing and developing brain via the placenta and breastfeeding. ARA uptake was found to be higher in early trimester trophoblast cells than EPA and DHA [239]. The ARA metabolism in the brain is suspected of having an altered profile in neurological, neurodegenerative, and psychiatric disorders. Using various knock-out models for enzymes involved in brain ARA metabolism, Bosetti showed that the ARA and its metabolites play a significant role in brain physiology via the PLA2/COX pathway [240]. The effects of DHA and ARA on body growth and brain functions were studied using delta-6-desaturase knock-out (D6D-KO) mice by feeding different combinations of PUFAs in milk formulations. The in vivo findings confirmed the complementary roles of ARA and DHA in body and brain development, respectively [241]. ARA may be required in a higher amount to support growth-promoting placental activities and the production of eicosanoids. In human milk, the amount of ARA typically exceeds the levels of DHA. Milk ARA content is also less varied than DHA, and, unlike DHA, ARA does not seem to be linked to maternal intake. There has been much discussion in recent years about the need for ARA and DHA in infant formula. Studies clearly show the requirement for both ARA and DHA in addition to the essential fatty acids (linoleic acid,18:2n-6 (LA), and ALA to support the optimal body, brain growth, and brain function. ARA is quantitatively the most common LCPUFA in the brain after DHA [242]. Although diverse roles of DHA are investigated, the roles of ARA in brain development and functions have not been investigated to a greater extent. Given that ARA and its precursor, LA, contribute significantly to the Western diet and its pleiotropic biological effects and its interactions with DHA, this n-6 LCPUFA is a crucial modifiable factor in brain development and preventive strategies of brain diseases. ARA corresponds to around 20% of the total amount of neuronal fatty acids and is mainly esterified in membrane phospholipids.

Several studies have suggested that the structure–function and metabolism of the brain depend on levels of ARA and DHA and interactions of their metabolites [29]. The recycling (de-esterification–re-esterification) of these two fatty acids in the brain are independently carried out by ARA- and DHA-selective enzymes. The ARA-mediated processes can be targeted or altered separately from the DHA-mediated processes by a dietary deficiency of n-3 PUFA or genetic manipulation. Therefore, in studies using n-3 PUFA deficiency models, the homeostatic mechanisms show DHA loss in the brain while increasing ARA metabolism. Further studies are required to understand the impact of the n-6/n-3 ratio on the regulation of DHA-selective iPLA2 and COX-1 or ARA-selective cPLA2, sPLA2, and COX-2 and their effects on brain function and neuroinflammation.

ARA must either be consumed in the diet or synthesized from its precursor LA in the liver. The brain contains relatively low LA levels, and its conversion into ARA is minimum in the brain. Thus, the growing brain depends on a steady supply of ARA [243] from the maternal circulation or via breast milk. Although lipoproteins and lysophospholipids of plasma may contribute to brain ARA levels, their quantitative contribution is unknown. Upon its entry into the brain, ARA is activated by a long-chain acyl-CoA synthetase and can be esterified into the sn-2 position of phospholipids. During neurotransmission, the brain ARA cascade is initiated when ARA is released from synaptic membrane phospholipid by the neuroreceptor-initiated activation of cPLA2. PLA2 is activated by dopaminergic, cholinergic, glutamatergic, and serotonergic stimulation via G-proteins or calcium [244]. Several PLA2 are activated via serotonergic (5-hydroxytryptaminergic), glutamatergic, dopaminergic, and cholinergic receptors [244,245]. Usually, calcium-dependent cytosolic PLA2 (cPLA2) resides at the postsynaptic terminals, selective for releasing ARA, whereas calcium-independent PLA2 is believed to release the DHA sn-2 position of phospholipids [13,14]. Upon its release, a portion of the unesterified ARA is converted to prostaglandins, leukotrienes, and lipoxins, a portion oxidized via β-oxidation, and the remainder (approximately 97% under basal conditions) is activated by ACSL and ultimately recycled and re-esterified into the sn-2 position of phospholipids [246]. An additional ARA is released by activated cytokine and glutamatergic N-methyl-d-aspartate receptors in conditions such as neuroinflammation and excitotoxicity. Although the signals that ARA and its derivatives relay are not entirely understood, they regulate blood flow, neuroinflammation, excitotoxicity, the sleep/wake cycle, and neurogenesis [247].

Like DHA, ARA is also directly involved in synaptic functions. The level of intracellular free ARA and the balance between the releasing and incorporating enzymes in membrane phospholipids may play critical roles in neuroinflammation and synaptic dysfunction. Both these events are observed in the murine model of Alzheimer's disease before the amyloid plaques and the neurofibrillary tangles, respectively formed by the two agents

known for Alzheimer's disease agents, A β peptide and hyperphosphorylated *tau.* Finally, western food, which contains excessive n-6/n-3 ratios, might favor more ARA levels and influence Alzheimer's disease mechanisms.

A better understanding of the complex relationships between ARA and DHA and their brain mechanisms is required. Free ARA contributes to Alzheimer's disease progression via different pathways. ARA and derivatives are pro-inflammatory and participate in neuroinflammation. ARA is directly involved in synaptic functions as a retrograde messenger and a regulator of neuro mediator exocytosis. ARA also influences tau phosphorylation, and polymerization can compete with DHA. Moreover, ARA has pleiotropic effects on brain disease, and it may be used in the fight against brain diseases. The dietary ARA and brain diseases about DHA should be investigated further to prevent the disease.

The transgenic Alzheimer's disease murine model showed that dietary ARA produced opposite Aβ production in Alzheimer's disease. Studies on the impact of dietary ARA on Alzheimer's disease are required to identify the underlying mechanisms of action. A reduced level of ARA in the temporal cortex of Alzheimer's patients was observed [248]; however, its relation with DHA and its metabolites is unknown. ARA involvement in Alzheimer's disease was mediated via cPLA2α. However, a DHA-rich diet did not show such effects [249]. A diet containing 2% ARA for 21 weeks increased Aβ1-42 production and deposition in 24-week-old CRND8 mice [250].

ARA is involved in cell division and signaling during brain growth and development [251]. In addition, ARA mediates neuronal firing [252], signaling [253] and long-term potentiation [254]. The absolute levels of n-3 PUFAs and the ratio of n-6 and n-3 PUFA affect gene expression of controlling neurogenesis and neural function.

ARA maintains structural order of the membrane and hippocampal plasticity [255]. ARA also protects the brain against oxidative stress in the hippocampus region via PPARγ and synthesizes new proteins [256]. Released intracellular ARA activates protein kinases and ion channels, inhibits the uptake of neurotransmitters, and enhances synaptic transmission, and modulates neuronal excitability [251]. As ARA is involved in intracellular signaling, the optimum levels of intracellular free ARA must be steadily maintained. ARA also activates syntaxin-3 (STX-3), a plasma membrane protein involved in the growth and repair of neurites [257]. Neurite growth closely correlates with the ARA-mediated activation of STX-3 in membrane expansion at growth cones [257]. The neurite growth from the cell body is a critical step in neuronal development. ARA stimulates exocytosis by allowing the attachment of STX-3 with the fusogenic soluble N-ethylmaleimide-sensitive factor receptors (SNARE complex) [258]. The SNARE proteins are involved in producing a fusion of vesicular and plasma membranes in the brain. The formation of this SNARE complex mediated by ARA drives membrane fusion, leading to the release of vesicular cargo into the extracellular spaces [258]. α-Synuclein plays a role in the development of Parkinson's disease, can sequester ARA and thus blocks the activation of the SNARE complex [258], suggesting the importance of ARA in synaptic transmission. All these data show the importance of ARA in cell signaling, trafficking, and the regulation of spatial–temporal interactions between cellular structures.

The most abundant prostaglandins in the brain are PGD2 and PGE2. They are synthesized from ARA by PGD2 and PGE2 synthases. COX-2 is overexpressed in the cortex and hippocampus of Alzheimer's disease patients [259]. PGE2 increases neuroinflammation and amplifies Alzheimer's disease pathology through various mechanisms.

Figure 4 describes different metabolites of ARA involved in brain function. ARAderived lipoxins are anti-inflammatory eicosanoids distinct from pro-inflammatory leukotriene and prostaglandin. Lipoxin biosynthesis occurs via two different pathways. Lipoxins mediate their action on endothelial cells to offer an inflammation resolution process. LipoxinA4 lowers neuroinflammation by reducing microglial activation. 5- Lipoxygenase (5-LOX) converts ARA into 5-HPETE and then 5-HETE or leukotriene A4 (LTA4). There is the increased expression of the dual enzyme 12/15-LOX and its products.

poral interactions between cellular structures.

**Figure 4.** Metabolites of ARA and their receptors. **Figure 4.** Metabolites of ARA and their receptors.

ARA is converted to lipoxinA4 or lipoxinB4 via two different pathways. Lipoxins mediate their action on endothelial cells to offer an inflammation resolution process. LipoxinA4 lowers neuroinflammation by reducing microglial activation. LTA4, LTB4, ARA is converted to lipoxinA4 or lipoxinB4 via two different pathways. Lipoxins mediate their action on endothelial cells to offer an inflammation resolution process. LipoxinA4 lowers neuroinflammation by reducing microglial activation. LTA4, LTB4, LTC4, LTD4, and LTE4 are leukotrienes A4, B4, C4, D4, and E4, respectively; DP1, EP1-EP4, FP, and IP, prostaglandin receptors; TP, thromboxane A2 receptor; BLT1 and BLT2, leukotriene B4 receptor; CysLT1 and CysLT2: cysteinyl leukotriene receptors; ALXR: lipoxins receptor.

ARA is involved in cell division and signaling during brain growth and development [251]. In addition, ARA mediates neuronal firing [252], signaling [253] and long-term potentiation [254]. The absolute levels of n-3 PUFAs and the ratio of n-6 and n-3 PUFA affect