The translation of lipid profiles to nutritional biomarkers in the study of infant metabolism

Introduction

Links between early life exposures and later health outcomes may, in part, be due to nutritional programming in infancy. This hypothesis is supported by observed long-term benefits associated with breastfeeding, such as better cognitive development in childhood, and lower risks of obesity and high blood pressure in later life. However, the possible underlying mechanisms are expected to be complex and may be difficult to disentangle due to the lack of understanding of the metabolic processes that differentiate breastfed infants compared to those receiving just formula feed.

Objective

Our aim was to investigate the relationships between infant feeding and the lipid profiles and to validate specific lipids in separate datasets so that a small set of lipids can be used as nutritional biomarkers.

Method

We utilized a direct infusion high-resolution mass spectrometry method to analyse the lipid profiles of 3.2 mm dried blood spot samples collected at age 3 months from the Cambridge Baby Growth Study (CBGS-1), which formed the discovery cohort. For validation two sample sets were profiled: Cambridge Baby Growth Study (CBGS-2) and Pregnancy Outcome Prediction Study (POPS). Lipidomic profiles were compared between infant groups who were either exclusively breastfed, exclusively formula-fed or mixed-fed at various levels. Data analysis included supervised Random Forest method with combined classification and regression mode. Selection of lipids was based on an iterative backward elimination procedure without compromising the class error in the classification mode.

Conclusion

From this study, we were able to identify and validate three lipids: PC(35:2), SM(36:2) and SM(39:1) that can be used collectively as biomarkers for infant nutrition during early development. These biomarkers can be used to determine whether young infants (3–6 months) are breast-fed or receive formula milk.

     

Metabolic analysis of infants with bronchopulmonary dysplasia under early nutrition therapy: An observational cohort study

To assess the amino acid and fatty acid metabolite patterns between infants with and without bronchopulmonary dysplasia in different nutritional stages after birth and identify metabolic indicators of bronchopulmonary dysplasia. This was an observational cohort of preterm infants born at a gestational age ≤32 + 6 weeks and with a body weight ≤2000 g. Amino acid and carnitine profiles were measured in dried blood spots (DBSs) during the early nutrition transitional phase using tandem mass spectrometry. Bronchopulmonary dysplasia was defined as oxygen dependence at 36 weeks of postmenstrual age or 28 days after birth. Metabolomic analysis was employed to define metabolites with significant differences, map significant metabolites into pathways, and identify metabolic indicators of bronchopulmonary dysplasia. We evaluated 45 neonates with and 40 without bronchopulmonary dysplasia. Four amino acids and three carnitines showed differences between the groups. Three carnitines (C0, C2, and C6:1) were high in the bronchopulmonary dysplasia group mostly; conversely, all four amino acids (threonine, arginine, methionine, and glutamine (Gln)) were low in the bronchopulmonary dysplasia group. Pathway analysis of these metabolites revealed two pathways with significant changes (p < 0.05). ROC analysis showed Gln/C6:1 at total parenteral nutrition phase had both 80% sensitivity and specificity for predicting the development of bronchopulmonary dysplasia, with an area under the curve of 0.81 (95% confidence interval 0.71–0.89). Amino acid and fatty acid metabolite profiles changed in infants with bronchopulmonary dysplasia after birth during the nutrition transitional period, suggesting that metabolic dysregulation may participate in the development of bronchopulmonary dysplasia. Our findings demonstrate that metabolic indicators are promising for forecasting the occurrence of bronchopulmonary dysplasia among preterm neonates.     

Perturbation of gene expression of the chromatin remodeling pathway in premature newborns at risk for bronchopulmonary dysplasia

Background

One-third to one-half of all infants born before the 28th week of gestation develop bronchopulmonary dysplasia (BPD). Inflammatory regulators appear to be involved in the pathogenesis of BPD, possibly beginning in fetal life. To evaluate the feasibility of using expression profiling in umbilical cord tissue to discover molecular signatures for developmental staging and for determining risk of BPD, we conducted a cross-sectional study of infants born at less than 28 weeks of gestation (n = 54). Sections of umbilical cord were obtained at birth from 20 infants who later developed BPD and from 34 of their peers who did not develop BPD.

Results

Umbilical cord expression profiles at birth exhibited systematic differences in bioenergetic pathways with respect to gestational age. Infants who subsequently developed BPD had distinct signatures involving chromatin remodeling and histone acetylation pathways, which have previously been implicated in several adult onset lung diseases. These findings are consistent with prior work on inflammatory processes and bioenergetics in prematurity.

Conclusion

This study of gene expression of the newborn umbilical cord implicates the chromatin remodeling pathways in those premature infants who subsequently develop BPD. Larger sample sizes will be required to generate prognostic markers from umbilical cord profiles.     

Aging and caloric restriction impact adipose tissue,adiponectin, and circulating lipids

Adipose tissue expansion has been associated with system‐wide metabolic dysfunction and increased vulnerability to diabetes, cancer, and cardiovascular disease. A reduction in adiposity is a hallmark of caloric restriction (CR), an intervention that extends longevity and delays the onset of these same age‐related conditions. Despite these parallels, the role of adipose tissue in coordinating the metabolism of aging is poorly defined. Here, we show that adipose tissue metabolism and secretory profiles change with age and are responsive to CR. We conducted a cross‐sectional study of CR in adult, late‐middle‐aged, and advanced‐aged mice. Adiposity and the relationship between adiposity and circulating levels of the adipose‐derived peptide hormone adiponectin were age‐sensitive. CR impacted adiposity but only levels of the high molecular weight isoform of adiponectin responded to CR. Activators of metabolism including PGC‐1a, SIRT1, and NAMPT were differentially expressed with CR in adipose tissues. Although age had a significant impact on NAD metabolism, as detected by biochemical assay and multiphoton imaging, the impact of CR was subtle and related to differences in reliance on oxidative metabolism. The impact of age on circulating lipids was limited to composition of circulating phospholipids. In contrast, the impact of CR was detected in all lipid classes regardless of age, suggesting a profound difference in lipid metabolism. These data demonstrate that aspects of adipose tissue metabolism are life phase specific and that CR is associated with a distinct metabolic state, suggesting that adipose tissue signaling presents a suitable target for interventions to delay aging.     

Polymorphism of class I alcohol dehydrogenase in French, Vietnamese and Niger populations: genotyping by PCR amplification and RFLP analysis on dried blood spots.

The polymorphism of human alcohol dehydrogenase (ADH) can contribute to the explanation of the important ethnic differences towards alcohol metabolism. Its assessment at the genomic DNA level with a procedure, excluding labelled probes, consisting of PCR (Polymerase chain reaction) amplification on dried blood spots and analysis of allele-specific RFLP (Restriction fragment length polymorphism) profiles, is well adapted to extensive studies in population samples. It can emphasize the importance of ADH as a genetic marker of population. Three ethnic groups (French Caucasians, Vietnamese Orientals, Black Africans from Niger) were studied. ADH2 and ADH3 genotypes were in equilibrium according to the Hardy-Weinberg law. Important differences were noted in the distribution of ADH2 and ADH3 alleles.     

Plasma diacylglycerol composition is a biomarker of metabolic syndrome onset in rhesus monkeys

Metabolic syndrome is linked with obesity and is often first identified clinically by elevated BMI and elevated levels of fasting blood glucose that are generally secondary to insulin resistance. Using the highly translatable rhesus monkey (Macaca mulatta) model, we asked if metabolic syndrome risk could be identified earlier. The study involved 16 overweight but healthy, euglycemic monkeys, one-half of which spontaneously developed metabolic syndrome over the course of 2 years while the other half remained healthy. We conducted a series of biometric and plasma measures focusing on adiposity, lipid metabolism, and adipose tissue-derived hormones, which led to a diagnosis of metabolic syndrome in the insulin-resistant animals. Plasma fatty acid composition was determined by gas chromatography for cholesteryl ester, FFA, diacylglycerol (DAG), phospholipid, and triacylglycerol lipid classes; plasma lipoprotein profiles were generated by NMR; and circulating levels of adipose-derived signaling peptides were determined by ELISA. We identified biomarker models including a DAG model, two lipoprotein models, and a multiterm model that includes the adipose-derived peptide adiponectin. Correlations among circulating lipids and lipoproteins revealed shifts in lipid metabolism during disease development. We propose that lipid profiling may be valuable for early metabolic syndrome detection in a clinical setting.     

Long-term programming of skeletal muscle and liver lipid and energy metabolism by resveratrol supplementation to suckling mice

Metabolic programming by dietary chemicals consumed in early life stages is receiving increasing attention. We here studied long-term effects of mild resveratrol (RSV) supplementation during lactation on muscular and hepatic lipid metabolism in adulthood. Newborn male mice received RSV or vehicle from day 2–20 of age, were weaned onto a chow diet on day 21, and were assigned to either a high-fat diet (HFD) or a normal-fat diet on day 90 of age for 10 weeks. RSV-treated mice showed in adulthood protection against HFD-induced triacylglycerol accumulation in skeletal muscle, enhanced muscular capacities for fat oxidation and mitochondria activity, signs of enhanced sirtuin 1 and AMP-dependent protein kinase signaling in muscle, and increased fat oxidation capacities and a decreased capacity for lipogenesis in liver compared with controls. Thus, RSV supplementation in early postnatal life may help preventing later diet-related disorders linked to ectopic lipid accumulation in muscle and liver tissues.     

Effects of age,sex, and genotype on high‐sensitivity metabolomic profiles in the fruit fly,Drosophila melanogaster

Researchers have used whole‐genome sequencing and gene expression profiling to identify genes associated with age, in the hope of understanding the underlying mechanisms of senescence. But there is a substantial gap from variation in gene sequences and expression levels to variation in age or life expectancy. In an attempt to bridge this gap, here we describe the effects of age, sex, genotype, and their interactions on high‐sensitivity metabolomic profiles in the fruit fly, Drosophila melanogaster. Among the 6800 features analyzed, we found that over one‐quarter of all metabolites were significantly associated with age, sex, genotype, or their interactions, and multivariate analysis shows that individual metabolomic profiles are highly predictive of these traits. Using a metabolomic equivalent of gene set enrichment analysis, we identified numerous metabolic pathways that were enriched among metabolites associated with age, sex, and genotype, including pathways involving sugar and glycerophospholipid metabolism, neurotransmitters, amino acids, and the carnitine shuttle. Our results suggest that high‐sensitivity metabolomic studies have excellent potential not only to reveal mechanisms that lead to senescence, but also to help us understand differences in patterns of aging among genotypes and between males and females.     

Adipose tissue proteomes of intrauterine growth-restricted piglets artificially reared on a high-protein neonatal formula

The eventuality that adipose tissues adapt to neonatal nutrition in a way that may program later adiposity or obesity in adulthood is receiving increasing attention in neonatology. This study assessed the immediate effects of a high-protein neonatal formula on proteome profiles of adipose tissues in newborn piglets with intrauterine growth restriction. Piglets (10th percentile) were fed milk replacers formulated to provide an adequate (AP) or a high (HP) protein supply from day 2 to the day prior weaning (day 28, n=5 per group). Adipocytes with small diameters were present in greater proportions in subcutaneous and perirenal adipose tissues from HP piglets compared with AP ones at this age. Two-dimensional gel electrophoresis analysis of adipose tissue depots revealed a total of 32 protein spots being up- or down-regulated (P<.10) for HP piglets compared with AP piglets; 18 of them were unambiguously identified by mass spectrometry. These proteins were notably related to signal transduction (annexin 2), redox status (peroxiredoxin 6, glutathione S-transferase omega 1, cyclophilin-A), carbohydrate metabolism (ribose-5-phosphate dehydrogenase, lactate dehydrogenase), amino acid metabolism (glutamate dehydrogenase 1) and cell cytoskeleton dynamics (dynactin and cofilin-1). Proteomic changes occurred mainly in dorsal subcutaneous adipose tissue, with the notable exception of annexin 1 involved in lipid metabolic process having a lower abundance in HP piglets for perirenal adipose tissue only. Together, modulation in those proteins could represent a novel starting point for elucidating catch-up fat growth observed in later life in growing animals having been fed HP formula.     

母代高脂饮食诱导子代在小鼠生命早期出现糖脂代谢紊乱且具有雄性易感性

目的：探讨孕期和哺乳期的高脂饮食能否导致子代在生命早期出现糖脂代谢紊乱。方法成年雌性C57BL/6J小鼠与正常饮食雄性小鼠进行交配,孕鼠随机分为高脂饮食组和正常饮食组,在孕期和哺乳期喂养高脂饲料或正常饲料,至交配后第一代鼠断乳时（3周龄）观察其糖脂代谢相关性指标以及肝脏病理表现。结果较正常饮食组子鼠相比,高脂饮食子鼠出生体重更低（ P＜0.05）。在断乳时,高脂饮食组雄性子鼠体重较重（ P ＝0.038）,腹腔糖耐量实验30 min和60 min血糖明显升高（P值分别为＜0.001和＜0.01）,糖耐量曲线下面积较大（P＝0.0016）,HOMA-IR值较大（P＜0.05）,雌性子鼠腹腔糖耐量实验在30 min血糖高于正常组（P＜0.01）,而糖耐量曲线下面积和HOMA-IR值在两组之间无明显统计学意义。雄性和雌性子代小鼠空腹胆固醇水平高脂饮食组均高于正常饮食组（ P值分别为＜0.0001和0.0004）,而两组雄性和雌性子代小鼠空腹胰岛素和甘油三酯水平差异均无显著性（ P均＞0.05）。另外,在断乳时高脂饮食子鼠出现肝脏脂肪变性,雌性和雄性子鼠无明显差异。结论母鼠孕期和哺乳期高脂饮食能够诱导子代在生命早期就能出现糖脂代谢紊乱并且雄性子鼠更易出现肥胖、糖耐量异常、胰岛素抵抗。     

Plasma free amino acid profiling of five types of cancer patients and its application for early detection

Background

Recently, rapid advances have been made in metabolomics-based, easy-to-use early cancer detection methods using blood samples. Among metabolites, profiling of plasma free amino acids (PFAAs) is a promising approach because PFAAs link all organ systems and have important roles in metabolism. Furthermore, PFAA profiles are known to be influenced by specific diseases, including cancers. Therefore, the purpose of the present study was to determine the characteristics of the PFAA profiles in cancer patients and the possibility of using this information for early detection.

Methods and Findings

Plasma samples were collected from approximately 200 patients from multiple institutes, each diagnosed with one of the following five types of cancer: lung, gastric, colorectal, breast, or prostate cancer. Patients were compared to gender- and age- matched controls also used in this study. The PFAA levels were measured using high-performance liquid chromatography (HPLC)–electrospray ionization (ESI)–mass spectrometry (MS). Univariate analysis revealed significant differences in the PFAA profiles between the controls and the patients with any of the five types of cancer listed above, even those with asymptomatic early-stage disease. Furthermore, multivariate analysis clearly discriminated the cancer patients from the controls in terms of the area under the receiver-operator characteristics curve (AUC of ROC >0.75 for each cancer), regardless of cancer stage. Because this study was designed as case-control study, further investigations, including model construction and validation using cohorts with larger sample sizes, are necessary to determine the usefulness of PFAA profiling.

Conclusions

These findings suggest that PFAA profiling has great potential for improving cancer screening and diagnosis and understanding disease pathogenesis. PFAA profiles can also be used to determine various disease diagnoses from a single blood sample, which involves a relatively simple plasma assay and imposes a lower physical burden on subjects when compared to existing screening methods.     

Neonatal environment exerts a sustained influence on the development of the intestinal microbiota and metabolic phenotype

The postnatal environment, including factors such as weaning and acquisition of the gut microbiota, has been causally linked to the development of later immunological diseases such as allergy and autoimmunity, and has also been associated with a predisposition to metabolic disorders. We show that the very early-life environment influences the development of both the gut microbiota and host metabolic phenotype in a porcine model of human infants. Farm piglets were nursed by their mothers for 1 day, before removal to highly controlled, individual isolators where they received formula milk until weaning at 21 days. The experiment was repeated, to create two batches, which differed only in minor environmental fluctuations during the first day. At day 1 after birth, metabolic profiling of serum by ¹H nuclear magnetic resonance spectroscopy demonstrated significant, systemic, inter-batch variation which persisted until weaning. However, the urinary metabolic profiles demonstrated that significant inter-batch effects on 3-hydroxyisovalerate, trimethylamine-N-oxide and mannitol persisted beyond weaning to at least 35 days. Batch effects were linked to significant differences in the composition of colonic microbiota at 35 days, determined by 16 S pyrosequencing. Different weaning diets modulated both the microbiota and metabolic phenotype independently of the persistent batch effects. We demonstrate that the environment during the first day of life influences development of the microbiota and metabolic phenotype and thus should be taken into account when interrogating experimental outcomes. In addition, we suggest that intervention at this early time could provide ‘metabolic rescue'' for at-risk infants who have undergone aberrant patterns of initial intestinal colonisation.     

NMR- and GC/MS-based metabolomics of sulfur mustard exposed individuals: a pilot study

Sulfur mustard (SM) is a potent alkylating agent and its effects on cells and tissues are varied and complex. Due to limitations in the diagnostics of sulfur mustard exposed individuals (SMEIs) by noninvasive approaches, there is a great necessity to develop novel techniques and biomarkers for this condition. We present here the first nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC/MS) metabolic profiling of serum from and healthy controls to identify novel biomarkers in blood serum for better diagnostics. Of note, SMEIs were exposed to SM 30 years ago and that differences between two groups could still be found. Pathways in which differences between SMEIs and healthy controls are observed are related to lipid metabolism, ketogenesis, tricarboxylic acid (TCA) cycle and amino acid metabolism.     

Time-dependent plasma protein changes in streptozotocin-induced diabetic rats before and after fungal polysaccharide treatments

Previous studies about protein modulation with chemically induced models of diabetes in animals have yielded conflicting results, in that many investigators have reported different regulation patterns for the same proteins. Therefore, it is reasonable to determine biomarkers for prognosis and diagnosis of diabetes with time profiling for the candidate proteins. In this regard, we examined the influence of hypoglycemic fungal polysaccharides (EPS) on the time-dependent plasma protein alterations in streptozotocin-induced diabetic rats. The 2-DE analysis of rat plasma demonstrated that about 50 proteins from about 900 visualized spots were found to be differentially regulated, of which 20 spots were identified as principal diabetes-associated proteins. The results of time profiling revealed that most of the identified proteins showed significant alterations in a time-dependent manner during 14 days, with notable trends. Nine out of the twenty proteins displayed very similar time profiles between normal healthy and EPS-treated diabetic rats. Interestingly, the altered profiles of several proteins by diabetes induction almost returned to control levels after EPS treatments. In particular, we found a clear distinction in differential expression of oxidative stress proteins (ceruloplasmin and transferrin) and lipid metabolism related proteins (Apo A-I, Apo A-IV, and Apo E) in the STZ-induced diabetic rats. The data presented here have identified and characterized the time-dependent changes in plasma proteins associated with EPS treatment in STZ-induced diabetic rats, thereby leading to the discovery of early-response and late-response biomarkers in diabetic and EPS-treated states.     

Blood-stained letter received by HIV/AIDS reference laboratory in 1993--a historical case report

We describe an interesting historical case of a blood-stained letter received in 1993 by Slovenian HIV/AIDS Reference Laboratory. According to the statement of the sender, the letter was spotted with HIV-infected blood. The polymerase chain reaction (PCR) amplification with two gag and env primer sets excluded the presence of HIV proviral DNA in the spot punches obtained from the dried blood spots. Although the presence of HV DNA was confirmed in 5 positive control blood spots using the same sets of primers, we still doubted in the accuracy of the HIV negative result. Fortunately, we obtained a serum sample of the author of the letter four months later from a psychiatric institution where he was hospitalized for paranoid schizophrenia. Since no anti HIV-1/2 antibodies were detected in his serum sample, our initial HIV negative findings based on PCR testing of blood spots were confirmed.     

Archived Guthrie blood spots as a novel source for quantitative DNA methylation analysis

Sodium bisulfite treatment followed by PCR and DNA sequencing is widely considered the gold standard for the analysis of DNA methylation patterns. However, this technique generally requires substantial quantities of genomic DNA as starting material and is often associated with degradation of DNA. Here, we assess the feasibility of performing bisulfite sequencing on DNA isolated from 3-mm diameter punches of dried blood Guthrie spots. We demonstrate that it is possible to perform bisulfite sequencing from both freshly prepared and archived dried blood spots, using a combination of high purity DNA extraction and efficient bisulfite conversion. With the number of new technologies available for DNA methylation studies, we have extended this analysis and have successfully used a high-throughput mass spectrometry method for DNA methylation analysis on these samples. This provides a new source of material for epigenetic analysis of birth samples and provides an invaluable reference point to track temporal change in epigenetic profiles possibly linked with health and disease.     

Preanalytical standardization of amino acid and acylcarnitine metabolite profiling in human blood using tandem mass spectrometry

Quantitative metabolite profiling in biological samples has the potential to reflect physiological status and to identify disease associated disturbances in metabolic networks. However, this approach is hampered by a wide range of preanalytical variables. Hence, the aim of our study was to develop a standardized preanalytical protocol for metabolite profiling of amino acids and acylcarnitines in human blood. Amino acids and acylcarnitines were simultaneous analyzed after butylation of 3 μL dried blood or 10 μL whole blood, serum and anticoagulated plasma using electrospray tandem-mass spectrometry. The influence of exogenous and endogenous preanalytical variables was investigated in healthy volunteers. Different sampling materials and anticoagulants for blood taking were investigated. Concentrations of long-chain acylcarnitines were 5-fold higher in EDTA-whole blood or dried whole blood compared to serum and anticoagulated plasma. Significant differences in amino acid concentrations were found for capillary versus venous blood taking. Fasting for 8 h before specimen collection minimized the nutritional influence. Physical activity significantly alters amino acid and short chain acylcarnitine concentrations. As a result of our preanalytical investigation we developed a pre-treatment protocol based on EDTA whole blood dried on filter paper to reduce the preanalytical variability and facilitate reproducible quantitative metabolite profiling in clinical trials.     

Effects of high-fat diet and age on the blood lipidome and circulating endocannabinoids of female C57BL/6 mice

Alterations in lipid metabolism play a significant role in the pathogenesis of obesity-associated disorders, and dysregulation of the lipidome across multiple diseases has prompted research to identify novel lipids indicative of disease progression. To address the significant gap in knowledge regarding the effect of age and diet on the blood lipidome, we used shotgun lipidomics with electrospray ionization-mass spectrometry (ESI-MS). We analyzed blood lipid profiles of female C57BL/6 mice following high-fat diet (HFD) and low-fat diet (LFD) consumption for short (6 weeks), long (22 weeks), and prolonged (36 weeks) periods. We examined endocannabinoid levels, plasma esterase activity, liver homeostasis, and indices of glucose tolerance and insulin sensitivity to compare lipid alterations with metabolic dysregulation. Multivariate analysis indicated differences in dietary blood lipid profiles with the most notable differences after 6 weeks along with robust alterations due to age. HFD altered phospholipids, fatty acyls, and glycerolipids. Endocannabinoid levels were affected in an age-dependent manner, while HFD increased plasma esterase activity at all time points, with the most pronounced effect at 6 weeks. HFD-consumption also altered liver mRNA levels of PPARα, PPARγ, and CD36. These findings indicate an interaction between dietary fat consumption and aging with widespread effects on the lipidome, which may provide a basis for identification of female-specific obesity- and age-related lipid biomarkers.     

Early intake of long-chain polyunsaturated fatty acids preserves brain structure and function in diet-induced obesity

Worldwide, the incidence of obesity is increasing at an alarming rate, and the number of children with obesity is especially worrisome. These developments raise concerns about the physical, psychosocial and cognitive consequences of obesity. It was shown that early dietary intake of arachidonic acid (ARA) and docosahexaenoic acid (DHA) can reduce the detrimental effects of later obesogenic feeding on lipid metabolism and adipogenesis in an animal model of mild obesity. In the present study, the effects of early dietary ARA and DHA on cognition and brain structure were examined in mildly obesogenic ApoE*3Leiden mouse model. We used cognitive tests and neuroimaging during early and later life. During their early development after weaning (4–13 weeks of age), mice were fed a chow diet or ARA and DHA diet for 8 weeks and then switched to a high-fat and high-carbohydrate (HFHC) diet for 12 weeks (14–26 weeks of age). An HFHC-diet led to increased energy storage in white adipose tissue, increased cholesterol levels, decreased triglycerides levels, increased cerebral blood flow and decreased functional connectivity between brain regions as well as cerebrovascular and gray matter integrity. ARA and DHA intake reduced the HFHC-diet-induced increase in body weight, attenuated plasma triglycerides levels and improved cerebrovasculature, gray matter integrity and functional connectivity in later life. In conclusion, an HFHC diet causes adverse structural brain and metabolic adaptations, most of which can be averted by dietary ARA and DHA intake early in life supporting metabolic flexibility and cerebral integrity later in life.