Iron Fortification and Inulin Supplementation in Early Infancy: Evaluating the Impact on Iron Metabolism and Trace Mineral Status in a Piglet Model.

Background: Infant formula in the United States contains abundant iron, raising health concerns about excess iron intake in early infancy. Objectives: Using a piglet model, we explored the impact of high iron fortification and prebiotic or synbiotic supplementation on iron homeostasis and trace mineral bioavailability. Methods: Twenty-four piglets were stratified and randomly assigned to treatments on postnatal day 2. Piglets were individually housed and received an iron-adequate milk diet (AI), a high-iron milk diet (HI), HI supplemented with 5% inulin (HI with a prebiotic [HIP]), or HIP with an oral gavage of Ligilactobacillus agilis YZ050, an inulin-fermenting strain, every third day (HI with synbiotic [HIS]). Milk was provided in 14 meals daily, mimicking formula feeding in infants. Fecal consistency score and body weight were recorded daily or every other day. Blood and feces were sampled weekly, and tissues collected on postnatal day 29. Data were analyzed using mixed model analysis of variance with repeated measures whenever necessary. Results: Diet did not affect growth. HI increased hemoglobin, hematocrit, and serum iron compared to AI. Despite marginal adequacy, AI upregulated iron transporter genes and maintained satisfactory iron status in most pigs. HI upregulated hepcidin gene expression in liver, caused pronounced tissue iron deposition, and markedly increased colonic and fecal iron. Inulin supplementation, regardless of L. agilis YZ050, not only attenuated hepatic iron overload but also decreased colonic and fecal iron without altering pH or the expression of iron regulatory genes. HI lowered zinc (Zn) and copper (Cu) in the duodenum and liver compared to AI, whereas HIP and HIS further decreased Zn and Cu in the liver and diminished colonic and fecal trace minerals. Conclusions: Early-infancy excessive iron fortification causes iron overload and compromises Zn and Cu absorption. Inulin decreases trace mineral absorption likely by enhancing gut peristalsis and stool frequency.

A randomized, controlled trial of a Nordic, protein-reduced complementary diet in infants: effects on body composition, growth, biomarkers, and dietary intake at 12 and 18 months.

BACKGROUND: High intake of protein and low intake of plant-based foods during complementary feeding can contribute to negative long-term health effects. OBJECTIVES: To investigate the effects of a protein-reduced, Nordic complementary diet on body composition, growth, biomarkers, and dietary intake, compared with current Swedish dietary recommendations for infants at 12 and 18 mo. METHODS: Healthy, term infants (n = 250) were randomly allocated to either a Nordic group (NG) or a conventional group (CG). From 4 to 6 mo, NG participants received repeated exposures of Nordic taste portions. From 6 to 18 mo, NG was supplied with Nordic homemade baby food recipes, protein-reduced baby food products, and parental support. CG followed the current Swedish dietary recommendations. Measurements of body composition, anthropometry, biomarkers, and dietary intake were collected from baseline and at 12 and 18 mo. RESULTS: Of the 250 infants, 82% (n = 206) completed the study. There were no group differences in body composition or growth. In NG, protein intake, blood urea nitrogen and plasma IGF-1 were lower compared to CG at 12 and 18 mo. Infants in NG consumed 42% to 45% more fruits and vegetables compared to CG at 12 and 18 mo, which was reflected in a higher plasma folate at 12 and 18 mo. There were no between-group differences in EI or iron status. CONCLUSIONS: Introduction of a predominantly plant-based, protein-reduced diet as part of complementary feeding is feasible and can increase fruit and vegetable intake. This trial was registered at clinicaltrials.gov as NCT02634749.

Low-Protein Formulas with Alpha-Lactalbumin-Enriched or Glycomacropeptide-Reduced Whey: Effects on Growth, Nutrient Intake and Protein Metabolism during Early Infancy: A Randomized, Double-Blinded Controlled Trial.

Protein intake is higher in formula-fed than in breast-fed infants during infancy, which may lead to an increased risk of being overweight. Applying alpha-lactalbumin (α-lac)-enriched whey or casein glycomacropeptide (CGMP)-reduced whey to infant formula may enable further reduction of formula protein by improving the amino acid profile. Growth, nutrient intake, and protein metabolites were evaluated in a randomized, prospective, double-blinded intervention trial where term infants received standard formula (SF:2.2 g protein/100 kcal; n = 83) or low-protein formulas with α-lac-enriched whey (α-lac-EW;1.75 g protein/100 kcal; n = 82) or CGMP-reduced whey (CGMP-RW;1.76 g protein/100 kcal; n = 80) from 2 to 6 months. Breast-fed infants (BF; n = 83) served as reference. Except between 4 and 6 months, when weight gain did not differ between α-lac-EW and BF (p = 0.16), weight gain was higher in all formula groups compared to BF. Blood urea nitrogen did not differ between low-protein formula groups and BF during intervention, but was lower than in SF. Essential amino acids were similar or higher in α-lac-EW and CGMP-RW compared to BF. Conclusion: Low-protein formulas enriched with α-lac-enriched or CGMP-reduced whey supports adequate growth, with more similar weight gain in α-lac-enriched formula group and BF, and with metabolic profiles closer to that of BF infants.

Trace Element Interactions, Inflammatory Signaling, and Male Sex Implicated in Reduced Growth Following Excess Oral Iron Supplementation in Pre-Weanling Rats.

Iron supplements are frequently provided to infants in high-income countries despite low incidence of iron deficiency. There is growing concern regarding adverse health and development outcomes of excess iron provision in early life. Excess iron may directly damage developing organs through the formation of reactive oxygen species, alter systemic inflammatory signaling, and/or dysregulate trace mineral metabolism. To better characterize the in vivo effects of excess iron on development, we utilized a pre-weanling rat pup model. Lewis rat litters were culled to eight pups (four males and four females) and randomly assigned to daily supplementation groups receiving either vehicle control (CON; 10% w/v sucrose solution) or ferrous sulfate (FS) iron at one of the following doses: 10, 30, or 90 mg iron/kg body weight-FS-10, FS-30, and FS-90, respectively-from postnatal day (PD) 2 through 9. FS-90 litters, but not FS-30 or FS-10, failed to thrive compared to CON litters and had smaller brains on PD 10. Among the groups, FS-90 liver iron levels were highest, as were white blood cell counts. Compared to CON, circulating MCP-1 and liver zinc were increased in FS-90 pups, whereas liver copper was decreased. Growth defects due to excess FS provision in pre-weanling rats may be related to liver injury, inflammation, and altered trace mineral metabolism.

Benefits and Risks of Early Life Iron Supplementation.

Infants are frequently supplemented with iron to prevent iron deficiency, but iron supplements may have adverse effects on infant health. Although iron supplements can be highly effective at improving iron status and preventing iron deficiency anemia, iron may adversely affect growth and development, and may increase risk for certain infections. Several reviews exist in this area; however, none has fully summarized all reported outcomes of iron supplementation during infancy. In this review, we summarize the risks and benefits of iron supplementation as they have been reported in controlled studies and in relevant animal models. Additionally, we discuss the mechanisms that may underly beneficial and adverse effects.

Correction: McMillen et al. Gut Microbiome Alterations following Postnatal Iron Supplementation Depend on Iron Form and Persist into Adulthood. Nutrients 2022, 14, 412.

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The role of orally ingested milk fat globule membrane on intestinal barrier functions evaluated with a suckling rat pup supplementation model and a human enterocyte model.

Milk fat globule membrane (MFGM), the membrane surrounding secreted fat droplets in milk, contains components involved in a wide range of bioprocesses including cell proliferation and differentiation. The intestine is relatively immature and permeable at birth. Since MFGM is partly resistant to digestion in infancy, we hypothesized that orally ingested MFGM promotes intestinal development by enhancing intestinal barrier functions in early life. An established suckling rat model was used; Sprague-Dawley rats were bred, and litters were culled to 10 pups/dam. Pups were supplemented orally with MFGM (0, 100, or 300 mg/kg/d) from postnatal day 1-20. Intestine samples were collected for histology, real-time quantitative PCR, immunoblotting, and immunohistochemistry analysis. Additionally, differentiated Caco-2 cells were used to assess effects of MFGM on the human intestinal barrier. Control and MFGM-supplemented rat pups showed similar growth. Intestinal differentiation and expression of tight junction proteins in jejunum and colon were significantly increased by orally ingested MFGM, and MFGM supplementation significantly activated PI3K/Akt/mTOR, mitogen-activated protein kinases, and myosin light chain kinase signaling pathways, suggesting that MFGM promotes intestinal development by triggering various signaling pathways. In human enterocytes (polarized Caco-2 cells), MFGM (400 µg/mL for 72 h) decreased permeability, as revealed by increased transepithelial electrical resistance. In Caco-2 cells, MFGM also enhanced expression of tight junction proteins, including claudin-4 and ZO-2. In conclusion, orally ingested MFGM may exert beneficial roles in intestinal development by activating various cell signaling pathways to upregulate tight junction proteins and thereby increasing intestinal barrier functions.

Gut Microbiome Alterations following Postnatal Iron Supplementation Depend on Iron Form and Persist into Adulthood.

The gut microbiota is implicated in the adverse developmental outcomes of postnatal iron supplementation. To generate hypotheses on how changes to the gut microbiota by iron adversely affect development, and to determine whether the form of iron influences microbiota outcomes, we characterized gut microbiome and metabolome changes in Sprague-Dawley rat pups given oral supplements of ferrous sulfate (FS), ferrous bis-glycinate chelate (FC), or vehicle control (CON) on postnatal day (PD) 2−14. Iron supplementation reduced microbiome alpha-diversity (p < 0.0001) and altered short-chain fatty acids (SCFAs) and trimethylamine (TMA) in a form-dependent manner. To investigate the long-term effects of iron provision in early life, an additional cohort was supplemented with FS, FC, or CON until PD 21 and then weaned onto standard chow. At ~8 weeks of age, young adult (YA) rats that received FS exhibited more diverse microbiomes compared to CON (p < 0.05), whereas FC microbiomes were less diverse (p < 0.05). Iron provision resulted in 10,000-fold reduced abundance of Lactobacilli in pre-weanling and YA animals provided iron in early life (p < 0.0001). Our results suggest that in pre-weanling rats, supplemental iron form can generate differential effects on the gut microbiota and microbial metabolism that persist into adulthood.

Human milk cholesterol is associated with lactation stage and maternal plasma cholesterol in Chinese populations.

BACKGROUND: Milk cholesterol concentrations throughout lactation were analyzed, and the relationship between maternal plasma cholesterol and milk cholesterol in various Chinese populations was examined. METHODS: A sub-sample of 1138 lactating women was randomly selected from a large cross-sectional study in China (n = 6481). Milk cholesterol concentrations were determined by HPLC, and concentrations of maternal plasma lipids were determined by an automated biochemical analyzer. RESULTS: The mean cholesterol concentrations were 200, 171, and 126 mg/L for colostrum, transitional milk, and mature milk, respectively. Cholesterol concentrations differed significantly between stages of lactation (colostrum vs. transitional milk, colostrum vs. mature milk, transitional milk vs. mature milk, all p < 0.001). Concentrations of maternal plasma total cholesterol (TC) (p = 0.02) and low-density lipoprotein cholesterol (LDL-C) (p = 0.03) were significantly associated with milk cholesterol. Milk cholesterol concentrations varied among different ethnicities (Tibetan vs. Hui: 164 vs. 131 mg/L, p = 0.027) but not among different geographic regions. CONCLUSIONS: The concentration of cholesterol in human milk changes dynamically throughout lactation. Milk cholesterol concentrations are significantly associated with maternal plasma concentrations of TC and LDL-C, and milk cholesterol concentrations vary across ethnicities in China. IMPACT: Concentrations of milk cholesterol were measured in various Chinese populations. Cholesterol concentrations differ significantly between stages of lactation. Maternal plasma total cholesterol and low-density lipoprotein cholesterol are associated with milk cholesterol. Milk cholesterol concentrations vary across ethnicities in China.

Postnatal Iron Supplementation with Ferrous Sulfate vs. Ferrous Bis-Glycinate Chelate: Effects on Iron Metabolism, Growth, and Central Nervous System Development in Sprague Dawley Rat Pups.

Iron-fortified formulas and iron drops (both usually ferrous sulfate, FS) prevent early life iron deficiency, but may delay growth and adversely affect neurodevelopment by providing excess iron. We used a rat pup model to investigate iron status, growth, and development outcomes following daily iron supplementation (10 mg iron/kg body weight, representative of iron-fortified formula levels) with FS or an alternative, bioavailable form of iron, ferrous bis-glycinate chelate (FC). On postnatal day (PD) 2, sex-matched rat litters (n = 3 litters, 10 pups each) were randomly assigned to receive FS, FC, or vehicle control until PD 14. On PD 15, we evaluated systemic iron regulation and CNS mineral interactions and we interrogated iron loading outcomes in the hippocampus, in search of mechanisms by which iron may influence neurodevelopment. Body iron stores were elevated substantially in iron-supplemented pups. All pups gained weight normally, but brain size on PD 15 was dependent on iron source. This may have been associated with reduced hippocampal oxidative stress but was not associated with CNS mineral interactions, iron regulation, or myelination, as these were unchanged with iron supplementation. Additional studies are warranted to investigate iron form effects on neurodevelopment so that iron recommendations can be optimized for all infants.

Neurodevelopment and growth until 6.5 years of infants who consumed a low-energy, low-protein formula supplemented with bovine milk fat globule membranes: a randomized controlled trial.

BACKGROUND: We previously reported results from a randomized controlled trial in which we found that Swedish infants consuming an experimental low-energy, low-protein formula (EF) supplemented with bovine milk fat globule membranes (MFGMs) until 6 mo of age had several positive outcomes, including better performance in the cognitive domain of Bayley Scales of Infant and Toddler Development 3rd Edition at 12 mo of age, and higher plasma cholesterol concentrations during the intervention, than infants consuming standard formula (SF). OBJECTIVES: We aimed to evaluate neurodevelopment, growth, and plasma cholesterol status at 6 and 6.5 y of age in the same study population. METHODS: We assessed cognitive and executive functions using the Wechsler Intelligence Scale for Children 4th Edition (WISC-IV), Brown Attention-Deficit Disorder Scales for Children and Adolescents (Brown-ADD), and Quantified Behavior (Qb) tests, and behavior using the Child Behavior Checklist (CBCL) and Teacher's Report Form (TRF), at 6.5 y of age. Anthropometrics and plasma lipids were assessed at 6 y of age. RESULTS: There were no differences between the EF and SF groups in any of the subscales in WISC-IV or Brown-ADD at 6.5 y of age, in the proportion of children with scores outside the normal range in the Qb test, nor in clinical or borderline indications of problems in adaptive functioning from parental and teacher's scoring using the CBCL and TRF. There were no differences between the EF and SF groups in weight, length, or head or abdominal circumferences, nor in plasma concentrations of homocysteine, lipids, insulin, or glucose. CONCLUSIONS: Among children who as infants consumed a low-energy, low-protein formula supplemented with bovine MFGMs, there were no effects on neurodevelopment, growth, or plasma cholesterol status 6-6.5 y later.

Milk Fat Globule Membrane as a Modulator of Infant Metabolism and Gut Microbiota: A Formula Supplement Narrowing the Metabolic Differences between Breastfed and Formula-Fed Infants.

SCOPE: Milk fat globule membrane (MFGM) is an important component of milk that has previously been removed in the manufacture of infant formulas, but has recently gained attention owing to its potential to improve immunological, cognitive, and metabolic health. The goal of this study is to determine whether supplementing MFGM in infant formula would drive desirable changes in metabolism and gut microbiota to elicit benefits observed in prior studies. METHODS AND RESULTS: The serum metabolome and fecal microbiota are analyzed using 1 H NMR spectroscopy and 16S rRNA gene sequencing respectively in a cohort of Chinese infants given a standard formula or a formula supplemented with an MFGM-enriched whey protein fraction. Supplementing MFGM suppressed protein degradation pathways and the levels of insulinogenic amino acids that are typically enhanced in formula-fed infants while facilitating fatty acid oxidation and ketogenesis, a feature that may favor brain development. MFGM supplementation did not induce significant compositional changes in the fecal microbiota but suppressed microbial diversity and altered microbiota-associated metabolites. CONCLUSION: Supplementing MFGM in a formula reduced some metabolic gaps between formula-fed and breastfed infants.

Reducing Iron Content in Infant Formula from 8 to 2 mg/L Does Not Increase the Risk of Iron Deficiency at 4 or 6 Months of Age: A Randomized Controlled Trial.

Many infant formulas are fortified with iron at 8-14 mg/L whereas breast milk contains about 0.3 mg/L. Another major difference between breast milk and infant formula is its high concentration of lactoferrin, a bioactive iron-binding protein. The aim of the present study was to investigate how reducing the iron content and adding bovine lactoferrin to infant formula affects iron status, health and development. Swedish healthy full-term formula-fed infants (n = 180) were randomized in a double-blind controlled trial. From 6 weeks to 6 months of age, 72 infants received low-iron formula (2 mg/L) fortified with bovine lactoferrin (1.0 g/L) (Lf+), 72 received low-iron formula un-fortified with lactoferrin (Lf-) and 36 received standard formula with 8 mg of iron/L and no lactoferrin fortification as controls (CF). Iron status and prevalence of iron deficiency (ID) were assessed at 4 and 6 months. All iron status indicators were unaffected by lactoferrin. At 4 and 6 months, the geometric means of ferritin for the combined low-iron groups compared to the CF-group were 67.7 vs. 88.7 and 39.5 vs. 50.9 µg/L, respectively (p = 0.054 and p = 0.056). No significant differences were found for other iron status indicators. In the low-iron group only one infant (0.7%) at 4 months and none at 6 months developed ID. Conclusion: Iron fortification of 2 mg/L is an adequate level during the first half of infancy for healthy term infants in a well-nourished population. Adding lactoferrin does not affect iron status.

A mouse model and 19 F NMR approach to investigate the effects of sialic acid supplementation on cognitive development.

Researchers have observed that a sialic acid (Sia)-supplemented neonatal diet leads to improved cognition in weanling piglets. However, whether cognitive improvement appears with different physiological backgrounds and persists into adulthood is not known. Here, we have established a convenient mouse model and used an 19 F NMR approach to address these questions, test the conditionally essential nutrient hypothesis about Sia supplementation, and assess the prospect of measuring Sia metabolism directly in vivo. Indeed, the neonatal mouse brain uptakes more Sia than the adult brain, and Sia supplementation of neonatal mice improves the cognitive performance of adult mice. The non-invasive 19 F NMR approach and viable mouse model opens unique opportunities for clarifying the interplay of nutritional supplementation, metabolism, and cognitive development.

Fecal microbiome and metabolome of infants fed bovine MFGM supplemented formula or standard formula with breast-fed infants as reference: a randomized controlled trial.

Human milk delivers an array of bioactive components that safeguard infant growth and development and maintain healthy gut microbiota. Milk fat globule membrane (MFGM) is a biologically functional fraction of milk increasingly linked to beneficial outcomes in infants through protection from pathogens, modulation of the immune system and improved neurodevelopment. In the present study, we characterized the fecal microbiome and metabolome of infants fed a bovine MFGM supplemented experimental formula (EF) and compared to infants fed standard formula (SF) and a breast-fed reference group. The impact of MFGM on the fecal microbiome was moderate; however, the fecal metabolome of EF-fed infants showed a significant reduction of several metabolites including lactate, succinate, amino acids and their derivatives from that of infants fed SF. Introduction of weaning food with either human milk or infant formula reduces the distinct characteristics of breast-fed- or formula-fed- like infant fecal microbiome and metabolome profiles. Our findings support the hypothesis that higher levels of protein in infant formula and the lack of human milk oligosaccharides promote a shift toward amino acid fermentation in the gut. MFGM may play a role in shaping gut microbial activity and function.

Protein-Reduced Complementary Foods Based on Nordic Ingredients Combined with Systematic Introduction of Taste Portions Increase Intake of Fruits and Vegetables in 9 Month Old Infants: A Randomised Controlled Trial.

Abstract: Fruits and vegetables are healthy foods but under-consumed among infants and children. Approaches to increase their intake are urgently needed. This study investigated the effects of a systematic introduction of taste portions and a novel protein-reduced complementary diet based on Nordic foods on fruit and vegetable intake, growth and iron status to 9 months of age. Healthy, term infants (n = 250) were recruited and randomly allocated to either a Nordic diet group (NG) or a conventional diet group (CG). Infants were solely breast- or formula-fed at study start. From 4 to 6 months of age, the NG followed a systematic taste portions schedule consisting of home-made purées of Nordic produce for 24 days. Subsequently, the NG was supplied with baby food products and recipes of homemade baby foods based on Nordic ingredients but with reduced protein content compared to the CG. The CG was advised to follow current Swedish recommendations on complementary foods. A total of 232 participants (93%) completed the study. The NG had significantly higher intake of fruits and vegetables than the CG at 9 months of age; 225 ± 109 g/day vs. 156 ± 77 g/day (p < 0.001), respectively. Energy intake was similar, but protein intake was significantly lower in the NG (-26%, p < 0.001) compared to the CG. This lower protein intake was compensated for by higher intake of carbohydrate from fruits and vegetables. No significant group differences in growth or iron status were observed. The intervention resulted in significantly higher consumption of fruits and vegetables in infants introduced to complementary foods based on Nordic ingredients.

Physiological Effects of Feeding Infants and Young Children Formula Supplemented with Milk Fat Globule Membranes.

Dietary supplementation with bovine milk fat globule membrane (MFGM) concentrates has recently emerged as a possible means to improve the health of infants and young children. Formula-fed infants are of special interest since infant formulas traditionally have lower concentrations of biologically active MFGM components than human milk. We identified 6 double-blind randomized controlled trials (DBRCT) exploring the effects of supplementing the diet of infants and children with bovine MFGM concentrates. Two studies found a positive effect on cognitive development in formula-fed infants. Three studies found a protective effect against infections at different ages during infancy and early childhood. We conclude that supplementation with MFGM during infancy and childhood appears safe, and the studies indicate positive effects on both neurodevelopment and defense against infections, especially in formula-fed infants. However, due to the small number of studies and the heterogeneity of interventions and outcomes, more high-quality DBRCTs are needed before firm conclusions can be drawn on the likely health benefits of MFGM supplementation to infants and children.

Iron Oversupplementation Causes Hippocampal Iron Overloading and Impairs Social Novelty Recognition in Nursing Piglets.

BACKGROUND: Iron oversupplementation in healthy term infants may adversely affect growth and cognitive development. OBJECTIVE: We hypothesized that early-life iron excess causes systemic and central nervous system iron overload, and compromises social behavior. METHODS: The nursing pig was used as a translational model in a completely randomized study. On postnatal day (PD) 1, 24 pigs (1.57 ± 0.28 kg mean ± standard deviation body wt) were assigned to the following treatment groups: 1) nonsupplemented iron-deficient group (NON); 2) control group (CON), intramuscularly injected with iron dextran (100 mg Fe) on PD2; 3) moderate iron group (MOD), orally administered ferrous sulfate at 10 mg Fe · kg body wt-1 · d-1; and 4) high iron group (HIG), orally administered ferrous sulfate at 50 mg Fe · kg-1 · d-1. Piglets were nursed by sows during the study from PD1 to PD21. Tissue iron was analyzed by atomic absorption spectrophotometry. Messenger RNA and protein expression of iron regulator and transporters were analyzed by quantitative reverse transcriptase-polymerase chain reaction and Western blot. A sociability test was performed on PD19-20. RESULTS: Both MOD and HIG treatments (5.51 and 9.85 µmol/g tissue), but not CON (0.54 µmol/g), increased hepatic iron as compared with NON (0.25 µmol/g, P < 0.05). Similarly, the hippocampal iron concentrations in the MOD and HIG groups were 14.9% and 31.8% higher than that of NON, respectively (P < 0.05). In comparison with NON, MOD and HIG treatment repressed DMT1 in duodenal mucosa by 4- and 46-fold, respectively (P < 0.05); HIG drastically induced HAMP in liver by 540-fold (P < 0.05); iron-supplemented groups reduced TFRC in the hippocampus by <1-fold (P < 0.05). However, duodenal expression of ferroportin, the predominant transporter in basal membrane, was not affected by treatment. Despite normal sociability, the MOD and HIG pigs displayed deficits in social novelty recognition (P = 0.004). CONCLUSIONS: Duodenal ferroportin was hyporesponsive to iron excess (MOD and HIG), which caused hippocampal iron overload and impaired social novelty recognition in nursing pigs.

The Role of Protein and Free Amino Acids on Intake, Metabolism, and Gut Microbiome: A Comparison Between Breast-Fed and Formula-Fed Rhesus Monkey Infants.

Background: Compared to breast-fed (BF), formula-fed (FF) infants exhibit more rapid weight gain, a different fecal microbial profile, as well as elevated serum insulin, insulin growth factor 1 (IGF-1), and branched chain amino acids (BCAAs). Since infant formula contains more protein and lower free amino acids than breast milk, it is thought that protein and/or free amino acids may be key factors that explain phenotypic differences between BF and FF infants. Methods: Newborn rhesus monkeys (Macaca mulatta) were either exclusively BF or fed regular formula or reduced protein formula either supplemented or not with a mixture of amino acids. Longitudinal sampling and clinical evaluation were performed from birth to 16 weeks including anthropometric measurements, intake records, collection of blood for hematology, serum biochemistry, hormones, and metabolic profiling, collection of urine for metabolic profiling, and collection of feces for 16s rRNA fecal microbial community profiling. Results: Reducing protein in infant formula profoundly suppressed intake, lowered weight gain and improved the FF-specific metabolic phenotype in the first month of age. This time-dependent change paralleled an improvement in serum insulin. All lower protein FF groups showed reduced protein catabolism with lower levels of blood urea nitrogen (BUN), urea, ammonia, albumin, creatinine, as well as lower excretion of creatinine in urine compared to infants fed regular formula. Levels of fecal microbes (Bifidobacterium and Ruminococcus from the Ruminococcaceae family), that are known to have varying ability to utilize complex carbohydrates, also increased with protein reduction. Adding free amino acids to infant formula did not alter milk intake or fecal microbial composition, but did significantly increase urinary excretion of amino acids and nitrogen-containing metabolites. However, despite the lower protein intake, these infants still exhibited a distinct FF-specific metabolic phenotype characterized by accelerated weight gain, higher levels of insulin and C-peptide as well as elevated amino acids including BCAA, lysine, methionine, threonine and asparagine. Conclusions: Reducing protein and adding free amino acids to infant formula resulted in growth and metabolic performance of infants that were more similar to BF infants, but was insufficient to reverse the FF-specific accelerated growth and insulin-inducing high BCAA phenotype.

Concentration of Lactoferrin in Human Milk and Its Variation during Lactation in Different Chinese Populations.

BACKGROUND: Lactoferrin (Lf) is a multifunctional protein and one of the most abundant proteins in human milk. Various factors may affect its concentration in human milk, such as stage of lactation, ethnicity, and diet. OBJECTIVES: The objectives of the present study were to examine the dynamic change in milk Lf throughout the course of lactation and explore factors associated with milk Lf concentrations in various Chinese populations. METHODS: This investigation was a part of a large cross-sectional study conducted in 11 provinces/autonomous regions/municipalities (Beijing, Gansu, Guangdong, Guangxi, Heilongjiang, Inner Mongolia, Shandong, Shanghai, Xinjiang, Yunnan, and Zhejiang) across China between 2011 and 2013. Lactating women (n = 6481) within 0⁻330 days postpartum were recruited in the original study. A sub-sample of 824 women was randomly selected, and milk Lf concentrations were determined by UPLC/MS. RESULTS: The Lf concentration in milk from women delivering at term was 3.16 g/L, 1.73 g/L and 0.90 g/L for colostrum, transitional milk, and mature milk, respectively. Lf concentrations differed significantly between stages of lactation (colostrum vs. transitional milk, colostrum vs. mature milk, transitional milk vs. mature milk, all p < 0.001). Maternal BMI, age, mode of delivery, parturition, protein intake, and serum albumin concentration were not correlated with milk Lf concentration. However, milk Lf concentrations varied among different geographical regions (Guangdong (1.91 g/L) vs. Heilongjiang (1.44 g/L), p = 0.037; Guangdong (1.91 g/L) vs. Gansu (1.43 g/L), p = 0.041) and ethnicities (Dai (1.80 g/L) vs. Tibetan (0.99 g/L), p = 0.007; Han (1.62 g/L) vs. Tibetan (0.99 g/L), p = 0.002) in China. CONCLUSIONS: The concentration of Lf in human milk changes dynamically throughout lactation. Few maternal characteristics affect the milk Lf concentration, but it varies across different geographical regions and ethnicities in China.