Anemia is a term that describes low hemoglobin concentrations and can result from micronutrient deficiencies, infection, or low birth weight. Early-life anemia, particularly iron-deficiency anemia (IDA) is associated with several negative metabolic, developmental, and cognitive outcomes, some of which persist into adulthood. The aim of this study was to investigate alterations in systemic metabolism and fecal microbial diversity and functionality associated with anemia and IDA in male and female infants from Iquitos, Peru. Cross-sectional stool and serum samples were collected from 95 infants (53 boys and 42 girls) at 12 mo of age. The fecal microbiome was assessed by using 16S ribosomal RNA gene sequencing, and the fecal and serum metabolomes were quantified using 1H-nuclear magnetic resonance. The prevalence of anemia was 64%, with a greater proportion of anemia in male infants attributed to iron deficiency. Metabolically, anemia was associated with decreased concentrations of tricarboxylic acid cycle metabolites in both sexes (males: succinate, P = 0.031; females: fumarate, P = 0.028). In addition, anemic male infants exhibited significantly lower serum concentrations of several amino acids compared with nonanemic male infants. Although no specific structural or functional differences in the microbiota were observed with anemia in general, likely due the heterogeneity of its etiology, IDA affected the microbiome both structurally and functionally. Specifically, the abundance of butyrate-producing bacteria was lower in IDA subjects of both sexes than in nonanemic, non-iron-deficient subjects of the same sex (females: Butyricicoccus, P = 0.041; males: Coprococcus, P = 0.010; Roseburia, P = 0.027). IDA male infants had higher concentrations of 4-hydroxyphenyllactate (P < 0.001) and putrescine (P = 0.042) than those without IDA, whereas IDA female infants exhibited higher concentrations of leucine (P = 0.011) and valine (P = 0.003). Sexually dimorphic differences associated with anemia and IDA are suggestive of greater mitochondrial dysfunction and oxidative stress in male infants compared with female infants, and alterations in microbial structure and function may further contribute. Differences in metabolic pathways associated with anemia and IDA in each sex point to potential mechanisms for the associated lasting cognitive deficits. This trial is registered at clinicaltrials.gov as NCT03377777.

A more comprehensive picture of tissue biology can be obtained through the application and integration of multiple omic technologies. However, the common challenge in working with a precious sample is having a sample too small to separately extract analytes of interest for each experiment. Considering the high heterogeneity that can be present in a single tissue sample, extracting all biomolecules from a single and undivided tissue is preferable because it allows direct comparison of results. Here, we combined a modified Folch extraction method with DNA, RNA, small RNA, and protein extraction using two commercial kits, which allowed us to extract polar metabolites and non-polar oxylipin metabolites, DNA, RNA, small RNA, and protein simultaneously from a small tissue sample. The method was validated in terms of quantity and quality of analytes for downstream analyses.

Infancy and childhood represent a high-risk period for developing iron deficiency (ID) and is a period of increased susceptibility to infectious disease. Antibiotic use is high in children from low-, middle-, and high-income countries, and thus we sought to determine the impact of antibiotics in the context of ID. In this study, a piglet model was used to assess the impact of ID and antibiotics on systemic metabolism. ID was induced by withholding a ferrous sulfate injection after birth to piglets in the ID group and providing an iron deficient diet upon weaning on postnatal day (PD) 25. Antibiotics (gentamicin and spectinomycin) were administered on PD34-36 to a set of control (Con*+Abx) and ID piglets (ID+Abx) after weaning. Blood was analyzed on PD30 (before antibiotic administration) and PD43 (7 days after antibiotic administration). All ID piglets exhibited growth faltering and had lower hemoglobin and hematocrit compared to control (Con) and Con*+Abx throughout. The metabolome of ID piglets at weaning and sacrifice exhibited elevated markers of oxidative stress, ketosis, and ureagenesis compared to Con. The impact of antibiotics on Con*+Abx piglets did not result in significant changes to the serum metabolome 7-days after treatment; however, the impact of antibiotics on ID+Abx piglets resulted in the same metabolic changes observed in ID piglets, but with a greater magnitude when compared to Con. These results suggest that antibiotic administration in the context of ID exacerbates the negative metabolic impacts of ID and may have long lasting impacts on development.

Background/Objectives: Iron deficiency (ID) is a common nutritional deficiency in infancy and early childhood associated with increased risk of infection and increased likelihood of receiving antibiotic intervention. In the context of ID, antibiotics have been shown to exaggerate the growth impairments and negative impacts on metabolic health of ID itself. The objective of this research was to assess the tissue-level impact of antibiotics when provided during ID. Methods: ID was induced in piglets by withholding an iron dextran shot shortly after birth, and iron deficiency was maintained after weaning by providing an iron-deficient diet starting on postnatal day (PD) 25. Half of the ID piglets received a 3-day antibiotic course (ID + Abx) consisting of spectinomycin and gentamicin from PD34-36. The kidney, liver, skeletal muscle, and hippocampal metabolomes, as well as activity of proteins in the mTOR signaling pathway, were assessed on PD43. Results: While ID had minimal impacts on the liver, kidney, and skeletal muscle metabolomes, ID + Abx impaired energy metabolism and increased ketosis and oxidative stress in peripheral tissues. Hippocampal metabolites involved in neurotransmitter synthesis pathways were affected by ID and ID + Abx to a greater extent. Additionally, the activities of several proteins in the mTOR pathway were upregulated in the hippocampi of ID + Abx piglets compared to both ID and control piglets. Abx provided to iron-sufficient piglets had minimal effects on tissue metabolomes and did not alter the activity of proteins in the mTOR pathway. Conclusions: These results highlight that antibiotic treatment in ID alters metabolism in peripheral tissues and the developing hippocampus beyond those induced by ID alone. Considering that infants and children are develop rapidly, the combination of ID and antibiotics may have lasting impacts on neurodevelopment and cognition.

Hormones are important signaling molecules controlling physiological homeostasis. ELISA kits are commonly used to measure hormones; however, few ELISA kits are multiplex, not all species-specific ELISA kits are commercially available, and ELISA kits typically require a significant volume of biological fluids. Pigs resemble humans in digestive physiology, making them an excellent model in preclinical research of nutrition and metabolism. In this study, we developed and validated a simple liquid–liquid extraction procedure and LC-MS/MS method for the simultaneous quantification of insulin, cortisol, glucagon-like peptide-1 (GLP-1) (7-37) and (7-36), acyl and des-acyl ghrelin, and carboxylated osteocalcin in pig serum. The proposed method is specific, highly sensitive (LOQ in ng/mL and pg/mL), reasonably accurate (more than 76.2% of all quality control samples within 20% error from nominal values), and precise (intra-day CV ≤ 10% and inter-day CV ≤ 23.1%). The recoveries of all analytes and corresponding internal standards ranged from 83.7 to 116.0%. The method also requires a low serum volume of 50–100 μL, which is invaluable when sample volume is limited. These methods could be easily extended for use in other mammalian species.