The ketogenic diet (KD) is a very low carbohydrate, high fat diet that reduces glucose catabolism and enhances β-oxidation and ketogenesis. While research in female rodents is limited, research in male rodents suggests that ketogenic interventions initiated at midlife may slow age-related cognitive decline, as well as preserve muscle mass and physical function later in life. Aging is accompanied by cellular metabolic changes, and thus investigations into the system-wide effects of a KD will inform potential mechanisms related to the anti-aging effects of this diet. Given the dietary restrictions of a KD, such a diet can be difficult to follow. Thus, investigations into the impact of a ketone ester (EST) supplement to mimic some of the metabolic changes of the KD would provide evidence of its efficacy as an alternative for those unable to follow a KD. This dissertation aimed to investigate the effect of a 2-month KD on metabolism in the major metabolic organs of 14-month-old (middle-aged) female C57BL/6 mice, and additionally to determine whether 6 weeks of ketone ester supplementation provided to aged (24-month-old) male and female mice would impact memory, physical function, body composition and metabolism. Serum, liver, kidney, gastrocnemius muscle, as well as cortex and hippocampal brain regions of female mice at 16 months of age (middle-age) were collected after consuming a KD for 2 months, and targeted 1H-NMR metabolomics was conducted. Analysis of the serum metabolome revealed that the 2-month KD resulted in increased concentrations of fatty acid catabolism metabolites, as well as system-wide elevations in ketones. Metabolites involved in the glucose-alanine cycle, antioxidant defenses and energy reserve were altered in skeletal muscle. Other tissue specific alterations were detected, including distinct effects on hepatic and renal one-carbon metabolism, as well as region specific differences in metabolism across hippocampal and cortical parts of the brain. Effects of a 6-week EST supplemented diet on age-related behavioral outcomes and circulating metabolites in 24-month-old (advanced age) male and female C57BL/6 mice revealed post-prandial elevations in ketones and reductions in glucose. Interestingly, in EST mice, greater elevations in ketone concentrations were observed in females compared to males. Sex-specific effects on body composition, memory, and serum metabolites were also detected. In female EST supplemented mice, muscle mass was conserved, while liver weights and the incidence of age-related spleen hypertrophy were reduced compared to controls. In contrast, only body fat percentage was reduced in EST supplemented males compared to controls. In females, EST supplemented mice had improved spatial memory and alterations to serum metabolites related to gut microbial metabolism, distinct from findings in the serum of middle-aged female mice on a KD. This dissertation contributes novel findings related to the systemic metabolic profile of middle-aged female mice on a KD, which is especially important given the lack of metabolomic studies in females. These results also show that a KD produces metabolic changes to serum that are not fully mimicked by a ketone ester supplement. Our findings demonstrate the distinct sex effects of an EST on memory and body composition in aged mice, with the EST preventing age-related memory loss, decline in muscle mass, and splenic hypertrophy in females.

Breast milk is the gold standard for infant nutrition because in addition to nutrients, it also provides bioactive components that benefit the health of the developing infant. Infants under six months are recommended to be exclusively breastfed (EBF). Infant feeding practices determined by parental recall methods have been reported to have discrepancies when compared with results measured by the deuterium dose-to-mother (DTM) technique. However, the DTM method is limited by its high workload of sample collection and its validity outside the sample collection window. Finding markers of infant feeding practice using a readily accessible biological sample (such as urine or feces) is a promising novel approach to determine infant feeding practices.Breast milk is dynamic and its composition is impacted by many factors among which maternal secretor status and Lewis blood type impacts the levels of several human milk oligosaccharides (HMOs). Previous research has demonstrated the influence of maternal HMO phenotype on the infant gut microbiome. However, these studies used recall methods to determine infant feeding practice and the sample size used were relatively small. Therefore, it is important to illustrate this impact in a larger infant population whose feeding practice were determined objectively. This dissertation investigates the impact of maternal HMO phenotype on the milk metabolome, infant metabolism and gut microbiome, and seeks to develop metabolic biomarkers of feeding practice from samples of infant feces and urine. Chapter 1 reviews the compositional differences between human milk and infant formula, as well as the recent efforts to narrow the gap between the two. Chapter 2 demonstrates the impact of maternal secretor status and Lewis blood type on breast milk metabolome. Fold change analysis showed that the non-secretor (Se-) Lewis negative (Le-) milk had major differences in free fatty acids, free amino acids, and metabolites related to energy metabolism. Chapter 3 shows the impact of maternal secretor status on the infant fecal metabolome and gut microbiome. Maternal secretor status did not alter the within-community (alpha) diversity, between-community (beta) diversity, or the relative abundance of bacterial taxa at the genus level. However, differences in infant gut microbial fermentation products were observed with succinate, amino acids and their derivatives, and 1,2-propanediol. Chapters 4 and 5 develop and validate metabolic biomarkers for the discrimination between EBF and nonexclusively breastfed (non-EBF) infants using fecal and urine samples, respectively. Metabolites with excellent prediction performance (area under the receiver operating curve (AUC) above 0.8) were identified as biomarkers, and these included HMOs, short-chain fatty acids, amino acids (AAs) and organic acids in infant feces, and AA derivatives, nicotinamide-adenine dinucleotide degradation products, and metabolites from dietary sources and/or host-microbial co-metabolism in infant urine. This work provides new insights into the impact of maternal secretor status and Lewis blood type on the breast milk metabolome, as well as a further knowledge on how maternal secretor status influences the EBF infant gut metabolome and microbiome. The results in this study pave the road to a deeper understanding of how the choice of feeding practices impacts infant metabolism and the gut microbiome, and demonstrates the power of utilizing metabolic biomarkers as a novel approach to determine infant feeding practice.

During pregnancy, maternal, placental, and fetal metabolism are highly coordinated to support healthy fetal development, including brain development. Prenatal environmental factors such as endocrine-disrupting chemicals or maternal nutritional status have been linked to increased risk of neurodevelopmental disorders, including autism spectrum disorder (ASD). Maternal, placental, and fetal metabolomics is a valuable tool for understanding both physiological and disrupted metabolism. Further, a growing body of evidence suggests that prenatal metabolic disruption may provide insight into mechanisms underlying ASD risk.

The purpose of this dissertation is to characterize the relationships between maternal, placental, and fetal metabolism; maternal prenatal exposures to endocrine-disrupting chemicals known as phthalates and prenatal vitamin supplements (PNV); and neurodevelopmental outcomes in a prospective birth cohort recruited from families with risk of ASD recurrence. In Chapter 2, investigation of the correlations between the maternal serum, placental, and fetal umbilical cord serum metabolomes revealed that placental and fetal metabolism were highly correlated with each other and with non-typically developing (Non-TD) outcomes. Across all three tissues, 3-hydroxybutyrate was identified as an important metabolite associated with Non-TD risk. In Chapter 3, the relationships between prenatal phthalate exposure, maternal and placental metabolism, and neurodevelopmental outcomes were evaluated. Phthalate exposure was associated with changes in the placental metabolome, including lipid-related metabolites, but these changes were not associated with neurodevelopmental outcomes. In Chapters 4 and 5, the relationships between PNV use in the first month of pregnancy, neurodevelopmental outcomes, and placental and fetal metabolism, respectively, were investigated. PNV use in the first month of pregnancy was associated with placental and fetal amino acid and energy metabolism and these metabolic changes partially explained the relationship between PNV use in the first month of pregnancy and Non-TD risk. Indeed, while the effects of PNV use on the placental metabolome were associated with reduced Non-TD risk, the effects of PNV use on the umbilical cord serum metabolome were associated with increased Non-TD risk.

Across these analyses, three key themes emerged. First, the placental metabolome serves as an important source of information related to neurodevelopment. Second, prenatal disruptions in lipid metabolism appear to be a promising predictor of Non-TD risk. Third, PNV use in the earliest stages of pregnancy exerts programming effects on placental and fetal metabolism that are associated with Non-TD risk and these effects are not entirely protective. These findings support a call-to-action to further investigate and ultimately regulate PNV formulations. This dissertation contributes new information on metabolism in the maternal-placental-fetal unit, the impacts of prenatal environmental exposures, and neurodevelopmental outcomes in a cohort with high-familial ASD risk.

The analysis of usual, or long-term average, intake of nutrients, foods, and other dietary components is of interest both for population health monitoring as well as for studies of diet-health relationships. Usual diet is not directly observable, given multiple sources of measurement error in dietary recall methods and the existence of day-to-day variation in dietary intakes, necessitating the use of statistical models to estimate population distributions of usual intakes or estimate epidemiological associations. A higher amount of bias is associated with food frequency questionnaires (FFQ) than 24-hour dietary recalls (24HDR), while the main source of measurement error in the latter is within-individual variation (WIV) in intakes. Biomarkers of dietary intake may address some of these issues but are subject to their own sources of measurement error, including WIV in some cases, but further research is needed to validate these biomarkers, particularly of food intakes. In order to accurately assess dietary adequacy at the population level and characterize diet-health relationships, the degree and nature of within-individual variability in dietary intakes and biomarkers should be understood and accounted for during the design and analysis stages of nutrition studies. The present studies address several gaps in current research in this area.The first study addresses the estimation of population usual nutrient intake distributions when only a single 24HDR or food record was collected per study participant. Determining the proportion of a population at risk of inadequate or excessive nutrient intake is a crucial step in planning and managing nutrition intervention programs. Multiple days of 24-hour dietary intake data per subject allow for adjustment of modeled usual nutrient intake distributions for the proportion of total variance in intake attributable to within-individual variation (WIV:total). When only single-day dietary data are available, an external adjustment factor can be used; however, WIV:total may vary by population, and use of incorrect WIV:total ratios may influence the accuracy of prevalence estimates and subsequent program impacts. In this study, we compiled WIV:total estimates from publications and from reanalyses of existing datasets to describe variation in WIV:total across populations and settings. The potential impact of variation in external WIV:total on estimates of prevalence of inadequacy was assessed through simulation analyses using the National Cancer Institute 1-day method. WIV:total were extracted from 40 publications from 24 countries, and additional values were calculated from 15 datasets from 12 nations. Wide variation in WIV:total (from 0.02 to 1.00) was observed in publications and reanalyses. Few patterns by population characteristics were apparent, but WIV:total varied by age in children (under vs. over 1 year) and between rural vs. urban settings. Simulation analyses indicated that estimates of the prevalence of inadequate intake are sensitive to the selected ratio in some cases. Thus, selection of an external WIV:total estimate should consider comparability between the reference and primary studies with regard to population characteristics, study design and statistical methods. Given wide variation of observed ratios with few discernible patterns, the collection of 2 or more days of intake data in at least a representative subsample in population dietary studies is strongly encouraged. In the case of single-day dietary studies, sensitivity analyses are recommended to determine the robustness of prevalence estimates to changes in the variance ratio. In the second study, the issue of WIV in a candidate dietary biomarker was examined in the context of an epidemiological pregnancy cohort. Proline betaine (Pro-B) has consistently been identified in biomarker discovery studies as a marker of citrus intake, yet gaps remain in understanding the nature of WIV of this largely short-term biomarker and its quantitative relationship with usual citrus intake in free-living populations, including in pregnant woman. The objective of this study was therefore to identify and quantify sources of within- and between-individual components of variance in urinary Pro-B concentration during pregnancy in a Northern California population and assess the correlation between repeated biomarker measurements and reported usual intake of citrus fruit and juice in this population. Up to 5 repeated spot or 24-h urine specimens throughout pregnancy (n = 255) from a nested case-control sample of women (n = 107) aged 22-47 years participating in the Markers of Autism in Babies: Learning Early Signs (MARBLES) were analyzed for Pro-B using 1H-NMR spectroscopy. Linear or log mixed effects regression models were used to quantify within- and between-individual variance components and test potential temporal predictors of continuous or elevated (i.e. > 100 µmol/L or > 30 µmol/mmol creatinine) Pro-B concentration, adjusting for covariates. The correlation between averaged repeated biomarker measures and usual citrus intake during pregnancy reported by Block food frequency questionnaires was assessed using Pearson or Spearman correlations. This population of pregnant women reported a median citrus intake of 0.32 (IQR, 0.50) servings/day and a median daily probability of consumption of 0.29 (IQR, 0.35). The overall proportion of samples with elevated Pro-B was 0.42 or 0.35 using a non-normalized or creatinine-normalized data threshold, respectively. The proportion of variance in Pro-B concentration attributable to WIV ranged from 0.69 to 0.74 in unadjusted and adjusted models of non-normalized or creatinine-normalized Pro-B. Citrus season was a significant predictor of urinary Pro-B in most analyses (p<0.05), while gestational age was a significant predictor only of non-normalized Pro-B. Moderate correlations were found between reported usual citrus intake and averaged repeated urinary Pro-B measurements, which were stronger compared to correlations using only a single measurement per person. In conclusion, a high degree of WIV in urinary Pro-B during pregnancy, which was partially explained by citrus season, and moderate correlations with usual reported citrus intake was observed. Multiple samples per participant are likely needed to account for random WIV and/or seasonal variation in citrus intake in studies assessing usual citrus intake at the population level or associations between citrus intake during pregnancy and maternal or child health outcomes. In the third study, the issue of WIV in a biomarker of oxidative stress was investigated in the same MARBLES subsample described above. Oxidative stress is associated with a number of medical pathologies, and excessive rises in pregnancy may be involved in adverse birth outcomes and neurodevelopmental challenges in offspring. F2-isoprostanes are a commonly measured biomarker of global oxidative stress produced from the free radical-mediated peroxidation of arachidonic acid on lipid membranes, but WIV in their synthesis and urinary excretion may affect their utility as a marker of usual oxidative stress exposure in epidemiological studies. This study aimed to quantify within- and between-individual components of variance in the 8-isoprostane urinary metabolite, 2,3-dinor-8-isoprostane (2,3-dinor-8-isop), investigate the relative contributions of gestation progression and diet to this variability, and determine the effect of the number of samples per person on the estimated association between prenatal urinary isoprostanes and autism spectrum disorder (ASD). Linear mixed effects models were constructed to assess variance components and potential temporal predictors, including gestational age and the diet-related urinary biomarkers hippurate and proline betaine, of urinary 2,3-dinor-8-isop from repeated spot or 24-hour urine measurements throughout pregnancy (n=254). The estimated association between child ASD and prenatal urinary 2,3-dinor-8-isop excretion was compared between models using 1) all available samples throughout pregnancy, 2) all available samples by trimester, and 3) trimester-specific analyses using 1 sample per participant. WIV constituted between 44% and 48% of total variance across repeated measurements of 2,3-dinor-8-isop, after accounting for variation in urinary creatinine. Corresponding intraclass correlation coefficients (ICCs) reflected moderate reproducibility. Gestational age was a significant predictor of creatinine-normalized 2,3-dinor-8-isop concentration but accounted for a small portion (1%) of total variance. Neither dietary biomarker was associated with 2,3-dinor-8-isop level. Isoprostane levels did not differ between pregnancies resulting in children with autism versus no developmental concern, regardless of the number of specimens per person included. In conclusion, moderate within-person variability was observed in a marker of oxidative stress during pregnancy, which is likely to cause some attenuation of risk estimates in epidemiological analyses. WIV in oxidative stress status and biomarkers during pregnancy should be further studied to properly account for this variation in studies investigating prenatal oxidative stress in relation to child outcomes. Overall, from these studies it can be concluded that the issue of within-person variability in dietary intake measures remains a significant challenge to usual dietary intake assessment and is not necessarily solved by the use of biomarkers, which also may be subject to substantial variability that may attenuate observed epidemiological associations. This issue also extends beyond dietary measures to other biomarkers, as exemplified in a marker of oxidative stress, which may be responsive to fluctuating environmental/biological exposures. Careful consideration of the degree and sources of WIV in dietary recall and biomarker measures, which may differ in pregnancy, is warranted during the design and analysis of studies of diet for population health monitoring and investigations of the impact of diet on health and neurodevelopmental outcomes.

The human gut microbiota is intimately linked to our overall health by performing various functions that augment our metabolic, immunologic and physiologic characteristics. A key commensal taxon that resides within the infant gut is the genus Bifidobacterium. For over a century bifidobacteria have been studied in the context of the infant gut microbiota with continual discoveries describing their mutualistic relationship with the host. Diet is a major determinant of bifidobacteria colonization during infancy by which human milk feeding enriches for Bifidobacterium through the prebiotic effect of human milk oligosaccharides. In contrast, conventional formulas typically lack prebiotic structures consumed specifically by bifidobacteria. As such, the microbiota of human milk-fed compared with formula-fed infants assemble into distinct community states leading to disparate effects on the infant’s metabolic and immunologic parameters. However, there are still knowledge gaps regarding the beneficial health effects conferred by Bifidobacterium species and the molecular basis of their enrichment in the gut. Through leveraging the strengths of different experimental designs centered on bifidobacteria, this body of work seeks to characterize the metabolic interactions of the host-Bifidobacterium relationship and better understand their role in the human gut microbiome. The first chapter provides an overview of the relationship between bifidobacteria and the host including factors affecting early gut colonization by Bifidobacterium and their metabolic interactions. Cell culture of Bifidobacterium pseudocatenulatum (B. pseudocatenulatum), an infant gut isolate, grown in 2’-fucosyllactose (2’-FL) or lactose reveal that the fermentation product profiles are substrate specific. The second chapter focuses on developing a mouse model of HMO driven bifidobacteria colonization in which a synbiotic of B. pseudocatenulatum and 2’-FL enable persistent populations of bifidobacteria. Comparing local and systemic differences in microbial derived metabolites in these mice provide insight into the dynamics of gut microbial perturbations that occur via persistent, rather than transient, enrichment of Bifidobacterium. Lastly, a prospective observational study in preterm infants supplemented with B. longum subsp. infantis (B. infantis) or Lactobacillus reuteri (L. reuteri) was conducted to contrast the changes in metabolite profiles in the gut from these probiotics and assess gut inflammation in relationship to these treatments. The results of these studies have provided important insights pertaining to the modulation of host metabolism in the presence of bifidobacteria. Fermentation products excreted in cell culture are driven by the starting substrate, in this case 2’-FL, which are observed to increase in the intestine and circulation in mice colonized with the probiotic. Furthermore, absorption of 1,2-propanediol produced by Bifidobacterium pseudocatenulatum in the gut is enriched in tissues including liver and brain. This novel finding demonstrates that modulation of the gut microbiome has the potential to influence peripheral tissue metabolism with the possibility of altering brain biochemistry independently of the gut-brain axis. In preterm infants, metabolites in the gut lumen are consistent with a Bifidobacterium-enriched microbiota showing significant increases in several fermentation products produced by bifidobacteria. Additionally, gut inflammation is reduced in preterm infants provided bifidobacteria and is associated with the anti-inflammatory metabolite indole-3-lactate. Together, these studies demonstrate the potential of pre- and probiotics, i.e. 2’-FL and bifidobacteria, to enrich the gut microbiota with commensal microorganisms and impact host metabolism.

Huanglongbing (HLB), also known as citrus greening disease or yellow dragon disease, is the most destructive disease of citrus worldwide. In the United States, Huanglongbing is associated with infection of the phloem-restricted bacterium Candidatus Liberibacter asiaticus (CLas). This pathogenic agent is transmitted by the Asian citrus psyllid (ACP; Diaphorina citri Kuwayama) which feeds directly on the phloem of diseased citrus plants, ingesting CLas in the process, and transmits the pathogen into the phloem of a new tree during subsequent feeding. Symptoms of infection with CLas can take months to years to develop which allows asymptomatic trees to act as sources of inoculum for dissemination of the pathogen. When symptoms do appear, the disease is associated with asymmetric leaf chlorosis (‘yellow mottle’), twig dieback, sparsely foliated trees, and production of fruit that is lopsided, bitter, and green even when ripe; symptoms ultimately culminate with premature tree death.

To date, most efforts to understand the plant’s early response to CLas introduce the pathogen by grafting diseased plant material onto healthy trees instead of using the pathogen’s natural ACP vector. To fully understand HLB, it is necessary to know how citrus responds to the psyllid vector and how that response differs when ACP are harboring CLas and introducing it into the plant. The purpose of this dissertation was to address this under-explored area of literature by characterizing the early leaf response of greenhouse citrus trees to exposure to ACP with or without CLas (Chapter 2) and to assess the translatability of ACP-inoculation greenhouse studies to trends observed in HLB field studies (Chapter 3).

In the first study (Chapter 2), we used transcriptomics, proteomics, and metabolomics to identify how exposure to ACP with or without CLas impacts the metabolism of citrus leaves for one year. Two hundred CLas-positive or CLas-negative ACP were introduced to pairs of Madam Vinous sweet orange (Citrus sinensis (L.) Osbeck) trees. Two weeks later, collection of leaf samples began and took place every two weeks for 52 weeks. The resulting analysis utilized transcriptomic data from four timepoints, proteomic data from six timepoints, and metabolomic data from 14 timepoints to gain insights into the response of trees over time. The results of this study indicated that the initial plant response of ACP-vectored CLas infection is primarily due to exposure to ACP and to a lesser extent, the presence or absence of CLas. Additionally, it demonstrated the complexity of this disease by identifying only 17 transcripts, one protein, and no metabolites that were consistently differentially accumulated between trees exposed to CLas-positive or CLas-negative ACP during the first 16 weeks post-exposure to psyllids. By using a controlled greenhouse environment to investigate the citrus response, we were able to clearly characterize changes in citrus leaf cellular metabolism due to these stimuli.

To understand the translatability of our results to real-world situations, a subsequent investigation was carried out to compare the metabolomic results of our greenhouse study to CLas-infected citrus grove trees from Texas (Chapter 3). Metabolomic data from symptomatic grove trees and non-infected screenhouse trees was previously acquired from southern Texas and reported as part of a multi-laboratory investigation to evaluate different early HLB detection techniques. By comparing the metabolic trends in response to ACP-inoculated CLas infection in greenhouse versus field trees, it was possible to understand how well controlled greenhouse studies reflect disease progression relative to what is actually occurring in the field. Overall, the results of this dissertation identified changes in citrus leaf metabolism due to ACP with or without CLas (Chapter 2) and examined the overlap and discrepancies between ACP-transmitted CLas infection between greenhouse and field studies (Chapter 3). The outcomes of the investigations conducted here can be used to refine molecular targets for early disease detection, development of resistant cultivars, and to inform a broader understanding of plant-microbe and plant-vector interactions.

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