Metabolomics Reveals New Mechanisms for Pathogenesis in Barth Syndrome and Introduces Novel Roles for Cardiolipin in Cellular Function

Barth Syndrome is the only known Mendelian disorder of cardiolipin remodeling, with characteristic clinical features of cardiomyopathy, skeletal myopathy, and neutropenia. While the primary biochemical defects of reduced mature cardiolipin and increased monolysocardiolipin are well-described, much of the downstream biochemical dysregulation has not been uncovered, and biomarkers are limited. In order to further expand upon the knowledge of the biochemical abnormalities in Barth Syndrome, we analyzed metabolite profiles in plasma from a cohort of individuals with Barth Syndrome compared to age-matched controls via 1H nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry. A clear distinction between metabolite profiles of individuals with Barth Syndrome and controls was observed, and was defined by an array of metabolite classes including amino acids and lipids. Pathway analysis of these discriminating metabolites revealed involvement of mitochondrial and extra-mitochondrial biochemical pathways including: insulin regulation of fatty acid metabolism, lipid metabolism, biogenic amine metabolism, amino acid metabolism, endothelial nitric oxide synthase signaling, and tRNA biosynthesis. Taken together, this data indicates broad metabolic dysregulation in Barth Syndrome with wide cellular effects

Maternal Early Pregnancy Serum Metabolomics Profile and Abnormal Vaginal Bleeding as Predictors of Placental Abruption: A Prospective Study

Background & Objective Placental abruption, an ischemic placental disorder, complicates about 1 in 100 pregnancies, and is an important cause of maternal and perinatal morbidity and mortality worldwide. Metabolomics holds promise for improving the phenotyping, prediction and understanding of pathophysiologic mechanisms of complex clinical disorders including abruption. We sought to evaluate maternal early pregnancy pre-diagnostic serum metabolic profiles and abnormal vaginal bleeding as predictors of abruption later in pregnancy. Methods Maternal serum was collected in early pregnancy (mean 16 weeks, range 15 to 22 weeks) from 51 abruption cases and 51 controls. Quantitative targeted metabolic profiles of serum were acquired using electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS/MS) and the Absolute IDQ® p180 kit. Maternal sociodemographic characteristics and reproductive history were abstracted from medical records. Stepwise logistic regression models were developed to evaluate the extent to which metabolites aid in the prediction of abruption. We evaluated the predictive performance of the set of selected metabolites using a receiver operating characteristics (ROC) curve analysis and area under the curve (AUC). Results Early pregnancy vaginal bleeding, dodecanoylcarnitine/dodecenoylcarnitine (C12 / C12:1), and phosphatidylcholine acyl-alkyl C 38:1 (PC ae C38:1) strongly predict abruption risk. The AUC for these metabolites alone was 0.68, for early pregnancy vaginal bleeding alone was 0.65, and combined the AUC improved to 0.75 with the addition of quantitative metabolite data (P = 0.003). Conclusion Metabolomic profiles of early pregnancy maternal serum samples in addition to the clinical symptom, vaginal bleeding, may serve as important markers for the prediction of abruption. Larger studies are necessary to corroborate and validate these findings in other cohorts

Untargeted metabolomics on first trimester serum implicates metabolic perturbations associated with BMI in development of hypertensive disorders: a discovery study

GoalBody mass index (BMI) in early pregnancy is a critical risk factor for hypertensive disorders of pregnancy (HDP). The pathobiology of the interplay between BMI and HDP is not fully understood and represents the focus of this investigation.MethodsBMI and 1st-trimester serum samples were obtained from the Global Alliance to Prevent Prematurity and Stillbirth repository for 154 women (105 without HDP and 49 with HDP). Metabotyping was conducted using ultra-high-performance liquid-chromatography high-resolution mass spectrometry (UHPLC HR-MS). Multivariable linear regression and logistic models were used to determine metabolites and pathway perturbations associated with BMI in women with and without HDP, and to determine metabolites and pathway perturbations associated with HDP for women in categories of obese, overweight, and normal weight based on the 1st trimester BMI. These outcome-associated signals were identified or annotated by matching against an in-house physical standards library and public database. Pathway analysis was conducted by the Mummichog algorithm in MetaboAnalyst.ResultVitamin D3 and lysine metabolism were enriched to associate with BMI for women with and without HDP. Tryptophan metabolism enrichment was associated with HDP in all the BMI categories. Pregnant women who developed HDP showed more metabolic perturbations with BMI (continuous) than those without HDP in their 1st-trimester serum. The HDP-associated pathways for women with normal weight indicated inflammation and immune responses. In contrast, the HDP-associated pathways for women of overweight and obese BMI indicated metabolic syndromes with disorders in glucose, protein, and amino acid, lipid and bile acid metabolism, and oxidative and inflammatory stress.ConclusionHigh first-trimester BMI indicates underlying metabolic syndromes, which play critical roles in HDP development. Vitamin D3 and tryptophan metabolism may be the targets to guide nutritional interventions to mitigate metabolic and inflammatory stress in pregnancy and reduce the onset of HDP

Validation of a Metallomics Analysis of Placenta Tissue by Inductively-Coupled Plasma Mass Spectrometry

Trace elements can play an important role in maternal health and fetal development, and deficiencies in some essential minerals including zinc and copper have been correlated in some individuals to the development of birth defects and adverse health outcomes later in life. The exact etiology of conditions like preeclampsia and the effects of fetal exposure to toxic metals has not been determined, making the assessment of trace element levels crucial to the elucidation of the causes of conditions like preeclampsia. Previous studies analyzing serum and placenta tissue have produced conflicting findings, suggesting the need for a robust, validated sample preparation and analysis method for the determination of trace elements in placenta. In this report, an acid digestion method and analysis by ICP-MS for a broad metallomics/mineralomics panel of trace elements is developed and validated over three experimental days for inter- and intraday precision and accuracy, linear range, matrix impact, and dilution verification. Spike recovery experiments were performed for the essential elements chromium (Cr), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), and zinc (Zn), and the toxic elements arsenic (As), cadmium (Cd), and lead (Pb) at levels equal to and in excess of native concentrations in control placenta tissue. The validated method will be essential for the development of scientific studies of maternal health and toxic metal exposure effects in childhood

Associations between the gut microbiome and metabolome in early life

Background: The infant intestinal microbiome plays an important role in metabolism and immune development with impacts on lifelong health. The linkage between the taxonomic composition of the microbiome and its metabolic phenotype is undefined and complicated by redundancies in the taxon-function relationship within microbial communities. To inform a more mechanistic understanding of the relationship between the microbiome and health, we performed an integrative statistical and machine learning-based analysis of microbe taxonomic structure and metabolic function in order to characterize the taxa-function relationship in early life. Results: Stool samples collected from infants enrolled in the New Hampshire Birth Cohort Study (NHBCS) at approximately 6-weeks (n = 158) and 12-months (n = 282) of age were profiled using targeted and untargeted nuclear magnetic resonance (NMR) spectroscopy as well as DNA sequencing of the V4-V5 hypervariable region from the bacterial 16S rRNA gene. There was significant inter-omic concordance based on Procrustes analysis (6 weeks: p = 0.056; 12 months: p = 0.001), however this association was no longer significant when accounting for phylogenetic relationships using generalized UniFrac distance metric (6 weeks: p = 0.376; 12 months: p = 0.069). Sparse canonical correlation analysis showed significant correlation, as well as identifying sets of microbe/metabolites driving microbiome-metabolome relatedness. Performance of machine learning models varied across different metabolites, with support vector machines (radial basis function kernel) being the consistently top ranked model. However, predictive R2 values demonstrated poor predictive performance across all models assessed (avg: − 5.06% -- 6 weeks; − 3.7% -- 12 months). Conversely, the Spearman correlation metric was higher (avg: 0.344–6 weeks; 0.265–12 months). This demonstrated that taxonomic relative abundance was not predictive of metabolite concentrations. Conclusions: Our results suggest a degree of overall association between taxonomic profiles and metabolite concentrations. However, lack of predictive capacity for stool metabolic signatures reflects, in part, the possible role of functional redundancy in defining the taxa-function relationship in early life as well as the bidirectional nature of the microbiome-metabolome association. Our results provide evidence in favor of a multi-omic approach for microbiome studies, especially those focused on health outcomes

Neonatal Metabolomic Profiles Related to Prenatal Arsenic Exposure

Prenatal inorganic arsenic (iAs) exposure is associated with health effects evident at birth and later in life. An understanding of the relationship between prenatal iAs exposure and alterations in the neonatal metabolome could reveal critical molecular modifications, potentially underpinning disease etiologies. In this study, nuclear magnetic resonance (NMR) spectroscopy-based metabolomic analysis was used to identify metabolites in neonate cord serum associated with prenatal iAs exposure in participants from the Biomarkers of Exposure to ARsenic (BEAR) pregnancy cohort, in GoÌmez Palacio, Mexico. Through multivariable linear regression, ten cord serum metabolites were identified as significantly associated with total urinary iAs and/or iAs metabolites, measured as %iAs, %monomethylated arsenicals (MMAs), and %dimethylated arsenicals (DMAs). A total of 17 metabolites were identified as significantly associated with total iAs and/or iAs metabolites in cord serum. These metabolites are indicative of changes in important biochemical pathways such as vitamin metabolism, the citric acid (TCA) cycle, and amino acid metabolism. These data highlight that maternal biotransformation of iAs and neonatal levels of iAs and its metabolites are associated with differences in neonate cord metabolomic profiles. The results demonstrate the potential utility of metabolites as biomarkers/indicators of in utero environmental exposure

Multi-Omics Analysis of Multiple Glucose-Sensing Receptor Systems in Yeast

The yeast Saccharomyces cerevisiae has long been used to produce alcohol from glucose and other sugars. While much is known about glucose metabolism, relatively little is known about the receptors and signaling pathways that indicate glucose availability. Here, we compare the two glucose receptor systems in S. cerevisiae. The first is a heterodimer of transporter-like proteins (transceptors), while the second is a seven-transmembrane receptor coupled to a large G protein (Gpa2) that acts in coordination with two small G proteins (Ras1 and Ras2). Through comprehensive measurements of glucose-dependent transcription and metabolism, we demonstrate that the two receptor systems have distinct roles in glucose signaling: the G-protein-coupled receptor directs carbohydrate and energy metabolism, while the transceptors regulate ancillary processes such as ribosome, amino acids, cofactor and vitamin metabolism. The large G-protein transmits the signal from its cognate receptor, while the small G-protein Ras2 (but not Ras1) integrates responses from both receptor pathways. Collectively, our analysis reveals the molecular basis for glucose detection and the earliest events of glucose-dependent signal transduction in yeast

Macronutrient-differential dietary pattern impacts on body weight, hepatic inflammation, and metabolism

IntroductionObesity is a multi-factorial disease frequently associated with poor nutritional habits and linked to many detrimental health outcomes. Individuals with obesity are more likely to have increased levels of persistent inflammatory and metabolic dysregulation. The goal of this study was to compare four dietary patterns differentiated by macronutrient content in a postmenopausal model. Dietary patterns were high carbohydrate (HC), high fat (HF), high carbohydrate plus high fat (HCHF), and high protein (HP) with higher fiber.MethodsChanges in body weight and glucose levels were measured in female, ovariectomized C57BL/6 mice after 15 weeks of feeding. One group of five mice fed the HCHF diet was crossed over to the HP diet on day 84, modeling a 21-day intervention. In a follow-up study comparing the HCHF versus HP dietary patterns, systemic changes in inflammation, using an 80-cytokine array and metabolism, by untargeted liquid chromatography-mass spectrometry (LCMS)-based metabolomics were evaluated.ResultsOnly the HF and HCHF diets resulted in obesity, shown by significant differences in body weights compared to the HP diet. Body weight gains during the two-diet follow-up study were consistent with the four-diet study. On Day 105 of the 4-diet study, glucose levels were significantly lower for mice fed the HP diet than for those fed the HC and HF diets. Mice switched from the HCHF to the HP diet lost an average of 3.7 grams by the end of the 21-day intervention, but this corresponded with decreased food consumption. The HCHF pattern resulted in dramatic inflammatory dysregulation, as all 80 cytokines were elevated significantly in the livers of these mice after 15 weeks of HCHF diet exposure. Comparatively, only 32 markers changed significantly on the HP diet (24 up, 8 down). Metabolic perturbations in several endogenous biological pathways were also observed based on macronutrient differences and revealed dysfunction in several nutritionally relevant biosynthetic pathways.ConclusionOverall, the HCHF diet promoted detrimental impacts and changes linked to several diseases, including arthritis or breast neoplasms. Identification of dietary pattern-specific impacts in this model provides a means to monitor the effects of disease risk and test interventions to prevent poor health outcomes through nutritional modification

Baseline Serum Biomarkers Predict Response to a Weight Loss Intervention in Older Adults with Obesity: A Pilot Study

Caloric restriction and aerobic and resistance exercise are safe and effective lifestyle interventions for achieving weight loss in the obese older population (>65 years) and may improve physical function and quality of life. However, individual responses are heterogeneous. Our goal was to explore the use of untargeted metabolomics to identify metabolic phenotypes associated with achieving weight loss after a multi-component weight loss intervention. Forty-two older adults with obesity (body mass index, BMI, ≥30 kg/m2) participated in a six-month telehealth-based weight loss intervention. Each received weekly dietitian visits and twice-weekly physical therapist-led group strength training classes with a prescription for aerobic exercise. We categorized responders’ weight loss using a 5% loss of initial body weight as a cutoff. Baseline serum samples were analyzed to determine the variable importance to the projection (VIP) of signals that differentiated the responder status of metabolic profiles. Pathway enrichment analysis was conducted in Metaboanalyst. Baseline data did not differ significantly. Weight loss was 7.2 ± 2.5 kg for the 22 responders, and 2.0 ± 2.0 kg for the 20 non-responders. Mummichog pathway enrichment analysis revealed that perturbations were most significant for caffeine and caffeine-related metabolism (p = 0.00028). Caffeine and related metabolites, which were all increased in responders, included 1,3,7-trimethylxanthine (VIP = 2.0, p = 0.033, fold change (FC) = 1.9), theophylline (VIP = 2.0, p = 0.024, FC = 1.8), paraxanthine (VIP = 2.0, p = 0.028, FC = 1.8), 1-methylxanthine (VIP = 1.9, p = 0.023, FC = 2.2), 5-acetylamino-6-amino-3-methyluracil (VIP = 2.2, p = 0.025, FC = 2.2), 1,3-dimethyl uric acid (VIP = 2.1, p = 0.023, FC = 2.3), and 1,7-dimethyl uric acid (VIP = 2.0, p = 0.035, FC = 2.2). Increased levels of phytochemicals and microbiome-related metabolites were also found in responders compared to non-responders. In this pilot weight loss intervention, older adults with obesity and evidence of significant enrichment for caffeine metabolism were more likely to achieve ≥5% weight loss. Further studies are needed to examine these associations in prospective cohorts and larger randomized trials

Identification of a New Class of Molecules, the Arachidonyl Amino Acids, and Characterization of One Member That Inhibits Pain

In mammals, specific lipids and amino acids serve as crucial signaling molecules. In bacteria, conjugates of lipids and amino acids (referred to as lipoamino acids) have been identified and found to possess biological activity. Here, we report that mammals also produce lipoamino acids, specifically the arachidonyl amino acids. We show that the conjugate of arachidonic acid and glycine (N-arachidonylglycine (NAGly)) is present in bovine and rat brain as well as other tissues and that it suppresses tonic inflammatory pain. The biosynthesis of NAGly and its degradation by the enzyme fatty acid amide hydrolase can be observed in rat brain tissue. In addition to NAGly, bovine brain produces at least two other arachidonyl amino acids: N-arachidonyl gamma-aminobutyric acid (NAGABA) and N-arachidonylalanine. Like NAGly, NAGABA inhibits pain. These findings open the door to the identification of other members of this new class of biomolecules, which may be integral to pain regulation and a variety of functions in mammals