In neonatal hypoxic-ischemic brain injury (HIBI), a common form of perinatal brain damage associated with mortality and neurological disability, the disruption of oxygen and nutrient supply severely impacts brain metabolism. Though therapeutic hypothermia reduces cerebral metabolic rate and improves outcomes, disruption of oxidative metabolism compromising neuronal survival often persists. The complex cerebral metabolic shifts in HIBI remain poorly understood. We directly analyzed the metabolome (LC-MS) of neonatal hypoxia-ischemia (HI)-affected brain tissue to gain further insight into HIBI pathophysiology, isolate the metabolic effects of ischemia and hypoxia, and identify potential therapeutic targets. Postnatal day 10 mice were subjected to five experimental conditions: HI (n = 9) by unilateral carotid artery ligation (UCAL) and hypoxia exposure; contralateral hemispheres; ischemia (UCAL, n = 8); hypoxia (n = 12); and naive (n = 9). Cerebral hemispheres were analyzed 24h post-HI to capture their acute metabolic state. HI resulted in marked alterations in energy production, amino acid and nucleotide metabolism, and pathways governing neuronal homeostasis. Metabolites and pathways linked to NAD⁺ signaling, glutamate regulation, PI3K/AKT signaling, arginine metabolism, neuroinflammation, and vascular regulation were significantly dysregulated. Importantly, these metabolic changes were largely reproduced by ischemia alone, revealing an ischemia-dominant metabolic phenotype. Overall, brain metabolomic profiling identified ischemia as a primary driver of metabolic dysfunction in neonatal HIBI and highlighted specific metabolic pathways involved in bioenergetic deficit, imbalance of neurodegenerative-neuroprotective mechanisms, inflammation, and vascular function, as candidate targets for future therapeutic strategies aimed at limiting secondary brain injury and mitigating neurodevelopmental sequelae.

Understanding metabolic evolution underlying pulmonary arterial hypertension (PAH) development may clarify pathobiology and reveal disease-specific biomarkers. Patients with systemic sclerosis (SSc) are regularly surveilled for PAH, presenting an opportunity to examine metabolic change as disease develops in an at-risk cohort. We performed mass spectrometry-based metabolomics on longitudinal serum samples collected before and near SSc-PAH diagnosis, compared with time-matched SSc subjects without PAH, in a SSc surveillance cohort. We validated metabolic differences in a second cohort and determined metabolite-phenotype relationships. In parallel, we performed serial metabolomic and hemodynamic assessments as the disease developed in a preclinical model. For differentially expressed metabolites, we investigated corresponding gene expression in human and rodent PAH lungs. Kynurenine and its ratio to tryptophan (kyn/trp) increased over the surveillance period in patients with SSc who developed PAH. Higher kyn/trp measured two years before diagnostic right heart catheterization increased the odds of SSc-PAH diagnosis (OR 1.57, 95% CI 1.05-2.36, P = 0.028). The slope of kyn/trp rise during SSc surveillance predicted PAH development and mortality. In both clinical and experimental PAH, higher kynurenine pathway metabolites correlated with adverse pulmonary vascular and RV measurements. In human and rodent PAH lungs, expression of TDO2, which encodes tryptophan 2,3 dioxygenase (TDO), a protein that catalyzes tryptophan conversion to kynurenine, was significantly upregulated and tightly correlated with pulmonary hypertensive features. Upregulated kynurenine pathway metabolism occurs early in PAH, localizes to the lung, and may be modulated by TDO2. Kynurenine pathway metabolites may be candidate PAH biomarkers and TDO warrants exploration as a potential novel therapeutic target.

AbstractAlthough PAH is partially attributed to disordered metabolism, previous human studies have mostly examined circulating metabolites at a single time point, potentially overlooking crucial disease biology. Current knowledge gaps include an understanding of temporal changes that occur within and across relevant tissues, and whether observed metabolic changes might contribute to disease pathobiology. We utilized targeted tissue metabolomics in the Sugen hypoxia (SuHx) rodent model to investigate tissue‐specific metabolic relationships with pulmonary hypertensive features over time using regression modeling and time‐series analysis. Our hypotheses were that some metabolic changes would precede phenotypic changes, and that examining metabolic interactions across heart, lung, and liver tissues would yield insight into interconnected metabolic mechanisms. To support the relevance of our findings, we sought to establish links between SuHx tissue metabolomics and human PAH ‐omics data using bioinformatic predictions. Metabolic differences between and within tissue types were evident by Day 7 postinduction, demonstrating distinct tissue‐specific metabolism in experimental pulmonary hypertension. Various metabolites demonstrated significant tissue‐specific associations with hemodynamics and RV remodeling. Individual metabolite profiles were dynamic, and some metabolic shifts temporally preceded the emergence of overt pulmonary hypertension and RV remodeling. Metabolic interactions were observed such that abundance of several liver metabolites modulated lung and RV metabolite‐phenotype relationships. Taken all together, regression analyses, pathway analyses and time‐series analyses implicated aspartate and glutamate signaling and transport, glycine homeostasis, lung nucleotide abundance, and oxidative stress as relevant to early PAH pathobiology. These findings offer valuable insights into potential targets for early intervention in PAH.

Breast cancer brain metastasis (BCBM) has an incidence of 10–30

Right ventricular (RV) adaptation is the principal determinant of outcomes in pulmonary arterial hypertension (PAH), however, RV function is challenging to assess. RV responses to hemodynamic stressors are particularly difficult to interrogate without invasive testing. This study sought to identify metabolomic markers of in vivo right ventricular function and exercise performance in PAH. Consecutive subjects with PAH (n = 23) underwent rest and exercise right heart catheterization with multibeat pressure volume loop analysis. Pulmonary arterial blood was collected at rest and during exercise. Mass spectrometry-based targeted metabolomics were performed, and metabolic associations with hemodynamics and comprehensive measures of RV function were determined using sparse partial least squares regression. Metabolite profiles were compared with N-terminal prohormone of B-type natriuretic peptide (NT-proBNP) measurements for accuracy in modeling ventriculo-arterial parameters. Thirteen metabolites changed in abundance with exercise, including metabolites reflecting increased arginine bioavailability, precursors of catecholamine and nucleotide synthesis, and branched-chain amino acids. Higher resting arginine bioavailability predicted more favorable exercise hemodynamics and pressure-flow relationships. Subjects with more severe PAH augmented arginine bioavailability with exercise to a greater extent than subjects with less severe PAH. We identified relationships between kynurenine pathway metabolism and impaired ventriculo-arterial coupling, worse RV diastolic function, lower RV contractility, diminished RV contractility with exercise, and RV dilation with exercise. Metabolite profiles outperformed NT-proBNP in modeling RV contractility, diastolic function, and exercise performance. Specific metabolite profiles correspond to RV functional measurements only obtainable via invasive pressure-volume loop analysis and predict RV responses to exercise. Metabolic profiling may inform discovery of RV functional biomarkers.NEW & NOTEWORTHY In this cohort of patients with pulmonary arterial hypertension (PAH), we investigate metabolomic associations with comprehensive right ventricular (RV) functional measurements derived from multibeat RV pressure-volume loop analysis. Our results show that tryptophan metabolism, particularly the kynurenine pathway, is linked to intrinsic RV function and PAH pathobiology. Findings also highlight the importance of arginine bioavailability in the cardiopulmonary system's response to exercise stress. Metabolite profiles selected via unbiased analysis outperformed N-terminal prohormone of B-type natriuretic peptide (NT-proBNP) in predicting load-independent measures of RV function at rest and cardiopulmonary system performance under stress. Overall, this work suggests the potential for select metabolites to function as disease-specific biomarkers, offers insights into PAH pathobiology, and informs discovery of potentially targetable RV-centric pathways.

Background Clinical risk factors in neonatal cardiac surgery do not fully capture discrepancies in outcomes. Targeted metabolomic analysis of plasma from neonates undergoing heart surgery with cardiopulmonary bypass was performed to determine associations with clinical outcomes. Methods and Result Samples and clinical variables from 149 neonates enrolled in the Corticosteroid Therapy in Neonates Undergoing Cardiopulmonary Bypass trial with surgical treatment for congenital heart disease between 2012 and 2016 were included. Blood samples were collected before skin incision, immediately after cardiopulmonary bypass, and 12 hours after surgery. Outcomes include composite morbidity/mortality (death, extracorporeal membrane oxygenation, cardiac arrest, acute kidney injury, and/or hepatic injury) and a cardiac composite (extracorporeal membrane oxygenation, cardiac arrest, or increase in lactate level), hepatic injury, and acute kidney injury. Targeted metabolite levels were determined by high‐resolution tandem liquid chromatography and mass spectrometry. Principal component and regression analyses were used to assess associations between metabolic profiles and outcomes, with 2 models created: a base clinical model and a base model+metabolites. Of the 193 metabolites examined, 40 were detected and quantified. The first principal component, principal component 1, was composed mostly of preoperative metabolites and was significantly associated with the composite morbidity/mortality, cardiac composite, and hepatic injury outcomes. In regression models, individual metabolites also improved model performance for the composite morbidity/mortality, cardiac composite, and hepatic injury outcomes. Significant disease pathways included myocardial injury (false discovery rate, 0.00091) and heart failure (false discovery rate, 0.041). Conclusions In neonatal cardiac surgery, perioperative metabolites were associated with postoperative outcomes and improved clinical model outcome associations. Preoperative metabolite levels alone may improve risk models and provide a basis for optimizing perioperative care.

D-Amino acids are regulatory molecules that affect biological processes. Therefore, being able to accurately detect and quantify these compounds is important for understanding their impact on nutrition and health. There is a paucity of information regarding D-amino acids in human milk. We developed a fast method for simultaneous analysis of amino acid enantiomers in human milk using liquid chromatography with tandem mass spectrometry. The method enables the separation of 41 amino acids without chemical derivatization. Our results revealed that human milk from mothers of preterm infants contains concentrations of D-amino acids that range from 0.5 to 45% that of their L-counterparts and that levels of most D-amino acids decrease as the milk production matures. Moreover, we found that Holder pasteurization of milk does not cause racemization of L-amino acids. To our knowledge, this is the first study to describe percentages of D-amino acid levels in human milk; changes in D-amino acid concentration as the milk matures; and the effect of Holder pasteurization on D- and L-amino acid concentrations in human milk.

To investigate mechanisms of injury and recovery in neonatal encephalopathy (NE), we performed targeted metabolomic analysis of plasma using liquid chromatography with tandem mass spectrometry (LC/MS/MS) from healthy term neonates or neonates with NE. Plasma samples from the NE (n = 45, day of life 0–1) or healthy neonatal (n = 30, ≥36 weeks gestation) cohorts had LC/MS/MS metabolomic profiling with a 193-plex targeted metabolite assay covering >366 metabolic pathways. Metabolite levels were compared to 2-year neurodevelopmental outcomes measured by the Bayley Scales of Infant and Toddler Development III (Bayley-III). Out of 193 metabolites, 57 met the pre-defined quality control criteria for analysis. Significant (after false discovery rate correction) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways included aminoacyl-tRNA biosynthesis, arginine biosynthesis, and metabolism of multiple amino acids. Significant disease pathways included seizures. In regression models, histidine and C6 sugar amine were significantly associated with cognitive, motor, and language and betaine with cognitive and motor Bayley-III composite scores. The addition of histidine, C6 sugar amine, and betaine to a Sarnat score-based clinical regression model significantly improved model performance (Akaike information criterion and adjusted r2) for Bayley-III cognitive, motor, and language scores. Plasma metabolites may help to predict neurological outcomes in neonatal brain injury and enhance current clinical predictors.

Introduction: Improving neurodevelopmental (ND) outcomes for children with congenital heart disease has become a priority. Known clinical and surgical risk factors do not fully capture differences in ND outcomes. The objective of this study was to determine if plasma metabolites at the time of neonatal cardiac surgery were association with ND outcomes at 1 year. Methods: A secondary analysis of a clinical trial in 110 neonates undergoing cardiac surgery was performed. Plasma samples obtained pre-op, immediately post-op and at 12 hours post op had LC/MS/MS metabolomic profiling with a 193-plex targeted metabolite assay covering >366 metabolic pathways. Metabolite levels were compared to one-year neurodevelopmental outcomes measured by the Bayley Scales of Infant and Toddler Development III (BSID). Results: 40/193 metabolites were detected and quantified for analysis. Regression models utilizing metabolites and metabolite features identified associations with cognitive (n=5), language (n=4) and motor (n=10) BSID composite scores. Enrichment analysis disease pathways included seizures (tyrosine and isoleucine) and indoxyl sulfate, a known neurotoxic product from tryptophan synthesis. Conclusions: Perioperative metabolites may serve as biomarkers for prediction of 1-year ND outcomes and provide a scientific basis for targeted interventions to optimize perioperative care.

The molecular determinants of PAH risk and disease severity in systemic sclerosis (SSc) are poorly understood. We hypothesized that metabolic profiling of SSc serum would enable 1) discrimination of PAH, and 2) discovery of metabolic patterns associated with distinct pulmonary vascular phenotypes. Serum from 24 adult subjects with SSc (12 with and 12 without PAH) underwent mass spectrometry for targeted metabolomics. Metabolite associations with clinical variables were examined with standard statistical and bioinformatic approaches. 3 clusters of subjects with unique metabolic patterns were formed on the basis of metabolites alone, and clinical differences were compared across clusters. Metabolites of purine catabolism (e.g. hypoxanthine, raw p 0.027) and bile acid synthesis (e.g. glycocholate, raw p 0.006) differed in SSc with versus without PAH in significance analysis of metabolites. In SSc-PAH, metabolites discriminated subjects whose NYHA functional class improved with PAH therapy. A trend for increasing disease severity was observed across metabolite clusters, with more adverse hemodynamics, shorter 6 minute walk distance, and shorter survival time in cluster 1 compared to clusters 2 and 3 (Figure). Bile acid and caffeine metabolite concentrations contributed to cluster differences. Metabolites discriminate pulmonary vascular phenotypes in SSc. Unique metabolic patterns may be novel biomarkers of PAH risk and severity in SSc.

Introduction: Neonatal heart surgery requiring cardiopulmonary bypass (CPB) is associated with significant morbidity and mortality. Methods to improve outcome prediction are lacking. Hypothesis: Targeted metabolic profiling can identify critical metabolites associated with important post-operative clinical outcomes. These in turn could be integrated into clinical prediction models. Methods: Secondary analysis of serum samples from the neonatal congenital cardiac surgery with CPB methylprednisolone trial, which evaluated effects of steroids on outcomes. Samples were from pre-op, immediate post-CPB, and 12 hrs post op. Outcomes were a cardiac dysfunction composite (death, ECMO, cardiac arrest and lactate) and low cardiac output syndrome (LCOS). Metabolite levels were determined with targeted mass spectrometry (LC/MS/MS). Principal component (PC) analysis and multivariable regression analysis with bootstrapping were used to assess the association between metabolic profiles and outcomes. Results: Total cohort had 149 patients. PC analysis identified 6 PCs (PC1-PC6) that explained 51% of variance. When PCs and metabolites were added to a base clinical model, model fit for the cardiac dysfunction composite outcome was significantly improved (c-stat 0.819 vs 0.902, p<0.001) as well as LCOS (c-stat 0.576 vs. 0.803, p=0.76 vs. p=0.01). Using adjusted regression models, metabolites (some of which are included in PCs) significantly associated with cardiac dysfunction composite outcome were proline (p=0.004) and leucine (p=0.01). Metabolites associated with LCOS were n-acetyltryptophan (p=0.01), glycocholic acid (p=0.001), cortisol (p=0.02), nicotinamide (p=0.04), methyladenosine (p=0.01) and methionine (p=0.01). For cardiac composite outcome, cumulative and post op values were significant, whereas all time points were for LCOS. Conclusions: Combining metabolic profiles with patient characteristics revealed significant pre-op metabolites associated with important clinical outcomes. Given that the association held true for all time points, these observations could be used to design molecular signature for post-operative cardiac complications or, to create prediction models based on pre-operative metabolic profiles.

Matrix-assisted laser/desorption ionization (MALDI) mass spectrometry imaging (MSI) is widely used as a unique tool to record the distribution of a large range of biomolecules in tissues. 2,6-Dihydroxyacetophenone (DHA) matrix has been shown to provide efficient ionization of lipids, especially gangliosides. The major drawback for DHA as it applies to MS imaging is that it sublimes under vacuum (low pressure) at the extended time necessary to complete both high spatial and mass resolution MSI studies of whole organs. To overcome the problem of sublimation, we used an atmospheric pressure (AP)-MALDI source to obtain high spatial resolution images of lipids in the brain using a high mass resolution mass spectrometer. Additionally, the advantages of atmospheric pressure and DHA for imaging gangliosides are highlighted. The imaging of [M–H]− and [M–H2O–H]− mass peaks for GD1 gangliosides showed different distribution, most likely reflecting the different spatial distribution of GD1a and GD1b species in the brain.

Mass spectrometry imaging (MSI) of tissue implanted with silver nanoparticulate (AgNP) matrix generates reproducible imaging of lipids in rodent models of disease and injury. Gas-phase production and acceleration of size-selected 8 nm AgNP is followed by controlled ion beam rastering and soft landing implantation of 500 eV AgNP into tissue. Focused 337 nm laser desorption produces high quality images for most lipid classes in rat brain tissue (in positive mode: galactoceramides, diacylglycerols, ceramides, phosphatidylcholines, cholesteryl ester, and cholesterol, and in negative ion mode: phosphatidylethanolamides, sulfatides, phosphatidylinositol, and sphingomyelins). Image reproducibility in serial sections of brain tissue is achieved within <10% tolerance by selecting argentated instead of alkali cationized ions. The imaging of brain tissues spotted with pure standards was used to demonstrate that Ag cationized ceramide and diacylglycerol ions are from intact, endogenous species. In contrast, almost all Ag cationized fatty acid ions are a result of fragmentations of numerous lipid types having the fatty acid as a subunit. Almost no argentated intact fatty acid ions come from the pure fatty acid standard on tissue. Graphical Abstract ᅟ.

Traumatic brain injury (TBI) is a serious public health problem and the leading cause of death in children and young adults. It also contributes to a substantial number of cases of permanent disability. As lipids make up over 50% of the brain mass and play a key role in both membrane structure and cell signaling, their profile is of particular interest. In this study, we show that advanced mass spectrometry imaging (MSI) has sufficient technical accuracy and reproducibility to demonstrate the anatomical distribution of 50 μm diameter microdomains that show changes in brain ceramide levels in a rat model of controlled cortical impact (CCI) 3 days post injury with and without treatment. Adult male Sprague-Dawley rats received one strike and were euthanized 3 days post trauma. Brain MS images showed increase in ceramides in CCI animals compared to control as well as significant reduction in ceramides in CCI treated animals, demonstrating therapeutic effect of a peptide agonist. The data also suggests the presence of diffuse changes outside of the injured area. These results shed light on the extent of biochemical and structural changes in the brain after traumatic brain injury and could help to evaluate the efficacy of treatments.

Background: Mild traumatic brain injury (TBI) is a common public health issue that may contribute to chronic degenerative disorders. Membrane lipids play a key role in tissue responses to injury, both as cell signals and as components of membrane structure and cell signaling. This study demonstrates the ability of high resolution mass spectrometry imaging (MSI) to assess sequences of responses of lipid species in a rat controlled cortical impact model for concussion.New method: A matrix of implanted silver nanoparticles was implanted superficially in brain sections for matrix-assisted laser desorption (MALDI) imaging of 50 mu m diameter microdomains across unfixed cryostat sections of rat brain. Ion-mobility time-of-flight MS was used to analyze and map changes over time in brain lipid composition in a rats after Controlled Cortical Impact (CCI) TBI.Results: Brain MS images showed changes in sphingolipids near the CCI site, including increased ceramides and decreased sphingomyelins, accompanied by changes in glycerophospholipids and cholesterol derivatives. The kinetics differed for each lipid class; for example ceramides increased as early as 1 day after the injury whereas other lipids changes occurred between 3 and 7 days post injury.Comparison with existing method(s): Silver nanoparticles MALDI matrix is a sensitive new tool for revealing previously undetectable cellular injury response and remodeling in neural, glial and vascular structure of the brain.Conclusions: Lipid biochemical and structural changes after TBI could help highlighting molecules that can be used to determine the severity of such injuries as well as to evaluate the efficacy of potential treatments. (C) 2016 Published by Elsevier B.V.

Alcohol abuse is a chronic disease characterized by the consumption of alcohol at a level that interferes with physical and mental health and causes serious and persistent changes in the brain. Lipid metabolism is of particular interest due to its high concentration in the brain. Lipids are the main component of cell membranes, are involved in cell signaling, signal transduction, and energy storage. In this study, we analyzed lipid composition of chronically ethanol exposed mouse brains. Juvenile (JUV) and adult (ADU) mice were placed on a daily limited-access ethanol intake model for 52 days. After euthanasia, brains were harvested, and total lipids were extracted from brain homogenates. Samples were analyzed using high resolution mass spectrometry and processed by multivariate and univariate statistical analysis. Significant lipid changes were observed in different classes including sphingolipids, fatty acids, lysophosphatidylcholines, and other glycerophospholipids.