Changes In Metabolite Levels Research Articles

The Urinary Metabolomic Fingerprint in Extremely Preterm Infants on Total Parenteral Nutrition vs. Enteral Feeds.

Total Parenteral Nutrition (TPN), which uses intravenous administration of nutrients, minerals and vitamins, is essential for sustaining premature infants until they transition to enteral feeds, but there is limited information on metabolomic differences between infants on TPN and enteral feeds. We performed untargeted global metabolomics on urine samples collected between 23-30 days of life from 314 infants born <29 weeks gestational age from the TOLSURF and PROP cohorts. Principal component analysis across all metabolites showed a separation of infants solely on TPN compared to infants who had transitioned to enteral feeds, indicating global metabolomic differences between infants based on feeding status. Among 913 metabolites that passed quality control filters, 609 varied in abundance between infants on TPN vs. enteral feeds at p < 0.05. Of these, 88% were in the direction of higher abundance in the urine of infants on enteral feeds. In a subset of infants in a longitudinal analysis, both concurrent and delayed changes in metabolite levels were observed with the initiation of enteral feeds. These infants had higher concentrations of essential amino acids, lipids, and vitamins, which are necessary for growth and development, suggesting the nutritional benefit of an enteral feeding regimen.

Carnitine metabolites and cognitive improvement in patients with schizophrenia treated with olanzapine: a prospective longitudinal study.

Objective: Cognitive impairment is one of the core symptoms of schizophrenia, which is stable and lifelong. L-carnitine has been shown to improve cognitive function and decrease the rate of cognitive deterioration in patients with Alzheimer's disease. However, it remains unclear regarding the role of L-carnitine and its metabolites in cognitive functions in schizophrenia after treatment with olanzapine. The purpose of this study was to evaluate the relationship between changes in plasma levels of L-carnitine metabolites and cognitive improvement after olanzapine treatment. Methods: This was a prospective longitudinal study. In this study, we recruited 25 female patients with first episode schizophrenia (FES) who were drug naïve at baseline and received 4 weeks of olanzapine monotherapy. Cognitive function was assessed at baseline and 4-week follow-up using the RBANS. Plasma L-carnitine metabolite levels were determined by a metabolomics technology based on untargeted ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Results: We found that the immediate memory index, delayed memory index and RBANS composite score were significantly increased at the 4-week follow-up after treatment. A total of 7 differential L-carnitine metabolites were identified in FES patients after olanzapine monotherapy. In addition, we found that changes in butyrylcarnitine were positively correlated with improvements in language index and RBANS composite score. Further regression analyses confirmed the association between reduced butyrylcarnitine levels and cognitive improvement after olanzapine monotherapy in FES patients. Conclusion: Our study shows that cognitive improvement after olanzapine treatment was associated with changes in L-carnitine metabolite levels in patients with FES, suggesting a key role of L-carnitine in cognition in schizophrenia.

Metabolic remission precedes possible weight regain after gastric bypass surgery.

Some patients regain weight to a variable extent from 1 year after Roux-en-Y gastric bypass surgery (RYGB), though rarely reaching preoperative values. The aim of the present study was to investigate whether, when, and to what extent metabolic remission occurs. Fasting metabolite and lipid profiles were determined in blood plasma collected from a nonrandomized intervention study involving 148 patients before RYGB and at 2, 12, and 60 months post RYGB. Both short-term and long-term alterations in metabolism were assessed. Anthropometric and clinical variables were assessed at all study visits. This study found that the vast majority of changes in metabolite levels occurred during the first 2 months post RYGB. Notably, thereafter the metabolome started to return toward the presurgical state. Consequently, a close-to-presurgical metabolome was observed at the time when patients reached their lowest weight and glucose level. Lipids with longer acyl chains and a higher degree of unsaturation were altered more dramatically compared with shorter and more saturated lipids, suggesting a systematic and reversible lipid remodeling. Remission of the metabolic state was observed prior to notable weight regain. Further and more long-term studies are required to assess whether the extent of metabolic remission predicts future weight regain and glycemic deterioration.

Amyloids of α-Synuclein Promote Chemical Transformations of Neuronal Cell Metabolites.

The assembly of α-synuclein into cross-β structured amyloid fibers results in Lewy body deposits and neuronal degeneration in Parkinson's disease patients. As the cell environment is highly crowded, interactions between the formed amyloid fibers and a range of biomolecules can occur in cells. Although amyloid fibers are considered chemically inert species, recent in vitro work using model substrates has shown α-synuclein amyloids, but not monomers, to catalyze the hydrolysis of ester and phosphoester bonds. To search for putative catalytic activity of α-synuclein amyloids on biologically relevant metabolites, we here incubated α-synuclein amyloids with neuronal SH-SY5Y cell lysates devoid of proteins. LC-MS-based metabolomic (principal component and univariate) analysis unraveled distinct changes in several metabolite levels upon amyloid (but not monomer) incubation. Of 63 metabolites identified, the amounts of four increased (3-hydroxycapric acid, 2-pyrocatechuic acid, adenosine, and NAD), and the amounts of seventeen decreased (including aromatic and apolar amino acids, metabolites in the TCA cycle, keto acids) in the presence of α-synuclein amyloids. Many of these metabolite changes match what has been reported previously in Parkinson's disease patients and animal-model metabolomics studies. Chemical reactivity of α-synuclein amyloids may be a new gain-of-function that alters the metabolite composition in cells and, thereby, modulates disease progression.

The Sizes and Composition of HDL-Cholesterol Are Significantly Associated with Inflammation in Rheumatoid Arthritis Patients.

Rheumatoid arthritis (RA), a chronic inflammatory disease, carries a significant burden of atherosclerotic cardiovascular diseases (ASCVD). With their heterogeneous composition, high-density lipoprotein (HDL) particles have varied athero-protective properties, and some may even increase ASCVD risk. In this prospective and cross-sectional study, we aimed to examine the relationship between HDL sizes/metabolites and inflammation in RA. Using 1H-NMR-based lipid/metabolomics, differential HDL-related metabolites were identified between RA patients and healthy control (HC) subjects and between RA patients with and without anti-citrullinated peptide antibodies (ACPA). The correlation between the discriminative HDL-related metabolites and C-reactive protein (CRP) was evaluated in RA patients. RA patients demonstrated higher particle number, lipids, cholesterol, cholesterol ester, free cholesterol, and phospholipids in large/very large-sized HDLs. ACPA-positive patients had higher L-HDL-C and L-HDL-CE but lower small-/medium-sized HDL-TG levels than ACPA-negative patients. An inverse correlation was found between CRP levels and small-sized HDLs. Janus kinase inhibitor treatment was associated with increased serum small-sized HDL-related metabolites and decreased CRP levels. We are the first to reveal the significant associations between RA inflammation and HDL sizes/metabolites. A potential link between ACPA positivity and changes in serum levels of HDL-related metabolites was also observed in RA patients.

Sall4 restricts glycolytic metabolism in limb buds through transcriptional regulation of glycolytic enzyme genes

Recent studies illustrate the importance of regulation of cellular metabolism, especially glycolysis and pathways branching from glycolysis, during vertebrate embryo development. For example, glycolysis generates cellular energy ATP. Glucose carbons are also directed to the pentose phosphate pathway, which is needed to sustain anabolic processes in the rapidly growing embryos. However, our understanding of the exact status of glycolytic metabolism as well as genes that regulate glycolytic metabolism are still incomplete. Sall4 is a zinc finger transcription factor that is highly expressed in undifferentiated cells in developing mouse embryos, such as blastocysts and the post-implantation epiblast. TCre; Sall4 conditional knockout mouse embryos exhibit various defects in the posterior part of the body, including hindlimbs. Using transcriptomics approaches, we found that many genes encoding glycolytic enzymes are upregulated in the posterior trunk, including the hindlimb-forming region, of Sall4 conditional knockout mouse embryos. In situ hybridization and qRT-PCR also confirmed upregulation of expression of several glycolytic genes in hindlimb buds. A fraction of those genes are bound by SALL4 at the promoters, gene bodies or distantly-located regions, suggesting that Sall4 directly regulates expression of several glycolytic enzyme genes in hindlimb buds. To further gain insight into the metabolic status associated with the observed changes at the transcriptional level, we performed a comprehensive analysis of metabolite levels in limb buds in wild type and Sall4 conditional knockout embryos by high-resolution mass spectrometry. We found that the levels of metabolic intermediates of glycolysis are lower, but glycolytic end-products pyruvate and lactate did not exhibit differences in Sall4 conditional knockout hindlimb buds. The increased expression of glycolytic genes would have caused accelerated glycolytic flow, resulting in low levels of intermediates. This condition may have prevented intermediates from being re-directed to other pathways, such as the pentose phosphate pathway. Indeed, the change in glycolytic metabolite levels is associated with reduced levels of ATP and metabolites of the pentose phosphate pathway. To further test whether glycolysis regulates limb patterning downstream of Sall4, we conditionally inactivated Hk2, which encodes a rate-limiting enzyme gene in glycolysis and is regulated by Sall4. The TCre; Hk2 conditional knockout hindlimb exhibited a short femur, and a lack of tibia and anterior digits in hindlimbs, which are defects similarly found in the TCre; Sall4 conditional knockout. The similarity of skeletal defects in Sall4 mutants and Hk2 mutants suggests that regulation of glycolysis plays a role in hindlimb patterning. These data suggest that Sall4 restricts glycolysis in limb buds and contributes to patterning and regulation of glucose carbon flow during development of limb buds.

Nitric oxide metabolites in heart failure patients with reduced or preserved ejection fraction and effects of different exercise modes

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): German Centre for Cardiovascular Research (DZHK). Background Alterations of endothelial function and nitric oxide (NO) release are involved in pathophysiology and clinical symptoms of heart failure (HF). Regular exercise training improves vascular and endothelial function and is closely linked to mechanisms of NO pathway by enhancing endothelial NO synthesis. However, it is not well investigated how exercise training affects metabolites of the NO pathway and whether different exercise modes have different effects on NO bioavailability in HF. Purpose The aims of this analysis were to 1) investigate differences in serum levels of the NO metabolites L-arginine, homoarginine, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) between patients with HF with reduced (HFrEF) or preserved ejection fraction (HFpEF) and 2) to investigate effects of high-intensity interval training (HIIT) and moderate continuous training (MCT) on these parameters. Methods Patients with HF from two clinical exercise intervention trials were included in this analysis. In both trials, patients were randomized (1:1:1) either to HIIT, MCT or guideline control (CG). Clinical data and blood samples were collected at baseline, after three months of supervised exercise training and after 12 months follow-up. NO metabolites were analysed from EDTA-plasma using liquid chromatography-tandem mass spectrometry (LC–MS/MS). Results Data from 366 patients with HF, including 206 HFrEF (median [1. and 3. quartile]: 61 [53;70] years, 18.9% females) and 160 HFpEF patients (71 [66;76] years, 65.6% females), was available for analysis. Patients with HFrEF or HFpEF showed no significant differences in NYHA classification (NYHA III: 30.6% vs. 26.2%, p=0.43) or presence of diabetes mellitus (23.8% vs. 26.2%, p=0.50), while HFpEF had a higher prevalence of hypertension (86.9% vs. 38.5%, p&amp;lt;0.001) but a smaller number of current smoker than HFrEF (4.4% vs. 18.0%, p&amp;lt;0.001). PeakVO2 (18.4 [14.5;22.6] ml/min/kg vs. 17.0 [14.3;20.4] ml/min/kg, p=0.03) and HDL-cholesterol (56 [46;65] mg/dl vs. 42 [36;54] mg/dl, p&amp;lt;0.001) were higher in HFpEF than in HFrEF. At baseline, homoarginine and ADMA levels were higher in HFrEF than in HFpEF, but no differences in L-arginine or SDMA could be observed (Table 1). Changes in metabolite levels from baseline to 3 and 12 months were not significantly different between groups (HIIT, MCT, CG) in either HF phenotype (all p&amp;gt;0.05). In all exercise groups, median adherence to training sessions was at 85% or above with no significant differences between groups (p&amp;gt;0.05). Conclusion At baseline, patients with HFrEF had significantly higher homoarginine and ADMA levels compared to patients with HFpEF. However, in both HF phenotypes neither MCT nor HIIT induced significant changes in serum levels of NO metabolites when compared to patients randomized to CG.

γ-Aminobutyric acid as a biomarker of the lateralizing and monitoring drug effect in patients with magnetic resonance imaging-negative temporal lobe epilepsy.

Despite verifying proton magnetic resonance spectroscopy (1H-MRS) for focal localization in magnetic resonance imaging (MRI)-negative temporal lobe epilepsy (TLE), it is necessary to illustrate metabolic changes and screen for effective biomarkers for monitoring therapeutic effect. We used 1H-MRS to investigate the role of metabolic levels in MRI-negative TLE. Thirty-seven patients (n = 37, 14 women) and 20 healthy controls (n = 20, 11 women) were investigated by 1H-MRS. We compared the metabolite level changes in the epileptic and contralateral sides on the mesial temporal and dorsolateral prefrontal cortices and analyzed their association with clinical symptoms. γ-Aminobutyric acid (GABA) levels were significantly lower on the epileptic side (2.292 ± 0.890) than in the contralateral side (2.662 ± 0.742, p = 0.029*) in patients on the mesial temporal lobe. N-acetylaspartate (NAA) levels were significantly lower on the epileptic side (7.284 ± 1.314) than on the contralateral side (7.655 ± 1.549, p = 0.034*). NAA + N-acetylaspartylglutamate levels were significantly lower on the epileptic side (7.668 ± 1.406) than on the contralateral side (8.086 ± 1.675, p = 0.032*). Glutamate levels were significantly lower on the epileptic side (7.773 ± 1.428) than on the contralateral side (8.245 ± 1.616, p = 0.040*). Moreover, a significant negative correlation was found between GABA levels in the epileptic mesial temporal lobe and tonic-clonic seizure frequency (r = -0.338, p = 0.046*). γ-Aminobutyric acid (GABA) is a potential biomarker for lateralization and monitoring seizure frequency in MRI-negative TLE.

Assays for profiling changes in T cell metabolism

Abstract Immune cell therapy, including engineered T cells, engineered NK cells, and treatments to enhance native T cell response, is a rapidly advancing field of research with great promise to treat multiple diseases. However, multiple challenges, from isolating the correct cell type, determining its mode of action, manufacturing and establishing appropriate quality control criteria, must be overcome to reach its potential. In recent years, cellular energy metabolism has emerged as an important consideration for efficacy and persistence when developing cell therapies. Here we evaluate a set of bioluminescent cell-based assays for measuring rate of metabolite uptake, monitoring activity for key metabolic pathways and determining changes in intracellular metabolite levels during T cell transition from naïve to activated to memory phenotypes. Metabolite levels measured include ATP, glucose uptake, malate, branched chain amino acids, and glycogen levels. Depending on the activation state of the cells, the cells respond differently to glycolysis and oxidative phosphorylation inhibitors, as demonstrated by changes to metabolite levels. Energy storage as measured by intracellular glycogen levels also differs depending on the T cell phenotype (naïve, activated, or memory). Understanding the processes governing dynamic metabolic changes during cell modification and expansion is required for next-generation cell therapy development. The developed bioluminescent cell-based assays provide the sensitivity, throughput and robustness required for rapid evaluation of such changes.

Characterization of the Key Compounds of Bell Pepper by Spectrophotometry and Gas Chromatography on the Effects of Induced Stress on the Concentration of Secondary Metabolite.

Sweet peppers are consumed worldwide, and traditional uses have sparked interest in their applications as dietary antioxidants, which can be enhanced in plants using elicitors. These are endowed with phytochemicals with potential health benefits such as antioxidants, bioavailability, and bioaccessibility. The trend in metabolomics shows us chemical fingerprints linking metabolomics, innovative analytical form, and bioinformatics tools. The objective was to evaluate the impact of multiple stress interactions, elicitor concentrations, and electrical conductivity on the concentration of secondary metabolites to relate their response to metabolic pathways through the foliar application of a cocktail of said elicitors in pepper crops under greenhouse conditions. The extracts were analyzed by spectrophotometry and gas chromatography, and it was shown that the PCA analysis identified phenolic compounds and low molecular weight metabolites, confirming this as a metabolomic fingerprint in the hierarchical analysis. These compounds were also integrated by simultaneous gene and metabolite simulants to obtain effect information on different metabolic pathways. Showing changes in metabolite levels at T6 (36 mM H2O2 and 3.6 dS/m) and T7 (0.1 mM SA and 3.6 dS/m) but showing statistically significant changes at T5 (3.6 dS/m) and T8 (0.1 mM SA, 36 mM H2O2, and 3.6 dS/m) compared to T1 (32 dS/m) or control. Six pathways changed significantly (p < 0.05) in stress-induced treatments: aminoacyl t-RNA and valine-leucine-isoleucine biosynthesis, and alanine-aspartate-glutamate metabolism, glycoxylate-dicarboxylate cycle, arginine-proline, and citrate. This research provided a complete profile for the characterization of metabolomic fingerprint of bell pepper under multiple stress conditions.

Benchtop NMR-Based Metabolomics: First Steps for Biomedical Application.

Nuclear magnetic resonance (NMR)-based metabolomics is a valuable tool for identifying biomarkers and understanding the underlying metabolic changes associated with various diseases. However, the translation of metabolomics analysis to clinical practice has been limited by the high cost and large size of traditional high-resolution NMR spectrometers. Benchtop NMR, a compact and low-cost alternative, offers the potential to overcome these limitations and facilitate the wider use of NMR-based metabolomics in clinical settings. This review summarizes the current state of benchtop NMR for clinical applications where benchtop NMR has demonstrated the ability to reproducibly detect changes in metabolite levels associated with diseases such as type 2 diabetes and tuberculosis. Benchtop NMR has been used to identify metabolic biomarkers in a range of biofluids, including urine, blood plasma and saliva. However, further research is needed to optimize the use of benchtop NMR for clinical applications and to identify additional biomarkers that can be used to monitor and manage a range of diseases. Overall, benchtop NMR has the potential to revolutionize the way metabolomics is used in clinical practice, providing a more accessible and cost-effective way to study metabolism and identify biomarkers for disease diagnosis, prognosis, and treatment.

Multinuclear Magnetic Resonance Spectroscopy at Ultra-High-Field: Assessing Human Cerebral Metabolism in Healthy and Diseased States.

The brain is a highly energetic organ. Although the brain can consume metabolic substrates, such as lactate, glycogen, and ketone bodies, the energy metabolism in a healthy adult brain mainly relies on glucose provided via blood. The cerebral metabolism of glucose produces energy and a wide variety of intermediate metabolites. Since cerebral metabolic alterations have been repeatedly implicated in several brain disorders, understanding changes in metabolite levels and corresponding cell-specific neurotransmitter fluxes through different substrate utilization may highlight the underlying mechanisms that can be exploited to diagnose or treat various brain disorders. Magnetic resonance spectroscopy (MRS) is a noninvasive tool to measure tissue metabolism in vivo. 1H-MRS is widely applied in research at clinical field strengths (≤3T) to measure mostly high abundant metabolites. In addition, X-nuclei MRS including, 13C, 2H, 17O, and 31P, are also very promising. Exploiting the higher sensitivity at ultra-high-field (>4T; UHF) strengths enables obtaining unique insights into different aspects of the substrate metabolism towards measuring cell-specific metabolic fluxes in vivo. This review provides an overview about the potential role of multinuclear MRS (1H, 13C, 2H, 17O, and 31P) at UHF to assess the cerebral metabolism and the metabolic insights obtained by applying these techniques in both healthy and diseased states.

Changes in brain metabolite levels across childhood

Metabolites play important roles in brain development and their levels change rapidly in the prenatal period and during infancy. Metabolite levels are thought to stabilize during childhood, but the development of neurochemistry across early-middle childhood remains understudied. We examined the developmental changes of key metabolites (total N-acetylaspartate, tNAA; total choline, tCho; total creatine, tCr; glutamate+glutamine, Glx; and myo-inositol, mI) using short echo-time magnetic resonance spectroscopy (MRS) in the anterior cingulate cortex (ACC) and the left temporo-parietal cortex (LTP) using a mixed cross-sectional/longitudinal design in children aged 2–11 years (ACC: N = 101 children, 112 observations; LTP: N = 95 children, 318 observations). We found that tNAA increased with age in both regions, while tCho decreased with age in both regions. tCr increased with age in the LTP only. Glx did not show linear age effects in either region, but a follow-up analysis in participants with ≥3 datapoints in the LTP revealed a quadratic effect of age following an inverted U-shape. These substantial changes in neurochemistry throughout childhood likely underlie various processes of structural and functional brain development.

Physical, chemical, and physiological characterization of the reactions preceding chilling injury–induced cell membrane damage in cucumber

Chilling injury in cucumber is commonly thought to begin with cell membrane damage caused by reactive oxygen species, which results in ion leakage from the tissue. However, additional short-term storage at ambient temperature after cold storage can accelerate the appearance of damage, and the different types of vegetables exhibit distinct symptoms. We hypothesized that cell membrane damage is a secondary reaction, and that preceding responses in the tissue lead to irreversible changes. To explore these responses in cucumber tissue during cold storage, we employed several techniques, including ion leakage measurements, bio-electrochemical impedance spectroscopy, X-ray computed tomography, nuclear magnetic resonance–based metabolite profiling, and transcriptome analysis. Our time-course comparison of different storage conditions (2 °С, 5 °С, 10 °С, and 15 °С for 0–14 d) indicated that several responses preceded detectable ion leakage–associated cell membrane damage. Specifically, the cold-stored samples exhibited abnormal electrolyte distribution and marked changes in metabolite levels and gene expression related to the tricarboxylic acid (TCA) cycle, which is critical for generating energy for homeostasis. Moreover, samples stored at 2 °С followed by 15 °С showed earlier initiation of tissue responses in metabolite and gene expression profiles, suggesting the occurrence of irreversible changes, possibly in the TCA cycle. These findings suggest that the critical response to chilling injury may occur prior to cell membrane damage, likely due to metabolic imbalances in the TCA cycle.

Profiling of metabolites, proteins, and protein phosphorylation in silica-exposed BEAS-2B epithelial cells.

Silicosis is an uncurable occupational disease induced by crystalline silica. Increased prevalence of silicosis has resulted in the increased need for development of treatment options. Although macrophages respond first to silica, epithelial cells are also involved in silicosis. However, changes in protein and metabolite levels have not been reported simultaneously. We used mass spectrometry to profile changes in metabolites, proteins, and phosphorylation in silica-exposed BEAS-2B epithelial cells. Silica exposure increased TCA cycle, alanine, aspartate and glutamate metabolism, and aerobic glycolysis activity. In addition, protein levels in the endoplasmic reticulum were significantly altered, and phosphorylation of MAPK signaling proteins was increased. The results of this study increased understanding the role of epithelial cells in silicosis.

Lipopolysaccharide-induced changes in the kynurenine pathway and symptoms of sickness behavior in humans

Metabolites of the kynurenine pathway are hypothesized to be implicated in inflammation-associated depression, but there is a lack of experimental studies in humans assessing the kinetics of kynurenine metabolites in relation to experimentally-induced sickness. The aim of the present study was to assess changes in the kynurenine pathway and to explore its relation to symptoms of sickness behavior during an acute experimental immune challenge.This double-blind placebo-controlled randomized cross-over study included 22 healthy human participants (n = 21 both sessions, Mage = 23.4, SD = 3.6, nine women) who received an intravenous injection of 2.0 ng/kg lipopolysaccharide (LPS) and saline (placebo) on two different occasions in a randomized order. Blood samples (0 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, 5 h, 7 h post-injection) were analyzed for kynurenine metabolites and inflammatory cytokines. The intensity of symptoms of sickness behavior was assessed using the 10-item Sickness Questionnaire at 0 h, 1.5 h, 3 h, 5 h, and 7 h post-injection.LPS induced significantly lower concentrations of plasma tryptophan (at 2 h, 4 h, 5 h, and 7 h post-injection), kynurenine (at 2 h, 3 h, 4 h, and 5 h post-injection), nicotinamide (at 4 h, 5 h, and 7 h post-injection), and higher levels for quinolinic acid at 5 h post-injection as compared to placebo. LPS did not affect kynurenic acid, 3-hydroxykynurenine, and picolinic acid. The development of the sickness symptoms was largely similar across items, with the highest levels around 1.5–3 h post-injection. Changes in plasma levels of kynurenine metabolites seem to coincide rather than precede or follow changes in subjective sickness. Exploratory analyses indicate that higher Sickness Questionnaire total scores at 1.5–5 h post-injection were correlated with lower kynurenic acid and nicotinamide levels.These results lend further support for LPS-induced changes in the kynurenine pathway, but may not, as interpreted from blood levels, causally link to LPS-induced acute symptoms of sickness behavior. Future research may consider a larger sample to further scrutinize the role of the kynurenine pathway in the sickness response.

Abstract 6050: Analysis of diverse metabolic pathways using bioluminescent cell-based assays

Abstract Cellular energy metabolism is a complex system of many interconnected metabolic pathways that govern both cell health and function. Cancer cells are adept at quickly switching between metabolic pathways to adapt to nutrient limitations in the environment, enhancing their growth advantages while negatively impacting other cells in the tumor microenvironment such as immune cells. To facilitate metabolic characterization of cancer cells, we developed assays for detection of metabolites involved in key pathways, such as glycolysis and glutaminolysis, using a bioluminescent NADH detection technology coupled to specific dehydrogenases. We have now extended these assays to include metabolites involved in glycogen, lipid, ketone body and amino acid metabolism, as well as intermediates in the TCA cycle such as malate, isocitrate, and 2-hydroxyglutarate. A second core bioluminescent hydrogen peroxide detection system was also used to develop an assay for pyruvate. Here we describe the use of these assays to measure metabolite levels in cell lysates and cell culture media collected from cancer cell lines plated in 96- or 384-well plates and in 2D or 3D cultures. Changes in metabolite levels were monitored in response to altered media composition and drug treatments. Levels were also compared between different cell lines. In comparative studies of glioblastoma cell lines, 2-hydroxyglutarate production was higher in cells expressing mutant isocitrate dehydrogenase. By reconfiguring the NADH detection technology to measure cellular enzyme activity rather than metabolite levels, we were able to detect activity of dehydrogenases that participate in the glycolysis pathway, the TCA cycle, and pentose phosphate pathway. Assays for cellular malate dehydrogenase, isocitrate dehydrogenase and other dehydrogenase enzymes were sensitive, requiring ≤ 400 lysed cells per assay. The expanded collection of bioluminescent assays provides a way to quickly characterize the metabolic states and behaviors of cancer cells to help identify pathways that may present therapeutic targets. Citation Format: Donna Leippe, Natasha Karassina, Mike Valley, Kayla Sylvester, Jolanta Vidugiriene. Analysis of diverse metabolic pathways using bioluminescent cell-based assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6050.

Quantification of Serum Metabolites in Early Colorectal Adenomas Using Isobaric Labeling Mass Spectrometry.

A major challenge in reducing the death rate of colorectal cancer is to screen patients using low-invasive testing. A blood test shows a high compliance rate with reduced invasiveness. In this work, a multiplex isobaric tag labeling strategy coupled with mass spectrometry is adopted to relatively quantify primary and secondary amine-containing metabolites in serum for the discovery of metabolite level changes of colorectal cancer. Serum samples from patients at different risk statuses and colorectal cancer growth statuses are studied. Metabolite identification is based on accurate mass matching and/or retention time of labeled metabolite standards. We quantify 40 metabolites across all the serum samples, including 18 metabolites validated with standards. We find significantly decreased levels of threonine and asparagine in the patients with growing adenomas or high-risk adenomas (p < 0.05). Glutamine levels decrease in patients with adenomas of unknown growth status or high-risk adenomas. In contrast, arginine levels are elevated in patients with low-risk adenoma. Receiver operating characteristic analysis shows high sensitivity and specificity of these metabolites for detecting growing adenomas. Based on these results, we conclude that a few metabolites identified here might contribute to distinguishing colorectal patients with growing adenomas from normal individuals and patients with unknown growth status of adenomas.

Arg-tRNA synthetase links inflammatory metabolism to RNA splicing and nuclear trafficking via SRRM2.

Cells respond to perturbations such as inflammation by sensing changes in metabolite levels. Especially prominent is arginine, which has known connections to the inflammatory response. Aminoacyl-tRNA synthetases, enzymes that catalyse the first step of protein synthesis, can also mediate cell signalling. Here we show that depletion of arginine during inflammation decreased levels of nuclear-localized arginyl-tRNA synthetase (ArgRS). Surprisingly, we found that nuclear ArgRS interacts and co-localizes with serine/arginine repetitive matrix protein 2 (SRRM2), a spliceosomal and nuclear speckle protein, and that decreased levels of nuclear ArgRS correlated with changes in condensate-like nuclear trafficking of SRRM2 and splice-site usage in certain genes. These splice-site usage changes cumulated in the synthesis of different protein isoforms that altered cellular metabolism and peptide presentation to immune cells. Our findings uncover a mechanism whereby an aminoacyl-tRNA synthetase cognate to a key amino acid that is metabolically controlled during inflammation modulates the splicing machinery.

Effects of Long-Term Storage on the Biobanked Neonatal Dried Blood Spot Metabolome.

Over 2.5 million neonatal dried blood spots (DBS) are stored at the Danish National Biobank. These samples offer extraordinary possibilities for metabolomics research, including prediction of disease and understanding of underlying molecular mechanisms of disease development. Nevertheless, Danish neonatal DBS have been little explored in metabolomics studies. One question that remains underinvestigated is the long-term stability of the large number of metabolites typically assessed in untargeted metabolomics over long time periods of storage. Here, we investigate temporal trends of metabolites measured in 200 neonatal DBS collected over a time course of 10 years, using an untargeted liquid chromatography tandem mass spectrometry (LC-MS/MS) based metabolomics protocol. We found that a majority (71%) of the metabolome was stable during 10 years of storage at -20 °C. However, we found decreasing trends for lipid-related metabolites, such as glycerophosphocholines and acylcarnitines. A few metabolites, including glutathione and methionine, may be strongly influenced by storage, with changes in metabolite levels up to 0.1-0.2 standard deviation units per year. Our findings indicate that untargeted metabolomics of DBS samples, with long-term storage in biobanks, is suitable for retrospective epidemiological studies. We identify metabolites whose stability in DBS should be closely monitored in future studies of DBS samples with long-term storage.