Untargeted Nuclear Magnetic Resonance Research Articles

Identification of Urine Metabolic Markers of Stroke Risk Using Untargeted Nuclear Magnetic Resonance Analysis

Stroke remains the second leading cause of mortality worldwide, and the third leading cause of death and morbidity combined, affecting more than 12 million people every year. Stroke pathophysiology results from complex interactions of several risk factors related to age, family history, gender, lifestyle, and the presence of cardiovascular and metabolic diseases. Despite all the evidence, it is not possible to fully prevent stroke onset. In recent years, there has been an exploration of innovative methodologies for metabolite analysis aimed at identifying novel stroke biomarkers. Utilizing Nuclear Magnetic Resonance (NMR) spectroscopy, we investigated small molecule variations in urine across different stages of stroke risk. The Framingham Stroke Risk Score was used in people over 63 years of age living in long-term care facilities (LTCFs) to calculate the probability of suffering a stroke: low stroke risk (LSR, control), moderate stroke risk (MSR), and high stroke risk (HSR). Univariate statistical analysis showed that urinary 4-hydroxyphenylacetate levels increased while glycolate levels decreased across the different stroke risk groups, from the LSR to the HSR groups. Trimethylamine N-oxide (TMAO) had average concentration values that were significantly higher in elderly people in the HSR group, while trigonelline levels were significantly lower in the MSR group. These metabolic markers can be used for early detection and to differentiate stages of stroke risk more efficiently.

#2985 Gut microbiome-dependent systemic alterations in experimental chronic kidney disease

Abstract Background and Aims Patients diagnosed with Chronic Kidney Disease (CKD) undergo a notable decline in both quality of life and life expectancy, primarily attributable to the heightened prevalence of cardiovascular diseases (CVD). These conditions not only contribute to the initiation of CKD but are also a consequence of inflammation associated with CKD. Despite optimal kidney replacement therapies, irreversible alterations of the CV system may persist. The responsible mechanisms in CKD leading to CVD are incompletely understood. CKD is characterized by gut microbial dysbiosis, with a significant proportion of uremic toxins suspected to have microbial origin. We hypothesize that the gut microbiota promotes inflammation and CV risk in states of impaired kidney function. Method Experimental CKD was induced in 129sv mice by 5/6 nephrectomy. Prior to nephrectomy, mice were assigned to receive either glucose (Ctrl) or a non-absorptive antibiotic cocktail (comprising Ampicillin, Metronidazole, Neomycin, and Vancomycin, Abx) via the drinking water to deplete their microbiomes throughout the 13-week study period. Ctrl and Abx-treated Sham-operated mice served as controls. Plasma, feces, and urine specimens were systematically collected at diverse time intervals, facilitating the longitudinal exploration of metabolite dynamics and CKD progression through an untargeted Nuclear Magnetic Resonance (NMR) approach. Simultaneously, echocardiography assessments were conducted at identical time points to assess cardiac function. Results Abx-treated CKD mice displayed improved kidney function, demonstrated by significantly reduced levels of plasma Cystatin C (0.2913 ± 0.1850 mg/L) compared to Ctrl (0.9133 ± 0.6616 mg/L)(p = 0.0256). Hearts from Abx-treated CKD mice exhibited significantly less remodeling, independent of their blood pressure (radiotelemetry measurements). The ratio of left ventricular (LV) mass to tibia length was 4.858 ± 0.3453 in the Abx-treated group compared to Ctrl (8.198 ± 0.3542) (p < 0.0001). Sirius red stained hearts revealed significantly less fibrosis in Abx-treated CKD mice (5.784 ± 5.465%) compared to Ctrl (11.98 ± 3.741%) (p = 0.0218). The cardiac expression of several damage-associated genes (Npbb, Ctgf, and Ngal) portrayed the same trend. To elucidate the interplay between the microbiome and the immune system underlying these observed effects, we provide a comprehensive atlas of the immune landscape in different organs (blood, spleen, heart, intestine), along with a catalogue of gut-derived metabolites, shedding light on the systemic alterations induced by microbiome-dependent mechanisms in CKD. Conclusion Our findings suggest that microbiome-dependent mechanisms drive kidney function decline and cardiac remodeling in experimental CKD. These results underscore the interplay between microbial alterations, metabolite dynamics, immune response, and cardiovascular complications in the context of CKD.

Comparison of Various Extraction Approaches for Optimized Preparation of Intracellular Metabolites from Human Mesenchymal Stem Cells and Fibroblasts for NMR-Based Study.

Metabolomics has proven to be a sensitive tool for monitoring biochemical processes in cell culture. It enables multi-analysis, clarifying the correlation between numerous metabolic pathways. Together with other analysis, it thus provides a global view of a cell's physiological state. A comprehensive analysis of molecular changes is also required in the case of mesenchymal stem cells (MSCs), which currently represent an essential portion of cells used in regenerative medicine. Reproducibility and correct measurement are closely connected to careful metabolite extraction, and sample preparation is always a critical point. Our study aimed to compare the efficiencies of four harvesting and six extraction methods. Several organic reagents (methanol, ethanol, acetonitrile, methanol-chloroform, MTBE) and harvesting approaches (trypsinization vs. scraping) were tested. We used untargeted nuclear magnetic resonance spectroscopy (NMR) to determine the most efficient method for the extraction of metabolites from human adherent cells, specifically human dermal fibroblasts adult (HDFa) and dental pulp stem cells (DPSCs). A comprehensive dataset of 29 identified and quantified metabolites were determined to possess statistically significant differences in the abundances of several metabolites when the cells were detached mechanically to organic solvent compared to when applying enzymes mainly in the classes of amino acids and peptides for both types of cells. Direct scraping to organic solvent is a method that yields higher abundances of determined metabolites. Extraction with the use of different polar reagents, 50% and 80% methanol, or acetonitrile, mostly showed the same quality. For both HDFa and DPSC cells, the MTBE method, methanol-chloroform, and 80% ethanol extractions showed higher extraction efficiency for the most identified and quantified metabolites Thus, preparation procedures provided a cell sample processing protocol that focuses on maximizing extraction yield. Our approach may be useful for large-scale comparative metabolomic studies of human mesenchymal stem cell samples.

Pd2Spermine as an Alternative Therapeutics for Cisplatin-Resistant Triple-Negative Breast Cancer.

Cisplatin (cDDP) resistance is a matter of concern in triple-negative breast cancer therapeutics. We measured the metabolic response of cDDP-sensitive (S) and -resistant (R) MDA-MB-231 cells to Pd2Spermine(Spm) (a possible alternative to cDDP) compared to cDDP to investigate (i) intrinsic response/resistance mechanisms and (ii) the potential cytotoxic role of Pd2Spm. Cell extracts were analyzed by untargeted nuclear magnetic resonance metabolomics, and cell media were analyzed for particular metabolites. CDDP-exposed S cells experienced enhanced antioxidant protection and small deviations in the tricarboxylic acid cycle (TCA), pyrimidine metabolism, and lipid oxidation (proposed cytotoxicity signature). R cells responded more strongly to cDDP, suggesting a resistance signature of activated TCA cycle, altered AMP/ADP/ATP and adenine/uracil fingerprints, and phospholipid biosynthesis (without significant antioxidant protection). Pd2Spm impacted more markedly on R/S cell metabolisms, inducing similarities to cDDP/S cells (probably reflecting high cytotoxicity) and strong additional effects indicative of amino acid depletion, membrane degradation, energy/nucleotide adaptations, and a possible beneficial intracellular γ-aminobutyrate/glutathione-mediated antioxidant mechanism.

Advancing Natural Product Discovery: A Structure-Oriented Fractions Screening Platform for Compound Annotation and Isolation.

Natural product discovery is hindered by the lack of tools that integrate untargeted nuclear magnetic resonance and mass spectrometry data on a library scale. This article describes the first application of the innovative NMR/MS-based machine learning tool, the "Structure-Oriented Fractions Screening Platform (SFSP)", enabling functional-group-guided fractionation and accelerating the discovery and characterization of undescribed natural products. The concept was applied to the extract of a marine fungus known to be a prolific producer of diverse natural products. With the assistance of SFSP, we isolated 24 flavipidin derivatives and five phenalenone analogues from Aspergillus sp. GE2-6, revealing 27 undescribed compounds. Compounds 7-22 were proposed as isomeric derivatives featuring a 5/6-ring fusion, formed by the dimerization of flavipidin E (5). Compounds 23 and 24 were envisaged as isomeric derivatives with a 6/5/6-ring fusion, generated through the degradation of two flavipidin E molecules. Furthermore, flavipidin A (1) and asperphenalenone E (28) exhibited potent anti-influenza (PR8) activities, with IC50 values of 21.9 ± 0.2 and 12.9 ± 0.1 μM, respectively. Meanwhile, asperphenalenone (26) and asperphenalenone P (27) treatments exhibited significant inhibition of HIV pseudovirus infection in 293FT cells, boasting IC50 values of 6.1 ± 0.9 and 4.6 ± 1.1 μM, respectively. Overall, SFSP streamlines natural product isolation through NMR and MS data integration, as showcased by the discovery of numerous undescribed flavipidins and phenalenones based on NMR olefinic signals and low-field hydroxy signals.

SAND: Automated Time-Domain Modeling of NMR Spectra Applied to Metabolite Quantification.

Developments in untargeted nuclear magnetic resonance (NMR) metabolomics enable the profiling of thousands of biological samples. The exploitation of this rich source of information requires a detailed quantification of spectral features. However, the development of a consistent and automatic workflow has been challenging because of extensive signal overlap. To address this challenge, we introduce the software Spectral Automated NMR Decomposition (SAND). SAND follows on from the previous success of time-domain modeling and automatically quantifies entire spectra without manual interaction. The SAND approach uses hybrid optimization with Markov chain Monte Carlo methods, employing subsampling in both time and frequency domains. In particular, SAND randomly divides the time-domain data into training and validation sets to help avoid overfitting. We demonstrate the accuracy of SAND, which provides a correlation of ∼0.9 with ground truth on cases including highly overlapped simulated data sets, a two-compound mixture, and a urine sample spiked with different amounts of a four-compound mixture. We further demonstrate an automated annotation using correlation networks derived from SAND decomposed peaks, and on average, 74% of peaks for each compound can be recovered in single clusters. SAND is available in NMRbox, the cloud computing environment for NMR software hosted by the Network for Advanced NMR (NAN). Since the SAND method uses time-domain subsampling (i.e., random subset of time-domain points), it has the potential to be extended to a higher dimensionality and nonuniformly sampled data.

Metabolic Evaluation of Lupin-Enriched Yogurt by Nuclear Magnetic Resonance Metabolomics.

Untargeted nuclear magnetic resonance (NMR) metabolomics was used to evaluate compositional changes during yogurt fermentation upon lupin enrichment compared to traditional conditions. Lupin significantly changed the sample metabolic profile and its time course dynamics, seemingly delaying microbial action. The levels of organic and amino acids were significantly altered, along with those of some sugars, nucleotides, and choline compounds. Lupin seemed to favor acetate and formate synthesis, compared to that of citrate and fumarate; a higher formate levels may suggest increased levels of Streptococcus thermophilus action, compared toLactobacillus bulgaricus. Lupin-yogurt was poorer in hippurate, lactose (and hence lactate), galactose, glucose-1-phosphate, and galactose-1-phosphate, containing higher orotate levels (possibly related to increased uridine derivatives), among other differences. Trigonelline was confirmed as a lupin marker, possibly together with glutamate and histidine. Other metabolite trajectories remained unchanged upon lupin addition, unveiling unaffected underlying processes. These results demonstrate the usefulness of untargeted NMR metabolomics to understand/develop new foodstuffs and their production processes, highlighting the identity of a variety of bioactive metabolites with importance for human health.

1H NMR Metabolomics on Pigs' Liver Exposed to Antibiotics Administration: An Explorative Study.

An untargeted Nuclear Magnetic Resonance (NMR) spectroscopy-based metabolomics approach was applied as a first attempt to explore the metabolome of pigs treated with antibiotics. The final goal was to investigate the possibility of discriminating between antibiotic-treated (TX group) and untreated pigs (CTRL group), with the further perspective of identifying the authentication tools for antibiotic-free pork supply chains. In particular, 41 samples of pig liver were subjected to a biphasic extraction to recover both the polar and the non-polar metabolites, and the 1H NMR spectroscopy analysis was performed on the two separate extracts. Unsupervised (principal component analysis) and supervised (orthogonal partial least squares discriminant analysis) multivariate statistical analysis of 1H NMR spectra data in the range 0-9 ppm provided metabolomic fingerprinting useful for the discrimination of pig livers based on the antibiotic treatment to which they were exposed. Moreover, within the signature patterns, significant discriminating metabolites were identified among carbohydrates, choline and derivatives, amino acids and some lipid-class molecules. The encouraging findings of this exploratory study showed the feasibility of the untargeted metabolomic approach as a novel strategy in the authentication framework of pork supply chains and open a new horizon for a more in-depth investigation.

Tumor Lipid Signatures Are Descriptive of Acquisition of Therapy Resistance in an Endocrine-Related Breast Cancer Mouse Model.

The lipid metabolism adaptations of estrogen and progesterone receptor-positive breast cancer tumors from a mouse syngeneic model are investigated in relation to differences across the transition from hormone-dependent (HD) to hormone-independent (HI) tumor growth and the acquisition of endocrine therapy (ET) resistance (HIR tumors). Results are articulated with reported polar metabolome results to complete a metabolic picture of the above transitions and suggest markers of tumor progression and aggressiveness. Untargeted nuclear magnetic resonance metabolomics was used to analyze tumor and mammary tissue lipid extracts. Tumor progression (HD-HI-HIR) was accompanied by increased nonesterified cholesterol forms and phospholipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelins, and plasmalogens) and decreased relative contents of triglycerides and fatty acids. Predominating fatty acids became shorter and more saturated on average. These results were consistent with gradually more activated cholesterol synthesis, β-oxidation, and phospholipid biosynthesis to sustain tumor growth, as well as an increase in cholesterol (possibly oxysterol) forms. Particular compound levels and ratios were identified as potential endocrine tumor HD-HI-HIR progression markers, supporting new hypotheses to explain acquired ET resistance.

Metabolic Disturbances in the Gut-brain Axis of a Mouse Model of MPTP-induced Parkinsonism Evaluated by Nuclear Magnetic Resonance

Parkinson's Disease is a synucleinopathy that primarily affects the dopaminergic cells of the central nervous system, leading to motor and gastrointestinal disturbances. However, intestinal peripheral neurons undergo a similar neurodegeneration process, marked by α-synuclein (αSyn) accumulation and loss of mitochondrial homeostasis. We investigated the metabolic alterations in different biometrics that compose the gut-brain axis (blood, brain, large intestine, and feces) in an MPTP-induced mouse model of sporadic Parkinson's Disease. Animals received escalating administration of MPTP. Tissues and fecal pellets were collected, and the metabolites were identified through the untargeted Nuclear Magnetic Resonance spectroscopic (1H NMR) technique. We found differences in many metabolites from all the tissues evaluated. The differential expression of metabolites in these samples mainly reflects inflammatory aspects, cytotoxicity, and mitochondrial impairment (oxidative stress and energy metabolism) in the animal model used. The direct evaluation of fecal metabolites revealed changes in several classes of metabolites. This data reinforces previous studies showing that Parkinson’s disease is associated with metabolic perturbation not only in brain-related tissues, but also in periphery structures such as the gut. In addition, the evaluation of the microbiome and metabolites from gut and feces emerge as promising sources of information for understanding the evolution and progression of sporadic Parkinson's Disease.

Untargeted Metabolomics Analysis Revealed the Difference of Component and Geographical Indication Markers of Panax notoginseng in Different Production Areas.

Panax notoginseng (P. notoginseng) has excellent medicinal and food dual-use characteristics. However, P. notoginseng with a unique origin label has become the target of fraud because of people confusing or hiding its origin. In this study, an untargeted nuclear magnetic resonance (NMR)-based metabolomics approach was used to discriminate the geographical origins of P. notoginseng from four major producing areas in China. Fifty-two components, including various saccharides, amino acids, saponins, organic acids, and alcohols, were identified and quantified through the NMR spectrum, and the area-specific geographical identification components were further screened. P. notoginseng from Yunnan had strong hypoglycemic and cardiovascular protective effects due to its high acetic acid, dopamine, and serine content, while P. notoginseng from Sichuan was more beneficial for diseases of the nervous system because of its high content of fumarate. P. notoginseng from Guizhou and Tibet had high contents of malic acid, notoginsenoside R1, and amino acids. Our results can help to distinguish the geographical origin of P. notoginseng and are readily available for nutritional recommendations in human consumption.

The influence of temperature rise on the metabolic response of Ruditapes philippinarum clams to 17-α-ethinylestradiol

Untargeted Nuclear Magnetic Resonance metabolomics was employed to study the effects of warming conditions (17–21 °C) and exposure to 17-α-ethinylestradiol (EE2) on the polar metabolome of Ruditapes philippinarum clams, to identify metabolic markers for monitoring/prediction of deviant environmental conditions. Warming alone triggered changes in alanine/aspartate/glutamate, aromatic amino acids, taurine/hypotaurine and homarine/trigonelline pathways, as well as in energy metabolism, suggesting osmoregulatory adaptations and glycolytic/tricarboxylic acid (TCA) cycle activation, possibly accompanied to some extent by gluconeogenesis to preserve glycogen reserves. At 17 °C, the lowest EE2 concentration (5 ng/L) specifically engaged branched-chain and aromatic amino acids to activate the glycolysis/TCA cycle. Notably, a partial metabolic recovery was observed at 25 ng/L, whereas higher EE2 concentrations (125 and 625 ng/L) again induced significant metabolic disturbances. These included enhanced glycogen biosynthesis and increased lipid reserves, sustained by low-level glutathione-based antioxidative mechanisms that seemed active. At 21 °C, response to EE2 was notably weak at low/intermediate concentrations, becoming particularly significant at the highest EE2 concentration (625 ng/L), suggesting higher protection capacity of Ruditapes philippinarum clams under warming conditions. At 625 ng/L, disturbances in alanine/aspartate/glutamate and taurine/hypotaurine metabolisms were observed, with no evidence of enhanced carbohydrate/protein catabolism. This low energy function profile was accompanied by marked antioxidative mechanisms and choline compounds modulation for cell membrane protection/repair. These results help monitor clams´ response to temperature rise and EE2 exposure, paving the way for future effective guidance and prediction of environmental damaging effects.

Nicotinamide as potential biomarker for Alzheimer's disease: A translational study based on metabolomics.

Introduction: The metabolic routes altered in Alzheimer's disease (AD) brain are poorly understood. As the metabolic pathways are evolutionarily conserved, the metabolic profiles carried out in animal models of AD could be directly translated into human studies. Methods: We performed untargeted Nuclear Magnetic Resonance metabolomics in hippocampus of McGill-R-Thy1-APP transgenic (Tg) rats, a model of AD-like cerebral amyloidosis and the translational potential of these findings was assessed by targeted Gas Chromatography-Electron Impact-Mass Spectrometry in plasma of participants in the German longitudinal cohort AgeCoDe. Results: In rat hippocampus 26 metabolites were identified. Of these 26 metabolites, nine showed differences between rat genotypes that were nominally significant. Two of them presented partial least square-discriminant analysis (PLS-DA) loadings with the larger absolute weights and the highest Variable Importance in Projection (VIP) scores and were specifically assigned to nicotinamide adenine dinucleotide (NAD) and nicotinamide (Nam). NAD levels were significantly decreased in Tg rat brains as compared to controls. In agreement with these results, plasma of AD patients showed significantly reduced levels of Nam in respect to cognitively normal participants. In addition, high plasma levels of Nam showed a 27% risk reduction of progressing to AD dementia within the following 2.5years, this hazard ratio is lost afterwards. Discussion: To our knowledge, this is the first report showing that a decrease of Nam plasma levels is observed couple of years before conversion to AD, thereby suggesting its potential use as biomarker for AD progression.

Plasma Metabolomic and Lipidomic Profiling of Metabolic Dysfunction-Associated Fatty Liver Disease in Humans Using an Untargeted Multiplatform Approach.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a complex disorder that is implicated in dysregulations in multiple biological pathways, orchestrated by interactions between genetic predisposition, metabolic syndromes and environmental factors. The limited knowledge of its pathogenesis is one of the bottlenecks in the development of prognostic and therapeutic options for MAFLD. Moreover, the extent to which metabolic pathways are altered due to ongoing hepatic steatosis, inflammation and fibrosis and subsequent liver damage remains unclear. To uncover potential MAFLD pathogenesis in humans, we employed an untargeted nuclear magnetic resonance (NMR) spectroscopy- and high-resolution mass spectrometry (HRMS)-based multiplatform approach combined with a computational multiblock omics framework to characterize the plasma metabolomes and lipidomes of obese patients without (n = 19) or with liver biopsy confirmed MAFLD (n = 63). Metabolite features associated with MAFLD were identified using a metabolome-wide association study pipeline that tested for the relationships between feature responses and MAFLD. A metabolic pathway enrichment analysis revealed 16 pathways associated with MAFLD and highlighted pathway changes, including amino acid metabolism, bile acid metabolism, carnitine shuttle, fatty acid metabolism, glycerophospholipid metabolism, arachidonic acid metabolism and steroid metabolism. These results suggested that there were alterations in energy metabolism, specifically amino acid and lipid metabolism, and pointed to the pathways being implicated in alerted liver function, mitochondrial dysfunctions and immune system disorders, which have previously been linked to MAFLD in human and animal studies. Together, this study revealed specific metabolic alterations associated with MAFLD and supported the idea that MAFLD is fundamentally a metabolism-related disorder, thereby providing new perspectives for diagnostic and therapeutic strategies.

Metabolic profiling of induced acute pancreatitis and pancreatic cancer progression in a mutant Kras mouse model.

Untargeted Nuclear Magnetic Resonance (NMR) metabolomics of polar extracts from the pancreata of a caerulin-induced mouse model of pancreatitis (Pt) and of a transgenic mouse model of pancreatic cancer (PCa) were used to find metabolic markers of Pt and to characterize the metabolic changes accompanying PCa progression. Using multivariate analysis a 10-metabolite metabolic signature specific to Pt tissue was found to distinguish the benign condition from both normal tissue and precancerous tissue (low grade pancreatic intraepithelial neoplasia, PanIN, lesions). The mice pancreata showed significant changes in the progression from normal tissue, through low-grade and high-grade PanIN lesions to pancreatic ductal adenocarcinoma (PDA). These included increased lactate production, amino acid changes consistent with enhanced anaplerosis, decreased concentrations of intermediates in membrane biosynthesis (phosphocholine and phosphoethanolamine) and decreased glycosylated uridine phosphates, reflecting activation of the hexosamine biosynthesis pathway and protein glycosylation.

Identification of chia, flax and sesame seeds authenticity markers by NMR-based untargeted metabolomics and their validation in bakery products containing them

Chia, flax, and sesame seeds are considered superseeds due to their beneficial nutritional properties, and they are frequently included as functional ingredients in foods. Authenticity markers of these seeds, including bakery products containing them, have been identified by both liquid and gas chromatography coupled to mass spectrometry (LC-MS/MS and GC–MS/MS, respectively) targeted metabolomics. However, there are no reports describing the use of nuclear magnetic resonance (NMR) spectroscopy based metabolomics to identify authenticity markers in either the raw seeds or foods containing them. We herein report the application of an untargeted NMR–based metabolomics workflow to the identification of authenticity markers for the three seeds. Seven markers, belonging to the families of polyphenols and cyanogenic glycosides, allowed good differentiation of the raw materials. Validation in cookies containing different seed percentages showed that two markers resisted the processing stage, making them feasible authenticity markers for the food trade.

Metabolic Regulation of Hepatitis B Virus Infection in HBV-Transgenic Mice.

Hepatitis B virus (HBV) infection is a worldwide health burden. Metabolomics analysis has revealed HBV-induced metabolism dysregulation in liver tissues and hepatocytes. However, as an infectious disease, the tissue-specific landscape of metabolic profiles of HBV infection remains unclear. To fill this gap, we applied untargeted nuclear magnetic resonance (NMR) metabolomic analysis of the heart, liver, spleen, lung, kidney, pancreas, and intestine (duodenum, jejunum, ileum) in HBV-transgenic mice and their wild-type littermates. Strikingly, we found systemic metabolic alterations induced by HBV in liver and extrahepatic organs. Significant changes in metabolites have been observed in most tissues of HBV-transgenic mice, except for ileum. The metabolic changes may provide novel therapeutic targets for the treatment of HBV infection. Moreover, tissue-specific metabolic profiles could speed up the study of HBV induced systemic metabolic reprogramming, which could help follow the progression of HBV infection and explain the underlying pathogenesis.

NMR Metabolomics Assessment of Osteogenic Differentiation of Adipose-Tissue-Derived Mesenchymal Stem Cells.

This Article presents, for the first time to our knowledge, an untargeted nuclear magnetic resonance (NMR) metabolomic characterization of the polar intracellular metabolic adaptations of human adipose-derived mesenchymal stem cells during osteogenic differentiation. The use of mesenchymal stem cells (MSCs) for bone regeneration is a promising alternative to conventional bone grafts, and untargeted metabolomics may unveil novel metabolic information on the osteogenic differentiation of MSCs, allowing their behavior to be understood and monitored/guided toward effective therapies. Our results unveiled statistically relevant changes in the levels of just over 30 identified metabolites, illustrating a highly dynamic process with significant variations throughout the whole 21-day period of osteogenic differentiation, mainly involving amino acid metabolism and protein synthesis; energy metabolism and the roles of glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation; cell membrane metabolism; nucleotide metabolism (including the specific involvement of O-glycosylation intermediates and NAD+); and metabolic players in protective antioxidative mechanisms (such as glutathione and specific amino acids). Different metabolic stages are proposed and are supported by putative biochemical explanations for the metabolite changes observed. This work lays the groundwork for the use of untargeted NMR metabolomics to find potential metabolic markers of osteogenic differentiation efficacy.

PSV-B-23 Metabolomic biomarker assessment of a Saccharomyces cerevisiae fermentate in exercise-stressed Labrador retrievers

Abstract Plasma metabolomic markers were evaluated in a population of 36 Labrador Retrievers (BW 31.32 ± 0.85 kg) studying the effects of a dietary Saccharomyces cerevisiae fermentation product (SCFP) at baseline and before and after two distance-defined exercise regimens (DDER; 6.4 km and 16.1 km). Canine subjects were blocked by BW and randomly assigned to one of two treatments: a control group receiving no supplement (CON) or a group receiving a single, daily oral dose of 250 mg SCFP. Each treatment group was comprised of 9 males and 9 females. During each DDER subjects were guided by an all-terrain vehicle and blood samples were collected at baseline and before and after each DDER. All metabolomic studies were performed blinded to treatment and DDER regimen. Metabolic signals were identified using untargeted nuclear magnetic resonance and mass spectroscopy. A total of 31 differentially relevant metabolites reflecting host metabolism were selected from a large array of signals along with an additional 24 metabolites associated with microbial or microbial-host co-metabolism. Predictive classification was performed using statistical causal inference to derive weights for probability-weighted principal components analysis, and statistical significance was subsequently calculated by using subsampling. Metabolomics data stratified by sex yielded a trend for a highly accurate predictive classification (P < 0.10) and between treatments for subjects born before 2012 (P < 0.05). However, differential metabolomics data without adjustment for age did not reveal differences in metabolic profiles. This study provides preliminary evidence that SCFP may elicit differential plasma metabolites with known relationships with various immune cell and gut microbiome derived functions that appear to be age dependent. Further study is needed to provide direct evidence that SCFP exerts immunomodulatory effects consistent with decreasing chronic inflammation while balancing pro-inflammatory and anti-inflammatory responses.

Pre- versus post-operative untargeted plasma nuclear magnetic resonance spectroscopy metabolomics of pheochromocytoma and paraganglioma

PurposePheochromocytomas and Paragangliomas (PPGL) result in chronic catecholamine excess and serious health complications. A recent study obtained a metabolic signature in plasma from PPGL patients; however, its targeted nature may have generated an incomplete picture and a broader approach could provide additional insights. We aimed to characterize the plasma metabolome of PPGL patients before and after surgery, using an untargeted approach, and to broaden the scope of the investigated metabolic impact of these tumors.DesignA cohort of 36 PPGL patients was investigated. Blood plasma samples were collected before and after surgical tumor removal, in association with clinical and tumor characteristics.MethodsPlasma samples were analyzed using untargeted nuclear magnetic resonance (NMR) spectroscopy metabolomics. The data were evaluated using a combination of uni- and multi-variate statistical methods.ResultsBefore surgery, patients with a nonadrenergic tumor could be distinguished from those with an adrenergic tumor based on their metabolic profiles. Tyrosine levels were significantly higher in patients with high compared to those with low BMI. Comparing subgroups of pre-operative samples with their post-operative counterparts, we found a metabolic signature that included ketone bodies, glucose, organic acids, methanol, dimethyl sulfone and amino acids. Three signals with unclear identities were found to be affected.ConclusionsOur study suggests that the pathways of glucose and ketone body homeostasis are affected in PPGL patients. BMI-related metabolite levels were also found to be altered, potentially linking muscle atrophy to PPGL. At baseline, patient metabolomes could be discriminated based on their catecholamine phenotype.