A multidimensional metabolomics workflow to image biodistribution and evaluate pharmacodynamics in adult zebrafish.

An integrated evaluation of the tissue distribution and pharmacodynamic properties of a therapeutic is essential for successful translation to the clinic. To date, however, cost-effective methods to measure these parameters at the systems level in model organisms are lacking. Here, we introduce a multidimensional workflow to evaluate drug activity that combines mass spectrometry-based imaging, absolute drug quantitation across different biological matrices, in vivo isotope tracing and global metabolome analysis in the adult zebrafish. As a proof of concept, we quantitatively determined the whole-body distribution of the anti-rheumatic agent hydroxychloroquine sulfate (HCQ) and measured the systemic metabolic impacts of drug treatment. We found that HCQ distributed to most organs in the adult zebrafish 24 h after addition of the drug to water, with the highest accumulation of both the drug and its metabolites being in the liver, intestine and kidney. Interestingly, HCQ treatment induced organ-specific alterations in metabolism. In the brain, for example, HCQ uniquely elevated pyruvate carboxylase activity to support increased synthesis of the neuronal metabolite, N-acetylaspartate. Taken together, this work validates a multidimensional metabolomics platform for evaluating the mode of action of a drug and its potential off-target effects in the adult zebrafish. This article has an associated First Person interview with the first author of the paper.

Metabolomic and lipidomic characterization of an X-chromosome deletion disorder in neural progenitor cells by UHPLC-HRMS.

INTRODUCTION: Intellectual disorders involving deletions of the X chromosome present a difficult task in the determination of a connection between symptoms and metabolites that could lead to treatment options. One specific disorder of X-chromosomal deletion, Fragile X syndrome, is the most frequently occurring of intellectual disabilities. Previous metabolomic studies have been limited to mouse models that may not have sufficiently revealed the full biochemical diversity of the disease in humans. OBJECTIVES: The primary objective of this study was to elucidate the human biochemistry in X-chromosomal deletion disorders through metabolomic and lipidomic profiling, using cells from a X-deletion patient as a representative case. METHODS: Metabolomic and lipidomic analysis was performed by UHPLC-HRMS on neural progenitor (NP) cells isolated from an afflicted female patient versus normal neural progenitor cells. RESULTS: Results showed perturbations in several metabolic pathways, including those of arginine and proline, that significantly impact both neurotransmitter generation and overall brain function. Coincidently, dysregulation was observed for lipids involved in both cellular structure and membrane integrity. The trends of observed metabolomic changes, as well as lipidomic profiling from identified features, are discussed. CONCLUSION: The lipidomic and metabolomic profiles of NP cell samples exhibited significant differentiation associated with partial deletion of the X chromosome. These findings suggest that rare X-chromosomal deletion disorders are not only a mental disorder limited to alterations in local neuronal functions, but are also metabolic diseases.

Extracellular Vesicle Analysis by Paper Spray Ionization Mass Spectrometry.

Paper spray ionization mass spectrometry (PSI-MS) is a direct MS analysis technique with several reported bacterial metabolomics applications. As with most MS-based bacterial studies, all currently reported PSI-MS bacterial analyses have focused on the chemical signatures of the cellular unit. One dimension of the bacterial metabolome that is often lost in such analyses is the exometabolome (extracellular metabolome), including secreted metabolites, lipids, and peptides. A key component of the bacterial exometabolome that is gaining increased attention in the microbiology and biomedical communities is extracellular vesicles (EVs). These excreted structures, produced by cells in all domains of life, contain a variety of biomolecules responsible for a wide array of cellular functions, thus representing a core component of the bacterial secreted metabolome. Although previously examined using other MS approaches, no reports currently exist for a PSI-MS analysis of bacterial EVs, nor EVs from any other organism (exosomes, ectosomes, etc.). PSI-MS holds unique analytical strengths over other commonly used MS platforms and could thus provide an advantageous approach to EV metabolomics. To address this, we report a novel application representing, to our knowledge, the first PSI-MS analysis of EVs from any organism (using the human gut resident Oxalobacter formigenes as the experimental model, a bacterium whose EVs were never previously investigated). In this report, we show how we isolated and purified EVs from bacterial culture supernatant by EV-specific affinity chromatography, confirmed and characterized these vesicles by nanoparticle tracking analysis, analyzed the EV isolate by PSI-MS, and identified a panel of EV-derived metabolites, lipids, and peptides. This work serves as a pioneering study in the field of MS-based EV analysis and provides a new, rapid, sensitive, and economical approach to EV metabolomics.

Metabolomics by UHPLC-HRMS reveals the impact of heat stress on pathogen-elicited immunity in maize.

INTRODUCTION: Studies investigating crop resistance to abiotic and biotic stress have largely focused on plant responses to singular forms of stress and individual biochemical pathways that only partially represent stress responses. Thus, combined abiotic and biotic stress treatments and the global assessment of their elicited metabolic expression remains largely unexplored. In this study, we employed targeted and untargeted metabolomics to investigate the molecular responses of maize (Zea mays) to abiotic, biotic, and combinatorial stress. OBJECTIVE: We compared the inducible metabolomes of heat-stressed (abiotic) and C. heterostrophus-infected (biotic) maize and examined the effects of heat stress on the ability of maize to defend itself against C. heterostrophus. METHODS: Ultra-high-performance liquid chromatography-high-resolution mass spectrometry was performed on plants grown under control conditions (28 °C), heat stress (38 °C), Cochliobolus heterostrophus infection, or combinatorial stress [heat (38 °C) + C. heterostrophus infection]. RESULTS: Multivariate analyses revealed differential metabolite expression between heat stress, C. heterostrophus infection, and their respective controls. In combinatorial experiments, treatment with heat stress prior to fungal inoculation negatively impacted maize disease resistance against C. heterostrophus, and distinct metabolome separation between combinatorial stressed plants and the non-heat-stressed infected controls was observed. Targeted analysis revealed inducible primary and secondary metabolite responses to abiotic/biotic stress, and combinatorial experiments indicated that deficiency in the hydroxycinnamic acid, p-coumaric acid, may contribute to the heat-induced susceptibility of maize to C. heterostrophus. CONCLUSION: These findings demonstrate that abiotic stress can predispose crops to more severe disease symptoms, underlining the increasing need to investigate defense chemistry in plants under combinatorial stress.

Oxalobacter formigenes produces metabolites and lipids undetectable in oxalotrophic Bifidobacterium animalis.

INTRODUCTION: In the search for new potential therapies for pathologies of oxalate, such as kidney stone disease and primary hyperoxaluria, the intestinal microbiome has generated significant interest. Resident oxalate-degrading bacteria inhabit the gastrointestinal tract and reduce absorption of dietary oxalate, thereby potentially lowering the potency of oxalate as a risk factor for kidney stone formation. Although several species of bacteria have been shown to degrade oxalate, select strains of Oxalobacter formigenes (O. formigenes) have thus far demonstrated the unique ability among oxalotrophs to initiate a net intestinal oxalate secretion into the lumen from the bloodstream, allowing them to feed on both dietary and endogenous metabolic oxalate. There is significant interest in this function as a potential therapeutic application for circulating oxalate reduction, although its mechanism of action is still poorly understood. Since this species-exclusive, oxalate-regulating function is reported to be dependent on the use of a currently unidentified secreted bioactive compound, there is much interest in whether O. formigenes produces unique biochemicals that are not expressed by other oxalotrophs which lack the ability to transport oxalate. Hence, this study sought to analyze and compare the metabolomes of O. formigenes and another oxalate degrader, Bifidobacterium animalis subsp. lactis (B. animalis), to determine whether O. formigenes could produce features undetectable in another oxalotroph, thus supporting the theory of a species-exclusive secretagogue compound. METHODS: A comparative metabolomic analysis of O. formigenes strain HC1 (a human isolate) versus B. animalis, another oxalate-degrading human intestinal microbe, was performed by ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). Bacteria were cultured independently in anaerobic conditions, harvested, lysed, and extracted by protein precipitation. Metabolite extracts were chromatographically separated and analyzed by UHPLC-HRMS using reverse phase gradient elution (ACE Excel 2 C18-Pentafluorophenyl column) paired with a Q Exactive™ mass spectrometer. OBJECTIVES: The purpose of this study was to assess whether O. formigenes potentially produces unique biochemicals from other oxalate degraders to better understand its metabolic profile and provide support for the theoretical production of a species-exclusive secretagogue compound responsible for enhancing intestinal oxalate secretion. RESULTS: We report a panel of metabolites and lipids detected in the O. formigenes metabolome which were undetectable in B. animalis, several of which were identified either by mass-to-charge ratio and retention time matching to our method-specific metabolite library or MS/MS fragmentation. Furthermore, re-examination of data from our previous work showed most of these features were also undetected in the metabolomes of Lactobacillus acidophilus and Lactobacillus gasseri, two other intestinal oxalate degraders. CONCLUSIONS: Our observation of O. formigenes metabolites and lipids which were undetectable in other oxalotrophs suggests that this bacterium likely holds the ability to produce biochemicals not expressed by at least a selection of other oxalate degraders. These findings provide support for the hypothesized biosynthesis of a species-exclusive secretagogue responsible for the stimulation of net intestinal oxalate secretion.

Metabolomic Alteration in the Mouse Distal Colonic Mucosa after Oral Gavage with Oxalobacter formigenes.

Oxalobacter formigenes has been investigated for years due to its proposed ability to produce a secretagogue compound that initiates net intestinal oxalate secretion, thereby theoretically reducing circulating oxalate and risk of kidney stone formation. Strains which have been shown to exhibit this function in vivo across native tissue include the human strain, HC1, and the wild rat strain, OxWR. While previous work on these secretagogue-relevant strains has focused on profiling their metabolome and lipidome in vitro, efforts to characterize their influence on host intestinal mucosal biochemistry in vivo are yet to be reported. Much work has been done over the years with O. formigenes in relation to the secretagogue hypothesis, but it has never been clearly demonstrated that this microorganism is capable of inducing metabolic changes in native host tissue, which would be expected with the production of a transport-inducing compound. In this work, we show how the distal colonic mucosal metabolomic profile in a mouse model exhibited significant changes in the levels of a variety of metabolites as a result of oral gavage with O. formigenes HC1. Among these significant metabolites was nicotinic acid, an essential nutrient shown in past work to be produced in the gut by the native microbiome. Our finding that the in vivo biochemical state of the distal colon was altered with O. formigenes lends support to the secretagogue hypothesis and serves as a pioneering step in characterizing the biochemical interplay between O. formigenes and the mammalian host.

Impact of matrix effects and ionization efficiency in non-quantitative untargeted metabolomics.

INTRODUCTION: LC-MS-based untargeted metabolomics has become increasingly popular due to the vast amount of information gained in a single analysis. Many studies utilize metabolomics to profile metabolic changes in various representative biofluids, tissues, or other sample types. Most analyses are performed measuring changes in the metabolic pool of a single biological matrix due to an altered phenotype, such as disease versus normal. Measurements in such experiments are typically highly reproducible with little variation due to analytical and technological advancements in mass spectrometry. With the expanded application of metabolomics into various non-analytical scientific disciplines, the emergence of studies comparing the signal intensities of specific analytes across different biological matrices (e.g. plasma vs. urine) is becoming more common, but the matrix effect between sample types is often neglected. Additionally, the practice of comparing the signal intensities of different analytes and correlating to relative abundance is also increasingly prevalent, but the response ratio between analytes due to differences in ionization efficiency is not always accounted for in data analysis. This report serves to communicate and raise awareness of these two well-recognized issues to prevent improper data interpretation in the field of metabolomics. OBJECTIVES: We demonstrate the impact of matrix effects and ionization efficiency with labeled analytical standards in human plasma, serum, and urine and describe how the direct comparison of non-quantitative signal intensities between biofluids, as well as between different analytes in the same biofluid, in untargeted metabolomics is inaccurate without proper response corrections. METHODS: Human plasma, serum, and urine (n = 4 technical replicates per biofluid) were spiked with a panel of labeled internal standards all at identical concentrations, simultaneously extracted, and analyzed by UHPLC-HRMS. Signal intensities were compared for demonstration of the impact of matrix effects in untargeted metabolomics. A neat mixture of two co-eluting, structurally-similar labeled standards at the same concentration was also analyzed to demonstrate the effect of ionization efficiency on signal intensity. RESULTS: Despite being spiked at identical concentrations, labeled standards we examined in this study showed significant differences in their signal intensities between biofluids, as well as from each other in the same biofluid, due to matrix effects. Co-eluting standards were also found to yield significantly different signal intensities at identical concentrations due to differences in ionization efficiency. CONCLUSIONS: Due to the presence of matrix effects in untargeted, non-quantitative metabolomics, the signal intensity of any single analyte cannot be directly compared to the signal intensity of that same analyte (or any other analyte) between any two different matrices. Due to differences in ionization efficiency, the signal intensity of any single analyte cannot be directly compared to the signal intensity of any other analyte, even in the same matrix.

Metabolomic profiling of oxalate-degrading probiotic Lactobacillus acidophilus and Lactobacillus gasseri.

Oxalate, a ubiquitous compound in many plant-based foods, is absorbed through the intestine and precipitates with calcium in the kidneys to form stones. Over 80% of diagnosed kidney stones are found to be calcium oxalate. People who form these stones often experience a high rate of recurrence and treatment options remain limited despite decades of dedicated research. Recently, the intestinal microbiome has become a new focus for novel therapies. Studies have shown that select species of Lactobacillus, the most commonly included genus in modern probiotic supplements, can degrade oxalate in vitro and even decrease urinary oxalate in animal models of Primary Hyperoxaluria. Although the purported health benefits of Lactobacillus probiotics vary significantly between species, there is supporting evidence for their potential use as probiotics for oxalate diseases. Defining the unique metabolic properties of Lactobacillus is essential to define how these bacteria interact with the host intestine and influence overall health. We addressed this need by characterizing and comparing the metabolome and lipidome of the oxalate-degrading Lactobacillus acidophilus and Lactobacillus gasseri using ultra-high-performance liquid chromatography-high resolution mass spectrometry. We report many species-specific differences in the metabolic profiles of these Lactobacillus species and discuss potential probiotic relevance and function resulting from their differential expression. Also described is our validation of the oxalate-degrading ability of Lactobacillus acidophilus and Lactobacillus gasseri, even in the presence of other preferred carbon sources, measuring in vitro 14C-oxalate consumption via liquid scintillation counting.

The proteome of extracellular vesicles released by clastic cells differs based on their substrate.

Extracellular vesicles (EVs) from osteoclasts are important regulators in intercellular communication. Here, we investigated the proteome of EVs from clastic cells plated on plastic (clasts), bone (osteoclasts) and dentin (odontoclasts) by two-dimensional high performance liquid chromatography mass spectrometry seeking differences attributable to distinct mineralized matrices. A total of 1,952 proteins were identified. Of the 500 most abundant proteins in EVs, osteoclast and odontoclast EVs were 83.3% identical, while clasts shared 70.7% of the proteins with osteoclasts and 74.2% of proteins with odontoclasts. For each protein, the differences between the total ion count values were mapped to an expression ratio histogram (Z-score) in order to detect proteins differentially expressed. Stabilin-1 and macrophage mannose receptor-1 were significantly-enriched in EVs from odontoclasts compared with osteoclasts (Z = 2.45, Z = 3.34) and clasts (Z = 13.86, Z = 1.81) and were abundant in odontoclast EVs. Numerous less abundant proteins were differentially-enriched. Subunits of known protein complexes were abundant in clastic EVs, and were present at levels consistent with them being in assembled protein complexes. These included the proteasome, COP1, COP9, the T complex and a novel sub-complex of vacuolar H+-ATPase (V-ATPase), which included the (pro) renin receptor. The (pro) renin receptor was immunoprecipitated using an anti-E-subunit antibody from detergent-solubilized EVs, supporting the idea that the V-ATPase subunits present were in the same protein complex. We conclude that the protein composition of EVs released by clastic cells changes based on the substrate. Clastic EVs are enriched in various protein complexes including a previously undescribed V-ATPase sub-complex.

Strain-Level Differentiation of Bacteria by Paper Spray Ionization Mass Spectrometry.

Paper spray ionization mass spectrometry (PSI-MS) is a relatively new analytical technique allowing for rapid mass spectrometric analysis of biological samples with little or no sample preparation. The expeditious nature of the analysis and minimal requirement for sample preparation make PSI-MS a promising avenue for future clinical assays with one potential application in the identification of different types of bacteria. Although past PSI-MS studies have demonstrated the ability to distinguish between bacteria of different species and morphological classes, achieving within-species strain-level differentiation has never been performed. In this report, we demonstrate the first strain-level bacterial differentiation by PSI-MS with the mammalian intestinal bacterium Oxalobacter formigenes ( Oxf). This novel application holds promising clinical significance as it could be used to differentiate between pathogenic bacteria and their harmless, commensal relatives, saving time and money in clinical diagnostics. Both whole cells and cell lysates of  Oxf strains HC1 and OxWR were analyzed using the Prosolia Velox 360TM PSI source coupled to a Thermo Scientific Q Exactive high-resolution mass spectrometer with a rapid 30 s analytical method. Multivariate statistical analysis followed by examination of significant features provided for and confirmed differentiation between Oxf HC1 and OxWR. We report a panel of strain-exclusive metabolites that could serve as potential strain-indicating biomarkers.

Metabolomic and lipidomic characterization of Oxalobacter formigenes strains HC1 and OxWR by UHPLC-HRMS.

Diseases of oxalate, such as nephrolithiasis and primary hyperoxaluria, affect a significant portion of the US population and have limited treatment options. Oxalobacter formigenes, an obligate oxalotrophic bacterium in the mammalian intestine, has generated great interest as a potential probiotic or therapeutic treatment for oxalate-related conditions due to its ability to degrade both exogenous (dietary) and endogenous (metabolic) oxalate, lowering the risk of hyperoxaluria/hyperoxalemia. Although all oxalotrophs degrade dietary oxalate, Oxalobacter formigenes is the only species shown to initiate intestinal oxalate secretion to draw upon endogenous, circulating oxalate for consumption. Evidence suggests that Oxalobacter regulates oxalate transport proteins in the intestinal epithelium using an unidentified secreted bioactive compound, but the mechanism of this function remains elusive. It is essential to gain an understanding of the biochemical relationship between Oxalobacter and the host intestinal epithelium for this microbe to progress as a potential remedy for oxalate diseases. This investigation includes the first profiling of the metabolome and lipidome of Oxalobacter formigenes, specifically the human strain HC1 and rat strain OxWR, the only two strains shown thus far to initiate net intestinal oxalate secretion across native gut epithelia. This study was performed using untargeted and targeted metabolomics and lipidomics methodologies utilizing ultra-high-performance liquid chromatography-mass spectrometry. We report our findings that the metabolic profiles of these strains, although largely conserved, show significant differences in their expression of many compounds. Several strain-specific features were also detected. Discussed are trends in the whole metabolic profile as well as in individual features, both identified and unidentified. Graphical abstract ᅟ.