Hyperpolarized 13 C†NMR Spectroscopy of Urine Samples at Natural Abundance by Quantitative Dissolution Dynamic Nuclear Polarization.

Hyperpolarized nuclear magnetic resonance (NMR) offers an ensemble of methods that remarkably address the sensitivity issues of conventional NMR. Dissolution Dynamic Nuclear Polarization (d-DNP) provides a unique and general way to detect 13 C NMR signals with a sensitivity enhanced by several orders of magnitude. The expanding application scope of d-DNP now encompasses the analysis of complex mixtures at natural 13 C abundance. However, the application of d-DNP in this area has been limited to metabolite extracts. Here, we report the first d-DNP-enhanced 13 C NMR analysis of a biofluid -urine- at natural abundance, offering unprecedented resolution and sensitivity for this challenging type of sample. We also show that accurate quantitative information on multiple targeted metabolites can be retrieved through a standard addition procedure.

Hyperpolarized NMR metabolomics.

Hyperpolarized NMR is a promising approach to address the sensitivity limits of conventional NMR metabolomics approaches, which currently fails to detect minute metabolite concentrations in biological samples. This review describes how tremendous signal enhancement offered by dissolution-dynamic nuclear polarization and parahydrogen-based techniques can be fully exploited for molecular omics sciences. Recent developments, including the combination of hyperpolarization techniques with fast multi-dimensional NMR implementation and quantitative workflows are described, and a comprehensive comparison of existing hyperpolarization techniques is proposed. High-throughput, sensitivity, resolution and other relevant challenges that should be tackled for a general application of hyperpolarized NMR in metabolomics are discussed.

Characterizing the metabolic effects of the selective inhibition of gut microbial ß-glucuronidases in mice.

The hydrolysis of xenobiotic glucuronides by gut bacterial glucuronidases reactivates previously detoxified compounds resulting in severe gut toxicity for the host. Selective bacterial ß-glucuronidase inhibitors can mitigate this toxicity but their impact on wider host metabolic processes has not been studied. To investigate this the inhibitor 4-(8-(piperazin-1-yl)-1,2,3,4-tetrahydro-[1,2,3]triazino[4',5':4,5]thieno[2,3-c]isoquinolin-5-yl)morpholine (UNC10201652, Inh 9) was administered to mice to selectively inhibit a narrow range of bacterial ß-glucuronidases in the gut. The metabolomic profiles of the intestinal contents, biofluids, and several tissues involved in the enterohepatic circulation were measured and compared to control animals. No biochemical perturbations were observed in the plasma, liver or gall bladder. In contrast, the metabolite profiles of urine, colon contents, feces and gut wall were altered compared to the controls. Changes were largely restricted to compounds derived from gut microbial metabolism. This work establishes that inhibitors targeted towards bacterial ß-glucuronidases modulate the functionality of the intestinal microbiota without adversely impacting the host metabolic system.

Nuclear Magnetic Resonance Spectroscopy in Clinical Metabolomics and Personalized Medicine: Current Challenges and Perspectives.

Personalized medicine is probably the most promising area being developed in modern medicine. This approach attempts to optimize the therapies and the patient care based on the individual patient characteristics. Its success highly depends on the way the characterization of the disease and its evolution, the patient's classification, its follow-up and the treatment could be optimized. Thus, personalized medicine must combine innovative tools to measure, integrate and model data. Towards this goal, clinical metabolomics appears as ideally suited to obtain relevant information. Indeed, the metabolomics signature brings crucial insight to stratify patients according to their responses to a pathology and/or a treatment, to provide prognostic and diagnostic biomarkers, and to improve therapeutic outcomes. However, the translation of metabolomics from laboratory studies to clinical practice remains a subsequent challenge. Nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) are the two key platforms for the measurement of the metabolome. NMR has several advantages and features that are essential in clinical metabolomics. Indeed, NMR spectroscopy is inherently very robust, reproducible, unbiased, quantitative, informative at the structural molecular level, requires little sample preparation and reduced data processing. NMR is also well adapted to the measurement of large cohorts, to multi-sites and to longitudinal studies. This review focus on the potential of NMR in the context of clinical metabolomics and personalized medicine. Starting with the current status of NMR-based metabolomics at the clinical level and highlighting its strengths, weaknesses and challenges, this article also explores how, far from the initial "opposition" or "competition", NMR and MS have been integrated and have demonstrated a great complementarity, in terms of sample classification and biomarker identification. Finally, a perspective discussion provides insight into the current methodological developments that could significantly raise NMR as a more resolutive, sensitive and accessible tool for clinical applications and point-of-care diagnosis. Thanks to these advances, NMR has a strong potential to join the other analytical tools currently used in clinical settings.

A targeted ultra performance liquid chromatography - Tandem mass spectrometric assay for tyrosine and metabolites in urine and plasma: Application to the effects of antibiotics on mice.

Tyrosine plays a key role in mammalian biochemistry and defects in its metabolism (e.g., tyrosinemia, alkaptonuria etc.) have significant adverse consequences for those affected if left untreated. In addition, gut bacterially-derived p-cresol and its metabolites are of interest as a result of various effects on host xenobiotic metabolism. A fit-for-purpose quantitative ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay was developed to target and quantify tyrosine and eleven metabolites in urine and plasma. Dansylation, using dansyl chloride, was used to improve chromatographic and mass spectral properties for tyrosine and nine phenolic metabolites, with detection using positive electrospray ionisation (ESI). The sulfate and glucuronide conjugates of p-cresol, where the phenol group was blocked, were quantified intact, using negative ESI via polarity switching during the same run. Sample preparation for urine and plasma involved deproteinization by solvent precipitation (of acetonitrile:isopropyl alcohol (1:1 v/v)) followed by in situ dansylation in 96 well plates. To minimize sample and solvent usage, and maximize sensitivity, analysis was performed using microbore reversed-phase gradient UPLC on a C8 phase with a 7.5 min. cycle time. The coefficients of variation obtained were <15%, with lower limits of quantification ranging from 5 to 250 nM depending upon the analyte. The method was applied to plasma and urine samples obtained from mice placed on a high tyrosine diet with one subgroup of animals subsequently receiving antibiotics to suppress the gut microbiota. Whilst plasma profiles were largely unaffected by antibiotic treatment clear reductions in the amount of p-cresol sulfate and p-cresol glucuronide excreted in the urine were observed for these mice.

A Two-Way Interaction between Methotrexate and the Gut Microbiota of Male Sprague-Dawley Rats.

Methotrexate (MTX) is a chemotherapeutic agent that can cause a range of toxic side effects including gastrointestinal damage, hepatotoxicity, myelosuppression, and nephrotoxicity and has potentially complex interactions with the gut microbiome. Following untargeted UPLC-qtof-MS analysis of urine and fecal samples from male Sprague-Dawley rats administered at either 0, 10, 40, or 100 mg/kg of MTX, dose-dependent changes in the endogenous metabolite profiles were detected. Semiquantitative targeted UPLC-MS detected MTX excreted in urine as well as MTX and two metabolites, 2,4-diamino-N-10-methylpteroic acid (DAMPA) and 7-hydroxy-MTX, in the feces. DAMPA is produced by the bacterial enzyme carboxypeptidase glutamate 2 (CPDG2) in the gut. Microbiota profiling (16S rRNA gene amplicon sequencing) of fecal samples showed an increase in the relative abundance of Firmicutes over the Bacteroidetes at low doses of MTX but the reverse at high doses. Firmicutes relative abundance was positively correlated with DAMPA excretion in feces at 48 h, which were both lower at 100 mg/kg compared to that seen at 40 mg/kg. Overall, chronic exposure to MTX appears to induce community and functionality changes in the intestinal microbiota, inducing downstream perturbations in CPDG2 activity, and thus may delay MTX detoxication to DAMPA. This reduction in metabolic clearance might be associated with increased gastrointestinal toxicity.

A comparison of collision cross section values obtained via travelling wave ion mobility-mass spectrometry and ultra high performance liquid chromatography-ion mobility-mass spectrometry: Application to the characterisation of metabolites in rat urine.

A comprehensive Collision Cross Section (CCS) library was obtained via Travelling Wave Ion Guide mobility measurements through direct infusion (DI). The library consists of CCS and Mass Spectral (MS) data in negative and positive ElectroSpray Ionisation (ESI) mode for 463 and 479 endogenous metabolites, respectively. For both ionisation modes combined, TWCCSN2 data were obtained for 542 non-redundant metabolites. These data were acquired on two different ion mobility enabled orthogonal acceleration QToF MS systems in two different laboratories, with the majority of the resulting TWCCSN2 values (from detected compounds) found to be within 1% of one another. Validation of these results against two independent, external TWCCSN2 data sources and predicted TWCCSN2 values indicated to be within 1-2% of these other values. The same metabolites were then analysed using a rapid reversed-phase ultra (high) performance liquid chromatographic (U(H)PLC) separation combined with IM and MS (IM-MS) thus providing retention time (tr), m/z and TWCCSN2 values (with the latter compared with the DI-IM-MS data). Analytes for which TWCCSN2 values were obtained by U(H)PLC-IM-MS showed good agreement with the results obtained from DI-IM-MS. The repeatability of the TWCCSN2 values obtained for these metabolites on the different ion mobility QToF systems, using either DI or LC, encouraged the further evaluation of the U(H)PLC-IM-MS approach via the analysis of samples of rat urine, from control and methotrexate-treated animals, in order to assess the potential of the approach for metabolite identification and profiling in metabolic phenotyping studies. Based on the database derived from the standards 63 metabolites were identified in rat urine, using positive ESI, based on the combination of tr, TWCCSN2 and MS data.

Para-cresol production by Clostridium difficile affects microbial diversity and membrane integrity of Gram-negative bacteria.

Clostridium difficile is a Gram-positive spore-forming anaerobe and a major cause of antibiotic-associated diarrhoea. Disruption of the commensal microbiota, such as through treatment with broad-spectrum antibiotics, is a critical precursor for colonisation by C. difficile and subsequent disease. Furthermore, failure of the gut microbiota to recover colonisation resistance can result in recurrence of infection. An unusual characteristic of C. difficile among gut bacteria is its ability to produce the bacteriostatic compound para-cresol (p-cresol) through fermentation of tyrosine. Here, we demonstrate that the ability of C. difficile to produce p-cresol in vitro provides a competitive advantage over gut bacteria including Escherichia coli, Klebsiella oxytoca and Bacteroides thetaiotaomicron. Metabolic profiling of competitive co-cultures revealed that acetate, alanine, butyrate, isobutyrate, p-cresol and p-hydroxyphenylacetate were the main metabolites responsible for differentiating the parent strain C. difficile (630Δerm) from a defined mutant deficient in p-cresol production. Moreover, we show that the p-cresol mutant displays a fitness defect in a mouse relapse model of C. difficile infection (CDI). Analysis of the microbiome from this mouse model of CDI demonstrates that colonisation by the p-cresol mutant results in a distinctly altered intestinal microbiota, and metabolic profile, with a greater representation of Gammaproteobacteria, including the Pseudomonales and Enterobacteriales. We demonstrate that Gammaproteobacteria are susceptible to exogenous p-cresol in vitro and that there is a clear divide between bacterial Phyla and their susceptibility to p-cresol. In general, Gram-negative species were relatively sensitive to p-cresol, whereas Gram-positive species were more tolerant. This study demonstrates that production of p-cresol by C. difficile has an effect on the viability of intestinal bacteria as well as the major metabolites produced in vitro. These observations are upheld in a mouse model of CDI, in which p-cresol production affects the biodiversity of gut microbiota and faecal metabolite profiles, suggesting that p-cresol production contributes to C. difficile survival and pathogenesis.