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Magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) allow the chemical analysis of physiological processes in vivo and provide powerful tools in the life sciences and for clinical diagnostics. Excellent homogeneity of the static B(0)magnetic field over the object of interest is essential for achieving high-quality spectral results and quantitative metabolic measurements. The experimental minimization of B(0)variation is performed in a process called B(0)shimming. In this article, we summarize the concepts of B(0)field shimming using spherical harmonic shimming techniques, specific strategies for B(0)homogenization and crucial factors to consider for implementation and use in both brain and body. In addition, experts' recommendations are provided for minimum requirements for B(0)shim hardware and evaluation criteria for the primary outcome of adequate B(0)shimming for MRS and MRSI, such as the water spectroscopic linewidth.

With a 40‐year history of use for in vivo studies, the terminology used to describe the methodology and results of magnetic resonance spectroscopy (MRS) has grown substantially and is not consistent in many aspects. Given the platform offered by this special issue on advanced MRS methodology, the authors decided to describe many of the implicated terms, to pinpoint differences in their meanings and to suggest specific uses or definitions. This work covers terms used to describe all aspects of MRS, starting from the description of the MR signal and its theoretical basis to acquisition methods, processing and to quantification procedures, as well as terms involved in describing results, for example, those used with regard to aspects of quality, reproducibility or indications of error. The descriptions of the meanings of such terms emerge from the descriptions of the basic concepts involved in MRS methods and examinations. This paper also includes specific suggestions for future use of terms where multiple conventions have emerged or coexisted in the past., This work defines terms and explains concepts used to describe all aspects of MR spectroscopy, starting from the definition of the MR signal and its theoretical basis to acquisition methods, processing and quantification procedures, as well as terms involved in describing results, including quality aspects, reproducibility or indications of error. Also featured are specific suggestions for future use of terms where multiple conventions have emerged or previously coexisted.

Purpose While PRESS is often employed to measure glutamate concentrations, MEGA-PRESS enables simultaneous Glx (glutamate and glutamine) and GABA measurements. This study aimed to compare validation, repeatability, and concordance of different approaches for glutamate quantification at 3 T to aid future studies in their selection of the appropriate sequence and quantification method. Materials and methods Nine phantoms with different glutamate and glutamine concentrations and five healthy participants were scanned twice to assess respectively the validation and repeatability of measurements with PRESS and MEGA-PRESS. To assess concordance between the different methods, results from 95 human participants were compared. PRESS, MEGA-PRESS (i.e. difference), and the MEGA-PRESS OFF spectra were analyzed with both LCModel and Gannet. Results In vitro, excellent agreement was shown between actual and measured glutamate concentrations for all measurements (r > 0.98). In vivo CVs were better for PRESS (2.9%) than MEGA-PRESS (4.9%) and MEGA-PRESS OFF (4.2%). However, the concordance between the sequences was low (PRESS and MEGA-PRESS OFF, r = 0.3) to modest (MEGA-PRESS versus MEGA-PRESS OFF, r = 0.8). Conclusion Both PRESS and MEGA-PRESS can be employed to measure in vivo glutamate concentrations, although PRESS shows a better repeatability. Comparisons between in vivo glutamate measures of different sequences however need to be interpreted cautiously.

Inborn errors of metabolism (IEMs) are rare diseases produced by the accumulation of abnormal amounts of metabolites, toxic to the newborn. When not detected on time, they can lead to irreversible physiological and psychological sequels or even demise. Metabolomics has emerged as an efficient and powerful tool for IEM detection in newborns, children, and adults with late onset. In here, we screened urine samples from a large set of neonates (470 individuals) from a homogeneous population (Basque Country), for the identification of congenital metabolic diseases using NMR spectroscopy. Absolute quantification allowed to derive a probability function for up to 66 metabolites that adequately describes their normal concentration ranges in newborns from the Basque Country. The absence of another 84 metabolites, considered abnormal, was routinely verified in the healthy newborn population and confirmed for all but 2 samples, of which one showed toxic concentrations of metabolites associated to ketosis and the other one a high trimethylamine concentration that strongly suggested an episode of trimethylaminuria. Thus, a non-invasive and readily accessible urine sample contains enough information to assess the potential existence of a substantial number (>70) of IEMs in newborns, using a single, automated and standardized H-1-NMR-based analysis. The authors thank the collaboration of the Basque Biobank/BioCruces Node www.biobancovasco.org for collecting the samples and data from the neonates included in this study. Support was provided from The Department of Industry, Tourism and Trade of the Government of the Autonomous Community of the Basque Country (Elkartek BG2015 & BG2017); grant from Agencia Estatal de Investigacion (Spain) CTQ2015-68756-R and for the Severo Ochoa Excellence Accreditation (SEV-2016-0644).

Background Rodent models are invaluable for studying biological processes in the context of whole organisms. The reproducibility of such research is based on an assumption of metabolic similarity between experimental animals, controlled for by breeding and housing strategies that minimise genetic and environmental variation. Here, we set out to demonstrate the effect of experimental uraemia on the rat urinary metabolome and gut microbiome but found instead that the effect of vendor shipment batch was larger in both areas than that of uraemia. Results Twenty four Wistar rats obtained from the same commercial supplier in two separate shipment batches underwent either subtotal nephrectomy or sham procedures. All animals undergoing subtotal nephrectomy developed an expected uraemic phenotype. The urinary metabolome was studied using 1H-NMR spectroscopy and found to vary significantly between animals from different batches, with substantial differences in concentrations of a broad range of substances including lactate, acetate, glucose, amino acids, amines and benzoate derivatives. In animals from one batch, there was a complete absence of the microbiome-associated urinary metabolite hippurate, which was present in significant concentrations in animals from the other batch. These differences were so prominent that we would have drawn quite different conclusions about the effect of uraemia on urinary phenotype depending on which batch of animals we had used. Corresponding differences were seen in the gut microbiota between animals in different batches when assessed by the sequencing of 16S rRNA gene amplicons, with higher alpha diversity and different distributions of Proteobacteria subtaxa and short-chain fatty acid producing bacteria in the second batch compared to the first. Whilst we also demonstrated differences in both the urinary metabolome and gut microbiota associated with uraemia, these effects were smaller in size than those associated with shipment batch. Conclusions These results challenge the assumption that experimental animals obtained from the same supplier are metabolically comparable, and provide metabolomic evidence that batch-to-batch variations in the microbiome of experimental animals are significant confounders in an experimental study. We discuss strategies for reducing such variability and the need for transparency in research publications about the supply of experimental animals. Electronic supplementary material The online version of this article (10.1186/s40168-019-0738-y) contains supplementary material, which is available to authorized users.

Large-scale metabolic profiling requires the development of novel economical high-throughput analytical methods to facilitate characterization of systemic metabolic variation in population phenotypes. We report a fit-for-purpose direct infusion nanoelectrospray high-resolution mass spectrometry (DI-nESI-HRMS) method with time-of-flight detection for rapid targeted parallel analysis of over 40 urinary metabolites. The newly developed 2 min infusion method requires 10 000 24 h urine samples from the INTERMAP study in 12 weeks and >2200 spot urine samples from the ARIC study in

Brain edema is a common feature associated with hepatic encephalopathy (HE). In patients with acute HE, brain edema has been shown to play a crucial role in the associated neurological deterioration. In chronic HE, advanced magnetic resonance imaging (MRI) techniques have demonstrated that low-grade brain edema appears also to be an important pathological feature. This review explores the different methods used to measure brain edema ex vivo and in vivo in animal models and in humans with chronic HE. In addition, an in-depth description of the main studies performed to date is provided. The role of brain edema in the neurological alterations linked to HE and whether HE and brain edema are the manifestations of the same pathophysiological mechanism or two different cerebral manifestations of brain dysfunction in liver disease are still under debate. In vivo MRI/magnetic resonance spectroscopy studies have allowed insight into the development of brain edema in chronic HE. However, additional in vivo longitudinal and multiparametric/multimodal studies are required (in humans and animal models) to elucidate the relationship between liver function, brain metabolic changes, cellular changes, cell swelling, and neurological manifestations in chronic HE.

Background Type 1 diabetes mellitus (T1DM) is one of the most common pediatric diseases and its incidence is rising in many countries. Recently, it has been shown that metabolites other than glucose play an important role in insulin deficiency and the development of diabetes. The aim of our study was to look for discriminating variation in the concentrations of small-molecule metabolites in the plasma of T1DM children as compared to non-diabetic matched controls using proton nuclear magnetic resonance (1H-NMR)-based metabolomics. Methods A cross-sectional study was set-up to examine the metabolic profile in fasting plasma samples from seven children with poorly controlled T1DM and seven non-diabetic controls aged 8–18 years, and matched for gender, age and BMI-SDS. The obtained plasma 1H-NMR spectra were rationally divided into 110 integration regions, representing the metabolic phenotype. These integration regions reflect the relative metabolite concentrations and were used as statistical variables to construct (train) a classification model in discriminating between T1DM patients and controls. Results The total amount of variation explained by the model between the groups is 81.0% [R2Y(cum)] and within the groups is 75.8% [R2X(cum)]. The predictive ability of the model [Q2(cum)] obtained by cross-validation is 50.7%, indicating that the discrimination between the groups on the basis of the metabolic phenotype is valid. Besides the expected higher concentration of glucose, the relative concentrations of lipids (triglycerides, phospholipids and cholinated phospholipids) are clearly lower in the plasma of T1DM patients as compared to controls. Also the concentrations of the amino acids serine, tryptophan and cysteine are slightly decreased. Conclusions The present study demonstrates that metabolic profiling of plasma by 1H-NMR spectroscopy allows to discriminate between T1DM patients and controls. The metabolites that significantly differ between both groups might point to disturbances in biochemical pathways including (1) choline deficiency, (2) increased gluconeogenesis, and (3) glomerular hyperfiltration. Although the sample size of this study is still somewhat limited and a validation should be performed, the proof of principle looks promising and justifies a deeper investigation of the diagnostic possibilities of 1H-NMR metabolomics in follow-up studies. Trial registration NCT03014908. Registered 06/01/2017. Retrospectively registered

The aetiopathogenesis of cardiovascular diseases (CVD) is multifactorial. Adverse blood pressure (BP) is a major independent risk factor for epidemic CVD affecting about 40% of the adult population worldwide and resulting in significant morbidity and mortality. Metabolic phenotyping of biological fluids has proven its application in characterisin g low molecule weight metabolites providing novel insights into gene -environmental -gut microbiome interaction in relations to a disease state. In this review, we synthesi s e key results from the International Study of Macro/Micronutrients and Blood Pressure (INTERMAP) Study, a cross - sectional epidemiological study of 4,680 men and women aged 40 -59 years from Japan, the People’s Republic of China, the United Kingdom, and the United States. We describe the advancements we have made on: 1) analytical techniques for high throughput metabolic phenotyping; 2) statistical analyses for biomarker identification; 3) discovery of unique food - specific biomarkers; and 4) application of metabolome -wide association (MWA) studies to gain a better understanding into the molecular mechanisms of cross cultural and regional BP differences.

Objective: To evaluate potential variations in the plasma metabolomic profile of endometriosis patients as a consequence of pathophysiologic alterations associated with this disorder. Design: Prospective study. For each subject, a plasma sample was collected after overnight fasting and before surgery. Setting: University medical center. Patient(s): The clinical cohort included 50 endometriosis patients, diagnosed at early (n = 6) and advanced (n = 44) stages of the disease, and 23 healthy women. All volunteers underwent diagnostic laparoscopy to visually confirm the presence or absence of endometriotic lesions. Intervention(s): Metabolomic profiling of plasma samples based on 1H-nuclear magnetic resonance (NMR) spectroscopy in combination with statistical approaches. Main Outcome Measure(s): Comparative identification of metabolites present in plasma from endometriosis patients and healthy women. Result(s): The plasma metabolomic profile of endometriosis patients was characterized by increased concentration of valine, fucose, choline-containing metabolites, lysine/arginine, and lipoproteins and decreased concentration of creatinine compared with healthy women. Metabolic alterations identified in the plasma metabolomic profile of endometriosis patients correlate with pathophysiologic events previously described in the progression of this disease. Conclusion(s): The results highlight the potential of H-1-NMR-based metabolomics to characterize metabolic alterations associated with endometriosis in plasma samples. This information could be useful to get a better understanding of the molecular mechanisms involved in the pathogenesis of endometriosis, thus facilitating the noninvasive diagnosis of this pathology at early stages. (C) 2016 by American Society for Reproductive Medicine.

Plasma lipid concentrations cannot properly account for the complex interactions prevailing in lipoprotein (patho)physiology. Sequential ultracentrifugation (UCF) is the gold standard for physical lipoprotein isolations allowing for subsequent analyses of the molecular composition of the particles. Due to labor and cost issues, however, the UCF-based isolations are usually done only for VLDL, LDL, and HDL fractions; sometimes with the addition of intermediate density lipoprotein (IDL) particles and the fractionation of HDL into HDL(2) and HDL(3) (as done here; n = 302). We demonstrate via these data, with the lipoprotein lipid concentration and composition information combined, that the self-organizing map (SOM) analysis reveals a novel data-driven in silico phenotyping of lipoprotein metabolism beyond the experimentally available classifications. The SOM-based findings are biologically consistent with several well-known metabolic characteristics and also explain some apparent contradictions. The novelty is the inherent emergence of complex lipoprotein associations; e.g., the metabolic subgrouping of the associations between plasma LDL cholesterol concentrations and the structural subtypes of LDL particles. Importantly, lipoprotein concentrations cannot pinpoint lipoprotein phenotypes. It would generally be beneficial to computationally enhance the UCF-based lipoprotein data as illustrated here. Particularly, the compositional variations within the lipoprotein particles appear to be a fundamental issue with metabolic and clinical corollaries.

The fungus Agaricus bisporus is commercially grown for the production of edible mushrooms. This cultivation occurs on compost, but not all of this substrate is consumed by the fungus. To determine why certain fractions remain unused, carbohydrate degrading enzymes, water-extracted from mushroom-grown compost at different stages of mycelium growth and fruiting body formation, were analyzed for their ability to degrade a range of polysaccharides. Mainly endo-xylanase, endo-glucanase, ß-xylosidase and ß-glucanase activities were determined in the compost extracts obtained during mushroom growth. Interestingly, arabinofuranosidase activity able to remove arabinosyl residues from doubly substituted xylose residues and a-glucuronidase activity were not detected in the compost enzyme extracts. This correlates with the observed accumulation of arabinosyl and glucuronic acid substituents on the xylan backbone in the compost towards the end of the cultivation. Hence, it was concluded that compost grown A. bisporus lacks the ability to degrade and consume highly substituted xylan fragments.

The prediction of embryo viability by usual morphological analysis is currently unsatisfactory. New non-invasive techniques such as high-resolution nuclear magnetic resonance (1H-NMR) spectroscopy that allows assessment of metabolic profiling in spent culture media might help embryologists to predict embryo development. Individual microdrops of culture media were analysed after 24 h of embryo culture (from day 3 to day 4) by spectroscopy using a 1 mm microliter probe allowing analysis without sample dilution. Embryos were divided into two groups on day 5: non-arrested embryos (n = 19) and arrested embryos unable to reach the blastocyst stage (n = 20). Multivariate analysis techniques such as Principal Component Analysis (PCA) and Orthogonal Partial Least Square Discriminant Analysis (OPLS-DA) were performed to compare extracellular metabolite balance. 1H-NMR used in combination with a 1 mm probe suggested that in vitro cultured human embryos that have a high developmental potential modify their environment slightly compared to embryos that cease to develop. However, differences between the two groups did not reach statistical significance and multivariate statistical analysis did not allow clustering of the two groups. This study indicated that this technique would not be sufficiently powerful alone to provide information that might help to assess the developmental potential of individual embryos for in vitro fertilisation (IVF).

Childhood obesity has become a prevalent risk to health of children and teenagers. To develop biomarkers in serum for altered lipid metabolism, genetically obese (Ningxiang strain) and lean (DurocxLandracexLarge Yorkshire strain) growing pigs were used as models to identify potential differences in the serum metabonome between the two strains of pigs after consuming the same diet for 46 days. At the end of the study, pigs were euthanized for analysis of the serum metabonome and determination of body composition. Obese pigs had higher fat mass (42.3 +/- 8.8% vs. 21.9 +/- 4.5%) and lower muscle mass (35.4 +/- 4.5% vs. 58.9 +/- 2.5%) than lean pigs (P

The metabolomic profiles of B16 melanoma cells were investigated in vitro with high resolution-magic angle spinning proton magnetic resonance spectroscopy and OPLS multivariate statistical analyse. We compared the profiles for untreated melanoma B16-F10 cells and Ca2+ chelating EGTA, doxorubicin or BP7033 bisphosphonate treated cells. The two last molecules are known to induce anti-proliferative effects by different mechanisms of action in cells. Untreated and EGTA treated cells had similar profiles and were considered together as control cells. Several spectral regions could discriminate control from doxorubicin as well as BP7033 treated cells. Doxorubicin and BP7033 displayed distinct metabolic profiles. Important changes in neutral lipids and inositol were related to doxorubicin activity whereas BP7033 affected essentially phospholipids and alanine/lactate metabolism. These results provide new putative targets for both drugs. Metabolomics by NMR is shown here to be a good tool for the investigation of the mechanisms of action of drugs in pre-clinical studies. Copyright © 2010 John Wiley & Sons, Ltd.

Commercial xylanase preparation Shearzyme®, which contains the glycoside hydrolase family 10 endo-1,4-ß-d-xylanase from Aspergillus aculeatus, was used to prepare short-chain arabinoxylo-oligosaccharides (AXOS) from rye arabinoxylan (AX). A major AXOS was formed as a hydrolysis product. Longer AXOS were also produced as minor products. The pure GH10 xylanase from A. aculeatus was used as a comparison to ensure that the formed AXOS were consequence of the endoxylanase`s function instead of some side enzymes present in Shearzyme. The major AXOS was purified and the structure confirmed with various analysis methods (TLC, HPAEC-PAD, MALDI-TOF-MS, and one- and two-dimensional NMR spectroscopy with nano-probe) as ¿-l-Araf-(1 ¿ 3)-ß-d-Xylp-(1 ¿ 4)-d-Xylp (arabinoxylobiose). This is the first report on 13C NMR data of pure arabinoxylobiose. The yield of arabinoxylobiose was 12% from the quantified hydrolysis products. In conclusion, GH10 endoxylanase from A. aculeatus is thus able to cut efficiently the xylosidic linkage next to the arabinofuranosyl-substituted xylose unit which is not typical for all the GH10 endoxylanases. Interestingly, pure A. aculeatus xylanase showed notably activity towards p-nitrophenyl-ß-d-xylopyranose. In previously studies longer AXOS have been produced with Shearzyme but the formation of short-chain AXOS by A. aculeatus GH10 xylanase has not been studied before. Keywords: Arabinoxylan; Arabinoxylobiose; Aspergillus aculeatus; Glycoside hydrolase family 10; Shearzyme; Xylanase; Xylo-oligosaccharides

The lipid–leukocyte (LL) module is associated with, and reactive to, a wide variety of serum metabolites. The LL module appears to be a link between metabolism, adiposity, and inflammation. Serum metabolite concentrations themselves determine the connectedness of LL module., Comprehensive characterization of human tissues promises novel insights into the biological architecture of human diseases and traits. We assessed metabonomic, transcriptomic, and genomic variation for a large population-based cohort from the capital region of Finland. Network analyses identified a set of highly correlated genes, the lipid–leukocyte (LL) module, as having a prominent role in over 80 serum metabolites (of 134 measures quantified), including lipoprotein subclasses, lipids, and amino acids. Concurrent association with immune response markers suggested the LL module as a possible link between inflammation, metabolism, and adiposity. Further, genomic variation was used to generate a directed network and infer LL module's largely reactive nature to metabolites. Finally, gene co-expression in circulating leukocytes was shown to be dependent on serum metabolite concentrations, providing evidence for the hypothesis that the coherence of molecular networks themselves is conditional on environmental factors. These findings show the importance and opportunity of systematic molecular investigation of human population samples. To facilitate and encourage this investigation, the metabonomic, transcriptomic, and genomic data used in this study have been made available as a resource for the research community.

International audience; In any metabolomics experiment, robustness and reproducibility of data collection is of vital importance. These become more important in collaborative studies where data is to be collected on multiple instruments. With minimisation of variance in sample preparation and instrument performance it is possible to elucidate even subtle differences in metabolite fingerprints due to genotype or biological treatment. In this paper we report on an inter laboratory comparison of plant derived samples by [1H]-NMR spectroscopy across five different sites and within those sites utilising instruments with different probes and magnetic field strengths of 9.4 T (400 MHz), 11.7 T (500 MHz) and 14.1 T (600 MHz). Whilst the focus of the study is on consistent data collection across laboratories, aspects of sample stability and the requirement for sample rotation within the NMR magnet are also discussed. Comparability of the datasets from participating laboratories was exceptionally good and the data were amenable to comparative analysis by multivariate statistics. Field strength differences can be adjusted for in the data pre-processing and multivariate analysis demonstrating that [1H]-NMR fingerprinting is the ideal technique for large scale plant metabolomics data collection requiring the participation of multiple laboratories.

Background & Aims: The sequence of events in hepatic encephalopathy (HE) remains unclear. Using the advantages of in vivo 1H-MRS (9.4T) we aimed to analyse the time-course of disease in an established model of type C HE by analysing the longitudinal changes in a large number of brain metabolites together with biochemical, histological and behavioural assessment. We hypothesized that neurometabolic changes are detectable very early, and that these early changes will offer insight into the primary events underpinning HE. Methods: Wistar rats underwent bile-duct ligation (BDL) and were studied before BDL and at post-operative weeks 2, 4, 6 and 8 (n = 26). In vivo short echo-time H-1-MRS (9.4T) of the hippocampus was performed in a longitudinal manner, as were biochemical (plasma), histological and behavioural tests. Results: Plasma ammonium increased early after BDL and remained high during the study. Brain glutamine increased (+47%) as early as 2-4 weeks post-BDL while creatine (-8%) and ascorbate (-12%) decreased. Brain glutamine and ascorbate correlated closely with rising plasma ammonium, while brain creatine correlated with brain glutamine. The increases in brain glutamine and plasma ammonium were correlated, while plasma ammonium correlated negatively with distance moved. Changes in astrocyte morphology were observed at 4 weeks. These early changes were further accentuated at 6-8 weeks post-BDL, concurrently with the known decreases in brain organic osmolytes. Conclusion: Using a multimodal, in vivo and longitudinal approach we have shown that neurometabolic changes are already noticeable 2 weeks after BDL. These early changes are suggestive of osmotic/oxidative stress and are likely the premise of some later changes. Early decreases in cerebral creatine and ascorbate are novel findings offering new avenues to explore neuroprotective strategies for HE treatment. Lay summary: The sequence of events in chronic hepatic encephalopathy (HE) remains unclear, therefore using the advantages of in vivo proton magnetic resonance spectroscopy at 9.4T we aimed to test the hypothesis that neurometabolic changes are detectable very early in an established model of type C HE, offering insight into the primary events underpinning HE, before advanced liver disease confounds the findings. These early, previously unreported neurometabolic changes occurred as early as 2 to 4 weeks after bile-duct ligation, namely an increase in plasma ammonium and brain glutamine, a decrease in brain creatine and ascorbate together with behavioural and astrocyte morphology changes, and continued to progress throughout the 8-week course of the disease. (C) 2019 European Association for the Study of the Liver. Published by Elsevier B.V.