Unravelling inulin molecules in food sources using a matrix-assisted laser desorption/ionization magnetic resonance mass spectrometry (MALDI-MRMS) pipeline.

Inulin, a polysaccharide characterized by a ß-2,1 fructosyl-fructose structure terminating in a glucosyl moiety, is naturally present in plant roots and tubers. Current methods provide average degrees of polymerization (DP) but lack information on the distribution and absolute concentration of each DP. To address this limitation, a reproducible (CV < 10 %) high throughput (<2 min/sample) MALDI-MRMS approach capable of characterizing and quantifying inulin molecules in plants using matched-matrix consisting of α-cyano-4-hydroxycinnamic acid butylamine salt (CHCA-BA), chicory inulin-12C and inulin-13C was developed. The method identified variation in chain lengths and concentration of inulin across various plant species. Globe artichoke hearts, yacón and elephant garlic yielded similar concentrations at 15.6 g/100 g dry weight (DW), 16.8 g/100 g DW and 17.7 g/100 g DW, respectively, for DP range between 9 and 22. In contrast, Jerusalem artichoke demonstrated the highest concentration (53.4 g/100 g DW) within the same DP ranges. Jerusalem artichoke (DPs 9-32) and globe artichoke (DPs 9-36) showed similar DP distributions, while yacón and elephant garlic displayed the narrowest and broadest DP ranges (DPs 9-19 and DPs 9-45, respectively). Additionally, qualitative measurement for all inulin across all plant samples was feasible using the peak intensities normalized to Inulin-13C, and showed that the ratio of yacón, elephant garlic and Jerusalem was approximately one, two and three times that of globe artichoke. This MALDI-MRMS approach provides comprehensive insights into the structure of inulin molecules, opening avenues for in-depth investigations into how DP and concentration of inulin influence gut health and the modulation of noncommunicable diseases, as well as shedding light on refining cultivation practices to elevate the beneficial health properties associated with specific DPs.

Odd Chain Fatty Acids Are Not Robust Biomarkers for Dietary Intake of Fiber.

SCOPE: Prior investigation has suggested a positive association between increased colonic propionate production and circulating odd-chain fatty acids (OCFAs; pentadecanoic acid [C15:0], heptadecanoic acid [C17:0]). As the major source of propionate in humans is the microbial fermentation of dietary fiber, OCFAs have been proposed as candidate biomarkers of dietary fiber. The objective of this study is to critically assess the plausibility, robustness, reliability, dose-response, time-response aspects of OCFAs as potential biomarkers of fermentable fibers in two independent studies using a validated analytical method. METHODS AND RESULTS: OCFAs are first assessed in a fiber supplementation study, where 21 participants received 10 g dietary fiber supplementation for 7 days. OCFAs are then assessed in a highly controlled inpatient setting, which 19 participants consumed a high fiber (45.1 g per day) and a low fiber diet (13.6 g per day) for 4 days. Collectively in both studies, dietary intakes of fiber as fiber supplementations or having consumed a high fiber diet do not increase circulating levels of OCFAs. The dose and temporal relations are not observed. CONCLUSION: Current study has generated new insight on the utility of OCFAs as fiber biomarkers and highlighted the importance of critical assessment of candidate biomarkers before application.

Iron status influences non-alcoholic fatty liver disease in obesity through the gut microbiome.

BACKGROUND: The gut microbiome and iron status are known to play a role in the pathophysiology of non-alcoholic fatty liver disease (NAFLD), although their complex interaction remains unclear. RESULTS: Here, we applied an integrative systems medicine approach (faecal metagenomics, plasma and urine metabolomics, hepatic transcriptomics) in 2 well-characterised human cohorts of subjects with obesity (discovery n = 49 and validation n = 628) and an independent cohort formed by both individuals with and without obesity (n = 130), combined with in vitro and animal models. Serum ferritin levels, as a markers of liver iron stores, were positively associated with liver fat accumulation in parallel with lower gut microbial gene richness, composition and functionality. Specifically, ferritin had strong negative associations with the Pasteurellaceae, Leuconostocaceae and Micrococcaea families. It also had consistent negative associations with several Veillonella, Bifidobacterium and Lactobacillus species, but positive associations with Bacteroides and Prevotella spp. Notably, the ferritin-associated bacterial families had a strong correlation with iron-related liver genes. In addition, several bacterial functions related to iron metabolism (transport, chelation, heme and siderophore biosynthesis) and NAFLD (fatty acid and glutathione biosynthesis) were also associated with the host serum ferritin levels. This iron-related microbiome signature was linked to a transcriptomic and metabolomic signature associated to the degree of liver fat accumulation through hepatic glucose metabolism. In particular, we found a consistent association among serum ferritin, Pasteurellaceae and Micrococcacea families, bacterial functions involved in histidine transport, the host circulating histidine levels and the liver expression of GYS2 and SEC24B. Serum ferritin was also related to bacterial glycine transporters, the host glycine serum levels and the liver expression of glycine transporters. The transcriptomic findings were replicated in human primary hepatocytes, where iron supplementation also led to triglycerides accumulation and induced the expression of lipid and iron metabolism genes in synergy with palmitic acid. We further explored the direct impact of the microbiome on iron metabolism and liver fact accumulation through transplantation of faecal microbiota into recipient's mice. In line with the results in humans, transplantation from 'high ferritin donors' resulted in alterations in several genes related to iron metabolism and fatty acid accumulation in recipient's mice. CONCLUSIONS: Altogether, a significant interplay among the gut microbiome, iron status and liver fat accumulation is revealed, with potential significance for target therapies. Video abstract.

Effects of Inulin Propionate Ester Incorporated into Palatable Food Products on Appetite and Resting Energy Expenditure: A Randomised Crossover Study.

Supplementation with inulin-propionate ester (IPE), which delivers propionate to the colon, suppresses ad libitum energy intake and stimulates the release of satiety hormones acutely in humans, and prevents weight gain. In order to determine whether IPE remains effective when incorporated into food products (FP), IPE needs to be added to a widely accepted food system. A bread roll and fruit smoothie were produced. Twenty-one healthy overweight and obese humans participated. Participants attended an acclimatisation visit and a control visit where they consumed un-supplemented food products (FP). Participants then consumed supplemented-FP, containing 10 g/d inulin or IPE for six days followed by a post-supplementation visit in a randomised crossover design. On study visits, supplemented-FP were consumed for the seventh time and ad libitum energy intake was assessed 420 min later. Blood samples were collected to assess hormones and metabolites. Resting energy expenditure (REE) was measured using indirect calorimetry. Taste and appearance ratings were similar between FP. Ad libitum energy intake was significantly different between treatments, due to a decreased intake following IPE-FP. These observations were not related to changes in blood hormones and metabolites. There was an increase in REE following IPE-FP. However, this effect was lost after correcting for changes in fat free mass. Our results suggest that IPE suppresses appetite and may alter REE following its incorporation into palatable food products.

Dietary supplementation with inulin-propionate ester or inulin improves insulin sensitivity in adults with overweight and obesity with distinct effects on the gut microbiota, plasma metabolome and systemic inflammatory responses: a randomised cross-over trial.

OBJECTIVE: To investigate the underlying mechanisms behind changes in glucose homeostasis with delivery of propionate to the human colon by comprehensive and coordinated analysis of gut bacterial composition, plasma metabolome and immune responses. DESIGN: Twelve non-diabetic adults with overweight and obesity received 20 g/day of inulin-propionate ester (IPE), designed to selectively deliver propionate to the colon, a high-fermentable fibre control (inulin) and a low-fermentable fibre control (cellulose) in a randomised, double-blind, placebo-controlled, cross-over design. Outcome measurements of metabolic responses, inflammatory markers and gut bacterial composition were analysed at the end of each 42-day supplementation period. RESULTS: Both IPE and inulin supplementation improved insulin resistance compared with cellulose supplementation, measured by homeostatic model assessment 2 (mean±SEM 1.23±0.17 IPE vs 1.59±0.17 cellulose, p=0.001; 1.17±0.15 inulin vs 1.59±0.17 cellulose, p=0.009), with no differences between IPE and inulin (p=0.272). Fasting insulin was only associated positively with plasma tyrosine and negatively with plasma glycine following inulin supplementation. IPE supplementation decreased proinflammatory interleukin-8 levels compared with cellulose, while inulin had no impact on the systemic inflammatory markers studied. Inulin promoted changes in gut bacterial populations at the class level (increased Actinobacteria and decreased Clostridia) and order level (decreased Clostridiales) compared with cellulose, with small differences at the species level observed between IPE and cellulose. CONCLUSION: These data demonstrate a distinctive physiological impact of raising colonic propionate delivery in humans, as improvements in insulin sensitivity promoted by IPE and inulin were accompanied with different effects on the plasma metabolome, gut bacterial populations and markers of systemic inflammation.

Integrated Analytical and Statistical Two-Dimensional Spectroscopy Strategy for Metabolite Identification: Application to Dietary Biomarkers.

A major purpose of exploratory metabolic profiling is for the identification of molecular species that are statistically associated with specific biological or medical outcomes; unfortunately, the structure elucidation process of unknowns is often a major bottleneck in this process. We present here new holistic strategies that combine different statistical spectroscopic and analytical techniques to improve and simplify the process of metabolite identification. We exemplify these strategies using study data collected as part of a dietary intervention to improve health and which elicits a relatively subtle suite of changes from complex molecular profiles. We identify three new dietary biomarkers related to the consumption of peas (N-methyl nicotinic acid), apples (rhamnitol), and onions (N-acetyl-S-(1Z)-propenyl-cysteine-sulfoxide) that can be used to enhance dietary assessment and assess adherence to diet. As part of the strategy, we introduce a new probabilistic statistical spectroscopy tool, RED-STORM (Resolution EnhanceD SubseT Optimization by Reference Matching), that uses 2D J-resolved 1H NMR spectra for enhanced information recovery using the Bayesian paradigm to extract a subset of spectra with similar spectral signatures to a reference. RED-STORM provided new information for subsequent experiments (e.g., 2D-NMR spectroscopy, solid-phase extraction, liquid chromatography prefaced mass spectrometry) used to ultimately identify an unknown compound. In summary, we illustrate the benefit of acquiring J-resolved experiments alongside conventional 1D 1H NMR as part of routine metabolic profiling in large data sets and show that application of complementary statistical and analytical techniques for the identification of unknown metabolites can be used to save valuable time and resources.

2-Furoylglycine as a Candidate Biomarker of Coffee Consumption.

Specific and sensitive food biomarkers are necessary to support dietary intake assessment and link nutritional habits to potential impact on human health. A multistep nutritional intervention study was conducted to suggest novel biomarkers for coffee consumption. (1)H NMR metabolic profiling combined with multivariate data analysis resolved 2-furoylglycine (2-FG) as a novel putative biomarker for coffee consumption. We relatively quantified 2-FG in the urine of coffee drinkers and investigated its origin, metabolism, and excretion kinetics. When searching for its potential precursors, we found different furan derivatives in coffee products, which are known to get metabolized to 2-FG. Maximal urinary excretion of 2-FG occurred 2 h after consumption (p = 0.0002) and returned to baseline after 24 h (p = 0.74). The biomarker was not excreted after consumption of coffee substitutes such as tea and chicory coffee and might therefore be a promising acute biomarker for the detection of coffee consumption in human urine.

Perturbations in fatty acid metabolism and apoptosis are manifested in calcific coronary artery disease: An exploratory lipidomic study.

BACKGROUND: Controversy exists concerning the beneficial or harmful effects of the presence of ectopic calcification in the coronary arteries. Additionally, further elucidation of the exact pathophysiological mechanism is needed. In this study, we sought to identify metabolic markers of vascular calcification that could assist in understanding the disease, monitoring its progress and generating hypotheses describing its pathophysiology. METHODS: Untargeted lipid profiling and complementary modeling strategies were employed to compare serum samples from patients with different levels of calcific coronary artery disease (CCAD) based on their calcium score (CS). Subsequently, patients were divided into three groups: no calcification (NC; CS=0; n=26), mild calcification (MC; CS:1-250; n=27) and severe (SC; CS>250; n=17). RESULTS: Phosphatidylcholine levels were found to be significantly altered in the disease states (p=0.001-0.04). Specifically, 18-carbon fatty acyl chain (FAC) phosphatidylcholines were detected in lower levels in the SC group, while 20:4 FAC lipid species were detected in higher concentrations. A statistical trend was observed with phosphatidylcholine lipids in the MC group, showing the same tendency as with the SC group. We also observed several sphingomyelin signals present at lower intensities in SC when compared with NC or MC groups (p=0.000001-0.01). CONCLUSIONS: This is the first lipid profiling study reported in CCAD. Our data demonstrate dysregulations of phosphatidylcholine lipid species, which suggest perturbations in fatty acid elongation/desaturation. The altered levels of the 18-carbon and 20:4 FAC lipids may be indicative of disturbed inflammation homeostasis. The marked sphingomyelin dysregulation in SC is consistent with profound apoptosis as a potential mechanism of CCAD.

Hippocampal Proteomic and Metabonomic Abnormalities in Neurotransmission, Oxidative Stress, and Apoptotic Pathways in a Chronic Phencyclidine Rat Model.

Schizophrenia is a neuropsychiatric disorder affecting 1% of the world's population. Due to both a broad range of symptoms and disease heterogeneity, current therapeutic approaches to treat schizophrenia fail to address all symptomatic manifestations of the disease. Therefore, disease models that reproduce core pathological features of schizophrenia are needed for the elucidation of pathological disease mechanisms. Here, we employ a comprehensive global label-free liquid chromatography-mass spectrometry proteomic (LC-MS(E)) and metabonomic (LC-MS) profiling analysis combined with the targeted proteomics (selected reaction monitoring and multiplex immunoassay) of serum and brain tissues to investigate a chronic phencyclidine (PCP) rat model in which glutamatergic hypofunction is induced through noncompetitive NMDAR-receptor antagonism. Using a multiplex immunoassay, we identified alterations in the levels of several cytokines (IL-5, IL-2, and IL-1ß) and fibroblast growth factor-2. Extensive proteomic and metabonomic brain tissue profiling revealed a more prominent effect of chronic PCP treatment on both the hippocampal proteome and metabonome compared to the effect on the frontal cortex. Bioinformatic pathway analysis confirmed prominent abnormalities in NMDA-receptor-associated pathways in both brain regions, as well as alterations in other neurotransmitter systems such as kainate, AMPA, and GABAergic signaling in the hippocampus and in proteins associated with neurodegeneration. We further identified abundance changes in the level of the superoxide dismutase enzyme (SODC) in both the frontal cortex and hippocampus, which indicates alterations in oxidative stress and substantiates the apoptotic pathway alterations. The present study could lead to an increased understanding of how perturbed glutamate receptor signaling affects other relevant biological pathways in schizophrenia and, therefore, support drug discovery efforts for the improved treatment of patients suffering from this debilitating psychiatric disorder.

Systems level metabolic phenotype of methotrexate administration in the context of non-alcoholic steatohepatitis in the rat.

Adverse drug reactions (ADRs) represent a significant clinical challenge with respect to patient morbidity and mortality. We investigated the hepatotoxicity and systems level metabolic phenotype of methotrexate (MTX) in the context of a prevalent liver disease; non-alcoholic steatohepatitis (NASH). A nuclear magnetic resonance spectroscopic-based metabonomic approach was employed to analyze the metabolic consequences of MTX (0, 10, 40, and 100 mg/kg) in the urine and liver of healthy rats (control diet) and in a model of NASH (methionine-choline deficient diet). Histopathological analysis confirmed baseline (0 mg/kg) liver necrosis, liver inflammation, and lipid accumulation in the NASH model. Administration of MTX (40 and 100 mg/kg) led to liver necrosis in the control cohort, whereas the NASH cohort also displayed biliary hyperplasia and liver fibrosis (100 mg/kg), providing evidence of the synergistic effect of MTX and NASH. The complementary hepatic and urinary metabolic phenotypes of the NASH model, at baseline, revealed perturbation of multiple metabolites associated with oxidative and energetic stress, and folate homeostasis. Administration of MTX in both diet cohorts showed dose-dependent metabolic consequences affecting gut microbial, energy, nucleobase, nucleoside, and folate metabolism. Furthermore, a unique panel of metabolic changes reflective of the synergistic effect of MTX and NASH was identified, including the elevation of hepatic phenylalanine, urocanate, acetate, and both urinary and hepatic formiminoglutamic acid. This systems level metabonomic analysis of the hepatotoxicity of MTX in the context of NASH provided novel mechanistic insight of potential wider clinical relevance for further understanding the role of liver pathology as a risk factor for ADRs.

Targeting the human genome-microbiome axis for drug discovery: inspirations from global systems biology and traditional Chinese medicine.

Most chronic diseases impairing current human public health involve not only the human genome but also gene-environment interactions, and in the latter case the gut microbiome is an important factor. This makes the classical single drug-receptor target drug discovery paradigm much less applicable. There is widespread and increasing international interest in understanding the properties of traditional Chinese medicines (TCMs) for their potential utilization as a source of new drugs for Western markets as emerging evidence indicates that most TCM drugs are actually targeting both the host and its symbiotic microbes. In this review, we explore the challenges of and opportunities for harmonizing Eastern-Western drug discovery paradigms by focusing on emergent functions at the whole body level of humans as superorganisms. This could lead to new drug candidate compounds for chronic diseases targeting receptors outside the currently accepted "druggable genome" and shed light on current high interest issues in Western medicine such as drug-drug and drug-diet-gut microbial interactions that will be crucial in the development and delivery of future therapeutic regimes optimized for the individual patient.

A comparison of self-reported analgesic use and detection of urinary ibuprofen and acetaminophen metabolites by means of metabonomics: the INTERMAP Study.

Information on dietary supplements, medications, and other xenobiotics in epidemiologic surveys is usually obtained from questionnaires and is subject to recall and reporting biases. The authors used metabolite data obtained from hydrogen-1 (or proton) nuclear magnetic resonance ((1)H NMR) analysis of human urine specimens from the International Study of Macro-/Micro-Nutrients and Blood Pressure (INTERMAP Study) to validate self-reported analgesic use. Metabolic profiling of two 24-hour urine specimens per individual was carried out for 4,630 participants aged 40-59 years from 17 population samples in Japan, China, the United Kingdom, and the United States (data collection, 1996-1999). (1)H NMR-detected acetaminophen and ibuprofen use was low (∼4%) among East Asian population samples and higher (>16%) in Western population samples. In a comparison of self-reported acetaminophen and ibuprofen use with (1)H NMR-detected acetaminophen and ibuprofen metabolites among 496 participants from Chicago, Illinois, and Belfast, Northern Ireland, the overall rate of concordance was 81%-84%; the rate of underreporting was 15%-17%; and the rate of underdetection was approximately 1%. Comparison of self-reported unspecified analgesic use with (1)H NMR-detected acetaminophen and ibuprofen metabolites among 2,660 Western INTERMAP participants revealed similar levels of concordance and underreporting. Screening for urinary metabolites of acetaminophen and ibuprofen improved the accuracy of exposure information. This approach has the potential to reduce recall bias and other biases in epidemiologic studies for a range of substances, including pharmaceuticals, dietary supplements, and foods.

Robust data processing and normalization strategy for MALDI mass spectrometric imaging.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) provides localized information about the molecular content of a tissue sample. To derive reliable conclusions from MSI data, it is necessary to implement appropriate processing steps in order to compare peak intensities across the different pixels comprising the image. Here, we review commonly used normalization methods, and propose a rational data processing strategy, for robust evaluation and modeling of MSI data. The approach includes newly developed heuristic methods for selecting biologically relevant peaks and pixels to reduce the size of a data set and remove the influence of the applied MALDI matrix. The methods are demonstrated on a MALDI MSI data set of a sagittal section of rat brain (4750 bins, m/z = 50-1000, 111 × 185 pixels) and the proposed preferred normalization method uses the median intensity of selected peaks, which were determined to be independent of the MALDI matrix. This was found to effectively compensate for a range of known limitations associated with the MALDI process and irregularities in MS image sampling routines. This new approach is relevant for processing of all MALDI MSI data sets, and thus likely to have impact in biomarker profiling, preclinical drug distribution studies, and studies addressing underlying molecular mechanisms of tissue pathology.

Application of NMR-based metabolomics to the investigation of salt stress in maize (Zea mays).

INTRODUCTION: High salinity, caused by either natural (e.g. climatic changes) or anthropic factors (e.g. agriculture), is a widespread environmental stressor that can affect development and growth of salt-sensitive plants, leading to water deficit, the inhibition of intake of essential ions and metabolic disorders. OBJECTIVE: The application of an NMR-based metabolic profiling approach to the investigation of saline-induced stress in Maize plants is presented. METHODOLOGY: Zea Maize seedlings were grown in either 0, 50 or 150 mM saline solution. Plants were harvested after 2, 4 and 6 days (n = 5 per class and time point) and (1) H NMR spectroscopy was performed separately on shoot and root extracts. Spectral data were analysed and interpreted using multivariate statistical analyses. RESULTS: A distinct effect of time/growth was observed for the control group with relatively higher concentrations of acetoacetate at day 2 and increased levels of alanine at days 4 and 6 in root extracts, whereas concentration of alanine was positively correlated with the shoot extracts harvested at day 2 and trans-aconitic acid increased at days 4 and 6. A clear dose-dependent effect, superimposed on the growth effect, was observed for saline treated shoot and root extracts. This was correlated with increased levels of alanine, glutamate, asparagine, glycine-betaine and sucrose and decreased levels of malic acid, trans-aconitic acid and glucose in shoots. Correlation with salt-load shown in roots included elevated levels of alanine, γ-amino-N-butyric acid, malic acid, succinate and sucrose and depleted levels of acetoacetate and glucose. CONCLUSIONS: The metabolic effect of high salinity was predominantly consistent with osmotic stress as reported for other plant species and was found to be stronger in the shoots than the roots. Using multivariate data analysis it is possible to investigate the effects of more than one environmental stressor simultaneously.

Metabolic profiling strategy for discovery of nutritional biomarkers: proline betaine as a marker of citrus consumption.

BACKGROUND: New food biomarkers are needed to objectively evaluate the effect of diet on health and to check adherence to dietary recommendations and healthy eating patterns. OBJECTIVE: We developed a strategy for food biomarker discovery, which combined nutritional intervention with metabolic phenotyping and biomarker validation in a large-scale epidemiologic study. DESIGN: We administered a standardized diet to 8 individuals and established a putative urinary biomarker of fruit consumption by using (1)H nuclear magnetic resonance (NMR) spectroscopic profiling. The origin of the biomarker was confirmed by using targeted NMR spectroscopy of various fruit. Excretion kinetics of the biomarker were measured. The biomarker was validated by using urinary NMR spectra from UK participants of the INTERMAP (International Collaborative Study of Macronutrients, Micronutrients, and Blood Pressure) (n = 499) in which citrus consumption was ascertained from four 24-h dietary recalls per person. Finally, dietary patterns of citrus consumers (n = 787) and nonconsumers (n = 1211) were compared. RESULTS: We identified proline betaine as a putative biomarker of citrus consumption. High concentrations were observed only in citrus fruit. Most proline betaine was excreted < or =14 h after a first-order excretion profile. Biomarker validation in the epidemiologic data showed a sensitivity of 86.3% for elevated proline betaine excretion in participants who reported citrus consumption and a specificity of 90.6% (P < 0.0001). In comparison with noncitrus consumers, citrus consumers had lower intakes of fats, lower urinary sodium-potassium ratios, and higher intakes of vegetable protein, fiber, and most micronutrients. CONCLUSION: The biomarker identification and validation strategy has the potential to identify biomarkers for healthier eating patterns associated with a reduced risk of major chronic diseases. The trials were registered at clinicaltrials.gov as NCT01102049 and NCT01102062.

Liquid chromatography-mass spectrometry methods for urinary biomarker detection in metabonomic studies with application to nutritional studies.

The effects of sample preparation and chromatographic method differences on the classification and recovery of metabolic biomarkers from UPLC-MS measurements on urine samples of humans exposed to different dietary interventions have been investigated. Eight volunteers consumed three high-fat meals (rich in saturated, monounsaturated and polyunsaturated fatty acids, respectively) in randomized order with a washout period in between. For each participant, urine samples were obtained prior to and at three timed intervals after each meal. Samples were processed either by dilution (1 : 4) or by liquid-liquid extraction and then run under two different gradient conditions. For each analysis method, a total of 96 observations (eight participants, four time points, three diets) were measured. The total ion count chromatograms were analyzed using partial-least-squares discriminant analysis. All three dietary classes could be discriminated irrespective of sample preparation and chromatographic method. However, the main discriminating metabolites varied according to sample preparation, indicating that sample treatment and chromatographic conditions influence the ability to extract biomolecular information. Diluted samples showed higher m/z compounds (ca 400 u) while liquid-liquid extraction samples showed low m/z at the same retention time span. Optimized methods for metabolite identification (e.g. organic acids) were statistically inferior to global screening for mixed compound identification, confirming that multiple compound class-based metabolic profiles are likely to give superior metabonomic (diagnostic) classification, although great care has to be taken in the interpretation in relation to matrix effects.

Identification of metabolites in human hepatic bile using 800 MHz 1H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS.

The first application of high field NMR spectroscopy (800 MHz for (1)H observation) to human hepatic bile (as opposed to gall bladder bile) is reported. The bile sample used for detailed investigation was from a donor liver with mild fat infiltration, collected during organ retrieval prior to transplantation. In addition, to focus on the detection of bile acids in particular, a bile extract was analysed by 800 MHz (1)H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS. In the whole bile sample, 40 compounds have been assigned with the aid of two-dimensional (1)H-(1)H TOCSY and (1)H-(13)C HSQC spectra. These include phosphatidylcholine, 14 amino acids, 10 organic acids, 4 carbohydrates and polyols (glucose, glucuronate, glycerol and myo-inositol), choline, phosphocholine, betaine, trimethylamine-N-oxide and other small molecules. An initial NMR-based assessment of the concentration range of some key metabolites has been made. Some observed chemical shifts differ from expected database values, probably due to a difference in bulk diamagnetic susceptibility. The NMR spectra of the whole extract gave identification of the major bile acids (cholic, deoxycholic and chenodeoxycholic), but the glycine and taurine conjugates of a given bile acid could not be distinguished. However, this was achieved by HPLC-NMR/MS, which enabled the separation and identification of ten conjugated bile acids with relative abundances varying from approximately 0.1% (taurolithocholic acid) to 34.0% (glycocholic acid), of which, only the five most abundant acids could be detected by NMR, including the isomers glycodeoxycholic acid and glycochenodeoxycholic acid, which are difficult to distinguish by conventional LC-MS analysis. In a separate experiment, the use of UPLC-MS allowed the detection and identification of 13 bile acids. This work has shown the complementary potential of NMR spectroscopy, MS and hyphenated NMR/MS for elucidating the complex metabolic profile of human hepatic bile. This will be useful baseline information in ongoing studies of liver excretory function and organ transplantation.

Fingerprinting of human bile during liver transplantation by capillary electrophoresis.

Increasing rates of success in liver transplantation have increased the number of cases considered. However, liver post-transplant graft dysfunction of liver transplants (TXs) is not fully understood and by applying holistic approaches we can investigate metabolic change deriving from confounding factors such as liver fat content, ischaemia time, donor age, recipient's health, etc. Twenty-six hepatic bile samples taken from liver donors and recipients were retrieved from a total of six TXs, from these one recipient underwent post-graft dysfunction. CE was employed to fingerprint bile collected at 10 min increments in the donors and in the recipients. The electropherograms of these samples were aligned and normalised using correlation optimised warping algorithms and modelled with multivariate techniques. The resulting metabolic signatures were compared; in general donors and recipients showed distinct fingerprints and clustered separately. When a partial least square discriminant analysis (PLS-DA) model was constructed between donor and recipient's samples, a recipient of a 32 year old liver with normal steatosis, and shortest cold ischaemia time showed as the observation nearest to its donor observation, denoting minimal metabolic change. This study proposes CE fingerprinting of human bile as a promising technique to help unravel the complex metabolic pathways involved during transplantation.

Temporal metabonomic modeling of l-arginine-induced exocrine pancreatitis.

The time-related metabolic responses to l-arginine (ARG)-induced exocrine pancreatic toxicity were investigated using single ip doses of 1,000 and 4,000 mg/kg body weight over a 7 day experimental period in male Sprague-Dawley rats. Sequential timed urine and plasma samples were analyzed using high resolution (1)H NMR spectroscopy together with complementary clinical chemistry and histopathology analyses. Principal components analysis (PCA) and orthogonal projection on latent structures discriminant analysis (O-PLS-DA) were utilized to analyze the (1)H NMR data and to extract and identify candidate biomarkers and to construct metabolic trajectories post ARG administration. Low doses of ARG resulted in virtually no histopathological damage and distinct reversible metabolic response trajectories. High doses of ARG caused pancreatic acinar degeneration and necrosis and characteristic metabolic trajectory profiles with several distinct phases. The initial trajectory phase (0-8 h) involved changes in the urea cycle and transamination indicating a homeostatic response to detoxify excess ammonia generated from ARG catabolism. By 48 h, there was a notable enhancement of the excretion of the gut microbial metabolites, phenylacetylglycine (PAG), 4-cresol-glucuronide and 4-cresol-sulfate, suggesting that compromised pancreatic function impacts on the activity of the gut microbiota giving potential rise to a novel class of surrogate extragenomic biomarkers of pancreatic injury. The implied compromise of microbiotal function may also contribute to secondary hepatic and pancreatic toxic responses. We show here for the first time the value of metabonomic studies in investigating metabolic disruption due to experimental pancreatitis. The variety of observed systemic responses suggests that this approach may be of general value in the assessment of other animal models or human pancreatitis.

Top-down systems biology integration of conditional prebiotic modulated transgenomic interactions in a humanized microbiome mouse model.

Gut microbiome-host metabolic interactions affect human health and can be modified by probiotic and prebiotic supplementation. Here, we have assessed the effects of consumption of a combination of probiotics (Lactobacillus paracasei or L. rhamnosus) and two galactosyl-oligosaccharide prebiotics on the symbiotic microbiome-mammalian supersystem using integrative metabolic profiling and modeling of multiple compartments in germ-free mice inoculated with a model of human baby microbiota. We have shown specific impacts of two prebiotics on the microbial populations of HBM mice when co-administered with two probiotics. We observed an increase in the populations of Bifidobacterium longum and B. breve, and a reduction in Clostridium perfringens, which were more marked when combining prebiotics with L. rhamnosus. In turn, these microbial effects were associated with modulation of a range of host metabolic pathways observed via changes in lipid profiles, gluconeogenesis, and amino-acid and methylamine metabolism associated to fermentation of carbohydrates by different bacterial strains. These results provide evidence for the potential use of prebiotics for beneficially modifying the gut microbial balance as well as host energy and lipid homeostasis.