Differences in Fecal Gut Microbiota, Short-Chain Fatty Acids and Bile Acids Link Colorectal Cancer Risk to Dietary Changes Associated with Urbanization Among Zimbabweans.

The incidence of colorectal cancer (CRC) is gradually rising in sub-Saharan Africa. This may be due to dietary changes associated with urbanization, which may induce tumor-promoting gut microbiota composition and function. We compared fecal microbiota composition and activity in 10 rural and 10 urban Zimbabweans for evidence of a differential CRC risk. Dietary intake was assessed by a food frequency questionnaire. Fecal microbiota composition, metabolomic profile, functional microbial genes were analyzed, and bile acids and short chain fatty acids quantified. Animal protein intake was higher among urban volunteers, but carbohydrate and fiber intake were similar. Bacteria related to Blautia obeum, Streptococcus bovis, and Subdoligranulum variabile were higher in urban residents, whereas bacteria related to Oscillospira guillermondii and Sporobacter termitidis were higher in rural volunteers. Fecal levels of primary bile acids, cholic acid, and chenodeoxycholic acid (P < 0.05), and secondary bile acids, deoxycholic acid (P < 0.05) and ursodeoxycholic acid (P < 0.001) were higher in urban residents. Fecal levels of acetate and propionate, but not butyrate, were higher in urban residents. The gut microbiota composition and activity among rural and urban Zimbabweans retain significant homogeneity (possibly due to retention of dietary fiber), but urban residents have subtle changes, which may indicate a higher CRC risk.

Application of 1H NMR spectroscopy to the metabolic phenotyping of rodent brain extracts: A metabonomic study of gut microbial influence on host brain metabolism.

1H NMR Spectroscopy has been applied to determine the neurochemical profiles of brain extracts from the frontal cortex and hippocampal regions of germ free and normal mice and rats. The results revealed a number of differences between germ free (GF) and conventional (CV) rats or specific pathogen-free (SPF) mice with microbiome-associated metabolic variation found to be both species- and region-dependent. In the mouse, the GF frontal cortex contained lower amounts of creatine, N-acetyl-aspartate (NAA), glycerophosphocholine and lactate, but greater amounts of choline compared to that of specific pathogen free (SPF) mice. In the hippocampus, the GF mice had greater creatine, NAA, lactate and taurine content compared to those of the SPF animals, but lower relative quantities of succinate and an unidentified lipid-related component. The GF rat frontal cortex contained higher relative quantities of lactate, creatine and NAA compared to the CV animals whilst the GF hippocampus was characterized by higher taurine and phosphocholine concentrations and lower quantities of NAA, N-acetylaspartylglutamate and choline compared to the CV animals. Of note is that, in both rat and mouse brain extracts, concentrations of hippocampal taurine were found to be greater in the absence of an established microbiome. The results provide further evidence that brain biochemistry can be influenced by gut microbial status, specifically metabolites involved in energy metabolism demonstrating biochemical dialogue between the microbiome and brain.

Metabolic Phenotypes of Carotid Atherosclerotic Plaques Relate to Stroke Risk: An Exploratory Study.

OBJECTIVE: Stroke is a major cause of death and disability. That three-quarters of stroke patients will never have previously manifested cerebrovascular symptoms demonstrates the unmet clinical need for new biomarkers able to stratify patient risk and elucidation of the biological dysregulations. In this study, the utility of comprehensive metabolic phenotyping is assessed to provide candidate biomarkers that relate to stroke risk in stenosing carotid plaque tissue samples. METHOD: Carotid plaque tissue samples were obtained from patients with cerebrovascular symptoms of carotid origin (n = 5), and from asymptomatic patients (n = 5). Two adjacent biological replicates were obtained from each tissue. Organic and aqueous metabolite extracts were obtained separately and analysed using two ultra performance liquid chromatography coupled to mass spectrometry metabolic profiling methods. Multivariate and univariate tools were used for statistical analysis. RESULTS: The two study groups demonstrated distinct plaque phenotypes using multivariate data analysis. Univariate statistics also revealed metabolites that differentiated the two groups with a strong statistical significance (p = 10(-4)-10(-5)). Specifically, metabolites related to the eicosanoid pathway (arachidonic acid and arachidonic acid precursors), and three acylcarnitine species (butyrylcarnitine, hexanoylcarnitine, and palmitoylcarnitine), intermediates of the ß-oxidation, were detected in higher intensities in symptomatic patients. However, metabolites implicated in the process of cell death, a process known to be upregulated in the formation of the vulnerable plaque, were unaffected. CONCLUSIONS: Discrimination between symptomatic and asymptomatic carotid plaque tissue is demonstrated for the first time using metabolic profiling technologies. Two biological pathways (eicosanoid and ß-oxidation) were implicated in differentiating symptomatic from asymptomatic patients and will be further investigated. These results indicate that metabolic phenotyping should be further explored to investigate the chemistry of the unstable plaque, in the pursuit of candidate biomarkers for risk-stratification and targets for pharmacotherapeutic intervention.

Cervical intraepithelial neoplasia disease progression is associated with increased vaginal microbiome diversity.

Persistent infection with oncogenic Human Papillomavirus (HPV) is necessary for cervical carcinogenesis. Although evidence suggests that the vaginal microbiome plays a functional role in the persistence or regression of HPV infections, this has yet to be described in women with cervical intra-epithelial neoplasia (CIN). We hypothesised that increasing microbiome diversity is associated with increasing CIN severity. llumina MiSeq sequencing of 16S rRNA gene amplicons was used to characterise the vaginal microbiota of women with low-grade squamous intra-epithelial lesions (LSIL; n = 52), high-grade (HSIL; n = 92), invasive cervical cancer (ICC; n = 5) and healthy controls (n = 20). Hierarchical clustering analysis revealed an increased prevalence of microbiomes characterised by high-diversity and low levels of Lactobacillus spp. (community state type-CST IV) with increasing disease severity, irrespective of HPV status (Normal = 2/20,10%; LSIL = 11/52,21%; HSIL = 25/92,27%; ICC = 2/5,40%). Increasing disease severity was associated with decreasing relative abundance of Lactobacillus spp. The vaginal microbiome in HSIL was characterised by higher levels of Sneathia sanguinegens (P < 0.01), Anaerococcus tetradius (P < 0.05) and Peptostreptococcus anaerobius (P < 0.05) and lower levels of Lactobacillus jensenii (P < 0.01) compared to LSIL. Our results suggest advancing CIN disease severity is associated with increasing vaginal microbiota diversity and may be involved in regulating viral persistence and disease progression.

Metabolic phenotype-microRNA data fusion analysis of the systemic consequences of Roux-en-Y gastric bypass surgery.

BACKGROUND/OBJECTIVES: Bariatric surgery offers sustained marked weight loss and often remission of type 2 diabetes, yet the mechanisms of establishment of these health benefits are not clear. SUBJECTS/METHODS: We mapped the coordinated systemic responses of gut hormones, the circulating miRNAome and the metabolome in a rat model of Roux-en-Y gastric bypass (RYGB) surgery. RESULTS: The response of circulating microRNAs (miRNAs) to RYGB was striking and selective. Analysis of 14 significantly altered circulating miRNAs within a pathway context was suggestive of modulation of signaling pathways including G protein signaling, neurodegeneration, inflammation, and growth and apoptosis responses. Concomitant alterations in the metabolome indicated increased glucose transport, accelerated glycolysis and inhibited gluconeogenesis in the liver. Of particular significance, we show significantly decreased circulating miRNA-122 levels and a more modest decline in hepatic levels, following surgery. In mechanistic studies, manipulation of miRNA-122 levels in a cell model induced changes in the activity of key enzymes involved in hepatic energy metabolism, glucose transport, glycolysis, tricarboxylic acid cycle, pentose phosphate shunt, fatty-acid oxidation and gluconeogenesis, consistent with the findings of the in vivo surgery-mediated responses, indicating the powerful homeostatic activity of the miRNAs. CONCLUSIONS: The close association between energy metabolism, neuronal signaling and gut microbial metabolites derived from the circulating miRNA, plasma, urine and liver metabolite and gut hormone correlations further supports an enhanced gut-brain signaling, which we suggest is hormonally mediated by both traditional gut hormones and miRNAs. This transomic approach to map the crosstalk between the circulating miRNAome and metabolome offers opportunities to understand complex systems biology within a disease and interventional treatment setting.

Chemical mapping of the colorectal cancer microenvironment via MALDI imaging mass spectrometry (MALDI-MSI) reveals novel cancer-associated field effects.

Matrix-assisted laser desorption ionisation imaging mass spectrometry (MALDI-MSI) is a rapidly advancing technique for intact tissue analysis that allows simultaneous localisation and quantification of biomolecules in different histological regions of interest. This approach can potentially offer novel insights into tumour microenvironmental (TME) biochemistry. In this study we employed MALDI-MSI to evaluate fresh frozen sections of colorectal cancer (CRC) tissue and adjacent healthy mucosa obtained from 12 consenting patients undergoing surgery for confirmed CRC. Specifically, we sought to address three objectives: (1) To identify biochemical differences between different morphological regions within the CRC TME; (2) To characterise the biochemical differences between cancerous and healthy colorectal tissue using MALDI-MSI; (3) To determine whether MALDI-MSI profiling of tumour-adjacent tissue can identify novel metabolic 'field effects' associated with cancer. Our results demonstrate that CRC tissue harbours characteristic phospholipid signatures compared with healthy tissue and additionally, different tissue regions within the CRC TME reveal distinct biochemical profiles. Furthermore we observed biochemical differences between tumour-adjacent and tumour-remote healthy mucosa. We have referred to this 'field effect', exhibited by the tumour locale, as cancer-adjacent metaboplasia (CAM) and this finding builds on the established concept of field cancerisation.

Relation of raw and cooked vegetable consumption to blood pressure: the INTERMAP Study.

Inverse associations have been reported of overall vegetable intake to blood pressure (BP); whether such relations prevail for both raw and cooked vegetables has not been examined. Here we report cross-sectional associations of vegetable intakes with BP for 2195 Americans ages 40-59 in the International Study of Macro/Micronutrients and Blood Pressure (INTERMAP) using four standardized multi-pass 24-h dietary recalls and eight BP measurements. Relations to BP of raw and cooked vegetables consumption, and main individual constituents were assessed by multiple linear regression. Intakes of both total raw and total cooked vegetables considered separately were inversely related to BP in multivariate-adjusted models. Estimated average systolic BP differences associated with two s.d. differences in raw vegetable intake (68 g per 1000 kcal) and cooked vegetable intake (92 g per 1000 kcal) were -1.9 mm Hg (95% confidence interval (CI): -3.1, -0.8; P=0.001) and -1.3 mm Hg (95% CI: -2.5, -0.2; P=0.03) without body mass index (BMI) in the full model; -1.3 mm Hg (95% CI: -2.4, -0.2; P=0.02) and -0.9 mm Hg (95% CI: -2.0, 0.2; P=0.1) with additional adjustment for BMI. Among commonly consumed individual raw vegetables, tomatoes, carrots, and scallions related significantly inversely to BP. Among commonly eaten cooked vegetables, tomatoes, peas, celery, and scallions related significantly inversely to BP.

Integrated histopathological and urinary metabonomic investigation of the pathogenesis of microcystin-LR toxicosis.

Patterns of change of endogenous metabolites may closely reflect systemic and organ-specific toxic changes. The authors examined the metabolic effects of the cyanobacterial (blue-green algal) toxin microcystin-LR by (1)H-nuclear magnetic resonance (NMR) analysis of urinary endogenous metabolites. Rats were treated with a single sublethal dose, either 20 or 80 µg/kg intraperitoneally, and sacrificed at 2 or 7 days post dosing. Changes in the high-dose, 2-day sacrifice group included centrilobular hepatic necrosis and congestion, accompanied in some animals by regeneration and neovascularization. By 7 days, animals had recovered, the necrotizing process had ended, and the centrilobular areas had been replaced by regenerative, usually hypertrophic hepatocytes. There was considerable interanimal variation in the histologic process and severity, which correlated with the changes in patterns of endogenous metabolites in the urine, thus providing additional validation of the biomarker and biochemical changes. Similarity of the shape of the metabolic trajectories suggests that the mechanisms of toxic effects and recovery are similar among the individual animals, albeit that the magnitude and timing are different for the individual animals. Initial decreases in urinary citrate, 2-oxoglutarate, succinate, and hippurate concentrations were accompanied by a temporary increase in betaine and taurine, then creatine from 24 to 48 hours. Further changes were an increase in guanidinoacetate, dimethylglycine, urocanic acid, and bile acids. As a tool, urine can be repeatedly and noninvasively sampled and metabonomics utilized to study the onset and recovery after toxicity, thus identifying time points of maximal effect. This can help to employ histopathological examination in a guided and effective fashion.

In-vitro identification of distinctive metabolic signatures of intact varicose vein tissue via magic angle spinning nuclear magnetic resonance spectroscopy.

OBJECTIVES: Nuclear magnetic resonance (NMR) spectroscopy is an established tool for metabolic profiling of tissues or biofluids with utility in identifying disease biomarkers and changes in enzymatic or gene expression. This pilot study aims to compare the metabolic profiles of intact varicose and non-varicose vein tissue via magic angle spinning (MAS) NMR spectroscopy with a view to promoting the understanding of the pathogenesis of varicose vein formation. METHODS: Varicose vein tissue (n = 8) was collected from patients undergoing varicose veins surgery. Control non-varicose great saphenous vein samples were collected from patients undergoing lower limb amputation (n = 3) and peripheral arterial bypass surgery (n = 5). Intact tissue samples (average weight 10.33 ± 0.8 mg) from each vein segment were analysed using 1D MAS (1)H NMR (600 MHz) spectroscopy. For selected vein samples, two-dimensional (2D) NMR experiments were performed. Differences between spectra from varicose and non-varicose tissues were elucidated using a variety of multivariate statistical analyses. RESULTS: The metabolic profiles of varicose veins samples were clearly differentiated from non-varicose veins samples. Lipid metabolites were present at a higher concentration in the non-varicose veins group whilst creatine, lactate and myo-inositol metabolites were more characteristic of the varicose veins group. CONCLUSION: We demonstrate differential metabolic profiles between varicose veins and non-varicose veins. Elucidating the metabolic signature underlying varicose veins can further improve our understanding of the biological mechanisms of disease initiation, progression, and aid in identifying putative therapeutic targets.

A metabolic phenotyping approach to understanding relationships between metabolic syndrome and breast tumour responses to chemotherapy.

PURPOSE: Breast cancer is associated with adverse outcomes in patients with the metabolic syndrome phenotype. To study this further, we examined the relationship between serum metabolite levels and the components of metabolic syndrome with treatment outcomes in breast cancer. METHODS: A total of 88 women with measurable breast cancer were studied; their serum metabolites as assessed by (1)H nuclear magnetic resonance spectroscopy, blood pressure, lipids, glucose, body mass index and waist circumference were recorded and correlated with treatment response. RESULTS: We identified metabolic syndrome in approximately half of our cohort (42 patients) and observed a significant trend (P = 0.03) of increased incidence of metabolic syndrome in partial response (33.3%), stable disease (42.9%) and progressive disease groups (66.1%). High blood sugar predicted a poor response (P < 0.001). Logistic regression of metabonomic data demonstrated that high lactate (P = 0.03) and low alanine (P = 0.01) combined with high glucose (P = 0.01) were associated with disease progression. CONCLUSIONS: Metabolic syndrome is commonly observed in metastatic breast cancer and these patients have poorer outcomes. These data, which support our previous findings, suggest that high blood glucose as part of metabolic syndrome is associated with a poor response in breast cancer. They also validate new therapeutic approaches that focus on metabolism.

UPLC-MS, HPLC-radiometric, and NMR-spectroscopic studies on the metabolic fate of 3-fluoro-[U-14C]-aniline in the bile-cannulated rat.

1. A study of the rates and routes of excretion of 3-fluoro-[U-(14)C]aniline following intraperitoneal administration to male bile-cannulated rats by liquid scintillation counting (LSC) gave a total recovery of approximately 90% in the 48 h following dosing, with the majority of the dose being excreted in the urine during the first 24 h (approximately 49%). 2. The total recovery as determined by (19)F-nuclear magnetic resonance ((19)F-NMR) was approximately 49%, with the majority of the dose excreted in the first 24 h (approximately 41%). The comparatively low recovery in comparison to that obtained from LSC was due to matrix effects in bile and a contribution from metabolic defluorination. 3. High-performance liquid chromatography with radiometric profiling of urine and bile revealed a complex pattern of metabolites with the bulk of the dose excreted as a single peak. 4. Ultra-performance liquid chromatography-orthogonal acceleration time of flight mass spectrometry profiling also showed a complex pattern of metabolites, detecting approximately 21 metabolites of 3-fluoroaniline (3-FA) with six of these detected only in urine and four solely in bile. 5. (19)F-NMR revealed the presence of the parent compound and 15 metabolites in urine collected during the first 24 h after -dosing. The matrix effects of bile on (19)F-NMR spectroscopy made metabolite profiling impractical for this biofluid. The major metabolite of 3-FA was identified as 2-fluoro-4-acetamidophenol-sulfate.

Integrated HPLC-MS and (1)H-NMR spectroscopic studies on acyl migration reaction kinetics of model drug ester glucuronides.

Acyl glucuronides (AGs) are common, chemically reactive metabolites of acidic xenobiotics. Concerns about the potential of this class of conjugate to cause toxicity in man require efficient methods for the determination of reactivity, and this is commonly done by measuring transacylation kinetics. High-performance liquid chromatography-mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy were applied to the kinetic analysis of AG isomerization and hydrolysis for the 1-beta-O-AGs of ibufenac, (R)- and (S)-ibuprofen, and an alpha,alpha-dimethylated ibuprofen analogue. Each AG was incubated in either aqueous buffer at pH 7.4 or human plasma at 37 degrees C. Aliquots of these samples, taken throughout the reaction time course, were analysed by HPLC-MS and (1)H-NMR spectroscopy and the results compared. For identification of the AGs incubated in pH 7.4 buffer and for analysis of kinetic rates, (1)H-NMR spectroscopy generally gave the most complete set of data, but for human plasma the use of (1)H-NMR spectroscopy was impractical and HPLC-MS was more suitable. HPLC-MS was more sensitive than (1)H-NMR spectroscopy, but the lack of suitable stable-isotope labelled internal standards, together with differences in response between glucuronides and aglycones, made quantification problematic. Using HPLC-MS a specific 1-beta-O-AG-related ion at m/z 193 (the glucuronate fragment) was noted enabling selective determination of these isomers. In buffer, transacylation reactions predominated, with relatively little hydrolysis to the free aglycone observed. In human plasma incubations the observed rates of reaction were much faster than for buffer, and hydrolysis to the free aglycone was the major route. These results illustrate the strengths and weaknesses of each analytical approach for this class of analyte.

Bidirectional correlation of NMR and capillary electrophoresis fingerprints: a new approach to investigating Schistosoma mansoni infection in a mouse model.

We demonstrate the statistical integration of nuclear magnetic resonance (NMR) spectroscopy and capillary electrophoresis (CE) data in order to describe a pathological state caused by Schistosoma mansoni infection in a mouse model based on urinary metabolite profiles. Urine samples from mice 53 days post infection with S. mansoni and matched controls were analyzed via NMR spectroscopy and CE. The two sets of metabolic profiles were first processed and analyzed independently and were subsequently integrated using statistical correlation methods in order to facilitate cross assignment of metabolites. Using this approach, metabolites such as 3-ureidopropionate, p-cresol glucuronide, phenylacetylglycine, indoxyl sulfate, isocitrate, and trimethylamine were identified as differentiating between infected and control animals. These correlation analyses facilitated structural elucidation using the identification power of one technique to enhance and validate the other, but also highlighted the enhanced ability to detect functional correlations between metabolites, thereby providing potential for achieving deeper mechanistic insight into the biological process.

A QSAR investigation of dermal and respiratory chemical sensitizers based on computational chemistry properties.

A wide range of physicochemical properties based on molecular topology, size and shape, and semi-empirical molecular orbital theory were calculated for a variety of dermal and respiratory sensitizers, as well as some non-active substances. Compounds were randomly selected to belong to a training set of substances (approximately 90%) for development of quantitative structure-activity relationship (QSAR) models or to a test set (approximately 10%) for testing the models. A choice was made of those descriptors which were related to sensitization using standard statistics. Pattern recognition methods were then utilized to identify the combination of properties that provided the greatest contribution to the observed biological effect. Principal components (PC) analysis was then performed on the most important properties. The models derived were then applied to a test set of known sensitizers to predict their class. For dermal and respiratory sensitizers respectively, the PC model classified five (100%) of the R-43 active and two (100%) of the R42-active test set compounds correctly. Analysis of the PC loadings showed that the most useful properties distinguishing respiratory and/or dermal sensitizers from inactive substances were the molecular orbital-based terms.

The role of gut microbiota in drug response.

Higher organisms such as mammals exist in a symbiotic relationship with their gut microbiota, formed from a diverse and highly metabolically active consortium of species. The gut microbiota, in addition to their ability to process dietary derived material, are also capable of performing a range of biotransformations on xenobiotics, such as drugs and their metabolites, in ways that can affect absorption and bioavailability. The potential for the gut microflora to influence drug metabolism and toxicity in unexpected ways is discussed.

Metabolism of 2-fluoro-4-iodoaniline in earthworm Eisenia veneta using (19)F-NMR spectroscopy, HPLC-MS, and HPLC-ICPMS ((127)I).

A combination of (19)F-NMR spectroscopy, HPLC-MS/MS, HPLC-MS with constant neutral loss scanning of 127, and HPLC-ICPMS with iodine detection has enabled the profiling, quantification, and limited characterization of the metabolites produced in the earthworm Eisenia veneta, following exposure to 2-fluoro-4-iodoaniline. Mass spectrometric analysis of the worm tissue and coelomic fluid afforded the identification of two Phase II metabolites, N-glutamyl and N-glucoside conjugates, indicating the importance of these pathways in the detoxification of xenobiotics for earthworms. Several further metabolites were observed and quantified by (19)F-NMR spectroscopy and HPLC-(127)I-ICPMS, although these were of low abundance and their structures were not unequivocally identified. The parent compound and the glutamyl conjugate were found to be the major xenobiotic components of both the coelomic fluid and the worm tissue, representing approximately 23 and approximately 35%, respectively, of the dose that was recovered from the earthworm tissue extract.

Subtle metabolic and liver gene transcriptional changes underlie diet-induced fatty liver susceptibility in insulin-resistant mice.

AIMS/HYPOTHESIS: Complex changes in gene expression are associated with insulin resistance and non-alcoholic fatty liver disease (NAFLD) promoted by feeding a high-fat diet (HFD). We used functional genomic technologies to document molecular mechanisms associated with diet-induced NAFLD. MATERIALS AND METHODS: Male 129S6 mice were fed a diet containing 40% fat (high-fat diet, HFD) for 15 weeks. Glucose tolerance, in vivo insulin secretion, plasma lipid profile and adiposity were determined. Plasma metabonomics and liver transcriptomics were used to identify changes in gene expression associated with HFD-induced NAFLD. RESULTS: In HFD-fed mice, NAFLD and impaired glucose and lipid homeostasis were associated with increased hepatic transcription of genes involved in fatty acid uptake, intracellular transport, modification and elongation, whilst genes involved in beta-oxidation and lipoprotein secretion were, paradoxically, also upregulated. NAFLD developed despite strong and sustained downregulation of transcription of the gene encoding stearoyl-coenzyme A desaturase 1 (Scd1) and uncoordinated regulation of transcription of Scd1 and the gene encoding sterol regulatory element binding factor 1c (Srebf1c) transcription. Inflammatory mechanisms appeared to be stimulated by HFD. CONCLUSIONS/INTERPRETATION: Our results provide an accurate representation of subtle changes in metabolic and gene expression regulation underlying disease-promoting and compensatory mechanisms, collectively contributing to diet-induced insulin resistance and NAFLD. They suggest that proposed models of NAFLD pathogenesis can be enriched with novel diet-reactive genes and disease mechanisms.

The mechanism of galactosamine toxicity revisited; a metabonomic study.

1H NMR spectroscopy was used to investigate the metabolic effects of the hepatotoxin galactosamine (galN) and the mechanism by which glycine protects against such toxicity. Rats were acclimatized to a 0 or 5% glycine diet for 6 days and subsequently administered vehicle, galN (500 mg/kg), glycine (5% via the diet), or both galN and glycine. Urine was collected over 12 days prior to administration of galN and for 24 hours thereafter. Serum and liver tissue were sampled on termination, 24 hours post-dosing. The metabolic profiles of biofluids and tissues were determined using high-field 1H NMR spectroscopy. Orthogonal-projection to latent structures discriminant analysis (O-PLS-DA) was applied to model the spectral data and enabled the hepatic, urinary, and serum metabolites that discriminated between control and treated animals to be determined. Histopathological data and clinical chemistry measurements confirmed the protective effect of glycine. The level of N-acetylglucosamine (glcNAc) in the post-dose urine was found to correlate strongly with the degree of galN-induced liver damage, and the urinary level of glcNAc was not significantly elevated in rats treated with both galN and glycine. Treatment with glycine alone was found to significantly increase hepatic levels of uridine, UDP-glucose, and UDP-galactose, and in view of the known effects of galactosamine, this suggests that the protective role of glycine against galN toxicity might be mediated by changes in the uridine nucleotide pool rather than by preventing Kupffer cell activation. Thus, we present a novel hypothesis: that administration of glycine increases the hepatic uridine nucleotide pool which counteracts the galN-induced depletion of these pools and facilitates complete metabolism of galN. These novel data highlight the applicability of NMR-based metabonomics in elucidating multicompartmental metabolic consequences of toxicity and toxic salvage.

Metabolism of [14C]-5-chloro-1,3-benzodioxol-4-amine in male Wistar-derived rats following intraperitoneal administration.

[14C]-5-chloro-1,3-benzodioxol-4-amine was administered intraperitoneally (i.p.) to bile duct-cannulated rats (Alpk:ApfSD, Wistar derived) at 25 mg kg-1 to determine the rates and routes of excretion of the compound and to investigate its metabolic fate. A total of 89.1% of the dose was excreted in the 48 h following administration, the majority being recovered in the urine during the first 12 h. The main metabolite in both urine and bile, detected by high-performance liquid chromatography (HPLC) with radioprofiling and mass spectrometry, was identified as a demethylenated monosulfate conjugate. Unchanged parent compound formed a major component of the radiolabel excreted in urine and, in addition to unchanged parent and demethylenated sulphate conjugate, a large number of minor metabolites were detected in urine and bile. The overall metabolic fate of 5-chloro-1,3-benzodioxol-4-amine in the rat was complex, with some similarities to previously studied methylenedioxyphenyl compounds.

Human metabolic phenotyping and metabolome wide association studies.

Metabolic phenotyping in large-scale population studies can yield crucial information regarding the impact and interaction of genetic and environmental factors with regard to the prevalence and risk of chronic diseases. Spectroscopic technologies such as nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) can be used to generate multi-parameter profiles of biological samples and together with automated sample delivery and mathematical modelling systems, can be used as a high throughput screening tool. The adaptation of these metabolic profiling tools from pre-clinical studies in animal models to population studies in man is explored and an overview of the current and future roles of metabolic phenotyping is described, including the idea of "Metabolome Wide Association Screening" focussing on key disease areas such as cardiovascular disease and metabolic syndrome, cancers and neurodegeneration.