Biochemical characterization of rat intestine development using high-resolution magic-angle-spinning 1H NMR spectroscopy and multivariate data analysis

We report details of metabolic profiles for small intestinal samples obtained using high-resolution magic-angle-spinning (HRMAS) (1)H NMR spectroscopy. Intact samples of jejunum and ileum from male Long Evans rats were analyzed on a 600 MHz spectrometer using standard one and two-dimensional (1)H NMR spectroscopic pulse sequences. The metabolic profiles of ileum and jejunum predominantly comprised a number of amino acids, lipids, glycerophosphocholine (GPC), choline, creatine, and ethanol, a number of carboxylic acids including acetate and lactate, and nucleoside bases including cytosine, isocytosine, and uracil. Principal component analysis (PCA) was applied to these NMR data to characterize the biochemical differences between jejunum and ileum tissues. Compared with ileum, jejunum contained higher levels of lipids, GPC, choline, lactate and creatinine, but lower levels of amino acids and acetate. In addition, the age dependence of the biochemical composition of intestinal tissues from young rats (15, 36 days and 3-4 months old) was studied. In general, levels of lipids, lactate, taurine and creatinine were positively correlated with age while amino acids and GPC decreased in the older age group. This study will provide a metabolic reference for further studies assessing the metabolic consequences of nutrition, stress and gut microbiota on intestinal composition.     

Topographical variation in metabolic signatures of human gastrointestinal biopsies revealed by high-resolution magic-angle spinning 1H NMR spectroscopy

Individual and topographical variation in the metabolic profiles of multiple human gastrointestinal tract (GIT) biopsies have been characterized using high-resolution magic-angle spinning (HRMAS) 1H NMR spectroscopy and pattern recognition. Samples from antrum, duodenum, jejunum, ileum, and transverse colon were obtained from 8 male and 8 female participants. Each gut region generated a highly characteristic metabolic profile consistent with the varying structural and functional properties of the tissue at different longitudinal levels of the gut. The antral (stomach) mucosa contained higher levels of choline, glycogen, phosphorylethanolamine, and taurine than other gut regions. The spatially close regions of the duodenum and jejunum were equivalent in terms of their gross biochemical composition with high levels of choline, glutathione, glycerophosphocholine (GPC), and lipids relative to other gut regions. The ileal mucosa showed poor discrimination from the duodenum and jejunum tissues and generated strong amino acids signatures but had relative low GPC signals. The colon (large intestine) was high in acetate, glutamate, inositols, and lactate and low in creatine, GPC, and taurine compared to the small intestine. These longitudinal metabolic variations in the human GIT could be attributed to functional variations in energy metabolism, osmoregulation, gut microbial activity, and oxidative protection. This work indicates that 1H HRMAS NMR studies may be of value in analyzing local metabolic variation due to pathological processes in gut biopsies.     

Effects of probiotic Lactobacillus paracasei treatment on the host gut tissue metabolic profiles probed via magic-angle-spinning NMR spectroscopy

We have used a simplified gnotobiotic mouse model to evaluate the effects of single bacterial species, Lactobacillus paracasei NCC2461, on the metabolic profiles of intact intestinal tissues using high-resolution magic-angle-spinning 1H NMR spectroscopy (HRMAS). A total of 24 female gnotobiotic mice were divided into three groups: a control group supplemented with water and two groups supplemented with either live L. paracasei or a gamma-irradiated equivalent. HRMAS was used to characterize the biochemical components of intact epithelial tissues from the duodenum, jejunum, ileum, proximal, and distal colons in all animals and data were analyzed using chemometrics. Variations in relative concentrations of amino acids, anti-oxidant, and creatine were observed relating to different physiological properties in each intestinal tissue. Metabolic characteristics of lipogenesis and fat storage were observed in the jejunum and colon. Colonization with live L. paracasei induced region-dependent changes in the metabolic profiles of all intestinal tissues, except for the colon, consistent with modulation of intestinal digestion, absorption of nutrients, energy metabolism, lipid synthesis and protective functions. Ingestion of gamma-irradiated bacteria produced no effects on the observed metabolic profiles. 1H MAS NMR spectroscopy was able to generate characteristic metabolic signatures reflecting the structure and function of intestinal tissues. These signals acted as reference profiles with which to compare changes in response to gut microbiota manipulation at the tissue level as demonstrated by ingestion of a bacterial probiotic.     

Hepatocytes of Wistar and Sprague Dawley rats differ significantly in their central metabolism

Wistar and Sprague‐Dawley (SD) rats are most commonly used experimental rats. They have similar genetic background and are therefore, not discriminated in practical research. In this study, we compared metabolic profiles of Wistar and SD rat hepatocytes from middle (6 months) and old (23 months) age groups. Principle component analysis (PCA) on the specific uptake and production rates of amino acids, glucose, lactate and urea indicated clear differences between Wistar and SD rat hepatocytes. SD rat hepatocytes showed higher uptake rates of various essential and non‐essential amino acids, particularly in early culture phases (0‐12 h) compared to later phases (12‐24 h). SD hepatocytes seem to be more sensitive to isolation procedure and in vitro culture requiring more amino acids for cellular maintenance and repair. Major differences between Wistar and SD rat hepatocytes were observed for glucose and branched chain amino acid metabolism. We conclude that the observed differences in the central carbon metabolism of isolated hepatocytes from these two rats should be considered when using one or the other rat type in studies on metabolic effects or diseases such as diabetes or obesity.     

Biochemical studies on the mechanism of difference in the renal toxicity of 5-hydroxy-L-tryptophan between Sprague Dawley and Wistar rats.

The biochemical mechanisms of the renal toxicity of 5-hydroxy-L-tryptophan to rats were studied using Wistar and Sprague Dawley rats, which had different LD50 values. When the amino acid was injected intraperitoneally, Wistar rats, which had a low LD50 value of 5-hydroxy-L-tryptophan, excreted larger amonts of serotonin and smaller amounts of 5-hydroxyindole acetic acid into the urine than Spraque Dawley rats, which had a high LD50 value. The activity of renal aromatic L-amino acid decarboxylase was higher in Wistar rats than in Sprague Dawley rats, while the activity of renal aromatic amino acid transaminase was in an opposite relationship. The activity of renal monoamine oxidase was almost the same in both strains and the activity of renal UDP glucuronyltransferase in Wistar rats was higher than in Sprague Dawley rats. Since the renal damage caused in rats by 5-hydroxy-L-tryptophan was very similar to that caused by serotonin, the amine formed from the administered amino acid was thought to be an important factor for the renal necroses, and difference in serotonin formation from the administered precursor amino acid may be one of the important factors leading to the difference in LD50 values in the two strains of rats.     

Metabolic profile associated with glucose and cholesterol lowering effects of berberine in Sprague–Dawley rats

In this study, we present an integrated strategy to deconvolute the metabolic signatures associated with the cholesterol lowering effect of berberine in the livers of Sprague?CDawley rats. The rats were dosed with berberine at 50?mg/kg. Urine samples and liver tissues were collected for the analysis of metabolite contents, while livers and kidneys were collected for histopathology. Metabolites such as fatty acids, cholesterol, glucose and others in liver were analyzed by gas chromatography/mass spectrometry. The urinary metabolites were analyzed using targeted profiling with liquid chromatography/tandem mass spectrometry and non-targeted profiling with proton nuclear magnetic resonance (¹H NMR). Our results demonstrated that analysis of metabolites in rat urine samples using liquid chromatography/mass spectrometry (LC/MS) and ¹H NMR produced complementary, consistent and reliable results. The administration of berberine resulted in a reduction of glucose, maltose, fatty acids (saturated and unsaturated) and cholesterol in the rat liver samples. The analysis of urinary metabolic profiles on different days showed that before the cholesterol reduction in the rat livers, a high rate of carbohydrate usage was found to be an early event (day 2). The results suggested that the animals utilized alternative energy sources by altering the synthesis and consumption of amino acids and fatty acids. In addition, changes in the level of glutamine for the treated animals on day 2 suggested that glutamine and glutamate metabolism could be affected. Since glutamine is a precursor for nucleotides synthesis and nucleotides are required for cell growth and replication, the results are consistent with the observed cholesterol lowering effect and weight reduction. Finally, our results demonstrated that the combination of LC/MS and ¹H NMR provided a unique metabolic profile associated with the cholesterol lowering effect of berberine in rat livers.     

Metabonomic and microbiological analysis of the dynamic effect of vancomycin-induced gut microbiota modification in the mouse

The effects of the antibiotic vancomycin (2 x 100 mg/kg/day) on the gut microbiota of female mice (outbred NMRI strain) were studied, in order to assess the relative contribution of the gut microbiome to host metabolism. The host's metabolic phenotype was characterized using (1)H NMR spectroscopy of urine and fecal extract samples. Time-course changes in the gut microbiotal community after administration of vancomycin were monitored using 16S rRNA gene PCR and denaturing gradient gel electrophoresis (PCR-DGGE) analysis and showed a strong effect on several species, mostly within the Firmicutes. Vancomycin treatment was associated with fecal excretion of uracil, amino acids and short chain fatty acids (SCFAs), highlighting the contribution of the gut microbiota to the production and metabolism of these dietary compounds. Clear differences in gut microbial communities between control and antibiotic-treated mice were observed in the current study. Reduced urinary excretion of gut microbial co-metabolites phenylacetylglycine and hippurate was also observed. Regression of urinary hippurate and phenylacetylglycine concentrations against the fecal metabolite profile showed a strong association between these urinary metabolites and a wide range of fecal metabolites, including amino acids and SCFAs. Fecal choline was inversely correlated with urinary hippurate. Metabolic profiling, coupled with the metagenomic study of this antibiotic model, illustrates the close inter-relationship between the host and microbial "metabotypes", and will provide a basis for further experiments probing the understanding of the microbial-mammalian metabolic axis.     

High-resolution magic angle spinning nuclear magnetic resonance analysis of metabolic changes in melanoma cells after induction of melanogenesis

Melanin synthesis affects melanoma behavior and tumor responsiveness to therapy; therefore, we investigated metabolic changes in melanoma cells after induction of melanogenesis. Amelanotic and melanotic melanoma cells were labeled with ¹³C precursors and changes in their metabolism was analyzed by high-resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR). HRMAS NMR demonstrated clear differences in the pattern of metabolic intermediates between amelanotic and melanotic cells. Although the exact nature of the metabolites requires further investigations, our comparative studies clearly show that induction of melanogenesis is associated with changes of glucose and sodium acetate metabolism, demonstrating HRMAS NMR as a powerful and noninvasive technique to investigate such process.     

Characterization of the biochemical effects of 1-nitronaphthalene in rats using global metabolic profiling by NMR spectroscopy and pattern recognition

Abstract

Metabolic fingerprints, in the form of patterns of high-concentration endogenous metabolites, of 1-nitronaphthalene (NN)-induced lung toxicity have been elucidated in bronchoalveolar lavage fluid (BALF), urine, blood plasma, and intact lung and liver tissue using NMR spectroscopy-based metabolic profiling. A single dose of NN (75?mg?kg^?1) was administered orally to Sprague–Dawley rats. BALF and lung tissue were obtained 24?h after dosing from these animals and matched control rats post-mortem. High-resolution ¹H-NMR spectroscopy of BALF samples indicated that NN caused increases in concentrations of choline, amino acids (leucine, isoleucine and alanine) and lactate together with decreased concentrations of succinate, citrate, creatine, creatinine and glucose. In addition, the intact lung weights were higher in the NN-treated group (p<0.01), consistent with pulmonary oedema. The NMR-detected perturbations indicated that NN induces a perturbation in energy metabolism in both lung and liver tissue, as well as surfactant production and osmolyte levels in the lungs. As well as reporting the first NMR spectroscopic combined examination of BALF and intact lung, this study indicates that such holistic approaches to investigating mechanisms of lung toxicity may be of value in evaluating disease progression or the effects of therapeutic intervention in pulmonary conditions such as surfactant disorders or asthma.     

Differential diagnosis of (inherited) amino acid metabolism or transport disorders

Summary Disorders of amino acid metabolism or transport are most clearly expressed in urine. Nevertheless the interpretation of abnormalities in urinary amino acid excretion remains difficult. An increase or decrease of almost every amino acid in urine can be due to various etiology. To differentiate between primary and secondary aminoacido-pathies systematic laboratory investigation is necessary. Early diagnosis of disorders of amino acid metabolism or transport is very important, because most of them can be treated, leading to the prevention of (further) clinical abnormalities. In those disorders, which cannot be treated, early diagnosis in an index-patient may prevent the birth of other siblings by means of genetic counseling and prenatal diagnosis.Primary aminoacidopathies can be due to genetically determined transport disorders and enzyme deficiencies in amino acid metabolism or degradation. Secondary aminoacidopathies are the result of abnormal or deficient nutrition, intestinal dysfunction, organ pathology or other metabolic diseases like organic acidurias.A survey of amino acid metabolism and transport abnormalities will be given, illustrated with metabolic pathways and characteristic abnormal amino acid chromatograms.     

Snow vole (Chionomys nivalis Martins) affects the redistribution of soil organic matter and hormone‐like activity in the alpine ecosystem: ecological implications

In alpine environments, colonies of snow vole (Chionomys nivalis Martins) cause strong pedoturbation, which may affect humification process and soil organic matter (SOM) cycling, with repercussions on the hormone‐like activity of organics. We investigated the effect of snow vole pedoturbation on the chemical and spectroscopic features of soil organic fractions, and the potential hormone‐like activity of humic and fulvic acids (HA, FA). The study site was located on the high‐mountain environment of the Majella massif (central Italy). Pedoturbated and regular soils were morphologically described and characterized for pH and content of total organic carbon, total extractable carbon, HA, and FA. Both HA and FA were extracted and investigated using attenuated total reflectance/Fourier transform infrared (ATR/FTIR), nuclear magnetic resonance with high‐resolution magic angle spinning (HRMAS‐NMR), and ¹H‐¹³C heteronuclear single quantum coherence (HSQC). HA and FA were also tested for their auxin‐like and gibberellin‐like activities. Results provide evidences that bioturbated and regular soils contain a poorly decomposed SOM, but HA and FA with a well‐defined molecular structure. The HA and FA from both bioturbated and regular soils show a hormone‐like activity with a different allocation along the soil profile. In the regular soil, the highest auxin‐like activity was shown by HA and FA from Oe1 horizon, while gibberellin‐like activity was expressed by FA from Oe2 horizon. Burrowing activity determines a redistribution of organics throughout the profile with a relatively high auxin‐like activity in the FA from straw tunnel wall (STW) and gibberellin‐like activity in the HA from vole feces (VF). The relative high presence of carboxylic acids, amides, proteins, and amino acids in the FA from STW and the aromatic moieties in the HA from VF put evidences for their different behavior. The fact that snow vole activity has modified the chemical and biological properties of SOM in these soils otherwise considered governed only by low temperature has important ecological implications such as the preservation of soil fertility and vegetal biodiversity.     

Metabonomic Profiling of Serum and Urine by 1H NMR-Based Spectroscopy Discriminates Patients with Chronic Obstructive Pulmonary Disease and Healthy Individuals

Chronic obstructive pulmonary disease (COPD) has seriously impacted the health of individuals and populations. In this study, proton nuclear magnetic resonance (¹H NMR)-based metabonomics combined with multivariate pattern recognition analysis was applied to investigate the metabolic signatures of patients with COPD. Serum and urine samples were collected from COPD patients (n = 32) and healthy controls (n = 21), respectively. Samples were analyzed by high resolution ¹H NMR (600 MHz), and the obtained spectral profiles were then subjected to multivariate data analysis. Consistent metabolic differences have been found in serum as well as in urine samples from COPD patients and healthy controls. Compared to healthy controls, COPD patients displayed decreased lipoprotein and amino acids, including branched-chain amino acids (BCAAs), and increased glycerolphosphocholine in serum. Moreover, metabolic differences in urine were more significant than in serum. Decreased urinary 1-methylnicotinamide, creatinine and lactate have been discovered in COPD patients in comparison with healthy controls. Conversely, acetate, ketone bodies, carnosine, m-hydroxyphenylacetate, phenylacetyglycine, pyruvate and α-ketoglutarate exhibited enhanced expression levels in COPD patients relative to healthy subjects. Our results illustrate the potential application of NMR-based metabonomics in early diagnosis and understanding the mechanisms of COPD.     

High-resolution magic angle spinning 1H NMR analysis of whole cells of Thalassiosira pseudonana (Bacillariophyceae): Broad range analysis of metabolic composition and nutritional value

Chemical composition of the microalga Thalassiosira pseudonana Hasle & Heimdalwas studied with different proton nuclearmagnetic resonance (¹H NMR)techniques, and by comparing NMR spectrafrom extraction samples with a spectrumfrom a sample of whole cells we show thathigh-resolution magic angle spinning (HRMAS) ¹H NMR can be used for broadrange analysis of metabolic composition inmicroalgal whole cells. Signals fromimportant metabolites such aspolyunsaturated fatty acids (PUFAs)eicosapentaenoic (EPA) and docosahexaenoic(DHA) acids were seen in a ¹H NMRspectrum of lipophilic extract, andpossibly also signals from the carotenoidfucoxanthin. In a spectrum of hydrophilicextract we assigned signals to amino acidssuch as glutamine (Gln) and glutamic acid(Glu), carbohydrate and ATP. These findingswere compared to a spectrum of HR MAS¹H NMR analysis of whole cells, whereit was possible to find signals coincidentwith the different metabolites seen inspectra of the extraction samples. Sincethe position of resonance peaks in a NMRspectrum depends on the chemicalsurroundings of each atom at the time ofanalysis some peak shift differencesbetween extract and whole cell samplespectra may occur, but signal shifts werenot significantly different between theanalyses here. In addition, application ofHR MAS highly increased spectral resolutionin the complex whole cell sample. Wetherefore suggest that HR MAS ¹H NMRanalysis is a suitable analysis tool tostudy metabolic composition directly onwhole cells of microalgae, making itpossible to study a broad range ofmetabolites simultaneously without tediousextraction procedures.     

A metabonomic investigation of the effects of 60 days exposure of rats to two types of pyrethroid insecticides

Type I and II pyrethroid insecticides display different neurotoxicity. To investigate the long-term (60 days exposure) metabolic effect of the two types of pyrethroid insecticides deltamethrin and permethrin, ¹H nuclear magnetic resonance (NMR) spectroscopy-based metabonomics was used to analyze the biochemical composition of urine and serum samples from rats administrated daily with deltamethrin or permethrin for 60 consecutive days, and principal component analysis used to visualize similarities and differences in the resultant biochemical profiles. Rats treated with either deltamethrin or permethrin displayed increased levels of urinary acetate, dimethylamine, dimethylglycine, trimethylamine and serum free amino acids, and decreased urinary 2-oxoglutarate, all of which are indicative of kidney lesions and nephrotoxicity. The reduced excretion of tricarboxylic acid cycle intermediates, together with increased 3-D-hydroxybutyrate, acetate, and lactate in treated rats could suggest disturbance of the energy metabolism, including an increased rate of anaerobic glycolysis, enhanced fatty acid β-oxidation and ketogenesis. These results show that these two types of insecticides have similarities in the urine and serum spectra, indicating that similar metabolic pathways are perturbed by the insecticides, which induced hepatotoxicity and nephrotoxicity. This approach may lead to the discovery of novel biomarkers of pyrethroids toxicity and thereby provide new insights into the toxicological mechanisms of pesticides pyrethroids.     

Metabolite profiles and the risk of developing diabetes

Emerging technologies allow the high-throughput profiling of metabolic status from a blood specimen (metabolomics). We investigated whether metabolite profiles could predict the development of diabetes. Among 2,422 normoglycemic individuals followed for 12 years, 201 developed diabetes. Amino acids, amines and other polar metabolites were profiled in baseline specimens by liquid chromatography-tandem mass spectrometry (LC-MS). Cases and controls were matched for age, body mass index and fasting glucose. Five branched-chain and aromatic amino acids had highly significant associations with future diabetes: isoleucine, leucine, valine, tyrosine and phenylalanine. A combination of three amino acids predicted future diabetes (with a more than fivefold higher risk for individuals in top quartile). The results were replicated in an independent, prospective cohort. These findings underscore the potential key role of amino acid metabolism early in the pathogenesis of diabetes and suggest that amino acid profiles could aid in diabetes risk assessment.     

Androgen-dependent proteins in the urine of bank voles (Clethrionomys glareolus)

The concentration of proteins in the urine of adult bank vole males was higher than that in urine of immature males and females. After separation of the urine by polyacrylamide gel electrophoresis in the presence of SDS, the principal urinary protein had a slightly lower mobility than cytochrome C. Urine from females or castrated males contained only trace amounts of this protein. Injection of testosterone into castrated males increased this protein band. We suggest that bank vole males, like those of rats and mice, synthesize and release in their urine an androgen-dependent protein fraction.     

Comparison of the urinary excretion time courses of pyrene-1,6-dione,pyrene-1,8-dione and 1-hydroxypyrene in rats intravenously exposed to pyrene

Abstract

The urinary excretion time courses of pyrene-1,6-dione (P16D), pyrene-1,8-dione (P18D) and 1-hydroxypyrene (1-OHP) were compared in Sprague–Dawley and Wistar rats. Groups of five male rats, of about 200 g of body weight, were injected intravenously with 0.05, 0.5, 5 and 50 µmol pyrene kg^?1 of body weight. Urine was collected at 2, 4, 6, 8, 10, 12, 18, 24, 30, 42 and 48 h post-dosing. Pyrene metabolites were measured by high-performance liquid chromatography (HPLC)/fluorescence after enzymatic hydrolysis of the glucurono- and sulfo-conjugates, extraction on Sep-Pak C18 cartridges and, for the analysis of dione metabolites, derivatization to stable diacetoxypyrene molecules. Over the 48-h sampling period, on average 17.4–25.6% of the injected pyrene was excreted overall as P16D, 6.4–8.8% as P18D and 0.6–0.8% as 1-OHP in the urine of Sprague–Dawley rats. By comparison, on average 10.3–14.7% of the intravenous pyrene dose was recovered as P16D, 4.8–6.4% as P18D and 0.3–0.4% as 1-OHP in the urine of Wistar rats. In both strains of rats there was no clear effect of the dose on the 0–48-h cumulative urinary excretion of P18D and 1-OHP over the entire dose range, while the percentage of dose recovered overall as P16D in urine at the highest dose (50 µmol kg^?1) was statistically lower than at the other doses. The 0–48-h cumulative percentage of pyrene dose excreted as metabolites in the urine of Sprague–Dawley rats was also significantly higher than in Wistar rats (p<0.01) exposed under identical conditions. As for the urinary excretion-time courses of the different metabolites, for a given dose and strain of rats, excretion curves of P16D, P18D and 1-OHP generally evolved in parallel. There was also no clear effect of the dose on the excretion rate, thus half-life, of pyrene metabolites, except for P16D in Sprague–Dawley rats at the highest dose where elimination tended to be slower compared with the other doses (p<0.01). The average first-order elimination half-life of P16D, P18D and 1-OHP was 4.0, 5.7 and 4.1 h, respectively, in Sprague–Dawley rats, and 5.1, 6.1 and 5.1 h, respectively, in Wistar rats (all doses combined but excluding the highest dose for P16D). This study showed the relative importance of metabolic pathways leading to diones compared with 1-OHP. These dioxygenated metabolites appear to be interesting biomarkers of pyrene exposure at environmentally and occupationally relevant doses. Their adequacy as biomarkers of human exposure has yet to be confirmed.     

Species variation in the fecal metabolome gives insight into differential gastrointestinal function

The metabolic composition of fecal extracts provides a window for elucidating the complex metabolic interplay between mammals and their intestinal ecosystems, and these metabolite profiles can yield information on a range of gut diseases. Here, the metabolites present in aqueous fecal extracts of humans, mice and rats were characterized using high-resolution (1)H NMR spectroscopy coupled with multivariate pattern recognition techniques. Additionally, the effects of sample storage and preparation methods were evaluated in order to assess the stability of fecal metabolite profiles, and to optimize information recovery from fecal samples. Finally, variations in metabolite profiles were investigated in healthy mice as a function of time. Interspecies variation was found to be greater than the variation due to either time or sample preparation. Although many fecal metabolites were common to the three species, such as short chain fatty acids and branched chain amino acids, each species generated a unique profile. Relatively higher levels of uracil, hypoxanthine, phenylacetic acid, glucose, glycine, and tyrosine amino acids were present in the rat, with beta-alanine being unique to the rat, and glycerol and malonate being unique to the human. Human fecal extracts showed a greater interindividual variation than the two rodent species, reflecting the natural genetic and environmental diversity in human populations. Fecal composition in healthy mice was found to change over time, which might be explained by altered gut microbial presence or activity. The systematic characterization of fecal composition across humans, mice, and rats, together with the evaluation of inherent variation, provides a benchmark for future studies seeking to determine fecal biomarkers of disease and/or response to dietary or therapeutic interventions.     

The comparative metabonomics of age-related changes in the urinary composition of male Wistar-derived and Zucker (fa/fa) obese rats

The global metabolite profiles of endogenous compounds excreted in urine by male Wistar-derived and Zucker (fa/fa) obese rats were investigated from 4 to 20 weeks of age using both 1H NMR spectroscopy and HPLC-TOF/MS with electrospray ionisation (ESI). Multivariate data analysis was then performed on the resulting data which showed that the composition of the samples changed with age, enabling age-related metabolic trajectories to be constructed. At 4 weeks it was possible to observe differences between the urinary metabolite profiles from the two strains, with the difference becoming more pronounced over time resulting in a marked divergence in their metabolic trajectories at 8-10 weeks. The changes in metabolite profiles detected using 1H NMR spectroscopy included increased protein and glucose combined with reduced taurine concentrations in the urine of the Zucker animals compared to the Wistar-derived strain. In the case of HPLC-MS a number of ions were found to be present at increased levels in the urine of 20 week old Zucker rats compared to Wistar-derived rats including m/z 71.0204, 111.0054, 115.0019, 133.0167 and 149.0454 (negative ion ESI) and m/z 97.0764 and 162.1147 (positive ion ESI). Conversely, ions m/z 101.026 and 173.085 (negative ion ESI) and m/z 187.144 and 215.103 (positive ion ESI) were present in decreased amounts in urine from Zucker compared to Wistar-derived rats. Metabolite identities proposed for these ions include fumarate, maleate, furoic acid, ribose, suberic acid, carnitine and pyrimidine nucleoside. The utility of applying metabonomics to understanding disease processes and the biological relevance of some of the findings are discussed.     

A study on human urine in a high-selenium area of China by 1H-NMR spectroscopy

In this study, high-resolution 600-MHz ¹H-NMR (nuclear magnetic resonance) spectroscopies were used to compare the urinary metabolic profiles of healthy humans and humans in a high-selenium area of China. NMR biomarkers for renal and liver lesions were observed by comparing the urine ¹H-NMR spectra. In urinary excretion, the concentrations in human urine samples of formate, lactate, acetate, hippurate, and alanine in overexposure to selenium were increased, whereas citrate, creatine, and TMAO excretion were decreased compared with that of the healthy human—some of them even disappeared. An interesting result was the appearance of formate in urine, which has previously been shown to lead to acidosis and chronic renal failure and interfere with the lumen and proximal tubular cells. The level of creatine was associated with the seminal activity. The changes of acetate and citrate may explain the disorder of the cellular energy metabolism caused by selenium, and the changes of other amino acids were a result of the reuptake of these compounds that had been blocked in the glomerulus and proximal tubule. The results elucidate the renal/liver lesion in humans in high-selenium area by ¹H-NMR spectroscopy and offer the molecular basic of selenium toxicity.