NMR Spectra Of Urine Research Articles

Nuclear magnetic resonance‐ and mass spectrometry‐based metabolomics to study maleic acid toxicity from repeated dose exposure in rats

We recently found the Figure S1 presented in the supplemental materials of our paper (Wu et al., 2017) may not be the most representative chromatogram generated from the analysis of four oxidative stress biomarkers (8-NO2Gua, 8-OHdG, HNE-MA and 8-isoPGF2α) from rat urine samples collected from repeated exposure to maleic acid in this study. The figure was put in the method section intended to demonstrate the feasibility of our analytical platform, which was cited and had been applied in previous research (Chen, Yuan, Shie, Wu, & Chan, 2017; Wu, Chen, Peng, Cheng, & Wu, 2016). Upon combing through our experimental raw data, this replacement chromatogram is more appropriate to include in the supplemental materials of this paper. In this diagram, only the four analytes and their respective isotopes are additionally marked. The corrected figure is provided in the Supporting Information. The replacement of this new figure in the supplementary material does not affect the main text and results of this paper. We apologize for the inconvenience that this replacement caused by this mistake. Table S1. The HPLC system comprised a quaternary pump, an autosampler, and a 3 μm, 100×2 mm Gemini-NX-C18 analytical column (Phenomenex, Torrance, CA). Table S2. Body weight gain and relative organ weights in low dose (LD), medium dose (MD), high dose (HD) group and the control group Figure S1. A representative chromatogram of four urinary oxidative stress biomarkers from a rat dosed with 60 mg/kg of maleic acid on day 14. Figure S2. The histopathology results of a representative frozen section of rat kidney from the (a) control and (b) high-dose (60 mg/kg) groups which treated with maleic acid daily for 28 days. Figure S3. The histopathology results of a representative frozen section of rat liver from the (a) control and (b) high-dose (60 mg/kg) groups which treated with maleic acid daily for 28 days Figure S4. A representative NMR spectrum of urinary metabolic profiling performed by 600 MHz 1H NMR from rats treated with maleic acid (high-dose) daily. Figure S5. PCA (a) scores plot from the analysis of 1H NMR spectra of urine collected from day and night from rats treated with control or maleic acid exposure; (b) loading plot from the analysis of 1H NMR spectra of urine collected from day and night from rats treated with control or maleic acid exposure. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Metabolic Profiling Analysis of the Alleviation Effect of the Fractions of Niuhuang Jiedu Tablet on Realgar Induced Toxicity in Rats.

Niuhuang Jiedu Tablet (NJT) is a classical formula in treating acute tonsillitis, pharyngitis, and so on. In the formula, significant level of Realgar as a potentially toxic element is contained. Our previous experiments revealed that it was less toxic for combined Realgar in NJT. However, the active fraction of this prescription with toxicity alleviation effect on Realgar was still obscure. NJT was divided into five different polar fractions (NJT-PET, NJT-25, NJT-50, NJT-75, and NJT-95), and we explored the toxicity alleviation effect on Realgar. Based on 1H NMR spectra of urine and serum from rats, PCA and PLS-DA were performed to identify different metabolic profiles. Liver and kidney histopathology examinations and serum clinical chemistry analysis were also performed. With pattern recognition analysis of metabolites in urine and serum, Realgar group showed a clear separation from control group, while the metabolic profiles of NJT-PET, NJT-25, NJT-50, and NJT-95 groups were similar to Realgar group, and the metabolic profiles of NJT and NJT-75 groups were very close to control group. Statistics results were confirmed by the histopathological examination and biochemical assay. The present work indicated that 75% EtOH fraction of NJT was the most valid fraction with the toxicity alleviation effect on Realgar.

1H NMR-Based Metabonomic Study of Functional Dyspepsia in Stressed Rats Treated with Chinese Medicine Weikangning.

1H NMR-based metabolic profiling combined with multivariate data analysis was used to explore the metabolic phenotype of functional dyspepsia (FD) in stressed rats and evaluate the intervention effects of the Chinese medicine Weikangning (WKN). After a 7-day period of model establishment, a 14-day drug administration schedule was conducted in a WKN-treated group of rats, with the model and normal control groups serving as negative controls. Based on 1H NMR spectra of urine and serum from rats, PCA, PLS-DA, and OPLS-DA were performed to identify changing metabolic profiles. According to the key metabolites determined by OPLS-DA, alterations in energy metabolism, stress-related metabolism, and gut microbiota were found in FD model rats after stress stimulation, and these alterations were restored to normal after WKN administration. This study may provide new insights into the relationship between FD and psychological stress and assist in research into the metabolic mechanisms involved in Chinese medicine.

A metabolic profiling analysis of the acute toxicological effects of the realgar (As2S2) combined with other herbs in Niuhuang Jiedu Tablet using 1H NMR spectroscopy

Ethnopharmacological relevanceNiuhuang Jiedu Tablet (NJT), composed of Realgar (As2S2), Bovis Calculus Artificialis, Borneolum Synthcticum, Gypsum Fibrosum, Rhei Radix et Rhizoma (RR), Scutellariae Radix (SR), Platycodonis Radix (PR) and Glycyrrhizae Radix et Rhizoma (GR), is an effective formula of traditional Chinese medicine (TCM) used in treating acute tonsillitis, pharyngitis, periodontitis and mouth ulcer. In the formula, significant level of realgar (As2S2) as a potentially toxic element is contained. In our pervious experiments, NJT was significantly less toxic than realgar (As2S2), and the material bases of toxicity alleviation effect to realgar (As2S2) were RR, SR, PR and GR. However, the toxicity alleviation effect of each above mentioned four herbs to realgar (As2S2) and their synergistic detoxification effects to realgar (As2S2) were still obscure. Materials and methodsMale Wistar rats were divided into 11 groups: control, group R (treated with Realgar), group RRSPG (treated with Realgar, RR, SR, PR and GR), group RRSP (treated with Realgar, RR, SR and PR), group RRSG (treated with Realgar, RR, SR and GR), group RRPG (treated with Realgar, RR, PR and GR), group RSPG (treated with Realgar, SR, PR and GR), group RR (treated with Realgar and RR), group RS (treated with Realgar and SR), group RP (treated with Realgar and PR) and group RG (treated with Realgar and GR). Based on 1H NMR spectra of urine and serum from rats, PCA and PLS-DA were performed to identify different metabolic profiles. Liver and kidney histopathology examinations and serum clinical chemistry analysis were also performed. ResultsThe metabolic profiles of groups RR, RS, RP and RG were similar to those of group R, while the metabolic profiles of groups RRSPG, RRSP, RRSG, RRPG and RSPG were almost in line with those of control group. Statistics results were confirmed by the histopathological examination and biochemical assay. ConclusionThe present work suggested that the toxicity alleviation effects of RR, SR, PR and GR to realgar (As2S2) were not obvious when combined with realgar (As2S2) respectively, but they had synergistic detoxification effects on realgar (As2S2) mutually.

1H NMR-based metabonomics study on the toxicity alleviation effect of other traditional Chinese medicines in Niuhuang Jiedu tablet to realgar (As2S2)

Ethnopharmacological relevanceNiuhuang Jiedu Tablet (NJT) is an effective prescription of traditional Chinese medicine (TCM) used in treating acute tonsillitis, pharyngitis, periodontitis and mouth ulcer. NJT is prepared from Xionghuang (Realgar, As2S2), Rengong Niuhuang (Bovis Calculus Artificialis), Bingpian (Borneolum Synthcticum), Shigao (Gypsum Fibrosum), Dahuang (Rhei Radix et Rhizoma), Huangqin (Scutellariae Radix), Jiegeng (Platycodonis Radix) and Gancao (Glycyrrhizae Radix et Rhizoma). In the prescription, significant level of realgar (As2S2) as a potentially toxic element is contained. Aim of the studyIn this study, 1H NMR-based metabonomics approach has been used to investigate the toxicity of realgar (As2S2) after being counterbalanced by other TCMs in NJT. Materials and methodsMale Wistar rats were divided into five groups: control, group I (treated with Realgar), group II (treated with Realgar, Bovis Calculus Artificialis, Borneolum Synthcticum, Gypsum Fibrosum, Rhei Radix et Rhizoma, Scutellariae Radix, Platycodonis Radix and Glycyrrhizae Radix et Rhizoma), group III (treated with Realgar, Bovis Calculus Artificialis, Borneolum Synthcticum and Gypsum Fibrosum) and group IV (treated with Realgar, Rhei Radix et Rhizoma, Scutellariae Radix, Platycodonis Radix and Glycyrrhizae Radix et Rhizoma). Based on 1H–NMR spectra of urine and serum from rats, PCA and PLS-DA were performed to identify different metabolic profiles. Liver and kidney histopathology examinations and serum clinical chemistry analysis were also performed. ResultsPLS-DA scores plots demonstrated that the cluster of group I was separated from that of control rats, while group II was located close to control rats, indicating that metabolic profiles of group II were restored toward those of control rats. The metabolic profiles of group III were similar to those of group I, while the metabolic profiles of group II were almost in line with those of group II. Statistics results were confirmed by the histopathological examination and biochemical assay. ConclusionOur results indicated that it was more secure and much less toxic for counterbalanced realgar (As2S2) in NJT. The effective material bases of toxicity alleviation to realgar (As2S2) were Dahuang (Rhei Radix et Rhizoma), Huangqin (Scutellariae Radix), Jiegeng (Platycodonis Radix) and Gancao (Glycyrrhizae Radix et Rhizoma), which regulated energy metabolism, choline metabolism, amino acid metabolism and gut flora disorder affected by realgar (As2S2) exposure.

1H NMR-based metabolite profiling of diet-induced obesity in a mouse mode

High-fat diets (HFD) and high-carbohydrate diets (HCD)- induced obesity through different pathways, but the metabolic differences between these diets are not fully understood. Therefore, we applied proton nuclear magnetic resonance ((1)H NMR)-based metabolomics to compare the metabolic patterns between C57BL/6 mice fed HCD and those fed HFD. Principal component analysis derived from (1)H NMR spectra of urine showed a clear separation between the HCD and HFD groups. Based on the changes in urinary metabolites, the slow rate of weight gain in mice fed the HCD related to activation of the tricarboxylic acid cycle (resulting in increased levels of citrate and succinate in HCD mice), while the HFD affected nicotinamide metabolism (increased levels of 1-methylnicotineamide, nicotinamide-N-oxide in HFD mice), which leads to systemic oxidative stress. In addition, perturbation of gut microflora metabolism was also related to different metabolic patterns of those two diets. These findings demonstrate that (1)H NMR-based metabolomics can identify diet-dependent perturbations in biological pathways.

Urinary metabolomic phenotyping of nickel induced acute toxicity in rat: an NMR spectroscopy approach

The metabolomic approach has been widely used in toxicology to investigate mechanisms of toxicity. To understand the mammalian system’s response to nickel exposure, we analysed the NiCl2 induced metabolomic changes in urine of rats using 1H nuclear magnetic resonance (1H NMR) spectroscopy together with clinically relevant biochemical parameters. Male Sprague–Dawley rats were administered intraperitoneally with NiCl2 at doses of 4, 10 and 20 mg/kg body weight. Urine samples were collected at 8, 16, 24, 72, 96 and 120 h post treatment. The metabolomic profile of rat urine showed prominent changes in citrate, dimethylamine, creatinine, choline, trimethylamine oxide (TMAO), phenyl alanine and hippurate at all doses. Principal component analysis of urine 1H NMR spectra demonstrated the dose and time dependent development of toxicity. The metabolomic time trajectory, based on pattern recognition analysis of 1H NMR spectra of urine, illustrated clear separation of pre and post treatments (temporal). Only animals treated with a low dose of NiCl2 returned to normal physiology. The 1H NMR spectral data correlated well with the clinically relevant nephrotoxic biomarkers. The urinary metabolomic phenotyping for NiCl2 induced nephrotoxicity was defined according to the predictive ability of the known metabolite biomarkers, creatinine, citrate and TMAO. The current approach demonstrates that metabolomics, one of the most important platform in system biology, may be a promising tool for identifying and characterizing biochemical responses to toxicity.

NMR‐based metabonomic approach on the toxicological effects of a Cimicifuga triterpenoid

Cimicifugae Rhizoma, a well-known botanical dietary supplement, has been the subject of intense interest due to its potential application for alleviating menopausal symptom. Although there are clinic data that the Cimicifuga extract should have hepatotoxicity, no evidence on the main chemical components has been reported. Cimicidol-3-O-β -d-xyloside (CX) is one of the main triterpenoids of the rhizome. This work studies the toxicological effects of CX after oral administration (50 mg kg(-1) per day) over a 7-day period in female SD rats using metabonomic analyses of (1) H NMR spectra of urine, serum and liver tissue extracts. Histopathological studies of liver and analyses of blood biochemical parameter, such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, blood urea nitrogen and creatinine revealed that CX had no negative impacts on liver and kidney. However, the metabolic signature of (1) H NMR-based urinalysis of daily samples displayed an increment in the levels of taurine, trimethylamine-N-oxide (TMAO), betaine and acetate. Elevated serum levels of creatinine, glucose, alanine, TMAO and betaine and lower levels of lactate were observed. Metabolic profiling on aqueous soluble extracts of liver showed simultaneously increases in succinate, glycogen, choline, glycerophosphorylcholine, TMAO and betaine levels and reduction in valine, glucose and lactate levels. Nevertheless, no changes in any metabonomic level were found in lipid-soluble extracts of liver. These findings indicate that CX has a slight toxicity in liver and kidney via disturbance of the metabolisms of energy and amino acids. The present study provides a reasonable explanation for the clinical hepatotoxicity of Cimicifuga extract.

Alterations in urine, serum and brain metabolomic profiles exhibit sexual dimorphism during malaria disease progression

BackgroundMetabolic changes in the host in response to Plasmodium infection play a crucial role in the pathogenesis of malaria. Alterations in metabolism of male and female mice infected with Plasmodium berghei ANKA are reported here.Methods1H NMR spectra of urine, sera and brain extracts of these mice were analysed over disease progression using Principle Component Analysis and Orthogonal Partial Least Square Discriminant Analysis.ResultsAnalyses of overall changes in urinary profiles during disease progression demonstrate that females show a significant early post-infection shift in metabolism as compared to males. In contrast, serum profiles of female mice remain unaltered in the early infection stages; whereas that of the male mice changed. Brain metabolite profiles do not show global changes in the early stages of infection in either sex. By the late stages urine, serum and brain profiles of both sexes are severely affected. Analyses of individual metabolites show significant increase in lactate, alanine and lysine, kynurenic acid and quinolinic acid in sera of both males and females at this stage. Early changes in female urine are marked by an increase of ureidopropionate, lowering of carnitine and transient enhancement of asparagine and dimethylglycine. Several metabolites when analysed individually in sera and brain reveal significant changes in their levels in the early phase of infection mainly in female mice. Asparagine and dimethylglycine levels decrease and quinolinic acid increases early in sera of infected females. In brain extracts of females, an early rise in levels is also observed for lactate, alanine and glycerol, kynurenic acid, ureidopropionate and 2-hydroxy-2-methylbutyrate.ConclusionsThese results suggest that P. berghei infection leads to impairment of glycolysis, lipid metabolism, metabolism of tryptophan and degradation of uracil. Characterization of early changes along these pathways may be crucial for prognosis and better disease management. Additionally, the distinct sexual dimorphism exhibited in these responses has a bearing on the understanding of the pathophysiology of malaria.

Taurine – a possible fingerprint biomarker in non-muscle invasive bladder cancer: A pilot study by 1H NMR spectroscopy

Urinary bladder cancer is a major epidemiological problem that continues to grow each year. It opens avenues for investigative research for the identification of new disease markers and diagnostic techniques. In this pilot study, utility of non-invasive (1)H NMR spectroscopy has been evaluated for probing the metabolic perturbations occurring in non-muscle invasive urinary bladder cancer. (1)H NMR spectra of urine of bladder cancer patients and controls (healthy and urinary tract infection/bladder stone) (n = 103) were acquired at 400MHz. The non-overlapping resonances of citrate, dimethylamine, phenylalanine, taurine and hippurate were first identified and then quantitated by (1)H NMR spectra, with respect to an external reference sodium-3-trimethylsilylpropionate (TSP). The concentrations of these metabolites were then statistically analyzed. The cancer patients showed significant (p < 0.05) variations in concentration of hippurate and citrate as compared with healthy controls and benign controls. The significant elevation in concentration of taurine was observed in urine of bladder cancer patients, which was below the sensitivity limit of 400MHz in control cases. However, stages Ta, T1 and carcinoma in situ (CIS) cannot be differentiated on the basis of altered metabolite indices but their composition may reflect the biochemical alterations in metabolism of cancer cells.

Large-scale human metabolic phenotyping and molecular epidemiological studies via 1H NMR spectroscopy of urine: investigation of borate preservation.

Borate is an antibacterial preservative widely used in clinical and large-scale epidemiological studies involving urine sample analysis. Since it readily forms covalent adducts and reversible complexes with hydroxyl and carboxylate groups, the effects of borate preservation in (1)H NMR-spectroscopy-based metabolic profiling of human urine samples have been assessed. Effects of various concentrations of borate (range 0-30 mM) on (1)H NMR spectra of urine were observed at sequential time points over a 12 month period. Consistent with known borate chemistry, the principal alterations in the (1)H resonance metabolite patterns were observed for compounds such as mannitol, citrate, and alpha-hydroxyisobutyrate and confirmed by ESI-MS analysis. These included line-broadening, T(1) and T(2) relaxation, and chemical shift changes consistent with complex formation and chemical exchange processes. To further investigate complexation behavior in the urinary metabolite profiles, a new tool for visualization of multicomponent relaxation variations in which the spectra were color-coded according to the T(1) and T(2) proton relaxation times respectively (T(1) or T(2) ordered projection spectroscopy, TOPSY) was also developed and applied. Addition of borate caused a general decrease in (1)H T(1) values, consistent with nonspecific effects such as solution viscosity changes. Minor changes in proton T(2) relaxation rates were observed for the most strongly complexing metabolites. From a molecular phenotyping and epidemiologic viewpoint, typical interpersonal biological variation was shown to be vastly greater than any variation introduced by the borate complexation, which had a negligible effect on the metabolic mapping and classification of samples. While caution is indicated in the assignment of biomarker signals where metabolites have diol groupings or where there are adjacent hydroxyl and carboxylate functions, it is concluded that borate preservation is "fit-for-purpose" for (1)H NMR-based epidemiological studies, since the essential biochemical classification features of the samples are robustly maintained.

Analytic Properties of Statistical Total Correlation Spectroscopy Based Information Recovery in 1H NMR Metabolic Data Sets

Structural assignment of resonances is an important problem in NMR spectroscopy, and statistical total correlation spectroscopy (STOCSY) is a useful tool aiding this process for small molecules in complex mixture analysis and metabolic profiling studies. STOCSY delivers intramolecular information (delineating structural connectivity) and in metabolism studies can generate information on pathway-related correlations. To understand further the behavior of STOCSY for structural assignment, we analyze the statistical distribution of structural and nonstructural correlations from 1050 (1)H NMR spectra of normal rat urine samples. We find that the distributions of structural/nonstructural correlations are significantly different (p < 10(-112)). From the area under the curve of the receiver operating characteristic (ROC AUC) we show that structural correlations exceed nonstructural correlations with probability AUC = 0.98. Through a bootstrap resampling approach, we demonstrate that sample size has a surprisingly small effect (e.g., AUC = 0.97 for a sample size of 50). We identify specific signatures in the correlation maps resulting from small matrix-derived variations in peak positions but find that their effect on discrimination of structural and nonstructural correlations is negligible for most metabolites. A correlation threshold of r > 0.89 is required to assign two peaks to the same metabolite with high probability (positive predictive value, PPV = 0.9), whereas sensitivity and specificity are equal at 93% for r = 0.22. To assess the wider applicability of our results, we analyze (1)H NMR spectra of urine from rats treated with 115 model toxins or physiological stressors. Across the data sets, we find that the thresholds required to obtain PPV = 0.9 are not significantly different and the degree of overlap between the structural and nonstructural distributions is always small (median AUC = 0.97). The STOCSY method is effective for structural characterization under diverse biological conditions and sample sizes provided the degree of correlation resulting from nonstructural associations (e.g., from nonstationary processes) is small. This study validates the use of the STOCSY approach in the routine assignment of signals in NMR metabolic profiling studies and provides practical benchmarks against which researchers can interpret the results of a STOCSY analysis.

Metabonomic profile of urine from rats administrated with different treatment period of rifampin

To study the effect of different treatment period, of isoniazid (INH) on the metabonomic profile of rat urine and its relationship with traditional toxicity evaluation of blood biochemical indicators and histopathology and to explore the feasibility of metabonomics in the application of drug toxicity. Sixty male Wistar rats were orally administrated with 0, 50, 100, 200, and 400 mg x kg(-1) INH for 3, 7, and 14 days, respectively. Rat urine was then collected and its 1H nuclear magnetic resonance (NMR) spectra were acquired. All animals underwent traditional toxicity evaluation. Hepatotoxicity was revealed by traditional toxicity evaluation in rats treated with higher dosage and longer treatment of INH. Time-response relationship existed during the treatment. Time-dependent metabonomics changes conformed with the results of traditional toxicity evaluation. The urine metabonomics showed a trajectory bias from those of the controls or pre-administration, and such bias exaggerated along with the prolongation of treatment, indicating a severer toxic injury. Along with the increase of the concentrations of urinary taurine and glucose and the decrease of the concentrations of urinary citrate and 2-oxoglutarate, the 1H NMR spectra of urine in rats treated with INH also changed. The metabonomics technique can distinguish the onset and development of toxicity, which helps track and identify biomarkers. The hepatic toxicity induced by INH is related to the injury of mitochondrial function, reduction of energy metabolism in tricarboxylic acid cycle, and perturbations in the metabolism of glucose and lipid. The effect of INH on the rat urine metabonomic profile is related with INH toxicology. Therefore, metabonomics can be recognized as an ideal technique to explore and evaluate the drug toxicities.

Metabolic profiling studies on the toxicological effects of realgar in rats by 1H NMR spectroscopy

The toxicological effects of realgar after intragastrical administration (1 g/kg body weight) were investigated over a 21 day period in male Wistar rats using metabonomic analysis of 1H NMR spectra of urine, serum and liver tissue aqueous extracts. Liver and kidney histopathology examination and serum clinical chemistry analyses were also performed. 1H NMR spectra and pattern recognition analyses from realgar treated animals showed increased excretion of urinary Kreb's cycle intermediates, increased levels of ketone bodies in urine and serum, and decreased levels of hepatic glucose and glycogen, as well as hypoglycemia and hyperlipoidemia, suggesting the perturbation of energy metabolism. Elevated levels of choline containing metabolites and betaine in serum and liver tissue aqueous extracts and increased serum creatine indicated altered transmethylation. Decreased urinary levels of trimethylamine- N-oxide, phenylacetylglycine and hippurate suggested the effects on the gut microflora environment by realgar. Signs of impairment of amino acid metabolism were supported by increased hepatic glutamate levels, increased methionine and decreased alanine levels in serum, and hypertaurinuria. The observed increase in glutathione in liver tissue aqueous extracts could be a biomarker of realgar induced oxidative injury. Serum clinical chemistry analyses showed increased levels of lactate dehydrogenase, aspartate aminotransferase, and alkaline phosphatase as well as increased levels of blood urea nitrogen and creatinine, indicating slight liver and kidney injury. The time-dependent biochemical variations induced by realgar were achieved using pattern recognition methods. This work illustrated the high reliability of NMR-based metabonomic approach on the study of the biochemical effects induced by traditional Chinese medicine.

Genetic algorithms for simultaneous variable and sample selection in metabonomics

Metabolic profiles derived from high resolution (1)H-NMR data are complex, therefore statistical and machine learning approaches are vital for extracting useful information and biological insights. Focused modelling on targeted subsets of metabolites and samples can improve the predictive ability of models, and techniques such as genetic algorithms (GAs) have a proven utility in feature selection problems. The Consortium for Metabonomic Toxicology (COMET) obtained temporal NMR spectra of urine from rats treated with model toxins and stressors. Here, we develop a GA approach which simultaneously selects sets of samples and spectral regions from the COMET database to build robust, predictive classifiers of liver and kidney toxicity. The results indicate that using simultaneous sample and variable selection improved performance by over 9% compared with either method alone. Simultaneous selection also halved computation time. Successful classifiers repeatedly selected particular variables indicating that this approach can aid defining biomarkers of toxicity. Novel visualizations of the results from multiple computations were developed to aid the interpretability of which samples and variables were frequently selected. This method provides an efficient way to determine the most discriminatory variables and samples for any post-genomic dataset. GA code available from http://www1.imperial.ac.uk/medicine/people/r.cavill/

Use of EDTA to minimize ionic strength dependent frequency shifts in the 1H NMR spectra of urine

The 1H NMR spectrum of urine exhibits a large number of detectable metabolites and is, therefore, highly suitable for the study of perturbations caused by disease, toxicity, nutrition or environmental factors in humans and animals. However, variations in the chemical shifts and intensities due to altered pH and ionic strength present a challenge in NMR-based studies. With a view towards understanding and minimizing the effects of these variations, we have extensively studied the effects of ionic strength and pH on the chemical shifts of common urine metabolites and their possible reduction using EDTA (ethylenediaminetetraacetic acid). 1H NMR chemical shifts for alanine, citrate, creatinine, dimethylamine, glycine, histidine, hippurate, formate and the internal reference, TSP (trimethylsilylpropionic acid-d4, sodium salt) obtained under different conditions were used to assess each effect individually. EDTA minimizes the frequency shifts of the metabolites that have a propensity for metal binding. Chelation of such metal ions is evident from the appearance of signals from EDTA complexed to divalent metal ions such as calcium and magnesium. Not surprisingly, increasing the buffer concentration or buffer volume also minimizes pH dependent frequency shifts. The combination of EDTA and an appropriate buffer effectively minimizes both pH dependent frequency shifts and ionic strength dependent intensity variations in urine NMR spectra.

Systemic multicompartmental effects of the gut microbiome on mouse metabolic phenotypes

To characterize the impact of gut microbiota on host metabolism, we investigated the multicompartmental metabolic profiles of a conventional mouse strain (C3H/HeJ) (n=5) and its germ-free (GF) equivalent (n=5). We confirm that the microbiome strongly impacts on the metabolism of bile acids through the enterohepatic cycle and gut metabolism (higher levels of phosphocholine and glycine in GF liver and marked higher levels of bile acids in three gut compartments). Furthermore we demonstrate that (1) well-defined metabolic differences exist in all examined compartments between the metabotypes of GF and conventional mice: bacterial co-metabolic products such as hippurate (urine) and 5-aminovalerate (colon epithelium) were found at reduced concentrations, whereas raffinose was only detected in GF colonic profiles. (2) The microbiome also influences kidney homeostasis with elevated levels of key cell volume regulators (betaine, choline, myo-inositol and so on) observed in GF kidneys. (3) Gut microbiota modulate metabotype expression at both local (gut) and global (biofluids, kidney, liver) system levels and hence influence the responses to a variety of dietary modulation and drug exposures relevant to personalized health-care investigations.

Heteronuclear 1H−31P Statistical Total Correlation NMR Spectroscopy of Intact Liver for Metabolic Biomarker Assignment: Application to Galactosamine-Induced Hepatotoxicity

As part of our ongoing development of methods for enhanced biomarker information recovery from spectroscopic data we present the first example of a new hetero-nuclear statistical total correlation spectroscopy (HET-STOCSY) approach applied to intact tissue samples collected as part of a toxicological study. One-dimensional 1H and 31P-{1H} magic angle spinning (MAS) NMR spectra of intact liver samples after galactosamine (galN) treatment to rats and after cotreatment of galN plus uridine were collected at 275 K. Individual samples were also followed by 1H and 31P-{1H} MAS NMR through time generating time dependent modulations in metabolite signatures relating to toxicity. High-resolution 1H NMR spectra of urine and plasma and clinical chemical data were also collected to establish a biological framework in which to place these novel statistical heterospectroscopic data. In HET-STOCSY, calculation of the covariance between the 31P-{1H} and 1H NMR signals of phosphorus containing metabolites allows their molecular connectivities to be established and the construction of virtual two-dimensional heteronuclear correlation spectra that connect all protons on the molecule to the heteroatom. We show how HET-STOCSY applied to MAS NMR spectra of liver samples can be used to augment biomarker detection. This approach is generic and can be applied to correlate the covarying signals for any spin-active nuclei where there is parallel or serial collection of data.

Multivariate techniques and their application in nutrition: a metabolomics case study

The post-genomic technologies are generating vast quantities of data but many nutritional scientists are not trained or equipped to analyse it. In high-resolution NMR spectra of urine, for example, the number and complexity of spectral features mean that computational techniques are required to interrogate and display the data in a manner intelligible to the researcher. In addition, there are often multiple underlying biological factors influencing the data and it is difficult to pinpoint which are having the most significant effect. This is especially true in nutritional studies, where small variations in diet can trigger multiple changes in gene expression and metabolite concentration. One class of computational tools that are useful for analysing this highly multivariate data include the well-known 'whole spectrum' methods of principal component analysis and partial least squares. In this work, we present a nutritional case study in which NMR data generated from a human dietary Cu intervention study is analysed using multivariate methods and the advantages and disadvantages of each technique are discussed. It is concluded that an alternative approach, called feature subset selection, will be important in this type of work; here we have used a genetic algorithm to identify the small peaks (arising from metabolites of low concentration) that have been altered significantly following a dietary intervention.

Detection of urinary drug metabolite (xenometabolome) signatures in molecular epidemiology studies via statistical total correlation (NMR) spectroscopy.

Western populations use prescription and nonprescription drugs extensively, but large-scale population usage is rarely assessed objectively in epidemiological studies. Here we apply statistical methods to characterize structural pathway connectivities of metabolites of commonly used drugs detected routinely in 1H NMR spectra of urine in a human population study. 1H NMR spectra were measured for two groups of urine samples obtained from U.S. participants in a known population study. The novel application of a statistical total correlation spectroscopy (STOCSY) approach enabled rapid identification of the major and certain minor drug metabolites in common use in the population, in particular, from acetaminophen and ibuprofen metabolites. This work shows that statistical connectivities between drug metabolites can be established in routine "high-throughput" NMR screening of human samples from participants who have randomly self-administered drugs. This approach should be of value in considering interpopulation patterns of drug metabolism in epidemiological and pharmacogenetic studies.