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?CDawley 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 spectroscopy based metabolic profiling of urine and serum for investigation of physiological perturbations during radiation sickness

Radiation accidents are rare events that induce radiation syndrome, a complex pathology which is difficult to treat. In medical management of radiation victims, life threatening damage to different physiological systems should be taken into consideration. The present study was proposed to identify metabolic and physiological perturbations in biofluids of mice during different phases of radiation sickness using ¹H nuclear magnetic resonance (¹H NMR) spectroscopy and pattern recognition (PR) technique. The ¹H NMR spectra of the biofluids collected from mice irradiated with 5 Gray (Gy) at different time points during radiation sickness were analysed visually and by principal components analysis. Urine and serum spectral profile clearly showed altered metabolic profiles during different phases of radiation sickness. Increased concentration of urine metabolites viz. citrate, α ketoglutarate, succinate, hippurate, and trimethylamine during prodromal and clinical manifestation phase of radiation sickness shows altered gut microflora and energy metabolism. On the other hand, serum nuclear magnetic resonance (NMR) spectra reflected changes associated with lipid, energy and membrane metabolism during radiation sickness. The metabonomic time trajectory based on PR analysis of ¹H NMR spectra of urine illustrates clear separation of irradiated mice group at different time points from pre dose. The difference in NMR spectral profiles depicts the pathophysiological changes and metabolic disturbances observed during different phases of radiation sickness, that in turn, demonstrate involvement of multiple organ dysfunction. This could further be useful in development of multiparametric approach for better evaluation of radiation damage as well as for medical management during radiation sickness.     

Evaluation of NMR spectral data of urine in conjunction with measured clinical chemistry and histopathology parameters to assess the effects of liver and kidney toxicants

Single low and high doses of several compounds with known renal toxic effects (para-aminophenol, puromycin aminonucleoside, sodium chromate, and hexachlorobutadiene,) or known liver toxic effects (galactosamine, allyl alcohol, and thioacetamide) were administered to male Wistar rats in groups of 4 or 8 for each compound. Predose urine samples (Day 0) and samples from post-dosing (Days 1–4) were collected for each rat and monitored by 1D ¹H NMR. Principal component analysis (PCA) of the NMR spectra was used to investigate differences between dose levels for each compound individually. The findings from PCA at both dose levels for each compound were examined in the context of the corresponding clinical chemistry and pathology data collected during the study. The PCA clustering of NMR spectra from rats dosed with each individual compound were shown to be associated with the measured levels of creatinine, BUN, AST, ALT and histopathology findings. Finally, scaled-to-maximum, aligned, and reduced trajectories (SMART) analysis was applied to compare the temporal metabolic trajectories obtained for each animal at each dose level of the administered compounds. By day 4, the SMART trajectories for allyl alcohol and hexachlorobutadiene had returned to predose levels indicating a recovery response, however, the high dose SMART trajectories for para-aminophenol, puromycin aminonucleoside, sodium chromate, and galactosamine did not appear to return to predose levels indicating a prolonged toxic effect.     

A metabonomic approach to the investigation of drug-induced phospholipidosis: an NMR spectroscopy and pattern recognition study

¹H NMR spectroscopy of urine and pattern recognition analysis have been used to study the metabolic perturbations caused following dosing of five novel drug candidates, two of which (GWA, GWB) caused mild lung and liver phospholipidosis, whilst the rest (GWC-GWE) did not cause any detectable toxicity. Urine samples were collected predose, 0-8 h, 8-16 h, 16-24 h and 24-32 h after single, oral dosing with each compound to Han Wistar rats (n = 3 per group), and liver and lung samples for were taken at 48 h for histology. ¹H NMR spectra of whole urine were acquired, processed and subsequently analysed using principal component analysis. All animals administered the drug candidates showed a significant reduction in serum triglycerides and those animals administered either GWA or GWB were observed to have foamy alveolar macrophages and the presence of multilamellar bodies in hepatocytes by electron microscopy. In the plot of the first two principal components, urinary spectra of those animals dosed with GWA or GWB mapped separately to controls, all pre-dose samples and animals dosed with GWC-GWE. Inspection of the principal components loadings indicated an increase in urinary phenylacetylglycine with a concomitant decrease in urinary citrate and 2-oxoglutarate, possibly constituting a novel urinary biomarker set for phospholipidosis. This work exemplifies the use of NMR spectroscopy and pattern recognition methods for the detection of novel biomarker combinations for poorly understood toxicity types and the potential in screening novel drugs for toxicity.     

Altered fatty acid metabolism in long duration road transport: An NMR-based metabonomics study in sheep

The physical, endocrine, and metabolic responses of livestock to road transport have been evaluated by conventional hematological and biochemistry parameters for more than 20 years. However, these measures are relatively insensitive to subtle metabolic adaptations. We applied NMR-based metabonomics to assess system-wide metabolic responses as expressed in urine and serum of a large cohort of animals (n = 80) subjected to 12 and 48 h road transport. The profiling of (1)H NMR spectra revealed that the transported animals experienced altered gut and energy metabolism, muscle catabolism, and possibly a renal response. The animals transported for 48 h exhibited a deeper metabolic response to the transport event and a complex and expanded metabolic trajectory over the 72 h recovery period. Intriguingly, excretion of acyl glycines and a dicarboxylic acid was observed after transport and during recovery, implicating peroxisomal fatty acid oxidation as a metabolic response to transport-induced stress.     

The metabonomics of aging and development in the rat: an investigation into the effect of age on the profile of endogenous metabolites in the urine of male rats using 1H NMR and HPLC-TOF MS

The effect of aging and development in male Wistar-derived rats on the profile of endogenous metabolites excreted in the urine was investigated using both (1)H NMR spectroscopy and HPLC-TOF MS using electrospray ionisation (ESI). The endogenous metabolites were profiled in samples collected from male rats every two weeks from just after weaning at 4 weeks up to 20 weeks of age. Multivariate data analysis enabled clusters to be visualised within the data according to age, with urine collected at 4 and 6 weeks showing the greatest differences by both analytical techniques. Markers detected by (1)H NMR spectroscopy included creatinine, taurine, hippurate and resonances associated with amino acids/fatty acids, which increased with age, whilst citrate and resonances resulting from glucose/myoinositol declined. A number of ions were detected by HPLC-MS that were only present in urine samples at 4 weeks of age in both positive and negative ESI, with a range of ions, including e.g. carnitine, increasing with age. Age predictions by PLS-regression modelling demonstrated an age-related trend within these data, between 4 and 12 weeks for HPLC-MS and 4-16 weeks for NMR. The possible utility of these techniques for metabonomic investigations of age-related changes in the rat is discussed and the importance of employing suitable control animals in pharmacological and toxicological studies is highlighted.     

Spectral editing and pattern recognition methods applied to high-resolution magic-angle spinning 1H nuclear magnetic resonance spectroscopy of liver tissues

Principal component analysis (PCA) has been applied to three nuclear magnetic resonance (NMR) spectral editing methods, namely, the Carr-Purcell-Meiboom-Gill spin-echo, diffusion editing, and skyline projection of a two-dimensional J-resolved spectrum, obtained from high-resolution magic-angle spinning NMR spectroscopy of liver tissues, to distinguish between control and hydrazine-treated rats. The effects of the toxin on rat liver biochemistry were directly observed and characterized by depleted levels of liver glycogen, choline, taurine, trimethylamine N-oxide, and glucose and by elevated levels of lipids and alanine. The highly unsaturated omega-3-type fatty acid was observed for the first time in hydrazine-treated rat liver. The contributions of the metabolites to the separation of control from dosed liver tissues varied depending on the type of spectral editing method used. We have shown that subtle changes in the metabolic profiles can be selectively amplified using a metabonomics approach based on the different NMR spectral editing techniques in conjunction with PCA.     

Characterization of data analysis methods for information recovery from metabolic 1H NMR spectra using artificial complex mixtures

The assessment of data analysis methods in ¹H NMR based metabolic profiling is hampered owing to a lack of knowledge of the exact sample composition. In this study, an artificial complex mixture design comprising two artificially defined groups designated normal and disease, each containing 30 samples, was implemented using 21 metabolites at concentrations typically found in human urine and having a realistic distribution of inter-metabolite correlations. These artificial mixtures were profiled by ¹H NMR spectroscopy and used to assess data analytical methods in the task of differentiating the two conditions. When metabolites were individually quantified, volcano plots provided an excellent method to track the effect size and significance of the change between conditions. Interestingly, the Welch t test detected a similar set of metabolites changing between classes in both quantified and spectral data, suggesting that differential analysis of ¹H NMR spectra using a false discovery rate correction, taking into account fold changes, is a reliable approach to detect differential metabolites in complex mixture studies. Various multivariate regression methods based on partial least squares (PLS) were applied in discriminant analysis mode. The most reliable methods in quantified and spectral ¹H NMR data were PLS and RPLS linear and logistic regression respectively. A jackknife based strategy for variable selection was assessed on both quantified and spectral data and results indicate that it may be possible to improve on the conventional Orthogonal-PLS methodology in terms of accuracy and sensitivity. A key improvement of our approach consists of objective criteria to select significant signals associated with a condition that provides a confidence level on the discoveries made, which can be implemented in metabolic profiling studies.     

Effect of Hypoglycemic Encephalopathy upon Amino Acids, High-Energy Phosphates, and pHi in the Rat Brain In Vivo: Detection by Sequential 1H and 31P NMR Spectroscopy

Metabolic alterations in amino acids, high-energy phosphates, and intracellular pH during and after insulin hypoglycemia in the rat brain was studied in vivo by 1H and 31P nuclear magnetic resonance (NMR) spectroscopy. Sequential accumulations of 1H and 31P spectra were obtained from a double-tuned surface coil positioned over the exposed skull of a rat while the electroencephalogram was recorded continuously. The transition to EEG silence was accompanied by rapid declines in phosphocreatine, nucleoside triphosphate, and an increase in inorganic orthophosphate in 31P spectra. In 1H spectra acquired during the same time interval, the resonances of glutamate and glutamine decreased in intensity while a progressive increase in aspartate was observed. Following glucose administration, glutamate and aspartate returned to control levels (recovery half-time, 8 min); recovery of glutamine was incomplete. An increase in lactate was detected in the 1H spectrum during recovery but it was not associated with any change in the intracellular pH as assessed in the corresponding 31P spectrum. Phosphocreatine returned to control levels following glucose administration, in contrast to nucleoside triphosphate and inorganic orthophosphate which recovered to only 80% and 200% of their control levels, respectively. These results show that the changes in cerebral amino acids and high-energy phosphates detected by alternating the collection of 1H and 31P spectra allow for a detailed assessment of the metabolic response of the hypoglycemic brain in vivo.     

Proton nuclear magnetic resonance spectra of excised rat brain. Assignment of resonances

We have recorded 1H NMR spectra of excised rat brain at 361 MHz using two different water suppression pulse sequences. The assignment of the resonances has been carried out in perchloric acid extracts and subcellular fractions. Our results show that cytosolic proteins, membrane phospholipids and 16 different metabolites contribute to the observed spectra. The new resonances assigned allow the direct observation of myo-inositol and urea. Moreover, changes in the spectral pattern upon anesthesia, ischemic exposure of the brain and age of the rat have been recorded and correlated with the compounds producing the spectra.     

Phosphorus-31 nuclear magnetic resonance study on the effects of endurance training in rat skeletal muscle

To evaluate changes in muscle energetics following endurance training, we measured phosphorus-31 nuclear magnetic resonance (31P NMR) spectra on rat muscle in vivo before and after training in the same animals. The endurance training lasted for 3 months. The 31P NMR spectra were obtained serially at rest, during exercise by electrical stimulation, and during recovery. Intramuscular phosphocreatine (PCr), inorganic phosphate (P(i)), adenosine 5'-triphosphate (ATP) and pH were determined from the NMR spectra. The ratio of PCr:(PCR + P(i) at rest showed no difference between the trained and control groups even after 3 months of training. During exercise, however, this ratio was significantly higher in the trained group than in the control group. The ratio also recovered more rapidly after exercise in the trained group. The intramuscular pH decreased slightly by approximately 0.1 pH unit during exercise but did not show a significant difference between the groups. These results indicated that endurance training of 3 months duration improved the ATP supply system in the muscle. They also demonstrated that 31P NMR is a potent method for evaluating the effects of training in the same individuals.     

Cell surface involvement in cancer metastasis: an NMR study

NMR spectroscopy is one of the few techniques which has the sensitivity to detect subtle changes to the surface chemistry of cells. It has previously been demonstrated that high resolution ¹H NMR methods can distinguish tumour cells with the capacity to metastasise and this information appears to arise from a type of proteolipid in or attached to the plasma membrane. Here we report that the ¹H NMR signal, which we have used to identify metastatic cells in rat tumours, is significantly reduced in intensity after cultured cells are treated with trypsin/EDTA. The long T₂ relaxation value (⪢ 350 ms) observed in metastatic cells is absent after enzyme treatment. 2D scalar correlated NMR (COSY) spectra of these treated cells show that a cross peak normally associated with malignancy and metastatic disease is markedly reduced. These findings indicate that the plasma membrane lipid particle which generates the high resolution spectrum is directly affected by trypsin/EDTA. Alterations to the cell surface properties were also demonstrated in vivo since reduced numbers of metastases were observed in animals injected with enzyme-treated cells. The correlation between the absence of a long T₂ relaxation value and the diminished numbers of metastases in animals suggests that the plasma membrane particle is involved in the metastatic process.     

Metabolomics and biomarker discovery: NMR spectral data of urine and hepatotoxicity by carbon tetrachloride, acetaminophen, and d-galactosamine in rats

The primary objective of this study was to discover biomarkers which are correlated with hepatotoxicity induced by chemicals using ¹H NMR spectral data of urine. A procedure of nuclear magnetic resonance (NMR) urinalysis using pattern recognition was proposed for early screening of the hepatotoxicity of CCl₄, acetaminophen (AAP), and d-galactosamine (GalN) in rats. The hepatotoxic compounds were expected to induce necrosis in hepatocytes. This was confirmed through blood biochemistry and histopathology. CCl₄ (1 ml/kg, po) or GalN (0.8 g/kg, ip) was single administered to Sprague–Dawley (S–D) rats and urine was collected every 24 h. Animals were sacrificed 24 h or 48 h post-dosing. AAP (2 g/kg, po) was administered for 2 days and then the animals were sacrificed 24 h after the last treatment. NMR spectroscopy revealed evidently different clustering between control groups and hepatotoxicant treatment groups in global metabolic profilings as indicated by partial least square (PLS)-discrimination analysis (DA). In targeted profilings, endogenous metabolites of allantoin, citrate, taurine, 2-oxoglutarate, acetate, lactate, phenylacetyl glycine, succinate, phenylacetate, 1-methylnicotinamide, hippurate, and benzoate were selected as putative biomarkers for hepatoxicity by CCl₄, AAP, and GalN. Comparison of our rat ¹H NMR PLS-DA data with histopathological changes suggests that ¹H NMR urinalysis can be used to predict hepatotoxicity induced by CCl₄, AAP, and GalN.     

A metabonomic approach to the investigation of drug-induced phospholipidosis: an NMR spectroscopy and pattern recognition study

¹H NMR spectroscopy of urine and pattern recognition analysis have been used to study the metabolic perturbations caused following dosing of five novel drug candidates, two of which (GWA, GWB) caused mild lung and liver phospholipidosis, whilst the rest (GWC-GWE) did not cause any detectable toxicity. Urine samples were collected predose, 0-8 h, 8-16 h, 16-24 h and 24-32 h after single, oral dosing with each compound to Han Wistar rats (n = 3 per group), and liver and lung samples for were taken at 48 h for histology. ¹H NMR spectra of whole urine were acquired, processed and subsequently analysed using principal component analysis. All animals administered the drug candidates showed a significant reduction in serum triglycerides and those animals administered either GWA or GWB were observed to have foamy alveolar macrophages and the presence of multilamellar bodies in hepatocytes by electron microscopy. In the plot of the first two principal components, urinary spectra of those animals dosed with GWA or GWB mapped separately to controls, all pre-dose samples and animals dosed with GWC-GWE. Inspection of the principal components loadings indicated an increase in urinary phenylacetylglycine with a concomitant decrease in urinary citrate and 2-oxoglutarate, possibly constituting a novel urinary biomarker set for phospholipidosis. This work exemplifies the use of NMR spectroscopy and pattern recognition methods for the detection of novel biomarker combinations for poorly understood toxicity types and the potential in screening novel drugs for toxicity.     

Role of myoglobin as a scavenger of cellular NO in myocardium

Recent studies have detected a (1)H nuclear magnetic resonance (NMR) reporter signal of metmyoglobin (metMb) during bradykinin stimulation of an isolated mouse heart. The observation has led to the hypothesis that Mb reacts with cellular nitric oxide (NO). However, the hypothesis depends on an unequivocal detection of metMb signals in vivo. In solution, nitrite oxidization of Mb produces a characteristic set of paramagnetically shifted (1)H NMR signals. In the upfield spectral region, MbO(2) and MbCO exhibit the gammaCH(3) Val E11 signals at -2.8 and -2.4 ppm, respectively. In the same spectral region, nitrite oxidation of Mb produces a set of signals at -3.7 and -4.7 ppm at 35 degrees C. Previous studies have confirmed the visibility of metMb signals in perfused rat myocardium. With bradykinin infusion, perfusion pressure and rate-pressure product decrease, consistent with endogenous NO formation. However, neither myocardial O(2) consumption nor high-energy phosphate levels, as reflected in the (31)P NMR signals, show any significant change. Bradykinin still triggers a similar physiological response even in the presence of CO that is sufficient to inhibit 86% Mb. In all cases, the (1)H NMR spectra from perfused rat myocardium reveal no metMb signals. The results suggest that bradykinin-induced NO does not interact significantly with cellular Mb to produce an NMR-detectable quantity of metMb in the perfused rat myocardium. As a consequence, the experiments cannot confirm the intriguing proposal that Mb acts as a cellular NO scavenger.     

The metal-binding properties of ovotransferrin. An investigation of cobalt(II) derivatives

Cobalt(II) ovotransferrin bicarbonate and oxalate ternary complexes were prepared and investigated in the pH range 7-10.5. Cobalt(II) provides an excellent and unique spectroscopic probe to monitor subtle structural differences in solution between the two sites of ovotransferrin and to investigate the structural dependence on pH. CD spectroscopy on one side and 1H NMR spectroscopy of isotropically shifted signals on the other are extremely sensitive techniques and are particularly suited for high spin cobalt(II)-containing compounds. In the case of the oxalate derivative the metal-binding ability of the protein is different at the two binding sites and is pH dependent; the CD spectra reveal two different sites, one of which is clearly pH dependent with a pKa of 9.5. On the contrary the bicarbonate analogue does not show any spectral difference between the two sites; both of them change with pH, the pKa being again 9.5. 1H NMR spectra of the oxalate derivatives at pH 7-8 reveal the presence of conformers, the distribution of which depends on the H2O/D2O ratio. Such conformers are not revealed in the bicarbonate system; at pH around 10 the NMR spectra of both systems show inequivalence between the two sites and/or the presence of different conformers for each site. Such differences are discussed in terms of the possible implications in mechanism and function. The overall spectral data are consistent with the donor groups being two histidines, two tyrosines, the synergistic anion, and possibly a solvent molecule.     

Biofluid 1H NMR-based metabonomic techniques in nutrition research - metabolic effects of dietary isoflavones in humans

A metabonomic approach to nutrition research may provide an insight into in vivo mechanisms of action following nutritional intervention. This approach was applied to investigate changes in the (1)H NMR spectral profile of urine collected from controlled dietary intervention studies conducted in premenopausal women before and following soy or miso consumption. The aim of the study was to identify the biochemical effects of a diet rich in soy isoflavones, phytochemicals which are receiving significant attention because of their potential importance to human health and wide bioactivity in vitro. By applying various chemometric techniques to the data the biochemical effects of conjugated and unconjugated isoflavones were determined. The biochemical changes observed suggest that soy isoflavone ingestion had significant effects on several metabolic pathways associated with osmolyte fluctuation and energy metabolism. These biochemical changes were more significant following ingestion of the unconjugated soy isoflavone (miso) diet suggesting that the chemical composition of the isoflavones present in soy-based foods may have an effect on their biological efficacy in vivo. This study describes a novel application for (1)H NMR analysis by determining subtle differences in biochemical profiles following dietary intervention and providing further insight into the mechanisms of action of phytochemicals in vivo.     

Nuclear magnetic resonance study on rabbit skeletal troponin C: calcium-induced conformational change

Rabbit skeletal muscle troponin C (TnC) was investigated by means of 1H NMR in the presence of dithiothreitol that prevents dimerization of the protein. Two-dimensional (2D) 1H NMR spectra were observed in order to assign resonances to specific amino acids. One-dimensional 1H NMR spectra were observed as a function of Ca2+ concentration. The Ca2+-induced spectral change is categorized into two types: type 1 corresponds to the conformational change of the C-terminal-half domain (Ca2+ high-affinity sites) and type 2 to that of the N-terminal-half domain (Ca2+ low-affinity sites). From the 2D NMR spectra and Ca2+ titration data, it was suggested that (1) amide protons of Gly-108, Ile-110, Gly-144, and Ile-146 are hydrogen-bonded when the C-terminal-half domain binds 2 mol of Ca2+ and (2) hydrogen bonds of Gly-108, Ile-110, Gly-144, and Ile-146 are destroyed or weakened when the C-terminal-half domain releases 2 mol of Ca2+. Nuclear Overhauser enhancement difference spectra as well as the Ca2+ titration data suggested that a hydrophobic cluster is formed in the C-terminal-half domain when the C-terminal-half domain binds 2 mol of Ca2+. A hydrophobic cluster exists in the N-terminal-half domain without regard to Ca2+ binding to the N-terminal-half domain. The spectra of Tyr-10 showed both types of spectral change during the Ca2+ titration. The results suggested that Tyr-10 of apo-TnC interacts with the C-terminal-half domain.     

Recommended strategies for spectral processing and post-processing of 1D <Superscript>1</Superscript>H-NMR data of biofluids with a particular focus on urine

¹H NMR spectra from urine can yield information-rich data sets that offer important insights into many biological and biochemical phenomena. However, the quality and utility of these insights can be profoundly affected by how the NMR spectra are processed and interpreted. For instance, if the NMR spectra are incorrectly referenced or inconsistently aligned, the identification of many compounds will be incorrect. If the NMR spectra are mis-phased or if the baseline correction is flawed, the estimated concentrations of many compounds will be systematically biased. Furthermore, because NMR permits the measurement of concentrations spanning up to five orders of magnitude, several problems can arise with data analysis. For instance, signals originating from the most abundant metabolites may prove to be the least biologically relevant while signals arising from the least abundant metabolites may prove to be the most important but hardest to accurately and precisely measure. As a result, a number of data processing techniques such as scaling, transformation and normalization are often required to address these issues. Therefore, proper processing of NMR data is a critical step to correctly extract useful information in any NMR-based metabolomic study. In this review we highlight the significance, advantages and disadvantages of different NMR spectral processing steps that are common to most NMR-based metabolomic studies of urine. These include: chemical shift referencing, phase and baseline correction, spectral alignment, spectral binning, scaling and normalization. We also provide a set of recommendations for best practices regarding spectral and data processing for NMR-based metabolomic studies of biofluids, with a particular focus on urine.