Urinary metabonomics study on the protective effects of ergosta-4,6,8(14),22-tetraen-3-one on chronic renal failure in rats using UPLC Q-TOF/MS and a novel MSE data collection technique

Ergosta-4,6,8(14),22-tetraen-3-one (ergone), isolated from the medicinal fungus Polyporus umbellatus, has been proven to prevent the progression of renal injury and the subsequent renal fibrosis. UPLC Q-TOF/MS was employed to investigate the metabonomic characteristics of adenine-induced chronic renal failure (CRF) and the proactive effects of ergone. The significant difference of the metabolic profiling was observed from ergone-treated group compared with the CRF model group during the 10-day and 20-day study periods by using the principal components analysis (PCA). The significant difference of the ergone-treated group in metabolic profiling was also observed between 10-day and 20-day study periods. The time-dependent tendency in ergone-treated group from day 10 to 20 was obtained, indicating the time-dependent recovery effect of ergone on CRF rats. Some significantly changed metabolites like creatinine, proline, adrenosterone, taurine, creatine, phenylalanine, ornithine, dopamine, kynurenine, kynurenic acid and 3-O-methyldopa have been identified during the 20-day study period. These biochemical changes are related to the disturbance in energy metabolism and amino acid metabolism, which are helpful to further understand the CRF and the therapeutic mechanism of ergone. This work suggests that this metabonomic approach could be used as a potentially powerful tool to investigate the biochemical changes of certain physiopathological conditions, such as metabolic syndrome, as an early diagnostic measure.     

Metabonomics analysis of urine and plasma from rats given long-term and low-dose dimethoate by ultra-performance liquid chromatography-mass spectrometry

This study assessed the effects of long-term, low-dose dimethoate administration to rats by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Dimethoate (0.04, 0.12, and 0.36mg/kg body weight/day) was administered daily to male Wistar rats through their drinking water for 24weeks. Significant changes in serum clinical chemistry were observed in the middle- and high-dose groups. UPLC-MS revealed evident separate clustering among the different dose groups using global metabolic profiling by supervised partial least squares-discriminant analysis. Metabonomic analysis showed alterations in a number of metabolites (12 from urine and 13 from plasma), such as l-tyrosine, dimethylthiophosphate (DMTP), dimethyldithiophosphate (DMDTP), citric acid, uric acid, suberic acid, glycylproline, allantoin, isovalerylglutamic acid and kinds of lipids. The results suggest that long-term, low-dose exposure to dimethoate can cause disturbances in liver function, antioxidant and nervous systems, as well as the metabolisms of lipids, glucose, fatty acids, amino acids, and collagen in rats. DMTP and DMDTP, which had the most significant changes among all other studied biomarkers, were considered as early, sensitive biomarkers of exposure to dimethoate. The other aforementioned proposed toxicity biomarkers in metabonomic analysis may be useful in the risk assessment of the toxic effects of dimethoate. Metabonomics as a systems toxicology approach was able to provide comprehensive information on the dynamic process of dimethoate induced toxicity. In addition, the results indicate that metabonomic approach could detect systemic toxic effects at an earlier stage compared to clinical chemistry. The combination of metabonomics and clinical chemistry made the toxicity of dimethoate on rats more comprehensive.     

Metabonomic study of aristolochic acid-induced nephrotoxicity in rats

This paper describes a metabonomic study characterizing the nephrotoxicity induced by aristolochic acid (AA), a suspected kidney toxicant. For these studies, we examined the biochemical compositions of AA-treated rat urine using LC-MS and pattern recognition methods. The biochemical and histological patterns of rat groups treated with different AA sources showed distinct differences from those of the control group. Certain metabolic pathways, such as homocysteine formation and the folate cycle were significantly accelerated, while others, including arachidonic acid biosynthesis, were decreased. A subset-validation procedure using linear discriminant analysis (LDA) and selected predictive variables indicated that approximately 95% of the treated and nontreated rat urine samples were classified correctly into their respective treatment groups. The results suggested that this metabonomic approach is a promising methodology for the rapid in vivo screening of nephrotoxicity associated with ingesting multi-ingredient medicinal herb supplements, and provides a valid method for comprehending the chemical-induced perturbations in the metabolic network and the networked lesions.     

A 1H NMR-Based Metabonomic Investigation of Time-Related Metabolic Trajectories of the Plasma,Urine and Liver Extracts of Hyperlipidemic Hamsters

The hamster has been previously found to be a suitable model to study the changes associated with diet-induced hyperlipidemia in humans. Traditionally, studies of hyperlipidemia utilize serum- or plasma-based biochemical assays and histopathological evaluation. However, unbiased metabonomic technologies have the potential to identify novel biomarkers of disease. Thus, to obtain a better understanding of the progression of hyperlipidemia and discover potential biomarkers, we have used a proton nuclear magnetic resonance spectroscopy (¹H-NMR)-based metabonomics approach to study the metabolic changes occurring in the plasma, urine and liver extracts of hamsters fed a high-fat/high-cholesterol diet. Samples were collected at different time points during the progression of hyperlipidemia, and individual proton NMR spectra were visually and statistically assessed using two multivariate analyses (MVA): principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). Using the commercial software package Chenomx NMR suite, 40 endogenous metabolites in the plasma, 80 in the urine and 60 in the water-soluble fraction of liver extracts were quantified. NMR analysis of all samples showed a time-dependent transition from a physiological to a pathophysiological state during the progression of hyperlipidemia. Analysis of the identified biomarkers of hyperlipidemia suggests that significant perturbations of lipid and amino acid metabolism, as well as inflammation, oxidative stress and changes in gut microbiota metabolites, occurred following cholesterol overloading. The results of this study substantially broaden the metabonomic coverage of hyperlipidemia, enhance our understanding of the mechanism of hyperlipidemia and demonstrate the effectiveness of the NMR-based metabonomics approach to study a complex disease.     

Application of biofluid 1H nuclear magnetic resonance-based metabonomic techniques for the analysis of the biochemical effects of dietary isoflavones on human plasma profile

This study describes the first metabonomic approach to determining biochemical modifications following dietary intervention in humans. Significant interest in the mechanisms of action of soy isoflavones has predominantly stemmed from in vitro experiments but to date the availability of analytical tools for studying the mechanisms of action in vivo have been limited. Here a metabonomic approach based on chemometric analysis of 1H nuclear magnetic resonance spectra of blood plasma has been used to investigate metabolic changes following dietary intervention with soy isoflavones in healthy premenopausal women under controlled environmental conditions. Clear differences in the plasma lipoprotein, amino acid, and carbohydrate profiles were observed following soy intervention, suggesting a soy-induced alteration in energy metabolism.     

Metabonomic alterations in hippocampus, temporal and prefrontal cortex with age in rats

The metabolic changes in hippocampus, temporal cortex and prefrontal cortex in SD rats along with aging were explored using a metabonomic approach, which based on high resolution “magic angle spinning” ¹H NMR spectroscopy. The metabolite profiles were analyzed by partial least squares-discriminant analysis, and the results showed that the metabolites of the above three brain regions in old rats were dramatically different from that in the adult and young rats. The old rats showed increased myo-inositol and lactate in all of the three brain regions, and decreased N-acetylaspartate in temporal and frontal cortex, Glutamate–GABA level became imbalance in temporal cortex of old rats. In addition, compared with the adult female rats, male rats had higher levels of N-acetylaspartate, taurine, and creatine in temporal or frontal cortex. The age-related metabolic changes may indicate the early functional alterations of neural cells in these brain regions, especially the temporal cortex. The gender-related metabolic changes suggest the significance of the hormonal regulation in brain metabolism. Our work highlights the potential of metabolic profiling to enhance our understanding of biological mechanisms of brain aging.     

A metabonomic investigation on the biochemical perturbation in liver failure patients caused by hepatitis B virus

A metabonomic study was performed to investigate the biochemical perturbation of the serum samples from liver failure patients induced by hepatitis B virus (HBV; n=24) and control normal subjects (n=23). The serum metabonome was detected using gas chromatography-mass spectrometry (GC-MS) technique integrated with a commercial mass spectral library for the peak identification. After peak deconvolution, identification, and matching, the acquired GC-MS data were normalized and processed by principal component analysis (PCA) and canonical discriminant analysis (CDA). Specific changes in the metabolic composition of serum samples from patients including amino acids (AAs) and glucose were shown in GC-MS total ion current (TIC) chromatograms. The distinctive biochemical difference between the healthy subjects and liver failure patients was displayed by the pattern recognition methods. We also found that the liver failure patients with different degree of severity categorized as MELD (model for end-stage of liver diseases) could be clearly classified by the corresponding metabonomic data. In comparison, the current routine clinical indices cannot characterize the global phenotyping of liver failure. The result demonstrated that the GC-MS technique is an alternative tool for the characterization of the metabolic perturbation and the metabonomic study promises to provide an integrative criterion to evaluate the severity and the prognosis of liver diseases.     

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.     

GC/MS analysis of the rat urine for metabonomic research

In this paper, an optimized protocol was established and validated for the metabonomic profiling in rat urine using GC/MS. The urine samples were extracted by methanol after treatment with urease to remove excessive urea, then the resulted supernatant was dried, methoximated, trimethylsilylated, and analyzed by GC/MS. Forty-nine endogenous metabolites were separated and identified in GC/MS chromatogram, of which 26 identified compounds were selected for quantitative analysis to evaluate the linearity, precision, and sensitivity of the method. It showed good linearity between mass spectrometry responses and relative concentrations of the 26 endogenous compounds over the range from 0.063 to 1.000 (v/v, urine/urine+water) and satisfactory reproducibility with intra-day and inter-days precision values all below 15%. The metabonomic profiling method based on GC/MS was successfully applied to urine samples from hyperlipidemia model rats. Obviously, separated clustering of model rats and the control rats were shown by principal components analysis (PCA); time-dependent metabonomic modification was detected as well. It was suggested that metabonomic profiling based on GC/MS be a robust method for urine samples.     

Metabonomic study of Chinese medicine Shuanglong formula as an effective treatment for myocardial infarction in rats

A UPLC/TOF-MS-based metabonomic study was conducted to assess the holistic efficacy of Traditional Chinese Medicine Shuanglong Formula (SLF) for myocardial infarction in rats. Thirty male Sprague-Dawley rats were randomly divided into five groups after surgery. The Panax ginseng group, Salvia miltiorrhiza group, and SLF group were treated with water extractions of Panax ginseng (PG), Salvia miltiorrhiza (SM), and SLF (the ratio of SM to PG was 3:7) at a dose of 5 g/kg·w·d for 21 consecutive days, respectively; the model group and sham surgery group were both treated with 0.9% saline solution. Urinary samples for metabonomic study, serum samples for biochemical measurement, and heart samples for histopathology were collected. As a result, metabonomics-based findings such as the PCA and PLS-DA plotting of metabolic state and analysis of potential biomarkers in urine correlated well to the assessment of serum biochemistry and histopathological assay, confirming that SLF exerted synergistic therapeutic efficacies to exhibit better effect on MI compared to PG or SM. The shifts in urinary TCA cycle as well as pentose phosphate pathway suggested that SLF may diminish cardiac injury of MI with its potential pharmacological effect in the regulation of myocardial energy metabolism.     

Metabolic profiling of 3-nitropropionic acid early-stage Huntington's disease rat model using gas chromatography time-of-flight mass spectrometry

3-Nitropropionic acid (3-NP), a potent irreversible inhibitor of mitochondrial complex II enzyme, leads to mitochondrial dysfunction and oxidative stress in Huntington's disease (HD) rat model. In this study, biochemical assays were used to demonstrate the presence of oxidative stress and mitochondrial dysfunction in 3-NP early stage HD rat models. Gas chromatography time-of-flight mass spectrometry (GC/TOFMS) was applied to analyze metabolites in brain and plasma of 3-NP-treated and vehicle-dosed rats. The orthogonal partial least-squares discriminant analysis (OPLS-DA) model generated using brain metabolic profiles robustly differentiated the 3-NP early stage HD rat model from the control. Metabonomic characterization of the 3-NP HD rat model facilitated the detection of biomarkers that define the physiopathological phenotype of early stage HD and elucidated the treatment effect of galantamine. Brain marker metabolites that were identified based on the OPLS-DA model were associated with altered glutathione metabolism, oxidative stress, and impaired energy metabolism. The treatment effect of galantamine in early stage HD could not be concluded mechanistically using the brain metabotype. Our study confirmed that GC/TOFMS is a strategic and complementary platform for the metabonomic characterization of 3-NP induced neurotoxicity in the early stage HD rat model.     

Integrated metabonomic analysis of bromobenzene-induced hepatotoxicity: novel induction of 5-oxoprolinosis

We present here a definitive metabonomic analysis in order to detect novel biomarker and metabolite information, implicating specific putative protein targets in the toxicological mechanism of bromobenzene-induced centrilobular hepatic necrosis. Male Han-Wistar rats were dosed with bromobenzene (1.5 g/kg, n = 25) and blood plasma, urine and liver samples were collected for NMR and magic angle spinning (MAS) NMR spectroscopy at various time-points postdose, with histopathology and clinical pathology performed in parallel. Liver samples were analyzed by 600 MHz 1H MAS NMR techniques and the resultant spectra were correlated to sequential 1H NMR measurements in urine and blood plasma using pattern recognition methods. 1D 1H NMR spectra were data-reduced and analyzed using principal components analysis (PCA) to show the time-dependent biochemical variations induced by bromobenzene toxicity. In addition to a holistic view of the effect of hepatic toxicity on the metabolome, a number of putative protein targets of bromobenzene and its metabolites were identified including those enzymes of the glutathione cycle, exemplified by the presence of a novel biomarker, 5-oxoproline, in liver tissue, blood plasma, and urine. As such, this work establishes the importance of metabonomics technology in resolving the mechanistic complexity of drug toxicity as well as the benefits of frontloading this approach in drug safety evaluation and biomarker discovery.     

An 1H NMR and UPLC�CMS-based plasma metabonomic study to investigate the biochemical changes in chronic unpredictable mild stress model of depression

Depression is a common and highly debilitating psychiatric illness. However, the pathophysiology of depression is not fully understood. In this study Sprague-Dawley rats were exposed to chronic unpredictable mild stress (CUMS) to induce depression. A metabonomic study on plasma of CUMS-induced depressive rats was performed to research the pathologic mechanism of depression by using ¹H nuclear magnetic resonance (NMR) spectroscopy and ultra performance liquid chromatography coupled to mass spectrometry (UPLC–MS). Clear separations between depressive rats and control rats were observed by principal component analysis (PCA) based on the data obtained using both analytical techniques and 18 significantly changed metabolites were identified as potential biomarkers of depression. Depressive rats were characterized by altered levels of plasma lysophosphatidylcholines, amino acids, cholic acid, choline, lactate, glycoproteins, glucose, ketone bodies, nucleosides and gut microflora metabolites, which were related to multiple perturbed metabolic pathways and contributed to the elucidation of the complex mechanism of depression. To the best of our knowledge, this is the first plasma metabonomic study on CUMS-induced depressive rats by using two complementary analytical technologies. Our results showed that metabonomic approach offers a useful tool to identify depression-specific biomarkers and provide new insights into the pathophysiology of depression.     

Metabolic profiling analysis of a D-galactosamine/lipopolysaccharide-induced mouse model of fulminant hepatic failure

The purpose of this study was to characterize the changes in metabolic intermediates and to investigate the metabolic profile of a mouse model of fulminant hepatic failure (FHF), induced by D-galactosamine/lipopolysaccharide (GalN/LPS). Plasma metabolite levels were detected using gas chromatography/time-of-flight mass spectrometry, and the acquired data were transferred into Simca-P and processed using principal components analysis (PCA). In total, 45 metabolites were identified from the 267 distinct compounds found in the study. Whereas significant differences were noted in the plasma levels of the control and FHF groups, no differences in gluconeogenesis or glycolysis were noted following GalN/LPS treatment. Our data also suggest that the production of ketone bodies, and the tricarboxylic acid and urea cycles, was inhibited. PCA data suggest that 5-hydroxyindoleacetic acid, glucose, beta-hydroxybutyrate, and phosphate parameters had the highest weights on each of the principal components, and that they were the most important metabolites contributing to the separation of groups. In conclusion, this metabonomic approach can be used as a powerful tool to characterize changes in metabolic intermediates and to search for metabolic markers under certain pathophysiological conditions, such as FHF. Our data also demonstrate that a combination of 5-hydroxyindoleacetic acid, glucose, beta-hydroxybutyrate, and phosphate concentrations in the plasma is a potential marker for FHF, as well as for the early prognosis of FHF.     

Investigations into biochemical changes due to diurnal variation and estrus cycle in female rats using high-resolution (1)H NMR spectroscopy of urine and pattern recognition

Metabonomic methods utilizing (1)H NMR spectroscopy and pattern recognition analysis (NMR-PR) have been applied to investigate biochemical variation in a control population of female rats over time in relation to diurnal and estrus cycle fluctuations. Urine samples were collected twice daily (6 AM-6 PM and 6 PM-6 AM) from female rats (n = 10) for a period of 10 days. (1)H NMR spectroscopic analysis and PR were performed on each sample. Subtle differences in the endogenous metabolite excretion profiles of urine samples at the various stages of the estrus cycle were observed. The main inherent metabolic clustering in the principal components analysis (PCA) maps was related to interrat variation and was observed in the first two principal components (PCs), accounting for 66% of the variance in these data. Separation of urinary data according to time of sampling (day and night) was achieved in the lower PCs. Some of the differences in the urinary profiles of day and night samples causing this separation were attributed to the increase in metabolic activity of the rat during the night. Individual rat data were also mapped as a function of time, using PCA, to produce a metabolic trajectory, which in a number of cases facilitated separation of one or more stages of the estrus cycle. Several of the fluctuations observed between urine samples collected during the different stages of the estrus cycle may be related to hormone levels. Although variation in metabolite profiles relating to both diurnal and hormonal variation could be detected these perturbations were minor compared with the effects observed due to interrat variation. This is the first time that a hormonal cycle has been described for individuals based on NMR spectroscopic and multivariate analysis of metabolic data and shows the value of metabonomic methods in the investigation of physiological variation and rhythms.     

The Metabolic Responses to Aerial Diffusion of Essential Oils

Anxiety disorders are the most prevalent psychiatric disorders and affect a great number of people worldwide. Essential oils, take effects through inhalation or topical application, are believed to enhance physical, emotional, and spiritual well-being. Although clinical studies suggest that the use of essential oils may have therapeutic potential, evidence for the efficacy of essential oils in treating medical conditions remains poor, with a particular lack of studies employing rigorous analytical methods that capture its identifiable impact on human biology. Here, we report a comprehensive gas chromatography time-of-flight mass spectrometry (GC-TOFMS) based metabonomics study that reveals the aromas-induced metabolic changes and the anxiolytic effect of aromas in elevated plus maze (EPM) induced anxiety model rats. The significant alteration of metabolites in the EPM group was attenuated by aromas treatment, concurrent with the behavioral improvement with significantly increased open arms time and open arms entries. Brain tissue and urinary metabonomic analysis identified a number of altered metabolites in response to aromas intervention. These metabolic changes included the increased carbohydrates and lowered levels of neurotransmitters (tryptophan, serine, glycine, aspartate, tyrosine, cysteine, phenylalanine, hypotaurine, histidine, and asparagine), amino acids, and fatty acids in the brain. Elevated aspartate, carbohydrates (sucrose, maltose, fructose, and glucose), nucleosides and organic acids such as lactate and pyruvate were also observed in the urine. The EPM induced metabolic differences observed in urine or brain tissue was significantly reduced after 10 days of aroma inhalation, as noted with the loss of statistical significance on many of the metabolites in the aroma-EPM group. This study demonstrates, for the first time, that the metabonomics approach can capture the subtle metabolic changes resulting from exposure to essential oils and provide the basis for pinpointing affected pathways in anxiety-related behavior, which will lead to an improved mechanistic understanding of anxiolytic effect of essential oils.     

Time-course changes in potential biomarkers detected using a metabonomic approach in Walker 256 tumor-bearing rats

A metabonomic approach based on complementary hydrophilic interaction chromatography and reversed-phase liquid chromatography combined with tandem mass spectrometry and time-course analysis of metabolites was implemented to find more reliable potential biomarkers in urine of Walker 256 tumor-bearing rats. A major challenge in metabonomics is distinguishing reliable biomarkers that are closely associated with the genesis and progression of diseases from those that are unrelated but altered significantly. In this study, these biomarkers were selected according to the change trends of discriminating metabolites during the genesis and progression of cancer. Seven consecutive batches of urine samples from preinoculation to 16 days after were collected and analyzed. Multivariate analysis revealed 87 discriminating metabolites. Time-course analysis of discriminating metabolites was used to select more reliable biomarkers with regular and reasonable change trends. Finally, 47 were found and 15 were identified including 12 carnitine derivatives, 2 amino acid derivatives, 1 nucleoside. On the basis of time-course behaviors of these potential biomarkers, we hypothesize such disruption might result from elevated cell proliferation, reduced β-oxidation of fatty acids, and poor renal tubular reabsorption. These studies demonstrate that this method can help to find more reliable potential biomarkers and provide valuable biochemical insights into metabolic alterations in tumor-bearing biosystems.     

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.