Metabolic profiles of sunflower genotypes with contrasting response to Sclerotinia sclerotiorum infection

We report a comprehensive primary metabolite profiling of sunflower (Helianthus annuus) genotypes displaying contrasting behavior to Sclerotinia sclerotiorum infection. Applying a GC-MS-based metabolite profiling approach, we were able to identify differential patterns involving a total of 63 metabolites including major and minor sugars and sugar alcohols, organic acids, amino acids, fatty acids and few soluble secondary metabolites in the sunflower capitulum, the main target organ of pathogen attack. Metabolic changes and disease incidence of the two contrasting genotypes were determined throughout the main infection period (R5.2-R6). Both point-by-point and non-parametric statistical analyses showed metabolic differences between genotypes as well as interaction effects between genotype and time after inoculation. Network correlation analyses suggested that these metabolic changes were synchronized in a time-dependent manner in response to the pathogen. Concerted differential metabolic changes were detected to a higher extent in the susceptible, rather than the resistant genotype, thereby allowing differentiation of modules composed by intermediates of the same pathway which are highly interconnected in the susceptible line but not in the resistant one. Evaluation of these data also demonstrated a genotype specific regulation of distinct metabolic pathways, suggesting the importance of detection of metabolic patterns rather than specific metabolite changes when looking for metabolic markers differentially responding to pathogen infection. In summary, the GC-MS strategy developed in this study was suitable for detection of differences in carbon primary metabolism in sunflower capitulum, a tissue which is the main entry point for this and other pathogens which cause great detrimental impact on crop yield.     

Serum metabolite signature predicts the acute onset of diabetes in spontaneously diabetic congenic BB rats

The clinical presentation of type 1 diabetes is preceded by a prodrome of beta cell autoimmunity. We probed the short period of subtle metabolic abnormalities, which precede the acute onset of diabetes in the spontaneously diabetic BB rat, by analyzing the serum metabolite profile detected with combined gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS). We found that the metabolite pattern prior to diabetes included 17 metabolites, which differed between individual diabetes prone (DP) BB rats and their age and sex matched diabetes resistant (DR) littermates. As the metabolite signature at the 40?days of age baseline failed to distinguish DP from DR, there was a brief 10-day period after which the diabetes prediction pattern was observed, that includes fatty acids (e.g. oleamide), phospholipids (e.g. phosphocholines) and amino acids (e.g. isoleucine). It is concluded that distinct changes in the serum metabolite pattern predict type 1 diabetes and precede the appearance of insulitis in spontaneously diabetic BB DP rats. This observation should prove useful to dissect mechanisms of type 1 diabetes.     

Metabolic profiling of isolated mitochondria and cytoplasm reveals compartment-specific metabolic responses

Introduction

Subcellular compartmentalization enables eukaryotic cells to carry out different reactions at the same time, resulting in different metabolite pools in the subcellular compartments. Thus, mutations affecting the mitochondrial energy metabolism could cause different metabolic alterations in mitochondria compared to the cytoplasm. Given that the metabolite pool in the cytosol is larger than that of other subcellular compartments, metabolic profiling of total cells could miss these compartment-specific metabolic alterations.

Objectives

To reveal compartment-specific metabolic differences, mitochondria and the cytoplasmic fraction of baker’s yeast Saccharomyces cerevisiae were isolated and subjected to metabolic profiling.

Methods

Mitochondria were isolated through differential centrifugation and were analyzed together with the remaining cytoplasm by gas chromatography–mass spectrometry (GC–MS) based metabolic profiling.

Results

Seventy-two metabolites were identified, of which eight were found exclusively in mitochondria and sixteen exclusively in the cytoplasm. Based on the metabolic signature of mitochondria and of the cytoplasm, mutants of the succinate dehydrogenase (respiratory chain complex II) and of the F_OF₁-ATP-synthase (complex V) can be discriminated in both compartments by principal component analysis from wild-type and each other. These mitochondrial oxidative phosphorylation machinery mutants altered not only citric acid cycle related metabolites but also amino acids, fatty acids, purine and pyrimidine intermediates and others.

Conclusion

By applying metabolomics to isolated mitochondria and the corresponding cytoplasm, compartment-specific metabolic signatures can be identified. This subcellular metabolomics analysis is a powerful tool to study the molecular mechanism of compartment-specific metabolic homeostasis in response to mutations affecting the mitochondrial metabolism.

     

Gas chromatography-mass spectrometry (GC/MS) reveals urine metabolites associated to light and heavy infections by Schistosoma mansoni in mice

High-throughput profiling of metabolites has been used to identify metabolic changes in murine models as a response to the infection by the parasitic trematode Schistosoma. These investigations have contributed to our understanding on the pathogenesis of this tropical neglected disease, with a potential of helping diagnosis. Here, our study aimed to investigate the application of gas chromatography–mass spectrometry (GC/MS) on the profiling of urine metabolites from mice carrying infections by Schistosoma mansoni. Two larval infection doses created distinctive infection intensities in mice, whereby the heavily infected animals were found to release 25 times more eggs in faeces than lightly infected animals. Over 200 urine metabolites were identified from these animals by GC/MS, following two complementary derivatisation methods. A list of 14 individual metabolites with altered relative abundances between groups were identified. Most of the altered metabolites showed a trend of increased abundances in response to infection intensity, indicating host-specific metabolic alterations as a result of the disease. Hippurate, a metabolite which concentration is intimately modulated by the gut microbiota, was found to be highly correlated to infection intensity. Our study showed that urine metabolic profiling by GC/MS can distinguish non-infected animals from those carrying light and heavy infections by S. mansoni, revealing metabolites associated to the infection and providing insights on the pathogenesis of schistosomiasis.     

Global urinary metabolic profiling procedures using gas chromatography-mass spectrometry

The role of urinary metabolic profiling in systems biology research is expanding. This is because of the use of this technology for clinical diagnostic and mechanistic studies and for the development of new personalized health care and molecular epidemiology (population) studies. The methodologies commonly used for metabolic profiling are NMR spectroscopy, liquid chromatography mass spectrometry (LC/MS) and gas chromatography-mass spectrometry (GC/MS). In this protocol, we describe urine collection and storage, GC/MS and data preprocessing methods, chemometric data analysis and urinary marker metabolite identification. Results obtained using GC/MS are complementary to NMR and LC/MS. Sample preparation for GC/MS analysis involves the depletion of urea via treatment with urease, protein precipitation with methanol, and trimethylsilyl derivatization. The protocol described here facilitates the metabolic profiling of ～400-600 metabolites in 120 urine samples per week.     

Metabolite profiling of maize grain: differentiation due to genetics and environment

A comparative metabolite profiling approach based on gas chromatography-mass spectrometry (GC/MS) was applied to investigate the impact of genetic background, growing location and season on the chemical composition of maize grain. The metabolite profiling protocol involved sub-fractionation of the metabolites and allowed the assessment of about 300 distinct analytes from different chemical classes (polar to lipophilic), of which 167 could be identified. A comparison, over three consecutive growing seasons, of the metabolite profiles of four maize cultivars which differed in their maturity classification, was carried out using principal component analysis (PCA). This revealed a strong separation of one cultivar in the first growing season, which could be explained by the immaturity of the kernels of this cultivar compared with others in the field trial. Further evaluations by pair-wise comparison using Student’s t-test and analysis of variance (ANOVA) showed that the growing season was the most prominent impact factor driving variation of the metabolite pool. An increased understanding of metabolic variation was achieved by analysis of a second sample set comprising one cultivar grown for 3 years at four locations. The applied GC/MS-based metabolite profiling demonstrated the natural variation in maize grain metabolite pools resulting from the interplay of environment, season, and genotype.     

Metabolic profiling of an alcoholic fatty liver in zebrafish (Danio rerio)

Zebrafish (Danio rerio) is becoming a popular developmental biology model to study diseases and for drug discovery. In this study, we performed proton nuclear magnetic resonance spectroscopy ((1)H-NMR)- and gas chromatography-mass spectrometry (GC/MS)-based metabolic profiling of an alcoholic fatty liver using a zebrafish disease model. We examined metabolic differences between the control and alcoholic fatty liver groups in zebrafish to determine how metabolism in an alcoholic fatty liver is regulated. Multivariate statistical analysis showed a significant difference between the control and alcoholic fatty liver groups. The alcoholic fatty liver group showed increased excretion of isoleucine, acetate, succinate, choline, creatine, acetoacetate, 3-hydroxybutyrate (3HB), ethyl glucuronide (EtG), lactate/pyruvate ratio, fatty acids, and cholesterol, and decreased excretion of citrate, aspartate, tyrosine, glycine, glucose, alanine, betaine, and maltose. Metabolites identified in the fatty liver groups were associated with long-term alcohol consumption, which causes both oxidation-reduction (redox) changes and oxidative stress. This study suggests that global metabolite profiling in a zebrafish model can provide insights into the metabolic changes in an alcoholic fatty liver.     

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

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

Metabolomics reveals the mechanism of (−)-hydroxycitric acid promotion of protein synthesis and inhibition of fatty acid synthesis in broiler chickens

(−)-Hydroxycitric acid (HCA), a major component of Garcinia cambogia extracts, has been shown to suppress BW gain and fat accumulation in animals and humans. However, the mechanism remains unknown. In this study, gas chromatography-mass spectrometry was used to analyse serum metabolites, and principal component analysis and partial least-squares-discriminant analysis models were generated to analyse serum metabolite changes in broiler chickens after the administration of (−)-HCA at 0, 1000, 2000 and 3000 mg/kg diets for 28 days. Metabolites showing significant changes were screened by ‘variable importance in the projection’ plots. The results showed that 20 metabolites in the 1000 mg/kg (−)-HCA treatment group and 16 metabolites in 3000 mg/kg (−)-HCA treatment group were significantly altered. Metabolites pathway enrichment analysis indicated that these metabolites were mainly associated with metabolism of amino acids, protein synthesis, citric acid cycle, and uric acid and fatty acid synthesis. The data indicated that (−)-HCA promoted protein synthesis by regulating the metabolic directions of amino acids. At the same time, (−)-HCA treatment inhibited fatty acid synthesis by promoting the citric acid cycle, resulting in reduced cytosolic acetyl-CoA content in broiler chickens. The present study identified global changes in metabolites and analysed the main canonical metabolic pathways in broiler chickens supplemented with (−)-HCA. These results will deepen our understanding of the mechanism of (−)-HCA’s effects in animals.     

Metabonomic characterization of early atherosclerosis in hamsters with induced cholesterol

Atherosclerosis is a complicated and multifactorial disease, induced not only by genotype, but also, even more importantly, by environmental factors. Study on the metabolic perturbation of endogenous compounds may offer deeper insight into development of atherosclerosis. Gas chromatography/mass spectrometry (GC/MS)-based metabonomics was used to profile a metabolic fingerprint of serum obtained from hamsters with induced cholesterol. The deconvoluted GC/MS data were processed by multivariate statistical analysis tools, such as principal component analysis (PCA) and partial least squares projection to latent structure and discriminant analysis (PLS-DA). For the first time we showed a time-dependent development of the model animal from normal to hypercholesterolaemia, and further to early atherosclerosis. Twenty-one compounds were identified as markers involved in the development to atherosclerosis. Identification of the compounds suggests that amino acid metabolism and fatty acid oxidation are significantly perturbed following cholesterol overloading. The data provide novel information to approach the pathophysiological processes of the hypercholesterolaemia and atherosclerosis disease continuum.     

Metabolic profiling of Medicago truncatula cell cultures reveals the effects of biotic and abiotic elicitors on metabolism

GC-MS-based metabolite profiling was used to analyse the response of Medicago truncatula cell cultures to elicitation with methyl jasmonate (MeJa), yeast elicitor (YE), or ultraviolet light (UV). Marked changes in the levels of primary metabolites, including several amino acids, organic acids, and carbohydrates, were observed following elicitation with MeJa. A similar, but attenuated response was observed following YE elicitation, whereas little response was observed following UV elicitation. MeJa induced the accumulation of the triterpene beta-amyrin, a precursor to the triterpene saponins, and LC-MS analysis confirmed the accumulation of triterpene saponins in MeJa-elicited samples. In addition, YE induced a slight, but significant accumulation of shikimic acid, an early precursor to the phenylpropanoid pathway, which was also demonstrated to be YE-inducible by LC-MS analyses. Correlation analyses of metabolite relationships revealed perturbation of the glycine, serine, and threonine biosynthetic pathway, and suggested the induction of threonine aldolase activity, an enzyme as yet uncharacterized from plants. Members of the branched chain amino acid pathway accumulated in a concerted fashion, with the strongest correlation being that between leucine and isoleucine (r2=0.941). While UV exposure itself had little effect on primary metabolites, the experimental procedure, as revealed by control treatments, induced changes in several metabolites which were similar to those following MeJa elicitation. Sucrose levels were lower in MJ- and YE-elicited samples compared with control samples, suggesting that a portion of the effects observed on the primary metabolic pool are a consequence of fundamental metabolic repartitioning of carbon resources rather than elicitor-specific induction. In addition, beta-alanine levels were elevated in all elicited samples, which, when viewed in the context of other elicitation responses, suggests the altered metabolism of coenzyme A and its esters, which are essential in secondary metabolism.     

MS-based metabolite profiling reveals time-dependent skin biomarkers in UVB-irradiated mice

We observed clinical and histological changes, including increased transepidermal water loss, epidermal thickness, and inflammatory cells, and changes in collagen fibers in the skin of mice chronically exposed to ultraviolet (UV) B radiation for 12 weeks. By using ultra-performance liquid chromatography-quadrupole time-of-flight (TOF) mass spectrometry (MS), gas chromatography (TOF–MS), and NanoMate tandem-MS-based metabolite profiling, we identified amino acids, organic compounds, fatty acids, lipids, nucleosides, carbohydrates, lysophosphatidylcholines, lysophosphatidylethanolamines, urocanic acids, and ceramides (CERs) as candidate biomarkers of the histological changes in mouse skin following UVB irradiation for 6 and 12 weeks. cis-Urocanic acid and cholesterol showed the most dramatic increase and decrease at 6 and 12 weeks, respectively. In addition, the changes in skin primary metabolites and lysophospholipids induced by UVB exposure were generally greater at 12 weeks than at 6 weeks. The results from primary metabolite, lysophospholipid, and CER profiles suggest that prolonged chronic exposure to UVB light has a great effect on skin by altering numerous metabolites. A comprehensive MS-based metabolomic approach for determining regulatory metabolites in UV-induced skin will lead to a better understanding of the relationship between skin and UV.     

Differences in the metabolite profiles of spinach (Spinacia oleracea L.) leaf in different concentrations of nitrate in the culture solution

The nitrogen (N) status of a plant determines the composition of its major components (amino acids, proteins, carbohydrates and organic acids) and, directly or indirectly, affects the quality of agricultural products in terms of their calorific value and taste. Although these effects are guided by changes in metabolic pathways, no overall metabolic analysis has previously been conducted to demonstrate such effects. Here, metabolite profiling using gas chromatography-mass spectrometry (GC-MS) was used to evaluate the effect of N levels on spinach tissue, comparing two cultivars that differed in their ability to use N. Wide variation in N content was observed without any distinct inhibition of growth in either cultivar. Principal component analysis (PCA) and self-organizing mapping (SOM) were undertaken to describe changes in the metabolites of mature spinach leaves. In PCA, the first component accounted for 44.5% of the total variance, the scores of which was positively correlated with the plant's N content, and a close relationship between metabolite profiles and N status was observed. Both PCA and SOM revealed that metabolites could be broadly divided into two types, correlating either positively or negatively with plant N content. The simple and co-coordinated metabolic stream, containing both general and spinach-specific aspects of plant N content, will be useful in future research on such topics as the detection of environmental effects on spinach through comprehensive metabolic profiling.     

GC/MS-based metabolomic studies reveal key roles of glycine in regulating silk synthesis in silkworm,Bombyx mori

Metabolic profiling of silkworm, especially the factors that affect silk synthesis at the metabolic level, is little known. Herein, metabolomic method based on gas chromatography-mass spectrometry was applied to identify key metabolic changes in silk synthesis deficient silkworms. Forty-six differential metabolites were identified in Nd group with the defect of silk synthesis. Significant changes in the levels of glycine and uric acid (up-regulation), carbohydrates and free fatty acids (down-regulation) were observed. The further metabolomics of silk synthesis deficient silkworms by decreasing silk proteins synthesis using knocking out fibroin heavy chain gene or extirpating silk glands operation showed that the changes of the metabolites were almost consistent with those of the Nd group. Furthermore, the increased silk yields by supplying more glycine or its related metabolite confirmed that glycine is a key metabolite to regulate silk synthesis. These findings provide important insights into the regulation between metabolic profiling and silk synthesis.     

Metabolic disturbances associated with systemic lupus erythematosus

The metabolic disturbances that underlie systemic lupus erythematosus are currently unknown. A metabolomic study was executed, comparing the sera of 20 SLE patients against that of healthy controls, using LC/MS and GC/MS platforms. Validation of key differences was performed using an independent cohort of 38 SLE patients and orthogonal assays. SLE sera showed evidence of profoundly dampened glycolysis, Krebs cycle, fatty acid β oxidation and amino acid metabolism, alluding to reduced energy biogenesis from all sources. Whereas long-chain fatty acids, including the n3 and n6 essential fatty acids, were significantly reduced, medium chain fatty acids and serum free fatty acids were elevated. The SLE metabolome exhibited profound lipid peroxidation, reflective of oxidative damage. Deficiencies were noted in the cellular anti-oxidant, glutathione, and all methyl group donors, including cysteine, methionine, and choline, as well as phosphocholines. The best discriminators of SLE included elevated lipid peroxidation products, MDA, gamma-glutamyl peptides, GGT, leukotriene B4 and 5-HETE. Importantly, similar elevations were not observed in another chronic inflammatory autoimmune disease, rheumatoid arthritis. To sum, comprehensive profiling of the SLE metabolome reveals evidence of heightened oxidative stress, inflammation, reduced energy generation, altered lipid profiles and a pro-thrombotic state. Resetting the SLE metabolome, either by targeting selected molecules or by supplementing the diet with essential fatty acids, vitamins and methyl group donors offers novel opportunities for disease modulation in this disabling systemic autoimmune ailment.     

Metabolic profiling of strawberry (Fragaria x ananassa Duch.) during fruit development and maturation

Strawberry (Fragaria × ananassa Duch), a fruit of economic and nutritional importance, is also a model species for fleshy fruits and genomics in Rosaceae. Strawberry fruit quality at different harvest stages is a function of the fruit's metabolite content, which results from physiological changes during fruit growth and ripening. In order to investigate strawberry fruit development, untargeted (GC-MS) and targeted (HPLC) metabolic profiling analyses were conducted. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were employed to explore the non-polar and polar metabolite profiles from fruit samples at seven developmental stages. Different cluster patterns and a broad range of metabolites that exerted influence on cluster formation of metabolite profiles were observed. Significant changes in metabolite levels were found in both fruits turning red and fruits over-ripening in comparison with red-ripening fruits. The levels of free amino acids decreased gradually before the red-ripening stage, but increased significantly in the over-ripening stage. Metabolite correlation and network analysis revealed the interdependencies of individual metabolites and metabolic pathways. Activities of several metabolic pathways, including ester biosynthesis, the tricarboxylic acid cycle, the shikimate pathway, and amino acid metabolism, shifted during fruit growth and ripening. These results not only confirmed published metabolic data but also revealed new insights into strawberry fruit composition and metabolite changes, thus demonstrating the value of metabolomics as a functional genomics tool in characterizing the mechanism of fruit quality formation, a key developmental stage in most economically important fruit crops.     

Metabolomic and proteomic analysis of a clonal insulin-producing beta-cell line (INS-1 832/13)

Metabolites generated from fuel metabolism in pancreatic beta-cells control exocytosis of insulin, a process which fails in type 2 diabetes. To identify and quantify these metabolites, global and unbiased analysis of cellular metabolism is required. To this end, polar metabolites, extracted from the clonal 832/13 beta-cell line cultured at 2.8 and 16.7 mM glucose for 48 h, were derivatized followed by identification and quantification, using gas chromatography (GC) and mass spectrometry (MS). After culture at 16.7 mM glucose for 48 h, 832/13 beta-cells exhibited a phenotype reminiscent of glucotoxicity with decreased content and secretion of insulin. The metabolomic analysis revealed alterations in the levels of 7 metabolites derived from glycolysis, the TCA cycle and pentose phosphate shunt, and 4 amino acids. Principal component analysis of the metabolite data showed two clusters, corresponding to the cells cultured at 2.8 and 16.7 mM glucose, respectively. Concurrent changes in protein expression were analyzed by 2-D gel electrophoresis followed by LC-MS/MS. The identities of 86 spots corresponding to 75 unique proteins that were significantly different in 832/13 beta-cells cultured at 16.7 mM glucose were established. Only 5 of these were found to be metabolic enzymes that could be involved in the metabolomic alterations observed. Anticipated changes in metabolite levels in cells exposed to increased glucose were observed, while changes in enzyme levels were much less profound. This suggests that substrate availability, allosteric regulation, and/or post-translational modifications are more important determinants of metabolite levels than enzyme expression at the protein level.