Metabolite profiling of potato (Solanum tuberosum L.) tubers during wound-induced suberization

Suberin is a specific cell wall-associated biopolymer characterized by the deposition of both a poly(phenolic) domain (SPPD) associated with the cell wall, and a poly(aliphatic) domain (SPAD) thought to be deposited between the cell wall and plasma membrane. In planta, suberin functions to prevent plants from desiccation and pathogen attack. Although the chemical identity of the monomeric components of the SPPD and SPAD are well known, their concerted biosynthesis and assembly into the suberin macromolecule is poorly understood. To expand our knowledge of suberin biosynthesis, a GC/MS-based metabolite profiling study was conducted, using wound healing potato (Solanum tuberosum L.) tubers as a model system. A time series of both non-polar and polar metabolite profiles were created, yielding a broad-based, dynamic picture of wound-induced metabolism, including suberization. Principal component analysis revealed a separation of metabolite profiles according to different suberization stages, with clear temporal differences emerging in the non-polar and polar profiles. In the non-polar profiles, suberin-associated aliphatics contributed the most to cluster formation, while a broader range of metabolites (including organic acids, sugars, amino acids and phenylpropanoids) influenced cluster formation amongst polar profiles. Pair-wise correlation analysis revealed strong correlations between known suberin-associated compounds, as well as between suberin-associated compounds and several un-identified metabolites in the profiles. These data may help to identify additional, as yet unknown metabolites associated with suberization process.     

Metabolomic analysis of the potato tuber life cycle

An analysis of the potato (Solanum tuberosum L.) tuber life cycle has been completed using a range of mass-spectrometry (MS) based approaches. Six stages have been examined which included developing and mature tubers, sprouting mature tubers and mature tubers stored at 5 or 10°C. The impact of excising developing tubers from the mother plant (source-sink manipulation) was also determined. Data was subjected to Principal Components Analysis, Analysis of Variance and Hierarchical Cluster Analysis to assess the potential for separating the life cycle stages, to define the major profiles for metabolite changes during the life cycle stages examined, and to inform on which metabolites underpinned these profiles. We have shown that it is possible to separate all of the stages using combined analytical approaches and that five major profiles can be used to describe the changes in metabolite levels. Data also indicate that, within a relatively short timeframe, manipulation of source-sink relations has a significant impact on metabolite pools beyond what is currently known for sugar–starch metabolism. We have also demonstrated that the metabolomics data can be mined to provide answers to specific questions––in this case to identify temporal changes in metabolites related to acrylamide-forming potential.     

Metabolism of auxin in pine tissues: Indole-3-acetic acid conjugation

Incubation of sections of various tissues of Pinus pinea L. with a relatively low concentration (3.6 μM) of indole-3-acetic acid-2-¹⁴C (IAA) resulted in the formation of two major metabolites. The first, which has not been identified, seemed to be a polar acidic compound and the second was identified as indole-3-acetylaspartic acid (IAAsp). The polar acidic metabolite has been found to be the major metabolite in needles, shoot wood and roots, while IAAsp has been found to be the major metabolite in shoot bark. Increasing the concentration of IAA in the incubation medium resulted in an increase in the formation of a third metabolite which proved to be l-O-(indole-3-acetyl)-β-d -glucose (IAGlu) and a concomitant decrease in the amount of the polar acidic metabolite. This phenomenon was prominent particularly in needles. IAGlu was isolated from needles and IAAsp was isolated from shoot bark by means of polyvinylpolypyrrolidone column chromatography and preparative thin-layer chromatography. IAGlu was identified by comparison with authentic material by co-chromatography in three different solvent systems and by ¹H-nuclear magnetic resonance analysis. IAAsp was identified by comparison with authentic material by gas-liquid chromatography and ¹H-nuclear magnetic resonance analysis. Several aspects of formation, separation and isolation of IAA metabolites are discussed.     

Metabolite changes with induction of Cuscuta haustorium and translocation from host plants

Cuscuta is a stem holoparasitic plant without leaves or roots, parasitizing various types of host plants and causing major problems for certain crops. Cuscuta is known as a generalist and, thus, must have unique parasite strategies to cope with different host plants. For elucidating metabolic responses and mechanisms of parasitization, metabolomic approaches using GC/MS were applied. We compared five stages of Cuscuta japonica: early stage seedlings, with far red light (FR) cue, with contact signal, haustorium induced seedlings by both signals and adult plant parasites on host plants. Sugars, amino acids, organic acids, nucleic acids, and polyols were identified from the polar phase fraction. The apical part contained metabolite profiles different from the haustorium induced part or the basal part. Amino acid and some organic acids were up-regulated for haustorium induction but decreased after parasitization. After attachment to different host plants, metabolite profiles of Cuscuta japonica changed dramatically due to the absorption of specific host plant metabolites such as pinitol. Cuscuta seedlings attached to pinitol rich host plants contained more pinitol and showed different profiles from those attached to plants having less or lacking pinitol.     

Development of a new sensitive and specific time-resolved fluoroimmunoassay (TR-FIA) of chlormadinone acetate in the serum of treated menopausal women

We describe the development of a serum chlormadinone acetate (CMA) time-resolved fluoroimmunoassay (TR-FIA). We prepared haptens (3-CMO-chlormadinone acetate and 6-chloropregna-4,6-dien-17,20-diol-3-one-20-hemisuccinate), biotinylated tracers (3(biotinylaminopropylamido) 3-CMO-chlormadinone acetate and 3-(6-chloropregna-4,6-dien-17,20-diol-3-one-20-hemisuccinylamino)1-biotinylaminopropane), and immunogens necessary for eliciting two antibodies (anti-chlormadinone acetate 3-CMO/BSA and anti-chlormadinone 20-hemisuccinate/BSA). The specificity of the assay was rigorously studied to eliminate possible interference by polar metabolites of CMA, particularly 17 alpha-acetoxy-6-chloro-3beta-hydroxypregna-4,6-diene-20-one (3beta-hydroxy metabolite), employing an easy-to-use ethylene glycol chromatographic step prior to immunoassay, so as to separate the polar metabolites, in particular the 3beta-hydroxy-CMA metabolite, from the intact CMA. The choice of the anti-CMA antibody was guided by the high assay sensitivity obtained with the anti-CMA 3-CMO/BSA antibody. The detection limit was 51pg/ml. Interassay reproducibility CVs were between 2.6 and 4.5%. This TR-FIA thus appeared to be a sensitive, specific, precise, and consequently well-suited method for measurement of serum CMA during a pharmacokinetic study in women.     

Metabolism of 1alpha,25-dihydroxyvitamin D(3) and its C-3 epimer 1alpha,25-dihydroxy-3-epi-vitamin D(3) in neonatal human keratinocytes

We previously reported that 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is metabolized into 1alpha,25-dihydroxy-3-epi-vitamin D(3) [1alpha,25(OH)(2)-3-epi-D(3)] in primary cultures of neonatal human keratinocytes. We now report that 1alpha,25(OH)(2)-3-epi-D(3) itself is further metabolized in human keratinocytes into several polar metabolites. One of the polar metabolite was unequivocally identified as 1alpha,23,25-trihydroxy-3-epi-vitamin D(3) by mass spectrometry and its sensitivity to sodium periodate. Three of the polar metabolites were identified as 1alpha,24,25-trihydroxy-3-epi-vitamin D(3), 1alpha,25-dihydroxy-24-oxo-3-epi-vitamin D(3) and 1alpha,23,25-trihydroxy-24-oxo-3-epi-vitamin D(3) by comigration with authentic standards on both straight and reverse phase HPLC systems. In addition to the polar metabolites, 1alpha,25(OH)(2)-3-epi-D(3) was also metabolized into two less polar metabolites. A possible structure of either 1alphaOH-3-epi-D(3)-20,25-cyclic ether or 1alphaOH-3-epi-D(3)-24,25-epoxide was assigned to one of the less polar metabolites through mass spectrometry. Thus, we indicate for the first time that 1alpha,25(OH)(2)-3-epi-D(3) is metabolized in neonatal human keratinocytes not only via the same C-24 and C-23 oxidation pathways like its parent, 1alpha,25(OH)(2)D(3); but also is metabolized into a less polar metabolite via a pathway that is unique to 1alpha,25(OH)(2)-3-epi-D(3).     

Responses to water stress of gas exchange and metabolites in Eucalyptus and Acacia spp

Studies of water stress commonly examine either gas exchange or leaf metabolites, and many fail to quantify the concentration of CO₂ in the chloroplasts (C_c). We redress these limitations by quantifying C_c from discrimination against ¹³CO₂ and using gas chromatography–mass spectrometry (GC–MS) for leaf metabolite profiling. Five Eucalyptus and two Acacia species from semi‐arid to mesic habitats were subjected to a 2 month water stress treatment (Ψ_pre‐dawn = ?1.7 to ?2.3 MPa). Carbohydrates dominated the leaf metabolite profiles of species from dry areas, whereas organic acids dominated the metabolite profiles of species from wet areas. Water stress caused large decreases in photosynthesis and C_c, increases in 17–33 metabolites and decreases in 0–9 metabolites. In most species, fructose, glucose and sucrose made major contributions to osmotic adjustment. In Acacia, significant osmotic adjustment was also caused by increases in pinitol, pipecolic acid and trans‐4‐hydroxypipecolic acid. There were also increases in low‐abundance metabolites (e.g. proline and erythritol), and metabolites that are indicative of stress‐induced changes in metabolism [e.g. γ‐aminobutyric acid (GABA) shunt, photorespiration, phenylpropanoid pathway]. The response of gas exchange to water stress and rewatering is rather consistent among species originating from mesic to semi‐arid habitats, and the general response of metabolites to water stress is rather similar, although the specific metabolites involved may vary.     

UHPLC-Q-Orbitrap HR-MS-Based Metabolomics for Profiling the Sida rhombifolia Metabolites with Different Plant Organs and Cultivation Ages

Plant organs and cultivation ages can result in different compositions and concentration levels of plant metabolites. The metabolite profile of plants can be determined using liquid chromatography. This study determined the metabolite profiles of leaves, stems, and roots of Sida rhombifolia at different cultivation ages at 3, 4, and 5 months post-planting (MPP) using liquid chromatography-mass spectrometry/mass spectrometry (LC/MS/MS). The results identified that 41 metabolites in S. rhombifolia extract for all plant organs and cultivation ages. We successfully identified approximately 36 (leaves), 22 (stems), and 18 (roots) compounds in all extract. Using principal component analysis (PCA) with peak area as the variable, we clustered all sample extracts based on plant organs and cultivation ages. As a result of PCA, S. rhombifolia extracts were grouped according to plant organs and cultivation ages. In conclusion, a clear difference in the composition and concentration levels of metabolites was observed in the leaves, stems, and roots of S. rhombifolia harvested at 3-, 4-, and 5-MPP.     

Subcellular reprogramming of metabolism during cold acclimation in Arabidopsis thaliana

Metabolite changes in plant leaves during exposure to low temperatures involve re‐allocation of a large number of metabolites between sub‐cellular compartments. Therefore, metabolite determination at the whole cell level may be insufficient for interpretation of the functional significance of cellular compounds. To investigate the cold‐induced metabolite dynamics at the level of individual sub‐cellular compartments, an integrative platform was developed that combines quantitative metabolite profiling by gas chromatography coupled to mass spectrometry (GC‐MS) with the non‐aqueous fractionation technique allowing separation of cytosol, vacuole and the plastidial compartment. Two mutants of Arabidopsis thaliana representing antipodes in the diversion of carbohydrate metabolism between sucrose and starch were compared to Col‐0 wildtype before and after cold acclimation to investigate interactions of cold acclimation with subcellular re‐programming of metabolism. A multivariate analysis of the data set revealed dominant effects of compartmentation on metabolite concentrations that were modulated by environmental condition and genetic determinants. While for both, the starchless mutant of plastidial phospho‐gluco mutase (pgm) and a mutant defective in sucrose‐phosphate synthase A1, metabolic constraints, especially at low temperature, could be uncovered based on subcellularly resolved metabolite profiles, only pgm had lowered freezing tolerance. Metabolic profiles of pgm point to redox imbalance as a possible reason for reduced cold acclimation capacity.     

Metabolism of gibberellins A1 and A3 in fruits and shoots of Prunus avium

Isotope-labelled GA metabolites were identified by GC--MS, following HPLC fractionation of extracts derived from fruits or shoots, that had been incubated with [2H]- and [3H]- GA1 or [2H]- and [3H]- GA3. GA1 (1) was converted into GA8 (10) by developing fruits and vegetative shoots of sweet cherry (Prunus avium cv. 'Stella'), while GA3 (4) was converted into GA3-isolactone (17). Other metabolites of each GA were detected but were not identified unequivocally. These included a metabolite of GA1 (1) in fruitlets that was more polar (by reverse phase HPLC) than GA8 (10) and a metabolite of similar polarity to GA87 (6), was obtained after incubating fruitlets with GA3 (4). However, no evidence was obtained to suggest that GA87 (6) was a metabolite of GA3 (4) or that GA85 (2) was a metabolite of GA1 (1) in these tissues, under the conditions used. The pattern of metabolites obtained from vegetative tissues was similar to that from fruitlets. However, the results suggested that GA1 (1) and GA3 (4) were metabolised at a greater rate in shoots from mature trees than in shoots from seedlings, and that GA1 (1) was metabolised more rapidly than GA3 (4) in juvenile and mature shoots. We conclude from these observations that GA3 (4) is not a precursor of GA87 (6) and GA32 (5), also, that GA1 (1) is not a precursor of GA85 (2) and GA86 (3) in developing fruits or in vegetative shoots of sweet cherry.     

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.     

Application of NMR based metabolomics for mapping metabolite variation in European wheat

In this study, we report on the use of NMR-based metabolomics to access variation in low molecular weight polar metabolites between the European wheat cultivars Apache, Charger, Claire and Orvantis. Previous unassigned resonances in the published NMR spectra of wheat extracts were identified using ¹³C NMR and two dimensional proton-carbon NMR. These included a peak for trans-aconitate (δ3.43) and resonances corresponding to fructose in the crowded carbohydrate region of the spectra. Large metabolite differences were observed between two different growth stages, namely the coleoptile and two week old leaf tissue extracts which were consistent across cultivars. Two week old leaf tissue extracts had higher abundances of glutamine, glutamate, sucrose and trans-aconitate and less glucose and fructose than were observed in the coleoptile extracts. Across both growth stages the cultivars Apache and Charger showed the greatest differences in metabolite profiles. Charger had higher abundances of betaine, the single most influential metabolite in the principal component analysis, in addition to fructose and sucrose. However, Charger had lower levels of aspartate, choline and glucose than Apache. These findings demonstrate the potential for a biochemical mapping approach using NMR, across European wheat germplasm, for metabolites of known importance to functional characteristics.     

Toward Arabidopsis thaliana hydrophilic metabolome: assessment of extraction methods and quantitative 1H NMR

Our goal was to establish the hydrophilic metabolome of heterotrophic Arabidopsis thaliana cells grown in suspension, a cellular model of plant sink tissues. Water‐soluble metabolites were extracted using four protocols: perchloric acid, boiling ethanol, methanol and methanol/chloroform (M/Chl). They were detected and quantified using ¹H nuclear magnetic resonance (NMR) spectroscopy at 400 MHz. Extraction yields and reproducibility of the extraction methods were investigated. The effects of cell harvest protocol, cell grinding and lyophilization and storage conditions on the measured metabolic profiles were also studied. These quantitative studies demonstrated for the first time that the four extraction protocols commonly used do lead to quite similar molecular compositions as analyzed by ¹H NMR. The M/Chl method proved effective and reliable to prepare series of physiologically significant extracts from plant cells for ¹H NMR analysis. Reproducibility of the detected metabolome was assessed over long periods of time by analyzing a large number of separate extracts prepared from independent cultures. Larger variations in the NMR metabolite profiles could be correlated to changes in physiological parameters of the culture medium. Quantitative resolved ¹H NMR of cell extracts proved to be robust and reliable for routine metabolite profiling of plant cell cultures.     

Metabolomic evaluation of rat liver and testis to characterize the toxicity of triazole fungicides

The effects of two triazole fungicides, myclobutanil and triadimefon, on endogenous rat metabolite profiles in blood serum, liver, and testis was assessed using proton nuclear magnetic resonance (¹H-NMR) spectroscopy. Adult male Sprague-Dawley rats were dosed daily by gavage for 14 days with myclobutanil or triadimefon, at two dose levels for each triazole. Following exposure, serum, liver, and testis were collected and processed for NMR analysis. Principal components analysis (PCA) and partial least squares discriminant analysis (PLS-DA) of the resulting spectra were used to determine changes in metabolite profiles as a result of exposure. Using this approach, responses common to both triazoles were identified, as well as responses indicative of differences in the toxicity of these two compounds. Although changes were observed in serum metabolites following exposure, none were robust enough to be considered a biomarker of exposure/effect. A number of metabolic changes were, however, observed in the liver with both triazoles, particularly in metabolites related to the methionine cycle. The testes of myclobutanil-exposed animals displayed altered levels of creatine and creatinine, consistent with testicular toxicity. Overall, the results of this study support the possible application of a metabolomics approach to assessing the toxicity of triazole fungicides and identifying biomarkers of exposure and/or effect.RNM supported by EPA/NCSU Cooperative Training Agreement #CT826512010 with the Department of Environmental and Molecular Toxicology, North Caroline State University, Raleigh.The research described in this article has been reviewed by the U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and the policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.     

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.     

Metabolomic analysis of polar metabolites in lipoprotein fractions identifies lipoprotein-specific metabolic profiles and their association with insulin resistance

While the molecular lipid composition of lipoproteins has been investigated in detail, little is known about associations of small polar metabolites with specific lipoproteins. The aim of the present study was to investigate the profiles of polar metabolites in different lipoprotein fractions, i.e., very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL) and two sub-fractions of the high-density lipoprotein (HDL). The VLDL, IDL, LDL, HDL(2), and HDL(3) fractions were isolated from serum of sixteen individuals having a broad range of insulin sensitivity and characterized using comprehensive two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GC×GC-TOFMS). The lipoprotein fractions had clearly different metabolite profiles, which correlated with the particle size and surface charge. Lipoprotein-specific associations of individual metabolites with insulin resistance were identified, particularly in VLDL and IDL fractions, even in the absence of such associations in serum. The results indicate that the polar molecules are strongly attached to the surface of the lipoproteins. Furthermore, strong lipoprotein-specific associations of metabolites with insulin resistance, as compared to their serum profiles, indicate that lipoproteins may be a rich source of tissue-specific metabolic biomarkers.     

Profiling lipid metabolites yields unique information on sex- and time-dependent responses of fathead minnows (Pimephales promelas) exposed to 17α-ethynylestradiol

Alterations in hepatic lipid profiles of fathead minnows (FHM) exposed to the synthetic estrogen 17α-ethynylestradiol (EE2) were determined using ¹H-NMR spectroscopy-based metabolite profiling. The exposures were conducted using either 10 ng/l or 100 ng/l EE2 via a continuous flow water delivery system. Livers were collected at 1, 4, and 8 days of the exposure and 8 days after the chemical was removed from the water (i.e. an 8 day depuration). The exposure resulted in a number of sex-specific changes in lipid profiles that were also highly time dependent. Those metabolites most affected by exposure included phosphatidylcholine, diglycerides, triglycerides and cholesterol. In addition, changes in the length and degree of unsaturation of hepatic fatty acids were observed. Lipid profiles in plasma for fish collected on the 4th day of exposure were also analyzed in order to provide further insights into changes observed in hepatic metabolite changes. Using validated partial least-squares discriminant analysis (PLS-DA), the response trajectories of the male liver lipid profiles at both exposure concentrations were compared. This analysis indicated that the males exposed to the low concentration of EE2 (10 ng/l) were largely able to recover from the exposure once the chemical was removed from the water. Conversely, the males exposed to the high concentration (100 ng/l) did not appear to recover from the exposure despite the 8 day depuration.     

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.     

Metabolic responses of Thellungiella halophila/salsuginea to biotic and abiotic stresses: Metabolite profiles and quantitative analyses

The metabolite profiles of the model crucifer Thellungiella salsuginea (salt cress) ecotype Shandong subjected to various biotic and abiotic stresses were analyzed using HPLC-DAD-ESI-MS. Two different cruciferous microbial pathogens, Albugo candida, a biotrophic oomycete, and Leptosphaeria maculans, a necrotrophic fungus, elicited formation of the phytoalexins wasalexins A and B without causing visual damage on inoculated leaves. Analyses of non-polar and polar metabolites led to elucidation of the chemical structures of five metabolites: 4′-O-(E)-sinapoyl-7-methoxyisovitexin-2″-O-β-d-glucopyranoside, 4′-O-(E)-sinapoylisovitexin-2″-O-β-d-glucopyranoside, 4-O-β-d-glucopyranosyl-7-hydroxymatairesinol, 5′-O-β-d-glucopyranosyldihydroneoascorbigen and 3-O-β-d-glucopyranosylthiane. 3-O-β-d-glucopyranosylthiane, an unique metabolite for which we suggest the name glucosalsuginin, is proposed to derive from the glucosinolate glucoberteroin. In addition, the identification of a broad range of polar metabolites identical to those of other crucifers was carried out. Quantification of several metabolites over a period of eight days showed that concentrations of the polar phytoanticipin 4-methoxyglucobrassicin increased substantially in leaves irradiated with UV light (λ_max 254 nm) relative to control leaves, but not in leaves subjected to other stresses.     

Effects of feeding and body weight loss on the 1H-NMR-based urine metabolic profiles of male Wistar Han rats: implications for biomarker discovery.

For almost two decades, 1H-NMR spectroscopy has been used as an 'open' system to study the temporal changes in the biochemical composition of biofluids, including urine, in response to adverse toxic events. Many of these in vivo studies have reported changes in individual metabolites and patterns of metabolites that correlated with toxicological changes. However, many of the proposed novel biomarkers are common to a number of different types of toxicity. These may therefore reflect non-specific effects of toxicity, such as weight loss, rather than a specific pathology. A study was carried out to investigate the non-specific effects on urinary metabolite profiles by administering four hepatotoxic compounds, as a single dose, to rats at two dose levels: hydrazine hydrate (0.06 or 0.08 g kg (1)), 1,2-dimethylhydrazine (0.1 or 0.3 g kg (-1)), alpha-napthylisothiocyanate (0.1 or 0.15 g kg(-1)) and carbon tetrachloride (1.58 or 3.16 g kg(-1)). The study included weight-matched control animals along with those that were dosed, which were then 'pair-fed' with the treated animals so they achieved a similar weight loss. The urinary metabolite profiles were investigated over time using 1H-NMR spectroscopy and compared with the pathology from the same animals. The temporal changes were analysed statistically using multivariate statistical data analysis including principal component analysis, partial least squares, parallel factor analysis and Fisher's criteria. A number of metabolites associated with energy metabolism or which are partially dietary in origin, such as creatine, creatinine, tricarboxylic acid (TCA) cycle intermediates, phenylacetylglycine, fumarate, glucose, taurine, fatty acids and N-methylnicotinamide, showed altered levels in the urine of treated and pair-fed animals. Many of these changes correlated well with weight loss. Interestingly, there was no increase in ketone bodies (acetate and beta-hydroxybutyrate), which might be expected if energy metabolism was switched from glycolysis to fatty acid beta-oxidation. In some instances, the metabolites that changed were considered to be non-specific markers of toxicity, but were also identified as markers of a specific type of toxicity. For example, taurine was raised significantly in carbon tetrachloride-treated animals but reduced in the pair-fed group. However, raised urinary bile acid levels were only seen after alpha-napthylisothiocyanate treatment. The methodology, statistical analysis used and the data generated will help improve the identification of specific markers or patterns of urinary markers of specific toxic effects.