Metabolomic Method: UPLC-q-ToF Polar and Non-Polar Metabolites in the Healthy Rat Cerebellum Using an In-Vial Dual Extraction

Unbiased metabolomic analysis of biological samples is a powerful and increasingly commonly utilised tool, especially for the analysis of bio-fluids to identify candidate biomarkers. To date however only a small number of metabolomic studies have been applied to studying the metabolite composition of tissue samples, this is due, in part to a number of technical challenges including scarcity of material and difficulty in extracting metabolites. The aim of this study was to develop a method for maximising the biological information obtained from small tissue samples by optimising sample preparation, LC-MS analysis and metabolite identification. Here we describe an in-vial dual extraction (IVDE) method, with reversed phase and hydrophilic liquid interaction chromatography (HILIC) which reproducibly measured over 4,000 metabolite features from as little as 3mg of brain tissue. The aqueous phase was analysed in positive and negative modes following HILIC separation in which 2,838 metabolite features were consistently measured including amino acids, sugars and purine bases. The non-aqueous phase was also analysed in positive and negative modes following reversed phase separation gradients respectively from which 1,183 metabolite features were consistently measured representing metabolites such as phosphatidylcholines, sphingolipids and triacylglycerides. The described metabolomics method includes a database for 200 metabolites, retention time, mass and relative intensity, and presents the basal metabolite composition for brain tissue in the healthy rat cerebellum.     

A core set of metabolite sink/source ratios indicative for plant organ productivity in Lotus japonicus

Plant growth is an important process in physiological as well as ecological respect and a number of metabolic parameters (elemental ratios as well as steady-state levels of individual metabolites) have been demonstrated to reflect this process on the whole plant level. Since plant growth is highly localized and is the result of a complex interplay of metabolic activities in sink and source organs, we propose that ratios in metabolite levels of sink and source organs are particularly well suited to characterize this process. To demonstrate such a connection, we studied organ-specific metabolite ratios from Lotus japonicus treated with mineral nutrients, salt stress or arbuscular mycorrhizal fungi. The plants were displaying a wide range of biomass and of flower/biomass ratios. In the analysis of our data we looked for correlations between shifts in sink/source metabolite ratios and plant productivity (biomass accumulated at the time of harvest). In addition we correlated shifts in metabolite ratios comparing competing generative and vegetative sink organs with shifts in productivity of the two organs (changes in flower/biomass ratios). In our analyses we observed clear shifts of carbohydrates and of compounds connected to nitrogen metabolism in favour of sink organs of particularly high productivity. These shifts were in agreement with general differences in metabolite steady-state levels when comparing sink and source organs. Our findings suggest that differentiation of sink and source organs during sampling for metabolomic experiments substantially increases the amount of information obtained from such experiments.     

Solid-phase microextraction and the human fecal VOC metabolome

The diagnostic potential and health implications of volatile organic compounds (VOCs) present in human feces has begun to receive considerable attention. Headspace solid-phase microextraction (SPME) has greatly facilitated the isolation and analysis of VOCs from human feces. Pioneering human fecal VOC metabolomic investigations have utilized a single SPME fiber type for analyte extraction and analysis. However, we hypothesized that the multifarious nature of metabolites present in human feces dictates the use of several diverse SPME fiber coatings for more comprehensive metabolomic coverage. We report here an evaluation of eight different commercially available SPME fibers, in combination with both GC-MS and GC-FID, and identify the 50/30 μm CAR-DVB-PDMS, 85 μm CAR-PDMS, 65 μm DVB-PDMS, 7 μm PDMS, and 60 μm PEG SPME fibers as a minimal set of fibers appropriate for human fecal VOC metabolomics, collectively isolating approximately 90% of the total metabolites obtained when using all eight fibers. We also evaluate the effect of extraction duration on metabolite isolation and illustrate that ex vivo enteric microbial fermentation has no effect on metabolite composition during prolonged extractions if the SPME is performed as described herein.     

High-throughput extraction and quantification method for targeted metabolomics in murine tissues

Introduction

Global metabolomics analyses using body fluids provide valuable results for the understanding and prediction of diseases. However, the mechanism of a disease is often tissue-based and it is advantageous to analyze metabolomic changes directly in the tissue. Metabolomics from tissue samples faces many challenges like tissue collection, homogenization, and metabolite extraction.

Objectives

We aimed to establish a metabolite extraction protocol optimized for tissue metabolite quantification by the targeted metabolomics AbsoluteIDQ? p180 Kit (Biocrates). The extraction method should be non-selective, applicable to different kinds and amounts of tissues, monophasic, reproducible, and amenable to high throughput.

Methods

We quantified metabolites in samples of eleven murine tissues after extraction with three solvents (methanol, phosphate buffer, ethanol/phosphate buffer mixture) in two tissue to solvent ratios and analyzed the extraction yield, ionization efficiency, and reproducibility.

Results

We found methanol and ethanol/phosphate buffer to be superior to phosphate buffer in regard to extraction yield, reproducibility, and ionization efficiency for all metabolites measured. Phosphate buffer, however, outperformed both organic solvents for amino acids and biogenic amines but yielded unsatisfactory results for lipids. The observed matrix effects of tissue extracts were smaller or in a similar range compared to those of human plasma.

Conclusion

We provide for each murine tissue type an optimized high-throughput metabolite extraction protocol, which yields the best results for extraction, reproducibility, and quantification of metabolites in the p180 kit. Although the performance of the extraction protocol was monitored by the p180 kit, the protocol can be applicable to other targeted metabolomics assays.

     

Physiological and environmental parameters associated with mass spectrometry-based salivary metabolomic profiles

Mass spectrometry (MS)-based metabolomic methods enable simultaneous profiling of hundreds of salivary metabolites, and may be useful to diagnose a wide range of diseases using saliva. However, few studies have evaluated the effects of physiological or environmental factors on salivary metabolomic profiles. Therefore, we used capillary electrophoresis-MS to analyze saliva metabolite profiles in 155 subjects with reasonable oral hygiene, and examined the effects of physiological and environmental factors on the metabolite profiles. Overall, 257 metabolites were identified and quantified. The global profiles and individual metabolites were evaluated by principle component analysis and univariate tests, respectively. Collection method, collection time, sex, body mass index, and smoking affected the global metabolite profiles. However, age also might contribute to the bias in sex and collection time. The profiles were relatively unaffected by other parameters, such as alcohol consumption and smoking, tooth brushing, or the use of medications or nutritional supplements. Temporomandibular joint disorders had relatively greater effects on salivary metabolites than other dental abnormalities (e.g., stomatitis, tooth alignment, and dental caries). These findings provide further insight into the diversity and stability of salivary metabolomic profiles, as well as the generalizability of disease-specific biomarkers.     

Validation of Metabolic Alterations in Microscale Cell Culture Lysates Using Hydrophilic Interaction Liquid Chromatography (HILIC)-Tandem Mass Spectrometry-Based Metabolomics

By standard convention, in order to increase the efficacy of metabolite detection from cell culture lysates, metabolite extracts from a large quantity of cells are utilized for multiple reaction monitoring-based metabolomic studies. Metabolomics from a small number of cell extracts offers a potential economical alternative to increased cell numbers, in turn increasing the utility of cell culture-based metabolomics. However, the effect of reduced cell numbers on targeted metabolomic profiling is relatively unstudied. Considering the limited knowledge available of the feasibility and accuracy of microscale cell culture metabolomics, the present study analyzes differences in metabolomic profiles of different cell numbers of three pancreatic cancer cell lines. Specifically, it examines the effects of reduced cell numbers on metabolite profiles by obtaining extracts either directly from microscale culture plates or through serial dilution of increased numbers of cellular metabolite extracts. Our results indicate reduced cell numbers only modestly affect the number of metabolites detected (93% of metabolites detected in cell numbers as low as 10⁴ cells and 97% for 10⁵ cells), independent of the method used to obtain the cells. However, metabolite peak intensities were differentially affected by the reduced cell numbers, with some peak intensities inversely proportional to the cell numbers. To help eliminate such potential inverse relationships, peak intensities for increased cell numbers were excluded from the comparative analysis. Overall, metabolite profiles from microscale culture plates were observed to differ from the serial dilution samples, which may be attributable to the medium-to-cell-number ratios. Finally, findings identify perturbations in metabolomic profiling for cellular extracts from reduced cell numbers, which offer future applications in microscale metabolomic evaluations.     

Procedure for tissue sample preparation and metabolite extraction for high-throughput targeted metabolomics

Reproducible quantification of metabolites in tissue samples is of high importance for characterization of animal models and identification of metabolic changes that occur in different tissue types in specific diseases. However, the extraction of metabolites from tissue is often the most labor-intensive and error-prone step in metabolomics studies. Here, we report the development of a standardized high-throughput method for rapid and reproducible extraction of metabolites from multiple tissue samples from different organs of several species. The method involves a bead-based homogenizer in combination with a simple extraction protocol and is compatible with state-of-the-art metabolomics kit technology for quantitative and targeted flow injection tandem mass spectrometry. We analyzed different extraction solvents for both reproducibility as well as suppression effects for a range of different animal tissue types including liver, kidney, muscle, brain, and fat tissue from mouse and bovine. In this study, we show that for most metabolites a simple methanolic extraction is best suited for reliable results. An additional extraction step with phosphate buffer can be used to improve the extraction yields for a few more polar metabolites. We provide a verified tissue extraction setup to be used with different indications. Our results demonstrate that this high-throughput procedure provides a basis for metabolomic assays with a wide spectrum of metabolites. The developed method can be used for tissue extraction setup for different indications like studies of metabolic syndrome, obesity, diabetes or cardiovascular disorders and nutrient transformation in livestock.     

Metabolomic Analysis of Cerebrospinal Fluid Indicates Iron Deficiency Compromises Cerebral Energy Metabolism in the Infant Monkey

Iron deficiency anemia affects many pregnant women and young infants worldwide. The health impact is significant, given iron’s known role in many body functions, including oxidative and lipid metabolism, protein synthesis and brain neurochemistry. The following research determined if ¹H NMR spectroscopy-based metabolomic analysis of cerebrospinal fluid (CSF) could detect the adverse influence of early life iron deficiency on the central nervous system. Using a controlled dietary model in 43 infant primates, distinct differences were found in spectra acquired at 600 MHz from the CSF of anemic monkeys. Three metabolite ratios, citrate/pyruvate, citrate/lactate and pyruvate/glutamine ratios, differed significantly in the iron deficient infant and then normalized following the consumption of dietary iron and improvement of clinical indices of anemia in the heme compartment. This distinctive metabolomic profile associated with anemia in the young infant indicates that CSF can be employed to track the neurological effects of iron deficiency and benefits of iron supplementation.     

Comparative Metabolic Profiling of Two Contrasting Date Palm Genotypes Under Salinity

Since specific metabolites may be associated with salinity tolerance, this study aimed to decipher the salinity tolerance mechanism in date palm based on the information encoded by the metabolomic profiles of the salt-tolerant “Umsila” and salt-susceptible “Zabad” cultivars when grown under salinity conditions. Changes in the metabolomic profiles of the leaf and root tissues were determined using hydrophilic interaction liquid chromatography (HILIC) and reverse-phase liquid chromatography (RPLC) mass spectrometry. The global untargeted metabolomic analysis showed the presence of 4878 metabolites accumulated in leaf and root tissues of the date palm seedlings. Principal component analysis (PCA) revealed the presence of unique groups of metabolites for each treatment and tissue type. Pathway analysis showed the involvement of some of these metabolites in the biosynthesis of several types of membranous lipids and glycolipids molecules such as 18:0-lyso-phosphatidylethanolamine (lysoPE), and also the synthesis of cell-wall components such as 16-hydroxy hexadecanoic acid, which is an intermediate metabolite in cutin, suberin, and wax biosynthesis. Moreover, antioxidant flavonoids such as (+)-catechin and epicatechin, vitamins such as B₉, phytohormone-associated compounds such as dihydrozeatin-9-N-glucoside-O-glucoside, and osmolytes such as the sulfonic amino acid taurine were all significantly (p?≤?0.05, FWER?≤?0.05) altered in response to salinity in both cultivars. These results indicate that salinity tolerance in date palm involved a multi-dimensional mechanism, which includes the modification of the cell-wall and cellular membranes, the production of oxygen species scavengers, the adjustment of cellular osmotic pressure, and probably the alteration of the hormonal balance. The intracultivar metabolomic profile comparison strategy performed in this study represents an approach that may pave the road toward the identification of salinity tolerance mechanisms in date palm based on the final protein products.

     

Design and synthesis of brain penetrant selective JNK inhibitors with improved pharmacokinetic properties for the prevention of neurodegeneration

The SAR of a series of brain penetrant, trisubstituted thiophene based JNK inhibitors with improved pharmacokinetic properties is described. These compounds were designed based on information derived from metabolite identification studies which led to compounds such as 42 with lower clearance, greater brain exposure and longer half life compared to earlier analogs.     

Metabolomic profiling of heat stress: hardening and recovery of homeostasis in Drosophila

Frequent exposure of terrestrial insects to temperature variation has led to the evolution of protective biochemical and physiological mechanisms, such as the heat shock response, which markedly increases the tolerance to heat stress. Insight into such mechanisms has, so far, mainly relied on selective studies of specific compounds or characteristics or studies at the genomic or proteomic levels. In the present study, we have used untargeted NMR metabolomic profiling to examine the biological response to heat stress in Drosophila melanogaster. The metabolite profile was analyzed during recovery after exposure to different thermal stress treatments and compared with untreated controls. Both moderate and severe heat stress gave clear effects on the metabolite profiles. The profiles clearly demonstrated that hardening by moderate heat stress led to a faster reestablishment of metabolite homeostasis after subsequent heat stress. Several metabolites were identified as responsive to heat stress and could be related to known physiological and biochemical responses. The time course of the recovery of metabolite homeostasis mirrored general changes in gene expression, showing that recovery follows the same temporal pattern at these two biological levels. Finally, our data show that heat hardening permits a quicker return to homeostasis, rather than a reduction of the acute metabolic perturbation and that the reestablishment of homeostasis is important for obtaining maximal heat-hardening effect. The results display the power of NMR metabolomic profiling for characterization of the instantaneous physiological condition, enabling direct visualization of the perturbation of and return to homeostasis.     

An Optimized Method of Metabolite Extraction from Formalin-Fixed Paraffin-Embedded Tissue for GC/MS Analysis

Formalin-fixed paraffin-embedded (FFPE) tissue specimens constitute a highly valuable source of clinical material for retrospective molecular studies. However, metabolomic assessment of such archival material remains still in its infancy. Hence, there is an urgent need for efficient methods enabling extraction and profiling of metabolites present in FFPE tissue specimens. Here we demonstrate the methodology for isolation of primary metabolites from archival tissues; either fresh-frozen, formalin-fixed or formalin-fixed and paraffin-embedded specimens of mouse kidney were analysed and compared in this work. We used gas chromatography followed by mass spectrometry (GC/MS approach) to identify about 80 metabolites (including amino acids, saccharides, carboxylic acids, fatty acids) present in such archive material. Importantly, about 75% of identified compounds were detected in all three types of specimens. Moreover, we observed that fixation with formalin itself (and their duration) did not affect markedly the presence of particular metabolites in tissue-extracted material, yet fixation for 24h could be recommended as a practical standard. Paraffin embedding influenced efficiency of extraction, which resulted in reduced quantities of several compounds. Nevertheless, we proved applicability of FFPE specimens for non-targeted GS/MS-based profiling of tissue metabolome, which is of great importance for feasibility of metabolomics studies using retrospective clinical material.     

Development of an NMR metabolomics-based tool for selection of flaxseed varieties

Flaxseed is an important source of lignans and ω-3 fatty acids, compounds which present interest in human health with many applications in food industry. It is therefore necessary to precisely know the metabolite content in flaxseed. A metabolomic approach using NMR was developed to achieve this goal. Due to particular characteristics of flaxseed (high level in oil, high amount in mucilage, and integration of the phenolics into a macromolecule), the extraction procedure had first to be optimized using an experimental design, based on the extraction time, in a water bath or an ultrasound bath, alkaline treatment, defatting, and centrifugation temperature. This methodology was then applied to several flaxseed varieties classified in function of their content in ω-3 fatty acid. The main differences in semi-polar metabolites between these varieties concern compounds of the phenylpropanoid pathway. Hydroxycinnamic acid glucoside and lignan content increase when ω-3 fatty acid content decrease whereas flavonoid content increase in the same way of ω-3 fatty acids.     

Human Breath Analysis May Support the Existence of Individual Metabolic Phenotypes

The metabolic phenotype varies widely due to external factors such as diet and gut microbiome composition, among others. Despite these temporal fluctuations, urine metabolite profiling studies have suggested that there are highly individual phenotypes that persist over extended periods of time. This hypothesis was tested by analyzing the exhaled breath of a group of subjects during nine days by mass spectrometry. Consistent with previous metabolomic studies based on urine, we conclude that individual signatures of breath composition exist. The confirmation of the existence of stable and specific breathprints may contribute to strengthen the inclusion of breath as a biofluid of choice in metabolomic studies. In addition, the fact that the method is rapid and totally non-invasive, yet individualized profiles can be tracked, makes it an appealing approach.     

Two strings to the systems biology bow: co-extracting the metabolome and proteome of yeast

Experimental samples are valuable and can represent a significant investment in time and resources. It is highly desirable at times to obtain as much information as possible from a single sample. This is especially relevant for systems biology approaches in which several ‘omics platforms are studied simultaneously. Unfortunately, each platform has a particular extraction methodology which increases sample number and sample volume requirements when multiple ‘omics are analyzed. We evaluated the integration of a yeast extraction method; specifically we explored whether fractions from a single metabolite extraction could be apportioned to multiple downstream ‘omics analytical platforms. In addition, we examined how variations to a chloroform/methanol yeast metabolite extraction regime influence metabolite recoveries. We show that protein suitable for proteomic analysis can be recovered from a metabolite extraction and that recovery of lipids, while reproducible, are not wholly quantitative. Higher quenching solution temperatures (?30 °C) can be used without significant leakage of intracellular metabolites when lower fermentation temperatures (20 °C) are employed. However, extended residence time in quenching solution, in combination with vigorous washing of quenched cell pellets, leads to extensive leakage of intracellular metabolites. Finally, there is minimal difference in metabolite amounts obtained when metabolite extractions are performed at 4 °C compared to extractions at ?20 °C. The evaluated extraction method delivers material suitable for metabolomic and proteomic analyses from the same sample preparation.     

A novel approach for nontargeted data analysis for metabolomics. Large-scale profiling of tomato fruit volatiles

To take full advantage of the power of functional genomics technologies and in particular those for metabolomics, both the analytical approach and the strategy chosen for data analysis need to be as unbiased and comprehensive as possible. Existing approaches to analyze metabolomic data still do not allow a fast and unbiased comparative analysis of the metabolic composition of the hundreds of genotypes that are often the target of modern investigations. We have now developed a novel strategy to analyze such metabolomic data. This approach consists of (1) full mass spectral alignment of gas chromatography (GC)-mass spectrometry (MS) metabolic profiles using the MetAlign software package, (2) followed by multivariate comparative analysis of metabolic phenotypes at the level of individual molecular fragments, and (3) multivariate mass spectral reconstruction, a method allowing metabolite discrimination, recognition, and identification. This approach has allowed a fast and unbiased comparative multivariate analysis of the volatile metabolite composition of ripe fruits of 94 tomato (Lycopersicon esculentum Mill.) genotypes, based on intensity patterns of >20,000 individual molecular fragments throughout 198 GC-MS datasets. Variation in metabolite composition, both between- and within-fruit types, was found and the discriminative metabolites were revealed. In the entire genotype set, a total of 322 different compounds could be distinguished using multivariate mass spectral reconstruction. A hierarchical cluster analysis of these metabolites resulted in clustering of structurally related metabolites derived from the same biochemical precursors. The approach chosen will further enhance the comprehensiveness of GC-MS-based metabolomics approaches and will therefore prove a useful addition to nontargeted functional genomics research.