Quantitative Metabolome Profiling Research Articles

P-158 Quantitative metabolomic profiling of follicular fluid identifies oocyte quality related biomarkers from infertile patients

Abstract Study question Is whether there is a metabolomic signature in the follicular fluid depending on oocyte quality and whether infertility may impact on this quality? Summary answer 46 metabolites were quantified via nuclear magnetic resonance spectroscopy in the follicular fluid, and each metabolite that was a biomarker of oocyte quality was pathology-specific. What is known already Literature confirmed that metabolomics had been valuable for exploring follicular fluid and the search for nutritive and non-nutritive biomarkers. There are currently no data in the literature concerning the homeostasis of this follicular fluid, and two options are possible: this fluid is a continuum of blood plasma, or it has its own homeostasis. Studies have highlighted follicular fluid qualitative and quantitative alterations in the metabolome due to one specific infertility pathology footprint and correlated in some instances to outcome measures of in vitro fertilization success but samples are pooled and there are no information concerning quality and competence oocyte assessment. Study design, size, duration 61 infertile patients who underwent in vitro fertilization (IVF) treatment with intracytoplasmic sperm injection (ICSI) from Femme Mère Enfant hospital (Bron, France) between 6th april 2022 and 12th may 2022 were included. Participants/materials, setting, methods 61 follicular fluids were collected individually and used for metabolomic profiling by nuclear magnetic resonance spectroscopy. A total of 46 metabolites were quantified. Metabolite concentrations in follicular fluid were compared with serum concentrations.Oocyte was considered mature if it has its first polar body and competent if it was able to develop into a blastocyst after culturing. In vitro fertilized oocytes were cultured and monitored with time-lapses incubation technology until day 5 or 6. Main results and the role of chance For 23 metabolites out of 46, follicular fluid exhibited lower concentrations than serum. Glucose and glutamine, which are among the most abundant metabolites, decreased by 54% (not significant) and 47% (P < 0.05) respectively. 7 metabolites exhibited higher concentrations in the follicular fluid than in the serum. An 85%-increase in lactate and a more-than-tenfold increase in pyruvate were noted (P < 0.05). The reverse ratios of lower substrates such as glucose and glutamine and higher waste products like lactate and pyruvate imply that the follicular fluid functions as a homeostatic compartment, being more influenced by exchange within follicular cells than by serum equilibration. The detected metabolic signatures of oocyte maturity and competence were all specific to the patient infertility-related pathology. The follicular fluid concentrations of aspartate, glucose, methionine and methylmalonate were significantly correlated to oocyte maturity in the control group (P < 0.05). For patients having endometriosis, asparagine concentration was significantly decreased by oocyte maturity (P < 0.05). For patients with diminished ovarian reserve, acetone concentration was significantly decreased by oocyte maturity (P < 0.05). The concentration of aspartate, that correlated with oocyte maturity, was also slightly decreased by oocyte competence, and for the patients with endometriosis the concentration of threonine was decreased by 30% by oocyte competence (P < 0.05). Limitations, reasons for caution Although 46 metabolites were well quantified, each metabolite that was a biomarker of oocyte maturity or competence was pathology specific. One major reason would be the high metabolite concentration variation observed between the different infertilities. This study indicates that the diagnosis of patients according to their infertility must be prior. Wider implications of the findings This study underlines the potential of follicular fluid metabolomics as a diagnostic avenue for oocyte-related becoming in order to enhance the selection of oocytes and improve outcomes in assisted reproductive and preservation fertility techniques. Trial registration number not applicable

Quantitative Metabolomic Analysis of the Rat Hippocampus: Effects of Age and of the Development of Alzheimer's Disease-Like Pathology.

Alzheimer's disease (AD) is the most common type of dementia in the elderly. Incomplete knowledge about the pathogenesis of this disease determines the absence of medications for the treatment of AD today. Animal models can provide the necessary knowledge to understand the mechanisms of biochemical processes occurring in the body in health and disease. To identify the most promising metabolomic predictors and biomarkers reflecting metabolic disorders in the development of AD signs. High resolution 1H NMR spectroscopy was used for quantitative metabolomic profiling of the hippocampus of OXYS rats, an animal model of sporadic AD, which demonstrates key characteristics of this disease. Animals were examined during several key periods: 20 days group corresponds to the "preclinical" period preceding the development of AD signs, during their manifestation (3 months), and active progression (18 months). Wistar rats of the same age were used as control. Ranges of variation and mean concentrations were established for 59 brain metabolites. The main metabolic patterns during aging, which are involved in energy metabolism pathways and metabolic shifts of neurotransmitters, have been established. Of particular note is the significant increase of scyllo-inositol and decrease of hypotaurine in the hippocampus of OXYS rats as compared to Wistars for all studied age groups. We suggest that the accumulation of scyllo-inositol and the reduction of hypotaurine in the brain, even at an early age, can be considered as predictors and potential biomarkers of the development of AD signs in OXYS rats and, probably, in humans.

Quantitative proteomic and metabolomic profiling reveals different osmoregulation mechanisms of tilapia cells coping with different hyperosmotic stress

This study aimed to investigate the different regulatory mechanisms of euryhaline fish under regular hyperosmotic and extreme hyperosmotic stress. The OmB (Oreochromis mossambicus brain) cells were exposed to three treatments: control, regular hyperosmotic stress and extreme hyperosmotic stress. After 12 h exposure, proteomics, metabolomics analyses and integrative analyses were explored.Both kinds of stress lead to lowering cell growth and morphology changes, while under regular hyperosmotic stress, the up-regulated processes related with compatible organic osmolytes synthesis are crucial strategy for the euryhaline fish cell line to survive; On the other hand, under extreme hyperosmotic stress, the processes related with cell apoptosis and cell cycle arrest are dominant. Furthermore, down-regulated pyrimidine metabolism and several ribosomal proteins partially participated in the lowered cell metabolism and increased cell death under both kinds of hyperosmotic stress. The PI3K-Akt and p53 signaling pathways were involved in the stagnant stage of cell cycles and induction of cell apoptosis under both kinds of hyperosmotic stress. However, HIF-1, FoxO, JAK-STAT and Hippo signaling pathways mainly contribute to disrupting the cell cycle, metabolism and induction of cell apoptosis under extreme hyperosmotic stress. SignificanceIn the past, the research on fish osmoregulation mainly focused on the transcription factors and ion transporters of osmoregulation, the processes between osmotic sensing and signal transduction, and the associations between signaling pathways and regulation processes have been poorly understood.Investigating fish cell osmoregulation and potential signal transduction pathways is necessary. With the advancements in omics research, it is now feasible to investigate the relationship between environmental stress and molecular responses.In this study, we aimed to explore the signaling pathways and substance metabolism mode during hyper-osmoregulation in OmB cell line, to reveal the key factors that are critical to cell osmoregulation.

A systematic evaluation of quenching and extraction procedures for quantitative metabolome profiling of HeLa carcinoma cell under 2D and 3D cell culture conditions.

Metabolic reprogramming has been coined as a hallmark of cancer, accompanied by which the alterations in metabolite levels have profound effects on gene expression, cellular differentiation, and the tumor environment. Yet a systematic evaluation of quenching and extraction procedures for quantitative metabolome profiling of tumor cells is currently lacking. To achieve this, this study is aimed at establishing an unbiased and leakage-free metabolome preparation protocol for HeLa carcinoma cell. We evaluated 12 combinations of quenching and extraction methods from three quenchers (liquid nitrogen, -40°C 50% methanol, 0.5°C normal saline) and four extractants (-80°C 80% methanol, 0.5°C methanol/chloroform/water [1:1:1 v/v/v], 0.5°C 50% acetonitrile, 75°C 70% ethanol) for global metabolite profiling of adherent HeLa carcinoma cells. Based on the isotope dilution mass spectrometry (IDMS) method, gas/liquid chromatography in tandem with mass spectrometry was used to quantitatively determine 43 metabolites including sugar phosphates, organic acids, amino acids (AAs), adenosine nucleotides, and coenzymes involved in central carbon metabolism. The results showed that the total amount of the intracellular metabolites in cell extracts obtained using different sample preparation procedures with the IDMS method ranged from 21.51 to 295.33nmol per million cells. Among 12 combinations, cells that washed twice with phosphate buffered saline (PBS), quenched with liquid nitrogen, and then extracted with 50% acetonitrile were found to be the most optimal method to acquire intracellular metabolites with high efficiency of metabolic arrest and minimal loss during sample preparation. In addition, the same conclusion was drawn as these 12 combinations were applied to obtain quantitative metabolome data from three-dimensional (3D) tumor spheroids. Furthermore, a case study was carried out to evaluate the effect of doxorubicin (DOX) on both adherent cells and 3D tumor spheroids using quantitative metabolite profiling. Pathway enrichment analysis using targeted metabolomics data showed that DOX exposure would significantly affect AA metabolism-related pathways, which might be related to the mitigation of redox stress. Strikingly, our data suggested that compared to two-dimensional (2D) cells the increased intracellular glutamine level in 3D cells benefited replenishing the tricarboxylic acid (TCA) cycle when the glycolysis was limited after dosing with DOX. Taken together, this study provides a well-established quenching and extraction protocol for quantitative metabolome profiling of HeLa carcinoma cell under 2D and 3D cell culture conditions. Based on this, quantitative time-resolved metabolite data can serve to the generation of hypotheses on metabolic reprogramming to reveal its important role in tumor development and treatment.

Quantitative structure-retention relationship for reliable metabolite identification and quantification in metabolomics using ion-pair reversed-phase chromatography coupled with tandem mass spectrometry

Hydrophilic metabolites are essential for all biological systems with multiple functions and their quantitative analysis forms an important part of metabolomics. However, poor retention of these metabolites on reversed-phase (RP) chromatographic column hinders their effective analysis with RPLC-MS methods. Herein, we developed a method for detecting hydrophilic metabolites using the ion-pair reversed-phase liquid-chromatography coupled with mass spectrometry (IPRP-LC-MS/MS) in scheduled multiple-reaction-monitoring (sMRM) mode. We first developed a hexylamine-based IPRP-UHPLC-QTOFMS method and experimentally measured retention time (tR) for 183 hydrophilic metabolites. We found that tRs of these metabolites were dominated by their electrostatic potential depending upon the numbers and types of their ionizable groups. We then systematically investigated the quantitative structure-retention relationship (QSRR) and constructed QSRR models using the measured tR. Subsequently, we developed a retention time predictive model using the random-forest regression algorithm (r2 = 0.93, q2 = 0.70, MAE = 1.28 min) for predicting metabolite retention time, which was applied in IPRP-UHPLC-MS/MS method in sMRM mode for quantitative metabolomic analysis. Our method can simultaneously quantify more than 260 metabolites. Moreover, we found that this method was applicable for multiple major biological matrices including biofluids and tissues. This approach offers an efficient method for large-scale quantitative hydrophilic metabolomic profiling even when metabolite standards are unavailable.

Altered amino acid metabolism between coronary heart disease patients with and without type 2 diabetes by quantitative 1H NMR based metabolomics

Coronary heart disease (CHD) is a multifactorial disease associated with complicated altered metabolic pathways, especially when it occurs with type 2 diabetes (T2D). Identification of metabolic changes could provide further understanding of the underlying pathological link between with CHD and T2D. In this study, 61 controls, 30 consecutive patients with only T2D, 188 with confirmed CHD among which 50 also having T2D were enrolled. Quantitative 1H nuclear magnetic resonance (1H NMR) based metabonomic approach was used to obtain the plasma metabolic profiles of the study samples. Multivariate principal component analysis and orthogonal partial least-squares discriminant analysis (OPLS-DA) were used to evaluate the differences of metabolites. Compared with controls, there were significant differences in CHD patient with T2D groups including BCAAs (isoleucine, leucine, and valine) and phenylalanine by using quantitative metabolomic profiling (all p < 0.05). With OPLS-DA, the results showed that, compared to CHD patients without T2D, there had higher levels of metabolites for leucine, valine, fumarate, tyrosine, and phenylalanine in the plasma of CHD patients with T2D. Moreover, the CHD patients with T2D showed modified phenylalanine, tyrosine and tryptophan biosynthesis, valine, leucine and isoleucine biosynthesis and degradation compared with CHD patients without T2D. By 1H NMR based quantitative metabolomic method, our findings suggest that T2D has important effects on BCAAs and AAAs metabolism in CHD patients indicating altered cardiac energy metabolism and activated signaling pathway could be potential targets for therapy in CHD disease combined with T2D.

Quantitative Proteomic and Metabolomic Profiling Reveals Altered Mitochondrial Metabolism and Folate Biosynthesis Pathways in the Aging Drosophila Eye

Aging is associated with increased risk of ocular disease, suggesting that age-associated molecular changes in the eye increase its vulnerability to damage. Although there are common pathways involved in aging at an organismal level, different tissues and cell types exhibit specific changes in gene expression with advanced age. Drosophila melanogaster is an established model system for studying aging and neurodegenerative disease that also provides a valuable model for studying age-associated ocular disease. Flies, like humans, exhibit decreased visual function and increased risk of retinal degeneration with age. Here, we profiled the aging proteome and metabolome of the Drosophila eye and compared these data with age-associated transcriptomic changes from both eyes and photoreceptors to identify alterations in pathways that could lead to age-related phenotypes in the eye. Of note, the proteomic and metabolomic changes observed in the aging eye are distinct from those observed in the head or whole fly, suggesting that tissue-specific changes in protein abundance and metabolism occur in the aging fly. Our integration of the proteomic, metabolomic, and transcriptomic data reveals that changes in metabolism, potentially due to decreases in availability of B vitamins, together with chronic activation of the immune response, may underpin many of the events observed in the aging Drosophila eye. We propose that targeting these pathways in the genetically tractable Drosophila system may help to identify potential neuroprotective approaches for neurodegenerative and age-related ocular diseases. Data are available via ProteomeXchange with identifier PXD027090.

Integrated Quantitative Proteomics and Metabolome Profiling Reveal MSMEG_6171 Overexpression Perturbing Lipid Metabolism of Mycobacterium smegmatis Leading to Increased Vancomycin Resistance.

In recent years, the treatment of tuberculosis is once again facing a severe situation because the existing antituberculosis drugs have become weaker and weaker with the emergence of drug-resistant Mycobacterium tuberculosis (Mtb). The studies of cell division and cell cycle-related factors in Mtb are particularly important for the development of new drugs with broad-spectrum effects. Mycobacterium smegmatis (Msm) has been used as a model organism to study the molecular, physiological, and drug-resistant mechanisms of Mtb. Bioinformatics analysis has predicted that MSMEG_6171 is a MinD-like protein of the septum site-determining protein family associated with cell division in Mycobacterium smegmatis. In our study, we use ultrastructural analysis, proteomics, metabolomics, and molecular biology techniques to comprehensively investigate the function of MSMEG_6171. Overexpression of MSMEG_6171 in Msm resulted in elongated cells, suggesting an important role of MSMEG_6171 in regulating cell wall morphology. The MSMEG_6171 overexpression could enhance the bacterial resistance to vancomycin, ethionamide, meropenem, and cefamandole. The MSMEG_6171 overexpression could alter the lipid metabolism of Msm to cause the changes on cellular biofilm property and function, which enhances bacterial resistance to antibiotics targeting cell wall synthesis. MSMEG_6171 could also induce the glyceride and phospholipid alteration in vivo to exhibit the pleiotropic phenotypes and various cellular responses. The results showed that amino acid R249 in MSMEG_6171 was a key site that can affect the level of bacterial drug resistance, suggesting that ATPase activity is required for function.

Post-mortem changes in metabolomic profiles of human serum, aqueous humor and vitreous humor.

Application of metabolomic methods to forensic studies may expand the limits of the post-mortem interval (PMI) estimation, and improve the accuracy of the estimation. To this end, it is important to determine which tissue is the most suitable for analysis, and which compounds are the most promising candidates for PMI estimation. This work is aimed at the comparison of human serum, aqueous humor (AH), and vitreous humor (VH) as perspective tissues for metabolomic-based PMI estimation, at the determination of most promising PMI biomarkers, and at the development of method of PMI estimation based on the measurement of concentrations of PMI biomarkers. Quantitative metabolomic profiling of samples of the human serum, AH, and VH taken at different PMIs has been performed with the use of NMR spectroscopy. It is found that the metabolomic changes in anatomically isolated ocular fluids are slower and smoother than that in blood. A good positive time correlation (Pearson coefficient r > 0.5) was observed for several metabolites, including hypoxanthine, choline, creatine, betaine, glutamate, and glycine. A model for PMI estimation based on concentrations of several metabolites in AH and VH is proposed. The obtained results demonstrate that the metabolomic analysis of AH and VH is more suitable for the PMI estimation than that of serum. The compounds with good positive time correlation can be considered as potential PMI biomarkers.

The Good, the Bad, and the Lethal: Gene Expression and Metabolomics Reveal Physiological Mechanisms Underlying Chronic Thermal Effects in Mayfly Larvae (Neocloeon triangulifer)

Temperature dictates the performance of aquatic ectotherms. However, the physiological and biochemical processes that drive thermally-mediated life history patterns (and limits) remain poorly understood because they are rarely studied simultaneously. In our previous work, we have established life history outcomes (e.g. survivorship, development time, growth rates and fitness) in mayflies (Neocloeon triangulifer) reared at static temperatures ranging from 14°C - 30°C at 2°C intervals. In this study, we conducted biochemical measurements (RT-qPCR of select genes and targeted, quantitative metabolomic profiling) on N. triangulifer mature larvae reared at temperatures associated with excellent survival and fitness (22-24°C), compromised survival and fitness (28°C), and chronic lethality (30°C -larvae survived for a few weeks but failed to emerge to adulthood). Patterns of gene expression were similar to those observed in acute ramping experiments reported previously: larvae reared at 30°C resulted in significant upregulation in the thermally responsive gene HEAT SHOCK PROTEIN 90 (HSP90) but no significant changes in hypoxia responsive genes (EGG LAYING DEFECTIVE 9 (EGL-9) and LACTATE DEHYDROGENASE (LDH)). Additionally, primers for genes associated with energy: INSULIN RECEPTOR (IR), mechanistic TARGET OF RAPAMYCIN (mTOR) and TREHALOSE 6 PHOSPHATE SYNTHASE (T6PS) were developed for this study. IR and mTOR were significantly upregulated while T6PS showed trend of downregulation in larvae reared at 30°C. Metabolomic profiles revealed general depletion of lipids and acylcarnitines in larvae exposed to chronic thermal stress, suggesting that larvae were energetically challenged despite continuous access to food. For example, concentrations of lysoPhosphatidylcholine (lysoPC) a C20:3 decreased as fitness decreased with increasing temperature (2.3 fold and 2.4 fold at 28 and 30°C relative to controls). Tissue concentrations of the biogenic amine histamine increased 2.1 and 3.1 fold with increasing temperature, and were strongly and negatively correlated with performance. Thus, both histamine and lysoPC a C20:3 are potential biomarkers of thermal stress. Taken together, our results primarily associate energetic challenge with thermally mediated fitness reduction in N. triangulifer.

Serially coupled reversed phase-hydrophilic interaction liquid chromatography–tailored multiple reaction monitoring, a fit-for-purpose tool for large-scale targeted metabolomics of medicinal bile

The acquisition of high-quality quantitative dataset is the prerequisite for large-scale targeted metabolomics. However, the goal is usually dampened by the complexity of the biological matrices in terms of structural diversity, concentration span, and polarity range. We attempted herein to propose an analytical approach being able to circumvent these technical obstacles, and medicinal bile was employed as a proof of concept. In the liquid chromatography (LC) domain, reversed phase LC and hydrophilic liquid interaction chromatography were coupled in series, namely RPLC-HILIC, to yield appropriate chromatographic behavior for each component regardless of the polarity. In-depth chemical characterization and widely quantitative analysis were successively conducted in the mass spectrometry (MS) domain. Hybrid ion trap-time of flight MS was jointly deployed with hybrid triple quadrupole-linear ion trap MS for metabolite searching because of their orthogonal natures. Afterwards, a tailored MRM strategy that integrated online parameter optimization, ion intensity tailoring, and scheduled monitoring, was subsequently constructed to accomplish comprehensive quantitation although authentic compounds weren't involved and concentration span was quite large. Calibration curve was constructed for each detected identity by preparing and serially diluting the universal metabolome standard (UMS) solution that merged chemical information from all bile samples. Quasi-contents of 164 components including bile acids, sterols, eicosanoids, amino acids, lipids, flavonoids, and so forth, were gained by applying those regressive calibration curves and replaced the role of peak areas to serve as the variables for multivariate statistical analysis. In particular, the concentration ratio between tauroursodeoxycholic acid (TUDCA) and taurohyodeoxycholic acid (THDCA) that were exactly co-eluted as a single peak was calculated from the intensity specific value of m/z 498 > 80 over 498 > 124. Different metabolome occurred among various animal bile samples, and significant variations were also observed for different batches of bear bile powders. Bile acids acted as the indicative components in either discrimination. Overall, RPLC-HILIC–tailored MRM enabled quantitative metabolome profiling of medicinal bile and was thereby a promising analytical tool for large-scale targeted metabolomics.

Overcoming Sample Matrix Effect in Quantitative Blood Metabolomics Using Chemical Isotope Labeling Liquid Chromatography Mass Spectrometry.

Blood is widely used for discovery metabolomics to search for disease biomarkers. However, blood sample matrix can have a profound effect on metabolome analysis, which can impose an undesirable restriction on the type of blood collection tubes that can be used for blood metabolomics. We investigated the effect of blood sample matrix on metabolome analysis using a high-coverage and quantitative metabolome profiling technique based on differential chemical isotope labeling (CIL) LC-MS. We used 12C-/13C-dansylation LC-MS to perform relative quantification of the amine/phenol submetabolomes of four types of samples (i.e., serum, EDTA plasma, heparin plasma, and citrate plasma) collected from healthy individuals and compare their metabolomic results. From the analysis of 80 plasma and serum samples in experimental triplicate, we detected a total of 3651 metabolites with an average of 1818 metabolites per run (n = 240). The number of metabolites detected and the precision and accuracy of relative quantification were found to be independent of the sample type. Within each sample type, the metabolome data set could reveal biological variation (e.g., sex separation). Although the relative concentrations of some individual metabolites might be different in the four types of samples, for sex separation, all 66 significant metabolites with larger fold-changes (FC ≥ 2 and p < 0.05) found in at least one sample type could be found in the other types of samples with similar or somewhat reduced, but still significant, fold-changes. Our results indicate that CIL LC-MS could overcome the sample matrix effect, thereby greatly broadening the scope of blood metabolomics; any blood samples properly collected in routine clinical settings, including those in biobanks originally used for other purposes, can potentially be used for discovery metabolomics.

Metabolites Linked to Bariatric Surgery Prolong Allograft Survival in Obese Recipients

Introduction: In transplantation, obesity has become a common co-morbidity linked to inferior transplant outcome through far-reaching effects on the complex network of metabolism, inflammation and immunity. Methods: Fully mismatched skin transplantations were performed in diet-induced obese (DIO) C57/Bl6 mice and lean littermates. Sleeve gastrectomies (SGx) were performed prior to transplantation to induce weight loss. Results: Obese graft recipients showed significantly accelerated allograft rejection when compared to lean littermates (p < 0.001), associated with increased IFN-γ expression in splenic CD4+ T cells. Bariatric surgery, on the other hand, prolonged allograft survival even beyond that of lean animals (p = 0.005) and promoted protective IL-10-skewed conditions with reduced alloreactivity as confirmed by ELISPOT. Of note, sham procedures and dietary weight loss showed inferior results in allograft survival and weight loss when compared to bariatric surgery. Quantitative metabolomic profiling identified a secondary bile acid and a proteinogenic amino acid as upregulated subsequent to bariatric surgery. Strikingly, combined application reduced obesity and resulted in significant prolongation of graft survival (p = 0.005; Fig.1). Metabolic treatment was also associated with reduced pro-inflammatory responses in splenic CD4+ T cells and a significant reduction in splenic M1-like antigen-presenting cells (Fig. 2). Mechanistically, metabolic treatment reduced M1-polarization of monocytes/macrophages through activation of the bile acid receptor TGR5. Activation of TGR5 through a specific agonist blocked M1-polarization in vitro and prolonged allograft survival to levels comparable to metabolic treatment, while disruption of intracellular signaling of TGR5 during metabolic treatment abrogated these effects. Of note, studies in metabolic cages demonstrated a reduced oral intake of treated obese animals while maintaining regular physical activity, indicating that weight loss was not induced through toxic side-effects of metabolic treatment. Conclusion: Obesity augments alloimmune responses resulting in accelerated graft rejection, while bariatric surgery induced a change in cytokine balances with transplant outcome superior even to lean controls. Two metabolic compounds were identified as key mediators skewing the interplay of metabolism and inflammation towards anti-inflammatory conditions, providing unique insights linking obesity, metabolism and immunity.FigureFigure

Integrated work-flow for quantitative metabolome profiling of plants, Peucedani Radix as a case

Universal acquisition of reliable information regarding the qualitative and quantitative properties of complicated matrices is the premise for the success of metabolomics study. Liquid chromatography-mass spectrometry (LC-MS) is now serving as a workhorse for metabolomics; however, LC-MS-based non-targeted metabolomics is suffering from some shortcomings, even some cutting-edge techniques have been introduced. Aiming to tackle, to some extent, the drawbacks of the conventional approaches, such as redundant information, detector saturation, low sensitivity, and inconstant signal number among different runs, herein, a novel and flexible work-flow consisting of three progressive steps was proposed to profile in depth the quantitative metabolome of plants. The roots of Peucedanum praeruptorum Dunn (Peucedani Radix, PR) that are rich in various coumarin isomers, were employed as a case study to verify the applicability. First, offline two dimensional LC-MS was utilized for in-depth detection of metabolites in a pooled PR extract namely universal metabolome standard (UMS). Second, mass fragmentation rules, notably concerning angular-type pyranocoumarins that are the primary chemical homologues in PR, and available databases were integrated for signal assignment and structural annotation. Third, optimum collision energy (OCE) as well as ion transition for multiple monitoring reaction measurement was online optimized with a reference compound-free strategy for each annotated component and large-scale relative quantification of all annotated components was accomplished by plotting calibration curves via serially diluting UMS. It is worthwhile to highlight that the potential of OCE for isomer discrimination was described and the linearity ranges of those primary ingredients were extended by suppressing their responses. The integrated workflow is expected to be qualified as a promising pipeline to clarify the quantitative metabolome of plants because it could not only holistically provide qualitative information, but also straightforwardly generate accurate quantitative dataset.

Metabolomics of the human aqueous humor

The optical elements of the eye—cornea, lens, and vitreous humor—are avascular tissues, and their nutrition and waste removal are provided by aqueous humor (AH). The AH production occurs through the active secretion and the passive diffusion/ultrafiltration of blood plasma. The comparison of the metabolomic profiles of AH and plasma is important for understanding of the mechanisms of biochemical processes and metabolite transport taking place in vivo in ocular tissues. The work is aimed at the determination of concentrations of a wide range of most abundant metabolites in the human AH, the comparison of the metabolomic profiles of AH and serum, and the analysis of the post-mortem metabolomic changes in these two biological fluids. The quantitative metabolomic profiling was carried out with the use of two independent methods—high-frequency 1H NMR spectroscopy and HPLC with high-resolution ESI-MS detection. The concentrations of 71 most abundant metabolites in blood serum and AH from living patients and human cadavers have been measured. It has been found that the level of ascorbate in AH is by two orders of magnitude higher than that in serum; the levels of other metabolites are either similar to that in serum, or differ from that by a factor of 2–5. The post-mortem metabolomic composition of both serum and AH undergoes rapid and strong changes. The differences between the metabolomic profiles of AH and serum for majority of metabolites can be attributed to the metabolic activity of the ocular tissues leading to the lack or excess of some metabolites, while the high concentration of ascorbate in AH demonstrates the activity of ascorbate-specific pumps at the blood-aqueous border. The post-mortem metabolomic changes are caused by the disruption of the major biochemical cycles and cell lysis. These changes should be taken into account in the analysis of disease-induced changes in post-mortem samples of the ocular tissues.

Quantitative Metabolomic Profiling of Plasma, Urine, and Liver Extracts by 1H NMR Spectroscopy Characterizes Different Stages of Atherosclerosis in Hamsters.

Atherosclerosis (AS) is a progressive disease that contributes to cardiovascular disease and shows a complex etiology, including genetic and environmental factors. To understand systemic metabolic changes and to identify potential biomarkers correlated with the occurrence and perpetuation of diet-induced AS, we applied 1H NMR-based metabolomics to detect the time-related metabolic profiles of plasma, urine, and liver extracts from male hamsters fed a high fat and high cholesterol (HFHC) diet. Conventional biochemical assays and histopathological examinations as well as protein expression analyses were performed to provide complementary information. We found that diet treatment caused obvious aortic lesions, lipid accumulation, and inflammatory infiltration in hamsters. Downregulation of proteins related to cholesterol metabolism, including hepatic SREBP2, LDL-R, CYP7A1, SR-BI, HMGCR, LCAT, and SOAT1 was detected, which elucidated the perturbation of cholesterol homeostasis during the HFHC diet challenge. Using "targeted analysis", we quantified 40 plasma, 80 urine, and 60 liver hydrophilic extract metabolites. Multivariate analyses of the identified metabolites elucidated sophisticated metabolic disturbances in multiple matrices, including energy homeostasis, intestinal microbiota functions, inflammation, and oxidative stress coupled with the metabolisms of cholesterol, fatty acids, saccharides, choline, amino acids, and nucleotides. For the first time, our results demonstrate a time-dependent metabolic progression of multiple biological matrices in hamsters from physiological status to early AS and further to late-stage AS, demonstrating that 1H NMR-based metabolomics is a reliable tool for early diagnosis and monitoring of the process of AS.

Quantitative metabolome profiling reveals the involvement of the kynurenine pathway in influenza-associated encephalopathy

Influenza-associated encephalopathy is a serious complication of influenza and is the most common form of acute encephalitis/encephalopathy in Japan. The number of reports from other countries is increasing, reflecting international recognition and concern. Identification of a specific biomarker could provide important clues about the pathophysiology of influenza-associated encephalopathy. During the 2009–2011 flu seasons, 34 pediatric patients hospitalized with influenza complications, including influenza-associated encephalopathy, were enrolled in the study. Serum samples were collected during the acute and convalescent phases of disease. Patients were classified into encephalopathy (n = 12) and non-encephalopathy (n = 22) groups. Serum metabolites were identified and quantified by capillary electrophoresis coupled with time-of-flight mass spectrometry. Quantified data were evaluated for comparative analysis. Subsequently, a total of 55 patients with or without encephalopathy were enrolled for absolute quantification of serum kynurenine and quinolinic acid. Based on m/z values and migration times, 136 metabolites were identified in serum samples. During the acute phase of disease, three metabolites (succinic acid, undecanoic acid, and kynurenine) were significantly higher, and two other metabolites (decanoic acid and cystine) were significantly lower, in the encephalopathy group compared to the non-encephalopathy group (p = 0.012, 0.022, 0.044, 0.038, 0.046, respectively). In a larger patient group, serum kynurenine and its downstream product in tryptophan metabolism, quinolinic acid, a known neurotoxin, were significantly higher in the encephalopathy than the non-encephalopathy without febrile seizure group. Comprehensive metabolite profiles revealed five metabolites as potential biomarkers for influenza-associated encephalopathy; the tryptophan–kynurenine metabolic process could be associated with its pathophysiology.

The Failing Heart Relies on Ketone Bodies as a Fuel.

Significant evidence indicates that the failing heart is energy starved. During the development of heart failure, the capacity of the heart to utilize fatty acids, the chief fuel, is diminished. Identification of alternate pathways for myocardial fuel oxidation could unveil novel strategies to treat heart failure. Quantitative mitochondrial proteomics was used to identify energy metabolic derangements that occur during the development of cardiac hypertrophy and heart failure in well-defined mouse models. As expected, the amounts of proteins involved in fatty acid utilization were downregulated in myocardial samples from the failing heart. Conversely, expression of β-hydroxybutyrate dehydrogenase 1, a key enzyme in the ketone oxidation pathway, was increased in the heart failure samples. Studies of relative oxidation in an isolated heart preparation using ex vivo nuclear magnetic resonance combined with targeted quantitative myocardial metabolomic profiling using mass spectrometry revealed that the hypertrophied and failing heart shifts to oxidizing ketone bodies as a fuel source in the context of reduced capacity to oxidize fatty acids. Distinct myocardial metabolomic signatures of ketone oxidation were identified. These results indicate that the hypertrophied and failing heart shifts to ketone bodies as a significant fuel source for oxidative ATP production. Specific metabolite biosignatures of in vivo cardiac ketone utilization were identified. Future studies aimed at determining whether this fuel shift is adaptive or maladaptive could unveil new therapeutic strategies for heart failure.

Comprehensive metabolome analyses reveal N-acetylcysteine-responsive accumulation of kynurenine in systemic lupus erythematosus: implications for activation of the mechanistic target of rapamycin.

Systemic lupus erythematosus (SLE) patients exhibit depletion of the intracellular antioxidant glutathione and downstream activation of the metabolic sensor, mechanistic target of rapamycin (mTOR). Since reversal of glutathione depletion by the amino acid precursor, N-acetylcysteine (NAC), is therapeutic in SLE, its mechanism of impact on the metabolome was examined within the context of a double-blind placebo-controlled trial. Quantitative metabolome profiling of peripheral blood lymphocytes (PBL) was performed in 36 SLE patients and 42 healthy controls matched for age, gender, and ethnicity of patients using mass spectrometry that covers all major metabolic pathways. mTOR activity was assessed by western blot and flow cytometry. Metabolome changes in lupus PBL affected 27 of 80 KEGG pathways at FDR p < 0.05 with most prominent impact on the pentose phosphate pathway (PPP). While cysteine was depleted, cystine, kynurenine, cytosine, and dCTP were the most increased metabolites. Area under the receiver operating characteristic curve (AUC) logistic regression approach identified kynurenine (AUC = 0.859), dCTP (AUC = 0.762), and methionine sulfoxide (AUC = 0.708), as top predictors of SLE. Kynurenine was the top predictor of NAC effect in SLE (AUC = 0.851). NAC treatment significantly reduced kynurenine levels relative to placebo in vivo (raw p = 2.8 × 10−7, FDR corrected p = 6.6 × 10−5). Kynurenine stimulated mTOR activity in healthy control PBL in vitro. Metabolome changes in lupus PBL reveal a dominant impact on the PPP that reflect greater demand for nucleotides and oxidative stress. The PPP-connected and NAC-responsive accumulation of kynurenine and its stimulation of mTOR are identified as novel metabolic checkpoints in lupus pathogenesis.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-015-0772-0) contains supplementary material, which is available to authorized users.

A quantitative metabolomic profiling and corresponding modeling approach of hepatocytes with activated Wnt/ß-Catenin signaling

The maintenance of liver homeostasis relies on the metabolic zonation, where pericentral (pc) and periportal (pp) zones differ in their enzyme composition, content and activity. To carry out a metabolic fingerprint along the porto-central axis of the sinusoid is of high relevance for establishing a suitable systems biology approach aiding a better mechanistic understanding of hepatocytes heterogeneity with their different cellular functions. Here, we present a metabolic modeling approach for mouse hepatocytes based on metabolite profiles in the cell and cell culture medium of of APCloxPneo mice, with an over-activated Wnt/β-Catenin signaling and control mice, with normal Wnt/β-Catenin activity. The pathway has been shown to play a key role in the functional heterogeneity of hepatocytes and fundamentally influence the metabolic zonation in adult liver. APCloxPneo mice develop an expanded zone of hepatocytes with pericentral functions, due to the partial reduction in the Adenomatous Polyposis Coli protein (APC) expression in hepatocytes. Therefore it is well suited for evaluation of the model. Hepatocytes were isolated and cultivated for up to 48 hours and medium was collected at different time points. The culture medium was analyzed with GC-MS and LC-MS methods for quantitative analyses of the compound classes' amino acids, bile acids and organic acids. A metabolic model consisting of 42 species and 67 interspecies interactions has been established to analyze the flux distribution patterns in different hepatocyte populations. The software system VANTED (Visualization and Analysis of Networks containing Experimental Data) has been used to interpret the different patterns of metabolites, occurring in various hepatocyte populations. To characterize the functional specialization of hepatocytes clustering with self-organizing maps (SOM) was performed. The flux balance analysis (FBA) was carried out to get a general idea of the metabolic capabilities of different hepatocyte populations. The model has been validated by comparison to published data of pp/pc and APCloxPneo hepatocytes. Conclusion: The model offers a valuable tool for understanding the role of hepatocyte heterogeneity in liver homeostasis under normal conditions and possibly also in diseased states. Further, it could serve as an important sub-module for a multi-scale liver model of the cellular metabolism bridging single-cell metabolism to the tissue and the whole-organ level.