Quantitative Metabolite Profiling Research Articles

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.

Biomarker discovery for tuberculosis using metabolomics.

Tuberculosis (TB) is the leading cause of death among infectious diseases, and the ratio of cases in which its pathogen Mycobacterium tuberculosis (Mtb) is drug resistant has been increasing worldwide, whereas latent tuberculosis infection (LTBI) may develop into active TB. Thus it is important to understand the mechanism of drug resistance, find new drugs, and find biomarkers for TB diagnosis. The rapid progress of metabolomics has enabled quantitative metabolite profiling of both the host and the pathogen. In this context, we provide recent progress in the application of metabolomics toward biomarker discovery for tuberculosis. In particular, we first focus on biomarkers based on blood or other body fluids for diagnosing active TB, identifying LTBI and predicting the risk of developing active TB, as well as monitoring the effectiveness of anti-TB drugs. Then we discuss the pathogen-based biomarker research for identifying drug resistant TB. While there have been many reports of potential candidate biomarkers, validations and clinical testing as well as improved bioinformatics analysis are needed to further substantiate and select key biomarkers before they can be made clinically applicable.

Differential Metabolomic Fingerprinting of the Crude Extracts of Three Asteraceae Species with Assessment of Their In Vitro Antioxidant and Enzyme-Inhibitory Activities Supported by In Silico Investigations

The Asteraceae is a large family, rich in ornamental, economical, and medicinally valuable plants. The current study involves the analytical and pharmacological assessment of the methanolic extracts of three less investigated Asteraceae plants, namely Echinops ritro, Centaurea deflexa, and Tripleurospermum decipiens, obtained by three different extraction methodologies viz. maceration (MAC), ultrasound-assisted extraction (UAE), and homogenizer-assisted extraction (HAE). LC-MS-MS analysis of E. ritro, C. deflexa, and T. decipiens extracts led to the identification of ca. 29, 20, and 33 metabolites, respectively, belonging to flavonoids, phenolic acids, and fatty acids/amides. Although there were significant differences in the quantitative metabolite profiles in the extracts of E. ritro and T. decipiens based on the used extraction method, no significant variation was observed in the extracts of C. deflexa in the three implemented extraction techniques. The antioxidant activities of the nine extracts were assessed in vitro using six different assays viz. DPPH, ABTS, CUPRAC, FRAP, PDA, and metal chelation assay (MCA). The HAE/UAE extracts of E. ritro and the UAE/ MAC extracts of C. deflexa displayed the highest antioxidant activity in the DPPH assay, while the UAE extract of T. decipiens showed the strongest antioxidant activity in both the CUPRAC and MCA assays. The enzyme inhibitory activities of the nine extracts were studied in vitro on five different enzymes viz. tyrosinase, α-amylase, α-glucosidase, acetylcholinesterase (AChE), and butyrylcholinestrase (BChE), affecting various pathological diseases. Concerning C. deflexa, its MAC /UAE extracts showed the strongest inhibition on α-amylase, while its UAE/HAE extracts displayed strong inhibitory power on AChE. However, no significant difference was observed on their effects on tyrosinase or BChE. For T. decipiens, its UAE/HAE showed potent inhibition to α-glucosidase, MAC/ HAE significantly inhibited AChE and BChE, while UAE could strongly inhibit tyrosinase enzyme. For E. ritro, all extracts equally inhibited α-amylase and α-glucosidase, MAC/HAE strongly affected tyrosinase, HAE/MAC best inhibited BChE, while HAE inhibited AChE to a greater extent. Chemometric analysis using PCA plot was able to discriminate between the plant samples and between the implemented extraction modes. The in vitro enzyme inhibitory activities of the extracts were supported by in silico data, where metabolites, such as the lignan arctiin and the flavonoid vicenin-2, dominating the extract of C. deflexa, displayed strong binding to AChE. Similarly, chlorogenic and dicaffeoyl quinic acids, which are some of the major metabolites in the extracts of E. ritro and T. decipiens, bound with high affinity to α-glucosidase.

Nutrient-depended metabolic switching during batch cultivation of Streptomyces coelicolor explored with absolute quantitative mass spectrometry-based metabolite profiling

The well-known secondary metabolite-producing bacterium Streptomyces coelicolor is a natural choice for the development of super-hosts optimized for the heterologous expression of antibiotic biosynthetic gene clusters (BGCs). In this study, we used S. coelicolor M145 and its derivative strain M1146 where all active BGCs have been deleted and generated high-resolution quantitative time series metabolite profiles under two cultivation conditions (phosphate and nitrogen limitation to cease growth and trigger secondary metabolism). Five targeted LC–MS/MS-based methods were used to quantify intracellular primary metabolites covering phosphorylated metabolites, amino acids, organic acids, (deoxy) nucleoside/sugar phosphates, Nicotinamide adenine dinucleotide (NAD), and Coenzyme A (CoA). The nitrogen limitation resulted in a sharp decline in respiration and an immediate drop in the cell mass concentration. Intracellularly, a reduction in the level of the metabolites next to α-ketoglutarate in the tricarboxylic acid cycle and a decrease in the NADH pool were among the most prominent adaptation to this nutrient limitation. Phosphate limitation evoked a different adaptation of the metabolite pools as most of the phosphorylated metabolite pools except 6-phosphogluconic acid (6PG) pool were downregulated. 13C-isotope-labeling experiments revealed the simultaneous activity of both glycolysis and gluconeogenesis during the co-utilization of glucose and glutamate. The S. coelicolor M1146 strain had similar time-series metabolite profile dynamics as the parent M145 strain, except for a visibly increased 6PG pool in the stationary phase. In general, the nutrient limitation had a larger effect on the metabolite pool levels than the absence of secondary metabolite production in M1146. This study provides new insight into the primary carbon metabolism and its link to the secondary metabolism which is needed for further optimization of both super-host genotype and cultivation conditions.

Adaptation of central metabolite pools to variations in growth rate and cultivation conditions in Saccharomyces cerevisiae

BackgroundSaccharomyces cerevisiae is a well-known popular model system for basic biological studies and serves as a host organism for the heterologous production of commercially interesting small molecules and proteins. The central metabolism is at the core to provide building blocks and energy to support growth and survival in normal situations as well as during exogenous stresses and forced heterologous protein production. Here, we present a comprehensive study of intracellular central metabolite pool profiling when growing S. cerevisiae on different carbon sources in batch cultivations and at different growth rates in nutrient-limited glucose chemostats. The latest versions of absolute quantitative mass spectrometry-based metabolite profiling methodology were applied to cover glycolytic and pentose phosphate pathway metabolites, tricarboxylic acid cycle (TCA), complete amino acid, and deoxy-/nucleoside phosphate pools.ResultsGlutamate, glutamine, alanine, and citrate were the four most abundant metabolites for most conditions tested. The amino acid is the dominant metabolite class even though a marked relative reduction compared to the other metabolite classes was observed for nitrogen and phosphate limited chemostats. Interestingly, glycolytic and pentose phosphate pathway (PPP) metabolites display the largest variation among the cultivation conditions while the nucleoside phosphate pools are more stable and vary within a closer concentration window. The overall trends for glucose and nitrogen-limited chemostats were increased metabolite pools with the increasing growth rate. Next, comparing the chosen chemostat reference growth rate (0.12 h−1, approximate one-fourth of maximal unlimited growth rate) illuminates an interesting pattern: almost all pools are lower in nitrogen and phosphate limited conditions compared to glucose limitation, except for the TCA metabolites citrate, isocitrate and α-ketoglutarate.ConclusionsThis study provides new knowledge-how the central metabolism is adapting to various cultivations conditions and growth rates which is essential for expanding our understanding of cellular metabolism and the development of improved phenotypes in metabolic engineering.

Combined Metabolome and Lipidome Analyses for In-Depth Characterization of Lipid Accumulation in the DHA Producing Aurantiochytrium sp. T66.

Thraustochytrids are marine heterotrophic microorganisms known for their potential to accumulate docosahexaenoic acid (DHA)-enriched lipids. There have been many attempts to improve thraustochytrid DHA bioprocesses, especially through traditional optimization of cultivation and media conditions. Nevertheless, thraustochytrid-based bioprocesses are still not commercially competitive for high volume-low cost production of DHA. Thus, it is realized that genetic and metabolic engineering strategies are needed for the development of commercially competitive thraustochytrid DHA cell factories. Here, we present an analytical workflow for high resolution phenotyping at metabolite and lipid levels to generate deeper insight into the thraustochytrid physiology, with particular focus on central carbon and redox metabolism. We use time-series sampling during unlimited growth and nitrogen depleted triggering of DHA synthesis and lipid accumulation (LA) to show-case our methodology. The mass spectrometric absolute quantitative metabolite profiling covered glycolytic, pentose phosphate pathway (PPP) and tricarboxylic acid cycle (TCA) metabolites, amino acids, complete (deoxy)nucleoside phosphate pools, CoA and NAD metabolites, while semiquantitative high-resolution supercritical fluid chromatography MS/MS was applied for the lipid profiling. Interestingly, trace amounts of a triacylglycerols (TG) with DHA incorporated in all three acyl positions was detected, while TGs 16:0_16:0_22:6 and 16:0_22:6_22:6 were among the dominant lipid species. The metabolite profiling data indicated that lipid accumulation is not limited by availability of the acyl chain carbon precursor acetyl-CoA nor reducing power (NADPH) but rather points to the TG head group precursor glycerol-3-phosphate as the potential cause at the metabolite level for the gradual decline in lipid production throughout the cultivation. This high-resolution phenotyping provides new knowledge of changes in the central metabolism during growth and LA in thraustochytrids and will guide target selection for metabolic engineering needed for further improvements of this DHA cell factory.

In vitro biotransformation of pyrrolizidine alkaloids in different species: part II\u2014identification and quantitative assessment of the metabolite profile of six structurally different pyrrolizidine alkaloids

Pyrrolizidine alkaloids (PA) exert their toxic effects only after bioactivation. Although their toxicity has already been studied and metabolic pathways including important metabolites were described, the quantification of the latter revealed a large unknown portion of the metabolized PA. In this study, the qualitative and quantitative metabolite profiles of structurally different PAs in rat and human liver microsomes were investigated. Between five metabolites for europine and up to 48 metabolites for lasiocarpine were detected. Proposals for the chemical structure of each metabolite were derived based on fragmentation patterns using high-resolution mass spectrometry. The metabolite profiles of the diester PAs showed a relatively good agreement between both species. The metabolic reactions were summarized into three groups: dehydrogenation, oxygenation, and shortening of necic acid(s). While dehydrogenation of the necine base is considered as bioactivation, both other routes are considered as detoxification steps. The most abundant changes found for open chained diesters were dealkylations, while the major metabolic pathway for cyclic diesters was oxygenation especially at the nitrogen atom. In addition, all diester PAs formed several dehydrogenation products, via the insertion of a second double bond in the necine base, including the formation of glutathione conjugates. In rat liver microsomes, all investigated PAs formed dehydropyrrolizidine metabolites with the highest amount formed by lasiocarpine, whereas in human liver microsomes, these metabolites could only be detected for diesters. Our findings demonstrate that an extensive analysis of PA metabolism can provide the basis for a better understanding of PA toxicity and support future risk assessment.

Mini-Review: Comprehensive Drug Disposition Knowledge Generated in the Modern Human Radiolabeled ADME Study.

The human radiolabeled absorption, distribution, metabolism, and excretion (ADME) study offers a quantitative and comprehensive overall picture of the disposition of a drug, including excretion pattern and metabolite profiles in circulation and excreta. The data gathered from the ADME study are highly informative for developing a cohesive strategy for clinical pharmacology studies. Elements of standard ADME study designs are described. An exciting new development in human ADME studies is the application of accelerator mass spectrometry (AMS) as the detection technique for carbon‐14, in replacement of radioactivity measurements. This technology permits administration of 100‐fold to 1,000‐fold lower amounts of carbon‐14, and thus opens the door to the application of new study designs. A new ADME study design, termed the AMS‐Enabled Human ADME study, is described. In this design, both oral and intravenous administration are assessed in a single clinical study with a two‐period crossover. In addition to all of the standard ADME study end points (e.g., mass balance and quantitative metabolite profiles), the AMS‐Enabled ADME study can provide the fundamental pharmacokinetic parameters of clearance, volume of distribution, absolute oral bioavailability, and even estimates of the fraction of the dose absorbed. Thus, we have entered a new era of human ADME study design that can yield vastly more informative and complete data sets enabling a superior understanding of overall drug disposition.

Advanced High-Coverage Targeted Metabolomics Method (SWATHtoMRM) for Exploring the Relationship of Follicular Fluid Components with Age

Background: The complexity of follicular fluid metabolome presents a huge challenge for qualitative and quantitative metabolite profiling and discovery of the comprehensive biomarkers. Objective: In order to address this challenge, novel SWATHtoMRM metabolomics method was used for providing broad coverage and excellent quantitative capability to discover the human follicular fluid metabolites related to age and evaluate their relationship with pregnancy outcome and oocyte senescence. Methods: The patients were divided into four groups according to age, including group A (28 cases, 21- 27 years old), group B (42 cases, 28-34 years old), group C (31 cases, 35-41 years old), and group D (24 cases, 42-48 years old). Follicular fluid samples from 125 IVF patients were analyzed. The differential ions among the four groups were identified by principal components analysis according to accurate mass, isotope ratio, and tandem mass spectroscopic spectra. Then, the differential metabolic pathways were further identified by a KEGG cluster analysis. Results: A total of 18 metabolites in the follicular fluid differed among the four groups, including amino acids, lipids, hormones, and vitamins. A total of 15 metabolites, including 6-oxohexanoate, phenylalanine, proline, hexadecanoic acid, linoleate, arachidonate, oleic acid, docosahexaenoic acid, LysoPC(16:1), LysoPC(20:5), LysoPC (20:3), 25-hydroxyvitamin D3, 5-dehydroepisterol, 27- hydroxycholesterol, and 5beta-cholestane-3alpha,7alpha,12alpha,23,25-pentol, were down-regulated with age and 3 metabolites, including LysoPC(18:3), LysoPC(18:1), and 13,14-dihydroretinol, were upregulated with age. Conclusion: Our study provides useful information for revealing the relationship between age and female reproductive capability.

A MALDI-TOF analysis of gliadins and glutenins in “Astana” wheat cultivar

Targeted, quantitative metabolomics can, in principle, provide precise information on intracellular metabolite levels, which can be applied to accurate modeling of intracellular processes required in systems biology and metabolic engineering. However, quantitative metabolite profiling is often hampered by biased mass spectrometry-based analyses caused by matrix effects, the degradation of metabolites and metabolite leakage during sample preparation, and unexpected variation in instrument responses. Isotope Dilution Mass Spectrometry (IDMS) has been proven as the most accurate method for high-throughput detection of intracellular metabolite concentrations, and the key has been the acquisition of the corresponding fully uniformly (U) -13C-labeled metabolites to be measured. Here, we have prepared U-13C-labeled cell extracts by cultivating P. chrysogenum in a fed-batch fermentation with fully U-13C-labeled substrates. Towards this goal, a dynamic fed-batch model describing P. chrysogenum growth and penicillin production was used to simulate the fermentation process and design the fed-batch fermentation media. Further, a case study with extensive intracellular metabolomics data from glucose-limited cultivation of Penicillium chrysogenum under both single and repetitive glucose pulses was illustrated by using the IDMS methods with the prepared U-13C-labeled cell extracts as internal standards. In conclusion, the IDMS method can be incorporated into well-established fast sampling and quenching protocols to obtain dynamic quantitative in vivo metabolome data at the timescales of (tens of) seconds and elucidate the underlying regulatory architecture. The case study revealed gross differences between single and repeated pulses, which suggests that single pulse studies have limited value for understanding of metabolic responses in large-scale bioreactors. Instead, intermittent feeding should be favored.

Novel high-coverage targeted metabolomics method (SWATHtoMRM) for exploring follicular fluid metabolome alterations in women with recurrent spontaneous abortion undergoing in vitro fertilization

The complexity of follicular fluid metabolome presents a significant challenge for qualitative and quantitative metabolite profiling, and for discovering the comprehensive biomarkers. In order to address this challenge, a novel SWATHtoMRM metabolomics method was used for providing broad coverage and excellent quantitative capability to discover the human follicular fluid metabolites related to recurrent spontaneous abortion (RSA) after in vitro fertilization and embryo transfer, and to evaluate their relationship with pregnancy outcome. The follicular fluid samples from the spontaneous abortion group (n = 22) and the control group (n = 22) were analyzed using ultra-performance liquid chromatography high-resolution mass spectrometry. A novel, high-coverage, targeted metabolomics method (SWATH to MRM) and a targeted metabolomics method were used to find and validate the differential metabolites between the two groups. A total of 18 follicular fluid metabolites, including amino acids, cholesterol, vitamins, fatty acids, cholic acid, lysophosphatidylcholine and other metabolites, were identified. In the RSA group, 8 metabolites, namely dehydroepiandrosterone, lysoPC(16:0), lysoPC(18:2), lysoPC(18:1), lysoPC(18:0), lysoPC(20:5), lysoPC(20:4), and lysoPC(20:3), were up-regulated, and 10 metabolites, namely phenylalanine, linoleate, oleic acid, docosahexaenoic acid, lithocholic acid, 25-hydroxyvitamin D3, hydroxycholesterol, 13-hydroxy-alpha-tocopherol, leucine, and tryptophan, were down-regulated. These differential metabolites related to RSA may provide a possible diagnostic basis and therapeutic target for RSA, as well as a scientific basis for elucidating the mechanism of RSA.

A dynamic model-based preparation of uniformly-13C-labeled internal standards facilitates quantitative metabolomics analysis of Penicillium chrysogenum

Targeted, quantitative metabolomics can, in principle, provide precise information on intracellular metabolite levels, which can be applied to accurate modeling of intracellular processes required in systems biology and metabolic engineering. However, quantitative metabolite profiling is often hampered by biased mass spectrometry-based analyses caused by matrix effects, the degradation of metabolites and metabolite leakage during sample preparation, and unexpected variation in instrument responses. Isotope Dilution Mass Spectrometry (IDMS) has been proven as the most accurate method for high-throughput detection of intracellular metabolite concentrations, and the key has been the acquisition of the corresponding fully uniformly (U) -13C-labeled metabolites to be measured. Here, we have prepared U-13C-labeled cell extracts by cultivating P. chrysogenum in a fed-batch fermentation with fully U-13C-labeled substrates. Towards this goal, a dynamic fed-batch model describing P. chrysogenum growth and penicillin production was used to simulate the fermentation process and design the fed-batch fermentation media. Further, a case study with extensive intracellular metabolomics data from glucose-limited cultivation of Penicillium chrysogenum under both single and repetitive glucose pulses was illustrated by using the IDMS methods with the prepared U-13C-labeled cell extracts as internal standards. In conclusion, the IDMS method can be incorporated into well-established fast sampling and quenching protocols to obtain dynamic quantitative in vivo metabolome data at the timescales of (tens of) seconds and elucidate the underlying regulatory architecture. The case study revealed gross differences between single and repeated pulses, which suggests that single pulse studies have limited value for understanding of metabolic responses in large-scale bioreactors. Instead, intermittent feeding should be favored.

Methodological consensus on clinical proton MRS of the brain: Review and recommendations.

Proton MRS (1 H MRS) provides noninvasive, quantitative metabolite profiles of tissue and has been shown to aid the clinical management of several brain diseases. Although most modern clinical MR scanners support MRS capabilities, routine use is largely restricted to specialized centers with good access to MR research support. Widespread adoption has been slow for several reasons, and technical challenges toward obtaining reliable good-quality results have been identified as a contributing factor. Considerable progress has been made by the research community to address many of these challenges, and in this paper a consensus is presented on deficiencies in widely available MRS methodology and validated improvements that are currently in routine use at several clinical research institutions. In particular, the localization error for the PRESS localization sequence was found to be unacceptably high at 3 T, and use of the semi-adiabatic localization by adiabatic selective refocusing sequence is a recommended solution. Incorporation of simulated metabolite basis sets into analysis routines is recommended for reliably capturing the full spectral detail available from short TE acquisitions. In addition, the importance of achieving a highly homogenous static magnetic field (B0 ) in the acquisition region is emphasized, and the limitations of current methods and hardware are discussed. Most recommendations require only software improvements, greatly enhancing the capabilities of clinical MRS on existing hardware. Implementation of these recommendations should strengthen current clinical applications and advance progress toward developing and validating new MRS biomarkers for clinical use.

Increased biosynthesis and accumulation of cholesterol in maternal plasma, but not amniotic fluid in pre-eclampsia

Preeclampsia is one of the most serious complications during pregnancy, defined as development of hypertension during late pregnancy affecting other organ systems (proteinuria, thrombocytopenia, renal insufficiency, liver involvement, cerebral symptoms or pulmonary edema). Preeclampsia is known to be associated with significant dyslipidemia, but the cause or mechanism of this metabolic aberration is not clear. Quantitative analysis of cholesterol precursors and metabolites can reveal metabolic signatures of cholesterol, and provide insight into cholesterol biosynthetic and degradation pathways. We undertook this study to compare the metabolic signatures of cholesterol in serum and amniotic fluid collected from women who delivered in the late preterm period. Matching serum and amniotic fluid samples were collected from women who delivered in the late preterm period (34-0/7–36-6/7 weeks), had undergone amniocentesis within 3 days of delivery, had no evidence of rupture of membranes or intra-amniotic infection/inflammation, and who had not received antenatal corticosteroid prior to amniocentesis. Patients were classified into 3 groups according to the etiology of their preterm birth: Group 1, preeclampsia; Group 2, spontaneous preterm labor; Group 3, other maternal medical indications for iatrogenic preterm birth. Quantitative metabolite profiling of cholesterols was performed using gas chromatography-mass spectrometry. A total of 39 women were included in the analysis (n = 14 in Group 1, n = 16 in Group 2, n = 9 in Group 3). In maternal blood, patients in Group 1 had significantly higher ratios of cholesterol/desmosterol and cholesterol/7-dehydrocholesterol (which represent 24- and 7-reductase enzyme activity, respectively) than those in Group 3 (p < 0.05 for each), which suggests increased cholesterol biosynthesis. In contrast, patients in Group 1 had significantly decreased ratios of individual cholesterol esters/cholesterol and total cholesterol esters/cholesterol than those in Groups 3 (p < 0.01 for each), suggesting increased reverse cholesterol transport. No differences in cholesterol ratios were found in amniotic fluid among the 3 groups. In conclusion, the metabolic signatures of cholesterol suggest increased cholesterol biosynthesis and accumulation in the maternal blood (but not amniotic fluid) of women with preeclampsia.

Development of a method for the quantitative metabolite profiling of pharmaceutical drugs using HPLC-ICP-MS following pre-column derivatization of their amino and hydroxyl groups using 4-aminopyridine as a model compound

Presence of amino and/or hydroxyl groups allows for derivatization of APIs and their metabolites with 4-iodobenzoylchloride, thus enabling HPLC-ICP-MS metabolite profiling.

Transcriptome Profiling of Two Ornamental and Medicinal Papaver Herbs.

The Papaver spp. (Papaver rhoeas (Corn poppy) and Papaver nudicaule (Iceland poppy)) genera are ornamental and medicinal plants that are used for the isolation of alkaloid drugs. In this study, we generated 700 Mb of transcriptome sequences with the PacBio platform. They were assembled into 120,926 contigs, and 1185 (82.2%) of the benchmarking universal single-copy orthologs (BUSCO) core genes were completely present in our assembled transcriptome. Furthermore, using 128 Gb of Illumina sequences, the transcript expression was assessed at three stages of Papaver plant development (30, 60, and 90 days), from which we identified 137 differentially expressed transcripts. Furthermore, three co-occurrence heat maps are generated from 51 different plant genomes along with the Papaver transcriptome, i.e., secondary metabolite biosynthesis, isoquinoline alkaloid biosynthesis (BIA) pathway, and cytochrome. Sixty-nine transcripts in the BIA pathway along with 22 different alkaloids (quantified with LC-QTOF-MS/MS) were mapped into the BIA KEGG map (map00950). Finally, we identified 39 full-length cytochrome transcripts and compared them with other genomes. Collectively, this transcriptome data, along with the expression and quantitative metabolite profiles, provides an initial recording of secondary metabolites and their expression related to Papaver plant development. Moreover, these profiles could help to further detail the functional characterization of the various secondary metabolite biosynthesis and Papaver plant development associated problems.

Structural and Functional Analysis of the Gut Microbiome for Toxicologists.

Characterizing the reciprocal interactions between toxicants, the gut microbiota, and the host, holds great promise for improving our mechanistic understanding of toxic endpoints. Advances in culture-independent sequencing analysis (e.g., 16S rRNA gene amplicon sequencing) combined with quantitative metabolite profiling (i.e., metabolomics) have provided new ways of studying the gut microbiome and have begun to illuminate how toxicants influence the structure and function of the gut microbiome. Developing a standardized protocol is important for establishing robust, reproducible, and importantly, comparative data. This protocol can be used as a foundation for examining the gut microbiome via sequencing-based analysis and metabolomics. Two main units follow: (1) analysis of the gut microbiome via sequencing-based approaches; and (2) functional analysis of the gut microbiome via metabolomics. © 2018 by John Wiley & Sons, Inc.

SWATHtoMRM: Development of High-Coverage Targeted Metabolomics Method Using SWATH Technology for Biomarker Discovery.

The complexity of metabolome presents a great analytical challenge for quantitative metabolite profiling, and restricts the application of metabolomics in biomarker discovery. Targeted metabolomics using multiple-reaction monitoring (MRM) technique has excellent capability for quantitative analysis, but suffers from the limited metabolite coverage. To address this challenge, we developed a new strategy, namely, SWATHtoMRM, which utilizes the broad coverage of SWATH-MS technology to develop high-coverage targeted metabolomics method. Specifically, SWATH-MS technique was first utilized to untargeted profile one pooled biological sample and to acquire the MS2 spectra for all metabolites. Then, SWATHtoMRM was used to extract the large-scale MRM transitions for targeted analysis with coverage as high as 1000-2000 metabolites. Then, we demonstrated the advantages of SWATHtoMRM method in quantitative analysis such as coverage, reproducibility, sensitivity, and dynamic range. Finally, we applied our SWATHtoMRM approach to discover potential metabolite biomarkers for colorectal cancer (CRC) diagnosis. A high-coverage targeted metabolomics method with 1303 metabolites in one injection was developed to profile colorectal cancer tissues from CRC patients. A total of 20 potential metabolite biomarkers were discovered and validated for CRC diagnosis. In plasma samples from CRC patients, 17 out of 20 potential biomarkers were further validated to be associated with tumor resection, which may have a great potential in assessing the prognosis of CRC patients after tumor resection. Together, the SWATHtoMRM strategy provides a new way to develop high-coverage targeted metabolomics method, and facilitates the application of targeted metabolomics in disease biomarker discovery. The SWATHtoMRM program is freely available on the Internet ( http://www.zhulab.cn/software.php ).

A pre-column derivatization method allowing quantitative metabolite profiling of carboxyl and phenolic hydroxyl group containing pharmaceuticals in human plasma via liquid chromatography-inductively coupled plasma-tandem mass spectrometry (LC-ICP-MS/MS)

Novel analytical strategies for investigating drug ADME (absorption, distribution, metabolism and excretion).

Metabolic changes in droplet vitrified semen of wild endangered Persian sturgeon Acipenser persicus (Borodin, 1997)

Comparative quantitative metabolite profiling can be used for better understanding of cell functions and dysfunctions in particular circumstances such as sperm banking which is an important approach for cryopreservation of endangered species. Cryopreservation techniques have some deleterious effects on spermatozoa which put the obtained results in controversy. Therefore, in the present study, quantitative 1H NMR (Nuclear Magnetic Resonance) based metabolite profiling was conducted to evaluate metabolite changes related to energetics and some other detected metabolites in vitrified semen of critically endangered wild Acipenser persicus. The semen was diluted with extenders containing 0, 5, 10, and 15 μM of fish antifreeze protein (AFP) type III as a cryoprotectant. Semen-extenders were vitrified and stored for two days. Based on post-thaw motility duration and motility percentage assessments, two treatments with 10 μM and 0 μM of AFP had the highest and the lowest motility percentages respectively and they were objected to 1H NMR spectroscopy investigations in order to reveal the extremes of the metabolites dynamic range. Univariate (ANOVA) and multivariate (PCA) analysis of the resulting metabolic profiles indicated significant changes (P > 0.05) in metabolites. The level of some metabolites including acetate, adenine, creatine, creatine phosphate, lactate, betaine, sarcosine, β-alanine and trimethylamine N-oxide significantly decreased in vitrified semen while some others such as creatinine, guanidinoacetate, N, N-dimethylglycine, and glycine significantly increased. There were also significant differences between vitrified treatments in levels of creatine, creatine phosphate, creatinine, glucose, guanidinoacetate, lactate, N, N-dimethylglycine, and glycine, suggesting how fish AFP type III can be effective as a cryoprotectant.