Recovery Of Metabolites Research Articles

The effect of liquid-liquid extraction on metabolite detection and analysis using NMR spectroscopy in human synovial fluid

The evaluation of joint disease using synovial fluid is an emerging field of metabolic profiling. The analysis is challenged by multiple macromolecules which can obscure the small molecule chemistry. The use of protein precipitation and extraction has been evaluated previously, but not in synovial fluid. We systematically review the published NMR spectroscopy methods of synovial fluid analysis and investigated the efficacy of three different protein precipitation techniques: methanol, acetonitrile and trichloroacetic acid. The trichloroacetic wash removed the most protein. However, metabolite recoveries were universally very poor. Acetonitrile liquid/liquid extraction gave metabolite gains from four unknown compounds with spectral peaks at δ = 1.91 ppm, 3.64 ppm, 3.95 ppm & 4.05 ppm. The metabolite recoveries for acetonitrile were between 1.5 and 7 times higher than the methanol method, across all classes of metabolite. The methanol method was more effective in removing protein as reported by the free GAG undefined peak (44 % vs 125 %). However, qualitative evaluation showed that acetonitrile and methanol provided good restoration of the spectra to baseline. The methanol extraction has issues of a gelatinous substrate in the samples. All metabolite recoveries had a CV of > 15 %. A recommendation of acetonitrile liquid/liquid extraction was made for human synovial fluid (HSF) analysis. This is due to consistency, effective protein precipitation, recovery of metabolites and additional compounds not previously visible.

Untargeted Lipidomics Revealed the Protective Effects of Cyanidin-3-O-glucoside on Bisphenol A-Induced Liver Lipid Metabolism Disorder in Rats.

Bisphenol A (BPA) is an estrogenic endocrine disruptor that induces metabolic disorders. Cyanidin-3-O-glucoside (C3G) has multiple functional activities and is the most abundant anthocyanin belonging to the flavonoid subgroup. This study aimed to investigate the protective effect of C3G on BPA-induced liver lipid metabolism disorder and explore its mechanism via lipidomics analysis. The results showed that C3G supplementation significantly ameliorated the serum levels of low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, total cholesterol, triacylglycerols (TG), and alanine and aspartate aminotransferase (ALT and AST). Furthermore, liver lipidomics indicated that C3G effectively facilitated the recovery of differential lipid metabolites, including TGs, phosphatidylethanolamines, phosphatidylcholines, lysophosphatidylcholines, phosphatidylinositol, cholesteryl esters, and phosphatidylserine, and reversed the levels of hepatic lipid synthesis-related genes. Our results suggest that C3G has an effective regulatory effect on BPA-induced disorders of lipid metabolism.

SiMeEx, a simplified method for metabolite extraction of adherent mammalian cells.

A reliable method for metabolite extraction is central to mass spectrometry-based metabolomics. However, existing methods are lengthy, mostly due to the step of scraping cells from cell culture vessels, which restricts metabolomics in broader application such as lower cell numbers and high-throughput studies. Here, we present a simplified metabolite extraction (SiMeEx) method, to efficiently and quickly extract metabolites from adherent mammalian cells. Our method excludes the cell scraping step and therefore allows for a more efficient extraction of polar metabolites in less than 30min per 12-well plate. We demonstrate that SiMeEx achieves the same metabolite recovery as using a standard method containing a scraping step, in various immortalized and primary cells. Omitting cell scraping does not compromise the performance of non-targeted and targeted GC-MS analysis, but enables metabolome analysis of cell culture on smaller well sizes down to 96-well plates. Therefore, SiMeEx demonstrates advantages not only on time and resources, but also on the applicability in high-throughput studies.

The enantioselective separation and quantitation of the hydroxy-metabolites of arachidonic acid by liquid chromatography – tandem mass spectrometry

Arachidonic acid (AA) is a polyunsaturated fatty acid with a structure of 20:4(ω-6). Cytochrome P450s (CYPs) metabolize AA to several regioisomers and enantiomers of hydroxyeicosatetraenoic acids (HETEs). The hydroxy-metabolites (HETEs) exist as enantiomers in the biological system. The chiral assays developed for HETEs are so far limited to a few assays reported for midchain HETEs. The developed method is capable of quantitative analysis for midchain, subterminal HETE enantiomers, and terminal HETEs in microsomes. The peak area or height ratios were linear over concentrations ranging (0.01 −0.6 µg/ml) with r2 > 0.99. The intra-run percent error and coefficient of variation (CV) were ≤ ± 12 %. The inter-run percent error and coefficient of variation (CV)were ≤ ± 13 %, and ≤ 15 %, respectively. The matrix effect for the assay was also within the acceptable limit (≤ ± 15 %). The recovery of HETE metabolites ranged from 70 % to 115 %. The method showed a reliable and robust performance for chiral analysis of cytochrome P450-mediated HETE metabolites.

Using heat-activated persulfate to accelerate short-chain fatty acids production from waste activated sludge fermentation triggered by sulfate-reducing microbial consortium

Persulfate has been applied extensively for waste activated sludge (WAS) decomposition due to the strong oxidizing sulfate radical generated as a product. However, the efficiency is not improved without activation to produce free radicals. In this study, a novel coupling strategy of heat-activated persulfate (Heat_PS) pretreatment and sulfate-reducing bacteria (SRB) triggering was explored to enhance short-chain fatty acids (SCFAs) produced by WAS fermentation. The remaining sulfate acts as an essential acceptor of electrons for the metabolism of synergistic SRB, thereby boosting WAS acidification by energetic cooperation with anaerobic fermenters. The results showed that SCFAs yield in the Heat_PS + SRB group peaked at 431.89 mg COD/gVSS, with the proportion of acetate reaching 57.8 %. This was 6.33 and 1.75 times higher than that in raw and single Heat_PS treated WAS, respectively. Carbon balance revealed a conversion rate of 26.1 % of carbon content in WAS to SCFAs, with 4.5 % lower CO2 equivalents emitted than that in raw WAS fermentation by the assessments of environmental impacts. This was partially attributed to the strong decomposition of WAS by SO4•− and •OH oxidation from heat-activated PS and the SRB trigger. In addition, the synergistic relationship among acidogenic/fermentative bacteria and SRB consortia was further verified by the positive correlation among Desulfovibrio, the hydrolytic Escherichia-Shigella, Morganella and the fermetative Macellibacteroides and Bacteroides, as revealed by molecular ecological networks (MENs) analysis. The results of this study may highlight the cooperation of the synergistic micribial consortia as an additional perspective for the recovery of value-added biological metabolites from complex biotransformation.

Antioxidants in Animal Nutrition: UHPLC-ESI-QqTOF Analysis and Effects on In Vitro Rumen Fermentation of Oak Leaf Extracts.

The genus Quercus supplies a large amount of residual material (e.g., bark, acorns, leaves, wood), the valorization of which can favor a supply of antioxidant polyphenols to be used in the pharmaceutical, nutraceutical, or cosmeceutical sector. The recovery of specialized metabolites could also benefit livestock feeding, so much so that polyphenols have gained attention as rumen fermentation modifiers and for mitigating the oxidative imbalance to which farm animals are subject. In this context, leaves of Quercus robur L. from Northern Germany were of interest and the alcoholic extract obtained underwent an untargeted profiling by means of ultra-high-performance liquid chromatography/high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) techniques. As triterpenes and fatty acids occurred, the alcoholic extract fractionation pointed out the obtainment of a polyphenol fraction, broadly constituted by coumaroyl flavonol glycosides and condensed tannins. Total phenol, flavonoid and condensed tannins content assays, as well as antiradical (DPPH● and ABTS+●) and reducing activity (PFRAP) were carried out on the alcoholic extract and its fractions. When the effects on rumen liquor was evaluated in vitro in terms of changes in fermentation characteristics, it was observed that oak leaf extract and its fractions promoted an increase in total volatile fatty acids and differently modulated the relative content of each fatty acid.

Green approach for the recovery of secondary metabolites from the roots of Nardostachys Jatamansi (D. Don) DC using microwave radiations: Process optimization and anti-alzheimer evaluation

Nardostachys jatamansi (D. Don) DC is a highly valued medicinal herb that has been used in traditional medicinal systems for its remedial effects. Owing to the over-exploitation and unethical trade of N. jatamansi, the accelerating global demand of herbal products from this plant cannot be satisfied by the conventional extraction approach. In view of the progressive demand and incredible biological potential of herb, the present research was designed to optimize various extraction parameters for microwave-assisted extraction (MAE). The extracts obtained from the traditional and green approach were also assessed for the recovery of secondary metabolites and anti-Alzheimer's potential. Various parameters like microwave power, temperature, and time of irradiation were optimized for MAE using Box Behkhen Design (BBD) The scanning electron microscopy of different plant samples was also done to observe the effect of microwave radiations. Further, the metabolite profiling of different extracts was also done by gas chromatography-mass spectrometry (GC-MS) analysis. Also the different behavioral and biochemical parameters along with acetylcholinesterase (AChE) inhibitory potential were assessed to evaluate the anti-Alzheimer's potential. Optimized parameters for MAE were found to be as microwave power 187.04 W, temperature 90°C, and irradiation time 20 min. The extract yield in MAE was significantly enhanced as compared to the conventional method. Also, the total phenolic content and total flavonoid content (TFC) were improved pointedly from 32.13 ± 0.55 to 72.83 ± 1.1 mg of GAE/g of extract and 21.7 ± 0.85 to 39.21 ± 0.7 mg of RUE/g of extract respectively. Later, the GC-MS analysis of various extracts confirmed the enhancement in the concentration of various sesquiterpenes like jatamansone, spirojatamol, valerenal, valeric acid, globulol, nootkatone and steroidal compounds such as sitosterol, ergosterol, stigmastanone, etc. in the optimized extract. A significant improvement in anti-Alzheimer's potential was also observed owing to the better concentration of secondary metabolites in the optimized microwave extract. From the current findings, it could be concluded that the MAE could be a successful and green alternative for the extraction and recovery of secondary metabolites from the selected medicinal herb.

Metabolic profiling workflow for cell extracts by targeted hydrophilic interaction liquid chromatography-tandem mass spectrometry

In this study, a targeted approach with wide metabolite coverage was developed for cellular metabolomic analysis using a UHPLC-QTrap-MS system operated in the scheduled multiple reaction monitoring (sMRM) mode. MRM ion pairs were acquired from HeLa cell samples through untargeted analysis using UHPLC-QTOF-MS with SWATH acquisition complemented by missing metabolites from pathway databases. Four different cell extraction protocols were studied and compared based on an experiment series involving the calculation of individual metabolite recoveries (pre/post extraction spiking U-13C isotope-labeled standards), with a Methanol/Water extraction mixture (1:1; v/v) showing the best results. Two HILIC-MS methods employing a Waters Premier BEH Amide column were developed, utilizing two different chromatographic conditions (20 mM ammonium formate as buffer additive adjusted to a pH = 3.5 with formic acid in ESI+ mode and 20 mM ammonium acetate adjusted to a pH = 7.5 with acetic acid in ESI− mode. One hundred sixty-one (161) metabolites were successfully detected in ESI+ mode, whereas 92 were detected in negative ionization mode, totaling to a number of 253 compounds in three different biological matrices covered by the analytical system employed. Both established HILIC methods were calibrated and validated based on 105 authentic chemical standards and U-13C-labeled Pichia pastoris (Komagataella phaffii) yeast extract as internal standards for cellular matrix (HeLa cells). Within-day and between-day precision was determined on three different QC concentration levels and was below 15% for the entirety of the analytes. Inter- and intra-day accuracies showed values in the range between 85 and 115% (assessed as % recovery) in the entire range. Matrix effects, extraction recoveries and process efficiencies were evaluated following the Matuszewski protocol with U-13C-labeled Pichia pastoris metabolite extract as internal standards. Eventually, the method was utilized to quantify metabolites in HeLa cell extracts.

Exogenous citrate restores the leaf metabolic profiles of navel orange plants under boron deficiency

Boron (B) is an essential micronutrient for higher plants, and its deficiency causes a change in the citrate concentration in leaves of young navel orange plants. Although citrate has been implicated in the regulation of gene expression for many transcripts, it is unclear whether citrate can affect metabolic profiling under B deficiency and if so, how many metabolites are affected. In this study, GC-TOF-MS-based untargeted metabolite profiling was used to identify the physiological effects of exogenous citrate on recovery of metabolites in B-deficient orange plants. There were 31 increased and 24 decreased metabolites in the boron-deficient (BD) group leaves relative to those of the boron-adequate (BA) group. Boron deficiency-induced changes in many metabolites were restored to the level of BA (control) group leaves or showed a recovery tendency at 1 week after citrate supply (foliar application of citrate, BDFC), including 11 organic acids, 9 sugars and polyols, 10 amino acids, and 4 other compounds. To compare with the metabolic recovery effects of exogenous citrate on B deficiency, exogenous application of B (borate) was also performed under same conditions (BDFB), and similar effects on the regulation of metabolic homeostasis under B deficiency were observed. Both the results of principal component analysis (PCA) and hierarchical cluster analysis (HCA) showed that BA, BDFC, and BDFB were relatively similar and clustered close to each other. There are different responsive and regulatory mechanisms to the additions of exogenous citrate in navel orange leaves under B adequate and deficient conditions. Based on these results, we suggest that citrate is an important component of the B deficiency stress response, and exogenous application of citrate generally restores the leaf metabolic profiles of navel orange plants under boron deficiency, which might play a positive role in this stress tolerance.

Ultrasound assisted cyanotoxin extraction for nematode inhibition in soil

Root-knot nematodes are one of the plant damaging nematodes in agriculture causing a projected annual yield loss of ∼12 % (∼$160 billion) worldwide. Conventional solutions to control these plant-parasitic nematodes involve chemical nematicides. To reduce the use of harmful chemicals, microalgal extracts can be used as greener alternatives for nematode management. Microalgae produce valuable metabolites, including cyanotoxins which can aid in nematode suppression.In this study, two microalgae species, Trichormus variabilis and Nostoc punctiforme, were treated with ultrasound for intensified recovery of secondary metabolites. Ultrasound results in cell wall disruption of the microalgal species, thus resulting in enhanced release of secondary metabolites. Microalgal biomass was treated with an ultrasound probe at 50 % amplitude, 20 kHz frequency, using water as the extraction medium, for 5–30 min. The extraction efficiency was determined in terms of the total chlorophyll (Chl) content of the extract. Microscopic images of the treated cells were also investigated to gain insight into the effect of the ultrasonication time on the cell morphology. Our results suggest that ultrasonication resulted in the intensified release of secondary metabolites, as established through the total chlorophyll content of the ultrasonicated microalgal samples as well as the microscopic images of the ruptured cells. The best extraction for Trichormus variabilis was achieved with 15 min extraction time where the Total Chl content increased by 29 times (compared to the non-ultrasonicated sample), and for the Nostoc punctiforme, 30 min extraction time gave the highest metabolite recovery of 6.4 times higher than the non-ultrasonicated sample.Ultrasonicated algal extracts were then tested for their nematicidal potential against root-knot nematode, Meloidogyne hapla, in infested field soil samples. Experimental study was conducted using different concentrations of each microalga, Trichormus sp. and Nostoc sp., individually, as well as in combination. The nematode count for the treated soil was compared with that of the control (untreated soil). Ultrasonicated microalgal extracts showed 66% to 100% inhibition on root-knot nematodes in the soil samples tested.

Microalgal biofuels: Pathways towards a positive energy balance

Current microalgae biofuel production pathways are still far from being energetically self-sufficient. Simulations scenarios (1 t·y−1 of biomass) under a purely energetic perspective, have revealed some bottlenecks mainly in the cultivation and metabolite recovery processes. Technologies such as intensified photobioreactors such as AlgoFilm©, infrared light filtering units, photovoltaic panels, solvent-free metabolite recovery processes, and high biomass concentration treatments at⩾145 kg·m−3 are suggested and discussed to also have a major contribution on the net energy ratio of the process. An energy-driven biorefinery approach has been proposed to increase the energy output by obtaining bioethanol and biodiesel liquid fuels, but also photovoltaic energy during the same production process. The latter may also compensate for the large amounts of energy consumed in typical operations of the wet-pathway processes like harvesting-concentration, cell disruption, or solvent recycling processes. This perspective increased the final volume of liquid biofuels up to 57%. It was found a maximum net energy ratio of 1.9 for a double biofuel process, and 8.5 if photovoltaic energy is also considered.

Untargeted Metabolomic Profiling and Antioxidant Capacities of Different Solvent Crude Extracts of Ephedra foeminea.

Ephedra foeminea is a traditional medicinal plant used in the Eastern Mediterranean region. This study aims to investigate the chemical profiles of different solvent extracts of E. foeminea via an untargeted metabolomics approach, alongside determining their antioxidant capacities. E. foeminea samples collected from Jordan were macerated in solvents of varying polarities; dichloromethane/methanol, methanol, ethanol, ethyl acetate, and acetone. The crude extracts were subjected to comprehensive chemical profiling and metabolomics study using Gas chromatography–Mass spectrometry (GC–MS), Liquid chromatography–Mass spectrometry (LC–MS), and Nuclear Magnetic Resonance (NMR). The obtained data were analyzed using Venn diagrams, Principle Component Analysis (PCA), and Metabolite Enrichment Set Analysis (MESA). ABTS assay was performed to measure the crude extracts’ antioxidant activity. MESA revealed the dominant chemical groups as amino acids, fatty acids, carboxylic acids, and carbohydrates. Results indicated that dichloromethane/methanol and methanolic extracts had the most distinct composition as well as the most unique compounds. The methanolic extract had the most potency (IC50 249.6 µg/mL) in the ABTS assay. However, no significant differences were found. In conclusion, solvents influenced the recovery of metabolites in E. foeminea and the antioxidant activity of the E. foeminea methanolic extract could be correlated to the abundant presence of diverse bioactive compounds.

Anti-obesity effect and UHPLC-QTOF-MS/MS based metabolite profiling of Solanum nigrum leaf extract

Objective: To evaluate the antioxidant potential and pancreatic lipase inhibitory action of optimized hydroethanolic extracts of Solanum nigrum. Methods: Optimized extraction for maximum recovery of metabolites was performed using a combination of freeze-drying and ultrasonication followed by determination of antioxidant and antiobesity properties. The ultra-high performance liquid chromatography equipped with mass spectrometry was used to analyze metabolite profiling of Solanum nigrum. Computational studies were performed using molecular docking and electrostatic potential analysis for individual compounds. The hypolipidemic potential of the most potent extract was assessed in the obese mice fed on fat rich diet. Results: The 80% hydroethanolic extract exhibited the highest extract yield, total phenolic contents, total flavonoid contents along with the strongest 2,2-diphenyl-1-picrylhydrazyl scavenging activity, total antioxidant power, and pancreatic lipase inhibitory properties. The 80% hydroethanolic extract not only regulated the lipid profile of obese mice but also restricted the weight gain in the liver, kidney, and heart. The 80% hydroethanolic extract also reduced alanine transaminase and aspartate transaminase concentrations in serum. The effects of plant extract at 300 mg/kg body weight were quite comparable with the standard drug orlistat. Conclusions: Solanum nigrum is proved as an excellent and potent source of secondary metabolites that might be responsible for obesity mitigation.

Understanding the Significance of Sample Preparation in Studies of the Nanoparticle Metabolite Corona.

The adsorption of metabolites to the surface of nanomaterials is a growing area of interest in the field of bionanointeractions. Like its more-established protein counterpart, it is thought that the metabolite corona has a key role in the uptake, distribution, and toxicity of nanomaterials in organisms. Previous research has demonstrated that nanomaterials obtain a unique metabolite fingerprint when exposed to biological matrices; however, there have been some concerns raised over the reproducibility of bionanointeraction research due to challenges in dispersion of nanomaterials and their stability. As such, this work investigates a much-overlooked aspect of this field, i.e., sample preparation, which is vital to the accurate, reproducible, and informative analysis of the metabolite corona. The impact of elution buffer pH, volume, and ionic strength on the metabolite corona composition acquired by uncapped and polyvinylpyrrolidone (PVP)-capped TiO2 from mixtures of cationic and anionic metabolites was studied. We demonstrate the temporal evolution of the TiO2 metabolite corona and the recovery of the metabolite corona, which resulted from a complex biological matrix, in this case human plasma. This work also demonstrates that it is vital to optimize sample preparation for each nanomaterial being investigated, as the metabolite recovery from Fe3O4 and Dispex-capped TiO2 nanomaterials is significantly reduced compared to the aforementioned uncapped and PVP-capped TiO2 nanomaterials. These are important findings for future bionanointeraction studies, which is a rapidly emerging area of research in nanoscience.

Optimization of the Recovery of Secondary Metabolites from Defatted Brassica carinata Meal and Its Effects on the Extractability and Functional Properties of Proteins.

The sustainable extraction of secondary metabolites from Brassica agro-industrial by-products often involves the use of high concentrations of ethanol, and/or high temperatures, which tends to decrease the efficiency of protein extraction (yield, profile, etc.). To understand the limits of the combination of these two extraction processes, aqueous ethanol extraction of secondary metabolites (e.g., phenolic compounds and glucosinolates) from Brassica carinata defatted meal was optimized using Response Surface Methodology. The validated models predicted that aqueous ethanol extraction of defatted Carinata meal, with a low aqueous EtOH concentration (22% EtOH) at moderate Te (50 °C), enables the efficient recovery of secondary metabolites (sinapine = 9.12 ± 0.05 mg/gDM, sinigrin = 86.54 ± 3.18 µmol/gDM) while maintaining good protein extractability (59.8 ± 2.1%) from successive alkaline extractions. The evaluation of functional properties of the resulting protein isolates revealed that aqueous extraction, under optimized conditions, improves foaming activity while preserving emulsion ability.

A Rapid LC-MS/MS Method for Simultaneous Determination of Ten Flavonoid Metabolites of Naringin in Rat Urine and Its Application to an Excretion Study.

Naringin is a dietary flavonoid glycoside with multiple bioactivities. It has been involved in numerous metabolism and excretion studies, and its metabolic properties are clear. However, information concerning the excretion profile of its original metabolites are still scarce, and few methods for simultaneous determination of multiple original metabolites of naringin in biological samples have been reported so far. In this study, a rapid and sensitive method for simultaneous determination of ten flavonoid metabolites of naringin in rat urine was developed with an UHPLC-Q-Trap-MS/MS system. One-step protein precipitation method with acetonitrile was used to extract analytes. A rapid chromatographic separation within 11 min was performed on an ACQUITY UPLC® BEH C18 column (2.1 mm × 50 mm, 1.7 μm) using gradient elution with a mobile phase of water and methanol, both with 0.1% formic acid (v/v). MS/MS detection was conducted in negative ion mode and multiple reactions monitoring scanning mode. The analytical method was fully validated and successfully applied to monitor the excretion profiles of naringin in rat urine. Quantitative results revealed the visible individual difference and low urinary recovery of flavonoid metabolites in the excretion of naringin, which may be helpful for further study to understand the in vivo behavior and action mechanism of naringin.

Recoupled-STOCSY-based co-expression network analysis to extract phenotype-driven metabolite modules in NMR-based metabolomics dataset

Nuclear magnetic resonance (NMR)-based metabolomics study usually involves spectral preprocessing, identification of biomarkers and interpretation of biological processes and pathogenesis, however, the traditional procedure is bound to inborn defects. In this study, a new analytical frame was proposed to assist spectral alignment and dimensionality reduction, screen the differential metabolites and get biological explanation of the metabolic network by combing weighted gene co-expression network analysis (WGCNA) and recoupled statistical total correlation spectroscopy (RSTOCSY). The performance of RSTOCSY-based WGCNA method was evaluated by the NMR dataset of serum from coronary heart disease with diabetes mellitus (CHDDM) patients. The statistical recoupling of variables (SRV) was successfully used to categorize the whole dataset into a number of superclusters of signals and served to spectral alignment, and its effectiveness was confirmed by the wine dataset with a larger spectral drift. Three phenotype-driven metabolite modules related to CHDDM were identified from the dataset by WGCNA, and 22 metabolites were further identified from the three modules according to the metabolic correlations within or between modules, and 40 significant metabolic correlations were observed from the intra- and inter-metabolites in the 2D pseudospectrum. These modules involve amino acid metabolism, microbial metabolism and glucose metabolism, and their analysis of metabolite network diffusion revealed a new discovery that the ferroptosis pathway is related to CHDDM. This RSTOCSY-based WGCNA approach provides an effective analysis workflow for information recovery and structure identification of metabolites and improving interpretability and understanding of the disease pathogenesis.

Ontogeny of Pediatric Pharmacogenetics: Celebrating the Past and Vision for the Future.

Ontogeny of Pediatric Pharmacogenetics: Celebrating the Past and Vision for the Future.

The oral bioavailability of di-2-ethylhexyl phthalate (DEHP), di-isononyl phthalate (DiNP) and di-(isononyl)-cyclohexane-1,2-dicarboxylate (DINCH®) in house dust

Phthalates and other plasticizers are detected in high amounts in the indoor environment and therefore house dust can be an exposure source. Especially children have a relatively high unintended uptake of house dust, thus a higher exposure to plasticizers compared to adults may be possible. As accurate as possible exposure assessment data of the oral bioavailability of these compounds are necessary, however only one in vivo study with piglets is available so far. The aim of this study was to examine the oral bioavailability of phthalates and DINCH® in humans, which occur in typical house dust samples. We focused on the high molecular weight phthalates DEHP and DINP and their substitute DINCH®. Eleven volunteers ingested 6 g of house dust sieved to 2 mm. The urine was collected over a period of 36 h. The excreted plasticizers metabolites were quantified by an LC–MS/MS method. The mean recovery of urine metabolites was 51 % ± 20 % for DEHP, 26 % ± 13 % for DINP and 19 % ± 6% for DINCH® based on the parent compounds administered as dust samples. The metabolites of DEHP, DINP and DINCH® reached their maximum concentration after 2–19 hours post dose in urine. The bioavailability of DEHP was in agreement among the different dust samples. For DEHP, we were able to confirm previous findings from the oral bioavailability study with piglets and we could not observe a significant difference between the dust particle size (65 μm vs 2 mm) and the bioavailability. Considering the observed bioavailability, an estimated dust intake of 50 mg/d for toddlers can substantially contribute to the total plasticizer exposure.

TAMI-53. CYSTEINE IS A LIMITING FACTOR FOR GLIOMA PROLIFERATION AND SURVIVAL

Abstract BACKGROUND Little is known about the mechanisms that render cancer cells dependent on certain nutrients from the microenvironment. Cysteine is a non-essential amino acid, since it can be synthetized from methionine through the transsulfuration pathway; moreover, cysteine is also uptake from the diet as cystine. We have investigated the metabolism of cysteine in glioma cell lines, and how cysteine/cystine-deprivation alters their antioxidant response in addition to the effect of this nutrient restriction to viability and proliferation in vitro and in vivo. METHODS Cysteine metabolism was investigated through LCMS-based 13C-tracing experiments and the expression levels of key enzymes in the transsulfuration pathway were also explored. Finally, a mouse model of IDH1 mutant glioma was subjected to a cysteine/cystine-free diet and tumor metabolism was analyzed by LCMS. RESULTS Herein, we report the dependence of glioma cells on exogenous cysteine/cystine, despite this amino acid being nonessential. Using several 13C-tracers and analysis of cystathionine synthase and cystathioninase levels, we revealed that glioma cells were not able to upregulate the transulfuration pathway cysteine, which allows methionine to be converted to cysteine in cysteine/cystine deprived conditions. We demonstrated that exogenous cysteine/cystine are crucial for glutathione synthesis, and impact growth and viability. Therefore, we explored the nutritional deprivation in a mouse model of glioma. Animals subjected to a cysteine/cystine-free diet survived longer, with concomitant reductions in glutathione and cysteine plasma levels. At the endpoint higher levels of oxidative stress were detected despite the systemic recovery of cysteine-related metabolites in the plasma. CONCLUSION The results presented herein reveal an alternative therapeutic approach combining cysteine/cysteine-deprivation diets and treatments involving ROS production by limiting the ability of glioma cells to quench oxidative stress through dietary interventions. Our study highlights a time window where cysteine deprivation can be exploited for additional therapeutic strategies.