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See detailQuantification of Stable Isotope Traces Close to Natural Enrichment in Human Plasma Metabolites Using Gas Chromatography-Mass Spectrometry
Krämer, Lisa; Jäger, Christian UL; Trezzi, Jean-Pierre UL et al

in Metabolites (2018), 8(1), 15

Currently, changes in metabolic fluxes following consumption of stable isotope-enriched foods are usually limited to the analysis of postprandial kinetics of glucose. Kinetic information on a larger ... [more ▼]

Currently, changes in metabolic fluxes following consumption of stable isotope-enriched foods are usually limited to the analysis of postprandial kinetics of glucose. Kinetic information on a larger diversity of metabolites is often lacking, mainly due to the marginal percentage of fully isotopically enriched plant material in the administered food product, and hence, an even weaker 13C enrichment in downstream plasma metabolites. Therefore, we developed an analytical workflow to determine weak 13C enrichments of diverse plasma metabolites with conventional gas chromatography-mass spectrometry (GC-MS). The limit of quantification was increased by optimizing (1) the metabolite extraction from plasma, (2) the GC-MS measurement, and (3) most importantly, the computational data processing. We applied our workflow to study the catabolic dynamics of 13C-enriched wheat bread in three human subjects. For that purpose, we collected time-resolved human plasma samples at 16 timepoints after the consumption of 13C-labeled bread and quantified 13C enrichment of 12 metabolites (glucose, lactate, alanine, glycine, serine, citrate, glutamate, glutamine, valine, isoleucine, tyrosine, and threonine). Based on isotopomer specific analysis, we were able to distinguish catabolic profiles of starch and protein hydrolysis. More generally, our study highlights that conventional GC-MS equipment is sufficient to detect isotope traces below 1% if an appropriate data processing is integrated. [less ▲]

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See detailAnalysis of mitochondrial metabolism in situ: Combining stable isotope labeling with selective permeabilization
Nonnenmacher, Yannic; Palorini, Roberta; Fouquier d'Hérouël, Aymeric UL et al

in Metabolic Engineering (2017)

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See detail3D organotypic HepaRG cultures as in vitro model for acute and repeated dose toxicity studies.
Mueller, Daniel; Kramer, Lisa; Hoffmann, Esther et al

in Toxicology in vitro : an international journal published in association with BIBRA (2014), 28(1), 104-12

Predictive in vitro models alternative to in vivo animal will have a significant impact in toxicology. Conventional 2D models do not reflect the complexity of a 3D organ resulting in discrepancies between ... [more ▼]

Predictive in vitro models alternative to in vivo animal will have a significant impact in toxicology. Conventional 2D models do not reflect the complexity of a 3D organ resulting in discrepancies between experimental in vitro and in vivo data. Using 3D HepaRG organotypic cultures we tested four drugs (aflatoxin B1, amiodarone, valproic acid and chlorpromazine) for toxic effects and compared the results with 2D HepaRG and HepG2 cultures. We show that 3D HepaRG cultures are more sensitive than the other tested cultures to aflatoxin B1 which is only toxic upon metabolic activation in the liver. We observed that CYP3A4 activity is higher in the 3D HepaRG cultures compared to the 2D HepaRG cultures. Furthermore, we investigated repeated dose toxicity of chlorpromazine and assessed its effects on glucose and lactate metabolism. Sub-toxic concentrations of chlorpromazine induced significant metabolic changes in both 2D and 3D HepaRG cultures upon acute and repeated dose (3 doses) exposure. In summary, our data support the hypothesis that 3D cell culture models better mimic the in vivo tissue and improve cellular functionality. The 3D HepaRG organotypic cultures represent a high throughput system for drug toxicity screening. This system is therefore a promising tool in preclinical testing of human relevance which can allow reducing and/or replacing animal testing for drug adverse effects. [less ▲]

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