Chemische Charakterisierung und Spurenanalytik
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Organisationseinheit der BAM
- 1.7 Organische Spuren- und Lebensmittelanalytik (71) (entfernen)
N-Myc is a transcription factor that is aberrantly expressed in many tumor types and is often correlated with poor patient prognosis. Recently, several lines of evidence pointed to the fact that oncogenic activation of Myc family proteins is concomitant with reprogramming of tumor cells to cope with an enhanced need for metabolites during cell growth. These adaptions are driven by the ability of Myc proteins to act as transcriptional amplifiers in a tissue-of-origin specific manner. Here, we describe the effects of N-Myc overexpression on metabolic reprogramming in neuroblastoma cells. Ectopic expression of N-Myc induced a glycolytic switch that was concomitant with enhanced sensitivity towards 2-deoxyglucose, an inhibitor of glycolysis. Moreover, global metabolic profiling revealed extensive alterations in the cellular metabolome resulting from overexpression of N-Myc. Limited supply with either of the two main carbon sources, glucose or glutamine, resulted in distinct shifts in steady-state metabolite levels and significant changes in glutathione metabolism. Interestingly, interference with glutamine-glutamate conversion preferentially blocked proliferation of N-Myc overexpressing cells, when glutamine levels were reduced. Thus, our study uncovered N-Myc induction and nutrient levels as important metabolic master switches in neuroblastoma cells and identified critical nodes that restrict tumor cell proliferation.
The presence of any aflatoxin contamination in exported figs needs to be monitored and measured through reliable and traceable methods, which require pure and matrix certified reference materials. On the other hand, certified reference materials (CRM) for determination of aflatoxins in dried fig are not yet available. Moreover, there is a lack of CRMs to be used in routine testing laboratories for method validation and quality control. The routine testing laboratories, participating in commercial proficiency testing (PT) programs, use the results available from consensus values to evaluate the performance of the participating laboratories, rather than metrologically traceable assigned values. This study initially proposed as a key comparison and presented at the EURAMET TC-MC SCOA meeting in Malta in 2015 and subsequently at the CCQM OAWG meeting in April 2015, proposes a CRM candidate for determination of levels of aflatoxins B1, B2, G1, G2 and their total in dried. Evidence of successful participation in formal, relevant international comparisons are needed to document measurement capability claims (CMCs) made by national metrology institutes (NMIs) and designated institutes (DIs).
In total nine NMI/DI participated in the Track C Key Comparison CCQM-K138 Determination of aflatoxins (AFB1, AFB2, AFG1, AFG2 and Total AFs) in Dried Fig. Participants were requested to evaluate the mass fractions expressed in ng/g units, of aflatoxins B1, B2, G1, G2 and total aflatoxin in a dried food matrix, dried fig. The CCQM-K138 results for the determination of aflatoxins (AFB1, AFB2, AFG1, AFG2 and total AFs) are ranging from 5.17 to 7.27 ng/g with an %RSD of 10.47 for AFB1, ranging from 0.60 to 0.871 ng/g with an %RSD of 11.69 for AFB2, ranging from 1.98 to 2.6 ng/g with an %RSD of 10.36 for AFG1, ranging from 0.06 to 0.32 ng/g with an %RSD of 35.6 for AFG2, and ranging from 8.29 to 10.31 ng/g with an %RSD of 7.69 for Total AFs. All participants based their analyses on LC-MS/MS, HPLC-FLD, HR-LC/MS, and IDMS. Brief descriptions of the analytical methods used by the participants, including sample preparation, analytical technique, calibrants, and quantification approach are summarized in Appendix F. Linear Pool was used to assign the Key Comparison Reference Values (KCRVs) for B1, B2, G1, G2 and total aflatoxins. Due to the traceability requirements for the calibrants not being met, results of KEBS, INTI, VNIIM and BAM were excluded from KCRV determination.
Successful participation in CCQM-K138 demonstrates the following measurement capabilities in determining mass fraction of organic compounds, with molecular mass of 100 g/mol to 500 g/mol, having high polarity (pKow > -2), in mass fraction range from 0.05 ng/g to 500 ng/g in dried food matrices.
Metabolites from Alternaria fungi exhibit a variety of biological properties such as phytotoxic, cytotoxic, or antimicrobial activity. Optimization of a literature procedure culminated in an efficient total synthesis of (−)-altenuene as well as a stable isotope-labeled derivative suitable for implementation in a LC-MS/MS method for mycotoxin analysis.
In this in vitro study, we got a first insight of a possible potential of Hypericin for the treatment of pediatric soft tissue sarcoma. By coupling with radioiodine, we developed a novel approach for a combined anti-tumor treatment. The in vitro experiments lay the foundation for further in vivo experiments, which are needed to study the effects of a sequential administration of 131I-HYP and HYP.
Tempeh is a common food in Indonesia, produced by fungal fermentation of soybeans using Rhizopus sp., as well as Aspergillus oryzae, for inoculation. Analogously, for economic reasons, mixtures of maize and soybeans are used for the production of so-called tempeh-like products. For maize, a contamination with the mycoestrogen zearalenone (ZEN) has been frequently reported. ZEN is a mycotoxin which is known to be metabolized by Rhizopus and Aspergillus species. Consequently, this study focused on the ZEN transformation during tempeh fermentation. Five fungal strains of the genera Rhizopus and Aspergillus, isolated from fresh Indonesian tempeh and authentic Indonesian inocula, were utilized for tempeh manufacturing from a maize/soybean mixture (30:70) at laboratory-scale. Furthermore, comparable tempeh-like products obtained from Indonesian markets were analyzed. Results from the HPLC-MS/MS analyses show that ZEN is intensely transformed into its metabolites alpha-zearalenol (alpha-ZEL), ZEN-14-sulfate, alpha-ZEL-sulfate, ZEN-14-glucoside, and ZEN-16-glucoside in tempeh production. alpha-ZEL, being significantly more toxic than ZEN, was the main metabolite in most of the Rhizopus incubations, while in Aspergillus oryzae fermentations ZEN-14-sulfate was predominantly formed. Additionally, two of the 14 authentic samples were contaminated with ZEN, alpha-ZEL and ZEN-14-sulfate, and in two further samples, ZEN and alpha-ZEL, were determined. Consequently, tempeh fermentation of ZEN-contaminated maize/soybean mixture may lead to toxification of the food item by formation of the reductive ZEN metabolite, alpha-ZEL, under model as well as authentic conditions.
The knowledge of transformation pathways and identification of transformation products (TPs) of veterinary drugs is important for animal health, food, and environmental matters. The active agent Monensin (MON) belongs to the ionophore antibiotics and is widely used as a veterinary drug against coccidiosis in broiler farming. However, no electrochemically (EC) generated TPs of MON have been described so far. In this study, the online coupling of EC and mass spectrometry (MS) was used for the generation of oxidative TPs. EC-conditions were optimized with respect to working electrode material, solvent, modifier, and potential polarity. Subsequent LC/HRMS (liquid chromatography/high resolution mass spectrometry) and MS/MS experiments were performed to identify the structures of derived TPs by a suspected target analysis. The obtained EC-results were compared to TPs observed in metabolism tests with microsomes and hydrolysis experiments of MON.
Five previously undescribed TPs of MON were identified in our EC/MS based study and one TP, which was already known from literature and found by a microsomal assay, could be confirmed.
Two and three further TPs were found as products in microsomal tests and following hydrolysis, respectively. We found decarboxylation, O-demethylation and acid-catalyzed ring-opening reactions to be the major mechanisms of MON transformation.
In this study, the thermal decomposition of 1,3,5-tris-(2,3-dibromopropyl)-1,3,5-triazine-2,4,6-trione (TDBPTAZTO) and 2,4,6-tris-(2,4,6-tribromo-phenoxy)-1,3,5-triazine (TTBP-TAZ) were investigated for the first time by thermogravimetric analysis. Both compounds were thermal degraded between 225 and 350 °C (TDBP-TAZTO) as well as 300 and 400 °C (TTBP-TAZ).
As a result, mass loss (%) of 98.5% for TTBP-TAZ and 95.4% for TDBP-TAZTO at 600 °C under N2 were observed. The major pyrolytic degradation products of TTBP-TAZ were formed in a single step and identified by FTIR analysis as 2,4,6-tribromophenol and further bromine-substituted aromatic compounds. In comparison, TDBP-TAZTO was pyrolytic degraded in two steps, whereby on the first step the release of hydrogen Bromide and 1,3,5-triallyl-1,3,5-triazine-2,4,6-trione could be detected. In the second minor step, isocyanic acid could be additionally identified.
Subsequently, the obtained products of the TGA-FTIR measurements were used for a targeted search for mass fragments in mass spectrometry measurements. For TTBP-TAZ, only the degradation product 1,3,5-tribromobenzene could be detected by MS/MS analyzes. No comparable thermal degradation products, except hydrogen bromide, were observed in the MS/MS spectra of TDBP-TAZTO. Therefore, the search of further mass fragments was not possible compared to the findings of the TGA-FTIR measurements.
Cancer cell lipid class homeostasis is altered under nutrient-deprivation but stable under hypoxia
(2019)
Background: Cancer cells modify the balance between fatty acid (FA) synthesis and uptake under metabolic stress, induced by oxygen/nutrient deprivation. These modifications were shown to alter the levels of individual triglyceride (TG) or phospholipid sub-species. To attain a holistic overview of the lipidomic profiles of cancer cells under stress we performed a broad lipidomic assay, comprising 244 lipids from six major classes. This assay allowed us to perform robust analyses and assess the changes in averages of broader lipid-classes, stratified on the basis of saturation index of their fatty-acyl side chains.
Methods: Global lipidomic profiling using Liquid Chromatography-Mass Spectrometry was performed to assess lipidomic profiles of biologically diverse cancer cell lines cultivated under metabolically stressed conditions.
Results: Neutral lipid compositions were markedly modified under serum-deprived conditions and, strikingly, the cellular level of triglyceride subspecies decreased with increasing number of double bonds in their fatty acyl chains.
In contrast and unexpectedly, no robust changes were observed in lipidomic profiles of hypoxic (2% O2) cancer cells despite concurrent changes in proliferation rates and metabolic gene expression.
Conclusions: Serum-deprivation significantly affects lipidomic profiles of cancer cells. Although, the levels of individual lipid moieties alter under hypoxia (2% O2), the robust averages of broader lipid classes remain unchanged.
Cancer cell lipid class homeostasis is altered under nutrient-deprivation but stable under hypoxia
(2019)
Background: Cancer cells modify the balance between fatty acid (FA) synthesis and uptake under metabolic stress, induced by oxygen/nutrient deprivation. These modifications were shown to alter the levels of individual triglyceride (TG) or phospholipid sub-species. To attain a holistic overview of the lipidomic profiles of cancer cells under stress we performed a broad lipidomic assay, comprising 244 lipids from six major classes. This assay allowed us to perform robust analyses and assess the changes in averages of broader lipid-classes, stratified on the basis of saturation index of their fatty-acyl side chains.
Methods: Global lipidomic profiling using Liquid Chromatography-Mass Spectrometry was performed to assess lipidomic profiles of biologically diverse cancer cell lines cultivated under metabolically stressed conditions.
Results: Neutral lipid compositions were markedly modified under serum-deprived conditions and, strikingly, the cellular level of triglyceride subspecies decreased with increasing number of double bonds in their fatty acyl chains.
In contrast and unexpectedly, no robust changes were observed in lipidomic profiles of hypoxic (2% O2) Cancer cells despite concurrent changes in proliferation rates and metabolic gene expression.
Conclusions: Serum-deprivation significantly affects lipidomic profiles of cancer cells. Although, the levels of individual lipid moieties alter under hypoxia (2% O2), the robust averages of broader lipid classes remain unchanged.