RESUMO
Printed circuit boards (PCBs) are the most complex and valuable component of electronic devices, but only 34% of them are recycled in an environmentally sound manner. Improving the recycling rate and efficiency requires a fast, reliable and uncostly analytical method. Although the X-ray fluorescence (XRF) shows high potential, it is often unreliable. In this study, we propose a novel XRF methodology for the elemental analysis of PCBs, using the certified reference material (CRM) to decrease uncertainty and enhance accuracy. The results show significant improvement in robustness and accuracy of portable XRF(pXRF) analyses for elements Cu, Pb, Ni, As and Au, with a relative average inaccuracy of approximately 5% compared to referenced values. The methodology validation carried out by comparing pXRF and inductively coupled plasma mass spectroscopy analyses of personal computer motherboard samples shows no statistically significant difference for elements Cu, Cr and Ag. The study shows that the calibration of pXRF by CRMs enables the necessary analysis of PCBs in an efficient and reliable manner and could be also be applied to different types of PCBs and other electronic components, batteries or contaminated soil samples.
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We introduce a new concept of yeast-derived biological matrix reference material for metabolomics research relying on in vivo synthesis of a defined biomass, standardized extraction followed by absolute quantification with isotope dilution. The yeast Pichia pastoris was grown using full control- and online monitoring fed-batch fermentations followed by fast cold methanol quenching and boiling ethanol extraction. Dried extracts served for the quantification campaign. A metabolite panel of the evolutionarily conserved primary metabolome (amino acids, nucleotides, organic acids, and metabolites of the central carbon metabolism) was absolutely quantified by isotope dilution utilizing uniformly labeled 13C-yeast-based internal standards. The study involved two independent laboratories employing complementary mass spectrometry platforms, namely hydrophilic interaction liquid chromatography-high resolution mass spectrometry (HILIC-HRMS) and gas chromatography-tandem mass spectrometry (GC-MS/MS). Homogeneity, stability tests (on a panel of >70 metabolites over a period of 6 months), and excellent biological repeatability of independent fermentations over a period of 2 years showed the feasibility of producing biological reference materials on demand. The obtained control ranges proved to be fit for purpose as they were either superior or comparable to the established reference materials in the field.
Assuntos
Saccharomyces cerevisiae , Espectrometria de Massas em Tandem , Cromatografia Gasosa-Espectrometria de Massas , Isótopos/metabolismo , Metaboloma , Metabolômica/métodos , Pichia/química , Espectrometria de Massas em Tandem/métodosRESUMO
The amyloid ß peptide, as one of the main components in senile plaque, represents a defining pathological feature for Alzheimer's disease, and is therefore commonly used as a biomarker for this disease in clinical analysis. However, the selection of suitable standards is limited here, since only a few are commercially available, and these suffer from varying purity. Hence, the accurate characterization of these standards is of great importance. In this study, we developed a method for the traceable quantification of the peptide content using species-specific isotope dilution and ICP-MS/MS detection. It is based on the separation of the sulfur-containing amino acids methionine and cysteine after oxidation and hydrolysis of the peptide. Using a strong anion exchange column, both amino acids could be separated from each other, as well as from their oxidized forms and sulfate. The sulfur content was determined via ICP-MS/MS using oxygen as reaction gas. Species-specific isotope dilution was enabled by using a 34S-labeled yeast hydrolysate, containing methionine sulfone and cysteic acid with different isotopic composition. The peptide contents of Aß standards (Aß40,42), as well as myoglobin and lysozyme with different degrees of purity, were determined. For validation purposes, the standard reference material NIST 2389a, which contains the amino acids in a similar concentration, was subjected to the developed sample preparation and analysis method. In addition to accounting for errors during sample preparation, high levels of accuracy and precision could be obtained using this method, making it fit-for-purpose for the characterization of peptide standards.
Assuntos
Peptídeos beta-Amiloides , Espectrometria de Massas em Tandem , Cromatografia Líquida de Alta Pressão , Isótopos , Fragmentos de Peptídeos , Espectrometria de Massas em Tandem/métodosRESUMO
Oxaliplatin is a very potent platinum(ii) drug which is frequently used in poly-chemotherapy schemes against advanced colorectal cancer. However, its benefit is limited by severe adverse effects as well as resistance development. Based on their higher tolerability, platinum(iv) prodrugs came into focus of interest. However, comparable to their platinum(ii) counterparts they lack tumor specificity and are frequently prematurely activated in the blood circulation. With the aim to exploit the enhanced albumin consumption and accumulation in the malignant tissue, we have recently developed a new albumin-targeted prodrug, which supposed to release oxaliplatin in a highly tumor-specific manner. In more detail, we designed a platinum(iv) complex containing two maleimide moieties in the axial position (KP2156), which allows selective binding to the cysteine 34. In the present study, diverse cell biological and analytical tools such as laser ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS), isotope labeling, and nano-scale secondary ion mass spectrometry (NanoSIMS) were employed to better understand the in vivo distribution and activation process of KP2156 (in comparison to free oxaliplatin and a non-albumin-binding succinimide analogue). KP2156 forms very stable albumin adducts in the bloodstream resulting in a superior pharmacological profile, such as distinctly prolonged terminal excretion half-life and enhanced effective platinum dose (measured by ICP-MS). The albumin-bound drug is accumulating in the malignant tissue, where it enters the cancer cells via clathrin- and caveolin-dependent endocytosis, and is activated by reduction to release oxaliplatin. This results in profound, long-lasting anticancer activity of KP2156 against CT26 colon cancer tumors in vivo based on cell cycle arrest and apoptotic cell death. Summarizing, albumin-binding of platinum(iv) complexes potently enhances the efficacy of oxaliplatin therapy and should be further developed towards clinical phase I trials.
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Non-targeted analysis by high-resolution mass spectrometry (HRMS) is an essential discovery tool in metabolomics. To date, standardization and validation remain a challenge. Community-wide accepted cost-effective benchmark materials are lacking. In this work, we propose yeast (Pichia pastoris) extracts derived from fully controlled fermentations for this purpose. We established an open-source metabolite library of >200 identified metabolites based on compound identification by accurate mass, matching retention times, and MS/MS, as well as a comprehensive literature search. The library includes metabolites from the classes of (1) organic acids and derivatives (2) nucleosides, nucleotides, and analogs, (3) lipids and lipid-like molecules, (4) organic oxygen compounds, (5) organoheterocyclic compounds, (6) organic nitrogen compounds, and (7) benzoids at expected concentrations ranges of sub-nM to µM. As yeast is a eukaryotic organism, key regulatory elements are highly conserved between yeast and all annotated metabolites were also reported in the human metabolome database (HMDB). Orthogonal state-of-the-art reversed-phase (RP-) and hydrophilic interaction chromatography mass spectrometry (HILIC-MS) non-targeted analysis and authentic standards revealed that 104 out of the 206 confirmed metabolites were reproducibly recovered and stable over the course of three years when stored at -80 °C. Overall, 67 out of these 104 metabolites were identified with comparably stable areas over all three yeast fermentation and are the ideal starting point for benchmarking experiments. The provided yeast benchmark material enabled not only to test for the chemical space and coverage upon method implementation and developments but also allowed in-house routines for instrumental performance tests. Transferring the quality control strategy of proteomics workflows based on the number of protein identification in HeLa extracts, metabolite IDs in the yeast benchmarking material can be used as metabolomics quality control. Finally, the benchmark material opens new avenues for batch-to-batch corrections in large-scale non-targeted metabolomics studies.
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We propose a fully automated novel workflow for lipidomics based on flow injection, followed by liquid chromatography-high-resolution mass spectrometry (FI/LC-HRMS). The workflow combined in-depth characterization of the lipidome achieved via reversed-phase LC-HRMS with absolute quantification by using a large number of lipid species-specific and/or retention time (RT)-matched/class-specific calibrants. The lipidome of 13C-labelled yeast (LILY) provided a large panel of cost-effective internal standards (ISTDs) covering triacylglycerols (TG), steryl esters (SE), free fatty acids (FA), diacylglycerols (DG), sterols (ST), ceramides (Cer), hexosyl ceramides (HexCer), phosphatidylglycerols (PG), phosphatidylethanolamines (PE), phosphatidic acids (PA), cardiolipins (CL), phosphatidylinositols (PI), phosphatidylserines (PS), phosphatidylcholines (PC), lysophosphatidylcholines (LPC) and lysophosphatidylethanolamines (LPE). The workflow in combination with the LILY lipid panel enables simultaneous quantification via (1) external multi-point calibration with internal standardization and (2) internal one-point calibration with LILY as a surrogate ISTD, increasing the coverage while keeping the accuracy and throughput high. Extensive measures on quality control allowed us to rank the calibration strategies and to automatically select the calibration strategy of the highest metrological order for the respective lipid species. Overall, the workflow enabled a streamlined analysis, with a limit of detection in the low femtomolar range, and provided validation tools together with absolute concentration values for >350 lipids in human plasma on a species level. Based on the selected standard panel, lipids from 7 classes (LPC, LPE, PC, PE, PI, DG, TG) passed stringent quality filters, which included QC accuracy, a precision and recovery bias of <30% and concentrations within the 99% confidence interval of the international laboratory comparison of SRM 1950, NIST, USA. The quantitative values are independent of common deuterated or non-endogenous ISTDs, thus offering cross-validation of different lipid methods and further standardizing lipidomics.
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In this work, a lipidomics workflow based on offline semi-preparative lipid class-specific fractionation by supercritical fluid chromatography (SFC) followed by high-resolution mass spectrometry was introduced. The powerful SFC approach offered separation of a wide polarity range for lipids, enabled enrichment (up to 3 orders of magnitude) of lipids, selective fractionation of 14 lipid classes/subclasses, and increased dynamic range enabling in-depth characterization. A significantly increased coverage of low abundant lipids improving lipid identification by numbers and degree (species and molecular level) was obtained in Pichia pastoris when comparing high-resolution mass spectrometry based lipidomics with and without prior fractionation. Proof-of-principle experiments using a standard reference material (SRM 1950, NIST) for human plasma showed that the proposed strategy enabled quantitative lipidomics. Indeed, for 70 lipids, the consensus values available for this sample could be met. Thus, the novel workflow is ideally suited for lipid class-specific purification/isolation from milligram amounts of sample while not compromising on omics type of analysis (identification and quantification). Finally, compared with established fractionation/pre-concentration approaches, semi-preparative SFC is superior in terms of versatility, as it involved only volatile modifiers and salt additives facilitating any follow-up use such as qualitative or quantitate analysis or further purification down to the single lipid species level. Graphical Abstract.
Assuntos
Cromatografia com Fluido Supercrítico/métodos , Lipidômica/métodos , Lipídeos/química , Espectrometria de Massas/métodos , Humanos , Metabolismo dos Lipídeos , Lipídeos/sangue , Pichia/química , Pichia/metabolismo , Plasma/químicaRESUMO
Resistance development is a major obstacle for platinum-based chemotherapy, with the anticancer drug oxaliplatin being no exception. Acquired resistance is often associated with altered drug accumulation. In this work we introduce a novel -omics workflow enabling the parallel study of platinum drug uptake and its distribution between nucleus/protein and small molecule fraction along with metabolic changes after different treatment time points. This integrated metallomics/metabolomics approach is facilitated by a tailored sample preparation workflow suitable for preclinical studies on adherent cancer cell models. Inductively coupled plasma mass spectrometry monitors the platinum drug, while the metabolomics tool-set is provided by hydrophilic interaction liquid chromatography combined with high-resolution Orbitrap mass spectrometry. The implemented method covers biochemical key pathways of cancer cell metabolism as shown by a panel of >130 metabolite standards. Furthermore, the addition of yeast-based 13C-enriched internal standards upon extraction enabled a novel targeted/untargeted analysis strategy. In this study we used our method to compare an oxaliplatin sensitive human colon cancer cell line (HCT116) and its corresponding resistant model. In the acquired oxaliplatin resistant cells distinct differences in oxaliplatin accumulation correlated with differences in metabolomic rearrangements. Using this multi-omics approach for platinum-treated samples facilitates the generation of novel hypotheses regarding the susceptibility and resistance towards oxaliplatin.
Assuntos
Antineoplásicos/farmacologia , Neoplasias do Colo/tratamento farmacológico , Oxaliplatina/farmacologia , Antineoplásicos/farmacocinética , Cromatografia Líquida/métodos , Neoplasias do Colo/metabolismo , Resistencia a Medicamentos Antineoplásicos , Células HCT116 , Humanos , Espectrometria de Massas/métodos , Metabolômica/métodos , Oxaliplatina/farmacocinéticaRESUMO
13C metabolite tracer and metabolic flux analyses require upfront experimental planning and validation tools. Here, we present a validation scheme including a comparison of different LC methods that allow for customization of analytical strategies for tracer studies with regard to the targeted metabolites. As the measurement of significant changes in labeling patterns depends on the spectral accuracy, we investigate this aspect comprehensively for high-resolution orbitrap mass spectrometry combined with reversed-phase chromatography, hydrophilic interaction liquid chromatography, or anion-exchange chromatography. Moreover, we propose a quality control protocol based on (1) a metabolite containing selenium to assess the instrument performance and on (2) in vivo synthesized isotopically enriched Pichia pastoris to validate the accuracy of carbon isotopologue distributions (CIDs), in this case considering each isotopologue of a targeted metabolite panel. Finally, validation involved a thorough assessment of procedural blanks and matrix interferences. We compared the analytical figures of merit regarding CID determination for over 40 metabolites between the three methods. Excellent precisions of less than 1% and trueness bias as small as 0.01-1% were found for the majority of compounds, whereas the CID determination of a small fraction was affected by contaminants. For most compounds, changes of labeling pattern as low as 1% could be measured. Graphical abstract.
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Isótopos de Carbono/análise , Cromatografia por Troca Iônica/métodos , Cromatografia de Fase Reversa/métodos , Espectrometria de Massas/métodos , Estudos de Validação como Assunto , Resinas de Troca Aniônica/química , Isótopos de Carbono/normas , Interações Hidrofóbicas e Hidrofílicas , Pichia/química , Padrões de Referência , Selênio/químicaRESUMO
A novel integrated metabolomics/lipidomics workflow is introduced enabling high coverage of polar metabolites and non-polar lipids within one analytical run. Dual HILIC and RP chromatography were combined to high-resolution mass spectrometry. As a major advantage, only one data file per sample was obtained by fully automated simultaneous analysis of two extracts per sample. Hence, the unprecedented high coverage without compromise on analytical throughput was not only obtained by the orthogonality of the chromatographic separations, but also by the implementation of dedicated sample preparation procedures resulting in optimum extraction efficiency for both sub-omes. Thus, the method addressed completely hydrophilic sugars and organic acids next to water-insoluble triglycerides. As for the timing of the dual chromatography setup, HILIC and RP separation were performed consecutively. However, re-equilibration of the HILIC column during elution of RP compounds and vice versa reduced the overall analysis time by one third to 32 min. Application to the Standard Reference Material SRM 1950 - Metabolites in Frozen Human Plasma resulted in >100 metabolite and >380 lipid identifications based on accurate mass implementing fast polarity switching and acquiring data dependent MS2 spectra with the use of automated exclusion lists. Targeted quantification based on external calibrations and 13C labeled yeast internal standards was successfully accomplished for 59 metabolites. Moreover, the potential for lipid quantification was shown integrating non-endogenous lipids as internal standards. In human plasma, concentrations ranging over 4 orders of magnitude (low nM to high µM) were assessed.
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Cromatografia Líquida/métodos , Cromatografia de Fase Reversa/métodos , Lipídeos/sangue , Metabolômica/métodos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas/métodosRESUMO
Lipid identification and quantification are essential objectives in comprehensive lipidomics studies challenged by the high number of lipids, their chemical diversity, and their dynamic range. In this work, we developed a tailored method for profiling and quantification combining (1) isotope dilution, (2) enhanced isomer separation by C30 fused-core reversed-phase material, and (3) parallel Orbitrap and ion trap detection by the Orbitrap Fusion Lumos Tribid mass spectrometer. The combination of parallelizable ion analysis without time loss together with different fragmentation techniques (HCD/CID) and an inclusion list led to higher quality in lipid identifications exemplified in human plasma and yeast samples. Moreover, we used lipidome isotope-labeling of yeast (LILY)-a fast and efficient in vivo labeling strategy in Pichia pastoris-to produce (nonradioactive) isotopically labeled eukaryotic lipid standards in yeast. We integrated the 13C lipids in the LC-MS workflow to enable relative and absolute compound-specific quantification in yeast and human plasma samples by isotope dilution. Label-free and compound-specific quantification was validated by comparison against a recent international interlaboratory study on human plasma SRM 1950. In this way, we were able to prove that LILY enabled quantification leads to accurate results, even in complex matrices. Excellent analytical figures of merit with enhanced trueness, precision and linearity over 4-5 orders of magnitude were observed applying compound-specific quantification with 13C-labeled lipids. We strongly believe that lipidomics studies will benefit from incorporating isotope dilution and LC-MSn strategies.
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Cromatografia de Fase Reversa/métodos , Lipídeos/sangue , Espectrometria de Massas/métodos , Isótopos de Carbono/análise , Isótopos de Carbono/sangue , Humanos , Técnicas de Diluição do Indicador , Lipídeos/análise , Fluxo de Trabalho , Leveduras/químicaRESUMO
METLIN originated as a database to characterize known metabolites and has since expanded into a technology platform for the identification of known and unknown metabolites and other chemical entities. Through this effort it has become a comprehensive resource containing over 1 million molecules including lipids, amino acids, carbohydrates, toxins, small peptides, and natural products, among other classes. METLIN's high-resolution tandem mass spectrometry (MS/MS) database, which plays a key role in the identification process, has data generated from both reference standards and their labeled stable isotope analogues, facilitated by METLIN-guided analysis of isotope-labeled microorganisms. The MS/MS data, coupled with the fragment similarity search function, expand the tool's capabilities into the identification of unknowns. Fragment similarity search is performed independent of the precursor mass, relying solely on the fragment ions to identify similar structures within the database. Stable isotope data also facilitate characterization by coupling the similarity search output with the isotopic m/ z shifts. Examples of both are demonstrated here with the characterization of four previously unknown metabolites. METLIN also now features in silico MS/MS data, which has been made possible through the creation of algorithms trained on METLIN's MS/MS data from both standards and their isotope analogues. With these informatic and experimental data features, METLIN is being designed to address the characterization of known and unknown molecules.
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Extratos Celulares/análise , Bases de Dados de Compostos Químicos/estatística & dados numéricos , Conjuntos de Dados como Assunto/estatística & dados numéricos , Metabolômica/métodos , Metabolômica/estatística & dados numéricos , Pichia/química , Pichia/metabolismo , Espectrometria de Massas em Tandem/estatística & dados numéricosRESUMO
The ruthenium drug and GRP78 inhibitor KP1339/IT-139 has already demonstrated promising anticancer activity in a phase I clinical trial. This study aimed to identify mechanisms underlying increased sensitivity to KP1339 treatment. Based on a screen utilizing 23 cell lines, a small panel was selected to compare KP1339-sensitive and low-responsive models. KP1339 sensitivity was neither based on differences in ruthenium accumulation, nor sensitivity to oxidative stress or constituents of KP1339 (ruthenium chloride and indazole). Subsequently, the biochemical response to KP1339 was analyzed using whole genome expression arrays indicating that, while sensitive cell lines were characterized by "response to chemical stimuli" and "regulation of cell death", low-responsive cells preferentially activated pathways controlling cell cycle, DNA repair, and metabolism. Cell culture experiments confirmed that, while low-responsive cells executed cell cycle arrest in G2 phase, pronounced apoptosis induction via activation of caspase 8 was found in sensitive cells. Cell death induction is based on a unique disruption of the ER homeostasis by depletion of key cellular chaperones including GRP78 in combination with enhanced KP1339-mediated protein damage.
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Antineoplásicos/farmacologia , Caspase 8/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Choque Térmico/antagonistas & inibidores , Compostos Organometálicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Reparo do DNA/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , RNA Mensageiro/metabolismo , Rutênio/metabolismoRESUMO
In this work, simultaneous targeted metabolic profiling by isotope dilution and non-targeted fingerprinting is proposed for cancer cell studies. The novel streamlined metabolomics workflow was established using anion-exchange chromatography (IC) coupled to high-resolution mass spectrometry (MS). The separation time of strong anion-exchange (2 mm column, flow rate 380 µL min-1, injection volume 5 µL) could be decreased to 25 min for a target list comprising organic acids, sugars, sugar phosphates, and nucleotides. Internal standardization by fully 13C labeled Pichia pastoris extracts enabled absolute quantification of the primary metabolites in adherent cancer cell models. Limits of detection (LODs) in the low nanomolar range and excellent intermediate precisions of the isotopologue ratios (on average <5%, N = 5, over 40 h) were observed. As a result of internal standardization, linear dynamic ranges over 4 orders of magnitude (5 nM-50 µM, R2 > 0.99) were obtained. Experiments on drug-sensitive versus resistant SW480 cancer cells showed the feasibility of merging analytical tasks into one analytical run. Comparing fingerprinting with and without internal standard proved that the presence of the 13C labeled yeast extract required for absolute quantification was not detrimental to non-targeted data evaluation. Several interesting metabolites were discovered by accurate mass and comparing MS2 spectra (acquired in ddMS2 mode) with spectral libraries. Significant differences revealed distinct metabolic phenotypes of drug-sensitive and resistant SW480 cells.
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Metabolômica , Isótopos de Carbono , Cromatografia por Troca Iônica , Humanos , Espectrometria de Massas , Pichia/metabolismo , Células Tumorais CultivadasRESUMO
Quantification is an essential task in comprehensive lipidomics studies challenged by the high number of lipids, their chemical diversity and their dynamic range of the lipidome. In this work, we introduce lipidome isotope labeling of yeast (LILY) in order to produce (non-radioactive) isotopically labeled eukaryotic lipid standards in yeast for normalization and quantification in mass spectrometric assays. More specifically, LILY is a fast and efficient in vivo labeling strategy in Pichia pastoris for the production of 13C labeled lipid library further paving the way to comprehensive compound-specific internal standardization in quantitative mass spectrometry based assays. More than 200 lipid species (from PA, PC, PE, PG, PI, PS, LysoGP, CL, DAG, TAG, DMPE, Cer, HexCer, IPC, MIPC) were obtained from yeast extracts with an excellent 13C enrichment >99.5%, as determined by complementary high resolution mass spectrometry based shotgun and high resolution LC-MS/MS analysis. In a first proof of principle study we tested the relative and absolute quantification capabilities of the 13C enriched lipids obtained by LILY using a parallel reaction monitoring based LC-MS approach. In relative quantification it could be shown that compound specific internal standardization was essential for the accuracy extending the linear dynamic range to four orders of magnitude. Excellent analytical figures of merit were observed for absolute quantification for a selected panel of 5 investigated glycerophospholipids (e.g. LOQs around 5 fmol absolute; typical concentrations ranging between 1 to 10 nmol per 108 yeast cell starting material; RSDs <10% (N = 4)).
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Isótopos de Carbono/química , Marcação por Isótopo , Lipídeos/análise , Cromatografia Líquida , Saccharomyces cerevisiae , Espectrometria de Massas em TandemRESUMO
To date, preclinical studies have addressed drug accumulation and intracellular distribution of cisplatin by determination of the total Pt content. In this work, the use of liquid chromatography in combination with inductively coupled plasma mass spectrometry (LC-ICP-MS) enabled accurate intact cisplatin quantification in cell model experiments. Hence, for the first time, intracellular drug degradation, drug accumulation and drug efflux were studied by actually quantifying the intact drug, along with the total Pt content of the cell nucleus, the cytosol and the low molecular weight fraction of the cytosol. The latter fraction was obtained by centrifugal filtration (cut-off filter of 10 kDa). Flow injection (FI)-ICP-MS was implemented for platinum quantification. In a first step, kinetics of intracellular cisplatin degradation was addressed by incubating cell extracts with sub-µM drug concentration levels. A half-life of 2 hours was observed in cell extracts of two different cancer cell lines (colon carcinoma and human mesothelioma), which was significantly shorter than that observed in sodium chloride. Hence, it was suggested that intact and nonaquated cisplatin was reacting with cellular components. Due to the large excess of potential binding partners pseudo first order kinetics were observed. The drug accumulation experiments revealed rapid uptake of the drug into the cytosol and the nucleus. Moreover, a significant fraction of Pt was bound to intracellular high molecular weight biomolecules after one hour of exposure. With ongoing time, the intracellular Pt concentration was increasing. However, the cisplatin concentration remained constant during 5 hours of continuous exposure. Assuming a cell volume of 10(-12) L, an intracellular concentration corresponding to the cisplatin concentration in the cell culture medium (5 µM) was estimated. At any time of investigation, intact cisplatin was the predominant species in the low molecular weight fraction of the cytosol. These findings support the hypothesis of passive diffusion as an uptake mechanism. Finally, a model experiment was designed resembling the situation of limited drug exposure time. Human mesothelioma cells were incubated with 5 µM cisplatin for 3 hours. Then the culture medium was replaced and the drug efflux was studied. The observed efflux was biphasic, with the intact cisplatin being removed within the first hour of investigation, while the Pt-protein adduct fraction was removed only partially (30% were still found in the cytosol after 24 hours). No net transfer of Pt from the cytosol to the nucleus fraction was observed after medium replacement.
Assuntos
Cromatografia Líquida/métodos , Cisplatino/farmacocinética , Espectrometria de Massas/métodos , Neoplasias/metabolismo , Cisplatino/metabolismo , Células HCT116 , HumanosRESUMO
Maleimide-functionalised Pt(IV) complexes with highly selective binding properties to thiol groups were synthesised as precursors for binding of thiol-containing tumour-targeting molecules like human serum albumin.
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Antineoplásicos/farmacologia , Neoplasias do Colo/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Maleimidas/química , Compostos Organoplatínicos/farmacologia , Pró-Fármacos/farmacologia , Albumina Sérica/análise , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/patologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Estrutura Molecular , Compostos Organoplatínicos/síntese química , Compostos Organoplatínicos/química , Pró-Fármacos/síntese química , Pró-Fármacos/química , Relação Estrutura-Atividade , Compostos de Sulfidrila/químicaRESUMO
Ruthenium-indazole complexes are promising anticancer agents undergoing clinical trials. KP1339 is administered intravenously (i.v.), where serum proteins are the first available biological binding partners. In order to gain a better insight into the mode of action, mice were treated with different doses of KP1339 i.v. and sacrificed at different time points. The blood plasma was isolated from blood samples and analyzed by capillary zone electrophoresis (CZE) and size exclusion/anion exchange chromatography (SEC-IC) both combined on-line to inductively coupled plasma-mass spectrometry (ICP-MS). The performance of the analytical methodology was compared and the interaction of KP1339 with mouse plasma proteins characterized in vivo. Interestingly, the samples of the mice treated with 50 mg kg(-1) and terminated after 24 h showed a ca. 4-fold lowered albumin content and increased ruthenation of albumin aggregates as compared to the untreated control group and the 40 mg kg(-1) group. The majority of Ru was bound to albumin and the stoichiometry of the KP1339 protein binding was determined through the molar Ru/S ratio. In general, good agreement of the data obtained with both techniques was achieved and the SEC-IC method was found to be more sensitive as compared to the CZE-ICP-MS approach, whereas the latter benefits from the shorter analysis time and lower sample consumption.