RESUMO
Well-absorbed iron-based nanoparticulated materials are a promise for the oral management of iron deficient anemia. In this work, a battery of in vitro and in situ experiments are combined for the evaluation of the uptake, distribution and toxicity of new synthesized ultrasmall (4 nm core) Fe2O3 nanoparticles coated with tartaric/adipic acid with potential to be used as oral Fe supplements. First, the in vitro simulated gastric acid solubility studies by TEM and HPLC-ICP-MS reveal a partial reduction of the core size of about 40% after 90 min at pH 3. Such scenario confirms the arrival of the nanoparticulate material in the small intestine. In the next step, the in vivo absorption through the small intestine by intestinal perfusion experiments is conducted using the sought nanoparticles in Wistar rats. The quantification of Fe in the NPs suspension before and after perfusion shows Fe absorption levels above 79%, never reported for other Fe treatments. Such high absorption levels do not seem to compromise cell viability, evaluated in enterocytes-like models (Caco-2 and HT-29) using cytotoxicity, ROS production, genotoxicity and lipid peroxidation tests. Moreover, regional differences in terms of Fe concentration are obtained among different parts of the small intestine as duodenum > jejunum > ileum. Complementary transmission electron microscopy (TEM) images show the presence of the intact particles around the intestinal microvilli without significant tissue damage. These studies show the high potential of these NP preparations for their use as oral management of anemia.
Assuntos
Compostos Férricos/farmacocinética , Compostos Férricos/toxicidade , Absorção Intestinal/efeitos dos fármacos , Intestino Delgado/metabolismo , Nanopartículas/toxicidade , Administração Oral , Animais , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Compostos Férricos/química , Células HT29 , Humanos , Intestino Delgado/efeitos dos fármacos , Masculino , Microscopia Eletrônica de Transmissão , Nanopartículas/química , Tamanho da Partícula , Ratos , Ratos Wistar , Distribuição TecidualRESUMO
Quantitative proteomics and absolute determination of proteins are topics of fast growing interest, since only the quantity of proteins or changes in their abundance reflect the status and extent of changes of a given biological system. Quantification of the desired proteins has been carried out by molecule specific MS techniques, but relative quantifications are commonplace so far even resorting to stable isotope labelling techniques such as ICAT and SILAC. In the last decade the idea of using element-selective mass spectrometric detection (e.g. ICP-MS instruments) to achieve absolute quantification has been realised and ICP-MS stands now as a new tool in the field of quantitative proteomics. In this review the emerging role of ICP-MS in protein and proteomic analysis is highlighted. The potential of ICP-MS methods and strategies for screening multiple heteroatoms (e.g. S, P, Se, metals) in proteins and their mixtures and extraordinary capabilities to tackle the problem of absolute protein quantifications, via heteroatom determinations, are discussed and illustrated. New avenues are also open derived from the use of ICP-MS for precise isotope abundance measurements in polyisotopic heteroatoms. The "heteroatom (isotope)-tagged proteomics" concept is focused on the use of naturally present element tags and also extended to any protein by resorting to bioconjugation reactions (i.e. labelling sought proteins and peptides with ICP-MS detectable heteroatoms). A major point of this review is displaying the possibilities of using a "hard" ion source, the ICP, to complement well-established "soft" ion sources for mass spectrometry to tackle present proteomic analysis.
Assuntos
Espectrometria de Massas/métodos , Proteoma , Proteômica/métodos , Animais , Bivalves , Calibragem , Eletroforese em Gel de Poliacrilamida , Humanos , Metaloproteínas/química , Metais/química , Peptídeos/química , Fósforo/química , Proteínas/química , Reprodutibilidade dos Testes , Selênio/química , Enxofre/químicaRESUMO
Iron is involved in the function of all living cells and, in fact, many diseases arise from imbalances in iron homeostasis. Therefore, the development of analytical methodologies to improve and automate the measurement of clinical indices of iron status has increased tremendously over the years. This work describes the development of two complementary methodologies to evaluate transferrin (Tf) saturation, total iron-binding capacity (TIBC), unsaturated iron-binding capacity (UIBC) and serum iron based on the use of iron-selective monitoring by inductively coupled plasma mass spectrometry (ICP-MS). The first methodology is based on the saturation of transferrin (Tf) with natural Fe3+ followed by separation of the different sialoforms in an anion exchange column (Mono-Q) to quantify the iron in each Tf sialoform and total Tf by ICP-MS using post-column isotope dilution analysis. In the second part, the saturation is done with an iron tracer (57Fe) and the application of pattern deconvolution analysis permits the direct quantification of the Tf saturation, the serum iron and the unsaturated iron-binding capacity. These strategies are validated by using a reference serum certified for total Tf and tested also in serum samples from individuals suffering from hemochromatosis and Fe-supplemented patients. The results obtained for all the parameters related to Fe status were in good agreement with those obtained by clinical tests. The use of stable isotope labelling in connection with the on-line coupling of fast protein liquid chromatography (FPLC) to ICP-MS allows the accurate determination of several parameters of great clinical relevance in iron homeostasis by means of two independent chromatographic runs. The main advantage of the proposed methodology is the number of parameters that can be simultaneously obtained.
Assuntos
Cromatografia por Troca Iônica/métodos , Ferro/sangue , Espectrometria de Massas/métodos , Anemia Hipocrômica/sangue , Hemocromatose/sangue , Humanos , Técnicas de Diluição do Indicador , Ferro/metabolismo , Isótopos de Ferro , Marcação por Isótopo/métodos , Transferrina/metabolismoRESUMO
Many essential cellular functions such as growth rate, motility, and metabolic activity are linked to reversible protein phosphorylation, since they are controlled by signaling cascades based mainly on phosphorylation/dephosphorylation events. Quantification of global or site-specific protein phosphorylation is not straightforward with standard proteomic techniques. The coupling of capillary liquid chromatography (microLC) with ICP-MS (inductively coupled plasma-mass spectrometry) is a method which allows a quantitative screening of protein extracts for their phosphorus and sulfur content, and thus provides access to the protein phosphorylation degree. In extension of a recent pilot study, we analyzed protein extracts from the model organisms Arabidopsis thaliana and Chlamydomonas reinhardtii as representatives for multicellular and unicellular green photosynthetically active organisms. The results indicate that the average protein phosphorylation level of the algae C. reinhardtii is higher than that of A. thaliana. Both the average phosphorylation levels were found to be between the extreme values determined so far for prokaryotes (C. glutamicum, lowest levels) and eukaryotes (Mus musculus, highest levels). Tissue samples of A. thaliana representing different stages of plant development showed varying levels of protein phosphorylation indicating a different adjustment of the kinase/phosphatase system. We also utilized the microLC-ICP-MS technology to estimate the efficiency of a novel phosphoprotein enrichment method based on aluminum hydroxide, since the enrichment of phosphorylated species is often an essential step for their molecular characterization.