Browsing by Author "Esteban Fernández, Diego"
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PublicationA shotgun approach for the identification of platinum–protein complexes(Springer, 2015) Moraleja, Irene; Moreno Gordaliza, Estefanía; Esteban Fernández, Diego; Mena Fernández, María Luz; Linscheid, Michael W.; Gómez Gómez, M.MilagrosA shotgun approach including peptide-based OFFGEL-isoelectric focusing (IEF) fractionation has been developed with the aim of improving the identification of platinum-binding proteins in biological samples. The method is based on a filter-aided sample preparation (FASP) tryptic digestion under denaturing and reducing conditions of cisplatin–, oxaliplatin–, and carboplatin–protein complexes, followed by OFFGEL-IEF separation of the peptides. Any risk of platinum loss is minimized throughout the procedure due to the removal of the reagents used after each stage of the FASP method and the absence of thiol-based reagents in the focusing buffer employed in the IEF separation. The platinum–peptide complexes stability after the FASP digestion and the IEF separation was confirmed by size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS). The suitability of peptide-based OFFGEL-IEF fractionation for reducing the sample complexity for further nano-liquid chromatographyelectrospray ionization-tandem mass spectrometry (nLC-ESIMS/MS) analysis has been demonstrated, allowing the detection of platinum-containing peptides, with significantly lower abundance and ionization efficiency than unmodified peptides. nLCMS/MS analysis of selected OFFGEL-IEF fractions from tryptic digests with different complexity degrees: standard human serum albumin (HSA), a mixture of five proteins (albumin, transferrin, carbonic anhydrase, myoglobin, and cytochrome-c) and human blood serum allowed the identification of several platinum–peptides from cisplatin–HSA. Cisplatin-binding sites in HSA were elucidated from the MS/MS spectra and assessed considering the protein three-dimensional structure. Most of the potential superficial binding sites available on HSA were identified for all the samples, including a biologically relevant cisplatin-cross-link of two protein domains, demonstrating the capabilities of the methodology PublicationAccumulation, Fractionation, and Analysis of Platinum in Toxicologically Affected Tissues after Cisplatin, Oxaliplatin, and Carboplatin Administration(Oxford Academic, 2008-03) Esteban Fernández, Diego; Verdaguer, J.M.; Ramírez, R.; Palacios Corvillo, María A.; Gómez Gómez, M. MilagrosAntitumoral Pt-containing drugs present side effects like nephrotoxicity and ototoxicity. Several systematic experiments have been carried out with Wistar rats treated with cisplatin, carboplatin, and oxaliplatin to study Pt-drugs accumulation and elimination, and Pt-biomolecule distribution in the cells and cytosols of ear, kidney, and liver. Inductively coupled plasma-mass spectrometry (ICP-MS) analysis shows a cisplatin accumulation capability between oxaliplatin (the highest) and carboplatin (the lowest). The maximum concentration of Pt in all the organs studied was achieved around the first week after cisplatin treatment. During the first 30 days, the elimination was very fast, decreasing in the subsequent 60 days in all the organs. Analysis of cytosols by liquid chromatography (LC)-ICP-MS showed an analogous behavior. In most samples, the distribution of the three drugs in the cellular and cytosolic fractions was similar for all the tissues. For kidney and ear, approximately 60% and 30%, respectively, of the metal accumulated was present in the cytosol, the cytosolic fractions smaller than 50 KDa being especially important. Cisplatin-biomolecule interaction strength under denaturing conditions was evaluated by LC-ICP-MS and showed a quite strong bond. PublicationAnalytical methodologies for metallomics studies of antitumor Pt-containing drugs(RSC, 2010) Esteban Fernández, Diego; Moreno Gordaliza, Estefanía; Cañas, Benito; Palacios Corvillo, M.Antonia; Gómez Gómez, M.MilagrosPt-containing drugs are nowadays essential components in cancer chemotherapy. However, drug resistance and side effects limit the efficiency of the treatments. In order to improve the response to Pt-based drugs, different administration strategies or new Pt-compounds have been developed with little success. The reason for this failure could be that the mechanism of action of these drugs is not completely understood. In this way, metallomics studies may contribute to clarify the interactions of Pt-containing drugs within the organism. This review is mainly focused on the role of Analytical Chemistry on the study of the interactions between Pt-based drugs and biomolecules. A summary of the analytical techniques and the most common sample treatment procedures currently used in metallomics studies of these drugs is presented. Both are of paramount importance to study these complex samples preserving the drug–biomolecule interaction. Separation and detection techniques must be carefully selected in order to achieve the intended goals. The use of multidimensional hyphenated techniques is usually necessary for a better understanding of the Pt-based drugs interactions in the organism. An overview of Pt-drugs biological interactions is presented, considering the different sample matrices and the drugs course through the organism. Samples analysed in the included studies are blood, urine, cell cytosol, DNA as well as the drugs themselves and their derivatives. However, most of these works are based on in vitro experiments or incubations of standards, leading in some cases to contradictory results depending on the experimental conditions used. Though in vivo experiments represent a great challenge due to the high complexity and the low concentrations of the Pt-adducts in real samples, these studies must be undertaken to get a deeper understanding of the real interactions concerning Pt-containing drugs. PublicationAtomic (HPLC-ICP-MS) and molecular mass spectrometry (ESI-Q-TOF) to study cis-platin interactions with serum proteins(RSC, 2008) Esteban Fernández, Diego; Montes Bayón, M.; Blanco González, E.; Gómez Gómez, M. Milagros; Palacios Corvillo, M. Antonia; Sanz Medel, A.The judicious use of cis-Pt as an intravenously administrable Pt(II) drug for chemotherapy requires the evaluation of its interactions with blood proteins. Therefore, the combined use of modern analytical chemical speciation and of analytical proteomics approaches to study these interactions is described here. The method involves incubation of cis-Pt with standard proteins and human serum samples. The separation of the proteins is conducted by liquid chromatography in an anion exchange column (Mono Q). Simultaneous molecular detection by UV absorption (280 nm) and elemental detection (195Pt) using inductively coupled plasma mass spectrometry (ICP-MS) are performed. Using this set-up, the effects of the incubation time as well as the drug concentration on cis-Pt interactions with transferrin, albumin and innmunoglobulin G were studied. In addition, the nature of interactions was also investigated by means of electrospray mass spectrometry (ESI-Q-TOF) of the intact protein. Transferrin and albumin showed different interactions, binding one and four cisplatin molecules, respectively. Also, some typical proteomic studies were initiated by tryptic digesting the transferrin and albumin cis-Pt complexes followed by capillary-LC-ICP-MS and ESI-Q-TOF parallel detection of the peptides obtained. The capLCICP-MS chromatogram provided clear evidence of Pt-containing peptides remaining after tryptic digestion. PublicationBridging the gap between molecular and elemental mass spectrometry: Higher energy collisional dissociation (HCD) revealing elemental information(ACS, 2014-12-22) Esteban Fernández, Diego; El-Khatib, Ahmed H.; Moraleja San José, Irene; Gómez Gómez, M.Milagros; Linscheid, Michael W.Molecular mass spectrometry has been applied to simultaneously obtain molecular and elemental information from metal-containing species. Energy tuning of the higher-energy collision dissociation (HCD) fragmentation cell allows the controlled production of typical peptide fragments or elemental reporter ions informing about the metallic content of the analyzed species. Different instrumental configurations and fragmentation techniques have been tested, and the efficiency extracting the elemental information has been compared. HCD fragmentation operating at very high energy led to the best results. Platinum, lanthanides, and iodine reporter ions from peptides interacting with cisplatin, peptides labeled with lanthanides-MeCAT-IA, and iodinated peptides, respectively, were obtained. The possibility to produce abundant molecular and elemental ions in the same analysis simplifies the correlation between both signals and open pathways in metallomics studies enabling the specific tracking of metal-containing species. The proposed approach has been successfully applied to in solution standards and complex samples. Moreover, interesting preliminary MALDI-imaging experiments have been performed showing similar metal distribution compared to laser ablation (LA)-ICPMS. PublicationCisplatin-induced hearing loss does not correlate with intracellular platinum concentration(Taylor and Francis, 2008) Ramírez Camacho, R.; Esteban Fernández, Diego; Verdaguer, J.M.; Gómez Gómez, M. Milagros; Trinidad, A.; García Berrocal, J.R.; Palacios Corvillo, M. AntoniaConclusion. Inductively coupled plasma mass spectrometry (ICP-MS) can be applied to organic tissues obtained from experimental animals. Hearing loss does not correlate with the platinum (Pt) concentration found in the inner ear. Drug structure and affinity to inner ear proteins could explain ototoxicity caused by cisplatin. Objectives. To analyse Pt affinity for brain and ear tissues (of similar embryologic origin) in the Wistar rat and clearance gradient after a single dose, and to correlate these findings with hearing changes. Materials and methods. Thirty-two Wistar rats were intraperitoneally injected with cisplatin at a dose of 5 mg/kg. Animals were sacrificed after obtaining auditory brain responses (ABRs) at 3, 7, 30 and 90 days (nine, seven, seven and nine animals, respectively). Brain and both temporal bones were extracted from each animal and analysed by ICP-MS to determine the absolute concentrations of the metal. Eight non-treated animals were employed as a control group. Results. The ABR thresholds were significantly elevated in animals from all groups after cisplatin treatment. A maximum accumulation of Pt for inner ear and brain was revealed around the first week: 3.175 (57%) and 0.342 (72%), respectively. Pt significantly accumulated in greater quantities in ear than in brain (pB0.01) and was cleared at a higher rate in brain than in ear (pB0.01) following cochlea/brain ratio analysis. No statistically significant correlation was found between amounts of Pt and hearing loss in the study animals. PublicationDesarrollo de métodos analíticos basados en técnicas cromatográficas y espectrometría de masas para estudios de metalómica de fármacos antitumorales de Pt en muestras biológicas(Universidad Complutense de Madrid, 2017-05-26) Esteban Fernández, Diego; Palacios Corvillo, María Antonia; Gómez Gómez, María MilagrosEl Platino (Pt) es un elemento químico de número atómico 78 y que pertenece al grupo 10 de la tabla periódica. Su configuración electrónica es [Xe] 4f14 5d9 6s1. Se trata de un metal de transición blanco grisáceo, precioso, pesado, maleable y dúctil. Es relativamente resistente al ataque químico, presenta buenas propiedades físicas a temperaturas altas y buenas propiedades eléctricas. Por estos motivos se emplea en contactos eléctricos, electrodos, equipamiento de laboratorio, empastes, catalizadores de automóviles y joyería. Fue descubierto en el siglo XVI por el español Antonio de Ulloa en unas minas de oro de Colombia. Es un metal escaso ya que su concentración en la corteza terrestre no supera los 15 ng Kg-1 y aparece asociado con Ru, Os, Rh, Ir y Pd, formando la llamada mena de platino. Alrededor de tres cuartas partes de la producción mundial de Pt procede de Sudáfrica, aunque Rusia, Canadá, Zimbabue y Estados Unidos también son productores destacados. En España existen pequeños yacimientos en la Serranía de Ronda (Málaga) y en Cabo Ortegal (La Coruña). Aun siendo un metal noble se disuelve en agua regia, es atacado lentamente por el ácido clorhídrico en presencia de aire y puede reaccionar, según las condiciones, con cianuros, azufre o halógenos como el Cl y el F. Puede formar compuestos en estados de oxidación 0, II, IV, V y VI, éstos dos últimos asociados a oxo y fluoroderivados. Los dos estados de oxidación más importantes en disolución acuosa son II y IV, y por lo tanto los más comunes en medios biológicos. La mayoría de la química del metal deriva de su química de coordinación. En estado de oxidación II la mayoría de los complejos son plano-cuadrados como corresponde a un sistema d8. Teniendo en cuenta que el Pt(II) se considera como un ácido de Lewis “blando”, los complejos que forme serán principalmente con bases blandas (S, Se, P,..) o con ligandos N-dadores. Uno de los complejos aniónicos más importantes es el [PtCl4]2- que en agua se hidroliza de forma rápida para dar [PtCl3(H2O)]- y [PtCl2(H2O)2], complejos mono y diacuo respectivamente. En estado de oxidación IV se forman complejos octaédricos... PublicationDual Internal Standards with Metals and Molecules for MALDI Imaging of Kidney Lipids(ACS, 2017) Aboulmagd, Sarah; Esteban Fernández, Diego; Moreno Gordaliza, Estefanía; Neumann, Boris; El-Khatib, A.H.; Lázaro, Alberto; Tejedor, Alberto; Gómez Gómez, M.Milagros; Linscheid, Michael W.The quest for internal standards useful in MALDI imaging studies goes on to get not only lateral distribution but also reliable relative quantitative information. We developed a method based on application of matrix and dual internal standards to allow intra- and intersample normalization of lipids intensities in kidney sections of control and cisplatin-treated Wistar rats. An inkjet printer was used to deposit a custom-prepared ink with DHB as MALDI matrix, a primary lipids-based internal standard, and a spiked lanthanide as a secondary internal standard. We applied different laser energy and varied the amounts of matrix-internal standards mixture to evaluate the normalization potential of the internal standards. Successful correction of intensity artifacts caused by instrumental drifts was possible, but not those resulting from uneven matrix application. ICP-MS absolute quantification of the lanthanide in the printed layer ensured the reproducibility of the matrix and internal standards application with RSD of 10−15%. Internal standard-normalized data allowed intrasample modification of the MALDI image to make it compatible with the optical image. Normalization to internal standards corrected a 2-fold difference in lipids intensity, which allowed a meaningful comparison of tissue lipids in control and cisplatin-treated kidneys. More importantly, normalization of lipid relative abundances based on the same adduct type (H+ , Na+ , and K+ ) for analyte and internal standard corrected for different ionization efficiencies showing a realistic signal level and enabling reliable comparison of different samples on relative quantitative basis. PublicationLA-ICP-MS and nHPLC-ESI-LTQ-FT-MS/MS for the analysis of cisplatin–protein complexes separated by two dimensional gel electrophoresis in biological samples(RSC, 2012) Moreno Gordaliza, Estefanía; Esteban Fernández, Diego; Giesen, Charlotte; Lehmann, Karola; Lázaro, Alberto; Tejedor, Alberto; Scheler, Christian; Cañas Montalvo, Benito; Jakubowski, Norbert; Linscheid, Michael W.; Gómez Gómez, M.MilagrosA method for the analysis of Pt–protein complexes in biological samples, previously subjected to cisplatin treatment, has been developed. Proteins were separated by gel electrophoresis, and those bound to Pt were detected with high sensitivity by LA-ICP-(SF)-MS. Pt-containing spots were in-gel digested with trypsin, and the peptides produced identified using nHPLC-ESI-LTQ-FT-MS/MS. The influence of protein separation conditions, staining and gel processing prior to laser ablation on Pt–protein bonds preservation have been evaluated using standard proteins incubated with cisplatin. 2-DE separation under non-reducing conditions followed by either Coomassie blue brilliant or silver staining is appropriate for Pt–protein complexes, achieving a good separating resolution of the proteins in biological samples. Direct LA-ICP-MS analysis of glycerol-treated dried gels for Pt–protein monitoring resulted in better sensitivity, more reliable relative Pt signals and a simpler and less timeconsuming approach compared to the analysis of blotted membranes. Ablation of gels allowed tackling protein identification of Pt-spots in the remaining non-ablated material in the gel, making it unnecessary to run several gels in parallel for separate Pt detection and protein identification. By using this approach, Pt coordinated to proteins, such as a-2-macroglobulin, transferrin, albumin or hemoglobin, was detected in the serum from a rat treated in vivo with cisplatin after nrSDS-PAGE separation. Furthermore, the first complete LA-ICP-MS metalloprotein contour map in a 2-DE gel has been produced, in this case for the detection of Pt–protein complexes in renal proximal tubule epithelial cells (RPTECs) incubated with cisplatin. Several proteins were identified in those spots containing Pt, which may have a connection with the drug-induced nephrotoxicity mainly affecting this cell type in the kidney. PublicationLipid imaging for visualizing cilastatin amelioration of cisplatin-induced nephrotoxicity(American Society for Biochemistry and Molecular Biology Inc., 2018-07) Moreno Gordaliza, Estefanía; Esteban Fernández, Diego; Lázaro, Alberto; Aboulmagd, Sarah; Humanes, Blanca; Linscheid, Michael W.; Gómez Gómez, M.MilagrosNephrotoxicity is a major limitation to cisplatin antitumor therapies. Cilastatin, an inhibitor of renal dehydropeptidase-I, was recently proposed as a promising nephroprotector against cisplatin toxicity, preventing apoptotic cell death. In this work, cilastatin nephroprotection was further investigated in a rat model, with a focus on its effect on 76 renal lipids altered by cisplatin, including 13 new cisplatin-altered mitochondrial cardiolipin species. Lipid imaging was performed with MALDI mass spectrometry imaging (MALDI-MSI) in kidney sections from treated rats. Cilastatin was proved to significantly diminish the lipid distribution alterations caused by cisplatin, lipid levels being almost completely recovered to those of control samples. The extent of recovery of cisplatin-altered lipids by cilastatin turned out to be relevant for discriminating direct or secondary lipid alterations driven by cisplatin. Lipid peroxidation induced by cisplatin was also shown to be reduced when cilastatin was administered. Importantly, significant groups separation was achieved during multivariate analysis of cortex and outer-medullary lipids, indicating that damaged kidney can be discerned from the nephroprotected and healthy groups and classified according to lipid distribution. Therefore, we propose MALDI-MSI as a powerful potential tool offering multimolecule detection possibilities to visualize and evaluate nephrotoxicity and nephroprotection based on lipid analysis. PublicationMALDI-LTQ-Orbitrap mass spectrometry imaging for lipidomic analysis in kidney under cisplatin chemotherapy(Elsevier, 2017) Moreno Gordaliza, Estefanía; Esteban Fernández, Diego; Lázaro, Alberto; Humanes, Blanca; Aboulmagd, Sarah; Tejedor, Alberto; Linscheid, Michael W.; Gómez Gómez, M.MilagrosImaging techniques for mapping molecular distributions in tissue sections can reveal valuable information on biomolecules involved in relevant biochemical processes. A method has been developed for comprehensive, reproducible and sensitive lipid imaging by matrix-assisted laser/desorption ionization-LTQ-Orbitrap mass spectrometry in kidney sections, showing the benefits of exact mass determination. Matrix deposition parameters for positive and negative lipid ion imaging using different matrices such as 2,5-dihydroxybenzoic acid (DHB), 9-aminoacridine (9-AA) or α-cyano-4-hydroxycinnamic acid (CHCA) have been optimized for the broadest detection and identification of renal lipids. The combination of 9-AA and DHB was found as the most suitable for negative and positive ion mode lipid imaging, respectively. Lipid mapping and related identification strategies and limitations have also been discussed. Production of 100-µm resolution images was proved to be enough for discerning lipid distribution in kidney substructures. Imaging reproducibility was assessed on parallel kidney slices with time. This method has been applied to the lipidomics analysis on kidney sections from rats treated with the antitumor drug cisplatin and compared to healthy control rats. Up to 66 different renal lipids out of 450 extracted ion images (mainly phospholipid species, in addition to sulfatides and cholesterol sulfate) have been found and identified showing a modified distribution pattern due to cisplatin-induced nephrotoxicity. These lipid species reflect either topographic, signaling or structural processes in damaged kidney and could potentially be used for nephrotoxicity assessment or as therapeutic targets. This is, to our knowledge, the first imaging lipidomics study for nephrotoxicity assessment of cisplatin chemotherapy. PublicationPharmacological inhibitors of extracellular signal-regulated protein kinases attenuate the apoptotic action of cisplatin in human myeloid leukemia cells via glutathione-independent reduction in intracellular drug accumulation(Elsevier, 2005) Amrán, Donna; Sancho, Patricia; Fernández, Carlos; Esteban Fernández, Diego; Ramos, Adrian M.; Blas, Elena de; Gómez Gómez, M.Milagros; Palacios Corvillo, María A.; Aller, PatricioIt has been reported that inhibition of extracellular signal-regulated protein kinases (ERKs) attenuates the toxicity cisplatin (cisplatinum (II)-diammine dichloride) in some cell types. This response was here investigated using human myeloid leukemia cells. Cisplatin stimulated ERK1/2 phosphorylation and caused apoptosis in U-937 promonocytic cells, an effect which was attenuated by the MEK/ERK inhibitors PD98059 and U0126. While ERK1/2 activation was a general phenomenon, irrespective of the used cell type or antitumour drug, the MEK/ERK inhibitors only reduced cisplatin toxicity in human myeloid cells (THP-1, HL-60 and NB-4), but not in RAW 264.7 mouse macrophages and NRK-52E rat renal tubular cells; and failed to reduce the toxicity etoposide, camptothecin, melphalan and arsenic trioxide, in U-937 cells. U0126 attenuated cisplatin–DNA binding and intracellular peroxide accumulation, which are important regulators of cisplatin toxicity. Although cisplatin decreased the intracellular glutathione (GSH) content, which was restored by U0126, treatments with GSH-ethyl ester and dl-buthionine-(S,R)-sulfoximine revealed that GSH does not regulate cisplatin toxicity in the present experimental conditions. In spite of it, PD98059 and U0126 reduced the intracellular accumulation of cisplatin. These results suggest that GSH-independent modulation of drug transport is a major mechanism explaining the anti-apoptotic action of MEK/ERK inhibitors in cisplatin-treated myeloid cells. PublicationPrinting metal-spiked inks for LA-ICP-MS bioimaging internal standardization: comparison of the different nephrotoxic behavior of cisplatin, carboplatin, and oxaliplatin(Springer, 2016) Moraleja San José, Irene; Esteban Fernández, Diego; Lázaro, Alberto; Humanes, Blanca; Neumann, Boris; Tejedor, Alberto; Mena, María Luz; Jakubowski, Norbert; Gómez Gómez, M.MilagrosThe study of the distribution of the cytostatic drugs cisplatin, carboplatin, and oxaliplatin along the kidney may help to understand their different nephrotoxic behavior. Laser ablation inductively coupled plasma mass spectrometry (LAICP-MS) allows the acquisition of trace element images in biological tissues. However, results obtained are affected by several variations concerning the sample matrix and instrumental drifts. In this work, an internal standardization method based on printing an Ir-spiked ink onto the surface of the sample has been developed to evaluate the different distributions and accumulation levels of the aforementioned drugs along the kidney of a rat model. A conventional ink-jet printer was used to print fresh sagittal kidney tissue slices of 4 μm. A reproducible and homogenous deposition of the ink along the tissue was observed. The ink was partially absorbed on top of the tissue. Thus, this approach provides a pseudo-internal standardization, due to the fact that the ablation sample and internal standard take place subsequently and not simultaneously. A satisfactory normalization of LA-ICP-MS bioimages and therefore a reliable comparison of the kidney treated with different Pt-based drugs were achieved even for tissues analyzed on different days. Due to the complete ablation of the sample, the transport of the ablated internal standard and tissue to the inductively coupled plasma-mass spectrometry (ICP-MS) is practically taking place at the same time. Pt accumulation in the kidney was observed in accordance to the dosages administered for each drug. Although the accumulation rate of cisplatin and oxaliplatin is high in both cases, their Pt distributions differ. The strong nephrotoxicity observed for cisplatin and the absence of such side effect in the case of oxaliplatin could explain these distribution differences. The homogeneous distribution of oxaliplatin in the cortical and medullar areas could be related with its higher affinity for cellular transporters such as MATE2-k. PublicationSEC-ICP-MS and ESI-MS as tools to study the interaction between cisplatin and cytosolic biomolecules(RSC, 2007) Esteban Fernández, Diego; Cañas, Benito; Pizarro, I.; Palacios Corvillo, M.Antonia; Gómez Gómez, M.MilagrosMost of cisplatin’s citotoxic properties are due to the interaction of the drug with DNA. However, other biological molecules present in the cell cytosol, such as MT (metallothionein) and GSH (glutathione), are potential targets for cisplatin and have been related to its side-effects or with the cellular resistance mechanisms to the drug. Experiments simulating physiological conditions have been performed to study the specific cisplatin metabolites which interact with GSH and MT and to characterize the different drug–biomolecule adducts over time. A combination of size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS) and electrospray ionization-mass spectrometry (ESI-MS) techniques has been used to provide sensible multi-elemental detection and structural information of the species of interest. Time dependent transformation of 10 mM cisplatin at neutral pH (7.4) produces different concentrations of the mono-aquo and oligomeric derivatives, as could be confirmed by ESI-MS. No di-aquo derivative was seen to be produced under these conditions at any of the incubation times used. Cisplatin and the oligomeric derivative were incubated with GSH and MT at different drug:biomolecule ratios. Adducts from cisplatin–GSH (1:500) and from cisplatin–MT (1:10) incubations were characterized by SEC-ICP-MS. While both GSH and MT reacted with cisplatin producing different compounds, only GSH reacted with the oligomeric derivative of cisplatin. SEC-ICP-MS experiments showed that, under neutral pH conditions, Cd atoms remained bound to the cisplatin:MT adducts, but Zn atoms were lost. Results were compared with those obtained by in vitro and in vivo experiments with rat kidney, liver and inner ear cytosols. PublicationSpeciation analysis of platinum antitumoral drugs in impacted tissues(Elsevier, 2007) Esteban Fernández, Diego; Gómez Gómez, M.Milagros; Cañas, Benito; Verdaguer, J.M.; Ramírez, R.; Palacios Corvillo, M.AntoniaChemical compounds containing platinum have been employed since 1978 as drugs to beat certain type of tumours. Nevertheless, besides of their exceptional antitumoral properties, these drugs also have important deleterious side effects, such as, nephrotoxicity and ototoxicity. A study of Pt accumulation and a speciation analysis has been performed by ICP-MS in samples from kidney and inner ear in a controlled population of Wistar rats treated with, either, cisplatin, carboplatin or oxaliplatin. The results on Pt accumulation point out to drug structure and not only to Pt content as the responsible for the alteration of organ functionality. Speciation studies in the samples from kidney and inner ear were performed coupling two-dimensional liquid chromatography (2D-LC) to ICP-MS. Size exclusion (SEC) and anion exchange fast protein liquid chromatography (FPLC) was employed for 2D orthogonal separation. After these separations, free drug peaks were not observed in any of the samples. The binding of Pt to biomolecules was demonstrated by SEC and, independently of the drug used, Pt eluted as two main bands with molecular weights of 12 kDa and 25–65 kDa for inner ear samples, and as two different bands with 20 kDa and 50–60 kDa in the samples from kidney. However, the relative band intensity presented important differences for the three drugs. Using the same chromatographic conditions, it was shown that a metallothionein (MT) standard eluted in the same position as some of the cytosolic Pt-biomolecules. High Pt-containing fractions eluting from the SEC column were analysed by anion exchange FPLC after a preconcentration step. Among the different preconcentration methods tested, sample focusing on the head of the FPLC column shows main advantages. In this way, the separation by 2D chromatography of the high molecular Pt-species has been considerably improved.