An approach for quantification of platinum distribution in tissues by LA-ICP-MS imaging using isotope dilution analysis.

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been revealed as a convenient technique for trace elemental imaging in tissue sections, providing elemental 2D distribution at a quantitative level. For quantification purposes, in the last years several approaches have been proposed in the literature such as the use of CRMs or matrix matched standards. The use of Isotope Dilution (ID) for quantification by LA-ICP-MS has been also described, being mainly useful for bulk analysis but not feasible for spatial measurements so far. In this work, a quantification method based on ID analysis was developed by printing isotope-enriched inks onto kidney slices from rats treated with antitumoral Pt-based drugs using a commercial ink-jet device, in order to perform an elemental quantification in different areas from bio-images. For the ID experiments 194Pt enriched platinum was used. The methodology was validated by deposition of natural Pt standard droplets with a known amount of Pt onto the surface of a control tissue, where could be quantified even 50pg of Pt, with recoveries higher than 90%. The amount of Pt present in the whole kidney slices was quantified for cisplatin, carboplatin and oxaliplatin-treated rats. The results obtained were in accordance with those previously reported. The amount of Pt distributed between the medullar and cortical areas was also quantified, observing different behavior for the three drugs.

Combining TBP-based rOFFGEL-IEF with FASP and nLC-ESI-LTQ-MS/MS for the analysis of cisplatin-binding proteins in rat kidney.

In this work, a methodology based on a reducing IEF separation in combination with a FASP tryptic digestion able to maintain the integrity of cisplatin-protein complexes has been developed. The method is based on OFFGEL-IEF under conditions provided by the thiol-free reducing agent TBP, which allowed the separation of cisplatin-binding proteins in liquid fractions. The FASP procedure is applied as an intermediate stage between the IEF separation and MS analysis where the proteins are retained and concentrated in a commercially available ultrafiltration device. The filter unit acts as a proteomic reactor for detergent removal, buffer exchange, chemical modification (reduction and alkylation) and protein digestion. Finally, purified peptides are recovered by centrifugation. This procedure provides efficiencies comparable to standard in-solution digestion and the risk of platinum-complexes loss is minimized due to the fact that reagents employed along the process are subsequently eliminated before the following step. The stability of platinum-protein complexes under the FASP tryptic digestion, either using TBP or DTT as reducing agents, was maintained, allowing the identification of several platinum-containing peptides from cisplatin-HSA. This methodology was applied to the separation of platinum-enriched protein fractions obtained by SEC-ICP-MS in a kidney tissue extract from a rat treated with cisplatin, followed by further identification by nLC-ESI-LTQ-MS/MS after FASP tryptic digestion of selected platinum-containing liquid fractions.

Thiol-free reducing agents in electrophoretic separations and FASP proteolytic digestions for the analysis of metal-binding proteins.

The analysis of the complexes between metal-based chemotherapeutic drugs and proteins in biological samples, such as cisplatin or oxaliplatin, can be a challenge due to metal strong reactivity towards S-donor molecules such as dithiothreitol (DTT) or ß-mercaptoethanol (BME), usually employed as reducing agents in electrophoretic separations and proteolytic digestions for LC-MS/MS analysis.•This protocol describes the use of the thiol-free reducing trialkylphosphines, such as tributylphosphine (TBP) and tris(2-carboxyethyl)phosphine (TCEP) as suitable reagents for the preservation of the metal-protein complexes during OFFGEL-IEF and SDS-PAGE separations, respectively.•Moreover, the filter-aided sample preparation (FASP) method is presented as an advantageous option to perform tryptic in-solution digestions of metal-protein complexes in combination with OFFGEL-IEF separations.•The FASP procedure allows including previous reduction and alkylation steps in addition to proteolysis, ensuring the preservation of the metal-protein complexes. The limited time that proteins remain in contact with the reducing agent, either TBP or even DTT, during FASP could be a key factor for its extraordinary performance on the digestion of metal-protein complexes.

Interleukin-9 in stable liver transplant recipients.

INTRODUCTION: Interleukin-9 (IL-9) has recently been described to be involved in the maintenance of a tolerant environment, but there is no evidence of its role in human liver transplantation. The aim of our study was to measure the serum levels of IL-9 in stable liver transplant recipients and examine their influence on immunosuppressant load. METHODS: Serum IL-9 levels were determined in 34 healthy subjects and 30 stable liver transplant recipients who were free of rejection episodes for at least 8 years. The results were analyzed according to the blood levels of calcineurin inhibitors (CNIs) at the time of the study: 13 patients showed high concentrations of either cyclosporine or tacrolimus (high CNI: cyclosporine > 80 ng/mL or tacrolimus > 5 ng/mL) and another 17 patients showed low CNI levels. RESULTS: The concentrations of IL-9 were significantly higher among liver transplant recipients compared with healthy subjects. In addition, patients with low CNI blood levels showed higher serum levels of IL-9, an effect that was greater with tacrolimus, albeit not significantly. CONCLUSIONS: These preliminary results indicated that increased serum IL-9 concentrations accompanied a lower immunosuppressive load. It remains to be established whether this relates to induction of tolerance in liver transplantation.