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Increased concentrations of circulating metal-degradation products derived from the use of Ti orthopaedic implants may have deleterious biological effects over the long term. Therefore, there is an increasing need to establish the basal level of Ti in the serum of the population (exposed and non-exposed) with appropriate highly sensitive techniques and strategies. With this aim, we have developed a quantitative strategy for the determination of total Ti concentration in human serum samples by isotope dilution analysis using a double-focussing inductively coupled plasma mass spectrometer. Minimizing sample handling and therefore contamination issues, we obtained detection limits of about 0.05 μg L Ti working at medium resolution (m/Δm 4000). Such extremely good sensitivity permitted us to establish the range of Ti concentration in serum of 40 control individuals (mean 0.26 μg L) and also to compare it with the level in exposed patients with different Ti metal implants. On the other hand, Ti transport 'in vivo' studies have been enabled by online coupling of liquid chromatography (anion-exchange) separation and double-focussing inductively coupled plasma mass spectrometry for sensitive detection of Ti. The development of a postcolumn isotope dilution strategy permitted quantitative characterization of the Ti-transporting biomolecules in human serum. The results for unspiked serum revealed that 99.8% of the Ti present in this fluid is bound to the protein transferrin, with column recoveries greater than 95%. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Aortic stenosis (AS) is the most prevalent degenerative valvular disease in western countries. Transthoracic echocardiography (TTE) is considered, nowadays, to be the main imaging technique for the work-up of AS due to high availability, safety, low cost, and excellent capacity to evaluate aortic valve (AV) morphology and function. Despite the diagnosis of AS being considered straightforward for a very long time, based on high gradients and reduced aortic valve area (AVA), many patients with AS represent a real dilemma for cardiologist. On the one hand, the acoustic window may be inadequate and the TTE limited in some cases. On the other hand, a growing body of evidence shows that patients with low gradients (due to systolic dysfunction, concentric hypertrophy or coexistence of another valve disease such as mitral stenosis or regurgitation) may develop severe AS (low-flow low-gradient severe AS) with a similar or even worse prognosis. The use of complementary imaging techniques such as transesophageal echocardiography (TEE), multidetector computed tomography (MDTC), or cardiac magnetic resonance (CMR) plays a key role in such scenarios. The aim of this review is to summarize the diagnostic challenges associated with patients with AS and the advantages of a comprehensive multimodality cardiac imaging (MCI) approach to reach a precise grading of the disease, a crucial factor to warrant an adequate management of patients.

The combined use of fluorescence-activated cell sorting (FACS) and single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS) is reported, for the first time, in this work. It is applied to evaluate the differences between the cellular uptake of ultrasmall iron oxide nanoparticles (FeNPs) loaded with cisplatin(IV) prodrug (FeNPs-Pt(IV)) and cisplatin regarding cell viability. For this aim, FACS is applied to separate viable, apoptotic, and necrotic A2780 ovarian cancer cells after exposing them to the nanotransported prodrug and cisplatin, respectively. The different sorted cell populations are individually analyzed using quantitative SC-ICP-MS to address the intracellular amount of Pt. The highest Pt intracellular content occurs in the apoptotic cell population (about 2.1 fg Pt/cell) with a narrow intercellular distribution when using FeNPs-Pt(IV) nanoprodrug and containing the largest number of cells (75% of the total). In the case of the cisplatin-treated cells, the highest Pt content (about 1.6 fg Pt/cell) could be determined in the viable sorted cell population. The combined methodology, never explored before, permits a more accurate picture of the effect of the intracellular drug content together with the cell death mechanisms associated with the free drug and the nanotransported prodrug, respectively, and opens the door to many possible single-cell experiments in sorted cell populations.

A systematic investigation on the cellular uptake, intracellular dissolution, and in vitro biological effects of ultra-small (<10 nm) iron hydroxide adipate/tartrate coated nanoparticles (FeAT-NPs) was carried out in intestinal Caco-2, hepatic HepG2 and ovarian A2780 cells, and the nucleotide excision repair (NER) deficient GM04312 fibroblasts. Quantitative evaluation of the nanoparticles uptake, as well as their transformation within the cell cytosol, was performed by inductively coupled plasma mass spectrometry (ICP-MS), alone or in combination with high performance liquid chromatography (HPLC). The obtained results revealed that FeAT-NPs are effectively taken up in a cell type-dependent manner with a minimum dissolution after 3 h. These results correlated with no effects on cell proliferation and minor effects on cell viability and reactive oxygen species (ROS) production for all the cell lines under study. Moreover, the comet assay results revealed significant DNA damage only in GM04312 cells. In vivo genotoxicity was further studied in larvae from Drosophila melanogaster, using the eye-SMART test. The obtained results showed that FeAT-NPs were genotoxic only with the two highest tested concentrations (2 and 5 mmol·L−1 of Fe) in surface treatments. These data altogether show that these nanoparticles represent a safe alternative for anemia management, with high uptake level and controlled iron release.

Purpose . Echocardiography assessment from apical five-chamber view (A5CV) is the standard technique for aortic stenosis (AS) grading. Data on non-apical views, such as right parasternal (RPV), subcostal (SCV) and suprasternal notch (SSNV), is scarce and constitutes the aim of our study. Methods. We designed an observational study that included patients with AS recruited prospectively in whom the stenosis was graded by echocardiography from A5CV and non-apical view. The value of non-apical views in up-grading the stenosis severity (primary objective), the prognostic relevance of such reclassification and the feasibility and reproducibility of non-apical views assessment (secondary objectives) was evaluated. Results. Feasibility of AS appraisal from RPV, SCV and SSNV was 78%, 81% and 56%, respectively (SCV vs SSNV, p  = .009). AS were up-graded from non-apical views according to peak gradient, mean gradient, area and indexed area by 24%, 17%, 24% and 22%, respectively ( p  < .0001). Non-apical views reclassified from non-severe to severe AS, from low gradient severe to high gradient severe AS and from non-critical to critical AS 19%, 23% and 3% of cases ( p  < .0001). The 4-years hard cardiac events rate was 41% in patients with non-severe AS, 67% in patients with severe AS from non-apical views, 68% in patients with severe AS from A5CV and 80% in patients with severe AS from A5CV and non-apical views ( p  < .001). Reproducibility of AS evaluation from non-apical views was fair to excellent (intraclass correlation coefficients: SSNV = 0.44, RPV = 0.61, SCV = 0.92). Conclusion. Assessment of AS from non-apical views is feasible, reproducible and valuable over A5CV; its use is encouraged.

One of the limitations in the use of cisplatin is its low penetration into cells. In addition, some cells develop the so called resistance, a multifactorial event that decreases significantly the intracellular cisplatin concentration. To circumvent these limitations, recent studies are focused on the use of nanocarriers that permit, among others, to achieve higher drug uptake. In this work, ferritin is evaluated as a nanostructured cisplatin-delivery system in cell models of ovarian cancer. One of the key aspects is the characterization of the encapsulated product, and for this aim, a battery of analytical techniques, including size exclusion chromatography (SEC) coupled to UV detection and to inductively coupled plasma mass spectrometry (ICP-MS) together with transmission electron microscopy (TEM), is conducted. Higher level of incorporation occurs when using initial concentrations of the Fe-containing form of the protein at 10 mg/mL and 1 mg/mL cisplatin solution. The incorporation of the free and encapsulated cisplatin is addressed in A2780 and A2780CIS, sensitive and cisplatin-resistant cell lines, respectively, showing a significantly higher uptake of the encapsulated form. These values ranged from 5- to 9-fold in the sensitive line and 2-4 in the resistant model, being always more pronounced at the lower doses. Functionality of the drug after encapsulation is addressed by monitoring the presence of Pt in DNA and normalizing DNA concentration through simultaneous P and Pt measurements by ICP-MS. Time elapsed between exposure and Pt detection in DNA proved to be critical in the encapsulated model, showing the slower drug release mechanism from the ferritin nanocage that could be advantageously used for a controlled therapy. Graphical abstract.

Cells are able to precisely control the amount of iron they acquire in the form of transferrin (TF)-bound iron by modulating the synthesis of transferrin receptor 1 (TfR1). In tumor cells, elevated TfR1 seems to be related to poorer outcome for patients. Thus, the direct measurement of this biomarker in breast cancer tissues and cells might serve as a prognosis biomarker. In this work, we have used Nd-labeled antibodies to tag the TfR1 present on the cell surface of two cell models of breast cancer with different malignancy (MCF7 and MDA-MB 231). For this aim, monoclonal antibody anti-TfR1 is first labeled with a polymeric chelator (MAXPAR) with the subsequent incorporation of several isotopic 143 Nd atoms. The characterization of the labeled antibody revealed a stoichiometry of 21 Nd atoms per antibody molecule that can be used for further quantification experiments. This antibody is used for cell tagging followed by single-cell analysis using inductively coupled plasma mass spectrometry (ICP-MS) detection. In this case, cell introduction is conducted using a high-efficiency nebulizer and spray chamber to achieve transport efficiencies of up to 55% for cells. Quantitative results revealed a number of receptors per cell significantly higher in the case of the most malignant phenotype (MDA-MB-231). Absolute and relative TfR1 concentration values are obtained in individual cells for the first time using the proposed system.

Cisplatin, one of the most extensively used metallodrugs in cancer treatment, presents the important drawback of patient resistance. This resistance is the consequence of different processes including those preventing the formation of DNA adducts and/or their quick removal. Thus, a tool for the accurate detection and quantitation of cisplatin-induced adducts might be valuable for predicting patient resistance. To prove the validity of such an assumption, highly sensitive plasma mass spectrometry (ICP-MS) strategies were applied to determine DNA adduct levels and intracellular Pt concentrations. These two metal-relative parameters were combined with an evaluation of biological responses in terms of genomic stability (with the Comet assay) and cell cycle progression (by flow cytometry) in four human cell lines of different origins and cisplatin sensitivities (A549, GM04312, A2780 and A2780cis), treated with low cisplatin doses (5, 10 and 20 μM for 3 hours). Cell viability and apoptosis were determined as resistance indicators. Univariate linear regression analyses indicated that quantitation of cisplatin-induced G-G intra-strand adducts, measured 1 h after treatment, was the best predictor for viability and apoptosis in all of the cell lines. Multivariate linear regression analyses revealed that the prediction improved when the intracellular Pt content or the Comet data were included in the analysis, for all sensitive cell lines and for the A2780 and A2780cis cell lines, respectively. Thus, a reliable cisplatin resistance predictive model, which combines the quantitation of adducts by HPLC-ICP-MS, and their repair, with the intracellular Pt content and induced genomic instability, might be essential to identify early therapy failure.

Analytical methods allowing sensitive determination of reduced homocysteine (rHcy), one of the so-called biothiols, in human serum is a topic of growing interest due to its close relation to several human disorders, mainly cardiovascular diseases. Although most widely used analytical strategies to determine total Hcy involve derivatization by means of fluorescent labels, this work proposes the use of ebselen, a Se-containing labelling agent to derivatize the reactive sulfhydryl group of the Hcy molecule in its "free" reduced form, which is more likely to play different roles in disease pathogenesis. Optimization of the derivatization and separation conditions by high-performance liquid chromatography (HPLC) to isolate the excess of derivatizing reagent is carried out here using UV/VIS detection. Further, the study of the Se labelling reaction by electrospray ionization tandem mass spectrometry (ESI-MS/MS) provides a stoichiometry of the derivative of 1:1. The main advantage of using ebselen as a labelling agent is the presence of the Se atom in the molecule that allows the use of inductively coupled plasma mass spectrometry (ICP-MS) as a sensitive and selective Se detector. The coupling of HPLC with ICP-MS provided excellent features for the determination of Se-derivatized rHcy (detection limit of 9.6 nM) in real samples. Quantification was accomplished by using post-column isotope dilution (ID) of Se in serum samples, after precipitation of the main serum proteins. Quantitative results for "free" rHcy turned out to be around 0.18-0.22 μM in serum samples from healthy individuals that could be directly analyzed without sample preconcentration.

Epigenetic alterations are increasingly implicated in the initiation and progression of cancer. Genome-wide (global) hypomethylation seems to occur in early neoplasia and is a feature of genomic DNA derived from solid tumour tissues like ovarian cancer. Thus, analytical methods that provide sensitive and quantitative information about cytosine methylation in DNA are currently required. In this work, we compare two different anion-exchange columns for the separation of methylated cytosine from the other DNA nucleotides: a silica-based (Tracer Extrasil SAX) column and a polystyrene/divinyl benzene-based (Mono-Q™) column. Under the optimised conditions, linearity range, precision and detection limits of the developed high-performance liquid chromatography (HPLC) method were evaluated and compared using conventional ultraviolet (UV) absorbance detection at 270 nm. Good separation of the five target nucleotides, including 5-methyl-2'-deoxycytidine monophosphate (5mdCMP) and 2'-deoxycytidine monophosphate (dCMP) was achieved on the Mono-Q™ column with a gradient elution of ammonium acetate buffer (1 M, pH 6.9) at a flow rate of 1 mL min(-1). The coupling of this column to inductively coupled plasma mass spectrometry (ICP-MS) permitted also phosphorous ((31)P) specific detection of the nucleotides. Both detection systems offered adequate analytical performance characteristics, with detection limits of 30 and 40 μg L(-1) for 5mdCMP by HPLC-UV and HPLC-ICP-MS, respectively. However, the latter method allowed the determination of the global DNA methylation level (%) without the need for external calibration. Different genomic DNA samples were analysed including calf thymus DNA and DNA from two human cancer cell lines (adenocarcinoma epithelial A549 and ovarian carcinoma A2780) using the proposed strategy. In the line A2780, the cisplatin-sensitive and cisplatin-resistant variants were analysed, finding no significant differences in the methylation percentage after treatment with cisplatin.

Detection of specific DNA sequences is nowadays an important tool in many scientific areas such as forensic science or clinical diagnosis. Although numerous approaches have been suggested for this challenging analysis, certain limitations still remain. In order to overcome these disadvantages, novel and alternative methodologies are required. In this work, we present a strategy based on elemental (lanthanide) labelling of DNA probes followed by size-exclusion chromatography (SEC) coupled with inductively coupled plasma-mass spectrometry (ICP-MS) for monitoring and determining complementary oligonucleotide sequences (oligonucleotide targets) and for visualising the corresponding hybridisation processes. The synthesis and characterisation of the DNA probes are described in detail. SEC was found to be suitable to discriminate between the DNA probe and the hybridised product. Using labelling of different probes with different lanthanides, multiplexed detection of the sought DNA sequences was possible as demonstrated here on three DNA probes (derivatised with Eu, Tb, and Ho, respectively). The achievable detection limits were in the range between 5 and 11 fmol absolute.