Towards single cell ICP-MS normalized quantitative experiments using certified selenized yeast.

In the search for a normalized procedure to replicate and compare single cell-inductively coupled plasma-mass spectrometry (SC-ICP-MS) experiments, SELM-1, a certified reference material containing selenium enriched yeast cells has been used. Selenium concentrations (both, intra- and extracellular) have been measured using either sequential or simultaneous procedures. Regarding quantitative results, the sequential procedure involving cell washing followed by freeze drying of the washed material and intracellular Se quantification using SC-ICP-MS provided best results. In this case, intracellular Se accounted for 1304 ± 48 mg kg-1 (corresponding to 64% of the certified Se content). The average mass of Se per yeast cell was 41.6 fg Se with a dispersion of 1.6-279 fg Se/cell. In the isolated extracellular Se fraction, the Se concentration accounted for 412 ± 48 mg kg-1 (about 21% of the total Se). Thus, the sequential procedure provided a total Se recovery of about 85% with respect to the certified value. The direct dilution and simultaneous measurement of intra- and extracellular Se by SC-ICP-MS provided results of 1024 ± 42 mg kg-1 for intracellular and 316 ± 30 mg kg-1 for extracellular Se representing a total recovery of about 66%. In both cases, an initial thorough characterization of the cell density per solid weighed material was conducted by flow cytometry and the cell integrity ensured using confocal microscopy. These results clearly demonstrated that with appropriate sample preparation, SC-ICP-MS is a unique tool, which is capable of providing quantitative information about intracellular and extracellular Se. In addition, SELM-1 seems the ideal tool to enable data normalization at the single cell level to replicate, benchmark, and improve new SC-ICP-MS studies by using the same material for data validation.

Evaluation of copper uptake in individual spores of Streptomyces coelicolor and endogenic nanoparticles formation to modulate the secondary metabolism.

Copper modulates secondary metabolism in Streptomyces. Although the cytosolic copper concentration is controlled by several chaperones and transporters, the formation of copper nanoparticles (NPs) and its relation to the antibiotic production has never been established in the model Streptomyces coelicolor. In this work, state-of-the-art analytical tools are used to evaluate the incorporation of copper in individual spores of S. coelicolor at different exposure concentrations (40, 80, and 160 µM Cu). Among them, the use of single cell-inductively coupled plasma-mass spectrometry revealed incorporation levels in the range of 2 to 2.5 fg/spore (median) increasing up to 4.75 fg/spore at the upper exposure concentrations. The copper storage within the spores in the form of NPs was evaluated using a combination of single particle-inductively coupled plasma-mass spectrometry and transmission electron microscopy. The obtained data confirmed the presence of NPs in the range of 8 to 40 (mean size 21 nm) inside S. coelicolor spores. The presence of the NPs was correlated with the actinorhodin production in liquid non-sporulating cultures amended with up to 80 µM Cu. However, further increase to 160 µM Cu, yielded to a significant decrease in antibiotic production. Secondary metabolism is activated under stressful conditions and cytosolic copper seems to be one of the signals triggering antibiotic production. Particularly, NP formation might contribute to modulate the secondary metabolism and prevent for copper toxicity. This work describes, for first time, the formation of endogenous copper NPs in S. coelicolor and reveals their correlation with the secondary metabolism.

Cellular Toxicity Mechanisms and the Role of Autophagy in Pt(IV) Prodrug-Loaded Ultrasmall Iron Oxide Nanoparticles Used for Enhanced Drug Delivery.

Ultrasmall iron oxide nanoparticles (<10 nm) were loaded with cis-diamminetetrachloroplatinum (IV), a cisplatin (II) prodrug, and used as an efficient nanodelivery system in cell models. To gain further insight into their behavior in ovarian cancer cells, the level of cellular incorporation as well as the platination of mitochondrial and nuclear DNA were measured using inductively coupled plasma mass spectrometry (ICP-MS) strategies. Quantitative Pt results revealed that after 24 h exposure to 20 µM Pt in the form of the Pt(IV)-loaded nanoparticles, approximately 10% of the incorporated Pt was associated with nuclear DNA. This concentration increased up to 60% when cells were left to stand in drug-free media for 3 h. These results indicated that the intracellular reducing conditions permitted the slow release of cisplatin (II) from the cisplatin (IV)-loaded nanoparticles. Similar results were obtained for the platination of mitochondrial DNA, which reached levels up to 17,400 ± 75 ng Pt/ mg DNA when cells were left in drug-free media for 3 h, proving that this organelle was also a target for the action of the released cisplatin (II). The time-dependent formation of Pt-DNA adducts could be correlated with the time-dependent decrease in cell viability. Such a decrease in cell viability was correlated with the induction of apoptosis as the main route of cell death. The formation of autophagosomes, although observed upon exposure in treated cells, does not seem to have played an important role as a means for cells to overcome nanoparticles' toxicity. Thus, the designed nanosystem demonstrated high cellular penetration and the "in situ" production of the intracellularly active cisplatin (II), which is able to induce cell death, in a sustained manner.

Targeting HER2 protein in individual cells using ICP-MS detection and its potential as prognostic and predictive breast cancer biomarker.

The human epidermal growth factor receptor 2 (HER2) is a transmembrane protein that has become one of the most specific prognostic and predictive biomarker of breast cancer. Its early detection is key for optimizing the patient clinical outcome. This work is focused on the detection of HER2 in individual cells using an antibody containing lutetium (Lu) as reporter group that is monitored by introducing the individual cells into the inductively coupled plasma mass spectrometer (ICP-MS). This Lu-containing antibody probe is used to label different breast cancer cell lines considered HER2 negative (MDA-MB-231) and positive (SKBR-3 and BT-474). Optimizations regarding the amount of the probe necessary to ensure complete labelling reactions are conducted in the different cell models. Concentrations in the range of 0.006 fg Lu/cell and 0.030 fg Lu/cell could be found in the HER2 negative and HER2 positive cells, respectively. In addition, the selectivity of the labelling reaction is tested by using two different metal-containing antibody probes for HER2 (containing Lu) and for transferrin receptor 1 (containing Nd), respectively, within the same cell population. Finally, the methodology is applied to the targeting of HER2 positive cells in complex cell mixtures containing variable amounts of BT-474 and MDA-MB-231 cells. The obtained results showed the excellent capabilities of the proposed strategy to discriminate among cell populations. This finding could help for scoring HER2 positive tumors improving existing technologies.

Ultrasmall iron oxide nanoparticles cisplatin (IV) prodrug nanoconjugate: ICP-MS based strategies to evaluate the formation and drug delivery capabilities in single cells.

Ultrasmall iron oxide nanoparticles (<10 nm) were explored here as nanotransporters of cis-diamminetetrachloroplatinum (IV) (a cisplatin prodrug) in cellular models. The coating of the particles containing reactive carboxylic acid groups enabled the formation of a stable conjugate between the prodrug and the nanoparticles using one pot reaction. The nanoconjugate was characterized by different techniques exhibiting diameters of about 6.6 ± 1.0 nm. The use of a hyphenated strategy based on high performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) permitted the quantitative evaluation of Fe and Pt in the nanoconjugate. Furthermore, the cellular uptake of the synthetic nanoconjugate was explored by single cell-ICP-MS (SC-ICP) which was used for the first time in this type of studies. The experiments in A2780 and A2780cis, sensitive and resistant ovarian cancer cell models respectively, revealed intracellular platinum concentrations of 12 fg/cell and 4 fg/cell, respectively which were 4-fold higher with respect to the uptake of cisplatin in both models. Intracellular drug release from the nanoconjugate was proved by measuring DNA platination in the same cells. In this case, levels of about 250 ng Pt/mg DNA were observed, about 5-fold higher when the nanoconjugate was used in comparison to cisplatin. Furthermore, the differences between the two lines turned to be significantly smaller than in the case of using cisplatin. The quantitative analytical tools developed here provided essential information required to fully characterize the developed nanoplatforms particularly important to overcome drug resistance.

Addressing the presence of biogenic selenium nanoparticles in yeast cells: analytical strategies based on ICP-TQ-MS.

Several organisms have demonstrated the ability of synthesising biogenic selenium-containing nanoparticles. Such particles from biological sources have attracted great attention due to several proven activities as antioxidants or antimicrobial agents. However, little is known in terms of size (distribution), shapes, chemical composition and number/amount/concentration of these particles. Therefore, in this work, we proposed the use of complementary analytical strategies that enabled the detection and characterization of selenium-containing nanoparticles in selenized yeast (Saccharomyces cerevisiae). The first strategy to address the intracellular presence of Se within yeast cells, involves the use of single cell ICP-TQ-MS (inductively coupled plasma-mass spectrometry). For this aim, selenium and phosphorous (as constitutive element) were measured as oxides (80Se16O+ and 31P16O+, resp.) in the triple-quadrupole mode. Then, a simple and fast cell lysis by mechanical disruption is conducted (approx. 30 min) in order to prove the presence of selenium-containing nanoparticles (SeNPs). The lysate is analysed by single particle ICP-TQ-MS and, complementarily, by liquid chromatography coupled to ICP-TQ-MS to cover a wider range of particle sizes. One of the samples revealed the presence of dispersed SeNPs with sizes between a few nm and up to 250 nm also confirmed by transmission electron microscopy (TEM) in the form of elemental selenium. The analysis of the certified reference material SELM-1 showed the presence of spherical SeNPs of 4 to 7 nm diameter. These biogenic particles, at least partially, were made of elemental selenium as well. The whole study reveals the excellent capabilities of "single" event ICP-MS methodologies in combination with HPLC-based strategies for a complete characterization of nanoparticulated material in biological samples.

Sensitive determination of the human epidermal growth factor receptor 2 (HER2) by immuno-polymerase chain reaction with inductively coupled plasma-mass spectrometry detection.

Sensitive and selective analytical methods are necessary for the determination of clinical biomarkers of breast cancer. The human epidermal growth factor receptor 2 (HER2) is an important breast cancer biomarker since tumors with HER2 protein overexpression (HER2-positive tumors) turn out to be more aggressive and likely to recur. Therefore, accurate determination of serum HER2 values is critical to optimize clinical outcomes in patients with breast cancer. To gain sensitivity and selectivity in the determination of HER2, a sandwich immune assay (highly selective) has been implemented using a detection antibody labelled with a DNA marker. Further amplification of the label using the polymerase chain reaction (PCR), followed by phosphorous quantification of the PCR product (amplicon) using inductively coupled plasma mass spectrometry (ICP-MS), completes this novel assay. Considering that the concentration of the amplicon is proportional to the amount of antigen (HER2) that is recognized by the labelled detection antibody, the concentration of HER2 can be directly obtained by P-analysis. For this aim, a DNA marker of 123 base pairs has been connected to the detection antibody of a sandwich immune assay conducted in pre-coated plates containing the capture antibody of HER2. After the recognition occurred, the PCR amplification was conducted and the PCR product analysed by ICP-MS. Detection limits of 2.5 pg mL-1 of HER2 could be achieved using 35 PCR cycles (7-fold lower than the commercial ELISA method). The developed methodology has been applied to the determination of HER2 in biological samples (human serum and cell culture supernatant of breast cancer cells, MDA-MB-231) obtaining mean method recoveries of about 87% and 81%, respectively.

An inductively coupled plasma-mass spectrometry (ICP-MS) linked immunoassay by means of iodinated antibodies for transferrin quantitative analysis in breast cancer cell lines.

The use of labelled antibodies in inductively coupled plasma-mass spectrometry (ICP-MS) linked immunoassays permits the accurate and sensitive determination of target analytes of biochemical importance. In this regard, the determination of the biomolecules responsible for iron homeostasis in cell cultures is crucial to understand the dysregulation of this element in breast cancer. For this aim, fast, simple, sensitive and accurate analytical strategies have to be developed. In this work, iodinated antibodies have been applied for the sensitive determination of transferrin in breast cancer cell lines using a novel immunoassay coupled to ICP-MS detection of iodine. Firstly, a study on the antibody iodination efficiency revealed an iodine: transferrin molar ratio of 27:1 which corresponds to the iodination of all the tyrosine residues present in the antibody. Secondly, the reactivity of the antibody after labelling was assessed in a quantitative immune subtraction experiment showing that the labelled species maintained their recognition capabilities by capturing 95% of the soluble transferrin. Finally, implementation of the iodinated antibody in a sandwich ICP-MS linked immunoassay was conducted in combination with a secondary biotinylated antibody and the use of streptavidin coated magnetic microparticles. Such set up was used for the determination of transferrin in cell cultures of breast cancer cells of different malignancy. Such determination revealed significant differences among lines with higher transferrin concentration in the line exhibiting higher iron levels, stressing the key aspects of this protein as importer of iron in cells.

Quantitative assessment of the metabolic products of iron oxide nanoparticles to be used as iron supplements in cell cultures.

Iron nanoparticles (NPs) metabolism is directly associated to human health due to their use as anemia treatment and should be studied in detail in cells. Here we present a speciation strategy for the determination of the metabolic products of iron oxide nanoparticles coated by tartaric and adipic acids in enterocytes-like cell models (Caco-2 and HT-29). Such methodology is based on the use of SDS-modified reversed phase high performance liquid chromatography (HPLC) separation using inductively coupled plasma-mass spectrometry (ICP-MS) as Fe selective detector. Post-column isotope dilution analysis is used as quantification tool by adding 57Fe as isotopically enriched standard. To assess the separation capability of the method, two different iron nanostructures: iron sucrose nanoparticles -Venofer®- used as model suspension and iron tartrate/adipate-modified nanoparticles, both of about 4 nm (core size) were evaluated. The two nanostructures were injected into the system showing good peak profiles and quantitative elution recoveries (>80%) in both cases. In addition, both nanoparticulate fractions could be based-line separated from ionic iron species, which needed to be complexed with 1 mM citrate to elute from the column. Exposed cells up to 0.5 mM of iron tartrate/adipate-modified nanoparticles were specifically treated to extract the internalized NPs and the extracts examined using the proposed strategy. The obtained results revealed the presence of three different fractions corresponding to nanoparticle aggregates, dispersed nanoparticles and soluble iron respectively in a single chromatographic run. Quantitative experiments (column recoveries ranging from 60 to 80%) revealed the presence of the majority of the Fe in the nanoparticulated form (>75%) by summing up the dispersed and aggregate particles. Such experiments point out the high uptake and low solubilization rate of the tartrate/adipate NPs making these structures highly suitable as Fe supplements in oral anemia treatments.

Multiplex polymerase chain reaction in combination with gel electrophoresis-inductively coupled plasma mass spectrometry: A powerful tool for the determination of gene copy number variations and gene expression changes.

During the last few years multiplex real-time or quantitative polymerase chain reaction PCR (qPCR) has become the method of choice for multiplex gene expression changes and gene copy number variations (CNVs) analysis. However, such determinations require the use of different fluorescent labels for the different amplified sequences, which increases significantly the costs of the analysis and limits the applicability of the technique for simultaneous amplification of many targets of interest in a single reaction. In this regard, the use of the coupling between gel electrophoresis (GE) separation with inductively coupled plasma mass spectrometry (ICP-MS) detection allows the label-free determination of multiplex PCR-amplified sequences (amplicons) by monitoring the P present in the DNA backbone. The quantitative dimension is obtained since under optimal and controlled multiplex PCR conditions the peak areas of the separated amplicons are directly proportional to the amount of DNA template in the original sample. Moreover, the calibration of the GE-ICP-MS system with a DNA ladder permits direct estimation of the size (bp) of the PCR products. The suitability of the proposed multiplex strategy has been evaluated addressing two different situations: determination of CNVs and gene expression changes in human ovarian cancer cells. In the first case, the results obtained for the simultaneous quantitation of CNVs of four genes (HER2, CCNE1, GSTM1, ACTB) on DNA obtained from OVCAR-3 cells were in accordance with the literature data, and also with the results obtained by conventional simplex qPCR. In the second case, multiplex gene expression changes of BAX, ERCC1 and CTR1 genes, using ACTB as constitutive gene, on A2780cis respect to A2780 cells, resistant and sensitive to cisplatin, respectively, provided the same information as single reaction reverse transcription (RT)-qPCR.

Single particle analysis of TiO2 in candy products using triple quadrupole ICP-MS.

Titanium dioxide (TiO2) belongs to the materials that have gained great importance in many applications. In its particulate form (micro- or nanoparticles), it has entered a huge number of consumer products and food-grade TiO2, better known as E171 within the European Union, represents an important food additive. Thus, there is an increasing need for analytical methods able to detect and quantify such particles. In this regard, inductively coupled-mass spectrometry (ICP-MS), in particular single particle ICP-MS (spICP-MS), has gained importance due to its simplicity and ease of use. Nevertheless, the number of applications for Ti nanoparticles is rather limited. In this study, we have applied the spICP-MS strategy by comparing different measuring modes available in triple quadrupole ICP-MS. First, single quadrupole mode using the collision/reaction cell system was selected for monitoring the isotope 47Ti. Different cell gases like He, O2 and NH3 were tested under optimised conditions for its applicability in spICP-MS of standard suspensions of TiO2. The determined analytical figures of merit were compared to those obtained by triple quadrupole mode using the 47Ti or 48Ti reaction products using O2 and NH3 as reaction gases. This comparison demonstrated that the triple quadrupole mode (TQ mode) was superior in terms of sensitivity due to the more efficient removal of spectral interferences. Particle size detection limits down to 26nm were obtained using the best instrumental conditions for TiO2 particles at a dwell time of 10ms. Finally, the different measuring modes were applied to the analysis of chewing gum samples after a simple extraction procedure using an ultrasonic bath. The obtained results showed a good agreement for the detected particle size range using the different TQ modes. The size range of TiO2 particles was determined to be between approximately 30 and 200nm, whereas roughly 40% of the particles were smaller than 100nm. For the determination of the particle number concentration in these real samples, we suggest CeO2 particles as internal standard.

Metformin regulates global DNA methylation via mitochondrial one-carbon metabolism.

The anti-diabetic biguanide metformin may exert health-promoting effects via metabolic regulation of the epigenome. Here we show that metformin promotes global DNA methylation in non-cancerous, cancer-prone and metastatic cancer cells by decreasing S-adenosylhomocysteine (SAH), a strong feedback inhibitor of S-adenosylmethionine (SAM)-dependent DNA methyltransferases, while promoting the accumulation of SAM, the universal methyl donor for cellular methylation. Using metformin and a mitochondria/complex I (mCI)-targeted analog of metformin (norMitoMet) in experimental pairs of wild-type and AMP-activated protein kinase (AMPK)-, serine hydroxymethyltransferase 2 (SHMT2)- and mCI-null cells, we provide evidence that metformin increases the SAM:SAH ratio-related methylation capacity by targeting the coupling between serine mitochondrial one-carbon flux and CI activity. By increasing the contribution of one-carbon units to the SAM from folate stores while decreasing SAH in response to AMPK-sensed energetic crisis, metformin can operate as a metabolo-epigenetic regulator capable of reprogramming one of the key conduits linking cellular metabolism to the DNA methylation machinery.

Quantitative Evaluation of Cisplatin Uptake in Sensitive and Resistant Individual Cells by Single-Cell ICP-MS (SC-ICP-MS).

One of the main limitations to the Pt-therapy in cancer is the development of associated drug resistance that can be associated with a significant reduction of the intracellular platinum concentration. Thus, intracellular Pt concentration could be considered as a biomarker of cisplatin resistance. In this work, an alternative method to address intracellular Pt concentration in individual cells is explored to permit the evaluation of different cell models and alternative therapies in a relatively fast way. For this aim, total Pt analysis in single cells has been implemented using a total consumption nebulizer coupled to inductively coupled plasma mass spectrometric detection (ICP-MS). The efficiency of the proposed device has been evaluated in combination with flow cytometry and turned out to be around 25% (cells entering the ICP-MS from the cells in suspension). Quantitative uptake studies of a nontoxic Tb-containing compound by individual cells were conducted and the results compared to those obtained by bulk analysis of the same cells. Both sets of data were statistically comparable. Thus, final application of the developed methodology to the comparative uptake of Pt-species in cisplatin resistant and sensitive cell lines (A2780cis and A2780) was conducted. The results obtained revealed the potential of this analytical strategy to differentiate between different cell lines of different sensitivity to the drug which might be of high medical interest.

Iron speciation, ferritin concentrations and Fe : ferritin ratios in different malignant breast cancer cell lines: on the search for cancer biomarkers.

Iron is an essential element for cell growth and division. Recent experiments have linked a deregulation of iron's metabolism with breast cancer progression, aggressiveness and recurrence. In fact, it is conceived that chronic failure in the redox balance due to the presence of a high intracellular concentration of this metal has the potential to modulate specific signaling networks associated with cancer malignancy. Thus, this work has been focused on the comparative evaluation of part of the Fe metallome in two breast cancer cell lines of different malignancies: MCF-7 and MDA-MB-231. Evaluation of the total cytosolic iron content as well as the ultrafiltrable iron content has been conducted using inductively coupled plasma mass spectrometry (ICP-MS) as a Fe selective detector. The obtained results revealed a significantly higher total Fe concentration in the less malignant phenotype. Additionally, Fe-fractionation experiments, conducted by coupling size exclusion chromatography (SEC) to ICP-MS showed a similar Fe distribution (speciation) in both cell phenotypes. However, further specific ferritin measurement using immunochemical based ICP-MS assays showed important differences regarding the total protein content among cell lines and, most importantly, significant differences in the Fe-content of the ferritin molecules between cell lines. This finding points out an iron-storage independent function also associated with ferritin in the most malignant phenotype of the evaluated breast cancer cells that stresses the interest in this molecule as a cancer biomarker.

New strategy to address DNA-methyl transferase activity in ovarian cancer cell cultures by monitoring the formation of 5-methylcytosine using HPLC-UV.

Methylation of mammalian genomic DNA is catalyzed by DNA methyltransferases (DNMTs). Aberrant expression and activity of these enzymes has been reported to play an important role in the initiation and progression of tumors and its response to chemotherapy. Therefore, there is a great interest in developing strategies to detect human DNMTs activity. We propose a simple, antibody-free, label-free and non-radioactive analytical strategy in which methyltransferase activity is measured trough the determination of the 5-methylcytosine (5mC) content in DNA by a chromatographic method (HPLC-UV) previously developed. For this aim, a correlation between the enzyme activity and the concentration of 5mC obtained by HPLC-UV is previously obtained under optimized conditions using both, un-methylated and hemi-methylated DNA substrates and the prokaryotic methyltransferase M.SssI as model enzyme. The evaluation of the methylation yield in un-methylated known sequences (a 623bp PCR-amplicon) turned to be quantitative (110%) in experiments conducted in-vitro. Methylation of hemi-methylated and low-methylated sequences could be also detected with the proposed approach. The application of the methodology to the determination of the DNMTs activity in nuclear extracts from human ovarian cancer cells has revealed the presence of matrix effects (also confirmed by standard additions) that hampered quantitative enzyme recovery. The obtained results showed the high importance of adequate sample clean-up steps.

Quantitative evaluation of cellular uptake, DNA incorporation and adduct formation in cisplatin sensitive and resistant cell lines: Comparison of different Pt-containing drugs.

The use of Pt-containing compounds as chemotherapeutic agents facilitates drug monitoring by using highly sensitive elemental techniques like inductively coupled plasma mass spectrometry (ICP-MS). However, methodological problems arise when trying to compare different experiments due to the high variability of biological parameters. In this work we have attempted to identify and correct such variations in order to compare the biological behavior of cisplatin, oxaliplatin and pyrodach-2 (a novel platinum-containing agent). A detailed study to address differential cellular uptake has been conducted in three different cell lines: lung adenocarcinoma (A549); cisplatin-sensitive ovarian carcinoma (A2780); and cisplatin-resistant ovarian carcinoma (A2780cis). The normalization of Pt results to cell mass, after freeze-drying, has been used to minimize the errors associated with cell counting. Similarly, Pt accumulation in DNA has been evaluated by referencing the Pt results to the DNA concentration, as measured by (31)P monitoring using flow-injection and ICP-MS detection. These strategies have permitted to address significantly lower Pt levels in the resistant cells when treated with cisplatin or oxaliplatin as well as an independent behaviour from the cell type (sensitive or resistant) for pyrodach-2. Similarly, different levels of incorporation in DNA have been found for the three drugs depending on the cell model revealing a different behavior regarding cell cisplatin resistance. Further speciation experiments (by using complementary HPLC-ICP-MS and HPLC-ESI-Q-TOF MS) have shown that the main target in DNA is still the N7 of the guanine but with different kinetics of the ligand exchange mechanism for each of the compounds under evaluation.

Reversed phase and cation exchange liquid chromatography with spectrophotometric and elemental/molecular mass spectrometric detection for S-adenosyl methionine/S-adenosyl homocysteine ratios as methylation index in cell cultures of ovarian cancer.

S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are essential compounds in the carbon metabolic cycle that have clinical implications in a broad range of disease conditions. The measurement of the ratio SAM/SAH also called methylation index, has become a way of monitoring the DNA methylation of a cell which is an epigenetic event with important clinical implications in diagnosis; therefore the development of suitable methods to accurately quantify these compounds is mandatory. This work illustrates the comparison of three independent methods for the determination of the methylation index, all of them based on the chromatographic separation of the two species (SAM and SAH) using either ion-pairing reversed phase or cation exchange chromatography. The species detection was conducted using either molecular absorption spectrophotometry (HPLC-UV) or mass spectrometry with electrospray (ESI-MS/MS) as ionization source or inductively coupled plasma (DF-ICP-MS) by monitoring the S-atom contained in both analytes. The analytical performance characteristics of the three methods were critically compared obtaining best features for the combination of reversed phase HPLC with ESI-MS in the MRM mode. In this case, detection limits of about 0.5ngmL(-1) for both targeted analytes permitted the application of the designed strategy to evaluate the effect of cisplatin on the changes of the methylation index among epithelial ovarian cancer cell lines sensitive (A2780) and resistant (A2780CIS) to this drug after exposition to cisplatin.

Enhanced detection of DNA sequences using end-point PCR amplification and online gel electrophoresis (GE)-ICP-MS: determination of gene copy number variations.

The design and evaluation of analytical methods that permit quantitative analysis of specific DNA sequences is exponentially increasing. For this purpose, highly sensitive methodologies usually based on labeling protocols with fluorescent dyes or nanoparticles are often explored. Here, the possibility of label-free signal amplification using end-point polymerase chain reaction (PCR) are exploited using on-column agarose gel electrophoresis as separation and inductively coupled plasma-mass spectrometry (ICP-MS) for the detection of phosphorus in amplified DNA sequences. The calibration of the separation system with a DNA ladder permits direct estimation of the size of the amplified gene fragment after PCR. With this knowledge, and considering the compound-independent quantification capabilities exhibited by ICP-MS for phosphorus (it is only dependent on the number of P atoms per molecule), the correlation of the P-peak area of the amplified gene fragment, with respect to the gene copy numbers (in the starting DNA), is then established. Such a relationship would permit the determination of copy number variations (CNVs) in genomic DNA using ICP-MS measurements. The method detection limit, in terms of the required amount of starting DNA, is ∼6 ng (or 1000 cells if 100% extraction efficiency is expected). The suitability of the proposed label-free amplification strategy is applied to CNVs monitoring in cells exposed to a chemical agent capable of deletion induction, such as cisplatin.

Incorporation of (57)Fe-isotopically enriched in apoferritin: formation and characterization of isotopically enriched Fe nanoparticles for metabolic studies.

The use of (57)Fe-isotopically enriched ferritin for the accurate measurement of Fe : ferritin ratios is proposed for metabolic studies. Thus, the synthesis of (57)Fe-isotopically enriched ferritin from horse apo-ferritin and isotopically enriched (NH4)2(57)Fe(II)(SO4)2 (Mohr's salt) is conducted. Size exclusion chromatography on-line with UV-VIS absorption (at 380 nm) is used in order to monitor the loading process of apo-ferritin. These studies revealed that the Fe-incorporation process involves also the formation of protein aggregates (oligomers) showing higher molecular mass than ferritin. A final optimized protocol involving incubation of the synthesized standard with guanidine hydrochloride (pH 3.5) has provided the best conditions for maintaining a stable protein structure without aggregates. Such (57)Fe-isotopically enriched ferritin was characterized and contained an average of 2200 atoms of Fe per mole of ferritin. The evaluation of the Fe-core after saturation with (57)Fe by Transmission Electron Microscopy (TEM) has revealed the formation of (57)Fe nanoparticles with a similar diameter to that of the commercial Fe-containing ferritin, confirming the process of Fe uptake, oxidation and mineralization within the protein cavity. The synthesized (57)Fe-ferritin shows great potential as a nanometabolic tracer to study the kinetics of Fe release in the cases of iron metabolic disorders.