RESUMEN
It is now well-established that dysregulation of the tricarboxylic acid (TCA) cycle enzymes succinate dehydrogenase, fumarate hydratase, and isocitrate dehydrogenase leads to the abnormal cellular accumulation of succinate, fumarate, and 2-hydroxyglutarate, respectively, which contribute to the formation and malignant progression of numerous types of cancers. Thus, these metabolites, called oncometabolites, could potentially be useful as tumour-specific biomarkers and as therapeutic targets. For this reason, the development of analytical methodologies for the accurate identification and determination of their levels in biological matrices is an important task in the field of cancer research. Currently, hyphenated gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) techniques are the most powerful analytical tools in what concerns high sensitivity and selectivity to achieve such difficult task. In this review, we first provide a brief description of the biological formation of oncometabolites and their oncogenic properties, and then we present an overview and critical assessment of the GC-MS and LC-MS based analytical approaches that are reported in the literature for the determination of oncometabolites in biological samples, such as biofluids, cells, and tissues. Advantages and drawbacks of these approaches will be comparatively discussed. We believe that the present review represents the first attempt to summarize the applications of these hyphenated techniques in the context of oncometabolite analysis, which may be useful to new and existing researchers in this field.
Asunto(s)
Neoplasias , Biomarcadores de Tumor , Cromatografía Liquida , Humanos , Espectrometría de MasasRESUMEN
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.
Asunto(s)
Neoplasias de la Mama , Inmunoconjugados , Biomarcadores de Tumor , Neoplasias de la Mama/diagnóstico , Línea Celular Tumoral , Femenino , Humanos , Lutecio , Pronóstico , Receptor ErbB-2RESUMEN
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 2.5â¯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.
Asunto(s)
Biomarcadores de Tumor/sangre , Inmunoensayo/métodos , Espectrometría de Masas/métodos , Reacción en Cadena de la Polimerasa/métodos , Receptor ErbB-2/sangre , Anticuerpos/inmunología , Biomarcadores de Tumor/inmunología , Biotina/química , Neoplasias de la Mama/diagnóstico , Línea Celular Tumoral , ADN/química , Humanos , Límite de Detección , Receptor ErbB-2/inmunología , Estreptavidina/químicaRESUMEN
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.
Asunto(s)
Anticuerpos/química , Neoplasias de la Mama/patología , Halogenación , Inmunoensayo/métodos , Espectrometría de Masas , Transferrina/análisis , Humanos , Células MCF-7 , Microesferas , Factores de TiempoRESUMEN
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-3â¯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 A2780â¯cells, resistant and sensitive to cisplatin, respectively, provided the same information as single reaction reverse transcription (RT)-qPCR.
Asunto(s)
ADN de Neoplasias/genética , Reacción en Cadena de la Polimerasa Multiplex , Neoplasias Ováricas/genética , Línea Celular Tumoral , Variaciones en el Número de Copia de ADN , Electroforesis Capilar , Femenino , Perfilación de la Expresión Génica , Humanos , Espectrometría de MasasRESUMEN
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.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Carbono/metabolismo , Neoplasias del Colon/tratamiento farmacológico , Metilación de ADN/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genoma Humano , Metformina/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Biomarcadores de Tumor , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Complejo I de Transporte de Electrón/metabolismo , Femenino , Estudios de Seguimiento , Humanos , Hipoglucemiantes/farmacología , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Neoplasias de la Mama/patología , Ferritinas/análisis , Hierro/análisis , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismo , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Ferritinas/metabolismo , Humanos , Hierro/metabolismoRESUMEN
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.
Asunto(s)
5-Metilcitosina/metabolismo , Cromatografía Líquida de Alta Presión/métodos , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Neoplasias Ováricas/enzimología , 5-Metilcitosina/análisis , Animales , Secuencia de Bases , Bovinos , Línea Celular Tumoral , Islas de CpG , ADN/química , ADN/metabolismo , ADN (Citosina-5-)-Metiltransferasas/análisis , Pruebas de Enzimas/métodos , Femenino , Humanos , Ovario/enzimologíaRESUMEN
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.
Asunto(s)
Metilación de ADN , Neoplasias Ováricas/metabolismo , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Antineoplásicos/farmacología , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión/métodos , Cromatografía por Intercambio Iónico/métodos , Cromatografía de Fase Inversa/métodos , Cisplatino/farmacología , Resistencia a Antineoplásicos , Femenino , Homocisteína , Humanos , Metilación , Peso Molecular , Neoplasias Ováricas/genética , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
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 â¼6 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.
Asunto(s)
Variaciones en el Número de Copia de ADN , ADN/genética , Internet , Reacción en Cadena de la Polimerasa , Secuencia de Bases , Línea Celular , Electroforesis , Humanos , Espectrometría de Masas , Reacción en Cadena de la Polimerasa/instrumentaciónRESUMEN
Serum transferrin (Tf) is an iron binding glycoprotein that plays a central role in the metabolism of this essential metal but it also binds other metal ions. Four main transferrin forms containing different iron binding states can be distinguished in human serum samples: monoferric (C-site or N-site), holotransferrin (with two Fe atoms) and apotransferrin (with no metal). Recently, it has been reported that Tf binds also Ti even more tightly than does Fe, in artificially Ti(iv) spiked solutions. However, very limited work has been done on the Ti binding to Tf at physiological concentrations in patients carrying intramedullary Ti nails. Here we report the chemical association of Ti to Tf "in vivo" under different chromatographic conditions by elemental mass spectrometry using double focusing inductively coupled plasma (DF-ICP-MS) as detector. For the separation of the Ti/Fe-Tf forms different gradient conditions have been explored. The observed results reveal that human serum Ti (from patients carrying intramedullary Ti nails) is uniquely associated to the N-lobe of Tf. The investigation of the influence of sialic acid in the carbohydrate chain of human serum Tf, studied by incubating the protein with neuraminidase (sialidase) to obtain the monosialilated species, revealed that the binding affinity of Ti was similar for monosialo-Tf and for native-Tf and occurs in the N-lobe. These results suggest that the species Fe(C)Ti(N)-TF might provide a route for Ti entry into cells via the transferrin receptors after the release of the metal from its implants.
Asunto(s)
Titanio/metabolismo , Transferrina/química , Transferrina/metabolismo , Sitios de Unión , Cromatografía , Humanos , Hierro/aislamiento & purificación , Unión Proteica , Ácidos Siálicos/análisis , Ácidos Siálicos/aislamiento & purificación , Titanio/aislamiento & purificaciónRESUMEN
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(-1) 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(-1)) 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%.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Cromatografía por Intercambio Iónico/métodos , Radioisótopos/sangre , Espectrofotometría Atómica/métodos , Titanio/sangre , Transferrina/análisis , Huesos/efectos de los fármacos , Huesos/cirugía , Calibración , Humanos , Límite de Detección , Prótesis e Implantes/efectos adversos , Unión Proteica , Técnica de Dilución de Radioisótopos , Radioisótopos/química , Estándares de Referencia , Reproducibilidad de los Resultados , Transferrina/metabolismoRESUMEN
A sample preparation strategy for the determination of the Fe-containing enzyme catalase (CAT) by Fe specific monitoring in human erythrocytes has been optimized. For this purpose, the combined use of elemental mass spectrometry (via inductively coupled plasma, ICP-MS), molecular mass spectrometry (via MALDI-TOF) and enzymatic activity measurements has been required. The procedure involved haemoglobin precipitation from cell lysate with a solution of ethanol-chloroform and preconcentration of the supernatant by using a Speed-Vac concentrator. Catalase recoveries of about 88 ± 15% could be measured by monitoring the protein enzymatic activity before and after precipitation. Further fractionation of Fe-containing proteins from the preconcentrated extract was achieved by size exclusion chromatography (Superdex 200) with a mobile phase of ammonium acetate (0.05 M, pH 7.4) coupled to ICP-MS (Fe monitoring) and UV/VIS detection (specific absorption of the heme-group at 408 nm). A second dimensional chromatography of the CAT-positive activity fraction was carried out by anion-exchange chromatography (Mono Q 5/50) using for elution a linear gradient of ammonium acetate (0-0.750 M in 15 min). This second step revealed a single Fe-containing species in the chromatogram and permitted the unambiguous characterization of the CAT in such fractions by MALDI-TOF. Column recoveries were evaluated and were quantitative, in terms of Fe bound to protein and CAT activity.
Asunto(s)
Catalasa/sangre , Eritrocitos/enzimología , Espectrometría de Masas/métodos , Resinas de Intercambio Aniónico , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Humanos , Espectrofotometría UltravioletaRESUMEN
The interest on accurate and precise determination of metalloproteins such as Cu, Zn-superoxide dismutase (Cu, Zn-SOD) involved in the redox balance of living cells is increasing. For this purpose, analytical strategies that provide absolute protein concentration measurements have to be developed. The determination of Cu, Zn-SOD through the measurement of the Cu associated to the protein, which provides its enzymatic activity, by liquid chromatography with online inductively coupled plasma mass spectrometric (ICPMS) detection is described here. Postcolumn isotope dilution analysis (IDA) of Cu has been applied for quantification after evaluation of the column recovery for the total Cu and also Cu-SOD that turned out to be quantitative. When the concentration results obtained via IDA using high-performance liquid chromatography (HPLC)-ICPMS are plotted versus the activity measurements (using the spectrophotometric pyrogallol autoxidation method) a good correlation curve is obtained. Such results permit us, from ICPMS measurements, to obtain simultaneously the Cu, Zn-SOD absolute concentration as well as its enzymatic activity by interpolation in the previously obtained curve. This possibility was explored in real samples (red blood cells of control individuals and patients with metallic total hip arthroplasty) obtaining a good match between direct enzymatic activity measurements and those obtained by interpolation in the correlation curve. The actual protein identification in the red blood cell extract was conducted by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and two matrixes were compared in order to preserve as much as possible the protein-metal interactions during the MALDI process. Interestingly, using a solution containing trihydroxyacetophenone in citrate buffer permitted us to observe some metal-protein interactions in the MS spectrum of the intact Cu, Zn-SOD from red blood cells.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Eritrocitos/enzimología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Superóxido Dismutasa/metabolismo , Animales , Bovinos , Humanos , Superóxido Dismutasa/análisisRESUMEN
Platinum compounds are the major group of metal-based chemotherapeutic drug used in current practice and still a topic of intense investigation. The relative contribution of structurally defined cisplatin adducts with DNA to induce apoptosis and the cellular processing of these lesions is still poorly understood mostly due to the lack of sensitive and accurate analytical tools for in vivo studies. In this regard, two novel sensitive and selective strategies are proposed here to quantify cisplatin-DNA adducts generated in Drosophila melanogaster larvae and in head and neck squamous cell carcinoma cultures. The methods involve the isolation and enzymatic digestion of the DNA in the samples exposed to cisplatin and further quantification by high-performance liquid chromatography with inductively coupled plasma mass spectrometric detection (HPLC-ICPMS). Two different strategies, based on isotope dilution analysis (IDA), have been attempted and evaluated for quantification: species-unspecific (the postcolumn addition of a 194Pt-enriched solution) and the species-specific (by means of a synthesized isotopically enriched cisplatin (194Pt) adduct). For the second approach, the synthesis and characterization of the cisplatin adduct in a custom oligonucleotide containing the sequence (5'-TCCGGTCC-3') was necessary. The adducted oligo was then added to the DNA samples either before or after enzymatic hydrolysis. The results obtained using these two strategies (mixing before and after enzymatic treatment) permit to address, quantitatively, the column recoveries as well as the efficiency of the enzymatic hydrolysis. Species-specific spiking before enzymatic digestion provided accurate and precise analytical results to clearly differentiate between Drosophila samples and carcinoma cell cultures exposed to different cisplatin concentrations.
Asunto(s)
Cisplatino/metabolismo , Aductos de ADN/metabolismo , Animales , Secuencia de Bases , Línea Celular Tumoral , Cisplatino/química , Aductos de ADN/genética , Drosophila melanogaster/metabolismo , Humanos , Técnicas de Dilución del Indicador , Isótopos , Espectrometría de Masas , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismoAsunto(s)
Aminoácidos/aislamiento & purificación , Cromatografía Capilar Electrocinética Micelar/métodos , Ciclodextrinas/química , Compuestos de Selenio/aislamiento & purificación , Dodecil Sulfato de Sodio/química , Ácido Taurodesoxicólico/química , beta-Ciclodextrinas , Aminoácidos/química , Tampones (Química) , Concentración de Iones de Hidrógeno , Indicadores y Reactivos , Compuestos de Selenio/química , EstereoisomerismoRESUMEN
HPLC was used to study the effectiveness of two different desferrioxamine (DFO) administration strategies (15 mg/kg DFO, 1 h or 44 h before dialysis) on generation of aluminoxamine and ferrioxamine in five hemodialysis patients. The percentage of ultrafilterable aluminum and iron in these patients was also investigated by electrothermal atomic absorption spectrometry. The administration of DFO in both schemes increased the ultrafilterable serum aluminum concentrations from a mean of 17.1 +/- 1.6% to a mean of 75.7 +/- 14.1%. However, 1 h after DFO infusion, only 38.8 +/- 7.7% of the total serum aluminum was bound to DFO; 44 h after DFO infusion, only 15.8 +/- 8.0% was bound. Similar results were obtained for ferrioxamine. These results suggest that the ultrafilterable serum fraction contains aluminum and iron chelated by DFO and by DFO metabolites, which retain similar metal-chelating abilities.
Asunto(s)
Aluminio/sangre , Quelantes , Deferoxamina/sangre , Compuestos Férricos/sangre , Compuestos Organometálicos/sangre , Uremia/sangre , Quelantes/administración & dosificación , Cromatografía Líquida de Alta Presión , Deferoxamina/administración & dosificación , Humanos , Diálisis Renal , Espectrofotometría Atómica , Factores de Tiempo , Uremia/terapiaRESUMEN
Fast protein liquid chromatography (FPLC) was used with electrothermal atomic absorption spectrometric (ETAAS) detection for quantitative studies of aluminium binding species in unspiked human uremic serum. A rapid and reproducible separation of human serum proteins and other aluminium binders (citrate and desferroxiamine) was achieved on a Mono Q (HR 5/5) anion-exchange column using a sodium chloride gradient (0-0.25 mol l-1) at the physiological human serum pH of 7.4 (0.05 mol l-1 buffer TRIS-HCl). The aluminium distribution in the column fractions was determined by ETAAS. Aluminium contamination was avoided by using an inert chromatographic system equipped with an on-line aluminium-chelating scavenger column (Kelex 100-impregnated silica C18). The sensitivity of the proposed method (detection limit for Al in serum = 5 micrograms l-1) allowed aluminium speciation studies at clinically relevant concentrations (unspiked serum from dialysis patients). The results obtained confirmed that transferrin is the only serum protein binding aluminium and it contains about 90% of total serum aluminium (post-elution aluminium recovery = 105 +/- 5%). It was also confirmed that in the presence of the chelating drug desferrioxamine (DFO) most of the serum aluminium (80%) is bound to DFO.