Quantification of modified nucleotides and nucleosides by isotope dilution mass spectrometry.

Epigenetic modifications are closely related to certain disorders of the organism, including the development of tumors. One of the main epigenetic modifications is the methylation of DNA cytosines, 5-methyl-2'-deoxycycytidine. Furthermore, 5-mdC can be oxidized to form three new modifications, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, and 5-carboxy-2'-deoxycytidine. The coupling of liquid chromatography with tandem mass spectrometry has been widely used for the total determination of methylated DNA cytosines in samples of biological and clinical interest. These methods are based on the measurement of the free compounds (e.g., urine) or after complete hydrolysis of the DNA (e.g., tissues) followed by a preconcentration, derivatization, and/or clean-up step. This review highlights the main advances in the quantification of modified nucleotides and nucleosides by isotope dilution using isotopically labeled analogs combined with liquid or gas chromatography coupled to mass spectrometry reported in the last 20 years. The different possible sources of labeled compounds are indicated. Special emphasis has been placed on the different types of chromatography commonly used (reverse phase and hydrophilic interaction liquid chromatography) and the derivatization methods developed to enhance chromatographic resolution and ionization efficiency. We have also revised the application of bidimensional chromatography and indicated significant biological and clinical applications of these determinations.

Titanium levels in the organs and blood of rats with a titanium implant, in the absence of wear, as determined by double-focusing ICP-MS.

Titanium (Ti) has long been regarded as an inert and biocompatible metal, ideal for biomedical applications such as dental implants or joint replacements. However, concerns about the biocompatibility of Ti have lately arisen. Unfortunately, information on reliable Ti baseline physiological levels in blood and organ tissues is still pending and the real effects of physiological corrosion as opposed to wear processes of Ti or Ti alloys implants is controversial so far. In this work a previously developed and validated methodology, based on using double-focusing inductively coupled plasma mass spectrometry (DF-ICP-MS) has been used to establish Ti basal levels in blood and organs (heart, liver, spleen, kidneys, and lungs) of Wistar rats. These data were compared with the levels found in three Wistar rats implanted with a Ti wire embedded in their femur for 18 months, in order to assign possible Ti released purely due to non-wear physiological mechanisms. Results showed that Ti content in all the selected organ tissues and blood was higher than previously determined Ti basal levels, clearly showing both corrosion of the Ti implant and systemic Ti accumulation in target tissues. These results indicate that Ti metal corrosion occurs. This seems to be the only mechanism responsible in the long term for the observed passive dissolution of Ti of the implant in the absence of wear. A comparative study of the systemic distribution of the soluble and particulate Ti potentially released from Ti implants was also carried out by intraperitoneally injection of soluble Ti(citrate)(3) and insoluble TiO(2) particles, respectively. Different systemic Ti storage was observed. Whereas soluble Ti was rapidly transported to all distal organs under study, TiO(2) particles were only accumulated in lung tissue.

HPLC-ICPMS and stable isotope-labeled approaches to assess quantitatively Ti(IV) uptake by transferrin in human blood serum.

Little is known about the effects of titanium found in patients wearing prostheses or about the biochemical pathways of this metal when used as an anticancer drug (e.g., titanocene dichloride). In this work, transferrin has been confirmed as the only carrier protein binding Ti in human blood serum samples by making use of different HPLC protein separations followed by element-specific Ti detection by ICPMS. Besides, isotope dilution analysis has been applied to the quantitative speciation of Ti-Tf in standards and human blood serum samples. Species-unspecific and species-specific isotope dilution modes have been explored. In the first case, very low Ti-Tf results were obtained even using two different chromatographic mechanisms, anion exchange (20-24%) and size exclusion (33-36%). Surprisingly, no major Ti species except Ti-Tf were observed in the chromatograms, suggesting that Ti(IV) hydrolysis and precipitation as inactive titanium oxide species could take place inside the chromatographic columns. These results demonstrate that chemical degradation of metalloproteins during analytical separations could ruin the sought speciation quantitative results. The isotope dilution species-specific mode, much more accurate in such cases, has been instrumental in demonstrating the possibility of gross errors in final metalloprotein quantification. For this purpose, an isotopically enriched standard of (49)Ti-Tf was synthesized and applied to the quantitative speciation of Ti-Tf again. Using this species-specific spike, Ti-Tf dissociation inside the chromatographic columns used could be corrected, and thus, quantitative Ti-Tf binding in serum (92-102%) was observed. In other words, the usefulness and potential of a species-specific isotope dilution analysis approach to investigate quantitatively metal-protein associations, which can be dissociated at certain experimental conditions, is demonstrated here for the first time.