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Elemental speciation analysis in biomolecules

Final Activity Report Summary - ELSA-BIM (Elemental speciation analysis in biomolecules)

Selenium is one of the most widely investigated of all the trace element nutrients in the last decades, mainly due to its role in cancer prevention. It is still largely accepted that most biological functions attributed to Se are mediated by the selenoproteins. In human serum two selenoproteins are most important, namely glutathione peroxidase (GPx) and selenoprotein P (SelP); Se is also incorporated into albumin forming a seleno-containing protein (selenoalbumin, SeAlb), via unspecified replacement of methionine with its seleno-isologue, namely selenomethionine. Accurate determination of selenoproteins in human blood/serum is of great importance, because their levels can provide valuable information regarding the risk of cancer for persons with selenium deficiency. Nevertheless, there is still a major lack in analytical methodologies for their accurate and fast determination. Therefore, the development of new analytical approaches for reliable speciation analysis of selenoproteins in human serum is extremely important.

The determination of serum GPx, SelP and SeAlb (for simplicity all refereed here as selenoproteins) is challenging because of their low concentration and the very complex serum matrix, which contains interfering components at very high levels. This study aimed at the development of simple, fast and highly reliable methods for simultaneous determination of serum selenoproteins (GPx, SelP and SeAlb).

In the first part of the work, such an approach was developed using affinity-liquid chromatography (AF-HPLC) hyphenated to a conventional inductively coupled plasma-quadrupole mass spectrometry (ICP-QMS). Anion exchange solid-phase extraction (AE-SPE) was carried out before the chromatographic separation (off-line) for the removal of chloride (Cl) and bromide (Br) (present in serum at high levels), which produce serious spectral interferences on Se determination by ICP-QMS. A system consisting of a double column AF-HPLC was used for separation of the selenoproteins in serum after its AE-SPE treatment. On-line external calibration (ON-EC) by using an inorganic selenium species as calibrant was also proven as a routine analytical tool for speciation analysis of selenoproteins in human serum (method published in J. Anal. At. Spectrom., 2008, 23, 402-406).

In the second part of the project, an on-line approach was used for the removal of Cl/Br interferences, by using an anion exchange column connected on-line approach with the affinity HPLC system. On-line (post-column) isotope dilution (ON-ID) and on-line external calibration (ON-EC)-ICP-QMS were used for the quantification of Se in GPx, SelP and SeAlb but external calibrationh using a Se-L-cystine standard was proven as a suitable approach for routine simultaneous speciation analysis of serum GPx, SelP and SeAlb, which can be useful for clinical laboratories (method published in Anal. Bioanal. Chem., 2008, 391, 661-669).

The last part of the work dealing with analytical method development focused on a new method for simultaneous speciation analysis of GPx, SelP and SeAlb in human serum on the basis of microbore AF-HPLC (5 microlitres of serum used for analysis) hyphenated to ICP-sector filed MS (ICP-SFMS). This is important because, besides the difficulties regarding the analysis of the serum selenoproteins by ICP-MS (due to spectral interferences), a major point to consider from a clinical point of view is also the limited availability of the (human) samples. In this context, microbore AF-HPLC, which requires only 5 microlitres of serum for analysis were made 'in house' for this project. In addition, given that ICP-SFMS is a high resolution technique, which does not suffer from spectral interferences (e.g. of Cl and Br on Se isotopes), there is no need for serum treatment for removal of Cl and Br, neither off-line nor on-line. This fact ensures a short analysis time compared with the two approaches mentioned above. This study is a novel one, as it employed for the first time microbore AF-HPLC hyphenated to ICP-SFMS and is a reliable approach for interference-free simultaneous speciation analysis of selenoproteins in human serum.

The very low volume of serum makes it particularly useful for clinical applications when the amount of sample available is commonly very limited. The manuscript dealing with the development of the microbore AF-HPLC-ICP-SFMS is currently in preparation. In conclusion, the studies carried out in the framework of the Marie Curie Individual Fellowship: 'Elemental speciation analysis in biomolecules' (ELSA-BIM) are innovative and hence valuable information is provided in terms of accurate assessment of selenoproteins in human serum.