Development of a Multidimensional Screening Technique for Metal Containing Proteins in Blood

Trace elements involved in the human metabolism have gained great importance for medical and physiological purposes; e.g. antioxidant enzymes involved in the metabolism of oxygen free radicals, superoxide dismutases and glutathion peroxidase, contain the essential trace elements Cu, Zn, Mn, and Se. Other problems deal with element speciation. Analytical methods for the determination of metals and their species in blood have to be established. For the separation of metal-protein complexes a combination of chromatographic techniques employing different separation principles involving hydrophobe interaction (HIC), size exclusion (SEC), ion exchange (IEC) and affinity type liquid chromatography (LC) but also electrophoretic separation techniques like two dimensional polyacrylamide gel electrophoresis (2-D PAGE), capillary zone electrophoresis (CZE), and isotachophoresis (ITP) will be used. Either atomic spectroscopy (inductively coupled plasma atomic emission spectrometry, ICP-AES, or graphite furnace atomic absorption spectrometry, GFAAS) as element specific detector, or mass spectrometry (MS-MS) as mass and fragment selective detector and separation methods will be hyphenated on-line and/or off-line and the analytical performance of the resulting coupled analytical techniques will be compared. The aim is to select and to develop an optimal combination of separation methods and specific detection for metal species and metal-protein complexes to obtain as much information as possible for their characterization. Combining multidimensional separation methods with selective and sensitive detection principles, which are set up for metal protein fractionation, results in a quite complex data matrix describing the metal protein composition. By the selected combination of methods both the status of metals and their species in blood and their distribution in blood fractions can be analyzed.

The metabolism of trace elements and antioxidants in human blood and in blood fractions (erythrocytes, plasma, lymphocytes) can lead to new understandings of chronic diseases (e.g. diabetes mellitus, Parkinson syndrome, Alzheimer disease). The essential elements Cu and Zn are constituents of antioxidant enzymes (superoxide dismutases) playing a role for the metabolism of the free radicals. The status of these elements in blood and the distribution of their species in blood fractions were investigated. The elements Zn and Cu could be related to SOD both in erythrocytes and blood plasma. Thereby the determination of SOD can be performed without the necessity of a complete separation of the proteins. For the separation of the metal containing proteins chromatographic techniques based on different separations principles (hydrophobic interaction, molecular size) were applied. Furthermore gel electrophoresis and capillary electrophoresis were tested. For the determination of the trace elements in question atomic spectrometric methods (ICP-AES and GFAAS) were applied enabling element specific detection down to the ultra trace level. The final determinations were optimized separately for separation method, since different and to a certain extend very low amounts of samples were investigated. In addition the exact determination of the trace elements was complicated by the different chemical surroundings (matrix) of the analytes. For the medical diagnostics it is interesting to develop a routinely performable hyphenation procedure for the separation and detection methods. The profiles obtained from ICP-AES after the chromatographic separations show that the main concentrations of the elements Cu, Mn and Zn are found in different fractions. Zn and Cu could be related to SOD (superoxide dismutases) both in erythrocytes and blood plasma, whereby the determination of SOD was possible by element detection without complete separation of the proteins. It follows that the element containing proteins can be separated by the hydrophobic interaction chromatography method applied. The tentacle SEC system was used for the separation of the proteins in real samples as well. An off-line detection of Cu, Fe, Zn, and Mn could be applied for the identification of Cu/Zn SOD in erythrocytes, while an off-line coupling could be performed successfully for the iron determination after separation of hemoglobin. The disadvantage of this method is the high dilution factor for the samples. The method developed enables the identification, i.e. qualitative determination, of the metal protein complexes but until now not the quantitative determination. Since all these separation techniques exhibit different advantages, they can be used for the clarification of different procedures. The different concentration ranges of the metals can be covered by two different atomic spectrometric methods. The total of the results obtained enables a comprehensive understanding of the functioning of biological systems.