Daria Martin, Schering Stiftung Projektraum


Schering Prize 2002

Dr. Nicole Richarz

Journal of Trace Elements in Medicine and Biology, Berlin

Speciation analysis of protein-bound elements in cytosols as biological markers for metabolic processes with special emphasis on metallothioneins in the brain

The physiological functions of most trace elements in the human body depend on their binding to different proteins: transport proteins distribute elements throughout the organism, storage proteins can either bind elements present in excess or compensate for an inadequate supply by remobilising them, metalloenzymes or metalloproteins are involved in various metabolic processes. Investigation of the different bound forms of the elements, i.e. the metal-protein-complexes, is crucial to an understanding of physiological and pathological processes in the organism.

The separation of the proteins and the determination of the bound trace elements is carried out by means of speciation analyses. A speciation analysis is an analysis performed to identify and/or quantify one or several chemical species in a sample. In the present work, the species investigated were elements bound to different proteins in tissue cytosols. The binding may comprise covalent integration of the element into the structure of a metalloprotein or complexation of the element by a protein.

Speciation analyses were conducted using hyphenated techniques: separation of the biomolecules by chromatography or capillary zone electrophoresis followed by detection of the bound elements in the eluate. The sensitive method of inductively coupled plasma mass spectrometry was used for multielement detection. The on-line hyphenation allowed detection of profiles of up to 36 isotopes in the same run. The first step was the optimisation of the analytical procedure for the investigation of metalloproteins in tissue cytosols. The signals from the element profiles were subsequently assigned to different proteins. This identification was achieved by probing the eluate of the chromatographical separations using specific detection methods.

In the next step, the element binding patterns were compared in different samples. Firstly, cytosols of different organs were investigated in order to find organ specific distributions of the metalloproteins. In addition, changes in the trace element patterns for a given organ with different pathologies were considered. Variations of the element binding patterns were found in liver cytosols of different patients.
The emphasis in this study was on the investigation of brain samples from patients who had suffered from Alzheimer's disease compared to a control group.

Special attention was paid to the metallothioneins (MT), proteins of low molecular weight, rich in cystein, that have a high capacity to bind metals like copper, zinc and cadmium. They have radical scavenging properties and are considered to play an important role in the homeostasis of the trace elements zinc and copper and to be involved in various vital metabolic processes. MT exist in several isoforms, the isoform MT-3 - or growth inhibition factor - is found especially in the brain and has been investigated in relation to Alzheimer's disease (AD). Preliminary studies had found a decreased expression of MT-3 in AD brains, which was regarded as a possible cause for the uncontrolled nerve growth occurring in AD. However, these findings could not be confirmed in all subsequent investigations. For this reason it was of particular interest to conduct a comparison of AD and control brains using a completely different approach - speciation analysis of elements.

The investigation of the brain cytosols showed lower MT copper and zinc signals in the element profiles of the AD samples compared to the controls. However, after adding a reducing agent to the same samples, all samples showed increased MT metal signals and the difference between AD and control brains were less pronounced. Therefore, the presence of oxidised MT in the cytosols of both patient groups can be assumed. The main difference between AD and control brains was not in the total amount of MT, but in an increased ratio of oxidised to reduced MT in the AD brains. A correlation between pathological changes occurring in Alzheimer's disease and oxidative processes could thereby be confirmed.

In conclusion, this study showed that the investigation of the distribution of trace elements between different proteins allows insights into physiological and pathological processes in different tissues. Speciation analysis with sensitive element detection is an appropriate method for this type of study.



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