Investigation of the metabolic changes caused by isocitrate dehydrogenase mutations using mass spectrometry

Isocitrate dehydrogenase mutations have previously been found to be associated with tumour cell growth, especially the mutation of arginine 132 to histidine (R132H) although other mutations have been observed. These mutations are found in the majority of grade 2 and grade 3 gliomas in humans, especially impacting those between the ages of 20 and 40 and currently have one of the lowest 5-year survival rates of all cancer types making them a prime clinical target. The R132H mutation disrupts the normal citric acid cycle conversion of isocitrate to 2-oxoglutarate and catalyses the production of 2-hydroxyglutarate from both species, possibly due to competitive binding to the isocitrate dehydrogenase active site which has been shown to be moderated by R132. The physiological purpose for the upregulation of 2-hydroxyglutarate is not currently known and requires further investigation as several competing mechanisms of action have been postulated. These include the inhibition of 2-oxoglutarate dependent oxygenases, which alters the expression of the genes involved in cell differentiation and posttranslational modification of proteins. The mutation has however, been observed in early glioma development suggesting a pro-oncogenic role. Loss of function mutations have also been observed in other cancer types which results in the accumulation of fumarate and succinate which are also citric acid cycle metabolites of prime interest.
Mass spectrometry remains the primary analytical method for metabolomics due to its high sensitivity and resolution which allow the detection of metabolite ions at femtomolar concentrations. This allows the identification of small molecules that are the products of cellular processes. Recent advances in metabolomics, especially ion-chromatography coupled with mass spectrometry, allow small polar and physiologically ionic metabolites, which are not observed using normal chromatographic techniques, to be identified in complex mixtures.
This work aims to improve the methods of quantitative and qualitative analysis of metabolites by electrospray ionisation mass spectrometry of a range of biological systems. The characterisation of metabolites from wild-type and mutant cell lines and various tissues. This will include the optimisation of ion chromatography and other chromatographic techniques to allow the separation and identification of metabolites with database comparison and quantification to measure cellular concentrations. Tissue culture of various cell lines with isocitrate dehydrogenase mutations will be required and the use of stable isotope labelled media will be used to follow glycolytic and citric acid cycle pathways by carbon flux analysis with the eventual aim of the identification of targets for pharmaceutical intervention to disrupt cancer cell growth.
This project falls within the EPSRC Physical Sciences - Analytical Science research area.