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dc.contributor.advisorKotzé, H.F.
dc.contributor.advisorMienie, L.J.
dc.contributor.authorMeeding, Lizette
dc.descriptionThesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2010.
dc.description.abstractDeficiencies of the pyruvate dehydrogenase complex enzyme and of the mitochondrial respiratory chain enzymes in humans both result in similar metabolic profiles in blood and urine. It is therefore almost impossible to distinguish between the two conditions based on the metabolic profile alone. Definitive diagnosis can only be made by assessing enzyme function in muscle biopsies. The aim of this study was to attempt to identify a method that is easy, non-invasive and definitive to distinguish between deficiencies in the two enzyme complexes. HeLa cells were treated with moniliformin and 3-bromopyruvate (inhibitors of pyruvate dehydrogenase) and rotenone (inhibitor of complex I) to induce pyruvate dehydrogenase complex and mitochondrial respiratory chain deficiencies respectively. After inhibition for 24 hours, the media were transferred to a clean tube and centrifuged. The cells were scraped off, sonified and centrifuged. Organic acid analyses were done on the media and cell extracts using gas chromatography-mass spectrometry. Identification of the organic acids in the chromatogram was done by using AMDIS software and a library compiled by Prof L.J. Mienie at the metabolic laboratory of the North-West University, Potchefstroom. I tested several hypotheses in order to achieve the aim of this study. The measured organic acid levels varied markedly which made it difficult to interpret. Organic acid comparisons of cell extracts were not significantly different, and were therefore not discussed. Not enough data was obtained to calculate the ratio limits of 4-hydroxyphenylpyruvic acid, 4-hydroxyphenyllactic acid and 4-hydroxyphenylacetic acid. This approach was therefore rejected. The calculation of the ratio limit of phenylpyruvic acid, phenyllactic acid and phenylacetic acid could also not be done because these molecules could not be detected in the medium. This approach was also rejected. No discernable pattern was observed in the principle component analysis (PCA). Our results therefore make it doubtful that PCA can be used as a tool to diagnose and distinguish between deficiencies in the pyruvate dehydrogenase complex enzymes and mitochondrial respiratory chain enzymes. With inhibition of pyruvate dehydrogenase, the ratios of citric acid to succinic acid and citric acid to fumaric acid were significantly decreased and fumaric acid to malic was significantly increased. Respiratory chain inhibition with rotenone had no marked effect on these three ratios. It is therefore likely that calculation of these ratios may distinguish between pyruvate dehydrogenase defect and a respiratory chain defect. This will have to be verified in patients with proven enzyme defects.
dc.publisherNorth-West University
dc.titleMetabolic markers to distinguish between moniliformin and 3-bromopyruvate induced pyruvate dehydrogenase and rotenone-induced respiratory chain complex I deficiencies in HeLa cellsen

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  • ETD@PUK [7579]
    This collection contains the original digitized versions of research conducted at the North-West University (Potchefstroom Campus)

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