Rafique, Roozina; (2000) Oxidative stress and mitochondrial function in progressive experimental vitamin e deficiency in the rat. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Free radicals and oxidative damage have been increasingly implicated in a wide range of human diseases, and at the same time have raised the importance of understanding the protective mechanism of antioxidant defences. Antioxidant depletion studies can provide useful models for studying the importance of specific antioxidants. Vitamin E, is a major lipid-soluble antioxidant, located in the hydrophobic interior of cellular membranes, where it protects membrane lipids against peroxidative damage. In rats, prolonged vitamin E deficiency gives rise to a neuromuscular syndrome characterised by a peripheral neuropathy and generalised muscle wasting. Oxidative damage and impairment of mitochondrial respiratory chain (MRC) function have been implicated in the muscle pathology. In this thesis, the vitamin E deficient rat model was used to study the complex interrelationship between oxidative stress, mitochondrial dysfunction and cell/tissue damage. To assess the evolution of the pathological processes, morphological and biochemical analyses were made at progressive stages of vitamin E deficiency (3, 6, 9 and 12 months). Investigations were performed on two different tissues, skeletal muscle and liver, to address the question of tissue specificity. In muscle, progressive vitamin E deficiency was associated with extensive skeletal muscle degeneration, significant increases in lipid peroxidation (14-45%) and oxidised glutathione (61%) and reduced GSH:GSSG ratios (21%), relative to controls. Prolonged vitamin E deficiency (12 months) induced a compensatory increase (64%) in the reduced glutathione pool. Polarographic analysis of isolated mitochondria revealed significantly reduced (40- 50%) respiration rates with NADH and FADH2-linked substrates, but normal respiratory control ratios (RCR). Enzyme analysis confirmed complex IV (cytochrome c oxidase) as the predominant site of MRC impairment. Mitochondrial membrane fluidity was marginally (6-8%) but significantly reduced. Evidence of increased oxidative DNA base damage or of apoptosis, was not found. Mitochondrial protein synthesis and the translation profiles obtained by electrophoresis of the labelled mtDNA-encoded polypeptides were normal, suggesting mtDNA integrity was not impaired. In liver, progressive vitamin E deficiency was not associated with any obvious morphological abnormalities. Lipid peroxidation was significantly elevated but only at the later stages of vitamin E deficiency (9 and 12 months). MRC function was not impaired and the levels of oxidised DNA bases, oxidised glutathione, the GSH:GSSG ratios and mitochondrial membrane fluidity were unchanged, relative to controls. As in muscle, a compensatory increase (32%) in the reduced glutathione pool was seen in response to prolonged vitamin E deficiency. These findings are discussed with respect to the inter-relating roles of oxidative stress, free radical damage and mitochondrial dysfunction in tissue damage.

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