Analysis Of F₂-isoprostanes By GC-MS And Characterization Of Oxidative Imbalance In Metabolic Disease And Neurodegenerative Disease

Increased oxidative stress is considered as an important contributing factor in pathogenesis of human diseases. The use of biomarkers for oxidative stress may provide further evidence of a causal relationship between oxidative damage to macromolecules (DNA, lipid and protein) and many diseases. F₂-isoprostanes are a complex family of compounds produced from arachidonic acid via a free-radical-catalyzed mechanism independent of the cyclooxygenase pathway. Measurement of F₂-isoprostanes or one specific biologically active isomer, iPF_2α-III, is increasingly being used as a novel marker of lipid peroxidation in vivo. In this thesis, the lipid peroxidation status in a rat model of simple steatosis and in PS cDKO mice, a model of familial Alzheimer's disease was studied.1. The analysis of iPF_2α-III in plasma and tissue samplesGas chromatography-negative ion chemical ionization-mass spectrometry (GC-NICI-MS) method for measuring iPF_2α-III was established. To extract iPF_2α-III from plasma and tissue smaples, a one-step solid phase extraction method was established. The average extraction yield of iPF_2α-III in the biological samples was 90%. The validation of methodology also showed a good linearity of iPF_2α-III quantification using peak height ratio between iPF_2α-III and the internal srandard iPF_2α-III-d₄, and both the intra- and inter-assay precisions were within 6%. The arachidonic acid (AA) oxidation in vitro further confirmed the formation of F2-isoprostanes via a free-radical-catalyzed mechanism.2. The lipid peroxidation in rats with steatosisSteatosis is an earlier stage of non-alcoholic fatty liver disease (NAFLD). To identify and understand oxidative imbalance as an important mechanism of NAFLD, especially at the simple steatotic satge, the hepatic and circulating iPF_2α-III and enzymatic antioxidant status in the rats with high-fat diet induced hepatic steatosis were studied. Significantly higher levels of iPF_2α-III and lower activities of SOD and CAT were observed in the livers of steatotic rats. Plasma total iPF_2α-III was significantly correlated with the abnormalities of blood lipids as well as ALT levels in the rats with simple steatosis. Enhancement of hepatic oxidative imbalance occurred at the steatotic stage of NAFLD suggests a possibility that manifestation of the local oxidative damage precedes that of systemic oxidative imbalance. Predominant metabolic features of the increased lipid peroxidation further reveal a close association of the oxidative imbalance and the dyslipidamia with functional deterioration of the steatotic liver.3. The lipid peroxidation in PS cDKO (Presenilin conditional double knockout) mice, a model of familial Alzheimer's disease (FAD)Forebrain-specific and conditional presenilin-1 and presenilin-2 double knock-out (PS cDKO) causes progressive memory dysfunction, forebrain degeneration and ventricle enlargement with recapitulation of most of the neurodegenerative phenotypes of Alzheimer's disease (AD). Oxidative stress mechanism has been long implicated predominantly in amyloidosis-mediated AD pathologies; however, its role in response to the loss of function pathogenic mechanism of AD remains unclear. To identify and understand oxidative imbalance as an important mechanism of AD in response to PS loss-of-function, we examined oxidative stress status, using F2-isoprostanes (iPF_2α-III) as the marker of lipid peroxidation in vivo, in both cerebral cortex and circulation of 2-, 4-, 7- and 15-month PS cDKO and the age- and gender-matched control mice (WT). Enhanced lipid peroxidation was occurred in a gender- and age-related manner in the PS cDKO mice independent of brain Aβdeposition. Such oxidative abnormalities predominantly in cerebral cortex at 2-4 months of age preceded the onset of many pronounced AD neuropathologies documented; suggesting that increased lipid peroxidation is not only an early pathophysiological response to PS inactivation but also the potential culprit in the AD-like neurodegenerative pathologies in the PS cDKO mice. The investigations into endogenous antioxidant defenses (CAT, SOD and GPx) revealed a systemic oxidative resistance mechanism during the early development of AD neuropathologies in response to the loss of PS functions. Western blot analysis of cortical glial fibrillary acidic protein confirmed an increased inflammatory response to PS inactivation, in particular in the PS cDKO mice at the age as early as 2-month, suggesting that synergic relationship between lipid peroxidation and inflammation may be closely associated with the loss of function neuropathogenic mechanism of AD.