Chan, Alvin C.,Tran, Khai T.2009-03-232009-03-2319931993Source: Dissertation Abstracts International, Volume: 55-08, Section: B, page: 3297.9780315897199http://hdl.handle.net/10393/6834http://dx.doi.org/10.20381/ruor-15035Our goal is to determine whether vitamin E, especially $\alpha$-tocopherol, has any role in the regulation of membrane phospholipid metabolism and whether it can be recycled after being oxidized. The mechanisms of vitamin E regeneration were delineated from the study using human platelet as a machinery to induce endogenous vitamin E oxidation and regeneration. Human endothelial cells were preincubated with vitamin E and then labelled with ($\sp3$H) -alkyl-GPC and ($\sp $C) -acyl-GPC. $\alpha$-Tocopherol was found to increase the formation of ($\sp3$H) -alkylacyl-GPC and ($\sp $C) -diacyl-GPC, whereas other vitamin E analogs showed only limited effect. Vitamin E enhanced the formation of alkylacyl-GPC by an indirect stimulation of the CoA-independent transacylase, whereas the increased acylation of acyl-GPC by vitamin E occurred at a level other than CoA-dependent acyltransferase. The regulation of vitamin E on the cellular diacylglycerol and phosphatidic acid levels in thrombin-stimulated endothelial cells was studied by determination of radiolabelled lipid products, by the quantitation of diacylglycerol mass and by the quantitation of enzyme activity. $\alpha$-Tocopherol incubation was found to attenuate the transient accumulation of diacylglycerol with a concomitant increase in the formation of phosphatidic acid. This effect resulted from an activation of diacylglycerol kinase which converted diacylglycerol to phosphatidic acid. Other enzyme pathways contributing to diacylglycerol and phosphatidic acid formation, including phospholipase C, phospholipase D and phosphatidate phosphohydrolase, were unaffected by vitamin E. Vitamin E was also found to modulate membrane protein kinase C (PKC) activity in both resting and phorbol ester (PMA)-stimulated endothelial cells. Incubation of endothelial cells with $\alpha$-tocopherol caused an increase in PKC activity in the membrane fraction. In response to PMA stimulation, the increase in PKC activity in the membrane was attenuated by vitamin E. Duroquinone, an oxyradical-generating substance, had a completely opposite effect on membrane PKC in both resting and PMA-stimulated cells. The effect of duroquinone on membrane PKC was totally abolished by vitamin E. Both vitamin E and duroquinone had no effect on cytosolic PKC. These results showed that vitamin E regulates resting and PMA-stimulated PKC by at least two different mechanisms. $\alpha$-Tocopherol in human platelet homogenates was readily oxidized by arachidonic acid (AA) via the 12-lipoxygenase pathway. This AA-induced oxidized tocopherol can be reduced back to tocopherol by water soluble reductants such as nordihydroguaiaretic acid (NDGA), ascorbate and reduced glutathione (GSH). Ascorbate and NDGA induced tocopherol regeneration by a non-enzymic mechanism whereas GSH recycled tocopherol via an enzymic pathway. Tocopherol regeneration was negatively correlated with oxidation time induced by AA, suggesting that the intermediate molecule is a short-lived tocopheroxyl radical. This finding provides evidence that vitamin E can be regenerated in human cells and that both membrane and cytosolic antioxidants operate in concert to protect cell membrane from radical induced injury. (Abstract shortened by UMI.)267 p.Chemistry, Biochemistry.Thesis