With the recognition of the central role of mitochondria in apoptosis, there is a need to develop specific tools to manipulate mitochondrial function within cells. Here we report on the development of a novel antioxidant that selectively blocks mitochondrial oxidative damage, enabling the roles of mitochondrial oxidative stress in different types of cell death to be inferred. This antioxidant, named mitoQ, is a ubiquinone derivative targeted to mitochondria by covalent attachment to a lipophilic triphenylphosphonium cation through an aliphatic carbon chain. Due to the large mitochondrial membrane potential, the cation was accumulated within mitochondria inside cells, where the ubiquinone moiety inserted into the lipid bilayer and was reduced by the respiratory chain. The ubiquinol derivative thus formed was an effective antioxidant that prevented lipid peroxidation and protected mitochondria from oxidative damage. After detoxifying a reactive oxygen species, the ubiquinol moiety was regenerated by the respiratory chain enabling its antioxidant activity to be recycled. In cell culture studies, the mitochondrially localized antioxidant protected mammalian cells from hydrogen peroxide-induced apoptosis but not from apoptosis induced by staurosporine or tumor necrosis factor-␣. This was compared with untargeted ubiquinone analogs, which were ineffective in preventing apoptosis. These results suggest that mitochondrial oxidative stress may be a critical step in apoptosis induced by hydrogen peroxide but not for apoptosis induced by staurosporine or tumor necrosis factor-␣. We have shown that selectively manipulating mitochondrial antioxidant status with targeted and recyclable antioxidants is a feasible approach to investigate the role of mitochondrial oxidative damage in apoptotic cell death. This approach will have further applications in investigating mitochondrial dysfunction in a range of experimental models.

Mitochondrial oxidative damage contributes significantly to a range of human disorders, including neurodegenerative diseases, ischaemia-reperfusion injury and ageing-associated dysfunction. To prevent this damage we have delivered a molecule containing the active antioxidant moiety of vitamin E to mitochondria. This was carried out by covalently coupling the antioxidant moiety to a lipophilic triphenylphosphonium cation. This mitochondrially targeted antioxidant, 2-[2-(triphenylphosphonio)ethyl]-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol bromide (TPPB), accumulated several-hundred fold within the mitochondrial matrix, driven by the organelle's large membrane potential. When cells were incubated with micromolar concentrations of TPPB, they accumulated millimolar concentrations within their mitochondria. The amount of TPPB taken up by mitochondria was < 80-fold greater than endogenous levels of vitamin E. Consequently the targeted derivative of vitamin E protected mitochondrial function from oxidative damage far more effectively than vitamin E itself. The mitochondrially targeted antioxidant TPPB has potential as an antioxidant therapy for disorders involving mitochondrial oxidative damage. It also suggests a new family of mitochondrially targeted antioxidants, redoxactive and pharmacologically active molecules designed to prevent damage or manipulate mitochondrial function.Keywords: mitochondria; antioxidant; targeting; vitamin E.Mitochondrial oxidative damage is a major factor in many human disorders, including neurodegenerative diseases, ischaemia-reperfusion injury, ageing and inflammatory damage [1±6]. Oxidative damage accumulates more in mitochondria than in the rest of the cell because electrons continually leak from the respiratory chain to form damaging reactive oxygen species [1,3,7±12]. This oxidative damage impairs mitochondrial ATP synthesis and calcium homeostasis and induces the mitochondrial permeability transition [1,4,5,13±15], leading to necrotic or apoptotic cell death [16±22]. The lethal potential of this damage was dramatically illustrated by the premature death of mice lacking mitochondrial superoxide dismutase [23,24], while mice lacking cytosolic superoxide dismutase developed normally [25].Selective prevention of mitochondrial oxidative damage should therefore be an effective therapy in a wide range of human diseases. This prediction has been confirmed by the overexpression of mitochondrial superoxide dismutase, which decreased liver reperfusion injury [26]. Here we have developed a strategy to deliver low molecular mass antioxidants to mitochondria within cells and thereby selectively prevent mitochondrial oxidative damage. Previously antioxidants such as vitamin E, ubquinol and N-acetyl cysteine have been shown to decrease mitochondrial oxidative damage, but because these compounds were not accumulated within mitochondria their effectiveness was limited [27±29]. To target an antioxidant to mitochondria we attached it to the lipophilic triphenylphosphonium cation which crosses ...

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HbA1c significantly underestimates glycaemic control in patients with diabetes and CKD stages 4 and 5. In severe CKD, GA more accurately reflects glycaemic control compared with fructosamine and HbA1c and should be the preferred marker of glycaemic control.

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