Targeted metabolomics to study lipid peroxidation in biological systems

During normal cellular metabolism reactive oxygen species (ROS) are inevitably formed as by-products of respiration. ROS are extremely reactive molecules and can react with and damage surrounding DNA, protein and lipid molecules and subsequently alter their normal function in the cell. This oxidative damage is counter balanced by antioxidant and repair mechanisms, which are present in every aerobic cell. When the levels of oxidative damage increase or the antioxidant and repair capacity is inefficient to protect against this damage a state of oxidative stress occurs. Oxidative stress is associated with various pathological conditions including cancer, diabetes, atherosclerosis, coronary heart disease and aging. Oxidative stress can be reversed through lifestyle and therapeutic intervention and it is therefore an important parameter to monitor to ensure good health. ROS are extremely short-lived molecules and directly measuring in vivo levels in biological samples is technically almost impossible and therefore requires indirect measurements like the use of molecular ROS probes and by measuring products of oxidative damage. ROS probes are very useful in in vitro settings to give information about ROS levels however, for in vivo studies products of oxidative damage to DNA, protein and lipids are more often measured. In order to answer important questions like the involvement of ROS and its damage in complex processes like aging and other age related diseases we need good methodologies for in vivo ROS and oxidative damage levels in biological systems like cell cultures, model organism and humans. Here we present an overview of current methods for measuring oxidative damage and reactive oxygen species in model organisms of aging. Reactive oxygen species have been associated with aging ever since the formulation of the Free Radical Theory of Aging, which explains aging as the accumulation of oxidatively damaged macro-molecules leading to an alteration in function and stability and ultimately manifesting as the phenotype of aging. We furthermore describe for the first time the formation of a new marker of oxidative damage in C. elegans, an important model organism for studying aging. This marker is F3-isoprostanes, which are formed upon oxidative damage to the polyunsaturated fatty acids in this worm. This is a novel method for accurate measurement of lipid peroxidation in C. elegans and we used it to look at oxidative damage during aging in a longitudinal manner. We found that long-lived C. elegans mutants have unexpectedly high levels of oxidative damage early in life, which possibly leads to the increased lifespan as a result of an adaptive response to these high levels of damage. Next we developed a protocol for measuring F2-isoprostanes in cellular systems. F2-isoprostanes are similar to the F3-isoprostanes described above, except they are formed from the polyunsaturated fatty acids present in mammalian cells. Cellular systems are important biological model systems, which are used to study various physiological processes and reliable methods to measure oxidative damage in these systems are important. This method for isoprostanes measurement was applied to assess levels of oxidative damage in patients with coronary heart disease that suffered from acute myocardial infarction (AMI). We found that coronary heart patients that suffered from AMI had higher levels of oxidative damage than coronary heart patients without AMI. Overall this thesis demonstrates that 1) F2- and F3- isoprostanes are reliable markers for oxidative damage quantification 2) the use of LC-MS for targeted metabolomics to be a powerful analytical combination particularly when applied on recently developed UPLC and ultra sensitive triple-quadropole mass analyzers

Quantification of in vivo oxidative damage in Caenorhabditis elegans during aging by endogenous F3-isoprostane measurement

Oxidative damage is thought to be a major cause in development of pathologies and aging. However, quantification of oxidative damage is methodologically difficult. Here, we present a robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach for accurate, sensitive, and linear in vivo quantification of endogenous oxidative damage in the nematode Caenorhabditis elegans, based on F3-isoprostanes. F3-isoprostanes are prostaglandin-like markers of oxidative damage derived from lipid peroxidation by Reactive Oxygen Species (ROS). Oxidative damage was quantified in whole animals and in multiple cellular compartments, including mitochondria and peroxisomes. Mutants of the mitochondrial electron transport proteins mev-1 and clk-1 showed increased oxidative damage levels. Furthermore, analysis of Superoxide Dismutase (sod) and Catalase (ctl) mutants uncovered that oxidative damage levels cannot be inferred from the phenotype of resistance to pro-oxidants alone and revealed high oxidative damage in a small group of chemosensory neurons. Longitudinal analysis of aging nematodes revealed that oxidative damage increased specifically with postreproductive age. Remarkably, aging of the stress-resistant and long-lived daf-2 insulin/IGF-1 receptor mutant involved distinct daf-16-dependent phases of oxidative damage including a temporal increase at young adulthood. These observations are consistent with a hormetic response to ROS