Accumulated evidence indicates oxidative stress plays important roles in disease and aging. Under oxidative stress, lipid peroxidation (LPO) leads to reactive carbonyl species (RCS) that can modify a wide range of biomolecules including protein, DNA and carbohydrate. In this dissertation, we investigate the modification of two model proteins, human serum albumin (HSA) and aconitase (ACO), by the LPO-relevant a, b-unsaturated aldehydes, acrolein (ACR) and 4-hydroxy-2-nonenal (HNE). The investigation is focused on the characterization and quantification ACR and HNE addition to the model proteins. A correlation between HNE modification and ACO activity is also determined. These results provide insights into the impact of oxidative stress at the molecular level and are relevant to aging and disease states. We finally investigate protein carbonylation in ischemic mouse heart mitochondria, and develop a quantitative method for detecting carbonylated protein in this system. The research is based on liquid chromatography/mass spectrometry (LC/MS), Western Blots, and enzymatic assay.

It is well established that free radical mediated oxidative stress plays a critical role in aging and age-related diseases. Among the post-translational protein modifications, carbonylation has attracted a great deal of attention due to its irreversible and irreparable nature. Despite the fact that protein carbonylation is associated with a series of physiological and pathological processes, there are still issues to be clarified such as why certain proteins are more vulnerable to modification, what are the locations of the protein modifications, and how does the nature of the oxidant affect the preferred site of modification. In this study, we will seek an answer to these questions and examine the global effect of oxidative stress on protein abundance. The study embraces three distinct specific aims. In the first, methods are developed for identifying sites of protein carbonylation. In the second specific aim, these methods are used to identify carbonylaytion sites in model proteins subjected to chemical oxidants. In the third aim, the focus is on a model organism, C. elegans, subjected to paraquat-induced oxidative stress. This is exploratory work and mass spectrometry is used to assess the impact of oxidative stress on the mitochondrial proteome.

The introduction of high fructose corn syrup into the diet has been proposed to be the cause of many illnesses related to the metabolic syndrome. Fructose and its metabolites can be metabolized into cytotoxic reactive dicarbonyls that can cause damage to macromolecules leading to deleterious consequences. Dihydroxyacetone, a fructose metabolite, was studied in this thesis. Its ability to autoxidize and cause protein carbonylation under standard (pH 7.4, 37°C) and oxidative stress conditions (Fentons reagent) was investigated. Dihydroxyacetone was able to form significant amounts of dicarbonyls and protein carbonylation. Several selected natural polyphenols were chosen for an in vitro toxicological study involving rat hepatocytes. The chosen dietary polyphenols were rutin, gallic acid, methylgallate, ethylgallate, propylgallate and curcumin. In this thesis, the polyphenols were found to be able to significantly protect against the deleterious effects of glyoxal and methylglyoxal. In summary, these polyphenols could be candidates for future in vivo studies.

The introduction of high fructose corn syrup into the diet has been proposed to be the cause of many illnesses related to the metabolic syndrome. Fructose and its metabolites can be metabolized into cytotoxic reactive dicarbonyls that can cause damage to macromolecules leading to deleterious consequences. Dihydroxyacetone, a fructose metabolite, was studied in this thesis. Its ability to autoxidize and cause protein carbonylation under standard (pH 7.4, 37°C) and oxidative stress conditions (Fentons reagent) was investigated. Dihydroxyacetone was able to form significant amounts of dicarbonyls and protein carbonylation. Several selected natural polyphenols were chosen for an in vitro toxicological study involving rat hepatocytes. The chosen dietary polyphenols were rutin, gallic acid, methylgallate, ethylgallate, propylgallate and curcumin. In this thesis, the polyphenols were found to be able to significantly protect against the deleterious effects of glyoxal and methylglyoxal. In summary, these polyphenols could be candidates for future in vivo studies.

High chronic fructose consumption has been linked to many diseases. Sugar metabolites, especially glyoxal and methylglyoxal can form advanced glycation products, which contribute to the pathology of diabetic complications. Our objective was to study the metabolism of these metabolites and the associated protein carbonyation and cytotoxicity in isolated hepatocytes. In addition, the effect of oxidative stress on the metabolism of these toxins was also investigated. Methylglyoxal and glyoxal can induce protein carbonylation, which contributes to hepatocyte toxicity. Methylglyoxal, but not glyoxal, was detoxified mainly by the glyoxalase system. Both toxins can be metabolized by mitochondrial aldehyde dehydrogenase. The detoxification of glyoxal was impaired under oxidative stress conditions (i.e. increased hydrogen peroxide level). Glyoxal was found to be a common autoxidation product from glyceraldehyde, hydroxypyruvate and glycolaldehyde. Glyoxal and the reactive oxygen species formation during the autoxidation process contributed to the hepatocyte toxicity of glyceraldehyde, hydroxypyruvate and glycolaldehyde.

High chronic fructose consumption has been linked to many diseases. Sugar metabolites, especially glyoxal and methylglyoxal can form advanced glycation products, which contribute to the pathology of diabetic complications. Our objective was to study the metabolism of these metabolites and the associated protein carbonyation and cytotoxicity in isolated hepatocytes. In addition, the effect of oxidative stress on the metabolism of these toxins was also investigated. Methylglyoxal and glyoxal can induce protein carbonylation, which contributes to hepatocyte toxicity. Methylglyoxal, but not glyoxal, was detoxified mainly by the glyoxalase system. Both toxins can be metabolized by mitochondrial aldehyde dehydrogenase. The detoxification of glyoxal was impaired under oxidative stress conditions (i.e. increased hydrogen peroxide level). Glyoxal was found to be a common autoxidation product from glyceraldehyde, hydroxypyruvate and glycolaldehyde. Glyoxal and the reactive oxygen species formation during the autoxidation process contributed to the hepatocyte toxicity of glyceraldehyde, hydroxypyruvate and glycolaldehyde.

Carbonyl and oxidative stress augment the development of diabetic complications. We evaluated the cytoprotectiveness of walnut and hazelnut extracts and catechins for decreasing cytotoxicity, lipid peroxidation, reactive oxygen species (ROS) formation, and protein carbonylation in cell death models of carbonyl and oxidative stress. Polar extracts (methanol or water) showed better cytoprotection than the non-polar (ethyl acetate) nut extracts against hydroperoxide-induced hepatocyte cell death and oxidative stress markers. Catechin flavonoids found in plants, including walnuts and hazelnuts, prevented serum albumin carbonylation in a carbonyl stress model (using glyoxal or methylglyoxal). Hepatocyte protein carbonylation and cell death were prevented and UV spectra data suggested a catechin:methylglyoxal adduct was formed. We conclude that (a) bioactive nut constituents in polar extracts were more protective than non-polar extracts against oxidative stress, and (b) catechins were effective under physiological temperature and pH, at preventing dicarbonyl induced cytotoxicity likely by trapping dicarbonyls or reversing early stage carbonylation.

Carbonyl and oxidative stress augment the development of diabetic complications. We evaluated the cytoprotectiveness of walnut and hazelnut extracts and catechins for decreasing cytotoxicity, lipid peroxidation, reactive oxygen species (ROS) formation, and protein carbonylation in cell death models of carbonyl and oxidative stress. Polar extracts (methanol or water) showed better cytoprotection than the non-polar (ethyl acetate) nut extracts against hydroperoxide-induced hepatocyte cell death and oxidative stress markers. Catechin flavonoids found in plants, including walnuts and hazelnuts, prevented serum albumin carbonylation in a carbonyl stress model (using glyoxal or methylglyoxal). Hepatocyte protein carbonylation and cell death were prevented and UV spectra data suggested a catechin:methylglyoxal adduct was formed. We conclude that (a) bioactive nut constituents in polar extracts were more protective than non-polar extracts against oxidative stress, and (b) catechins were effective under physiological temperature and pH, at preventing dicarbonyl induced cytotoxicity likely by trapping dicarbonyls or reversing early stage carbonylation.

Free radicals are the compounds having one or more unpaired electrons in their outer orbital and this unpaired electron make these compounds very reactive. Especially as their concentration increases, they initiate a chain oxidation reaction of lipids, proteins and nucleic acids. The condition, in which the production of free radicals exceeds their elimination or tissue defense mechanism decrease against them or both occur together, is called oxidative stress. In diabetes mellitus which is a glucose metabolism disorder, there occurs excessive non-enzymatic protein oxidation, glucose autoxidation and enhanced activity of polyol pathway enzymes, which are the possible sources of the oxidative stress in this disease. In this study, the conditions of the activity measurements of major antioxidant enzymes, namely superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), glutathione peroxidase (GPx, 1.11.1.9) and glutathione S-transferase (GST, EC 2.5.1.18) were studied and the optimum conditions (pH, temperature and substrate concentrations) for each assay were determined. Further objectives of the study were to characterize the enzymatic antioxidant systems (catalase, superoxide dismutase, glutathione peroxidase and glutathione S-transferase), tissue oxidation status (concentrations of TBARS, protein carbonylation, and lipid/protein ratios) and nonenzymatic antioxidant (reduced glutathione) levels of the diabetic rat liver tissues. According to our results, the hepatic SOD and GPx activities significantly increased whereas CAT activity markedly decreased in diabetic rats compared to control group. Also, GST activities did not change in diabetes. As a result of oxidative stress, TBARS concentration, lipid/protein ratios and protein carbonylation increased and GSH levels decreased in diabetic rats compared to control rats. This increase in tissue damage, in spite of the increase in antioxidant enzyme activities, could have been due to the overproduction of reactive oxygen species that exceeded the capacity of the antioxidant enzymes during the eight week of diabetes.

No description available.