Stress conditions such as heat shock, UV, alkylating agents, and H2O2 have been shown to result in the modification of a variety of protein targets via the production of reactive electrophiles. These modifications can directly impact protein function or can alter posttranslational modifications, thus leading to a disruption of cellular regulatory processes. Recent studies have shown that stress-induced protein modifications can modulate posttranslational modification by the small ubiquitin related modifier (SUMO) family of proteins. Unlike ubiquitination, which primarily targets proteins for proteasomal degradation, sumoylation exerts a variety of effects including protein stabilization, subcellular localization, and the alteration of protein-protein interactions and transcriptional activity. To investigate the effects of alkylation and oxidative stress on sumoylation, HEK293 cells were treated with iodoacetamide, hydroquinone, benzoquinone, Texas Red C5 bromoacetamide, hydrogen peroxide, and 4-hydroxynonenal (HNE), a highly reactive product of lipid peroxidation associated with oxidative stress. Western blot analysis revealed that the agents tested resulted in concentration-dependent changes in the patterns of SUMO-1 and SUMO-2/3 protein conjugation. Localization studies using western blot analysis and confocal immunofluorescence microscopy demonstrated that SUMO-1 protein conjugates were located primarily in the nucleus, whereas SUMO-2/3 protein conjugates were more equally distributed between the nucleus and the cytoplasm. SUMO-associated proteins were harvested from vehicle- and HNE-treated non-transfected HEK293 cells using agarose conjugated anti-SUMO-1 antibodies or from HA-SUMO-1- and HA-SUMO-3-expressing HEK293 cells using immunoaffinity chromatography. Multidimensional liquid chromatography-tandem mass spectrometry analyses resulted in the identification of 54 HA-SUMO-1-associated proteins and 37 HA-SUMO-3-associated proteins in vehicle-treated cells and 21 HA-SUMO-1- and HA-SUMO-3-associated proteins in HNE treated cells. Additionally, 27 SUMO-1-associated proteins were identified in the HNE-treated non-transfected cells. The functional classes of proteins targeted included RNA binding and processing proteins, metabolic enzymes, cytoskeletal regulators, and chaperone proteins. HNE treatment resulted in a near complete redistribution of both SUMO-1 and SUMO-3 to different targets. There was a 15% overlap in SUMO-1 and SUMO-3 associated proteins in vehicle-treated cells and a 10% overlap in HNE-treated cells indicating that SUMO proteins target distinct protein groups. These results indicate that protein modifying reactive electrophiles can regulate protein functions through the indirect alteration of endogenous posttranslational modifications.

1). The effect of temperature, P450 inhibitors (pyrazole and imidazole), sulphydryl reagents (iodoacetamide and N-ethyl maleimide) and glutathione on the activation of CCl4 in rat liver microsomes was studied. Spin trapping of CCI3', covalent binding of CCl4 to protein and CCl4-dependent MDA formation were used as indices of CCl4 metabolism. Formation of PBN-CCI3' adduct, 14CCl4 covalent binding to protein and CCl4-dependent :MDA production were dependent on temperature range from 15-40°C. The transition temperature was at 26.7 -27 .5°C when the activation was measured by formation of PBNCCl3' adduct and specific 14CCl4 covalent binding. The transition temperature was found to be 34.3°C when CCl4 -dependent MDA production was taken as the index of the activation of CCI4. Pyrazole, imidazole and iodoacetamide inhibited CC14 -dependent MDA formation only at high concentrations (10-20 mM), whereas glutathione showed a strong inhibitory effect on CCl4-stimulated lipid peroxidation. MDA formation was nearly 100°;6 inhibited by 1 roM GSH. GSH also delayed the onset of lipid peroxidation. N-ethyl maleimide (NEM) exerted biphasic effects on CCl4 -dependent MDA formation. The lower concentration of NEM (0.5 mM-l mM) reduced the :MDA prodUction, while the higher concentration of NEM (5-10 mM) enhanced the MDA formation. 2). Ribonucleotide reductase was partially purified from juvenile normal rat liver. The enzyme was purified 30 fold after DEAE-cellulose chromatography. The CDP reductase activity in tissues with different growth states or rates was compared. The enzyme activity was developed well in juvenile rat liver, regenerating liver and hepatoma (cells), while the enzyme activity was undetectable in adult rat liver and sham-operated rat liver. The enzyme activity in Yoshida cells was 3-fold of the activity in Morris 5123tc tumours. Dithiothreitol (DIT) activated the activity of CDP reductase from 48h and 60h regenerating liver, but DIT did not activate the enzyme activity of juvenile 'normal rat liver. The possible mechanism of the activation of enzyme activity by DIT was discussed and a mechanism of regulation of the ribonucleotide reductase activity in regenerating liver was suggested. 3). The effect of the lipid peroxidation product 4-hydroxynonenal (HNE) on CDP reductase from juvenile normal rat liver was investigated. HNE inhibited the CDP reductase activity. The inhibition was dependent on the concentration of HNE and the incubation time. The enzyme activity was reduced 500/0 by 0.1 roM HNE. The inhibitory effect of HNE was irreversible. DIT protected the enzyme against HNE suggesting that HNE inhibited the activity of ribonucleotide reductase from rat liver through the mechanism of blockage of functional SH groups in the enzyme protein.

Introduction : Les enfants prématurés ont la particularité de naître alors que leur développement est souvent incomplet et nécessite la mise en œuvre de soins intensifs visant à poursuivre leur croissance en dehors de l’environnement utérin. Souvent cependant, le stade développemental de l’enfant ne lui permet pas d’assimiler une alimentation entérale du fait de l’immaturité de son système digestif. Le recours à une voie centrale délivrant les nutriments assurant le développement devient alors une nécessité. Ce type de nutrition, appelée nutrition parentérale (NP, ou total parenteral nutrition TPN), permet l’administration de molécules simples, directement dans le sang du prématuré. Il n’est toutefois pas exempt de risques puisqu’exposée à la lumière, la NP peut s’oxyder et générer des molécules oxydantes telles que des hydroperoxydes lipidiques susceptibles de se fragmenter par la suite en hydroxy-alkénals. Ceci devient problématique au vu de l’immaturité des systèmes de défenses antioxydants du nouveau-né prématuré. L’utilisation prolongée de la NP est d’ailleurs à l’origine de maladie hépatiques dans lesquelles le stress oxydant et la nécro-inflammation sont des composantes majeures. Nous avons émis l’hypothèse que l’infusion chez les enfants prématurés, d’aldéhydes d’origine lipidique est en relation avec le développement du stress oxydant et de l’inflammation hépatique. Objectif : Notre étude a consisté à évaluer la relation entre les quantités d’hydroxy-alkénals dans la NP et les effets hépatiques engendrés sur les marqueurs de stress oxydant et les voies de signalisation responsables d’une induction de processus inflammatoire. Dans ce but, nous avons cherché à mesurer la peroxydation lipidique dans l’émulsion lipidique de la NP et la conséquence de l’infusion en continue d’hydroxy-alkénals sur les marqueurs de stress oxydant, sur la voie de signalisation médiée par le Nuclear Factor κB et sur le déclenchement du processus inflammatoire hépatique. A la suite de ce travail, nous avons également travaillé sur des alternatives à la photoprotection, qui est la seule méthode réellement optimale pour réduire la peroxydation des lipides de la NP, mais cliniquement difficilement praticable. Résultats : Nos résultats ont mis en évidence la génération de 4-hydroxynonenal in vitro dans la NP, ce phénomène est augmenté par une exposition lumineuse. Dans ce cadre, nous avons montré l’inefficacité de l’ajout de multivitamines dans l’émulsion lipidique comme alternative à la photoprotection. Dans la validation biologique qui a suivi sur un modèle animal, nos résultats ont permis de démontrer que l’augmentation des adduits glutathion-hydroxynonenal était imputable à l’augmentation de 4-hydroxynonenal (4-HNE) dans la NP, et non à une peroxydation endogène. Nos données indiquent que la probable augmentation hépatique des niveaux de 4-HNE a conduit à une activation du NFκB responsable de l’activation de la transcription des gènes pro-inflammatoires du Tumour Necrosis Factor-α (TNF-α) et de l’interleukine-1 (IL-1). Nous avons alors évalué la capacité d’une émulsion lipidique enrichie en acides gras polyinsaturés (AGPI) n-3 à baisser les concentrations de 4-HNE dans la NP, mais également à moduler le stress oxydant et les marqueurs pro-inflammatoires. Enfin, nous avons démontré, en collaboration avec l’équipe du Dr Friel, que certains peptides isolés du lait humain (par un processus mimant la digestion) permettent également une modulation du stress oxydant et du processus inflammatoire. Conclusion : Le stress oxydant exogène issu de la NP a conduit par activation de facteurs de transcription intra-hépatiques au déclenchement d’un processus inflammatoire potentiellement responsable du développement de maladies hépatiques reliées à la NP telle que la cholestase. Dans ce sens, les AGPI n-3 et les peptides antioxydants peuvent se poser en tant qu’alternatives crédibles à la photoprotection. / Introduction: Premature infants usually born before full term require intensive care to continue to grow up outside the uterine environment. Premature newborns are born with gastrointestinal systems that are too immature to absorb nutrients safely. Therefore they receive their initial nutrients through intravenous feeding, called total parenteral nutrition which delivers simple nutrients directly into bloodstream. However, light exposed-TPN can generate oxidant molecules such as lipid hydroperoxides, which can potently break up into hydroxy-alkenals. Prolonged use of TPN is also a cause of liver disease in which oxidative stress and necro-inflammation are major components. Thus, we hypothesize that lipid aldehydes contained in TPN are associated with oxidative stress and hepatic inflammation developments. Objectives: The aim of our study is to assess the relationship between quantities of hydroxyl-alkenals generated in TPN and effects on oxidative stress biomarkers and cell-signalling pathways molecules implicated in hepatic inflammation induction. To this end, we measure lipid peroxidation in the TPN lipid emulsion in and the consequence of continuous infusion of hydroxy-alkenals on markers of oxidative stress, on cell-signaling pathway mediated by the NFkB, and on liver inflammation induction. Following these data, we also worked on alternatives of photoprotection, which is the only optimal method for preventing lipid peroxidation, but unfortunately clinically impractical. Results: In vitro studies have highlighted the generation of 4-HNE in the TPN, increased under light exposure. In this context, we have demonstrated that the addition of multivitamins in the lipid emulsion cannot be a valuable alternative to photoprotection. Concerning the biological validation in our guinea pig animal model, our results demonstrated that the increase of GS-HNE adducts was due to increased 4-HNE in the TPN, and does not provide from endogenous peroxidation. Our data also indicate that the increase of hepatic 4-HNE led to an activation of NFkB, responsible for the activation of the transcription of proinflammatory genes TNF-α, IL-1. In the next study, we have evaluated the ability of a lipid emulsion enriched with n-3 polyunsaturated fatty acids (PUFA) to reduce 4-HNE concentrations generated in TPN, and to modulate oxidative stress markers and pro-inflammatory process on the same animal model. We also have demonstrated, in collaboration with Dr Friel’s team, that two antioxidant peptides (derived from a process mimicking digestion process of human milk) allow also a modulation of oxidative stress and inflammatory process in the liver. Conclusion: This form of exogenous oxidative stress from the TPN led to an inflammatory process resulting from the activation of intrahepatic transcription, which is potentially responsible of liver disease development such as cholestasis. In this sense, the n-3 PUFA and antioxidant peptides may arise as a valuable alternative of photoprotection.

Introduction : Les enfants prématurés ont la particularité de naître alors que leur développement est souvent incomplet et nécessite la mise en œuvre de soins intensifs visant à poursuivre leur croissance en dehors de l’environnement utérin. Souvent cependant, le stade développemental de l’enfant ne lui permet pas d’assimiler une alimentation entérale du fait de l’immaturité de son système digestif. Le recours à une voie centrale délivrant les nutriments assurant le développement devient alors une nécessité. Ce type de nutrition, appelée nutrition parentérale (NP, ou total parenteral nutrition TPN), permet l’administration de molécules simples, directement dans le sang du prématuré. Il n’est toutefois pas exempt de risques puisqu’exposée à la lumière, la NP peut s’oxyder et générer des molécules oxydantes telles que des hydroperoxydes lipidiques susceptibles de se fragmenter par la suite en hydroxy-alkénals. Ceci devient problématique au vu de l’immaturité des systèmes de défenses antioxydants du nouveau-né prématuré. L’utilisation prolongée de la NP est d’ailleurs à l’origine de maladie hépatiques dans lesquelles le stress oxydant et la nécro-inflammation sont des composantes majeures. Nous avons émis l’hypothèse que l’infusion chez les enfants prématurés, d’aldéhydes d’origine lipidique est en relation avec le développement du stress oxydant et de l’inflammation hépatique. Objectif : Notre étude a consisté à évaluer la relation entre les quantités d’hydroxy-alkénals dans la NP et les effets hépatiques engendrés sur les marqueurs de stress oxydant et les voies de signalisation responsables d’une induction de processus inflammatoire. Dans ce but, nous avons cherché à mesurer la peroxydation lipidique dans l’émulsion lipidique de la NP et la conséquence de l’infusion en continue d’hydroxy-alkénals sur les marqueurs de stress oxydant, sur la voie de signalisation médiée par le Nuclear Factor κB et sur le déclenchement du processus inflammatoire hépatique. A la suite de ce travail, nous avons également travaillé sur des alternatives à la photoprotection, qui est la seule méthode réellement optimale pour réduire la peroxydation des lipides de la NP, mais cliniquement difficilement praticable. Résultats : Nos résultats ont mis en évidence la génération de 4-hydroxynonenal in vitro dans la NP, ce phénomène est augmenté par une exposition lumineuse. Dans ce cadre, nous avons montré l’inefficacité de l’ajout de multivitamines dans l’émulsion lipidique comme alternative à la photoprotection. Dans la validation biologique qui a suivi sur un modèle animal, nos résultats ont permis de démontrer que l’augmentation des adduits glutathion-hydroxynonenal était imputable à l’augmentation de 4-hydroxynonenal (4-HNE) dans la NP, et non à une peroxydation endogène. Nos données indiquent que la probable augmentation hépatique des niveaux de 4-HNE a conduit à une activation du NFκB responsable de l’activation de la transcription des gènes pro-inflammatoires du Tumour Necrosis Factor-α (TNF-α) et de l’interleukine-1 (IL-1). Nous avons alors évalué la capacité d’une émulsion lipidique enrichie en acides gras polyinsaturés (AGPI) n-3 à baisser les concentrations de 4-HNE dans la NP, mais également à moduler le stress oxydant et les marqueurs pro-inflammatoires. Enfin, nous avons démontré, en collaboration avec l’équipe du Dr Friel, que certains peptides isolés du lait humain (par un processus mimant la digestion) permettent également une modulation du stress oxydant et du processus inflammatoire. Conclusion : Le stress oxydant exogène issu de la NP a conduit par activation de facteurs de transcription intra-hépatiques au déclenchement d’un processus inflammatoire potentiellement responsable du développement de maladies hépatiques reliées à la NP telle que la cholestase. Dans ce sens, les AGPI n-3 et les peptides antioxydants peuvent se poser en tant qu’alternatives crédibles à la photoprotection. / Introduction: Premature infants usually born before full term require intensive care to continue to grow up outside the uterine environment. Premature newborns are born with gastrointestinal systems that are too immature to absorb nutrients safely. Therefore they receive their initial nutrients through intravenous feeding, called total parenteral nutrition which delivers simple nutrients directly into bloodstream. However, light exposed-TPN can generate oxidant molecules such as lipid hydroperoxides, which can potently break up into hydroxy-alkenals. Prolonged use of TPN is also a cause of liver disease in which oxidative stress and necro-inflammation are major components. Thus, we hypothesize that lipid aldehydes contained in TPN are associated with oxidative stress and hepatic inflammation developments. Objectives: The aim of our study is to assess the relationship between quantities of hydroxyl-alkenals generated in TPN and effects on oxidative stress biomarkers and cell-signalling pathways molecules implicated in hepatic inflammation induction. To this end, we measure lipid peroxidation in the TPN lipid emulsion in and the consequence of continuous infusion of hydroxy-alkenals on markers of oxidative stress, on cell-signaling pathway mediated by the NFkB, and on liver inflammation induction. Following these data, we also worked on alternatives of photoprotection, which is the only optimal method for preventing lipid peroxidation, but unfortunately clinically impractical. Results: In vitro studies have highlighted the generation of 4-HNE in the TPN, increased under light exposure. In this context, we have demonstrated that the addition of multivitamins in the lipid emulsion cannot be a valuable alternative to photoprotection. Concerning the biological validation in our guinea pig animal model, our results demonstrated that the increase of GS-HNE adducts was due to increased 4-HNE in the TPN, and does not provide from endogenous peroxidation. Our data also indicate that the increase of hepatic 4-HNE led to an activation of NFkB, responsible for the activation of the transcription of proinflammatory genes TNF-α, IL-1. In the next study, we have evaluated the ability of a lipid emulsion enriched with n-3 polyunsaturated fatty acids (PUFA) to reduce 4-HNE concentrations generated in TPN, and to modulate oxidative stress markers and pro-inflammatory process on the same animal model. We also have demonstrated, in collaboration with Dr Friel’s team, that two antioxidant peptides (derived from a process mimicking digestion process of human milk) allow also a modulation of oxidative stress and inflammatory process in the liver. Conclusion: This form of exogenous oxidative stress from the TPN led to an inflammatory process resulting from the activation of intrahepatic transcription, which is potentially responsible of liver disease development such as cholestasis. In this sense, the n-3 PUFA and antioxidant peptides may arise as a valuable alternative of photoprotection.

Organic nitrates, such as glyceryl trinitrate (GTN), have been used clinically for more than a century. However optimal nitrate therapy is hindered by the development of tolerance, which is associated with a desensitized response to GTN, oxidative stress, and the inactivation of aldehyde dehydrogenase 2 (ALDH2). This thesis evaluated the ALDH2 inactivation hypothesis of GTN tolerance and investigated the role of oxidative stress in GTN tolerance mediated by the lipid peroxidation product, 4-hydroxynonenal (HNE). Evidence for a direct role of ALDH2 in nitrate action was sought using a stably transfected cell line that overexpressed ALDH2, or siRNA to deplete endogenous ALDH2. Neither manipulation altered GTN-induced cGMP formation, indicating that ALDH2 does not mediate GTN bioactivation and tolerance. In a second study using an in vivo GTN tolerance model and a cell culture model of nitrate action, a marked increase in HNE adduct formation was detected in GTN-tolerant tissues, and treatment with HNE reduced the cGMP and vasodilator responses to GTN, thus mimicking GTN-tolerance. Together, the results suggest a primary role for HNE in the development of GTN tolerance, and provide the framework for a unified hypothesis that accommodates the previous findings of sulfhydryl depletion, ALDH2 inactivation and oxidative stress that are associated with nitrate tolerance. Studies have implicated oxidative stress and increased HNE formation in the pathogenesis of Alzheimer’s disease (AD). It was hypothesized that the gene deletion of ALDH2 would result in increased HNE-adduct formation leading to impaired cognitive function, and AD-like pathological changes. We observed a marked increase in HNE-adduct formation in Aldh2-/- mouse hippocampi as well as hyperphosphorylated tau, activated caspases, age-related changes in hippocampal amyloid βeta1-42 (Aβ1-42), post-synaptic density protein 95 (PSD95) and phosphorylated cyclic adenosine monophosphate response element binding protein (pCREB) expression, endothelial dysfunction and other vascular pathologies. These data provide further evidence for the importance of HNE and oxidative stress in AD pathogenesis, and establish Aldh2-/- mice as a new, oxidative stress-based animal model of age-related cognitive impairment and AD. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2013-05-31 11:10:58.145

Organic nitrates, such as glyceryl trinitrate (GTN), have been used clinically for more than a century. However optimal nitrate therapy is hindered by the development of tolerance, which is associated with a desensitized response to GTN, oxidative stress, and the inactivation of aldehyde dehydrogenase 2 (ALDH2). This thesis evaluated the ALDH2 inactivation hypothesis of GTN tolerance and investigated the role of oxidative stress in GTN tolerance mediated by the lipid peroxidation product, 4-hydroxynonenal (HNE). Evidence for a direct role of ALDH2 in nitrate action was sought using a stably transfected cell line that overexpressed ALDH2, or siRNA to deplete endogenous ALDH2. Neither manipulation altered GTN-induced cGMP formation, indicating that ALDH2 does not mediate GTN bioactivation and tolerance. In a second study using an in vivo GTN tolerance model and a cell culture model of nitrate action, a marked increase in HNE adduct formation was detected in GTN-tolerant tissues, and treatment with HNE reduced the cGMP and vasodilator responses to GTN, thus mimicking GTN-tolerance. Together, the results suggest a primary role for HNE in the development of GTN tolerance, and provide the framework for a unified hypothesis that accommodates the previous findings of sulfhydryl depletion, ALDH2 inactivation and oxidative stress that are associated with nitrate tolerance. Studies have implicated oxidative stress and increased HNE formation in the pathogenesis of Alzheimer’s disease (AD). It was hypothesized that the gene deletion of ALDH2 would result in increased HNE-adduct formation leading to impaired cognitive function, and AD-like pathological changes. We observed a marked increase in HNE-adduct formation in Aldh2-/- mouse hippocampi as well as hyperphosphorylated tau, activated caspases, age-related changes in hippocampal amyloid βeta1-42 (Aβ1-42), post-synaptic density protein 95 (PSD95) and phosphorylated cyclic adenosine monophosphate response element binding protein (pCREB) expression, endothelial dysfunction and other vascular pathologies. These data provide further evidence for the importance of HNE and oxidative stress in AD pathogenesis, and establish Aldh2-/- mice as a new, oxidative stress-based animal model of age-related cognitive impairment and AD. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2013-05-31 11:10:58.145

Lipid peroxidation is a key contributor to the pathophysiology of traumatic brain injury (TBI). Traditional antioxidant therapies are intended to scavenge the free radicals responsible for either the initiation or propagation of lipid peroxidation (LP). However, targeting free radicals after TBI is difficult as they rapidly react with other cellular macromolecules, and thus has a limited post-injury time window in which they may be intercepted by a radical scavenging agent. In contrast, our laboratory has begun testing an antioxidant approach that scavenges the final stages of LP i.e. formation of carbonyl-containing breakdown products. By scavenging breakdown products such as the highly reactive and neurotoxic aldehydes (often referred to as “carbonyls”) 4-hydroxynonenal (4-HNE) and acrolein (ACR), we are able to prevent the covalent modification of cellular proteins that are largely responsible for posttraumatic neurodegeneration. Without intervention, carbonyl additions render cellular proteins non-functional which initiates the loss of ionic homeostasis, mitochondrial failure, and subsequent neuronal death. Phenelzine (PZ) is an FDA-approved monoamine oxidase (MAO) inhibitor traditionally used for the treatment of depression. Phenelzine also possesses a hydrazine functional group capable of covalently binding neurotoxic carbonyls. The hypothesis of this dissertation is that carbonyl scavenging with PZ will exert an antioxidant neuroprotective effect in the traumatically injured rat brain mechanistically related to PZ’s hydrazine moiety reacting with the lipid peroxidation (LP)-derived reactive aldehydes 4-hydroxynonenal (4-HNE) and acrolein (ACR). Data from our ex vivo experiments demonstrate that the exogenous application of 4-HNE or ACR significantly reduced respiratory function and increased markers of oxidative damage in isolated non-injured rat cortical mitochondria, whereas PZ pre-treatment significantly prevented mitochondrial dysfunction and oxidative modification of mitochondrial proteins in a concentration-related manner. Additionally, PZ’s neuroprotective scavenging mechanism was confirmed to require the presence of a hydrazine moiety based on experiments with a structurally similar MAO inhibitor, pargyline, which lacks the hydrazine group and did not protect the isolated mitochondria from 4-HNE and ACR. Our in vivo work demonstrates that subcutaneous injections of PZ following TBI in the rat are able to significantly protect brain mitochondrial respiratory function, decrease markers of oxidative damage, protect mitochondrial calcium buffering capacity, and increase cortical tissue sparing without decreasing neuronal cytoskeletal spectrin degradation. These results confirm that PZ is capable of protecting mitochondrial function and providing neuroprotection after experimental TBI related to scavenging of neurotoxic LP degradation products.

No description available.

<p>Oxidative stress can be defined as a state of disturbance in the pro-oxidant/antioxidant balance in favour of the former, leading to potential damage. Processes associated with oxidative stress involve reactive oxygen species and radicals and can result in elevated levels of oxidatively modified or toxic molecules that can cause cellular malfunction, and even cell death. Destruction of membrane lipids, lipid peroxidation, caused by reactive oxygen species and radicals has been coupled to many diseases and also normal ageing. </p><p>The measurement of low molecular weight biomarkers of oxidative stress present in complex matrices such as brain tissue, plasma, urine or cerebrospinal fluid is a delicate and difficult task and there is a need for improved analytical tools in this field of research. </p><p>The major foci of this thesis and the work underlying it are the development of analytical techniques and methods for determining biomarkers for oxidative stress and lipid peroxidation. Aspects of particular concern include the effects of sample treatments prior to analysis, evaluation of the developed methods with respect to possible artefacts, and the scope for results to be misinterpreted. The specific research goals and issues addressed are detailed in five papers, which this thesis is based upon.</p><p><b>Paper I</b> focuses on malondialdehyde, describing and evaluating two new simplified sample pre-treatment regimes for the determination of malondialdehyde in rat brain tissue by capillary electrophoresis with UV detection. The effects of sample storing and handling are also considered.</p><p><b>Paper II</b> describes the synthesis, characterization and implementation of a new internal standard for the determination of malondialdehyde in biological samples using electrophoretic or chromatographic separation techniques. The usefulness of the internal standard is demonstrated in analyses of rat brain tissue samples.</p><p><b>Paper III</b> presents a method for the determination of 4-hydroxynon-2-enal in brain tissue from rats employing micellar electrokinetic chromatography separation and laser-induced fluorescence detection. </p><p><b>Paper IV</b> is focused on the development of a new methodology for determining the stereoisomeric F2-isoprostanes in human urine samples employing chromatographic separation on porous graphitic carbon and detection by electrospray ionization-tandem mass spectrometry. The results from this study conflict with the hypothesis that peripheral isoprostanes are elevated in patients with Alzheimer’s disease.</p><p><b>Paper V</b> describes porous graphitic carbon chromatography-tandem mass spectrometry for the determination of isoprostanes in human cerebrospinal fluid. A new simplified sample pre-treatment regime, involving a column switching technique, is presented that allows direct injection of a relatively large volume of CSF into the chromatographic system.</p>

Oxidative stress can be defined as a state of disturbance in the pro-oxidant/antioxidant balance in favour of the former, leading to potential damage. Processes associated with oxidative stress involve reactive oxygen species and radicals and can result in elevated levels of oxidatively modified or toxic molecules that can cause cellular malfunction, and even cell death. Destruction of membrane lipids, lipid peroxidation, caused by reactive oxygen species and radicals has been coupled to many diseases and also normal ageing. The measurement of low molecular weight biomarkers of oxidative stress present in complex matrices such as brain tissue, plasma, urine or cerebrospinal fluid is a delicate and difficult task and there is a need for improved analytical tools in this field of research. The major foci of this thesis and the work underlying it are the development of analytical techniques and methods for determining biomarkers for oxidative stress and lipid peroxidation. Aspects of particular concern include the effects of sample treatments prior to analysis, evaluation of the developed methods with respect to possible artefacts, and the scope for results to be misinterpreted. The specific research goals and issues addressed are detailed in five papers, which this thesis is based upon. <b>Paper I</b> focuses on malondialdehyde, describing and evaluating two new simplified sample pre-treatment regimes for the determination of malondialdehyde in rat brain tissue by capillary electrophoresis with UV detection. The effects of sample storing and handling are also considered. <b>Paper II</b> describes the synthesis, characterization and implementation of a new internal standard for the determination of malondialdehyde in biological samples using electrophoretic or chromatographic separation techniques. The usefulness of the internal standard is demonstrated in analyses of rat brain tissue samples. <b>Paper III</b> presents a method for the determination of 4-hydroxynon-2-enal in brain tissue from rats employing micellar electrokinetic chromatography separation and laser-induced fluorescence detection. <b>Paper IV</b> is focused on the development of a new methodology for determining the stereoisomeric F2-isoprostanes in human urine samples employing chromatographic separation on porous graphitic carbon and detection by electrospray ionization-tandem mass spectrometry. The results from this study conflict with the hypothesis that peripheral isoprostanes are elevated in patients with Alzheimer’s disease. <b>Paper V</b> describes porous graphitic carbon chromatography-tandem mass spectrometry for the determination of isoprostanes in human cerebrospinal fluid. A new simplified sample pre-treatment regime, involving a column switching technique, is presented that allows direct injection of a relatively large volume of CSF into the chromatographic system.