Acne is a common disease characterised by follicular hyperkeratinisation, bacterial hipercolonisation as well as immune reactions and inflammation. In acne, reactive oxygen species (ROS) may be released from the damaged follicular walls, which could cause the advancement of inflammation in the pathogenesis of the disease. The topical application of antioxidants is a promising approach to support the endogenous antioxidant defence and avoid oxidative injury that may lead to acne. The skin provides a painless and patient-friendly approach for systemic drug administration. Transdermal drug delivery not only improves patient compliance, but also avoids the first-pass effect. The major hurdle to penetration of matter through the skin is provided by an outward layer of the skin, the stratum corneurm (SC). Overcoming this barrier safely and reversibly is a fundamental problem in the field of transdermal drug delivery. Alpha-lipoic acid was utilised as the cosmeceutical active and can be classified in a mixed category of compounds that lie between cosmetics and drugs. Alpha-lipoic acid and its reduced form, dihydrolipoic acid, have been described as the "universal antioxidants" because of their capacity to quench a number of free radicals in both aqueous and lipid environments, their metal-chelating properties and ability to restore other antioxidants from their inactive form. The Pheriod™ system is a new manner of drug delivery aimed at overcoming the barrier function of the skin. It consists of vesicular structures, the sizes of which vary from 200-440 nm. These vesicles, prepared from customised essential fatty acids, were found to advance the efficacy of topically administered compounds. The aim of this study was to determine whether the Pheroid™ delivery system would enhance the transdermal delivery of formulations containing alpha-lipoic acid to the target site by performing Franz cell diffusion studies over a 12 hour period, followed by tape-stripping experiments. The results of the formulations containing Pheroid™ were compared to those of the formulations without Pheroid™. Experimental determination of transdermal flux of the alpha-lipoic acid formulations revealed that Pheroid™ improved the transdermal delivery of alpha-lipoic acid. The average flux of Pheroid™ cream from 0 to 2 hours wass 58.01 ± 6.63 ug/cm2.h. The average flux of Pheroid™ gel from 4 to 12 hours was 22.18 ± 3.33 ug/cm2.h. Tape-stripping experiments proved that the concentrations of alpha-lipoic acid in Pheroid™ cream and cream that remained in the epidermis after application to the skin were 569.10 ug/ml and 764.93 ug/ml respectively. The concentrations of alpha-lipoic acid in Pheroid gel and gel that diffused into the dermis were 23.62 ug/ml and 61.06 ug/ml respectively. Aqueous solubility and log D partition coefficient of alpha-lipoic acid were determined. Inspection of the log D value of -0.78 indicated that the compound was unfavourable to penetrate the skin, whereas the aqueous solubility of 8.602 mg/ml in PBS at a temperature of 32 °C indicated favourable penetration. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.

Acne is a common disease characterised by follicular hyperkeratinisation, bacterial hipercolonisation as well as immune reactions and inflammation. In acne, reactive oxygen species (ROS) may be released from the damaged follicular walls, which could cause the advancement of inflammation in the pathogenesis of the disease. The topical application of antioxidants is a promising approach to support the endogenous antioxidant defence and avoid oxidative injury that may lead to acne. The skin provides a painless and patient-friendly approach for systemic drug administration. Transdermal drug delivery not only improves patient compliance, but also avoids the first-pass effect. The major hurdle to penetration of matter through the skin is provided by an outward layer of the skin, the stratum corneurm (SC). Overcoming this barrier safely and reversibly is a fundamental problem in the field of transdermal drug delivery. Alpha-lipoic acid was utilised as the cosmeceutical active and can be classified in a mixed category of compounds that lie between cosmetics and drugs. Alpha-lipoic acid and its reduced form, dihydrolipoic acid, have been described as the "universal antioxidants" because of their capacity to quench a number of free radicals in both aqueous and lipid environments, their metal-chelating properties and ability to restore other antioxidants from their inactive form. The Pheriod™ system is a new manner of drug delivery aimed at overcoming the barrier function of the skin. It consists of vesicular structures, the sizes of which vary from 200-440 nm. These vesicles, prepared from customised essential fatty acids, were found to advance the efficacy of topically administered compounds. The aim of this study was to determine whether the Pheroid™ delivery system would enhance the transdermal delivery of formulations containing alpha-lipoic acid to the target site by performing Franz cell diffusion studies over a 12 hour period, followed by tape-stripping experiments. The results of the formulations containing Pheroid™ were compared to those of the formulations without Pheroid™. Experimental determination of transdermal flux of the alpha-lipoic acid formulations revealed that Pheroid™ improved the transdermal delivery of alpha-lipoic acid. The average flux of Pheroid™ cream from 0 to 2 hours wass 58.01 ± 6.63 ug/cm2.h. The average flux of Pheroid™ gel from 4 to 12 hours was 22.18 ± 3.33 ug/cm2.h. Tape-stripping experiments proved that the concentrations of alpha-lipoic acid in Pheroid™ cream and cream that remained in the epidermis after application to the skin were 569.10 ug/ml and 764.93 ug/ml respectively. The concentrations of alpha-lipoic acid in Pheroid gel and gel that diffused into the dermis were 23.62 ug/ml and 61.06 ug/ml respectively. Aqueous solubility and log D partition coefficient of alpha-lipoic acid were determined. Inspection of the log D value of -0.78 indicated that the compound was unfavourable to penetrate the skin, whereas the aqueous solubility of 8.602 mg/ml in PBS at a temperature of 32 °C indicated favourable penetration. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.

MOLECULAR INVESTIGATION OF THE EFFECTS OF ANTIOXIDANTS ON RAT BRAIN TISSUES

Oxidative and nitrosative stress are deleterious physiological processes caused by an imbalance between reactants such as reactive oxygen and nitrogen species and antioxidants. Due to the links between oxidative and nitrosative stress and disease, there is much interest in accurately quantifying these in biological and physiological samples. There are numerous methods to quantify the in vivo oxidative and nitrosative damage to lipids, DNA and proteins however they are generally time-consuming, expensive and difficult. Furthermore, due to the complex nature of oxidative and nitrosative stress it would be appropriate to measure a number of different biomarkers, however this is rarely done. The first section of this thesis contains research aimed at developing a bioassay to simultaneously detect markers of oxidative and nitrosative stress. This includes; 1) a review of the studies investigating the ability of these biomarkers to predict the onset of disease, 2) a description of the attempts to develop the bioassay, 3) a study designed to test the sensitivity of the bioassay to detect changes in oxidative stress. Unfortunately, the attempts to develop the bioassay were not as successful as hoped and, in the interests of completing the PhD in the time allowed, the PhD changed focus to look at the effects of oxidant:antioxidant balance on mitochondrial biogenesis. The second section of the thesis contains a review of the literature on this topic and two original investigations. It is well documented that oxidative and nitrosative stress contributes to the progression of many diseases including; cardiovascular disease, type 2 diabetes, Alzheimer’s disease, kidney disease and cancer. To determine which biomarkers would have the greatest efficacy in the bioassay, a comprehensive review was undertaken. The aim of the review was to investigate studies which have measured oxidative and nitrosative biomarkers to determine whether they are independent predictors of cardiovascular events (Chapter two). From the review, fifty-one studies were identified with twenty-six of these measuring oxidised (Ox)-LDL, fifteen assessing myeloperoxidase (MPO), seven using lipid peroxidation measures and three quantifying protein oxidation in plasma/serum. The recommendation of the review was that all areas require further investigation, however, it was determined that Ox-LDL and MPO would be beneficial for inclusion in the bio-assay. Other biomarkers considered for the bio-assay were nitrotyrosine, superoxide dismutase and glutathione peroxidase. Chapter three outlines method development used to measure the oxidative and nitrosative markers simultaneously. Recent technology allows multiple analytes to be detected simultaneously from the one sample. The Mulit-plex system is used to detect analytes that have been sandwiched between primary capture and secondary biotinylated detection antibodies. The secondary antibody attaches to streptavidin-phycoerythrin and is used by the Mulit-plex analyser to quantify the analyte. During development of the bio-assay, clumping of microspheres, high background, no detection of standard curve or samples, matrix effects, mislabeling of antibodies by manufacturers and lack of commercial available antibodies were obstacles that limited the success of this method. MPO was the only biomarker that was successful. Chapter four contains a study that investigated the sensitivity of the MPO mulitplex bio-assay. Nine highly trained cyclists underwent an extensive exercise protocol designed to induce dehydration by 4 % body mass, rehydration of 150 % fluid loss and a performance time-trial. Plasma samples were taken at five time points; baseline, post dehydration, post rehydration, pre time-trial and post time-trial and analysed using the mulitplex bio-assay. The results showed that there was a significant increase in MPO post dehydration and post time-trial compared with all other time points (P<0.05), thereby demonstrating that the mulitplex bio-assay is sensitive to detect changes in exercise and appropriate rehydration reduces oxidative stress. The MPO mulitplex bio-assay requires further testing on patients with diseases to further validate its future applications. As mentioned above, due to time constraints it was decided to stop the attempts to create a multi-analyte bioassay and focus on another important area of cellular oxidative stress. Currently, there is much interest in the involvement of oxidant:antioxidant balance in mitochondrial biogenesis. The increase of mitochondrial content within the skeletal muscle, termed mitochondrial biogenesis, provides an increased capacity to generate ATP during exercise and is recognized as one of major cellular adaptations to exercise. Reactive oxygen species are produced during exercise and have been shown to induce mitochondrial biogenesis. One of the key instigators of mitochondrial biogenesis is peroxisome proliferator activated receptor gamma coactivator-1α (PGC-1α). PGC-1α is central to the transcription of mitochondrial and nuclear encoded genes, which regulate downstream pathways such as oxidative phosphorylation and fatty acid oxidation. Antioxidant supplementation is common among athletes and healthy individuals; however, antioxidant supplements suppress reactive oxygen species and could therefore could hinder mitochondrial biogenesis and the positive adaptations associated with exercise. To establish whether antioxidant supplementation reduced mitochondrial biogenesis in skeletal muscle, male Wistar rats were supplemented with α-tocopherol and α-lipoic acid for fourteen weeks (Chapter six). Animals were separated into four groups: 1) sedentary control diet, 2) sedentary antioxidant diet, 3) exercise control diet and 4) exercise antioxidant diet. The exercise animals were trained 5 days/week for 14 weeks. Consistent with increased mitochondrial biogenesis and antioxidant defences following training, there were significant increases in PGC-1α mRNA and protein, COX IV and Cyt C protein abundance, citrate synthase activity, Nfe2l2 and SOD2 protein (P<0.05). Antioxidant supplementation reduced PGC-1α mRNA, PGC-1α and COX IV protein, and citrate synthase enzyme activity (P<0.05) in both sedentary and exercise-trained rats. In summary, antioxidants α-tocopherol and -lipoic acid supplementation suppresses beneficial adaptations in skeletal muscle such as markers of mitochondrial biogenesis and mitochondrial proteins, regardless of training status. The reduction in mitochondrial biogenesis may affect exercise training adaptations and reduce the ability of healthy individuals to attain optimal exercise adaptations. The last investigation (Chapter seven) studied the effect of reduced glutathione, through diethyl maleate (DEM) administration, on upstream regulators of mitochondrial biogenesis, markers of mitochondrial biogenesis and downstream signalling. Glutathione is a key antioxidant that reduces the amount of hydrogen peroxide. Male Wistar rats were divided into six groups 1) sedentary control, 2) sedentary DEM, 3) post-exercise control, 4) post-exercise DEM, 5) exercise-recovery and 6) exercise-recovery DEM. After an exercise bout to fatigue, animals were euthanized directly after exercise (post-exercise) or four hours post exercise (exercise-recovery). Exercising animals given DEM had significantly (P<0.05) decreased glutathione in skeletal muscle and had a significantly (P<0.05) greater increase in PGC-1α gene expression. There were also main interaction effects between exercise and DEM administration on SOD2 activity. Exercise altered the gene expression of GPx and the phosphorylation of p38 MAPK. Glutathione depletion decreased GPX activity and oxidised glutathione levels. These novel findings represent important in vivo evidence of the involvement of glutathione and oxidant:antioxidant balance in mitochondrial biogenisis. Overall this thesis has provided 1) the first comprehensive review on the prognostic ability of oxidative stress biomarkers to predict the onset of cardiovascular disease, 2) detailed information to assist in the further development of a multi-analyte bioassay to quantify oxidative and nitrosative stress, 3) data indicating that the MPO Mulit-plex bioassay is sensitive to detect physiological perturbations to oxidative stress, 4) evidence that antioxidant supplementation suppresses mitochondrial biogenesis and 5) proof that glutathione is important in the regulation of exercise-induced mitochondrial biogenesis.

Thesis (Ph. D.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 128-139). Also available on the World Wide Web.

Thesis (Ph. D.)--University of Missouri-Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 87-99). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 87-99). Also available on the Internet.

Thesis (Ph. D.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 177-202). Also available on the World Wide Web.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Este trabalho teve como objetivo investigar os efeitos comportamentais e neuroquÃmicos do Ãcido &#945;-lipÃico sozinho ou associado com LDOPA no modelo animal de doenÃa de Parkinson induzido pela injeÃÃo estereotÃxica da neurotoxina 6-hidroxidopmina (6-OHDA) em corpo estriado de rato. Os animais (ratos Wistar machos, 250-300g) foram tratados com Ãcido lipÃico nas doses de 100mg/Kg ou 200mg/Kg sozinho ou associado com LDOPA na dose de 50mg/Kg 1h antes da injeÃÃo estereotÃxica com 6-OHDA e diariamente por 14 dias apÃs a lesÃo. O comportamento motor foi testado atravÃs da avaliaÃÃo rotacional induzida pela apomorfina, da atividade exploratÃria horizontal no teste de campo aberto e da assimetria no uso das patas dianteiras no teste do cilindro. Os testes neuroquÃmicos visaram avaliar o efeito antioxidante e neuroprotetor do Ãcido &#945;-lipÃico atravÃs da determinaÃÃo da peroxidaÃÃo lipÃdica no TBARS, determinaÃÃo da concentraÃÃo de nitrito/nitrato e avaliaÃÃo da atividade da catalase. O Ãcido &#945;-lipÃico promoveu uma diminuiÃÃo significativa no nÃmero de rotaÃÃes contralaterais induzidas pela apomorfina, bem como promoveu uma melhora do desempenho motor, aumentando a atividade exploratÃria horizontal no teste de campo aberto e a utilizaÃÃo da pata contralateral à lesÃo produzida pela 6-OHDA no teste do cilindro. Quanto ao efeito antioxidante, o Ãcido &#945;-lipÃico promoveu uma reduÃÃo da peroxidaÃÃo lipÃdica ao reduzir o TBARS, reduziu a produÃÃo de nitrito/nitrato, bem como interagiu com o sistema antioxidante endÃgeno promovendo uma reduÃÃo na atividade da enzima catalase. Portanto, o Ãcido &#945;-lipÃico preveniu a lesÃo produzida nos neurÃnios dopaminÃrgicos pela 6-OHDA e pelo uso prolongado da LDOPA, demonstrando que este antioxidante teve efeito neuroprotetor, sendo um importante alvo terapÃutico para a doenÃa de Parkinson. / This study aimed to investigate the behavioral and neurochemical effects of &#945;-lipoic acid alone or in combination with LDOPA in an animal model of Parkinson Disease induced by stereotactic injection of the neurotoxin 6-hydroxydopamine (6 -OHDA) in rat striatum. The animals (male Wistar rats, 250-300g) were treated with doses of lipoic acid or 100mg/kg 200mg/Kg alone or in combination with a dose of 50mg/Kg LDOPA 1h before stereotactic injection of 6-OHDA and daily for 14 days after injury. Motor behavior was tested by evaluating apomorphine-induced rotational, horizontal exploratory activity in open field test and asymmetry in the use of the forelegs in the cylinder test. The neurochemical tests were intended to evaluate the antioxidant and neuroprotective effect of &#945;-lipoic acid by determining the lipid peroxidation in the TBARS and determining the concentration of nitrite/nitrate and evaluation of catalase activity. &#945;-Lipoic acid promoted a significant decrease in the number of rotations induced by apomorphine, as well as promoted an improvement in motor performance by increasing the horizontal exploratory activity in open field test and use the paw of the opposite side of the lesion produced by 6-OHDA in the test cylinder. The antioxidant effect of the &#945;-lipoic acid promoted reduction of lipid peroxidation by reducing the TBARS reduced the production of nitrite / nitrate and interacted with the antioxidant system promoting a reduction in endogenous catalase activity. Therefore, &#945;-lipoic acid prevented the damage produced in the dopaminergic neurons by 6-OHDA and the chronic use of LDOPA, indicating that this antioxidant had a neuroprotective effect, being an important therapeutic target for Parkinson disease.

In these studies antioxidant supplementation and oxidative stress measures were the basis for determining the health and welfare of the equine endurance athlete. The first study determined that lipoic acid (LA) orally administered to horses is effective in diminishing the levels of lipid peroxidation in the plasma, and is non-toxic at a dose of 10-mg/kg body weight. The second study showed muscle leakage measured by plasma creatine kinase (CK) and aspartate aminotransferase (AST) positively correlated to oxidative stress (measured by lipid hydroperoxides) during an endurance race (Research Ride 2001; R1). Also higher plasma ascorbate (ASC) status was found in the vitamin E plus C supplemented group versus the group receiving vitamin E alone. In the third study, the Research Ride 2002 (R2) determined that horses that did not finish the ride had higher CK and AST before, during and after (P < 0.05) the ride compared to horses that finished. These results were compared to the finishers of the Old Dominion 2000 (OD) and R1 and found that oxidative stress and muscle enzymes were greater during R2 due to the difficulty of terrain and ambient temperature. A higher level of horses' fitness in OD also could have explained the diminished oxidative stress. The fourth study calculated that horses at R2 were receiving 2265 ± 114 IU/d of vitamin E in the total diet prior to the race. These levels negatively correlated to plasma CK and AST and positively correlated to plasma a-tocopherol (TOC; r = 0.21; P = 0.005) throughout the 80-km race. In the final study the LA supplemented group had similar increases in antioxidant status (TOC, ASC, and total glutathione) as the vitamin E supplemented group of horses exercising for 55 km on a treadmill to simulate an endurance race. Both groups also had lower (P < 0.050) white blood cell apoptosis throughout exercise then the control group. These studies prove the need for antioxidant supplementation, specifically vitamin E or LA, during heavy endurance exercise to improve the health and welfare of our equine athlete. / Ph. D.