The present study determined whether dietary supplementation with reduced glutathione (GSH), glutathione ester (GSHE) or N-acetylcysteine (NAC) increased the mitochondrial level of GSH, the major antioxidant inside cells, in rat kidney cortex and medulla. Nine month-old female Lewis rats were given daily intraperitoneal injections of isotonic saline (n=6), or saline containing GSH (250mg or 0.81mmol/Kg of body wt; n=7), GSHE (12mg or 0.03mmol/Kg; n=8), or NAC (200mg or 1.22mmol/Kg; n=8) for four weeks. At the end of the injection period, the rats were anesthetized and the kidneys removed. The kidneys were separated into cortical and medullary sections, weighed, and homogenized. The sections were separated into cytosolic and mitochondrial fractions by differential centrifugation. The GSH levels were determined by a colorimetric assay. Cortical and medullary mitochondrial GSH levels were significantly increased by all three supplements. Cytosolic GSH levels were also significantly increased in both cortical and medullary sections. Thus, dietary supplementation can significantly increase the mitochondrial pool of GSH in the rat kidney. / Department of Physiology and Health Science

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Contusion injuries cause significant muscle damage, activating a series of cellular events. Satellite cells (SC), the key role players in muscle regeneration, are activated to proliferate and develop into mature myoblasts, which could fuse to form new myotubes or to repair damaged fibres. Evidence suggests that anti-oxidants, such as those found in grape seed extract (GSE), enhance repair, but their effect on SCs is still unclear. This study aimed to harvest and culture primary rat myoblasts to investigate the effect of chronic in vivo GSE supplementation on SCs following a standardised crush injury. Using a modified pre-plate technique, myoblasts were harvested from rat muscle and then compared with the immortal C2C12 cell line for proliferation and differentiation competence. Several media options were compared: i) DMEM with or without L-glutamine, ii) Ham‘s F10 or iii) DMEM with L-glutamine and Ham‘s F10 combined. Primary myoblasts proliferated and differentiated at a much slower rate than C2C12 cells. The combined media was selected for further use. To investigate the effects of GSE on the recovery, rats were supplemented daily with GSE or placebo 14 days prior to a standardised mass-drop crush injury to the gastrocnemius. SCs were isolated and cultured from uninjured (NI, baseline) and from injured rats 4 hours (4h), 3 days (3d) or 14 days (14d) post-injury. Expression of myogenic proteins Pax7, M-cadherin, MyoD, CD56, desmin and CD34 was determined by flow cytometry. Myoblasts were sorted according to their CD56 and CD34 expression and three sub-sets were collected and re-cultured, namely CD56+/CD34-, CD56-/CD34+ and CD56+/CD34+. After 24 hours, sorted cells were stained for desmin expression. Pax7, M-cadherin and MyoD were present in 100% of isolated cells from all groups confirming their myogenic SC identity. For all groups, desmin was expressed only in ~80% of SCs. Lower adhesion competency in GSE supplemented groups resulted in lower yield obtained for culturing. Expression of CD56 increased significantly 3d post-injury in the placebo group. In contrast, with GSE, CD56 already increased 4h post-injury and decreased again 3d post-injury. Although CD34 expression did not differ dramatically, expression pattern resembled that of CD56. Immunocytochemistry revealed a range in morphology and desmin expression of sorted myoblasts. More myoblasts with high desmin expression were observed in the two CD56+ sub-sets (irrespective of CD34 expression), indicating that CD56 is still expressed in more mature myoblasts. Flow cytometry revealed a population of myoblasts expressing particularly high levels of desmin, primarily in the non-injured baseline GSE group. We hypothesise that this result is an indication of preparedness of myoblasts to respond earlier to injury, enabling quicker repair. This cell population with high desmin content is restored in skeletal muscle after repair (14d), only when supplemented with GSE. In conclusion, GSE attenuated adhesion competence of primary myoblasts in culture, but resulted in earlier maturation of SCs, possibly due to baseline preparedness of myoblasts in uninjured muscle for a quick response. Both reduced adhesion competence and early progression of myoblasts could enhance wound healing in skeletal muscle. / AFRIKAANSE OPSOMMING: Kneuswonde veroorsaak aansienlike skade aan skeletspier, wat ‘n reeks sellulêre prosesse in werking stel. Satellietselle, die hoofrolspelers tydens spierregenerasie, vermenigvuldig en ontwikkel tot volwasse mioblaste, wat saamsmelt om nuwe spiervesels te vorm. Antioksidante, soos die wat in druiwepit-ekstrak voorkom, bespoedig herstel, maar hul uitwerking op satellietselle is steeds onduidelik. Die doel van hierdie studie was om mioblaste uit rotspiere te isoleer en te kweek om die effek van langdurige in vivo aanvulling van druiwepit-ekstrak op satellietselle na ‘n kneusbesering te bepaal. 'n Aangepaste protokol is gebruik om primêre mioblaste te isoleer, wat daarna met C2C12 selle, ten opsigte van hul vermenigvuldigings- en differensiasievermoë vergelyk is. Verskeie groeimedia is gebruik: i) DMEM met of sonder L-glutamien, ii) Ham F10 en iii) ‘n kombinasie van DMEM, L-glutamien en Ham F10. Primêre mioblaste het stadiger vermenigvuldig en gedifferensieer as C2C12 selle. Die gekombineerde medium is vir verdere gebruik gekies. Om die uitwerking van druiwepit-ekstrak op spierherstel te ondersoek, is rotte vir 14 dae onderwerp aan daaglikse aanvullings van druiwepit-ekstrak of placebo voor ‘n gestandardiseerde kneusbesering aan die gastrocnemius. Satellietselle is geïsoleer vanuit onbeseerde spier (basiskontrole) en vanuit beseerde spier 4 ure (4h), 3 dae (3d) en 14 dae (14d) na die besering. Die uitdrukking van spierverwante proteïene Pax7, M-cadherin, MyoD, CD56, desmin en CD34 is vasgestel met 'n vloeisitometer. Mioblaste is daarna gesorteer op grond van hul CD56- en CD34-uitdrukking. Drie sub-groepe is versamel en verder gekweek, nl. CD56+/CD34-, CD56-/CD34+ en CD56+/CD34+. Na 24 uur is gesorteerde selle gekleur om desmin-uitdrukking te bepaal. Pax7, M-cadherin en MyoD is deur 100% satellietselle in alle groepe uitgedruk, wat hul spierverwante identiteit bevestig, alhoewel slegs 80% selle in alle groepe desmin uitgedruk. Druiwepit-ekstrak het die vermoë van selle om aan plate te heg onderdruk, wat gelei het tot ‘n laer opbrengs van mioblaste. Drie dae na die besering in die placebo groep het die CD56-uitdrukking beduidend toegeneem. In teenstelling hiermee het CD56-uitdrukking in die druiwepit-ekstrak groep 4 ure na die besering beduidend toegeneem en weer afgeneem na 3 dae. Hoewel daar nie sulke dramatiese verskille was tussen groepe ten opsigte van CD34-uitdrukking nie, was daar ‘n soortgelyke tendens as vir CD56-uitdrukking. Immunositochemie het ‘n verskeidenheid van morfologieë en variërende desminvlakke in gesorteerde mioblaste blootgestel. In die twee CD56+ groepe is meer mioblaste wat hoë desmin vlakke uitdruk gevind, wat aandui dat CD56 uitgedruk word deur meer volwasse mioblaste, ongeag van CD34-uitdrukking. Tydens vloeisitometrie is ‘n populasie selle wat hoë desminvlakke uitdruk, hoofsaaklik in die onbeseerde en 14d druiwepit-ekstrak groepe gevind. Dit is ‘n aanduiding dat sommige mioblaste voorbereid is om na 'n besering vinniger te reageer. Na die herstelproses word hierdie groep selle hernu in die teenwoordigheid van druiwepit-ekstrak-aanvulling. Die resultate het gevolglik daartoe gelei dat druiwepit-ekstrak die hegtingsvemoë van mioblaste verlaag, maar dat die aanvulling in vivo tot vroeër ontwikkeling van mioblaste lei, waarskynlik deur satellietselle voor te berei vir 'n vinnige respons na ‘n besering. Beide die onderdrukking van aanhegting aan kultuurplate en die vroeë ontwikkeling van mioblaste, kan die herstel van die skeletspier verbeter. / NRF and the Harry Crossley bursary for funding

[Truncated abstract. Please see the pdf format for the complete text.] Interest in the role of peroxynitrite in the pathogenesis of atherosclerosis has increased due to many in vitro studies which have demonstrated its potent oxidising and nitrating capability and immunohistochemical staining studies which demonstrate nitration of tyrosine in vivo. It is frequently suggested that the production of nitric oxide and superoxide at sites of inflammation implicates peroxynitrite as the major damaging reactive nitrogen species in vivo. Evidence for a role for peroxynitrite is often demonstrated by measurement of 3-nitrotyrosine yet even this cannot distinguish peroxynitrite from other nitrating species. Clearly, however, if peroxynitrite is important in atherogenesis, then identification of mechanisms for its detoxification could provide a means of preventing such effects. Therefore, this Thesis has sought to determine whether phenolic compounds of dietary origin can be preferentially nitrated by reactive nitrogen species thereby protecting endogenous structures, such as low density lipoproteins, from atherogenic modifications. This Thesis focuses upon phenolic acids as they have received relatively less attention than other classes of phenolic compounds, such as flavonoids, yet they are quite abundant in socially important beverages such as red wine. In order to complete the required analyses, the development of methods to detect phenolic acids and their nitration products together with 3-nitrotyrosine, dityrosine and 5-nitro-γ-tocopherol was necessary. The initial in vitro experiments described herein sought to determine the products of reaction of peroxynitrite with phenolic acids of the 4-hydroxy and 3,4-dihydroxy type and then to examine whether these products could account for a protective effect upon tyrosine, lipids and endogenous anti-oxidants, if any was observed, when isolated LDL was treated with SIN-1, which releases peroxynitrite through the simultaneous generation of nitric oxide and superoxide. A concurrent minor focus was to examine the relationship between structure and activity of these phenolic acids under various regimes of oxidative insult. These experiments indicate that, at least in this in vitro model, oxidation is a dominant mechanism over nitration. Peroxynitrite was shown to nitrate coumaric acid in moderate yields but exclusive oxidation of caffeic acid appeared to occur. Although a potential role for γ-tocopherol as an anti-nitration agent was inferred, all types of chemical treatment of LDL in the presence of phenolic acids yielded oxidation as the primary end point. In fact, nitration of tyrosine was not detected and nitration of coumaric acid was at the limit of detection. Since nitration of tyrosine is generally regarded as important in many disease states, a more physiological nitrating mechanism involving artificially stimulated neutrophils was used. This system demonstrated that although physiologically relevant reactive nitrogen species can result in nitration of phenolic compounds, in a complex system including biological structures (LDL) and phenolic compounds, oxidation but not nitration of all species appears to occur. As a consequence of the results above, an examination of carotid plaque was undertaken to determine to what extent nitration occurred relative to oxidation in atherosclerotic tissue. These studies applied methods developed herein to detect 3-nitrotyrosine and dityrosine in complex biological matrices as markers of nitration and oxidation respectively. The data obtained demonstrated that nitration was a minor modification of protein (0.01%) compared to oxidation (0.3%) even in a highly diseased tissue such as carotid artery plaque. A secondary study examining plasma revealed that dityrosine, which has been implicated in irreversible albumin aggregation in chronic renal failure and more recently in heart disease, is elevated in chronic renal failure subjects compared to well matched controls. A separate examination of plasma from healthy subjects revealed that in both the fasting and post prandial state 3-nitrotyrosine could not be detected and, in fact, interfering species could be problematic in the GC-MS analysis of 3-nitrotyrosine. The lack of nitration of any substrate observed in vitro using reactive nitrogen species generated in the aqueous phase, the relative lack of nitration of tyrosine in plaque proteins and the lipophilicity of nitric oxide, the precursor of all reactive nitrogen species, suggested that nitration could be more closely associated with lipid structures. The known ability of γ-tocopherol to form 5-nitro-γ-tocopherol was used to probe this concept. The 5-nitro-γ-tocopherol content of lipid extracts obtained from carotid artery plaques was very high (30%). This indicated that nitration is predominantly a lipid phase phenomenon and that nitrating species are present in much greater abundance than oxidising species in vivo.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The human body has developed an integrated antioxidant defence system to protect against free radical damage. Acute exercise may result in the increased generation of free radicals, including reactive oxygen species, and this may overwhelm antioxidant defence systems resulting in oxidative stress. However, it has been shown that individuals who undergo regular exercise training may have improved antioxidant capacity when compared to sedentary controls. Results from research regarding the association between antioxidant capacity and exercise training are however not conclusive and further investigation is required. Therefore, the aim of this study was to investigate the association between the total plasma antioxidant status and selected plasma indicators of antioxidant status and the MnSOD Ala-9Val (-28C®T) polymorphism in trained male athletes (rugby players) and sedentary male students while controlling for dietary intake of the major antioxidants using a validated dietary assessment method. In order to address the potential confounding effect of dietary antioxidant intake on antioxidant status in the main study, a FFQ that measures vitamin C, vitamin E, carotenoid and flavonoid intake was developed. The reproducibility was assessed by the repeat administration of the FFQ (n = 38), while the va lidity was assessed using a 28-day closeended dietary record and repeated plasma vitamin C values (n = 18). Several statistical tests were conducted to compare the values obtained from the FFQ with values obtained from the various reference methods. While results from Bland-Altman plots suggested that the reproducibility and validity of FFQ was not completely satisfactory, similar mean values, moderate to strong correlation coefficients, and a high percentage of individuals classified correctly according to quartiles of intake indicated satisfactory reproducibility and validity of the FFQ in assessing antioxidant intake. Furthermore, moderate to strong validity coefficients obtained from the method of triads also indicated satisfactory validity for the FFQ. The main study involved a cross-sectional study that compared plasma vitamin C and carotenoid levels as well as total plasma antioxidant status in trained rugby players (n = 76) and sedentary male subjects (n = 39) with different MnSOD genotypes, while controlling for dietary antioxidant intake. Rugby players had significantly higher plasma vitamin C and carotenoid levels compared to sedentary students, which indicated more satisfactory plasma antioxidant status. This was also reflected in the tendency for total plasma antioxidant status (ORAC assay) to be higher in rugby players than sedentary students. MnSOD genotype did not influence plasma vitamin C and carotenoid levels or plasma total antioxidant status, with or without control for dietary antioxidant intake. Dietary vitamin C, vitamin E, carotenoid an flavonoid intake (from foods + supplements) was similar for rugby players and sedentary students and was adequate for both groups. Thus the association between antioxidant status and MnSOD genotype in rugby players and sedentary students seemed not to be influenced by dietary antioxidant intake. In conclusion therefore, rugby players undergoing regular exercise training had a more satisfactory antioxidant status compared to sedentary students. Based on this conclusion, the widespread use of antioxidant supplements by athletes is questioned. / AFRIKAANSE OPSOMMING: Die menslike liggaam beskik oor ‘n geintegreerde antioksidantmeganisme om dit teen vryradikaalskade te beskerm. Akute oefening kan bydra tot ‘n verhoogde produksie van vry radikale, insluitend reaktiewe suurstofspesies, wat kan veroorsaak dat die antioksidantbeskermingsmeganisme oorlaai word, wat dan kan aanleiding gee tot die ontstaan van oksidatiewe stress. Dit is aangetoon dat persone wat gereeld oefening doen verbeterde antioksidantkapasiteit toon in vergelyking met persone wat geen oefening doen nie. Die resultate van navorsingstudies wat die verband tussen antioksidantkapasiteit en oefening ondersoek is egter teenstrydig en verdere navorsing op hierdie gebied is essensieël om uitsluitsel te kry oor kontensieuse vraagstukke. Die doel van hierdie studie was dus om ondersoek in te stel na die verband tussen plasma antioksidant status, die MnSOD Ala-9Val (-28C T) polimorfisme en geselekteerde plasma antioksidantmerkers in geoefende manlike atlete (rugby spelers) en ‘n onaktiewe manlike kontrolegroep terwyl gekontroleer word vir die dieetinname van die vernaamste antioksidante. Om vir die potensiële invloed van dieetantioksidantinname op die antioksidantstatus van proefpersone in die hoofstudie te kontroleer, is ‘n voedsel frekwensievraelys wat vitamien C-, vitamien E-, karotenoïed- en flavinoïedinname meet, ontwikkel. Die herhaalbaarheid (betroubaarheid) van die vraelys is getoets deur herhaalde voltooiing daarvan deur ‘n toetsgroep (n=38), terwyl die geldighied getoets is deur gebruik te maak van ‘n 28-dag geslote dieetrekord en herhaalde plasma vitamien C bepalings as verwysingswaardes (n=18). Verskeie statistiese toetse is uitgevoer om die frekwensievraelys waardes met die verskillende verwysingswaardes te vergelyk. Alhoewel die Bland -Altman grafieke nie dui op bevredigende herhaalbaarheid en geldigheid van die voedselfrekwensie vraelys nie, dui gelyke gemiddelde waardes, matig tot sterk en betekenisvolle korrelasiekoeffisiënte en ‘n hoë persentasie individue korrek geklassifiseer volgens kwartiele van inname, wel op bevredigende herhaalbaarheid en geldigheid. Matige tot sterk geldigheidskoeffisiënte is ook verkry met die toepassing van “The method of Triads”, wat verdere steun bied vir bevredigende geldigheid. In die hoofstudie is plasma vitamien C, karotenoïedvlakke en totale plasma antioksidantstatus in manlike rugby spelers (n=76) vergelyk met dié van onaktiewe manlike kontroles (n=39). Vergelykings tussen MnSOD genotipes binne die aktiwiteitsgroepe is ook getref. Al genoemde analises is gekontroleer vir dieet antioksidantinname. Resultate dui daarop dat die plasma vitamien C en karotenoïedvlakke van rugby spelers betekenisvol hoër was as dié van die kontrolegroep, wat dui op ‘n meer bevredigende antioksidantstatus. Hierdie resultaat is ook weerspieël in die feit dat totale plasma antioksidantstatus (ORAC) in die rugby spelers oog geneig was om hoër te wees as dié van die kontrole groep. Dit het ook geblyk dat MnSOD genotipe nie ‘n effek gehad het op plasma vitamien C-, karotenoïed- of totale antioksidantstatus nie, met of sonder kontrole vir dieet antioksidantinname. Die dieet vitamien C-, vitamien E-, karotenoïed- en flavinoïedinname (vanaf voedsel en supplemente) was dieselfde vir rugby spelers en kontrole en was toereikend vir beide groepe. Dit blyk dus dat dat die verband tussen antioksidantstatus en MnSOD genotipe in die twee groepe nie beinvloed is deur antioksidantinname nie. Ten slotte kan die gevolgtrekking gemaak word dat manlike rugby spelers ‘n meer bevredigende antioksidant status het as onaktiwe manlike kontroles. Op grond van hierdie gevolgtrekking word die algemene gebruik van antioksidant supplemente deur atlete bevraagteken.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Human studies on the response of muscle to contusion injury are limited, probably due to the large variability in injury severity and the non-specificity of clinical symptoms reported. To circumvent this problem, several experimental animal models have been designed to study muscle damage and regeneration after contusion injuries. However, the majority of techniques currently used to induce contusion injury are very invasive and therefore not optimal. Furthermore, published studies regarding clinical treatment of such injuries are limited. The main aims of this study were therefore: a) to establish and characterise an in vivo model of non-invasive contusion injury, and b) to assess the effect of pre-injury chronic administration of the antioxidant supplement Oxiprovin™ - a natural grape seed extract (GSE) - on skeletal muscle recovery after experimentallyinduced injury. Two groups of male Wistar rats were subjected to 14 days of oral administration of isovolaemic placebo (sterile isotonic saline) or GSE (20 mg/kg/day) prior to induced contusion. Contusion injury was induced with the mass-drop technique, and recovery parameters assessed for up to 14 days post-injury. Placebotreated rats on average exhibited a 56 % higher creatine kinase (CK) activity when compared to the GSE-treated rats when area under the curve (AUC) was calculated for 14 days post-injury (p < 0.001). In the placebo group, plasma oxygen radical absorbance capacity (ORAC) was unchanged over time, but muscle ORAC was significantly increased by day 7 post-injury (p < 0.001). In the GSE group, a significant decrease in both plasma (p < 0.01) and muscle ORAC (p < 0.001) was evident 4 hr after injury, followed by a significant increase by day 3 (p < 0.05 and p < 0.001 respectively). CD34+ satellite cell (SC) numbers (quiescent and activated) peaked earlier in GSE-treated rats when compared to placebo-treated rats (4 hours vs. day 7 post-injury). Total satellite cell number (CD56+) also peaked earlier in GSE-treated rats than in placebo-treated rats (4 hours vs. 3 days post-injury), while M-cadherin+ SC numbers (quiescent, activated or proliferating) in both treatment groups were significantly increased 4 hours post-injury (p < 0.001), but more so in the placebo group. In GSE-treated rats when compared to placebo-treated rats, newly generated muscle fibres (displaying central nuclei and MHCf +) both appeared (day 3 vs. day 7 post-injury) and peaked in number (day 3 vs. day 7 post-injury; increase from baseline p < 0.001 for both) earlier. The results of this study demonstrate that we have successfully established an in vivo model for non-invasive contusion injury in rats. Furthermore, we have shown that Oxiprovin™: a) increased the ability to scavenge reactive species generated after injury and b) resulted in the activation of satellite cells and formation of newly generated muscle fibres at an earlier time point, thus accelerating the recovery of skeletal muscle after a standardised contusion injury. / AFRIKAANSE OPSOMMING: Eksperimente aangaande die reaksie van spier op kneusings in mense is beperk, waarskynlik as gevolg van die groot verskeidenheid simptome wat mag voorkom en die verskille in die ernstigheid van beserings. Ten einde hierdie problem te oorbrug, is verskeie eksperimentele diermodelle opgestel om kneusings en die herstel van spier daarna te ondersoek. Die tegnieke wat grootendeels vandag gebruik word om kneusings te veroorsaak, maak inbraak op die spier deur die spier te ontbloot voor besering, en is dus nie ideaal nie. Daar is ook nie baie bewyse aangaande die mees geskikte manier om so ‘n besering klinies te behandel nie. Die doel van hierdie studie was dus om: a) ‘n in vivo model van kneusings op te stel en te omskryf, en b) die effek van chroniese toediening van die antioksidant Oxiprovin™ - ‘n natuurlike druifsaad ekstrak (DSE) – op die herstel van skeletspier na ‘n kneusing te ondersoek. Twee groepe manlike Wistar rotte is onderwerp aan mondelikse toediening van isovolemiese plasebo (steriele isotoniese soutoplossing) of DSE (20 mg/kg/dag) vir ‘n tydperk van 14 dae voor kneusing. Kneusing is geïnduseer met die “massdrop” tegniek, en parameters van herstel is ondersoek tot en met 14 dae na besering. Plasebo-behandelde rotte het gemiddeld 56 % hoër kreatien kinase (KK) aktiwiteit in vergelyking met DSE-behandelde rotte (p < 0.001), toe die oppervlak onder die kurwe (OOK) tot en met 14 dae na besering bereken is. Geen verskil oor tyd is in die plasebo groep opgemerk toe plasma suurstof radikaal absorpsie kapasiteit (SRAK) bepaal is nie, maar ‘n betekenisvolle toename in spier SRAK teen dag 7 (p < 0.001) is waargeneem. ‘n Betekenisvolle afname in beide plasma (p < 0.01) en spier (p < 0.001) SRAK van die DSE is teen 4 hr waargeneem, gevolg deur ‘n betekenisvolle toename teen dag 3 na besering (p < 0.05 en p < 0.001 onderskeidelik). Die aantal CD34+ satelliet selle (SS – rustend en geaktiveerd) het beduidend vroeër in die DSE groep gestyg in vergelyking met die plasebo groep (4 uur vs. 7 dae na besering). Die totale aantal SS (CD56+) het ook vroeër in die DSE-behandelde rotte as die plasebobehandelde rotte gestyg (4 uur vs. 3 dae na besering), terwyl die aantal Mcadherin+ SS (rustend, geaktiveerd of prolifererend) betenisvol gestyg het in beide groepe teen 4 uur (p < 0.001) na besering, maar hoër in die plasebo groep was. Die aantal nuutgevormde spiervesels (met sentraal geleë nukleï en MHCf +) het beide vroeër verskyn en gepiek in die DSE-behandelde rotte in vergelyking met die plasebo-behandelde rotte (dag 3 vs. dag 7 na besering). Die resultate van hierdie studie dui aan dat ons instaat was om ‘n in vivo model van nie-indringende kneusing in rotte op te stel. Verder, het ons ook bewys dat Oxiprovin™ toediening die vermoë verleen het om: a) reaktiewe spesies wat na beserings gevorm word, meer doeltreffend te verwyder en b) satelliet selle vroeër te aktiveer en die vorming van nuwe skeletspiervesels te vervroeg, om sodoende die herstel van skeletspier na ‘n gestandardiseerde kneusing vinniger te bewerkstellig.

Wong, Wai Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 92-108). / Abstracts in English and Chinese. / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Atherosclerosis --- p.1 / Chapter 1.1.1 --- Pathogenesis of Atherosclerosis --- p.2 / Chapter 1.1.2 --- Atherosclerosis and Cardiovascular Disease --- p.4 / Chapter 1.2 --- Cardiovascular Disease (CVD) --- p.5 / Chapter 1.2.1 --- Term Definition --- p.5 / Chapter 1.2.2 --- Risk Factors --- p.6 / Chapter 1.2.3 --- Current Western Medications --- p.7 / Chapter 1.3 --- Reactive Oxygen Species (ROS) --- p.8 / Chapter 1.3.1 --- Impact of ROS --- p.8 / Chapter 1.3.2 --- "Superoxide Anion Radical, Hydrogen Peroxide, Hydroxyl Radical, Nitric Oxide" --- p.9 / Chapter 1.3.3 --- ROS Production by NAD(P)H Oxidases --- p.11 / Chapter 1.3.4 --- ROS Production by Mitochondria --- p.12 / Chapter 1.3.5 --- Lipid Peroxidation --- p.13 / Chapter 1.3.6 --- Other Sources of ROS --- p.15 / Chapter 1.4 --- Antioxidants --- p.16 / Chapter 1.4.1 --- Superoxide Dismutase (SOD) --- p.16 / Chapter 1.4.2 --- Catalase (CAT) --- p.17 / Chapter 1.4.3 --- Glutathinoe Peroxidase (GPx) --- p.17 / Chapter 1.4.4 --- Glutathione-S-Transferase (GST) --- p.18 / Chapter 1.4.5 --- Vitamin E --- p.18 / Chapter 1.4.6 --- Vitamin C --- p.19 / Chapter 1.5 --- Ageing --- p.19 / Chapter 1.6 --- Antioxidants and CVD --- p.21 / Chapter 1.7 --- Traditional Chinese Medicine (TCM) --- p.22 / Chapter 1.7.1 --- Danshen --- p.23 / Chapter 1.7.2 --- Gegen --- p.25 / Chapter 1.7.3 --- Danshen-Gegen Compound Formula (DG) --- p.26 / Chapter 1.8 --- Aim of Study --- p.27 / Chapter Chapter 2 --- In vitro Foam Cells Formation --- p.29 / Chapter 2.1 --- Materials and Methods --- p.29 / Chapter 2.1.1 --- Materials --- p.29 / Chapter 2.1.2 --- Methods --- p.30 / Chapter 2.1.2.1 --- Herbal Preparation by Hot Water Extraction --- p.30 / Chapter 2.1.2.2 --- Resident Peritoneal Macrophages Preparation --- p.31 / Chapter 2.1.2.3 --- "Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl Tetrazolium Bromide (MTT) Assay" --- p.31 / Chapter 2.1.2.4 --- DG Effect on in vitro Foam Cells Formation --- p.32 / Chapter 2.2 --- Results and Discussion --- p.32 / Chapter 2.3 --- Summary --- p.39 / Chapter Chapter 3 --- In vivo Antioxidant Level --- p.40 / Chapter 3.1 --- DG Effect on in vivo Antioxidant Levels on Young-adult Wistar Rats --- p.40 / Chapter 3.1.1 --- Materials and Methods --- p.40 / Chapter 3.1.1.1 --- Herbal Preparation by Hot Water Extraction --- p.40 / Chapter 3.1.1.2 --- Assay Kits --- p.41 / Chapter 3.1.1.3 --- Antibodies for Protein Expression Determination in Organs --- p.41 / Chapter 3.1.1.4 --- Animals and Experimental Design --- p.41 / Chapter 3.1.1.5 --- Plasma Antioxidants --- p.42 / Chapter 3.1.1.6 --- Lipid Peroxidation and Protein Expression in Organs --- p.46 / Chapter 3.1.1.7 --- Statistics --- p.52 / Chapter 3.1.2 --- Results and Discussion --- p.53 / Chapter 3.2 --- DG Effect on in vivo Antioxidant Levels on Middle-aged Wistar Rats --- p.74 / Chapter 3.2.1 --- Materials and Methods --- p.75 / Chapter 3.2.2 --- Results and Discussion --- p.75 / Chapter 3.3 --- Summary --- p.87 / Chapter Chapter 4 --- Conclusion and Future Work --- p.90 / Chapter 4.1 --- Conclusion --- p.90 / Chapter 4.2 --- Future work --- p.90 / Reference --- p.92 / Related Publication --- p.109

During extreme exercise, athletes experience increased inflammation that is similar to the acute phase response. Endurance athletes, distance runners in particular, are also more susceptible to compromised iron stores. This study evaluated inflammation, immune function and iron status in athletes completing a 50K ultramarathon. Twenty-two well-trained distance runners, 11 males and 11 females, were randomized in a double blind manner into--1) those who consumed 300 mg vitamin E and 1000 mg vitamin C (500 mg twice daily) or 2) placebos--for six weeks before and one week following a 50K ultramarathon race. Blood samples were obtained on 13 separate occasions throughout the study: before supplementation, during supplementation, the day before the race, pre-race, mid-race, immediately post-race, 2 hours following the race, and daily for six days following the race. Plasma levels of ascorbic acid and ��-tocopherol were measured by HPLC with electrochemical detection. Inflammatory cytokines, interleukin-6 (IL-6), tumor necrosis factor-�� (TNF-��), and interleukin-1�� (IL-1��) were measured using standard clinical assays. Each subject recorded immune function in an activity log and incidence of illness was tabulated as number of days ill. Ferritin was measured by enzyme immunoassay. Hemoglobin, hematocrit, and total-iron binding capacity (TIBC) and serum total iron were analyzed by standard procedures. Plasma concentrations of ascorbic acid and ��-tocopherol increased significantly in supplemented subjects (p<0.0001). Although the ultramarathon race elicited an inflammatory response, antioxidant supplementation did not alter the responses of IL-6 and TNF-��, which both increased from pre-race to mid-race, post- and post-2 h (Scheffe post-hoc analysis, p<0.0001) and returned to pre-race concentrations by 1 day after the race. Male supplemented subjects had lower IL-1�� concentrations compared to females consuming the supplement or to males consuming the placebo (ANCOVA, gender/time/treatment interaction; p<0.01) at mid-race (p<0.05 females, p<0.005 males), post 1 and 2 days (all p<0.002). Males had significantly higher ferritin levels than the female subjects (ANOVA, p<0.0001); supplementation resulted in lower ferritin concentrations at post-5 days (p<0.02, ANCOVA treatment time interaction, p<0.005). Supplementation did not reduce the days illness among those consuming antioxidants compared to those consuming the placebos. Ferritin not only increases during inflammation, it also is a measure of iron stores. Females had significantly lower levels of iron than the male subjects for each of the iron parameters measured (hemoglobin and hematocrit both p<0.0001, ferritin p<0.001, TIBC p<0.02) excluding serum total iron. The ferritin concentrations measured in the women were indicative of depleted iron stores (<12 ��g/l), and antioxidant supplementation increased hematocrit levels in the female subjects (p<0.05). This investigation indicates that female distance runners need to be aware of an increased susceptibility to iron depletion compared to their male counterparts. Antioxidant supplementation improved hematocrit levels (p<0.05) among female runners and may improve iron status among females with depleted stores. Although other investigations have suggested that antioxidant vitamins decrease exercise induced inflammation, no profound benefit of supplementation was found in this investigation though a response similar to the acute phase response was elicited by the ultramarathon race. Improvements in IL-i and ferritin in response to antioxidant supplementation may indicate that the supplementation was beneficial, but more research is needed to draw definitive conclusions. / Graduation date: 2003

Thesis (PhD) -- Macquarie University, Division of Environmental and Life Sciences, Dept. of Chemistry and Biomolecular Sciences, 2007. / Bibliography: leaves 295-322. / Generation of peroxide groups in proteins exposed to a wide variety of reactive oxygen species (ROS) requires an initial formation of protein carbon-centred or peroxyl free radicals, which can be reduced to hydroperoxides. Both protein radicals and protein hydroperoxides are capable of oxidizing important biomolecules and thus initiate biological damage. In this study, we investigated the inhibition of protein hydroperoxide formation by ascorbate and GSH in gamma-irradiated HL-60 cells.--We used HL-60 cells as a model for general protection of living organisms by ascorbate (Asc) and glutathione (GSH) from the deleterious effects of protein hydroperoxides generated by radicals produced by gamma radiation. Measurement by HPLC indicated that incubation of HL-60 cells with Asc in the presence of ascorbate oxidase resulted in the accumulation of intracellular Asc. The intracellular Asc levels were lowered by irradiation, demonstrating intracellular consumption of Asc by the radiation-generated radicals. Exposure of HL-60 cells to increasing gamma irradiation doses resulted in increasing accumulation of protein peroxides in the cells. This was measured by the FOX assay. A significant decrease in intracellular protein hydroperoxides was noted when the cells were treated with ascorbic acid before irradiation. A dose-dependent protective effect of Asc was observed. Asc loading also provided strong protection from radiation-generated protein hydroperoxides independently of the composition of the external medium, showing that only the radicals formed within the cells were effective in oxidizing the cell proteins. Similarly, protein peroxidation was inhibited in cells with enhanced levels of GSH and increased when the intracellular GSH concentration was reduced. These findings indicate that ascorbate and GSH are important antioxidants in protecting cells from oxidative stress associated with the generation of protein hydroperoxide. / Mode of access: World Wide Web. / xxix, 322 leaves ill