Plasma neopterin is an excellent marker of inflammation and is found in elevated levels in plasma of patients with cardiovascular disease. Neopterin originates as the oxidation product of 7,8-dihydroneopterin (7,8-NP), which is secreted by human macrophages when stimulated with interferon-y during inflammation. 7,8-NP has been shown to be a very efficient free radical scavenger and a potent antioxidant which can protect macrophages from a range of oxidative stresses. The uptake of oxidised low density lipoprotein (oxLDL) by macrophages which lead to the formation of foam cells is a hallmark of early atherosclerotic lesions. OxLDL-induced cell death is also considered to be an important process in the formation of necrotic lipid rich plaques and in atherosclerotic plaque destabilisation. This thesis examined the extent of oxLDL-induced damaged to HMDMs and whether 7,8-NP can inhibit oxLDL-mediated cell death in HMDMs. Foam cells had previously been defined as cholesteryl ester (CE) macrophages that stained positive with oil red-O. This thesis shows that the foamy appearance and presence of lipid droplets stained with oil red-O was not dependent on accumulation of CE which raises the suitability of using oil-red-O staining to identify the foam cells. In addition, HPLC but not GC analysis showed an increased in CE levels of the macrophages when the macrophages were incubated with oxLDL. The HPLC approach spared the samples of lengthy manipulations that might cause ex vivo oxidation. It also avoided subjecting the samples to high temperature treatment that could alter the lipid composition and therefore quantification of the lipid contents. Previous studies showed that 7,8-NP is a potent antioxidant and cytoprotective agent. Exposure of HMDMs to 1 mg/ml oxLDL caused 50% loss of cell viability as measured by the MTT reduction and trypan blue exclusion assays. The development of apoptotic features including caspase-3 activity, cytochrome c release from mitochondria and phophatidyserine (PS) exposure was examined. OxLDL did not cause caspase-3 activation as shown by Western Blot analysis and did not cause DEVD-AMC cleavage in HMDMs. However, cytochrome c release and phosphatidylserine exposure were observed when HMDMs were incubated with oxLDL as shown by Western Blot analysis and Annexin V-FITC staining respectively. Dihydroethidium (DHE) staining showed that oxLDL treatment caused mitochondrial superoxide generation in HMDMs. OxLDL-induced oxidative stress appeared to cause a rapid loss of HMDMs' intracellular glutathione (GSH) as analysed by HPLC technique. Incubation of HMDMs' with buthionine sulfoximine (BSO) and diethyl maleate (DEM) caused similar loss in GSH as incubation with oxLDL but did not result in HMDMs' death. This showed that oxLDL-induced decrease in GSH alone was not sufficient to cause cell death. The loss of cell viability by oxLDL was inhibited by 7,8-NP in the concentration range of 50 to 200 lM. HMDMs' GSH loss caused by oxLDL was similarly inhibited by 7,8-NP supporting the idea that preventing the cellular GSH loss will protect the HMDMs from death. Incubation of HMDMs with 7,8-NP showed reduction in DHE fluorescence intensity staining suggesting that 7,8-NP inhibited or scavenged oxLDL-dependent generation of superoxide. 7,8-NP also effectively inhibited oxLDL-induced PS externalisation to the outer membrane but failed to inhibit the oxLDL-induced release of cytochrome c from mitochondria to the cytosol. The labelling of oxLDL with DiI showed that 7,8-NP significantly inhibited the uptake of oxLDL. However, the inhibitory effect was only measured at non-toxic concentration of oxLDL. The ability of 7,8-NP to inhibit oxLDL uptake raised the possibility that 7,8-NP protective effect against oxLDL involved modulation of the scavenger receptors'expression in particular SRA and CD36. The Western Blot analysis showed that incubation of HMDMs with 7,8-NP did not affect HMDMs' SRA protein expression. In 50% of the experiments, it was demonstrated that certain isoforms of CD36 protein were significantly down regulated by 7,8-NP suggesting that various factors might interact with 7,8-NP or CD36. The ability of 7,8-NP to protect HMDMs from oxLDL-induced death provides further evidence that this antioxidant is secreted by HMDMs to protect them against the oxidative damage in the highly oxidative environment of atherosclerotic plaque.

Any lipoproteins and cells present in the inflammatory environment of atherosclerotic plaques are likely to be exposed to high levels of oxidative stress. As 7,8-dihydroneopterin (7,8-NP) is synthesized by interferon-γ (IFN-γ)-activated macrophages, this pteridine is also thought to exist at sites of inflammation. 7,8-NP s in vivo role remains controversial, but numerous in vitro studies have identified a radical scavenging activity. The possibility of 7,8-NP protecting against oxidative damage in inflammatory environments like plaque was investigated in this thesis. Both human monocyte-derived macrophages (HMDMs) and low density lipoprotein (LDL) were used as substrates. The extent of protein hydroperoxide formation in each model, and 7,8-NP s effect on this process, were specifically studied since most previous research has focussed on lipid rather than protein peroxidation. For the first time, neopterin (including oxidized 7,8-NP) was also directly detected by high performance liquid chromatography in the inflammatory environments of 19 pus and two atherosclerotic plaque samples. Peak concentrations even reached the low micromolar range. The positive correlation identified in the pus between neopterin and a well known antioxidant, vitamin E, further hinted at a potential antioxidant function. However, no significant association was noted between neopterin and markers of protein or lipid oxidation. Exposure of HMDMs to the AAPH peroxyl radical generator resulted in significant quantities of lipid hydroperoxides but not protein hydroperoxides, as detected by the FOX assays. This is likely due to the large accumulation of polyunsaturated fatty acidrich lipid in the primary HMDMs during differentiation in 10% human serum and is of relevance to atherosclerotic plaque, where macrophages also become lipid-loaded. The addition of up to 200μM 7,8-NP failed to prevent AAPH-induced lipid peroxidation and was also unable to inhibit a loss of cellular thiols or viability. This lack of effect suggests the damaging peroxyl radicals are not being scavenged by 7,8-NP. The high lipid content of HMDM cells appears to cause the AAPH and/or 7,8-NP to localize to a cellular site, where they are unable to interact. Macrophage-mediated oxidation of LDL in iron(II)-supplemented Hams F10 was associated with the formation of 30-40 moles of protein hydroperoxides per mole of LDL. The close parallel between protein and lipid peroxidation supports the theory that lipid-derived radicals are involved in protein hydroperoxide formation on LDL and indicates that protein hydroperoxides are an early product of LDL oxidation. Their detection during exposure of LDL to both the THP-1 macrophage cell line and primary HMDM cells confirms that protein hydroperoxides are also a normal consequence of macrophage-mediated LDL oxidation. Incubation of LDL with micromolar 7,8-NP prevented macrophage-mediated protein hydroperoxide formation in a concentration-dependent manner. Lipid oxidation and vitamin E loss were similarly inhibited by 7,8-NP during the cell-mediated attack of LDL. Kinetic analysis revealed protection due to extension of the lag phase, with 7,8-NP depletion and initiation of the propagation phase coinciding. This supports a radical scavenging activity for 7,8-NP, resulting in protection of the entire LDL particle. By contrast, the release of nanomolar quantities of 7,8-NP by IFN-γ-stimulated THP-1 macrophages failed to prevent LDL oxidation. HMDMs activated by IFN-γ did significantly inhibit LDL oxidation, including protein hydroperoxide formation, for up to 48 hours but this antioxidant effect was not due to the de novo synthesis of 7,8-NP. These results indicate that both the prevalence of protein hydroperoxides, and the ability of 7,8-NP to act as an antioxidant, depend on the system under investigation. Neopterin exists in inflammatory environments but, considering the lack of protection against AAPH-mediated HMDM oxidation and the 7,8-NP concentration required to inhibit macrophage-mediated LDL oxidation, strong evidence for an antioxidant activity of 7,8-NP in atherosclerotic plaque is currently lacking.

Intracellular oxidative stress is induced by oxidised low density lipoprotein (oxLDL) in macrophages. In the atherosclerotic lesions, this oxLDL dependent oxidative stress appears to cause macrophage cell death, a key process in the development of the necrotic core within the complex plaque. Macrophages are activated by γ-interferon to synthesise and release a potent antioxidant, 7,8-dihydroneopterin (7,8-NP), which has been previously shown to protect human monocyte-like U937 cells and human monocyte-derived macrophage (HMDM) cells from oxLDL cytotoxicity. This study examined whether oxLDL causes the loss of cellular metabolic function and whether 7,8-dihydroneopterin can prevent this loss of metabolic activity in U937 cells and HMDM cells. OxLDL prepared by copper oxidation caused cell death in both U937 and HMDM cells at concentrations of 0.5 and 2.0 mg/ml, respectively. Cell morphology showed the oxLDL caused a necrotic like death in both cells as indicated by cell swelling and lysis. The decrease in cell viability was only observed after the loss of intracellular glutathione (GSH) which occurred in the first 3 hours in U937 cells following oxLDL addition. The loss of GSH appeared to be due to the production of intracellular oxidants generated in response to the presence of the oxLDL. Within 3 hours of oxLDL addition to both cell types, there was a rapid and progressive shutdown of cell metabolism indicated by a significant decrease in the enzymatic activity of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a fall in lactate production and intracellular ATP levels. GAPDH activity was found to be inactivated rather than being lost from the cell. Gel electrophoresis with specific staining for oxidised proteins showed that the GAPDH had been oxidatively inactivated in the cells when oxLDL was present. Unlike GAPDH, lactate dehydrogenase (LDH) was not inactivated by the oxidation but was lost from the cells due to cell lysis. The observed rate of glycolysis failure was similar in both cell types except the HMDM cells did not lose lactate, LDH activity and cell viability until 6 hours compared to 3 hours with the U937 cells. The rate of oxygen consumption (VO2) was measured in U937 cells by taking cells at set time points and placing them in the respirometers to measure the VO2. U937 cells were found to increase their VO2 with incubation but this increase was inhibited in the presence of oxLDL within 3 hours. The addition of the 7,8 dihydroneopterin above 100 μM to both the U937 and HMDM cells significantly inhibited the oxLDL-induced loss of cell viability. GAPDH activity loss was also inhibited while lactate production was maintained. The 7,8-dihydroneopterin also prevented the decrease in the VO2 in oxLDL-treated U937 cells. OxLDL was labelled with fluorescent DiI to measure the uptake of oxLDL by HMDM cells. The incorporation of DiI into oxLDL was found to make it non-cytotoxic, possibly due to DiI’s antioxidant properties. Studies were therefore conducted using either a mixture of oxLDL and DiI labelled oxLDL (DiI-oxLDL) at non-protective concentrations or low concentration of DiI-oxLDL alone. These studies showed that 7,8-dihydroneopterin downregulated the oxLDL uptake in oxLDL-treated HMDM cells. Surprisingly the uptake rates also suggested that there was no relationship between oxLDL uptake and cell death assuming oxLDL and DiI-oxLDL are taken up by the same mechanism. This research showed that oxLDL-induced oxidative stress in macrophage cells causes a rapid oxidative loss of GAPDH activity which leads to the loss of glycolytic activity and a fall in ATP levels. The failure of cell metabolism appears to be a key event in the death mechanism triggered by the oxLDL. The radical scavenging activity of 7,8-dihydroneopterin appears to prevent the oxidative stress as indicated by the protection of the GSH pool. Without the oxidative stress, GAPDH remains functioning, glycolytic activity is maintained and both the U937 cells and HMDM cells did not die. This suggests that within the atherosclerotic plaque, 7,8-dihydroneopterin may act to stabilise the metabolism of macrophage cells in the presence of oxLDL and downregulate the oxLDL uptake.

Any lipoproteins and cells present in the inflammatory environment of atherosclerotic plaques are likely to be exposed to high levels of oxidative stress. As 7,8-dihydroneopterin (7,8-NP) is synthesized by interferon-γ (IFN-γ)-activated macrophages, this pteridine is also thought to exist at sites of inflammation. 7,8-NP s in vivo role remains controversial, but numerous in vitro studies have identified a radical scavenging activity. The possibility of 7,8-NP protecting against oxidative damage in inflammatory environments like plaque was investigated in this thesis. Both human monocyte-derived macrophages (HMDMs) and low density lipoprotein (LDL) were used as substrates. The extent of protein hydroperoxide formation in each model, and 7,8-NP s effect on this process, were specifically studied since most previous research has focussed on lipid rather than protein peroxidation. For the first time, neopterin (including oxidized 7,8-NP) was also directly detected by high performance liquid chromatography in the inflammatory environments of 19 pus and two atherosclerotic plaque samples. Peak concentrations even reached the low micromolar range. The positive correlation identified in the pus between neopterin and a well known antioxidant, vitamin E, further hinted at a potential antioxidant function. However, no significant association was noted between neopterin and markers of protein or lipid oxidation. Exposure of HMDMs to the AAPH peroxyl radical generator resulted in significant quantities of lipid hydroperoxides but not protein hydroperoxides, as detected by the FOX assays. This is likely due to the large accumulation of polyunsaturated fatty acidrich lipid in the primary HMDMs during differentiation in 10% human serum and is of relevance to atherosclerotic plaque, where macrophages also become lipid-loaded. The addition of up to 200μM 7,8-NP failed to prevent AAPH-induced lipid peroxidation and was also unable to inhibit a loss of cellular thiols or viability. This lack of effect suggests the damaging peroxyl radicals are not being scavenged by 7,8-NP. The high lipid content of HMDM cells appears to cause the AAPH and/or 7,8-NP to localize to a cellular site, where they are unable to interact. Macrophage-mediated oxidation of LDL in iron(II)-supplemented Hams F10 was associated with the formation of 30-40 moles of protein hydroperoxides per mole of LDL. The close parallel between protein and lipid peroxidation supports the theory that lipid-derived radicals are involved in protein hydroperoxide formation on LDL and indicates that protein hydroperoxides are an early product of LDL oxidation. Their detection during exposure of LDL to both the THP-1 macrophage cell line and primary HMDM cells confirms that protein hydroperoxides are also a normal consequence of macrophage-mediated LDL oxidation. Incubation of LDL with micromolar 7,8-NP prevented macrophage-mediated protein hydroperoxide formation in a concentration-dependent manner. Lipid oxidation and vitamin E loss were similarly inhibited by 7,8-NP during the cell-mediated attack of LDL. Kinetic analysis revealed protection due to extension of the lag phase, with 7,8-NP depletion and initiation of the propagation phase coinciding. This supports a radical scavenging activity for 7,8-NP, resulting in protection of the entire LDL particle. By contrast, the release of nanomolar quantities of 7,8-NP by IFN-γ-stimulated THP-1 macrophages failed to prevent LDL oxidation. HMDMs activated by IFN-γ did significantly inhibit LDL oxidation, including protein hydroperoxide formation, for up to 48 hours but this antioxidant effect was not due to the de novo synthesis of 7,8-NP. These results indicate that both the prevalence of protein hydroperoxides, and the ability of 7,8-NP to act as an antioxidant, depend on the system under investigation. Neopterin exists in inflammatory environments but, considering the lack of protection against AAPH-mediated HMDM oxidation and the 7,8-NP concentration required to inhibit macrophage-mediated LDL oxidation, strong evidence for an antioxidant activity of 7,8-NP in atherosclerotic plaque is currently lacking.

Atherosclerosis (Heart Disease) is an inflammatory disease caused by the formation of plaque within the arterial wall. In response to inflammation, monocytes enter the artery wall, differentiate into macrophages and take up altered low-density-lipoprotein (such as oxidised-LDL). This oxLDL is taken up into the phagocytotic macrophages via the action of the scavenger receptors. If more oxLDL is engulfed than the cell can process, they further differentiate into lipid-loaded foam cells. These are the main cell type found in atherosclerotic plaques. The scavenger receptor CD36 is responsible for 70% of oxLDL uptake by macrophages. Previous studies show that CD36 expression can be down regulated by the antioxidant, 7,8-dihydroneopterin. This research focuses on the effect of CD36 down regulation by 7,8-dihydroneopterin on foam cell formation. Human macrophages prepared from monocytes purified from human blood were incubated with copper oxidised LDL for up to 48 hours. Macrophage accumulation of the sterols was measured using a high performance chromatograph (HPLC) method developed as part of this project. The HPLC analysis measured: cholesterol, cholesteryl-oleate and -palmitate and 7-ketocholesterol accumulation within human macrophages. A flow cytometry procedure was developed where the strongly adherent macrophages could be lifted from the tissue culture plates before immuno staining for CD36. Effect of incubating macrophages with 7,8-dihydroneopterin on the formation of foam cells was studied by measuring the lipid content by HPLC and flow cytometry measurement of CD36. HPLC analysis showed non-cytotoxic levels of oxLDL produced a large accumulation of cholesterol and cholesteryl esters in the macrophages. Cholesterol, 7-ketocholesterol and cholesteryl-oleate and -palmitate concentrations in the cells rose significantly over the first 24 hours and stayed at a steady level for the following 24 hours. CD36 levels was further analysed on human macrophages. This study shows that foam cell formation can be measured using human macrophages. 7,8-Dihydroneopterin treatment resulted in a reduction of cholesterol and oxysterol uptake back to basal levels. It also reduced CD36 cell surface expression by a third. These results suggest that even a small reduction in CD36 cell surface expression may have a large effect on foam cell formation. This is another mechanism by which 7,8-dihydroneopterin protects against atherosclerosis developing.

Hypochlorous acid (HOCl) is a powerful oxidant produced by activated phagocytes at sites of inflammation to kill a wide range of pathogens. Yet, it may also damage and kill the neighbouring host cells. The abundance of dead macrophages in atherosclerotic plaques and their colocalization with HOCl-modified proteins implicate HOCl may play a role in killing macrophages, contributing to disease progression. The first part of this research was to investigate the cytotoxic effect and cell death mechanism(s) of HOCl on macrophages. Macrophages require efficient defense mechanism(s) against HOCl to function properly at inflammatory sites. The second part of the thesis was to examine the antioxidative effects of glutathione (GSH) and 7,8-dihydroneopterin (7,8-NP) on HOCl-induced cellular damage in macrophages. GSH is an efficient scavenger of HOCl and a major intracellular antioxidant against oxidative stress, whereas 7,8-NP is secreted by human macrophages upon interferon-γ (IFN-γ) induction during inflammation and can also scavenge HOCl. HOCl caused concentration-dependent cell viability loss in human monocyte derived macrophage (HMDM) cells above a specific concentration threshold. HOCl reacted with HMDMs to cause viability loss within the first 10 minutes of treatment, and it posed no latent effect on the cells afterwards regardless of the HOCl concentrations. The lack of caspase-3 activation, rapid influx of propidium iodide (PI) dye, rapid loss of intracellular ATP and cell morphological changes (cell swelling, cell membrane integrity loss and rupture) were observed in HMDM cells treated with HOCl. These results indicate that HOCl caused HMDM cells to undergo necrotic cell death. In addition to the loss of intracellular ATP, HOCl also caused rapid loss of GAPDH enzymatic activity and mitochondrial membrane potential, indicating impairment of the metabolic energy production. Loss of the mitochondrial membrane potential was mediated by mitochondrial permeability transition (MPT), as blocking MPT pore formation using cyclosporin A (CSA) prevented mitochondrial membrane potential loss. HOCl caused an increase in cytosolic calcium ion (Ca2+) level, which was due to both intra- and extra-cellular sources. However, extracellular sources only contributed significantly above a certain HOCl concentration. Preventing cytosolic Ca2+ increase significantly inhibited HOCl-induced cell viability loss. This suggests that cytosolic Ca2+ increase was associated with HOCl-induced necrotic cell death in HMDM cells, possibly via the activation of Ca2+-dependent calpain cysteine proteases. Calpain inhibitors prevented HOCl-induced lysosomal destabilisation and cell viability loss in HMDM cells. Calpains induced HOCl-induced necrotic cell death possibly by degrading cytoskeletal and other cellular proteins, or causing the release of cathepsin proteases from ruptured lysosomes that also degraded cellular components. The HOCl-induced cytosolic Ca2+ increase also caused mitochondrial Ca2+ accumulation and MPT activation-mediated mitochondrial membrane potential loss. MPT activation, like calpain activation, was also associated with the HOCl-induced necrotic cell death, as preventing MPT activation completely inhibited HOCl-induced cell viability loss. The involvement of both calpain activation and MPT activation in HOCl-induced necrotic cell death in HMDM cells implies a cause and effect relationship between these two events. HMDM cells depleted of intracellular GSH using diethyl maleate showed increased susceptibility towards HOCl insult compared to HMDM cells with intact intracellular GSH levels, indicating that intracellular GSH played an important role in protecting HMDM cells against HOCl exposure. Intracellular GSH level in each HMDM cell preparation directly correlated with HOCl concentration required to kill 50% of population for each cell preparation, indicating intracellular GSH concentrations determine the efficiency of GSH in preventing HOCl-induced damage to HMDM cells. Intracellular GSH and cell viability loss induced by 400 μM HOCl were significantly prevented by 300 μM extracellular 7,8-NP, indicating that added 7,8-NP is an efficient scavenger of HOCl and out-competed intracellular GSH for HOCl. The amount of 7,8-NP synthesized by HMDM cells upon IFN-γ induction was too low to efficiently prevent HOCl-mediated intracellular GSH and cell viability loss. HOCl clearly causes HMDM cells to undergo necrosis when the concentration exceeds the intracellular GSH concentrations. Above this concentration HOCl causes oxidative damage to the Ca2+ ion channels on cell and ER membranes, resulting in an influx of Ca2+ ions into the cytosol and possibly the mitochondria. The rise in Ca2+ ions triggers calpain activation, resulting in the MPT-mediated loss of mitochondrial membrane potential, lysosomal instability and cellular necrosis.

Cholesterol crystals have been linked to activation of the NLRP3 inflammasome and the formation of foreign body giant cells (FBGCs). It has been hypothesized that FBGCs have a role in advanced atherosclerotic plaque formation. This thesis examined the feasibility of producing stable cultures of FBGCs starting with human monocytes with the goal to examine pterin production by these cells in comparison to human monocyte derived macrophages (HMDMs). The study also investigated the effect of cholesterol crystals on 7,8-dihydroneopterin (7,8-NP) production and modulation of IL-1β levels in macrophages. 7,8-Dihydroneopterin is a potent antioxidant generated by macrophages which also down regulates the expression of macrophage scavenger receptor CD36. The use of alpha-tocopherol and IL-4 as FBGC fusion mediators was explored. Using these mediators, large numbers of FBGC were successfully cultured. The rates of fusion achieved in the cultures were low, and the cells had poor adhesion, which prevented pterin measurement. FBGC, which are thought to remove crystallized cholesterol from the plaque, cleared 21% of cholesterol crystal compared to 50% cleared by HMDM cells. Due to this result, the effect of cholesterol crystals on pterin production in monocytes and macrophages was explored. Cholesterol crystals cause inflammation through the activation of the NLRP3 inflammasome, however, it was unknown whether they could modulate 7,8-NP production. Cholesterol crystals caused an intracellular dose-dependent loss of 7,8-NP to its oxidized form, neopterin, in HMDM cells. Cholesterol crystals induced intracellular synthesis of 7,8-NP in HMDMs. 7,8-NP was released into the supernatant and oxidized to neopterin in media. Monocytes treated with cholesterol crystals released up to 100 nM of neopterin and 120 nM of 7,8-NP in the media after 48 hours. The combination of IFN- and cholesterol crystals appeared to inhibit the release of 7,8-NP into the media for the first 48 hours, after this time 7,8-NP release rapidly increased. The addition of exogenous 200 μM 7,8-NP showed that in the presence of monocytes, cholesterol crystals did not cause the oxidation of 7,8-NP to neopterin, as seen in HMDMs but possibly to 7,8-dihydroxanthopterin or xanthopterin. The presence of 7,8-NP increased IL-1β expression in the presence of cholesterol crystals after 24 hours incubation. FBGCs and the removal of cholesterol crystals may be a key process in the resolution of atherosclerotic plaques. It appears that cholesterol crystals are able to modulate inflammatory processes including activation of the inflammasome and balance of 7,8-dihydroneopterin to the oxidized neopterin. The infiltrating monocytes may provide antioxidant protection against the inflammation induced by cholesterol crystals and the activity of the infammasome.

Atherosclerosis is a chronic inflammatory disease leading to plaque buildup in the major arteries. The plaques consist of cholesterol, calcium, inflammatory cells, extracellular matrix and fibrous material. Under inflammatory conditions IFN-• stimulation of human monocytes and macrophages generates reduced pteridine, 7,8-dihydroneopterin (78NP) which has been shown to be an effective cytoprotective agent to some cell types against oxidative damage by reactive oxygen species (ROS). 7,8-dihydroneopterin is oxidized to fluorescent neopterin in the presence of hypochlorite (HOCl). Although a considerable amount of work has been published on the composition of neopterin in atherosclerotic plaques, very little is known about the variation of 78NP and other oxidative biomarkers across the length of the carotid and femoral and their contribution to plaque progression, which was researched in this work. Atherosclerotic plaques excised from patients with carotid and femoral plaques were sliced into 3-5 mm sections, and each section was analyzed for concentrations of neopterin, 7,8- dihydroneopterin, •-tocopherol, TBARS, DOPA, cholesterol, dityrosine, protein carbonyls •- aminoadipic semialdehyde (AAS) and •-glutamic semialdehyde (GGS), free and esterified 7- ketocholesterol (7-KC). Cultured live plaque as a source of 7,8-dihydroneopterin and neopterin was also investigated in this study. It was shown that carotid plaques significantly vary from femoral plaques, in the levels and range of most oxidative biomarkers. Carotid plaques showed a high variation in the biomarker concentrations between plaques but also between sections of an individual plaque. Femoral plaques on the other hand showed lower amounts of biomarkers with very little variation in biomarker concentrations. High variation with pterin concentrations and other biomarkers suggests dynamic and active changes in inflammation within the plaque. Collectively, it was observed that every plaque was unique with respect to its composition and correlations between the biomarkers. Though shown to be a well-known antioxidant and a radical scavenger, there is no published literature on 7,8-dihydroneopterin’s mode of entry into and out of the cell. To understand how it enters the cells could explain the difference in its protective ability of different cell types Abstract xxviii against oxidative stress-mediated cell death. Knowledge of transport of 7,8-dihydroneopterin will provide insights about its protection of monocyte/macrophage cell death which could potentially reduce atherosclerotic plaque growth and progression. As 7,8-dihydroneopterin is produced from guanosine, a nucleoside that is transported using specialized nucleoside transporters (equilibrative nucleoside transporters (ENT's) and concentrative nucleoside transporters (CNT's), their role was examined and characterized for 7,8-dihydroneopterin transport. It was found that 7,8-dihydroneopterin and neopterin are transported via nucleoside transporters in U937 cells, THP-1 cells and human monocytes. ENT 2 was the major transporter in U937 cells while ENT 1 transported bulk of 7,8-dihydroneopterin in THP-1 cells. Both ENT's and CNT's are involved in 7,8-dihydroneopterin uptake in human monocytes. In all the cell lines tested, 7,8-dihydroneopterin protection against AAPH mediated oxidative cell death was inhibited by nucleoside transport inhibitors, suggesting that nucleoside transporters are indispensible for 7,8-dihydroneopterin mediated intracellular protection against oxidative stress. Accurate measurement of neopterin, as a biomarker of inflammation in plaques and cells is critical aspect to assess disease progression. The current C18 HPLC method used in our laboratory for neopterin measurement lacks sensitivity due to interference of acetonitrile (ACN) over time. Acidic tri-iodide conversion of 7,8-dihydroneopterin to neopterin was also variable at times giving inconsistent measurement of neopterin so the manganese oxide (MnO2) method was looked at as an alternative. Electrochemical detector (ECD) was another option studied as it did not require any precolumn oxidation of 7,8-dihydroneopterin to neopterin. A new method using strong cation exchange (SCX) column was developed for a precise, sensitive neopterin assay which got rid of the ACN interference completely. The MnO2 method of 7,8-dihydroneopterin oxidation did not work with biological samples such as serum or plaque homogenates. Electrochemical detection was also found to be very unreliable and inconsistent.

Hypochlorous acid (HOCl) is a powerful oxidant produced by activated phagocytes at sites of inflammation to kill a wide range of pathogens. Yet, it may also damage and kill the neighbouring host cells. The abundance of dead macrophages in atherosclerotic plaques and their colocalization with HOCl-modified proteins implicate HOCl may play a role in killing macrophages, contributing to disease progression. The first part of this research was to investigate the cytotoxic effect and cell death mechanism(s) of HOCl on macrophages. Macrophages require efficient defense mechanism(s) against HOCl to function properly at inflammatory sites. The second part of the thesis was to examine the antioxidative effects of glutathione (GSH) and 7,8-dihydroneopterin (7,8-NP) on HOCl-induced cellular damage in macrophages. GSH is an efficient scavenger of HOCl and a major intracellular antioxidant against oxidative stress, whereas 7,8-NP is secreted by human macrophages upon interferon-γ (IFN-γ) induction during inflammation and can also scavenge HOCl. HOCl caused concentration-dependent cell viability loss in human monocyte derived macrophage (HMDM) cells above a specific concentration threshold. HOCl reacted with HMDMs to cause viability loss within the first 10 minutes of treatment, and it posed no latent effect on the cells afterwards regardless of the HOCl concentrations. The lack of caspase-3 activation, rapid influx of propidium iodide (PI) dye, rapid loss of intracellular ATP and cell morphological changes (cell swelling, cell membrane integrity loss and rupture) were observed in HMDM cells treated with HOCl. These results indicate that HOCl caused HMDM cells to undergo necrotic cell death. In addition to the loss of intracellular ATP, HOCl also caused rapid loss of GAPDH enzymatic activity and mitochondrial membrane potential, indicating impairment of the metabolic energy production. Loss of the mitochondrial membrane potential was mediated by mitochondrial permeability transition (MPT), as blocking MPT pore formation using cyclosporin A (CSA) prevented mitochondrial membrane potential loss. HOCl caused an increase in cytosolic calcium ion (Ca2+) level, which was due to both intra- and extra-cellular sources. However, extracellular sources only contributed significantly above a certain HOCl concentration. Preventing cytosolic Ca2+ increase significantly inhibited HOCl-induced cell viability loss. This suggests that cytosolic Ca2+ increase was associated with HOCl-induced necrotic cell death in HMDM cells, possibly via the activation of Ca2+-dependent calpain cysteine proteases. Calpain inhibitors prevented HOCl-induced lysosomal destabilisation and cell viability loss in HMDM cells. Calpains induced HOCl-induced necrotic cell death possibly by degrading cytoskeletal and other cellular proteins, or causing the release of cathepsin proteases from ruptured lysosomes that also degraded cellular components. The HOCl-induced cytosolic Ca2+ increase also caused mitochondrial Ca2+ accumulation and MPT activation-mediated mitochondrial membrane potential loss. MPT activation, like calpain activation, was also associated with the HOCl-induced necrotic cell death, as preventing MPT activation completely inhibited HOCl-induced cell viability loss. The involvement of both calpain activation and MPT activation in HOCl-induced necrotic cell death in HMDM cells implies a cause and effect relationship between these two events. HMDM cells depleted of intracellular GSH using diethyl maleate showed increased susceptibility towards HOCl insult compared to HMDM cells with intact intracellular GSH levels, indicating that intracellular GSH played an important role in protecting HMDM cells against HOCl exposure. Intracellular GSH level in each HMDM cell preparation directly correlated with HOCl concentration required to kill 50% of population for each cell preparation, indicating intracellular GSH concentrations determine the efficiency of GSH in preventing HOCl-induced damage to HMDM cells. Intracellular GSH and cell viability loss induced by 400 μM HOCl were significantly prevented by 300 μM extracellular 7,8-NP, indicating that added 7,8-NP is an efficient scavenger of HOCl and out-competed intracellular GSH for HOCl. The amount of 7,8-NP synthesized by HMDM cells upon IFN-γ induction was too low to efficiently prevent HOCl-mediated intracellular GSH and cell viability loss. HOCl clearly causes HMDM cells to undergo necrosis when the concentration exceeds the intracellular GSH concentrations. Above this concentration HOCl causes oxidative damage to the Ca2+ ion channels on cell and ER membranes, resulting in an influx of Ca2+ ions into the cytosol and possibly the mitochondria. The rise in Ca2+ ions triggers calpain activation, resulting in the MPT-mediated loss of mitochondrial membrane potential, lysosomal instability and cellular necrosis.

Intense exercise is known to cause alterations in the psychophysiological status of an athlete. Monitoring the health and recovery of an athlete is imperative for the maintenance of performance and reduced fatigue and injury incidence. The physicality associated with select sports results in significant elevations and suppression of psychophysiological biomarkers that are often modulated by game-related impacts, intense training regimes and psychosocial aspects associated with the professional era. The aim of the studies outlined in this thesis were to determine the effectiveness of selected “stress” markers in several sports that result in significant “stress”, and quantify the level of acute and chronic “stress” following individual games and competitions to improve athlete management and recovery. Study one aimed at developing a new strong-cation exchange high performance liquid chromatography (SCX-HPLC) method for the detection and quantification of urinary pterins and creatinine in a body-building cohort completing high intensity resistance training. The method had an intra- and inter-assay variability of 3.04 % and 5.42 % respectively, with visibly clear peaks and no tailing. Urinary neopterin (NP) and 7,8-dihydroneopterin during a week of competitive natural body-building did not significantly change indicating no alteration in immune system function and oxidative stress. It did provide evidence for the use of specific gravity as a similarly reliable method for urine volume correction following exercise. Study two focused on a playoff game of elite amateur rugby. The time course changes of NP, cortisol, salivary immunoglobulin A (sIgA) and myoglobin in 11 elite amateur rugby players were measured up to 86 hours post-game. Cortisol increased 4-fold, myoglobin 2.85-fold, NP 1.75-fold and total NP 2.3-fold, all significant, whilst sIgA did not change. All markers returned to baseline within 17 hours providing valuable information for sample collection schedule optimization. Respiratory elastance was also measured by ventilation for the assessment of exercise induced lung inflammation/injury following the game (Chapter three). There was an increase in elastance in selected individuals that did not correlate with either global positioning system (GPS) or impact data. It was shown however, that a ventilator is capable of measuring respiratory changes in a conscious and healthy individual. Study three focused on the final three games of professional rugby in the 2013 ITM Cup. The acute and cumulative changes in the same four markers were analysed following three home games. There were significant increases in NP, total NP, cortisol and myoglobin along with significant suppression of sIgA (p < 0.05). Large intra- and inter-individual variation existed between players with changes associated with total impacts. Moreover, impact induced muscle damage may account for changes in oxidative status. Specific gravity (SG) was shown to be a more reliable marker for urine volume correction in comparison to creatinine; while some players showed signs of cumulative stress. Study four examined stress in a professional team throughout the 22 week 2014 Super 15 competition. Part one investigated changes in oxidative stress and muscle damage markers to solidify the muscle damage/oxidative status theory postulated in the previous study. Experimental evidence showed iron and myoglobin are separately capable of oxidizing 7,8-dihydroneopterin to NP in vitro. It was then identified that players who suffered the greatest muscle damage as a result of impacts also had the greatest change in oxidative status (NP). This evidence suggests rugby union induces significant alterations in oxidative status that may be exacerbated by the impact induced release of myoglobin. Part two measured urinary NT-proBNP during the last two consecutive home games to identify whether rugby union causes significant cardiovascular stress and if the pre to post-game change can be explained by GPS technology. Significant individualized elevations were observed in games one and two which did not correlate with any GPS measurements or impacts. Concentrations returned to normal ~ 36 hours post-game suggesting no permanent damage to cardiac muscle had occurred. The lack of correlation suggests GPS technology is not an accurate measure of cardiovascular stress in professional rugby union. Part three involved the measurement of cortisol, total NP and sIgA throughout the season to assess the degree of cumulative stress. Samples were taken at regular intervals ~ 36 hours post-game for 22 weeks. Extreme inter-individual variation was present. Select individuals showed continual elevation in immune system activation and psychophysiological stress, whilst others presented with a continual decline in immune system function. Collectively however, minor deviations from baseline in all markers were observed and participation in long distance travel did not significantly affect the psychophysiological status of the group. Together this suggests a season does not cause an accumulation in psychophysiological stress, although careful individual player analysis is warranted. Understanding rugby union positional demands is essential for training program specification and position specific development of players. Part four used GPS, video-analysis and biochemical analysis to identify positional demands in five regular season games. Forwards tended to be involved in more impacts and covered less distance, while backs covered more distance and carried the ball into contact more regularly. There was no difference in the psychophysiological status between positions indicating both aspects of stress (impacts and distance covered) may induce a similar response. Alternatively, individual biological variation may be solely responsible for this change suggesting careful consideration should be given when using traditional work-load measures such as GPS when quantifying “stress”. Part five assessed the effectiveness of varied recovery interventions. Total NP, cortisol, myoglobin and sIgA were measured pre- post- and ~ 36 hours post game to identify which intervention was most effective at returning players to a psychophysiological state that allowed for the resumption of normal training. Findings concluded the immediate post-game strategy employed by the team (cold bath, consumption of protein and carbohydrates, compression garments and eight hours sleep) seemed to provide the greatest psychophysiological improvement regardless of the “next-day” intervention. There was large inter-individual variation and players were still in a state of recovery ~ 36 hours post-game as indicated by the elevated total NP and sIgA concentrations. Study five had four aspects. Develop a new, cost-effective and simple reverse phase HPLC (RP-HPLC) method for the quantification of urinary myoglobin in a clinically relevant range, quantify the level of structural stress following a simulated mixed martial arts (MMA) contest, determine whether cold water immersion attenuates the level of inflammation and muscle damage following a contest, and whether this hypothesized attenuation may be explained by cryotherapy induced mononuclear cell activation suppression in vitro. The RP-HPLC method had an intra- and inter-assay variations from 0.32 - 2.94 %. Linearity was in the range of 5 – 1000 µg/mL which detected significant increases in urinary myoglobin following the MMA contest. Total NP was found to significantly increase following the contest and return to approximately pre-contest levels 24 hours later for the passive group only. Cold water immersion was further found to attenuate the total NP increase in the first two hours post-contest solidifying its use as a recovery technique following intense exercise, while cryotherapy significantly suppressed T-cell activation. This study provides a reliable and repeatable assay for muscle damage quantification in a clinically relevant range, evidence of the physicality associated with MMA, and indicates cold water immersion is a reliable recovery intervention that may impart its positive benefits through T-cell suppression. The data generated by these investigations highlights the necessity for individual physiological analysis. Group data often masks the extreme variation that exists in clinical and exercise trials where treatment and management of athletes is conducted for recovery and performance. Biochemical analysis provides an added sophistication of work-load and psychophysiological assessment that common technological methods cannot emulate. With a lack of correlation between the quantitative changes in specific non-overlapping biomarkers and GPS, video-analysis and questionnaires, it would seem pertinent to develop a non-invasive quantitative approach in elite sport to understand the level of exercise-induced psychophysiological stress for the precise management of athletes.