Elevated levels of homocysteine (HCy) are a known risk factor in several disease states (1). HCy has several other metabolites, homocysteine thiolactone (HCy-T) and homocysteic acid (HCA). Whilst HCy-mediated neurotoxicity has been extensively studied, the underlying mechanisms of HCy-T and HCA mediated neuronal damage remain largely unknown. This thesis aims to explore the underlying mechanisms, triggered by HCy and metabolites which result in neuronal cell death, and may be appropriate targets for future research on disease-modifying interventions in neurodegenerative disorders. As ageing is the greatest risk factor for neurodegeneration, a novel model of human neuronal ageing was established, permitting investigation of the pathways triggered by HCy in ageing. Using SH-SY5Y cells, a novel differentiation protocol was established and categorised, once fully differentiated, these cells were shown to be fully functional neurons and could be maintained for a month in culture. Using a range of concentrations of HCy and HCy-T, the concentration cell death occurs at was determined using crystal violet and lactate dehydrogenase assays. Mechanisms of toxicity were determined using pharmacological intervention at the NMDA receptor, nitric oxide scavengers and antioxidants. Using a combination of immunocytochemistry, live cell imaging and ELISA, alterations in markers of cell damage could be examined. The results showed HCy and HCy-T have distinct mechanisms of toxicity. Whilst both are neurotoxic, HCy directly acts via the NMDA receptor, however HCy-T appears to be less potent. Additionally, HCy-T caused a greater increase in reactive oxygen species generation than HCy, and each metabolite also displayed distinct mitochondrial network abnormalities. Finally, using the long-term culture methods, the chronic effects of HCy, HCy-T and HCA were examined. However, extensive cell death was apparent at low doses in all metabolites therefore no definitive mechanisms could be determined. This culture method was deemed not appropriate for toxicity experiments.

M. Tech (Department of Biotechnology, Faculty of Applied and Computer Sciences) Vaal University of Technology. / Background: Cardiovascular diseases are one of the most common causes of death worldwide. This is not only a problem in developed countries, it is of major concern for public health in developing countries as well. Increased homocysteine is an independent risk factor for cardiovascular diseases. Nutritional deficiencies of folate, vitamin B6 and vitamin B12 are associated with hyperhomocysteinemia. MTHFR A1298C, a single nucleotide polymorphism, is similarly linked with higher concentrations of homocysteine. The aim of this study was to determine the prevalence of MTHFR A1298C in a black elderly population, along with folate, vitamin B6 and vitamin B12 and to evaluate the effect on homocysteine levels. Methodology: The research design was an observational cross-sectional study and was ethically approved. A total of 84 elderly who attend a day-care centre (also met inclusion criteria) were purposively selected. DNA was extracted and frozen on the day of blood collection. The MTHFR A1298C genotype was determined with real time PCR. Homocysteine, folate, vitamin B6 and vitamin B12 serum levels were detected with commercial assay kits. Results: Homocysteine was found to be elevated with a median of 17.78 µmol/L (interquartile range 13.98-21.03 µmol/L). Serum folate, vitamin B6 and vitamin B12 medians were in the normal range. Although, 5.95% and 22.62% of the population were deficient and possibly deficient for vitamin B12, respectively. MTHFR A1298C frequency was as follow: 89.29% (AA), 9.52% (AC) and 1.19% (CC), with no significant correlation (p>0.05) with homocysteine. Vitamin B12 correlated significantly with homocysteine levels. Conclusion: Vitamin B12 deficiency had an effect on homocysteine levels. Overall, nutritional deficiencies are not responsible for the hyperhomocysteinemia in this population. In conclusion from this study showed MTHFR A1298C frequency in black South Africans does not contribute to homocysteine as a risk factor for cardiovascular disease. Keywords: Cardiovascular disease, elderly, folate, homocysteine, MTHFR A1298C, vitamin B6, vitamin B12.

[Truncated abstract] An elevated total plasma homocysteine concentration (tHcy) is associated with an increased risk of myocardial infarction and ischemic stroke. Folic acid, vitamin B6 and B12 supplements significantly reduce tHcy even in people who are not overtly vitamin deficient. If homocysteine is a causal risk factor for atherothrombotic events, treatment with B-vitamins might prove a simple and cost-effective means to reduce cardiovascular risk. However, it remains unclear whether elevated tHcy causes atherosclerosis or is simply a risk marker. To prove that homocysteine is a modifiable risk factor for cardiovascular disease it is necessary to show that lowering tHcy reduces vascular risk. The aim of this study was to determine whether long-term homocysteine-lowering with B-vitamins would improve vascular structure and function in people with a history of stroke. This study was a cross-sectional sub-study of the Vitamins TO Prevent Stroke trial (VITATOPS), a multi-centre, randomised, double-blind, placebo-controlled clinical trial designed to test the efficacy and safety of B-vitamins (folic acid 2mg, vitamin B6 25mg and vitamin B12 0.5mg) in the prevention of vascular events in patients with a recent history of stroke or transient ischemic attack. 173 VITATOPS participants were recruited for the current study. Age, sex, stroke type, medications, cardiovascular risk factors and smoking history were recorded and blood pressure, height, weight, waist and hip girth were measured in all subjects at least two years after randomisation. ... After a mean treatment period of 3.9 ± 0.9 years, the subjects randomised to vitamin treatment had significantly lower tHcy than the subjects randomised to placebo (7.9mol/L, 95%CI 7.5, 8.4 versus 11.8mol/L, 95%CI 10.9, 12.8; p<0.001). There were no significant differences between groups in CIMT (0.84 ± 0.17mm vitamins versus 0.83 ± 0.18mm placebo; p=0.74) or FMD (median of 4.0%, IQR 0.9, 7.2, vitamins versus 3.0%, IQR 0.6, 6.6 placebo; p=0.48). Pooled estimates from the meta-analyses showed that B-vitamin treatment reduces CIMT by 0.10mm (95%CI –0.20, -0.01mm) and increases FMD by 1.4%, (95%CI 0.7, 2.2), although these estimates may have been influenced by positive publication bias. The improvement in FMD was significant in studies of less than eight weeks duration but not in studies with longer treatment periods. The association between tHcy and CIMT and FMD was eliminated by adjustment for renal function and long-term B-vitamin treatment did not alter the strong linear relationship between tHcy and cystatin C. Lowering tHcy did not alter arterial wall inflammation assessed by 18FDG-PET, although small subject numbers meant we were unable to exclude a minor treatment effect. Long-term homocysteine-lowering with B-vitamin treatment did not improve CIMT or FMD or reduce arterial wall inflammation in people with a history of stroke. The relationship between tHcy and these markers of vascular risk was eliminated by adjustment for renal function. Our data are consistent with the hypothesis that elevated tHcy is a risk marker for cardiovascular disease rather than a modifiable causal risk factor.