Thesis (Ph.D.)--University of Nebraska-Lincoln, 2007. / Title from title screen (site viewed Aug. 1, 2007). PDF text: 292 p. : ill. (some col.) UMI publication number: AAT 3256642. Includes bibliographical references. Also available in microfilm and microfiche formats.

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Homocysteine, an intermediate metabolite biosynthesized from the methionine cycle, is a homologue of cysteine. Homocysteine differs from cysteine by an additional methylene group, which makes it more reactive. Elevated homocysteine level is a risk factor for cardiovascular disease and cerebrovascular disease, brain atrophy, neurodegenerative diseases and cognitive dysfunctions. Recent studies suggest a bi-directional relationship between homocysteine levels and immune-inflammatory activation. Our studies sought to determine if hyperhomocysteinemia affects cell infiltrates in the Central Nervous System (CNS). Inflammatory monocytes recruitment into the CNS and microglia proliferation have been shown in several inflammatory models, and Ly-6Chi CCR2+ monocytes have been shown to be the precursor for microglia. Based on these findings, we hypothesized that hyperhomocysteinemia (HHcy) would alter CNS infiltrate composition. We investigated whether HHcy affected the total mononuclear cells composition in the CNS. We also determined whether HHcy altered the inflammatory monocyte subsets composition in the CNS. In order to determine the effects of HHcy in the CNS mononuclear cells composition, we genotyped the mice, and isolated mononuclear cells from the CNS using percoll gradient method. Then we simultaneously stained the cells with three antibodies, PE-labeled anti-mouse CD11b, PE-Cy5-labeled anti-mouse CD45, and FITC-labeled anti-mouse Ly-6C and analyzed the samples by flow cytometry method. HHcy made no difference in the percentage of lymphocytes, infiltrating monocytes and microglia in the total CNS mononuclear cells, but within infiltrating monocytes, HHcy decreased Ly-6Clo and increased Ly-6Chi subsets. These findings demonstrate that HHcy has effects on the CNS mononuclear cell composition. In summary, HHcy decreased Ly-6Clo and increased Ly-6Chi subsets of infiltrating monocytes in the CNS. There is a potential role of HHcy in increasing inflammatory monocytes infiltration. / Pharmacology

Thesis (Ph. D.)--University of Canterbury, 2006. / Typescript (photocopy). "20-July-2006." Includes bibliographical references. Also available via the World Wide Web.

published_or_final_version / Pharmacology / Master / Master of Philosophy

by Cheng Sau-kwan. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 98-106). / LIST OF TABLES / LIST OF FIGURES / ACKNOWLEDGEMENTS / ABSTRACT --- p.1 / Chapter CHAPTER ONE --- p.3 / Chapter 1.1 --- Introduction --- p.3 / Chapter 1.1.1 --- Sources of homocysteine and origins of deficiency or excess in the human body --- p.3 / Chapter 1.1.2 --- Homocysteine metabolism --- p.4 / Chapter 1.2 --- Causes of and clinical syndromes in homocysteinaemia --- p.12 / Chapter 1.2.1 --- Deficiency of cystathionine β-synthase --- p.12 / Chapter 1.2.1.1a --- Homozygous homocysteinaemia --- p.13 / Chapter 1.2.1.1b --- Heterozygous hyperhomocysteinaemia --- p.17 / Chapter 1.2.2 --- "Deficiency of 5, 10 methylenetetrahydrofolate reductase" --- p.20 / Chapter 1.2.3 --- Defects of cobalamin synthesis --- p.21 / Chapter 1.3 --- Standardised oral methionine load test --- p.23 / Chapter 1.4 --- Treatment and prospects for homocysteinaemia --- p.25 / Chapter 1.4.1 --- Homozygous homocysteinaemia --- p.25 / Chapter 1.4.2 --- Heterozygous homocysteinaemia --- p.27 / Chapter 1.5 --- Pathogenesis of vascular disease in homocystinuria --- p.28 / Chapter 1.6 --- Aim of the study --- p.30 / Chapter CHAPTER TWO --- p.31 / Chapter 2.1 --- Patient's criteria --- p.31 / Chapter 2.2 --- Control' s criteria --- p.32 / Chapter 2.3 --- Exclusion criteria for patients and controls --- p.32 / Chapter 2.4 --- The methionine loading test and additional investigations carried out --- p.33 / Chapter 2.5 --- Statistics used for data analyses --- p.35 / Chapter CHAPTER THREE --- p.38 / Chapter 3.1 --- Sample collection --- p.38 / Chapter 3.2 --- Analytical methods for homocysteine determination --- p.39 / Chapter 3.2.1 --- Cyanide nitroprusside test --- p.39 / Chapter 3.2.2 --- Radioenzymic Assays --- p.40 / Chapter 3.2.3 --- Gas chromatography - Mass spectrometry --- p.41 / Chapter 3.2.4 --- HPLC with Electrochemical detection --- p.42 / Chapter 3.2.5 --- HPLC and postcolumn derivatization --- p.43 / Chapter 3.2.6 --- "Precolumn derivatization, HPLC and fluorescence detection" --- p.44 / Chapter 3.3 --- The method used in this study --- p.47 / Chapter 3.3.1 --- Materials --- p.48 / Chapter 3.3.2 --- Reagents --- p.49 / Chapter 3.3.3 --- Instrumentation --- p.49 / Chapter 3.3.4 --- Sample preparation --- p.50 / Chapter 3.3.4.1 --- Reduction --- p.50 / Chapter 3.3.4.2 --- Derivatization --- p.50 / Chapter 3.3.5 --- Chromatographic conditions --- p.51 / Chapter 3.3.6 --- Standard preparation --- p.51 / Chapter 3.4 --- Method Optimization --- p.52 / Chapter 3.4.1 --- Choice of reducing agent --- p.52 / Chapter 3.4.1.1 --- Dithiotreitol (DTT) --- p.52 / Chapter 3.4.1.2 --- Sodium borohydride --- p.53 / Chapter 3.4.2 --- Choice of precipitating reagent --- p.56 / Chapter 3.4.3 --- Optimization of chromatographic conditions --- p.56 / Chapter 3.4.3.1 --- "Flow rate, temperature and organic composition of mobile phase" --- p.56 / Chapter 3.4.3.2 --- pH of the mobile phase --- p.59 / Chapter 3.4.4 --- Confirmation of homocysteine peak --- p.60 / Chapter 3.5 --- Analysis of results --- p.60 / Chapter 3.6 --- Method validation --- p.60 / Chapter 3.6.1 --- Linearity --- p.60 / Chapter 3.6.2 --- Precision --- p.63 / Chapter 3.6.3 --- Recovery --- p.64 / Chapter CHAPTER FOUR --- RESULTS --- p.66 / Chapter 4.1 --- The pre- and post-methionine loading plasma homocysteine concentrations in patients and controls --- p.66 / Chapter 4.2 --- The frequency distributions of hyperhomocysteinaemia in patients and controls --- p.68 / Chapter 4.2.1 --- The distributions of homocysteinaemia in patients and controls --- p.68 / Chapter 4.2.2 --- The frequency distributions of fasting hyper-homocysteinaemia in patients and controls --- p.68 / Chapter 4.2.3 --- The frequency distributions of post-methionine hyperhomocysteinaemia in patients and controls --- p.73 / Chapter 4.2.4 --- The frequency distributions of the abnormal methionine tolerance in patients and controls --- p.75 / Chapter 4.3 --- "The frequency distributions of hypertension and hyperlipidaemia in controls and, including smoking,in patients without and with hyperhomocysteinaemia" --- p.77 / Chapter 4.3.1 --- The frequency distribution of hypertension and hyperlipidaemia in patients and controls --- p.77 / Chapter 4.3.2 --- "The frequency distributions of hyper-lipidaemia, hypertension, smoking and gender in patients with vascular disease with and without hyperhomocysteinaemia" --- p.79 / Chapter 4.4 --- "The comparison of the age, haematological and biochemical indices and the blood pressure between the patients and controls" --- p.81 / Chapter 4.4.1 --- The comparison of the patients' age at presentation and plasma lipids following recovery from the acute episode with those in controls at the time of methionine loading --- p.81 / Chapter 4.4.2 --- The comparison of the age at presentation and the plasma lipids in patients with and without hyperhomocysteinaemia --- p.83 / Chapter 4.4.3 --- "The comparison of the B12, serum folate and RBC folate in patients and controls at the time of presentation" --- p.84 / Chapter 4.4.4 --- "The comparison of the B12, serum folate and RBC folate levels in patients with and without hyperhomocysteinaemia and in controls" --- p.85 / Chapter CHAPTER FIVE --- DISCUSSION --- p.87 / REFERENCES --- p.98

In periodontitis, there is a disruption in the homeostasis of the oral microbiome by peridontopathogenic bacteria. However, while bacteria is essential for periodontitis to occur, the severity, pattern and progression of the disease is not solely determined by the microbial burden, and in fact has a lot to do with the overwhelming host inflammatory response. The response can vary even in two individuals with similar periodontopathogenic profiles. The host response leads to extracellular matrix (ECM) destruction, loss of attachment, alveolar bone resorption and eventually, edentulism. The host's reaction is orchestrated by proinflammatory cytokines and chemokines and matrix metalloproteinases (MMPs). MMPs are proteolytic enzymes capable of degrading collagen fibers from the extracellular matrix and are the main responsible for tissue damage and gingival recession in periodontitis. As a response to the limitations of the traditional therapies, new agents have been used in preclinical and clinical studies, namely host-modulatory agents, including anti-proteinase agents, anti-inflammatory agents and anti-resorptive agents. Focusing on changing the inflammatory process, as opposed to the microbial insult, can slow down the disease progression, improve clinical outcomes and even prevent tooth loss in severely compromised patients. This work examines the role of pro-inflammatory markers homocysteine in chronic inflammation and periodontitis. Homocysteine (Hcy) is a non-protein amino acid derived from the metabolism of the essential amino acid methionine via methyl group metabolism. Controlling Homocysteine as a potential inductor of MMPs, and hence of tissue destruction, can lead to new adjuvant therapies to improve clinical outcomes and prevent activation of the disease

Three biologically-active aminothiols cysteamine (CA), DL-cysteine (CYSH) and DL-homocysteine, were studied in this thesis. These aminothiols react with nitrous acid (HNO2), prepared in situ, to produce S-nitrosothiols (RSNOs): S-nitrosocyteamine (CANO), S-nitrosocysteine (CYSNO) and S-nitrosohomocysteine (HCYSNO). They also react with S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) through a transnitrosation reaction to produce their corresponding RSNOs. A detailed kinetics and mechanistic study on the formation of these RSNOs and their subsequent decomposition to release nitric oxide (NO) were studied. For all three aminothiols the stoichiometry of their reaction with nitrous acid is strictly 1:1 with the formation of one mole of RSNO from one mole of HNO2. In all cases, the nitrosation reaction is first order in nitrous acid, thus implicating it as a nitrosating agent in mildly acidic pH conditions. Acid catalyzes nitrosation after nitrous acid has saturated, implicating another nitrosating agent, the nitrosonium cation, NO+ ( which is produced from the protonation of nitrous acid) as a contributing nitrosating species in highly acidic environments. The acid catalysis at constant nitrous acid concentrations suggests that the nitrosonium cation nitrosates at a much higher rate than nitrous acid. Nitric oxide itself was not detected as a nitrosant. Bimolecular rate constants for the nitrosation of CA, CYSH and HCYSH were deduced to be 17.9, 6.4, 0.09 M-1 s-1 for the nitrosation by nitrous acid and 8.25 x 1010, 2.89 x 1010 and 6.57 x 1010 M-1 s-1 for the nitrosation by nitrosonium cation respectively. A linear correlation was obtained between the rate constants and the pKa of the sulfur center of the aminothiols for nitrosation by NO+. The stabilities of the three RSNOs were found to be affected by metal ions. They were unstable in the presence of metal ions, with half-lives of few seconds. However, in the presence of metal ion chelators, they were found to be relatively stable with half-lives of 10, 30 and 198 hours for CYSNO, CANO and HCYSNO respectively. The relative stability of HCYSNO may be an advantage in the prevention of its metabolic conversion to homocysteine thiolactone, the major culprit in HCYSH pathogenesis. This dissertation has thus revealed new potential therapeutic way for the modulation of HCYSH related cardiovascular diseases.

Several studies have found a positive correlation between hypertension and hyperhomocysteinemia. Increasing evidence implicates oxidative stress as one of the initiating events closely linked to the homocysteines ability to damage endothelium, subsequently causing vascular dysfunction. We previously found that heparin protects cultured endothelial cells from free radical injury and oral heparin at 1 mg/kg/48h prevents venous thrombosis in a rat model in vivo. Our objective was to study the protective effects of oral heparin in a rat model with elevated plasma homocysteine (Hcy) concentrations, and begin to elucidate whether the pathophysiological effects of Hcy are mediated through an oxidative mechanism causing endothelial dysfunction.<p> Elevated plasma Hcy levels were induced by feeding male Wistar Kyoto rats a diet containing an additional 1.7% methionine for 8 weeks. Groups included rats fed additional methionine, methionine plus oral heparin (1 mg/kg/48h by gastric feeding tube), and age-matched controls fed normal rat chow. At the end of 8 weeks of treatment, rats were anesthetized using 1.5% isoflurane in 100% oxygen. Hemodynamics parameters were assessed by inserting a Millar Mikro Tip pressure transducer into the left ventricular chamber and the thoracic aorta. Fasting plasma total Hcy levels were measured using a Hcy immunoassay kit with an Abbott IMx instrument. Malondialdehyde (MDA) concentrations, a lipid peroxidation product and marker for oxidative stress, was measured by a spectrophotometric method in serum and tissue samples. Glutathione (GSH) concentrations, an important antioxidant for low-level oxidative stress was measured by HPLC in plasma and tissues samples. Lastly, tissue samples from each experimental group were stained with the TUNEL method to assess their respective percentage of apoptotic endothelial cells. Results were expressed as mean ± S.E. Unpaired Students two-tailed t-test was employed to assess the difference between groups with p < 0.05 considered significant.<p> Plasma Hcy was significantly elevated after 8 weeks in the methionine (7.17 ± 0.46 umol/L) and methionine plus heparin treated rats (7.02 ± 0.40 umol/L) compared to control (5.46 ± 0.36 umol/L). All measures of arterial pressure, systolic (SP) and diastolic pressure (DP) and mean arterial pressure (MAP), were significantly elevated in rats fed the methionine diet without heparin (119.9 ± 3.9 mmHg; 90.3 ± 3.5 mmHg; 97.7 ± 2.9 mmHg, respectively) compared to controls (107.8 ± 2.5 mmHg; 79.2 ± 2.1 mmHg; 88.8 ± 2.2 mmHg, respectively) but not compared to heparin (114.7 ± 3.3 mmHg; 83.4 ± 2.4 mmHg; 93.8 ± 2.7 mmHg, respectively). Left ventricular end diastolic pressure (LVEDP) was significantly elevated with the methionine diet without heparin (14.2 ± 2.5 mmHg) but not with heparin treatment (8.4 ± 1.9 mmHg) versus controls (7.1 ± 1.1 mmHg). Also, left ventricular systolic pressure (LVSP) was significantly elevated in the methionine fed rats after 8 weeks (122.6 ± 3.2 mmHg) compared to controls (112.3. ± 2.9 mmHg). Heparin treatment had no effect on LVSP (119.9 ± 3.2 mmHg). <p> Additionally, the results of this study showed that oral heparin treatment significantly decreased liver MDA concentrations (2.42 ± 0.28 nmol/mg protein) compared to the methionine treated group (5.10 ± 0.96 nmol/mg protein) and methionine treatment alone significantly reduced MDA concentrations in kidney tissue (1.59 ± 0.12 nmol/mg protein) compared with controls (3.26 ± 0.66 nmol/mg protein). Methionine diet significantly decreased GSH concentrations in plasma (0.59 ± 0.59 µmol/L) compared with controls (4.24 ± 0.94 µmol/L) and oral heparin treatment significantly attenuated the decrease in GSH concentrations in left ventricle tissue samples (0.0229 ± 0.0023 µmol/mg protein) compared with methionine treatment alone (0.0135 ± 0.0016 µmol/mg protein). <p> Elevated plasma homocysteine levels, induced by methionine diet feeding significantly increased the percent of apoptotic endothelial cells in the aortas (17.04 ± 3.74%) and superior mesenteric arteries (17.99 ± 1.90%) of WKY rats compared with control aortas and mesenteric arteries (6.08 ± 3.24%; 7.43 ±1.62%, respectively) and compared to oral heparin treated mesenteric arteries (7.31 ± 1.18%). <p> The results of this study showed that elevated plasma levels of Hcy correlate with the development of hypertension, defined as significantly increased arterial pressure. Oral heparin treatment prevented the significant increase in arterial pressures and LVEDP, decreased MDA concentrations and therefore the oxidative stress on the liver, attenuated the decrease caused by elevated plasma Hcy in left ventricle GSH concentrations, and significantly reduced the number of apoptotic endothelial cells in the superior mesenteric artery of high methionine fed rats. We conclude that elevated levels of plasma Hcy contributes to the development of hypertension and furthermore towards the onset of heart failure likely through an oxidative mechanism and that oral heparin reduces the overall oxidative stress in specific physiological environments, preventing Hcy mediated endothelial cell apoptosis.

Several studies have found a positive correlation between hypertension and hyperhomocysteinemia. Increasing evidence implicates oxidative stress as one of the initiating events closely linked to the homocysteines ability to damage endothelium, subsequently causing vascular dysfunction. We previously found that heparin protects cultured endothelial cells from free radical injury and oral heparin at 1 mg/kg/48h prevents venous thrombosis in a rat model in vivo. Our objective was to study the protective effects of oral heparin in a rat model with elevated plasma homocysteine (Hcy) concentrations, and begin to elucidate whether the pathophysiological effects of Hcy are mediated through an oxidative mechanism causing endothelial dysfunction.<p> Elevated plasma Hcy levels were induced by feeding male Wistar Kyoto rats a diet containing an additional 1.7% methionine for 8 weeks. Groups included rats fed additional methionine, methionine plus oral heparin (1 mg/kg/48h by gastric feeding tube), and age-matched controls fed normal rat chow. At the end of 8 weeks of treatment, rats were anesthetized using 1.5% isoflurane in 100% oxygen. Hemodynamics parameters were assessed by inserting a Millar Mikro Tip pressure transducer into the left ventricular chamber and the thoracic aorta. Fasting plasma total Hcy levels were measured using a Hcy immunoassay kit with an Abbott IMx instrument. Malondialdehyde (MDA) concentrations, a lipid peroxidation product and marker for oxidative stress, was measured by a spectrophotometric method in serum and tissue samples. Glutathione (GSH) concentrations, an important antioxidant for low-level oxidative stress was measured by HPLC in plasma and tissues samples. Lastly, tissue samples from each experimental group were stained with the TUNEL method to assess their respective percentage of apoptotic endothelial cells. Results were expressed as mean ± S.E. Unpaired Students two-tailed t-test was employed to assess the difference between groups with p < 0.05 considered significant.<p> Plasma Hcy was significantly elevated after 8 weeks in the methionine (7.17 ± 0.46 umol/L) and methionine plus heparin treated rats (7.02 ± 0.40 umol/L) compared to control (5.46 ± 0.36 umol/L). All measures of arterial pressure, systolic (SP) and diastolic pressure (DP) and mean arterial pressure (MAP), were significantly elevated in rats fed the methionine diet without heparin (119.9 ± 3.9 mmHg; 90.3 ± 3.5 mmHg; 97.7 ± 2.9 mmHg, respectively) compared to controls (107.8 ± 2.5 mmHg; 79.2 ± 2.1 mmHg; 88.8 ± 2.2 mmHg, respectively) but not compared to heparin (114.7 ± 3.3 mmHg; 83.4 ± 2.4 mmHg; 93.8 ± 2.7 mmHg, respectively). Left ventricular end diastolic pressure (LVEDP) was significantly elevated with the methionine diet without heparin (14.2 ± 2.5 mmHg) but not with heparin treatment (8.4 ± 1.9 mmHg) versus controls (7.1 ± 1.1 mmHg). Also, left ventricular systolic pressure (LVSP) was significantly elevated in the methionine fed rats after 8 weeks (122.6 ± 3.2 mmHg) compared to controls (112.3. ± 2.9 mmHg). Heparin treatment had no effect on LVSP (119.9 ± 3.2 mmHg). <p> Additionally, the results of this study showed that oral heparin treatment significantly decreased liver MDA concentrations (2.42 ± 0.28 nmol/mg protein) compared to the methionine treated group (5.10 ± 0.96 nmol/mg protein) and methionine treatment alone significantly reduced MDA concentrations in kidney tissue (1.59 ± 0.12 nmol/mg protein) compared with controls (3.26 ± 0.66 nmol/mg protein). Methionine diet significantly decreased GSH concentrations in plasma (0.59 ± 0.59 µmol/L) compared with controls (4.24 ± 0.94 µmol/L) and oral heparin treatment significantly attenuated the decrease in GSH concentrations in left ventricle tissue samples (0.0229 ± 0.0023 µmol/mg protein) compared with methionine treatment alone (0.0135 ± 0.0016 µmol/mg protein). <p> Elevated plasma homocysteine levels, induced by methionine diet feeding significantly increased the percent of apoptotic endothelial cells in the aortas (17.04 ± 3.74%) and superior mesenteric arteries (17.99 ± 1.90%) of WKY rats compared with control aortas and mesenteric arteries (6.08 ± 3.24%; 7.43 ±1.62%, respectively) and compared to oral heparin treated mesenteric arteries (7.31 ± 1.18%). <p> The results of this study showed that elevated plasma levels of Hcy correlate with the development of hypertension, defined as significantly increased arterial pressure. Oral heparin treatment prevented the significant increase in arterial pressures and LVEDP, decreased MDA concentrations and therefore the oxidative stress on the liver, attenuated the decrease caused by elevated plasma Hcy in left ventricle GSH concentrations, and significantly reduced the number of apoptotic endothelial cells in the superior mesenteric artery of high methionine fed rats. We conclude that elevated levels of plasma Hcy contributes to the development of hypertension and furthermore towards the onset of heart failure likely through an oxidative mechanism and that oral heparin reduces the overall oxidative stress in specific physiological environments, preventing Hcy mediated endothelial cell apoptosis.