Thesis (Ph. D.)--University of Sydney, 2009. / Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Discipline of Pathology, Faculty of Medicine. Title from title screen (viewed Sept. 30, 2009) Includes bibliography. Also available in print form.

Two studies were conducted to determine how HDL cholesteryl ester and apoprotein catabolism might contribute to the observed hypercholesterolemia of copper-deficient rats. Weanling male Sprague-Dawley rats were divided into two dietary treatments; copper-adequate (control, 5-7 mg Cu/kg diet) and copper-deficient (0.6-0.8 mg Cu/kg diet). Deionized water and diet were provided ad libitum. Dietary copper deficiency resulted in enlarged intravascular pools of HDL cholesteryl esters and total protein. HDL were isolated from rats of both treatment groups, radiolabeled, and injected into animals of the respective groups. In Study I, HDL apoproteins were labeled by iodination, whereas HDL in Study II were doubly labeled by additionally incorporating into the particle core [³H]cholesteryl linoleyl ether, which served as a nondegradable analog of HDL cholesteryl ester. At specific time intervals up to 12 hours after injection, blood and tissue samples were removed and analyzed for radioactivity. Plasma disappearance curves indicated that HDL cholesteryl esters were preferentially catabolized 1.6-fold faster than HDL protein in controls and 2.5-fold faster in copper-deficient animals. Clearance of individual apoproteins did not occur at significantly different rates in either treatment group. Absolute mass removal of HDL cholesteryl ester and total protein from the plasma was significantly increased in copper-deficient rats. Virtually all of the increased removal of HDL cholesteryl ester was attributed to the liver, whereas most of the increased uptake of HDL protein was attributed to the bulk tissues and not the liver. Since previous studies indicate that copper deficiency may not result in increased cholesterol excretion, these data suggest that cholesteryl esters delivered to the liver of copper-deficient rats are possibly reassembled into new HDL particles at an increased rate. The observed hypercholesterolemia in this animal model, then, appears to be the result of an imbalance in the net flux of cholesterol between the tissues and the plasma.

M.Phil. / Numerous studies have demonstrated the favourable effects of aerobic training on blood lipid profiles. However, few studies have generated conclusive data on the effects of dynamic resistance training (DRT) on blood lipid profiles. In order to evaluate the effect of DRT on lipoprotein-lipid profiles, a group of 28 sedentary but healthy males (mean age 28 years and 7 months) were matched and randomly assigned into a control/non-exercising (n = 15) or an experimental (n = 13) group. To control for variations in lipoprotein-lipid profiles, the present investigation recorded dietary intake and smoking behaviour in an attempt to account for any changes in lipoprotein-lipid profiles over the eight-week period. The experimental group (EG) exercised using DRT for a period of eight weeks and was monitored for changes in lipoprotein-lipid profiles. The control group (CG) took part in no structured exercise throughout the eight-week period. The experimental training programme consisted of nine exercises (dumbbell (D/B) shoulder shrugs, D/B lateral shoulder raises, seated chest press, latissimus dorsi pulldowns, seated pulley rows, biceps curls, triceps extensions, crunchies and unilateral leg press). These exercises were performed at 60% of one repetition maximum (1-RM) and were performed three times per week on non-consecutive days. Serum was analyzed for total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C). In addition to this, the TC: HDL-C and LDL-C: HDL-C ratios were calculated. The Independent t-Test and the Paired t-Test were utilized to determine the significance (at a 95% confidence level (p ¡Ü 0.05)) of the lipoprotein-lipid profile changes from pre- to post-test. These student t-Tests demonstrated no statistically significant changes in TC, TG, LDL-C, HDL-C, TC: HDL-C ratios and LDL-C: HDL-C ratios in the EG. However, the present investigation did demonstrate the following changes: a 0.50% decrease in TC, a 1.74% increase in TG, a 2.95% decrease in LDL-C, a 4.61% increase in HDL-C, a 4.12% decrease in the TC: HDL-C ratio and a 5.96% decrease in the LDL-C: HDL-C ratio. The lack of statistically significant changes in the individual lipoprotein-lipid parameters could not have been affected by diet, cigarettes smoked daily, aerobic fitness and/or body mass, since these parameters did not change significantly from pre- to post-test. Specifically, both the EG and CG demonstrated no statistically significant changes in intake in total calories consumed, carbohydrates, proteins, fats (monounsaturated, polyunsaturated and saturated fatty acids), cholesterol and fibre. Although the present investigation findings suggest that this study¡¯s eight-week combination of dose, workload, number of repetitions and order and number of exercises may not have been sufficient to elicit significant improvements in lipoprotein-lipid parameters in this population of sedentary but healthy males, it is the opinion of the author that DRT should be included with aerobic modes of exercise. DRT should be used in conjunction with aerobic modes of exercise for its additional benefits. Such additional benefits include inter alia: increased strength, increased lean tissue mass, increased maintenance of metabolically active tissue in the elderly and increased muscle control. / Prof. J.M. Loots Mr. L. Lategan

Les macrophages jouent un rôle clé dans l'athérosclérose. Après la captation massive des LDL oxydées (oxLDL), les macrophages sous-endothéliaux sont chargés en cholestérol et se transforment ainsi en cellules spumeuses qui contribuent à la formation de la plaque d'athérome. Les oxystérols, produits d'oxydation du cholestérol, sont retrouvés en quantité importante dans les oxLDL. Au niveau cellulaire, ils sont impliqués dans la régulation de l'homéostasie du cholestérol, l'induction du stress oxydatif cellulaire et de la cytotoxicité. Notre travail montre que le cholestérol, associé aux LDL et d'origine cellulaire, est fortement oxydé par les macrophages lors d'une exposition aux oxLDL. Les principaux produits d'oxydation sont le 7-cétocholesterol et 7α/β-hydroxycholestérol. De plus, nous démontrons que ces oxystérols sont exportés hors des macrophages via les HDL, mais pas l'apoA1. Nous avons aussi caractérisé les oxystérols formés dans les HDL suite à des modifications oxydatives et les HDL issues de patients diabétiques. Nous avons en outre montré que ces modifications sont associées à une diminution de la capacité des HDL à exporter les oxystérols. / Macrophages play a key role in atherosclerosis. After massive uptake of oxidized LDL (oxLDL), subendothelial macrophages are overloaded with cholesterol thereby leading to the formation of foam cells, which is one characteristic of atherogenesis. Oxysterols, the oxidation products of cholesterol, are one of major components of oxLDL; they are involved in the regulation of cholesterol homeostasis, induction of cellular oxidative stress and cytotoxicity. Our works show that both LDL derived-cholesterol and cellular cholesterol can be strongly oxidized in human THP1 and murine RAW macrophages, especially during exposure of oxLDL. The major oxidative products are 7-ketocholesterol and 7α/β-hydroxycholesterol. Moreover, we demonstrate that both oxysterols derived from LDL cholesterol and cellular cholesterol can be exported to HDL, whereas not to apoA1. Then, we studied the functionality of modified HDL and diabetic HDL on oxysterols efflux. A decrease of oxysterols efflux was observed with oxidized and glycoxidized HDL. Compared to the HDL of healthy controls, the HDL of diabetic subjects are less efficient to efflux oxysterols. Taken together, the increased production of oxysterols in presence of oxLDL and their lower efflux by modified HDL support the detrimental role of these oxidative compounds in pathophysiological conditions like diabetes.

[Truncated abstract] The metabolic syndrome is characterized by cardiovascular risk factors including dyslipidemia, insulin resistance, visceral obesity, hypertension and diabetes. The dyslipidemia of the metabolic syndrome includes elevated plasma triglyceride and apolipoprotein (apo) B levels, accumulation of small, dense low-density lipoprotein (LDL) particles and low high-density lipoprotein (HDL) cholesterol concentration. However, the precise mechanisms for this dyslipoproteinemia, specifically low plasma HDL cholesterol, are not well understood. This thesis therefore, focuses on HDL, its structure, function and metabolism. However, lipoprotein metabolism is a complex interconnected system, which includes forward and reverse cholesterol transport pathways. Hence, this thesis also examines and discusses the metabolism of apoB-containing lipoproteins. This thesis tests the general hypothesis that apolipoprotein kinetics are altered in the metabolic syndrome, and that lipid regulating therapies can improve these kinetic abnormalities. The aims were first, to compare and establish the clinical, metabolic and kinetic differences between metabolic syndrome and lean subjects; and second, to determine the regulatory effects of statin therapy, specifically, rosuvastatin on lipoprotein transport in the metabolic syndrome. Five observation statements were derived from the general hypothesis and examined in the studies described below. The findings are presented separately as a series of original publications. Study 1 Twelve men with the metabolic syndrome and ten lean men were studied in a case-control setting. ... These findings explain the HDL raising effects of rosuvastatin in the metabolic syndrome. Collectively, these studies suggest that the dyslipidemia of the metabolic syndrome results from increased production rates of VLDL and LDL particles, reduced fractional catabolic rates of these lipoproteins, together with accelerated catabolism of HDL particles. Treatment with rosuvastatin increases the catabolic rates of all apoB-containing lipoproteins and at a higher dose, decreases LDL apoB production. These effects are consistent with inhibition of cholesterol synthesis leading to an upregulation of LDL receptors. Rosuvastatin decreases the fractional catabolism of HDL particles. The effects of rosuvastatin on HDL kinetics may be related to a reduction in triglyceride concentration and cholesterol ester transfer protein activity. These findings are consistent with the general hypothesis that apolipoprotein kinetics are altered in the metabolic syndrome, and that statin therapy improves these kinetic abnormalities.

The physiological role of LCAT has been the subject of a number of recent articles (Glomset, 1979; Nilsson-Ehle et al., 1980). According to most current theories, the enzyme functions in combination with high-density lipoproteins in the reverse cholesterol transport pathway which presumably returns peripheral cholesterol to the liver where cholesterol catabolism takes place. Despite the exciting potential for studies on the catalytic function and the nature of the enzyme-substrate complex, the mechanism of action of LCAT remains largely unexplored. The relatively slow progress in the elucidation of the LCAT reaction mechanism is likely to be due to the difficulties in the isolation of the enzyme in sufficient quantities. Consequently, considerably less is known about the physical and chemical properties of the enzyme. Therefore, the first objective of this investigation was to isolate and purify sufficient amount of enzyme for subsequent characterization studies. The second objective of this investigation was to characterize the physical properties of the enzyme by techniques including analytical ultracentrifugation, ultraviolet spectroscopy, and circular dichroism and fluorescence spectroscopy. The third objective of this investigation was to characterize the chemical properties of the enzyme which deals with the amino acid and carbohydrate composition and with some basic structural features that are related to the chemical composition of LCAT.

Total and high-density lipoprotein cholesterol (TC and HDL-C) and TC/HDL-C ratio were compared in 30 adult male recreational swimmers and 21 sedentary controls. Percentage of body fat, number of cigarettes smoked daily, and daily alcohol consumption were assessed for both groups. Maximum workout heartrate, weekly swim duration and weekly swim distance of the swimmers were also measured. Maximum workout heartrate (mean ± S. D. ) was 140 ± 24 beats per minute . Mean weekly swim duration was 142 ± 84 minutes, and mean weekly swim distance was 5317 ± 3217 yards. Swimmers and controls were nonsignificantly different in age, number of cigarettes smoked daily, and percent body fat. In this sample, the swimmers consumed significantly higher levels of alcohol than the non-swimmers. TC and HDL-C concentrations of swimmers were not significantly different than controls, (204 vs 199 mg/dl, and 48 vs 47 mg/dl, respectively). TC/HDL-C ratio of swimmers was 4.69, while that of controls was 4.65. This study showed that adult male recreational swimmers who train at low intensity do not differ significantly in total and HDL-C or TC/HDL-C ratio from male sedentary controls. / M.S.

Research has reported that increased levels of plasma TC are directly related, while low levels of plasma HDL-C are inversely related, to coronary heart disease. Regular physical exercise has been suggested as a method for reducing plasma TC and increasing plasma HDL-C. Thirty-one healthy, sedentary women (ages 18-30) were studied to determine the effects of a jogging, weight training, or a combined jogging and weight training program on plasma total cholesterol, high-density lipoproteins, body composition. Experimental subjects were randomly assigned to the treatment conditions. The subjects trained three days a week for nine weeks. The R group ran for 30 minutes a session at 75% predicted maximum HR. The W group trained with weights utilizing exercises to strengthen all major muscle groups for one hour at 60% one repetition maximum the first 3 weeks and 75% one repetition maximum weeks 4 - 9. The RW group ran for 25 minutes a session at 75% predicted maximum HR, then lifted weights using the leg-strengthening exercises for 30 minutes, similar to the W group. Preceding and following the treatment period, plasma TC, HDL-C, body weight, and percent body fat was assessed for all four groups. Plasma TC was not significantly altered, although a downward trend was observed for all three treatment groups. Plasma HDL-C did not change over the treatment period for any group. The plasma TC/HDL-C ratio changed significantly among groups over the treatment period, with the R group decreasing their ratio from 3.5 to 2.9 (p < .05). No changes were noted In percent body fat, fat-free mass, or body weight for any of the groups. The Pearson product-moment correlations performed between the changes in blood lipids and the changes in body composition found no significant relationships. The results of this study indicate that an exercise program consisting of endurance training for 30 minutes, 3 times per week, or weight training for one hour, 3 times per week, or a combination aerobic/weight training program 3 times per week is not adequate to significantly improve plasma TC or HDL-C in young females over a nine week period. However, significant improvements may be made in the plasma TC/HDL-C ratio which may decrease the risk for CHD. / Master of Science

Recent research has shown that the combination of high triglyceride (TG) levels and low high density lipoprotein (HDL) levels, significantly increases the incidence of coronary artery disease (CAD). The incidence of CAD is also increased in abdominally obese individuals. To assess differences in postprandial TG clearance patterns between abdominally obese (AO) and controls (C), fourteen healthy, normolipidemic males (seven controls and seven abdominally obese) completed an oral fat loading test (78 grams of fat). Blood samples were collected every hour for eight hours. Abdominally obese individuals had significantly greater TG values, significantly lower total HDL and HDL2 values and significantly greater area under the TG curve (p = 0.03). Time to reach peak TG and time to reach baseline TG values did not differ between the two groups, even though fewer AO individuals reached baseline within eight hours. The data from the present investigation indicate that increased time to clear TG in AO individuals may be one pathway that increases the incidence of CAD in this group. / School of Physical Education

Enzymatic and lipid transfer reactions involved in reverse cholesterol transport were studied in HDL deficient plasma systems. Fasting plasma samples were obtained from control and cholesterol fed guinea pigs as well as from a fish eye disease patient and were used to localize the enzyme LCAT among plasma lipoproteins (VLDL, LDL, and HDL). In both guinea pig and fish eye disease patient plasma, the LCAT activity was found in association with the HDL type particles. Cholesterol feeding in guinea pigs altered the properties of lipoprotein substrates for LCAT resulting in some changes, specifically: 1) decreased fractional rate of plasma cholesterol esterification and, 2) lower transfer of free cholesterol (FC) and esterified cholesterol (CE) within the lipoprotein fractions.