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High-density lipoprotein (HDL) with its cardio- protective effects has provided remarkable optimism to the ever-increasing incidences of coronary artery disease. Therefore, the aim of this randomized, comparative, research trial was to determine whether endurance exercise training, weight training and/or a combination of aerobic and weight training can be utilized in the management of high-density lipoprotein cholesterol (HDL-C). Subsequent to the 16-week intervention period, dependant t-Tests revealed that the non-exercising and weight training groups demonstrated non- significant mean 1.3% (p = 0.754) and 11.1% (p = 0.069) increases in fasting serum HDL-C, respectively. Conversely, there was a significant increase in HDL-C following the 16 weeks of endurance training (p = 0.003) and combination training (p = 0.005) (22.4% and 37.9%, respectively). Further, Spearman’s rho indicated no correlations between HDL-C and BMI (r = -0.131), percentage body fat (r = - 0.141), cholesterol intake (r = - 0.026) and total fat intake (r = - 0.239). The absence of changes in these inter-correlations indicated that changes in these parameters had no effect on the HDL-C. On the contrary, moderate correlations were established between HDL-C and number of cigarettes smoked daily (r = - 0.344) and intake of saturated fat (r = - 0.317) indicating that exercise effect on these variables could have indirectly contributed significantly in altering HDL- C in the endurance and combination training groups. As such, endurance and combination training can be utilized as an effective method in the management of HDL-C in sedentary male smokers.

The high density lipoprotein (HDL) receptor Scavenger Receptor, Class B, Type I (SRBI) is a 509 amino acid integral membrane protein which has been shown to have an important role in HDL-mediated reverse cholesterol transport. SR-BI has been shown to mediate selective uptake of cholesterol, and also mediates efflux of cholesterol to HDL as seen in in vitro cell culture studies. SR-BI is abundant in the liver and steroidogenic tissues, and is also present in macrophages, which play an important role in the initial stages of atherosclerotic development. SR-BI has been shown to be protective against atherosclerosis by way of overexpression and knockout (KO) studies in murine atherosclerosis models, including low density lipoprotein receptor (LDLR) knockout mice, apolipoprotein E (ApoE) knockout mice, and human apolipoprotein B (ApoB) transgenic mice. SR-BI/LDLR double knockout (dKO) mice show a 6-fold increase in diet-induced atherosclerosis compared to LDLR single KO controls, and SR-BI/ApoE dKO mice show severe coronary occlusion, myocardial infarction, and premature death on a normal chow diet. In both, plasma total cholesterol levels are significantly elevated, and associated with abnormally large HDL particles. The majority ofSR-BI's atheroprotective effect has been shown to result from plasma cholesterol clearance by way of selective uptake in the liver. Recently, Covey et al showed that elimination of SRBI expression in macrophages of LDLR KO mice resulted in increased diet-induced atherosclerosis. To see if SR-BI in macrophages contributes to the overall atheroprotective effect of SR-BI in ApoE KO mice, presumably by mediating cellular cholesterol efflux to HDL, selective deletion ofSR-BI was induced in bone marrow derived cells of ApoE KO mice using bone marrow transplantation. Female ApoE -/recipient mice were transplanted with either SR-BI +/+ ApoE -/-or SR-BI -/- ApoE -/bone marrow from male donor mice, and fed a high fat diet for 12 weeks. This resulted in significantly increased atherosclerosis in mice transplanted with SR-BI -/- ApoE -/-bone marrow, with a concomitant decrease in cholesterol associated with HDL-sized lipoproteins. No significant differences were seen in plasma total cholesterol levels or levels of cholesterol associated with non-HDL lipoproteins. These data suggest that SRBI in macrophages contributes to SR-BI's overall protective effect against atherosclerosis, and also plays a role in the regulation ofHDL cholesterol, in ApoE deficient mice. / Thesis / Master of Science (MSc)

Predominance of small dense Low Density Lipoprotein (LDL) is associated with a two to threefold increase in risk for Coronary Heart Disease (CVD). Small, dense HDL (High Density Lipoprotein) particles protect small dense LDL from oxidative stress. Technological advancements have introduced an array of techniques for measuring diameters of LDL and HDL as well as estimating overall particle heterogeneity. However, there is lack of comparative studies between these techniques, and, hence, no conclusive evidence to establish the merits of one method relative to others. The primary purpose of this study was to compare Nondenaturing Gradient Gel Electrophoresis (NDGGE) and Dynamic Laser Light Scattering (DLLS) methods in determining LDL and HDL particle diameter. Our comparison entailed: 1) Evaluating the two methods in terms of their reproducibility 2) Correlating the two methods(in future studies method selection would be driven by time and cost considerations if the two methods correlate), and 3) Evaluating the two methods in terms of their ability to identify bi-modal samples. A secondary purpose of this research was to investigate the effect of refrigerated plasma storage on particle diameter. Reproducibility was measured as Coefficient of Variance (CV). Within and between runs, CV for LDL and HDL for NDGGE were <6% and <15%, respectively and for DLLS, CV within runs were <3% and <5.5%, respectively. No correlation was observed between LDL diameter from the two methods. NDGGE showed two bands for 157 HDL samples of which only 24 samples showed bimodal peaks in DLLS. In order to study the effect of storage, three sample sets of LDL and two sample sets of HDL were used. NDGGE showed a significant difference between mean diameter of fresh and stored LDL and HDL sample for all sets, whereas DLLS showed a significant difference in only one LDL sample set and none for HDL sample sets. We conclude that DLLS may be a better method for measuring LDL diameter because NDGGE overestimated LDL diameter. However, NDGGE was able to resolve subpopulation better in an HDL sample than DLLS. Thus, NDGGE may be a better choice for measuring HDL diameter than DLLS.

Elevated plasma concentrations of lipoprotein(a) [Lp(a)] have been identified as an independent risk factor for vascular diseases including coronary heart disease and stroke. In the current study, we have examined the binding and degradation of recombinant forms of apolipoprotein(a) [r-apo(a)], the unique kringle-containing moiety of Lp(a), using a cultured cell model. We found that the incubation of human hepatoma (HepG2) cells with an iodinated 17 kringle-containing (17K) recombinant form of apo(a) resulted in a two-component binding system characterized by a high affinity (Kd = 12 nM), low capacity binding site, and a low affinity (Kd = 249 nM), high capacity binding site. We subsequently determined that the high affinity binding site on HepG2 cells corresponds to the LDL receptor. In the HepG2 cell model, association of apo(a) with the LDL receptor was shown to be dependent on the formation of Lp(a) particles from endogenous LDL. Using an apo(a) mutant incapable of binding to the high affinity site through its inability to form Lp(a) particles (17KΔLBS7,8), we further demonstrated that the LDL receptor does not participate in Lp(a) catabolism. The low affinity binding component observed on HepG2 cells, familial hypercholesterolemia (FH) fibroblasts and human embryonic kidney (HEK) 293 cells may correspond to a member(s) of the plasminogen receptor family, as binding to this site(s) was decreased by the addition of the lysine analogue epsilon-aminocaproic acid. The lysine-dependent nature of the low affinity binding site was further confirmed in HepG2 binding studies utilizing r-apo(a) species with impaired lysine binding ability. We observed a reduction maximum binding capacity for 17K r-apo(a) variants lacking the strong lysine binding site (LBS) in kringle IV type 10 (17KΔAsp) and the very weak LBS in kringle V (17KΔV). Degradation of Lp(a)/apo(a) was found to be mediated exclusively by the low affinity component on both HepG2 cells and FH fibroblasts. Fluorescence confocal microscopy, using the 17K r-apo(a) variant fused to green fluorescent protein, further confirmed that degradation by the low affinity component on HepG2 cells does not proceed by the activity of cellular lysosomes. Taken together, these data suggest a potentially significant route for Lp(a)/apo(a) clearance in vivo. / Thesis (Master, Biochemistry) -- Queen's University, 2009-09-26 02:15:50.754

Magister Pharmaceuticae - MPharm / Cardiovascular diseases (CVDs) such as ischaemic heart diseases, heart failure and stroke remain a major cause of death globally. Various deep-rooted factors influence CVD development; these include but are not limited to elevated blood lipids, high blood pressure, obesity and diabetes. A considerable number of proteins are involved directly and indirectly in the transport, maintenance and elimination of plasma lipids, including high and low-density lipoprotein cholesterol (HDL-C and LDL-C). There are several mechanisms involved in the removal of LDL particles from systemic circulation. One such mechanism is associated with the gene that encodes proprotein convertase subtilisin/kexin type 9 (PCSK9), which has become an exciting therapeutic target for the reduction of residual risk of CVDs. Currently, statins are the mainstay treatment to reduce LDL-C, and a need exists to further develop more effective LDL-C-lowering drugs that might supplement statins.

Background Elevated concentrations of low-density lipoprotein cholesterol (LDL-c) are a major risk factor for the development of coronary artery disease (CAD) because of their role in the progression of atherosclerosis. The black South African population is known to have had historically low LDL-c and in the past there was almost no CAD in the population. However, as this population moves through the nutrition transition, LDL-c levels are increasing. LDL-c levels are regulated by the LDL receptors, which is the major protein involved with transporting cholesterol across cell membranes in humans. Proprotein convertase subtilisinlike/kexin type 9 (PCSK9) is another protein involved with the regulation of LDL-c through its role in assisting with the degradation of the LDL receptor. Variants in both genes can cause elevated or lowered LDL-c levels. Very little information is available on the frequency or presence of variants in the low-density lipoprotein receptor (LDLR) and PCSK9 gene in the black South African population and on how these variants associate with LDL-c. The main aim of the study was thus to determine novel and existing genetic variants in these two genes and to describe the manner in which they associate with plasma LDL-c levels in a black South African population undergoing an epidemiological transition. Methods The 2005 baseline data from the Prospective Urban and Rural (PURE) study population were used in this study. The study population consisted of apparently healthy black volunteers form the North West province of South Africa, aged 35 to 60 years. Thirty individuals were randomly chosen from the 1860 volunteers to determine the presence of known and novel variants in these genes by automated bidirectional sequencing. The promoter region, exons and flanking regions were sequenced and variants were identified utilising CLC DNA Workbench. Deoxyribonucleic acid (DNA) samples for 1500 individuals of the PURE study population were genotyped by means of a Golden Gate Genotyping Assay. Analyses of covariance (ANCOVA) were used to test for associations between the different genotypes in both the LDLR and PCSK9 genes and LDL-c levels. Haplotypes were generated by using the confidence intervals on the software programme, HaploView. A genetic risk score (GRS) was determined by including variants which associated significantly with LDL-c. The GRS, the haplotypes and the variants that associated significantly with LDL-c were used in separate linear regression models with variants which correlated with LDL-c to determine how all these variables contribute to the differences in LDL-c levels. Results and discussion Novel and known variants were identified in both the genes and in total 52 variants were genotyped. Rare variants such as rs17249141 and rs28362286 were detected in the study population and are associated with low levels of LDL-c. The variants identified in the LDLR gene were situated largely in regulatory regions such as the promoter, intron and 3‟untranslated regions. Haplotypes in the LDLR gene with the highest frequency associated with lower LDL-c levels, which could contribute to the study population‟s low mean LDL-c level. Haplotypes identified in the PCSK9 gene had a weaker association with LDL-c levels. The minor allele frequencies of many of the variants differed from those of the European population and therefore the importance of population-specific research cannot be sufficiently emphasised. The GRS, haplotypes and variants used in the regression models to determine whether they contributed to predicting the variance in LDL-c in the study population made a small contribution to explaining this. BMI best explained the variance in LDL-c levels. Older women with a body mass index (BMI)>25kg/m2 were identified as being at greater risk of developing elevated LDL-c levels than the rest of the study population. Heterozygote carriers of variant, rs28362286, had 0.787 mmol/L lower LDL-c than carriers of the wild type and this is associated with a reduced risk of developing CAD. Conclusion and recommendation When considering the results mentioned above, adding genetic analysis to explaining the variance in LDL-c levels seems to have its limitations, but the study included only two of many genes that play a role in the metabolism and regulation of LDL-c levels. Incorporating more genes and more variants into analyses and prediction models will add greater value to defining LDL-c levels. Rarer variants with a large impact on protein function, such as rs28362286, have a greater effect on LDL-c levels and could predict the variance better than the common variants. Risk factors such as BMI can also still be trusted to indicate which individuals or groups are at risk of developing elevated LDL-c levels. Health advice should be given to appropriate target groups such as older women with a BMI >25kg/m2 in order to prevent CAD from becoming a burden in this population. / PhD (Dietetics), North-West University, Potchefstroom Campus, 2014

Background Elevated concentrations of low-density lipoprotein cholesterol (LDL-c) are a major risk factor for the development of coronary artery disease (CAD) because of their role in the progression of atherosclerosis. The black South African population is known to have had historically low LDL-c and in the past there was almost no CAD in the population. However, as this population moves through the nutrition transition, LDL-c levels are increasing. LDL-c levels are regulated by the LDL receptors, which is the major protein involved with transporting cholesterol across cell membranes in humans. Proprotein convertase subtilisinlike/kexin type 9 (PCSK9) is another protein involved with the regulation of LDL-c through its role in assisting with the degradation of the LDL receptor. Variants in both genes can cause elevated or lowered LDL-c levels. Very little information is available on the frequency or presence of variants in the low-density lipoprotein receptor (LDLR) and PCSK9 gene in the black South African population and on how these variants associate with LDL-c. The main aim of the study was thus to determine novel and existing genetic variants in these two genes and to describe the manner in which they associate with plasma LDL-c levels in a black South African population undergoing an epidemiological transition. Methods The 2005 baseline data from the Prospective Urban and Rural (PURE) study population were used in this study. The study population consisted of apparently healthy black volunteers form the North West province of South Africa, aged 35 to 60 years. Thirty individuals were randomly chosen from the 1860 volunteers to determine the presence of known and novel variants in these genes by automated bidirectional sequencing. The promoter region, exons and flanking regions were sequenced and variants were identified utilising CLC DNA Workbench. Deoxyribonucleic acid (DNA) samples for 1500 individuals of the PURE study population were genotyped by means of a Golden Gate Genotyping Assay. Analyses of covariance (ANCOVA) were used to test for associations between the different genotypes in both the LDLR and PCSK9 genes and LDL-c levels. Haplotypes were generated by using the confidence intervals on the software programme, HaploView. A genetic risk score (GRS) was determined by including variants which associated significantly with LDL-c. The GRS, the haplotypes and the variants that associated significantly with LDL-c were used in separate linear regression models with variants which correlated with LDL-c to determine how all these variables contribute to the differences in LDL-c levels. Results and discussion Novel and known variants were identified in both the genes and in total 52 variants were genotyped. Rare variants such as rs17249141 and rs28362286 were detected in the study population and are associated with low levels of LDL-c. The variants identified in the LDLR gene were situated largely in regulatory regions such as the promoter, intron and 3‟untranslated regions. Haplotypes in the LDLR gene with the highest frequency associated with lower LDL-c levels, which could contribute to the study population‟s low mean LDL-c level. Haplotypes identified in the PCSK9 gene had a weaker association with LDL-c levels. The minor allele frequencies of many of the variants differed from those of the European population and therefore the importance of population-specific research cannot be sufficiently emphasised. The GRS, haplotypes and variants used in the regression models to determine whether they contributed to predicting the variance in LDL-c in the study population made a small contribution to explaining this. BMI best explained the variance in LDL-c levels. Older women with a body mass index (BMI)>25kg/m2 were identified as being at greater risk of developing elevated LDL-c levels than the rest of the study population. Heterozygote carriers of variant, rs28362286, had 0.787 mmol/L lower LDL-c than carriers of the wild type and this is associated with a reduced risk of developing CAD. Conclusion and recommendation When considering the results mentioned above, adding genetic analysis to explaining the variance in LDL-c levels seems to have its limitations, but the study included only two of many genes that play a role in the metabolism and regulation of LDL-c levels. Incorporating more genes and more variants into analyses and prediction models will add greater value to defining LDL-c levels. Rarer variants with a large impact on protein function, such as rs28362286, have a greater effect on LDL-c levels and could predict the variance better than the common variants. Risk factors such as BMI can also still be trusted to indicate which individuals or groups are at risk of developing elevated LDL-c levels. Health advice should be given to appropriate target groups such as older women with a BMI >25kg/m2 in order to prevent CAD from becoming a burden in this population. / PhD (Dietetics), North-West University, Potchefstroom Campus, 2014