UBC Theses and Dissertations
Pharmacologically-induced changes in the concentration of high density lipoprotein cholesterol and the relationship with cholesterol efflux Tancon, Scott MacKenzie Alber
Low concentrations of high-density lipoprotein cholesterol (HDL-C) have been associated with increased risk of atherosclerosis in clinical trials, and pathological, genetic, and epidemiological studies. High-density lipoproteins (HDL) have numerous anti-atherogenic properties; however, the process of removing excess cholesterol from lipid-laden macrophages (Reverse Cholesterol Transport (RCT)) is believed to be the predominant mechanism of cardioprotection. Cholesterol efflux, the first step in RCT, is presumed to be the rate-limiting step and has been extensively studied. Cholesterol efflux is influenced by both the quality and quantity of HDL. Physicians use the clinical measure of HDL-C in assessing patient risk of coronary artery disease. Numerous pharmacological agents can alter HDL-C; however, the effect of these changes on cholesterol efflux has been poorly studied. The present study attempts to measure the functional properties (defined herein as "quality") of HDL in patients with pharmacologically-induced changes in HDL-C, in terms of each particle's ability to promote cholesterol efflux from cells. Most studies have measured cholesterol efflux potential at only a single serum concentration, which fails to differentiate between potential differences in quantity and quality. We use serum dose-response curves to measure cholesterol efflux potential from Fu5AH cells and define the affinity constant, apparent Km, as being a good measure of quality. Twenty-three healthy control subjects were recruited to develop normal ranges for the parameter apparent Km. Finally, forty-three experimental subjects were assigned to one of four experimental groups: high pharmacologically-induced HDL-C (>1.55 mmol/L), high non-pharmacologically-induced HDL-C, low pharmacologically-induced HDL-C (<1.04 mmol/L) and low non-pharmacologicallyinduced HDL-C. Sera were assayed for fractional esterification rates of the HDL fraction (FERHDL) and serum dose-response curves in cholesterol efflux studies. The intra-assay and inter-assay coefficients of variation (CV) for apparent Km measurements were 6.5% and 20.5%, respectively. A pooled sera standard was included to control for differences between assay days; and the maximal rate of efflux (apparent V[ sub max]) and apparent K m values were calculated relative to this standard. Relative to a pooled sera standard, the apparent V[ sub max] values ranged from 83% -112% in healthy subjects. Two patients with familial LCAT deficiency exhibited 67.4% and 62.6% reductions in apparent V[ sub max] , and serum treated with DTNB (an effective LCAT inhibitor) exhibited a 90.2% reduction in apparent V[ sub max], suggesting a possible role of apparent V[ sub max] in diagnosing HDL-associated genetic defects. These studies demonstrated that the quality of HDL particles changes with HDLC. Relative apparent Km values (per particle) increase with increasing HDL-C, whereas FERHDL decreases as HDL-C increases. These are compatible findings; smaller HDL have been shown to promote cholesterol efflux from Fu5AH cells to a greater extent than larger HDL. It was hypothesized that HDL quality is a more predominant factor than HDL quantity in determining the rate of cholesterol efflux in vivo; however, this does not seem to be the case if RCT is the major anti-atherogenic property of HDL, since quality cannot account for the epidemiological data. The data suggest that HDL-C is still a good predictor of the cholesterol efflux potential of serum and quality of HDL particles when HDL-C>1.04 mmol/L, regardless of pharmacological intervention. However, patients with pharmacologically-induced changes in HDL-C when HDL-C<1.04 mmol/L, have qualitatively different HDL particles than subjects with matched HDL-C and no pharmacologically-induced change, and may have to be evaluated in a different context.
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