Abstract
High density lipoprotein (HDL) plays an important role in the prevention of cardiovascular disease. Apolipoprotein A-I (apoA-I), the major apolipoprotein of HDL is mainly secreted by the liver. Once secreted, apoA-I acquires lipid to form nascent discoidal prebetaHDL which by further acquisition of lipid are converted into spherical alphaHDL particles (HDL2 and HDL3). Current understanding of HDL metabolism is limited due to technical difficulties in measuring the kinetics of HDL subclasses. Traditionally, HDL is separated by ultracentrifugation (1.063<d<1.21 g/ml), however, this process may damage the integrity of certain HDL particles leading to the dissociation of apoA-I. The aim of this study was to develop methods to separate and determine the kinetics of alphaHDL and prebetaHDL using stable isotopic techniques. Separation of HDL2 and HDL3 by sequential ultracentrifugation aimed to investigate the kinetic difference between HDL2 and HDL3. A two dimensional electrophoresis technique was developed and optimised to separate apoA-I from alphaHDL and prebetaHDL. Using this methodology apoA-I kinetics were measured in healthy subjects. Subjects (3F+1M) were given a primed constant infusion of 1-13C-leucine to label apoA-L Plasma was separated by agarose gel electrophoresis in the first dimension. alphaHDL and prebetaHDL bands were removed and apolipoproteins were extracted and separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis in second dimension. The isotopic enrichment of apoA-I in HDL subclasses was measured by gas chromatography mass spectrometry. The results indicate this protocol can be used to separate and measure the kinetics of alphaHDL and prebetaHDL. The fractional secretion rate (FSR) of apoA-I from alphaHDL and prebetaHDL was 0.15+/-0.017 pools/day and 0.19+/-0.022 pools/day respectively (P-0.039). HDL2 and HDL3 FSR was 0.16+/-0.018 pools/day and 0.14+/-0.017 pools/day respectively (P=0.003) and their production rates were 4.07+/-0.61 and 2.82+/-0.37 mg/kg/day respectively. This novel technique allows the measurement of prebetaHDL and alphaHDL apoA-I kinetics.