The Development and Application of a Novel Method for the Quantification of Prebeta-1 HDL in Plasma

Abstract

Prebeta-1 HDL was one of the distinct molecular subspecies of HDL identified in the mid to late eighties . It is, now recognised as having a pivotal role in reverse cholesterol transport accounting for up to 90% of cholesterol trafficking from the periphery. Prebeta-1 HDL does so by acting as the quantum particle and primary acceptor of free cholesterol effluxed by the ATP-binding cassette A1 transporter from arterial macrophages, which are central to the atherosclerotic process .The ability to measure circulating levels of prebeta-1HDL may provide a novel biomarker of cardiovascular risk. The need for a novel, reliable, easy to use method to quantify prebeta-1 HDL in large numbers of patient samples is underscored by the number of technically difficult methods previously described, many of which tend to be slow and semi quantitative, only catering for small numbers of samples at any one time . The method developed and described in this thesis provides a robust technique for quantifying plasma prebeta-1 HDL, utilising ultrafiltration of plasma, isotope dilution and an enzyme-linked immunosorbent assay modified to allow the equal detection of apolipoprotein A-I in prebeta-1 HDL and in other plasma HDL particle species. This method quantifies the fraction of total apolipoprotein A-I that is present in prebeta-1 HDL particle species. In brief, using low-velocity centrifugation in ultrafilters with a 100 KDa cut off, it was demonstrated that prebeta-1 HDL, could be separated categorically from larger-diameter HDL particles in plasma and quantified by application of the isotope dilution principle and measurement of Apo A-I by ELISA. Levels of prebeta-1-HDL are reported as prebeta-1-HDL?A-I. This method was then utilised to measure prebeta-1 HDL levels: Firstly in 136 normal males and females, which found that, lower levels of prebeta-1 HDL were associated with female gender. Secondly, as exercise is associated with prevention of coronary artery disease, and is thought to be mechanistically related to its HDL raising effect, the effects of acute physical exercise on prebeta-1 HDL was also examined in normolipidemic subjects, demonstrating that acute exercise significantly increased absolute plasma prebeta-1HDL levels. Finally prebeta-1 HDL levels were also measured in a large well characterised clinical cohort of 1255 subjects from the University of California San Francisco Lipid and Cardiovascular Clinics and collaborating Cardiologists. An association was found between, increased absolute levels of prebeta-1 HDL and structural coronary disease and myocardial infarction. This was the first study in which prebeta -1 HDL was identified as a novel and independent predictor of MI above and beyond traditional CHD risk.