Development of a method to measure preßHDL and αHDL apoA-I enrichment for stable isotopic studies of HDL kinetics.

ABSTRACT

Our understanding of HDL metabolism would be enhanced by the measurement of the kinetics of preßHDL, the nascent form of HDL, since elevated levels have been reported in patients with coronary artery disease. Stable isotope methodology is an established technique that has enabled the determination of the kinetics (production and catabolism) of total HDL apoA-I in vivo. The development of separation procedures to obtain a preßHDL fraction, the isotopic enrichment of which could then be measured, would enable further understanding of the pathways in vivo for determining the fate of preßHDL and the formation of αHDL. A method was developed and optimised to separate and measure preßHDL and αHDL apoA-I enrichment. Agarose gel electrophoresis was first used to separate lipoprotein subclasses, and then a 4-10 % discontinuous SDS-PAGE used to isolate apoA-I. Measures of preßHDL enrichment in six healthy subjects were undertaken following an infusion of L-[1-¹³C-leucine]. After isolation of preß and αHDL, the isotopic enrichment of apoA-I for each fraction was measured by gas chromatography-mass spectrometry. PreßHDL apoA-I enrichment was measured with a CV of 0.51 % and αHDL apoA-I with a CV of 0.34 %. The fractional catabolic rate (FCR) of preßHDL apoA-I was significantly higher than the FCR of αHDL apoA-I (p < 0.005). This methodology can be used to selectively isolate preß and αHDL apoA-I for the measurement of apoA-I isotopic enrichment for kinetics studies of HDL subclass metabolism in a research setting.