Quantification of HDL Particle Concentration by Calibrated Ion Mobility Analysis

BACKGROUND: It is critical to develop new metrics to determine whether HDL is cardioprotective in humans. One promising approach is HDL particle concentration (HDL-P), the size and concentration of HDL in plasma. However, the 2 methods currently used to determine HDL-P yield concentrations that differ >5-fold. We therefore developed and validated an improved approach to quantify HDL-P, termed calibrated ion mobility analysis (calibrated IMA). METHODS: HDL was isolated from plasma by ultracentrifugation, introduced into the gas phase with electrospray ionization, separated by size, and quantified by particle counting. We used a calibration curve constructed with purified proteins to correct for the ionization efficiency of HDL particles. RESULTS: The concentrations of gold nanoparticles and reconstituted HDLs measured by calibrated IMA were indistinguishable from concentrations determined by orthogonal methods. In plasma of control (n = 40) and cerebrovascular disease (n = 40) participants, 3 subspecies of HDL were reproducibility measured, with an estimated total HDL-P of 13.4 (2.4) μmol/L. HDL-C accounted for 48% of the variance in HDL-P. HDL-P was significantly lower in participants with cerebrovascular disease ( P = 0.002), and this difference remained significant after adjustment for HDL cholesterol concentrations ( P = 0.02). CONCLUSIONS: Calibrated IMA accurately determined the concentration of gold nanoparticles and synthetic HDL, strongly suggesting that the method could accurately quantify HDL particle concentration. The estimated stoichiometry of apolipoprotein A-I determined by calibrated IMA was 3–4 per HDL particle, in agreement with current structural models. Furthermore, HDL-P was associated with cardiovascular disease status in a clinical population independently of HDL cholesterol.