Quantification of HDL Particle Concentration by Calibrated Ion Mobility Analysis

Abstract

Background It is critical to develop new metrics to determine whether high density lipoprotein (HDL) is cardioprotective in humans. One promising approach is HDL particle concentration (HDL-P) – the size and concentration of HDL in plasma or serum. However, the two methods currently used to determine HDL-P yield concentrations that differ more than 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. A calibration curve constructed with purified proteins was used 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) subjects, three subspecies of HDL were reproducibility measured, with an estimated total HDL-P of 13.4±2.4 µM (mean±SD). HDL-C accounted for 48% of the variance in HDL-P. HDL-P was significantly lower in subjects with cerebrovascular disease, and this difference remained significant after adjustment for HDL cholesterol levels. Conclusions Calibrated IMA accurately and reproducibly determined the concentration of gold nanoparticles and synthetic HDL, strongly suggesting the method could accurately quantify HDL particle concentration. Importantly, the estimated stoichiometry of apoA-I determined by calibrated IMA was 3–4 per HDL particle, in excellent agreement with current structural models. Furthermore, HDL-P associated with cardiovascular disease status in a clinical population independently of HDL cholesterol.

The Changing Face of HDL and the Best Way to Measure It

“…The best example of this is the use of nuclear magnetic resonance (NMR) spectroscopy for measuring HDL particle number, which has been shown in several large studies to be superior to HDL-C in predicting future cardiovascular events (4,183,184 ). NMR can also be used to quantify specific HDL subfractions, such as apoB-containing lipoproteins, as well as LDL and HDL particle size and distribution, which may also be predictive of increased mortality in a wide range of disease states (185)(186)(187)(188)(189)(190). Correlations between specific HDL subfractions particle concentration and other cardiovascular measures have also been identified using NMR.…”

“…Recently, a mass spectrometry-based test called ion mobility analysis has been developed as an alternative way to measure HDL particle number, as well as HDL composition and size subfractions (190,192 ). Finally, a test based on electron paramagnetic resonance spectroscopy has also been described for measuring the integration of apoA-I into HDL in samples, which in small-scale studies appears to be related to HDL function and CVD risk (4,189 ).…”

HDL-C vs HDL-P: How Changing One Letter Could Make a Difference in Understanding the Role of High-Density Lipoprotein in Disease

“…The report by Hutchins et al in this issue of Clinical Chemistry directly addresses this HDL-P accuracy problem (14 ). Building on the IM approach, they used a set of purified proteins of known size and concentration to internally calibrate the method.…”

Ion Mobility in Clinical Analysis: Current Progress and Future Perspectives

“…This methodology allows for separation of HDLs on the basis of their size in ESI-produced aerosols by differential ion mobility, followed by quantification using a particle counter. Results from a clinically validated method indicate that total HDL-P, grouped into categories of small (approximately 120 kDa), medium (approximately 160 kDa), and large HDL (approximately 270 kDa), can be reproducibly analyzed (59 ). This application reveals the potential for simple, standalone IMS instrumentation to be implemented for common clinical tests, with limited analytical and interpretative requirements.…”