Hutchins Patrick M, Ronsein Graziella E, Monette Jeffrey S, Pamir Nathalie, Wimberger Jake, He Yi, Anantharamaiah G M, Kim Daniel Seung, Ranchalis Jane E, Jarvik Gail P, Vaisar Tomas, Heinecke Jay W
Department of Medicine, University of Washington, Seattle, WA;
Atherosclerosis Unit, University of Alabama Medical Center, Birmingham, AL.
Clin Chem. 2014 Nov;60(11):1393-401. doi: 10.1373/clinchem.2014.228114. Epub 2014 Sep 15.
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).
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.
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).
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.
开发新的指标以确定高密度脂蛋白（HDL）对人类是否具有心脏保护作用至关重要。一种有前景的方法是HDL颗粒浓度（HDL-P），即血浆中HDL的大小和浓度。然而，目前用于测定HDL-P的两种方法所产生的浓度相差超过5倍。因此，我们开发并验证了一种改进的HDL-P定量方法，称为校准离子淌度分析（校准IMA）。
通过超速离心从血浆中分离HDL，用电喷雾电离将其引入气相，按大小分离，并通过颗粒计数进行定量。我们使用由纯化蛋白构建的校准曲线来校正HDL颗粒的电离效率。
通过校准IMA测量的金纳米颗粒和重组HDL的浓度与通过正交方法测定的浓度无明显差异。在对照组（n = 40）和脑血管疾病组（n = 40）参与者的血浆中，可重复性地测量到3种HDL亚类，估计总HDL-P为13.4（2.4）μmol/L。HDL胆固醇占HDL-P变异的48%。脑血管疾病参与者的HDL-P显著较低（P = 0.002），在调整HDL胆固醇浓度后，这种差异仍然显著（P = 0.02）。
校准IMA准确地测定了金纳米颗粒和合成HDL的浓度，强烈表明该方法能够准确地定量HDL颗粒浓度。通过校准IMA确定的载脂蛋白A-I的估计化学计量为每个HDL颗粒3 - 4个，与当前的结构模型一致。此外，在临床人群中，HDL-P与心血管疾病状态相关，且独立于HDL胆固醇。
