Cell-free, high-density lipoprotein–specific phospholipid efflux assay predicts incident cardiovascular disease
Masaki Sato, Edward B. Neufeld, Martin P. Playford, Yu Lei, Alexander V. Sorokin, Angel M. Aponte, Lita A. Freeman, Scott M. Gordon, Amit K. Dey, Kianoush Jeiran, Masato Hamasaki, Maureen L. Sampson, Robert D. Shamburek, Jingrong Tang, Marcus Y. Chen, Kazuhiko Kotani, Josephine L.C. Anderson, Robin P.F. Dullaart, Nehal N. Mehta, Uwe J.F. Tietge, Alan T. Remaley
Masaki Sato, Edward B. Neufeld, Martin P. Playford, Yu Lei, Alexander V. Sorokin, Angel M. Aponte, Lita A. Freeman, Scott M. Gordon, Amit K. Dey, Kianoush Jeiran, Masato Hamasaki, Maureen L. Sampson, Robert D. Shamburek, Jingrong Tang, Marcus Y. Chen, Kazuhiko Kotani, Josephine L.C. Anderson, Robin P.F. Dullaart, Nehal N. Mehta, Uwe J.F. Tietge, Alan T. Remaley
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Clinical Research and Public Health Vascular biology

Cell-free, high-density lipoprotein–specific phospholipid efflux assay predicts incident cardiovascular disease

Abstract

BACKGROUND Cellular cholesterol efflux capacity (CEC) is a better predictor of cardiovascular disease (CVD) events than HDL-cholesterol (HDL-C) but is not suitable as a routine clinical assay.METHODS We developed an HDL-specific phospholipid efflux (HDL-SPE) assay to assess HDL functionality based on whole plasma HDL apolipoprotein–mediated solubilization of fluorescent phosphatidylethanolamine from artificial lipid donor particles. We first assessed the association of HDL-SPE with prevalent coronary artery disease (CAD): study I included NIH severe-CAD (n = 50) and non-CAD (n = 50) participants, who were frequency matched for sex, BMI, type 2 diabetes mellitus, and smoking; study II included Japanese CAD (n = 70) and non-CAD (n = 154) participants. We also examined the association of HDL-SPE with incident CVD events in the Prevention of Renal and Vascular End-stage Disease (PREVEND) study comparing 340 patients with 340 controls individually matched for age, sex, smoking, and HDL-C levels.RESULTS Receiver operating characteristic curves revealed stronger associations of HDL-SPE with prevalent CAD. The AUCs in study I were as follows: HDL-SPE, 0.68; apolipoprotein A-I (apoA-I), 0.62; HDL-C, 0.63; and CEC, 0.52. The AUCs in study II were as follows: HDL-SPE, 0.83; apoA-I, 0.64; and HDL-C, 0.53. Also longitudinally, HDL-SPE was significantly associated with incident CVD events independent of traditional risk factors with ORs below 0.2 per SD increment in the PREVEND study (P < 0.001).CONCLUSION HDL-SPE could serve as a routine clinical assay for improving CVD risk assessment and drug discovery.TRIAL REGISTRATION ClinicalTrials.gov NCT01621594.FUNDING NHLBI Intramural Research Program, NIH (HL006095-06).

Authors

Masaki Sato, Edward B. Neufeld, Martin P. Playford, Yu Lei, Alexander V. Sorokin, Angel M. Aponte, Lita A. Freeman, Scott M. Gordon, Amit K. Dey, Kianoush Jeiran, Masato Hamasaki, Maureen L. Sampson, Robert D. Shamburek, Jingrong Tang, Marcus Y. Chen, Kazuhiko Kotani, Josephine L.C. Anderson, Robin P.F. Dullaart, Nehal N. Mehta, Uwe J.F. Tietge, Alan T. Remaley

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Figure 3

Exchangeable HDL apolipoproteins mediate HDL-SPE.

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Exchangeable HDL apolipoproteins mediate HDL-SPE. (A) Assay to identify ...
(A) Assay to identify plasma proteins that mediate HDL-SPE. (B) Time course of LC-CSH *PE efflux to HP versus saline after incubation at 37°C for the indicated durations. (C) Release of *PE from LC-CSH in A, after washing and subsequent incubation with saline at 37°C for 1 hour. (D) Agarose gel electrophoresis: D-HP, HP directly labeled with *PE; SUP, supernatant after incubation of HP with fluorescent *PE-labeled LC-SCH for 30 minutes; R, plasma proteins released to saline from LC-CSH preincubated with HP. (E) Fluorescent native gel lipoprotein electrophoresis and (F) Kymograph analysis of *PE fluorescence revealed that the supernatant and released plasma proteins contained mostly small *PE-tagged HDL-like particles. Red arrow indicates small HDL/albumin band. HP, unlabeled HP. (G–I) iBAQ analyses revealed the relative distribution of the identified plasma proteins. (G) HP proteins, (H) LC-CSH–bound proteins, and (I) LC-CSH–released proteins. All data are the mean ± SD from triplicate assays.