We describe simple, sensitive and robust methods to monitor lipoprotein remodeling and cholesterol and apolipoprotein exchange, using fluorescent Lissamine Rhodamine B head-group tagged phosphatidylethanolamine (*PE) as a lipoprotein reference marker. Fluorescent Bodipy cholesterol (*Chol) and *PE directly incorporated into whole plasma lipoproteins in proportion to lipoprotein cholesterol and phospholipid mass, respectively. *Chol, but not *PE, passively exchanged between isolated plasma lipoproteins. Fluorescent apoA-I (*apoA-I) specifically bound to high-density lipoprotein (HDL) and remodeled *PE-and *Chol-labeled synthetic lipoprotein-X multilamellar vesicles (MLV) into a pre-β HDL-like particle containing *PE, *Chol, and *apoA-I. Fluorescent MLV-derived *PE specifically incorporated into plasma HDL, whereas MLV-derived *Chol incorporation into plasma lipoproteins was similar to direct *Chol incorporation, consistent with apoA-I-mediated remodeling of fluorescent MLV to HDL with concomitant exchange of *Chol between lipoproteins. Based on these findings, we developed a model system to study lipid transfer by depositing fluorescent *PE and *Chol-labeled on calcium silicate hydrate crystals, forming dense lipid-coated donor particles that are readily separated from acceptor lipoprotein particles by low-speed centrifugation. Transfer of *PE from donor particles to mouse plasma lipoproteins was shown to be HDL-specific and apoA-I-dependent. Transfer of donor particle *PE and *Chol to HDL in whole human plasma was highly correlated. Taken together, these studies suggest that cell-free *PE efflux monitors apoA-I functionality.
|State||Published - Sep 2019|
Bibliographical noteFunding Information:
Acknowledgments: This research was supported in part by the Intramural Research Program of the NIH and NHLBI. The content is solely the responsibility of the authors and does not represent the official views of the National Institutes of Health.
Funding: This research was supported by the Intramural Research Program of the National Heart, Lung, and Blood Institute (NHLBI) (HL006095) at the National Institutes of Health.
© 2019 by the authors. Licensee MDPI, Basel, Switzerland.
- HDL (High-density lipoprotein)/metabolism
- Low-density lipoprotein (LDL)
- Mass spectrometry
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology (all)
- Immunology and Microbiology (all)
- Agricultural and Biological Sciences (all)