Abstract 430: A Novel Nonradioactive Method Documents Strong Elevation of the Cholesterol Esterification Rate by CSL112, a Novel Formulation of Human Apolipoprotein A-I
Background: CSL112 was designed as an infusible formulation of human apoA-I to rapidly remove plaque cholesterol and reduce risk after an ACS. Following the initial efflux of unesterified cholesterol from cells, the second step of reverse cholesterol transport is the esterification of cholesterol by LCAT. Unfortunately, the current methods for measuring cholesterol esterification rate (CER) require radioactive labeling, are impractical for clinical laboratories and are unable to quantify individual cholesteryl esters (CE). In this study, we developed a novel non-radioactive method using D7-cholesterol as tracer and liquid chromatography-mass spectrometry (LC-MS) to detect and quantify seven different CE formed by LCAT esterification in plasma samples.
Methods: Plasma from normal healthy subjects was spiked with a range of concentrations (0 - 2.8 mg/mL) and formulations of human apoA-I (purified HDL; CSL112 (formulated with soy phosphatidyl choline (PC)); or formulated with sphingomyelin (SM). Dithionitrobenzoic acid was added to block esterification, and D7-cholesterol tracer was allowed to diffuse into the lipoproteins by 24 h, 4°C incubation with filter disks containing hydrous D7-cholesterol crystals. The esterification reaction was conducted at 37°C for 1 h in the presence of β-mercaptoethanol to reactivate LCAT. Newly formed D7-CE were quantitated by LC-MS following extraction from the plasma.
Results: As expected, native HDL did not increase CER and was used as control. The addition of CSL112 elevated CER in a concentration dependent manner. CSL112 is formulated with soy PC, a good substrate for LCAT, and the fatty acids appearing in the CE reflect the fatty acid composition of the soy PC. To confirm a role for the added PC in elevation of CER we showed that substitution of SM for PC caused CER to decrease rather than increase.
Conclusion: A sensitive non-radioactive method for CER has been developed that allows the quantification of LCAT-mediated formation of seven individual CE. CSL112, an apoA-I formulation containing soy PC supported concentration-dependent increases in CER, while a formulation of apoA-I with SM inhibited CER in a concentration-dependent manner.
Author Disclosures: D.J. Boerema: Employment; Significant; CSL Behring. K.L. Martin: Employment; Significant; CSL Limited. P.J. Meikle: None. S.D. Wright: Employment; Significant; CSL Behring. A. Gille: Employment; Significant; CSL Limited.
- © 2014 by American Heart Association, Inc.