In-vivo metabolism of VLDL-apolipoprotein-B, -CIII and -E in normolipidemic subjects

Abstract

Background and aim ApoE and apoC-III are important components of lipoprotein metabolism. While the function of both apoproteins is relatively well understood, little is known about the in vivo metabolism of these proteins, partly because of the lack of a standardized method to isolate these apoproteins in large sample numbers. Methods and results We developed a new reverse phase HPLC method (acetonitril/phosphate gradient; Aquapore RP-300, 7 μm, 220 × 4.6 mm) to isolate a number of different apoproteins, including apoC-III and apoE from VLDL. This method was then used in a study which aimed at determining VLDL-apoE-3 and VLDL-apoC-III metabolism. In addition VLDL-apoB and LDL-apoB metabolism was determined. Endogenous labeling with d 3-leucine, mass spectrometry and multicompartmental modeling was used in 6 normolipidemic healthy male subjects. Tracer/tracee ratios of free plasma leucine, VLDL-apoE, -apoC-III, -apoB, and LDL-apoB leucine were determined over 60 h following a bolus of d 3-leucine (5 mg kg −1). In all subjects sufficient apoC-III could be isolated by reverse phase HPLC to derive metabolic parameters, while apoE metabolic parameters could only be determined if apoE plasma concentration was 0.75 mg dl −1 or higher. Compared to VLDL-apoB (FCR 10.4 ± 3.3 d −1, production 17.8 ± 4.5 mg kg −1 d −1), VLDL-apoE-3 (FCR 1.03 ± 0.11 d −1, production 0.50 ± 0.29 mg kg −1 d −1) and VLDL-apoC-III (FCR 1.67 ± 1.22 d −1, production 0.44 ± 0.24 mg kg −1 d −1) parameters were much lower. This indicates that apoE-3 and apoC-III recirculate in plasma and that only a small fraction of apoE and apoC-III on VLDL is newly synthesized. Conclusions We conclude that HPLC methodology can be used to isolate VLDL-apoC-III and apoE for metabolic studies and that the metabolic fate of apoC-III and apoE is different from that of apoB because both apoproteins recycle through the VLDL fraction.