² H ₂ O-Based High-Density Lipoprotein Turnover Method for the Assessment of Dynamic High-Density Lipoprotein Function in Mice

Abstract

Objective High-density lipoprotein (HDL) promotes reverse cholesterol transport (RCT) from peripheral tissues to the liver for clearance. Reduced HDL-cholesterol (HDLc) is associated with atherosclerosis; however, as a predictor of cardiovascular disease, HDLc has limitations as it is not a direct marker of HDL functionality. Our objective was to develop a mass spectrometry based method for the simultaneous measurement of HDLc and ApoAI kinetics in mice using a single 2H2O tracer, and use it to examine genetic and drug perturbations on HDL turnover in vivo. Approach and Results Mice were given 2H2O in the drinking water and serial blood samples were collected at different time points. HDLc and ApoAI gradually incorporated 2H, allowing experimental measurement of fractional catabolic rates (FCR) and production rates (PR) for HDLc and ApoA1. ApoE−/− mice displayed increased FCR (p<0.01) and reduced PR of both HDLc and ApoAI (p<0.05) compared to controls. In human ApoAI transgenic mice, levels and PRs of HDLc and human ApoAI were strikingly higher than in wild type mice. Myriocin, an inhibitor of sphingolipid synthesis, significantly increased both HDL flux and macrophage-to-feces RCT, indicating compatibility of this HDL turnover method with the macrophage specific RCT assay. Conclusions 2H2O-labeling can be used to measure HDLc and ApoAI flux in vivo, and to assess the role of genetic and pharmacological interventions on HDL turnover in mice. Safety, simplicity, and low cost of the 2H2O-based HDL turnover approach suggest that this assay can be scaled for human use to study effects of HDL targeted therapies on dynamic HDL function.

HDL from apoA1 transgenic mice expressing the 4WF isoform is resistant to oxidative loss of function

“…Our data are consistent with fi ndings from previous studies that have reported that mouse apoA1 levels represent only a small fraction of total apoA1 in human apoA1 transgenic mice ( 25,37,38 ). …”

“…Plasma human and mouse apoA1 levels in wild-type mice and human apoA1 and 4WF transgenic mice were determined as previously described ( 25 ). In addition, a turnover study for HDL-C and human apoA1 was performed, as previously described ( 25 ), and fractional catabolic rates (FCRs) and production rates (PRs) were determined for each analyte.…”

“…In addition, a turnover study for HDL-C and human apoA1 was performed, as previously described ( 25 ), and fractional catabolic rates (FCRs) and production rates (PRs) were determined for each analyte. The method takes advantage of the integrated peak ratio(s) of the endogenous apoA1 peptides generated upon protein digestion to known quantities of a stable heavy-isotope-labeled internal standard.…”

“…The pellets were saved for the analysis of apoA1 by LC/MS/MS analysis. The supernatant was used for the analysis of HDL-C. After the evaporation of acetone, total HDL-C was analyzed by GC/MS as previously described ( 25 ). HDL-C turnover data were normalized for water body labeling prior to performing statistical analysis.…”

“…Mouse apoA1 levels were determined by mass spectrometry after protease digestion and the addition of a heavy-isotope-labeled mouse apoA1 peptide as internal standard ( 25 ). The plasma mouse apoA1 levels in C57BL/6 mice were ‫ف‬ 90.2 ± 16 mg/dl ( Fig.…”