Cholesterol efflux analyses using stable isotopes and mass spectrometry

Cholesterol efflux from macrophages and the vascular wall is the initial step of the cardiovascular protective reverse cholesterol transport process. This study demonstrates a mass spectrometry based assay to measure the cellular and medium content of [d(7)]cholesterol and unlabeled cholesterol that can be used to measure cholesterol efflux from cell lines. Using a triple-quadrupole electrospray ionization-MS instrument in direct infusion mode, product ion scanning for m/z 83, neutral loss (NL) 375.5 scanning, and NL 368.5 scanning were used to detect cholesterol (as an acetylated derivative), [d(7)]cholesteryl ester (CE), and unlabeled CE, respectively. The same mass of [d(7)]cholesterol was substituted for [(3)H]cholesterol under standard efflux assay conditions. At the end of [d(7)]cholesterol loading, the intracellular mass of [d(7)]cholesterol was twofold greater than that of unlabeled cholesterol, and the intracellular [d(7)]CE profile was similar to that of unlabeled CE. Efflux of cholesterol to apolipoprotein A-I and high-density lipoproteins was similar comparing efflux of either [d(7)]cholesterol or [(3)H]cholesterol as measured by following efflux of the tracers only. This technique also can be used to assess the efflux of unlabeled cholesterol to acceptors in medium that are initially cholesterol-free (e.g., apolipoprotein A-I). Taken together, this mass spectrometry-based assay provides new molecular detail to assess cholesterol efflux.