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Abstract To date, several methods have been developed to determine the activity of plasma lipid transfer proteins. These methods have largely involved the addition of the transfer protein in question to labeled substrates, followed by prolonged incubation (4 to 18 hours) and subsequent evaluation of the radioactivity transferred to precipitated low-density lipoprotein (LDL). While adequate for determining the activity of cholesteryl ester transfer protein (CETP), these methods generally do not take into account the composition or levels of lipoproteins present within a given individual plasma because pools of high-density lipoprotein (HDL) are labeled and used for the transfer experiments. Both the direction and the extent of lipid transfer are dependent on the composition and relative abundance of both donor and acceptor particles as well as the activity of the lipid transfer protein(s). Here we describe a new method for the determination of the capacity of plasma samples to facilitate cholesteryl ester transfer from HDL to LDL and very-low-density lipoprotein (VLDL), a method that has several advantages. First, the subject's HDL is labeled and used for transfer. Second, the labeled HDL, in a quantity equivalent to 1% of the plasma HDL mass, is added to the subject's plasma, and therefore the relative abundance of both donor and acceptor particles is preserved at their physiological levels. Third, both cholesteryl ester mass and radioactivity are determined, allowing the net mass transfer of cholesteryl ester and cholesteryl ester exchange to be quantified separately. Fourth, the application of an ultracentrifugal density gradient for the subsequent reisolation of the lipoproteins permits estimation of the transfer of cholesteryl esters to various subfractions of LDL; such measurement is not possible when precipitation techniques are used to determine total LDL radioactivity. The method allows estimation of the total physiological capacity of a plasma sample to mediate cholesteryl ester transfer, which may represent a more relevant measurement than that of CETP activity alone. Cholesteryl ester exchange and mass transfer were determined in four normolipidemic and two moderately hypertriglyceridemic subjects by this new in vitro method. Net transfer of cholesteryl ester from HDL to VLDL was increased twofold in mildly hypertriglyceridemic subjects (triglycerides > 100 and < 150 mg/dL). Cholesteryl ester mass transfer predominated for the first 6 hours of incubation, after which cholesteryl ester exchange predominated.