The maintenance of proper intracellular levels of cholesterol is dependent on a dynamic equilibrium between cholesterol efflux and uptake of the lipid component after having bound to an extracellular acceptor in serum. To study this phenomenon, a method was developed to assay the combination of "released" cholesterol with serum acceptor molecules. The objective of the present research is to extend the current assay in order to obtain more information on other parameters that can influence cholesterol efflux. These factors include intracellular cholesterol content and the relative activities of serum components, other than those lipoproteins already known to regulate cholesterol homeostasis such as lecithin cholesterol ester hydrolase (LCAT) and cholesterol ester transfer protein (CETP). To begin to monitor the movement of cholesterol after entry into the extracellular acceptor pool, methods will be developed to assay intracellular acyl cholesterol ester transferase (ACAT). Changes in ACAT activity have been shown to correlate with cell cholesterol in numerous cell systems. With the ACAT enzyme assay in place, cholesterol efflux values will be correlated with changes in serum components such as high density lipoprotein (HDL) subclasses, other lipoproteins, and standard parameters of LCAT and CETP. Finally, the new assay method will be scaled down for screening small volume serum samples from genetically engineered mice, that have extra, or missing copies, of genes for serum proteins that play a crucial role in cholesterol metabolism. %%% A better method will be developed to measure changes in the circulating total concentration of cholesterol in serum. This is difficult to do because cholesterol exists in combination with a variety of different circulating serum components, and in this combined state, it is not detected by ordinary procedures now in use. Development of such an assay system and the identification of more of the different extracellular cholesterol-absorbing components in serum will provide a better understanding of cholesterol metabolism in vertebrate animals.
