Physical state of cholesteryl esters deposited in cultured macrophages

Abstract

J774 macrophages load with cholesteryl ester (CE) when incubated with acetylated low-density lipoprotein and cholesterol-rich liposomes; the CE accumulates as cytoplasmic inclusions 1-2 .mu.m in diameter. The CE core of the droplet comprises about 90% of its mass; the predominant CE species present are cholesteryl palmitate (CP, 41%) and cholesteryl oleate (CO, 37%). The thermotropic properties of the inclusions, both in intact cells and after isolation, have been characterized by differential scanning calorimetry. On heating, the inclusions exhibit two endothermic transitions at about 41 and 53.degree. C with a total enthalpy of 7.7 .+-. 1.2 cal/g of CE. Very similar thermal behavior is exhibited by a binary mixture containing equal weights of CO and CP; this indicates that these two species dominate the phase behavior of CE in J774 inclusions. A phase diagram for the CO/CP system has been generated, and this reflects simple eutectic behavior. The eutectic is 83% w/w CO, and it melts at 49-50.degree. C. Below this temperature, CO and CP form two immiscible crystalline phases due to the very limited ability of the unsaturated oleate and saturated palmitate acyl chains to mix in the crystal phase. On heating a 1/1 w/w CO/CP mixture, an isotropic liquid of eutectic composition forms at 49.degree. C, and the remaining crystalline cholesteryl palmitate melts over the temperature range 50-69.degree. C. The phase diagram indicates that bulk mixtures of CE molecules in J774 inclusions should be crystalline at 37.degree. C, the growth temperature of the cells. CO and CP are miscible at all ratios in either the liquid or the liquid-crystalline state where the hydrocarbon chains are melted. The smectic liquid-crystal phase is metastable at all ratios of CO and CP, whereas the cholesteric phase is metastable only in mixtures containing 15-70% and 90-100% CO by weight. These metastable liquid-crystal phases which form on cooling eventually crystallize on storage. The enthalpies associated with the two thermal transitions of the CE in J774 inclusions suggest that the core of this particle comprises microcrystals of CP suspended in a fluid CO-rich phase. The fluid phase could be either metastable, smectic liquid-crystal or metastable, isotropic liquid.