Assay for Lecithin: Cholesterol Acyltransferase

This chapter describes the different aspects of assay for lecithin. The enzyme can be assayed by determining the disappearance of unesterified cholesterol, by measuring the conversion of radioactive cholesterol to radioactive cholesteryl ester, or by observing the incorporation of radiolabeled fatty acids from lecithin into cholesteryl esters. The reaction is usually run near pH 7 in the presence of a chelating agent to inhibit heavy metal-catalyzed peroxidation of the lecithin and in the presence of a reducing agent, which is usually necessary for maximum enzyme activity. Radiolabeled lecithin is incubated with cholesterol, and incorporation of the labeled fatty acids into cholesteryl esters is observed. The products are separated from the labeled substrate by a solvent partition step. The blank is reduced by treating the neutral lipid fraction after the partition with silicic acid and separation of fatty acids from cholesteryl esters is obtained by extraction of the washed neutral lipid fraction with a basic aqueous solution. It is found that the assay in plasma is complicated by the simultaneous presence of variable amounts of both enzyme and substrate and the possibility that variable amounts of activating cofactor proteins might also be present.