An optimized method for measuring lecithin : cholesterol acyltransferase activity, independent of the concentration and quality of the physiological substrate.
The aim of the study was to achieve the measurement of lecithin : cholesterol acyltransferase (EC 2.3.1.43) activity independent of and uninfluenced by equilibration of lipids between different lipoproteins, their molar ratios or by possible differences in their substrate quality. A mixture of sodium phosphotungstic acid/MgCl2 was added to serum samples to achieve total precipitation of all plasma lipoproteins. The filtrate containing the total plasma lecithin : cholesterol acyltransferase activity, apolipoprotein A-I and major plasma proteins was used for the assay. Liposomes comprised of phosphatidylcholine, cholesterol and dicetylphosphate served as substrate. The decrease in free cholesterol was determined enzymatically after incubation for 60 min at 37 degrees C. The assay followed zero-order kinetics and was linear for more than 60 min. The following Km values for various substrates were obtained: liposomes, 0.43 mM; HDL, 0.63 mM; LDL, 0.0; VLDL, 0.0; abnormal lipoprotein, found in cholestasis, (LP-X), 0.0. Comparison with generally used lecithin : cholesterol acyltransferase assay methods revealed similar activities for healthy controls, but in different forms of dyslipoproteinemia higher values were obtained with our method and, in special cases, the activity could be enhanced by addition of apolipoprotein A-I.
D002779:Cholestasis / Q000201:enzymology; D002784:Cholesterol / Q000378:metabolism; D006505:Hepatitis / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008722:Methods; D010195:Pancreatitis / Q000201:enzymology; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000097:blood*; D013379:Substrate Specificity
D008075:Lipoproteins, HDL; D008081:Liposomes; D002784:Cholesterol; D007862:Phosphatidylcholine-Sterol O-Acyltransferase