Determination of cholesterol esterase

Abstract

Advantage is taken of macrolide antibiotics' complexation with free cholesterol to yield fluorescent complexes to determine the levels of free cholesterol, total cholesterol, or lecithin: cholesterol acyl transferase (LCAT) in serum or plasma or fractions thereof or to determine the level of cholesterol esterase in a sample.

1 . A method to detect and quantitate total cholesterol (TC) in a sample which method comprises:
 adding to the sample an amount of cholesteryl esterase effective to hydrolyze any cholesteryl ester (CE) in said sample; and   contacting said sample with a macrolide antibiotic that forms a complex with free cholesterol (FC) which complex is fluorescent upon excitation; and   quantitating the level of fluorescence as a measure of the concentration of FC in said sample whereby the total cholesterol (TC) in said sample is thereby determined.   
     
     
         2 . The method of  claim 1  wherein the sample comprises serum, plasma or fraction thereof 
     
     
         3 . The method of  claim 2  wherein the sample consists essentially of high-density lipoprotein particles (HDL) from said serum or plasma. 
     
     
         4 . The method of  claim 1  wherein the macrolide antibiotic is pimaricin, amphotericin B, nystatin, lucensomycin, fungichromin (lagosin) or filipin. 
     
     
         5 . The method of  claim 1  wherein said sample is maintained at a temperature below room temperature or wherein an inhibitor of lecithin: cholesterol acyl transferase (LCAT) is added to the sample. 
     
     
         6 . The method of  claim 5  wherein the inhibitor is iodoacetate, an anti-LCAT antibody or an aptamer specific for LCAT. 
     
     
         7 . A method to measure the activity of cholesterol esterase (CE) in a sample which method comprises:
 a) incubating said sample for a period of time with a macrolide antibiotic that forms a complex with FC which complex exhibits fluorescence upon excitation; and   b) quantitating the fluorescence at a plurality of times within said period so as to measure the concentration of FC as a function of time over said period; or   c) incubating said sample for a period of time;   d) withdrawing aliquots from said sample at a plurality of times within said period;   e) adding to said aliquots a macrolide antibiotic that forms a complex with FC which complex exhibits fluorescence upon excitation; and   f) quantitating the quantity of fluorescence in said aliquots so as to measure the concentration of FC as a function of time;   wherein an increase in the concentration of FC over said time period is a measure of the activity of CE in said sample.   
     
     
         8 . The method of  claim 7  wherein the macrolide antibiotic is pimaricin, amphotericin B, nystatin, lucensomycin, fungichromin (lagosin) or filipin. 
     
     
         9 . The method of  claim 7  wherein the incubating is for 20 minutes.