Reagent for the enzymatic determination of serum total cholesterol with improved lipolytic efficiency

We describe a sensitive method for quantifying the extent of cholesterol ester cleavage during enzymatic assay of total cholesterol in serum. Lipids are extracted from the assay mixture with chloroform/methanol (1/1 by vol), concentrated, then quantified by "high-performance" thin-layer chromatography. Although with conventional enzymatic reagents for determination of serum total cholesterol the hydrolysis of the cholesterol esters may be incomplete, a new enzymatic cholesterol reagent (Monotest Cholesterol, High Performance, Boehringer Mannheim) gives virtually complete cholesterol ester cleavage (i.e., greater than or equal to 99.5%). Use of this reagent with its improved lipolytic efficiency yields results for serum total cholesterol that are identical to those measured with a candidate reference procedure involving alkaline cholesterol ester saponification.