Kinetic enzymic method for automated determination of total cholesterol in serum

We describe a rapid, kinetic, fixed-time method for determining serum total cholesterol by use of cholesterol esterase, cholesterol oxidase, and the indicator reaction with peroxidase, 4-aminophenazone, and phenol. On addition of the competitive inhibitor 3,4-dichlorophenol the Michaelis constant of cholesterol oxidase is apparently increased, which extends the linear relation between absorbance change and cholesterol concentration to 20.7 to 25.9 mmol/L, depending on the analyzer being used. For calibration, a single standard is used. Total analysis time is in the range of 80 to 210 s. Incubation temperature is 25 degrees C or 37 degrees C. The single-reagent procedure has been adapted to three different centrifugal analyzers and to the Eppendorf ACP 5040 analyzer. It yields precise and accurate results and is insensitive to potential interferences.