Oxidase determination of plasma cholesterol as cholest-4-en-3-one using iso-octane extraction

Cholesterol esters in 20 microliter of plasma are hydrolysed with hot ethanolic KOH. Preformed cholesterol and cholesterol released by hydrolysis is reacted with cholesterol oxidase to form hydrogen peroxide and cholest-4-en-3-one, which is extracted from alkaline 50% v/v ethanolic solution with iso-octane. The absorbance of the ketone in iso-octane at the 232 nm peak is used to measure the cholesterol originally present. Plasma blanks obtained by omitting the cholesterol oxidase from the reagent shown negligible absorbance even when samples are grossly icteric, lipaemic, or haemolysed. The test is carried out in a single glass screw-capped tube, and the absorbance given by a sample containing 6.25 mmol/1 cholesterol is approximately 0.44, corresponding to a molar absorbance of approximately 17 500. The conversion of cholesterol to cholest-4-en-3-one is stoichiometric, and the absorbance of the iso-octane layer is stable for at least 48 hours. A single determination occupies 30 minutes, 30 samples can be analysed in 1 1/2 hours.