Enzymatic assay for cholesterol ester hydrolase activity

A rapid and accurate method is described for the assay of cholesterol ester hydrolase (CEH) activity. Aliquots of the enzyme-substrate incubation mixture are extracted into isopropanol. The free cholesterol concentration in each extract is determined enzymatically using a single aqueous reagent containing cholesterol oxidase and peroxidase. The free cholesterol remaining after the cholesterol ester hydrolase-catalyzed esterification is converted to delta 4-cholestenone and hydrogen peroxide (H2O2); peroxidase couples H2O2 with phenol and 4-amino-antipyrine to yield a stable rose-colored product absorbing at 500 nm. The method is highly reproducible and the values correlate well with those obtained with the chromatographic radioassay of CEH activity.