Abstract
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.
Highlights
Aliquots of the enzyme-substrate incubation mixture are extracted into isopropanol
One unit of cholesterol ester hydrolase (CEH) activity is defined as the amount of enzyme catalyzing the disappearance of 1 pmolof cholesterol per hr
A comparison of thedetermination of CEH activity (0-200 units) with the chromatographic radioassay of Vahouny et al [4] and the enzymatic assay described in the present paper is given inFig. 3
Summary
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 A'-cholestenone and hydrogen peroxide (H202)p;eroxidase couples H202 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
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