Assay Procedure for Cholesterol Esterase

Cholesterol esterase is a reversible enzyme that can hydrolyze or synthesize fatty acid esters of cholesterol and other sterols. It also hydrolyzes tri-, di-, and mono-acylglycerols, phospholipids, lysophospholipids, and ceramide. Cholesterol esterase may have multiple functions in lipid and lipoprotein metabolism, and atherosclerosis.

PRINCIPLE

The appearance of quinoneimine dye formed when coupled with 4-aminoantipyrine and phenol is measured at 500 nm by spectrophotometry.

Unit definition

One unit causes the formation of one micromole of hydrogen peroxide (half a micromole of quinoneimine dye) per minute under the conditions described below.

Method

Reagents
A. 0.2 M Potassium-Phosphate buffer, pH 7.0
B. Cholesterol linoleate solution	To 39 mg of cholesterol linoleate, add 2 mL of isopropanol and dissolve completely by heating slightly. Mix with ∼ 80 mL of 1.0% (v/v) hot Triton X-100 solution (preheated to 72-74 ℃) to the cholesterol linoleate solution and keep the solution in a hot water bath (72-74 ℃), stirring for 30 minutes. The solution will turn clear and then cloudy. Cool under running water with gentle agitation until temperature of the solution adjusts to room temperature. Add 600 mg of Na-cholate and dissove. Fill to 100 mL with 1.0% Triton X-100 solution. This solution is stable at 4 ℃ for at least 5 days.
C. 4-AA solution	1.76% (1.76 g 4-aminoantipyrine/100 mL of H2O) – store at 4 ℃ in a brownish bottle
D. Phenol solution	6.0% (6.0 g phenol/100 mL of H2O) – store at 4 ℃ in a brown bottle
E. POD solution	Horseradish peroxidase 7,500 purpurogallin units/50 mL of 0.1 M potassium-phosphate buffer, pH 7.0 (150 PU/mL) – prepare fresh
F. COD solution	Streptomyces sp. cholesterol oxidase 1,500U/5.0 mL of ice-cold H2O (300 U/mL) – should be prepared fresh
G. Enzyme diluent	20 mM potassium-phosphate buffer, pH 7.5 containing 2mM MgCl2, 0.5mM EDTA-Na3 and 0.2% BSA

Procedure

1. Prepare the following working solution (50 tests) in a brown bottle.
   75 mL Buffer solution (A)
   50 mL Substrate solution (B)
   2.5 mL 4-AA solution (C)
   5.0 mL Phenol solution (D)
   5.0 mL POD solution (E)

Concentration in assay mixture

Potassium-Phosphate buffer	0.11 M
Cholesterol linoleate	0.20 mM
4-Aminoantipyrine	1.5 mM
Phenol	22 mM
EDTA	17 μM
Isopropanol	0.68 %
COD	ca.10 U/mL
POD	ca. 5.1 U/mL

1. Pipette 2.75 mL of working solution into a cuvette (d＝1.0 cm) and equilibrate at 37 ℃ for about 5 minutes. Add 0.1 mL of COD solution (F), mix and keep at 37 ℃ for another 2 minutes.
   
2. Add 0.1 mL of the enzyme solution* and mix with gentle inversion.
   
3. Record the increase in optical density at 500nm against water for 3 to 4 minutes in a spectrophotometer thermostated at 37 ℃, and calculate the ΔOD per minute from the initial linear portion of the curve (ΔOD test).
   

At the same time, measure the blank rate (ΔOD blank) using the same method as the test except that the enzyme diluent (G) is added instead of the enzyme solution.

* Dissolve the enzyme preparation in ice-cold enzyme diluent (G), and dilute to 0.08－0.22U/mL with the same buffer, immediately before assay.

Calculation

Activity can be calculated by using the following formula：

Vt	Total volume (2.95 mL)
Vs	Sample volume (0.1 mL)
13.78	Millimolar extinction coefficient of quinoneimine dye under the assay conditions (F/micromole)
1/2	Factor based on the fact that one mole of H2O2 produces half a mole of quinoneimine dye
1.0	Light path length (cm)
df	Dilution factor
C	Enzyme concentration in dissolution (c mg/mL)