Cholesterol Esterase Assay

Method: The initial rate of hydrolysis of cholesterol ester is determined by following the increase in absorbance at 505 nm. One unit hydrolyzes 1 μmole of cholesterol ester per minute at 37°C and pH 7.0 under the specified conditions.

Reagents

• 0.1 M Phosphate buffer, pH 7.0
• 0.7 mM 4-aminoantipyrine in 0.1 M Phosphate buffer, pH 7.0. Discard after 8 hours.
• 5.0 mM Phenol in 0.1 M Phosphate buffer, pH 7.0. Discard after 8 hours. Use only fresh, white phenol crystals.
• 5% Triton X-100. Store at 4°C.
• Sodium Cholate: Dissolve 3.51 g Sodium cholate in 10.0 ml reagent grade water. Discard after 8 hours.
• Cholesterol Oxidase: Dissolve 28.5 units cholesterol oxidase/ml in reagent grade water. Discard after 8 hours.
• Peroxidase: 8200 u/ml in reagent grade water. Discard after 8 hours.
• Cholesteryl Acetate. Dissolve 220 mg Cholesteryl Acetate (Eastman) in 50.0 ml Isopropanol (ACS Grade). Stir vigorously to dissolve. Store at 4° C.

Enzyme

Dissolve one mg/ml in 0.1 M Phosphate buffer and then prepare further dilutions (usually 2-3X) to achieve a rate of approximately 0.045/minute.

Procedure

Adjust spectrophotometer to 505 nm and 37°C. Prepare master batch, sufficient for 5 tests, by pipetting into a beaker:

0.7 mM 4-aminoantipyrine	7.0 ml
5.0 mM Phenol	7.0 ml
Sodium Cholate	0.5 ml
5% Triton X-100	0.5 ml
Cholesterol Oxidase	0.25 ml
Peroxidase	0.05 ml

Mix gently. Stored at 4°C, master batch is stable for 8 hours.

Pipette into test and blank cuvettes 3.0 ml master batch. Add 0.10 ml cholesterol acetate. Mixture should be used within 1 hour. Equilibrate temperature to 37°C. Add to test cuvettes, 0.1 ml enzyme dilution and 0.1 ml reagent grade water to the blank. If the blank rate exceeds 0.005 ΔA per minute, reincubate for 5 more minutes. Determine ΔA₅₀₅ of test and blank cuvettes using the linear portion of the first 5 minutes of the reaction. Subtract blank rates, if any.

Calculation