The amount of enzyme causing the liberation of one micromole of glycerol per minute at 37°C and pH 7.0.
Assay Method
The assay is based on a 4-step enzyme-coupled reaction. In the final step of the sequence, a dye is formed which can be measured spectrophotometrically at 490 nm. The increase in absorbance at 490 nm is directly proportional to lipase activity.
Applications
Lipase (EC 3.1.1.3) hydrolyses emulsified triglycerides of the long chain fatty acids. Lipase is active at the interface between the oil drops and the aqueous phase. In normal serum the concentration of lipase is low. In acute pancreatitis and in pancreatic carcinoma a rise in serum lipase activity occurs, with a mean increase being 50 times that of normal values. A rise in the serum lipase content is also found in acute and chronic renal diseases.
Reagents
0.1 M Potassium phosphate buffer, pH 7.0.
0.05 M 4-Aminoantipyrine-HCl (12 mg/ml) in distilled H2O.
0.05 M 1,7-Dihydroxynaphthalene (8 mg/ml absolute ethanol).
0.05 M Magnesium sulfate (6.02 g/1000 ml) in distilled H2O.
0.05 M ATP (30.3 mg/ml) in distilled H2O.
Peroxidase solution (588 U/ml). Dilute in 0.05 M phosphate buffer, pH 7.0 to a final concentration of 588 U/ml.
Glycerokinase solution (36 U/ml). Dilute in 0.05 M phosphate buffer, pH 7.0 to a final concentration of 36 U/ml.
a-Glycerophosphate Oxidase solution (700 U/ml). Dilute in distilled H2O to a final concentration of 700 U/ml.
Triolein emulsion. Mix 300 mg triolein/10.6 g Triton X-100 and heat until a single phase appears. Add 90 ml of distilled H2O and mix.
Reaction mixture. Mix together 0.128 ml 4-Aminoantipyrine, 0.064 ml 1,7-Dihydroxynaphthalene, 0.128 ml peroxidase, 12.4 ml 0.1 M phosphate buffer, pH 7.0, 0.150 ml glycerokinase, 0.32 ml a-glycerophosphate oxidase, 0.375 ml triolein emulsion, 0.375 ml magnesium sulfate and 0.375 ml ATP. Equilibrate at 4¡C for one hour before use. Stable for 8 hours at 4¡C.
Procedure
Set spectrophotometer (equipped with strip chart recorder and temperature control) at 490 nm and 37°C.
Into a cuvette pipette 2.95 ml of reaction mixture and equilibrate at 37°C for 10 min.
Dissolve 10 mg of lyophilized lipase in 10 ml of distilled H2O and dilute 1:100 with distilled H2O.
Add 0.05 ml of the lipase solution to the equilibrated cuvette and mix.
Record the change in absorbance at 490 nm for 10 min.
Measure the slope after 5 min. If (delta)A/min. is greater than 0.015, a larger dilution is required. If the (delta)A/min. is less than 0.005, a smaller dilution is required.
