Apr 1, 1970
Clinica chimica acta; international journal of clinical chemistry
Abstract A method is described for the photometric determination of lipase activity by measuring the release of H+ ion from a micellar solubilized substrate. The H+ generation is measured photometrically by the use of an indicator. 1. 1. The change in pH in an ammonium acetate buffer is directly proportional to H+ ion addition. The change in pH is measured between pH 7.3 and 7.0. 2. 2. Bromcresolpurple is used as an indicator at a concentration of 0.125 mM. The change of light absorption is measured at 436 nm wave length. The addition of 0.595 μequiv. H+ ion to the incubation medium is followed by a change in extinction (ΔE) of 0.125. 3. 3. Di-olein at a concentration of 4.3 μnmole/ml is used as substrate. It is solubilized in a solution containing 20 μmole/ml of sodium taurodesoxycholate and 18 μmole/ml of mono-olein. The velocity of the reaction varies with the enzyme concentration and is constant for a long period of time. The pH optimum is 7.2. Enzyme activity varies with temperature and substrate concentration. Enzyme saturation cannot be reached within the solubility range of the substrate. An increase in ionic strength lowers the rate of hydrolysis. Addition of calcium ions stimulates enzyme activity. 4. 4. By using esterase inhibitors and highly purified lipase it has been shown that the hydrolytic activity is due to the action of a pancreatic lipase. 5. 5. Esterase from rat liver, carboxypeptidase A, chymotrypsin and trypsin are completely inactive in hydrolysing solubilized di-olein and mono-olein.