Lipoxygenic micromethod for specific determination of lipase activity in serum and duodenal fluid.
We propose a rapid enzymatic micromethod for the specific determination of lipase (EC 3.1.1.3) activity in serum and duodenal fluid. Free linoleic acid produced during 10-min incubation of 10 mul of sample with 1 ml of substrate (trillinolein emulsion) at 30 degrees C is converted by lipoxygenase (EC 1.99.2.1), in a coupled reaction, to its hydroperoxide, which is measured photometrically after solubilizing the reaction mixture in ethanol. Lipase activity is calculated from the rate of hydroperoxide formation, with linoleic acid as primary standard. The velocity of the reaction is greatest at pH 8.8, 35-37 degrees C, and a deoxycholate concentration of 3.6 mmol/liter. The energy of activation is 6.7 kcal/mol. The differing "apparent" Km values obtained for lipase in undiluted serum (4 X 10(-5) mol/liter) and in albumin-based diluents (1 X 10(-5) mol/liter) indicate the presence of a competitive inhibitor in the serum matrix. We detected no lipase activity in urine. Results by the proposed method correlate well with those by a copper soap extraction method (r = 0.95), but values are significantly higher for pancreatitis patients' sera (slope 1.6). The linear dynamic range extends to 1000 U/liter. Hemolysis, lipemia, and hyperbilirubinemia do not interfere. The normal range is 40-60 U/liter. Lipase activity of pancreatitis patients generally exceed 1000 U/liter during the acute phase and 250 U/liter for as long as 10 days after it.
Lipoxygenic micromethod for specific determination of lipase activity in serum and duodenal fluid. We propose a rapid enzymatic micromethod for the specific determination of lipase (EC 3.1.1.3) activity in serum and duodenal fluid. Free linoleic acid produced during 10-min incubation of 10 mul of sample with 1 ml of substrate (trillinolein emulsion) at 30 degrees C is converted by lipoxygenase (EC 1.99.2.1), in a coupled reaction, to its hydroperoxide, which is measured photometrically after solubilizing the reaction mixture in ethanol. Lipase activity is calculated from the rate of hydroperoxide formation, with linoleic acid as primary standard. The velocity of the reaction is greatest at pH 8.8, 35-37 degrees C, and a deoxycholate concentration of 3.6 mmol/liter. The energy of activation is 6.7 kcal/mol. The differing "apparent" Km values obtained for lipase in undiluted serum (4 X 10(-5) mol/liter) and in albumin-based diluents (1 X 10(-5) mol/liter) indicate the presence of a competitive inhibitor in the serum matrix. We detected no lipase activity in urine. Results by the proposed method correlate well with those by a copper soap extraction method (r = 0.95), but values are significantly higher for pancreatitis patients' sera (slope 1.6). The linear dynamic range extends to 1000 U/liter. Hemolysis, lipemia, and hyperbilirubinemia do not interfere. The normal range is 40-60 U/liter. Lipase activity of pancreatitis patients generally exceed 1000 U/liter during the acute phase and 250 U/liter for as long as 10 days after it.