Ottimizzazione di un metodo di misura della lipasi pancreatica con 1,2-o-dilauril-rac-glicero-3-glutarico (6-metilresorufina) estere come substrato

AUTORI

ABSTRACT

Optimization of a 1,2-o-dilauryl-rac-glycero-3-glutaric acid (6-methylresorufin) ester method for the measurement of pancreatic lipase catalytic activity.

The measure of the catalytic activity of lipase in serum is a good indicator of acute pancreatitis. Today there is not an accepted reference method. Aim of this work was to optimize a method for measurement of catalytic activity of pancreatic lipase in serum, which could have the necessary characteristics to serve as a candidate reference method. Serum pools with different concentrations of lipase in native and inactivated form (heat inactivation at 56 °C for 1 h) and control materials were used as samples. Optimal concentrations in the final reaction mixture were obtained through a series of experiments. Optimization was reached when higher catalytic activity and lower nonspecific signal were obtained. The following conditions were tested: buffer type and concentration, concentration of bile salts (taurodeoxycholate and deoycholate), concentration of calcium chloride, effect of different surfactants, substrate concentration, colipase concentration, and pH. The optimization experiments lead to the following reaction conditions (concentrations in the final reaction mixture): pH, 8.0; taurodeoxycholate, 21,9 mmol/L; deoxycholate, 5,28 mmol/L; calcium chloride, 6,43 mmol/L; colipase, 2,24 mg/L; 1,2-o-dilauryl-rac-glycero-3-glutaric acid (6-methylresorufin) ester, 0,11 mmol/L; Triton X-100, 0,75 g/L. Using the herein described measurement conditions, CV ranging from 1,4% to 2,9% were obtained. A comparison with the assay used in our clinical laboratory using the same substrate gave the following regression equation: y=1.56x + 2,4 U/L, r2=0.982.

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