Influence of colipase on the turbidimetric determination of pancreatic lipase catalytic activity.

The influence of colipase on the turbidimetric measurement of the catalytic activity of pure human pancreatic lipase (EC 3.1.1.3) and of sera from pancreatitis patients was studied. A deoxycholate-stabilized triolein emulsion served as substrate. It was found that the activity of the pure, colipase-free lipase is strongly inhibited by deoxycholate, and can be blocked completely if normal serum, pure human albumin, or the globulin fraction of normal serum is present. The inhibition by serum is competitive. This finding largely excludes the existence of a specific lipase inhibitor in human serum and explains the non-linear response of activity to the amount of serum added, a frequently observed problem with various turbidimetric lipase methods. A high molar excess of colipase (greater than 250-fold) completely abolishes the inhibition of lipase, irrespective of the inhibitory factor studied. Sera of pancreatitis patients, when measured turbidimetrically without addition of colipase, exhibit elevated lipase activity only if they contain colipase. However, the activity measured is not a function of the serum lipase concentration alone but of the molar ratio of colipase to lipase. Since this ratio varies considerably and is usually too low to ensure complete activation of lipase, erroneously low or even false negative results are obtained. For this reason it is strongly recommended that an excess of colipase is used in turbidimetric lipase assays. It therefore also appears important to study the influence of the serum colipase level on non-turbidimetric lipase methods.

Detection of colipase in serum and urine of pancreatitis patients.

Colipase, like other pancreatic proteins, is liberated into the circulation in acute pancreatitis. Its concentration was measured in serum by a turbidimetric and in urine by a titrimetric method. The principle of both assays is based on the reactivation of bile acid inhibited, pure human pancreatic lipase by colipase. Whereas in healthy individuals colipase was found neither in serum nor urine (detection limit approximately 6.5 micrograms/1), a wide concentration range was observed in 29 patients with acute pancreatitis. Urine values varied between 3.8 and 7121 micrograms colipase/g creatinine; in serum levels up to 664 micrograms/1 were found. There was no correlation with serum lipase activity: On a molar basis, the ratio of serum colipase to serum lipase ranged between less than 0.04 and 2.14, but was below 1 in most sera. Colipase is rapidly removed from the circulation by glomerular filtration, its elimination rate from serum being more than twice as fast as that of lipase. This results in a constant decrease of the colipase/lipase ratio during the course of the disease. Probably determination of colipase is of no direct diagnostic value in pancreatic disorders, but our findings are of considerable significance for the measurement of serum lipase in the presence of bile acids, particularly with regard to turbidimetric assays. We conclude that lipase activity values obtained by these methods are mainly dependent on the degree of saturation of the enzyme with its cofactor and not on the true lipase concentration.

Identification of a non-specific carboxylesterase in human pancreas using vinyl 8-phenyloctanoate as a substrate.

Human pancreatic tissue, pancreatic juice and sera of patients suffering from acute pancreatitis contain a vinyl 8-phenyloctanoate hydrolysing activity which was separated from true pancreatic lipase (EC 3.1.1.3). The enzyme, preliminary called "non-specific pancreatic carboxylesterase, was partially purified from human pancreatic tissue by DEAE-cellulose chromatography. Its molecular weight was found to be 54 000 by gel filtration on Sephadex G-100. The isoelectric point was estimated as 4.65 by isoelectric focusing. The results explain the poor correlation obtained when determinations of "serum lipase activity" using triolein and vinyl 8-phenyloctanoate as substrates are compared. However, since non-specific pancreatic carboxylesterase is liberated into the serum, determination of this new enzyme provides additional information in the diagnosis of pancreatic diseases.

[A glucose dehydrogenase for the determination of glucose concentrations in body fluids (author's transl)]. / Eine Glucose-Dehydrogenase für die Glucose-Bestimmung in Körperflüssigkeiten

The isolation of glucose dehydrogenase from Bacillus megaterium M 1286 is outlined. Data on the specificity of the enzyme towards carbohydrates are given. A specific method for glucose determination using this enzyme was developed. Methods and results of four variants of this glucose determination are presented: End point determination in the UV range, determination with formazan as reaction product, kinetic determination in the UV range, and continuous flow analysis in the UV range (AutoAnalyzer method).