Combined effect of a lecithin and a bile salt on pancreatic lipase activity.

Lipolysis is regulated by the presence of amphiphilic compounds such as bile salts and lecithin, which are adsorbed at the triglyceride-water interface and therefore influence the approach of water-soluble pancreatic lipase to its insoluble substrate (emulsified triglycerides). The partition of bile salts between the lipid and the aqueous phase is of prime importance in the expression of lipase activity. Lipase activity was determined as a function of different combinations of concentrations of deoxycholate and dipalmitoylphosphatidyl choline. From zeta-potential measurements, it is evident that lecithin affects the partition of bile salts, most probably by displacing deoxycholate molecules. Our results indicate that lecithin cannot be classified a priori as inhibitor or activator of pancreatic lipase. As an amphiphilic compound, lecithin exerts a synergistic effect with bile salts via the formation of mixed micelles. The final effect on lipolysis depends on the ratio of lecithin to bile salt: low ratios enhance enzyme activity, whereas high ratios lead to inhibition.

Kinetics of the two-step hydrolysis of triacylglycerol by pancreatic lipases.

Pancreatic lipases catalyze the hydrolysis of triacylglycerol in a sequential manner. First, triacylglycerol is hydrolyzed to 1,2-diacylglycerol, which is subsequently converted to 2-monoacylglycerol. We studied the kinetics of trioleoylglycerol hydrolysis by rabbit and human pancreatic lipases. The products (acylglycerols and fatty acid) were analyzed by extraction from the reaction mixture, separation by thin-layer chromatography, and quantification by capillary gas chromatography. The first-order rate constants of trioleoylglycerol and dioleoylglycerol hydrolysis were calculated showing that both enzymes hydrolyze dioleoylglycerol faster than trioleoylglycerol. Using rabbit pancreatic lipase, we found that deoxycholate enhanced dioleoylglycerol hydrolysis to a higher degree than trioleoylglycerol hydrolysis. Colipase increased both rate constants similarly at high deoxycholate concentrations (35 mM), while at low concentrations (5 mM) a selectivity toward trioleoylglycerol was observed. From the variation of the rate constants with respect to temperature, we calculated the apparent activation energies of trioleoylglycerol and dioleoylglycerol hydrolysis to be 59.8 kJ.mol-1 and 53.5 kJ.mol-1, respectively. Upon storage, both rabbit and human pancreatic lipases showed a greater loss of activity toward dioleoylglycerol as compared to trioleoylglycerol, suggesting that different conformational elements of the enzyme molecule are responsible for the interaction with each substrate.

Pancreatic lipase assays with triglycerides as substrate: contribution of each sequential reaction to product formation.

Human pancreatic lipase assays are usually performed in the presence of either emulsified triglycerides or diglycerides within the limits of their solubility. Two reactions are catalyzed in the presence of triglycerides: hydrolysis of triglycerides to diglycerides, and diglycerides to monoglycerides. The contribution of each reaction to the final result was determined after extensive kinetic studies on the appearance and/(or) accumulation of intermediates and/(or) products. Acylated glycerides were analyzed after extraction from the reaction mixture, separation of lipid classes by thin-layer chromatography, and quantification by capillary gas chromatography. The results show that after 10 min of reaction in the presence of high concentrations of triolein, 75% of the released fatty acids arise from the first reaction. Relative merits and disadvantages of each substrate (triglyceride or diglyceride) are discussed in terms of practicability.

Transferability of lipase titrimetric assays: deductions from an interlaboratory study.

Following the selection of the most appropriate method for emulsification and the optimization of the reaction medium, interlaboratory studies were conducted to check the effect of preparing substrates and measuring the catalytic concentration of lipase at different sites as well as the effect of transport on emulsion. The determinations of lipase activity in an abnormal chemistry control against emulsions prepared by two laboratories (and used by both laboratories) and, also, against five separate emulsions prepared by one laboratory (and used by five different laboratories) resulted in average enzyme activity values (2234 +/- 125 and 2263 +/- 204 U/l respectively) which are not statistically different. Standard preparations of lipase, control sera and reference materials can therefore be titrated according to the procedure followed by at least two laboratories for at least 3 days against two separate emulsions.

[Titrimetric measurement of catalytic concentration of pancreatic lipase: state of the art]. / La mesure titrimétrique de la concentration catalytique de la lipase pancréatique: état de l'art.

Most lipase routine assays are carried out using either soluble substrates or emulsified substrates (triglycerides or olive oil) at low concentrations. Many of these techniques require a secondary standard, which must be titrated beforehand; the need for a reference method is thus compelling. Titrimetric assays have several advantages such as the possibility of employing high substrate concentrations allowing the direct determination of the product of lipolysis in the absence of interfering phenomena. In a recent study it was demonstrated that human lipase activity depends on the zeta-potential of the lipid droplets, the number of hydroxy groups present in each individual bile salt, the aggregation number and the conjugation of bile salts with taurine or glycine. Hydroxypropyl methylcellulose proposed by Tietz et al is to be preferred to gum arabic for being a pure, well defined emulsifier. Ultrasonic homogenizers enable volumes of oil-in-water emulsions, characterized by fine lipid droplets with good homogeneity to be obtained without overheating. Lipolytic activity is completely inhibited by 70 mmol/l of bile salt (regardless of the type) in the absence of colipase. Variable concentrations of colipase are needed to restore the lipase activity in the presence of different bile salts: optimal cofactor concentrations vary from 0.1 mg/l with deoxycholate or cholate to 6 mg/l with taurocholate or glycocholate. Even after optimization of the medium with colipase, marked differences in enzyme activity are noted depending on the bile salt used.(ABSTRACT TRUNCATED AT 250 WORDS)

Interlaboratory study of the IFCC method for alanine aminotransferase performed with use of a partly purified reference material.

We present the results of a study on performance of a reference material for alanine aminotransferase (ALT, EC 2.6.1.2) and the corresponding IFCC-approved method in an interlaboratory trial involving 13 laboratories. The ALT material was partly purified from pig heart (specific activity, 150 kU/g) and was essentially free of six potentially contaminating enzyme activities, including aspartate aminotransferase (EC 2.6.1.1). The partly purified ALT was lyophilized in a triethanolamine-buffered matrix, pH 6.4, containing bovine serum albumin and saccharose. Under these conditions, the predicted yearly loss of activity was 0.02% at 4 degrees C and < 0.01% at -20 degrees C. The final blank-corrected results of the accepted set of data gave a mean (SD) of 128.5 (5.1) U/L. The among-laboratory SD was 4.6 U/L and the within-laboratory SD was 2.0 U/L. The certified ALT catalytic concentration in the reconstituted material was 129 U/L with a 0.95 confidence interval of +/- 4 U/L.

A mathematical approach to lipolysis based on the interrelationship of physicochemical and biochemical data.

A novel approach is described for the quantitation of the parameters involved in lipolytic activity. The activity of lipase purified from human pancreatic juice against an oil-water emulsion depends on the degree of emulsification, the type of emulsifier used, the type and the concentration of bile salt, the concentration of colipase and, finally, on the zeta-potential of the interface. The zeta-potential, in turn, depends on the partition of bile salt molecules between the micellar state in the bulk and their adsorption on the oil-water interface. None of these parameters correlates straightforward with lipase activity. However, if several parameters are taken into account, the enzyme activity determined in our system obeys a multiparametric equation where the variables are: the conjugation of bile salts, the number of hydroxy groups, their aggregation number and, finally, the zeta-potential of the oil droplets.

Spectrophotometric determination of lipase activity in the presence of increased triolein concentration.

A novel approach for the determination of pancreatic lipase (EC 3.1.1.3) activity by a spectrophotometric method is described. Enzyme activity is measured in the presence of 1 mmol/l triolein, a concentration much higher than usually employed in turbidimetry. The primary reaction medium is optimized as regards bile salt and colipase concentrations. The released fatty acids are enzymatically determined via a secondary reaction scheme using the constituents of a commercially available kit. The proposed method is easy to perform and may prove useful in determining lipase activity of standard lipase preparations, which is required for indirect assays. In addition to satisfactory precision and linearity as well as close correlation with other lipase assays, the procedure described in the present paper by-passes a number of short-comings (eg uncontrolled increase in absorbance) inherent to the turbidimetric methods.

Evidence for multiple lipase forms in the rabbit pancreas.

Evidence is presented of the existence of at least two forms of lipase (A and B) in homogenized rabbit pancreas. These forms are separated by means of gel filtration and anion-exchange chromatography. Both forms are colipase-dependent, but lipase A is activated to a significant extent by 140 mmol/l NaCl even in the absence of the protein cofactor. Lipase A exhibits greater affinity towards emulsified triolein than does lipase B, as evidenced by the respective apparent Km values. Lipase B appears to be more colipase-dependent and resembles more closely the 'pancreatitis' lipase of human plasma. Form B is to be preferred as internal standard in turbidimetric and nephelometric indirect lipase assays.

Lipase activity and properties in serum of chronic alcoholics.

Serum lipase levels are much greater in cases of chronic alcoholics (without any marked symptom of pancreatitis) than in healthy subjects. In fact, about 43 p. cent of the studied samples from alcoholics exhibit a lipase activity above the reference interval (0-160 U/l). On the other hand, the lipase activity present in the serum of alcoholics exhibits different properties than lipoprotein lipase or post-heparin plasma lipase. Furthermore the enzyme from alcoholics presents similar properties to those of the serum lipase released in cases of pancreatitis mainly concerning the sensitivity versus colipase and biliary salt concentration. This similarity with the "pancreatitis" enzyme suggests a pancreatic disorder in number of cases of chronic alcoholics. Therefore, the authors think that serum lipase activity could be taken into account in the evaluation of the risk of any abnormality in the pancreatic function in all alcoholics.

[Urinary lipase: properties and value of its determination]. / Lipase urinaire: propriétés et intérêt de sa détermination.

Lipase activity was detected by nephelometry in urine specimens from 23 patients. Urinary lipase and plasma activities present different sensitivities to changes in ionic strength and copper ion concentration. Experiments with mixtures of urine specimens with and without lipasic activity showed that there is no lipase inhibitor in urine. Urinary lipase is generally seen in patients presenting signs of renal impairment (increase in uremia and creatinemia) and hematuria. The highest urinary lipase levels in such patients are usually seen when blood lipase is increased. High urinary lipase in the absence of hepatic aliments could, therefore, be indicative of renal disorder.

Plasma lipase properties as related to pancreatic condition.

We examined the sensitivity to colipase of two types of lipase (EC 3.1.1.3) activity in plasma. Results were very similar for plasma lipase corresponding to that found in cases of acute pancreatitis and for swine pancreas lipase, whereas we found some differences between "pancreatitis lipase" and lipase from healthy subjects. Gel-filtration experiments suggest that the two forms of lipase in plasma have different relative molecular masses; moreover, their avidity for antibodies against human pancreatic lipase differs. Guided by these studies, we propose optimal conditions for nephelometry of "pancreatitis" lipase.

Differentiating two forms of plasma lipase by use of media with different ionic strengths.

Three different techniques of lipase (EC 3.1.1.3) determination (titrimetry, nephelometry, and enzyme immunoassay) were used to investigate an interesting effect of ionic strength on enzyme activity. Both activation in the presence of NaCl (140 mmol/L) and strong immunoinhibition were observed for lipase from plasma of subjects with acute pancreatitis. Another type of lipase, not associated with pancreatitis and only weakly immunoinhibited, showed maximum activity in the presence of 80 mmol/L NaCl. The pancreatitis-type lipase was activated by 140 mmol/L NaCl solution only if colipase and sodium deoxycholate were also present, which suggests that these components are cofactors in the activating effect and confirms the specificity of this property. These findings can be exploited to improve the diagnosis of acute pancreatitis.

[Value of blood lipase determination in pancreatic diseases. Comparison with blood amylase assay]. / Intérêt de la détermination de la lipasémie au cours des affections pancréatiques. Comparaison avec le dosage de l'amylasémie.

The comparative diagnostic value of plasma lipase and amylase levels was evaluated in 90 patients with various symptoms of pancreatic disease. Comparison with healthy subjects showed that the lipase assay was more sensitive than the amylase assay in patients with acute pancreatitis or post-traumatic pancreatic injury, but not in patients with chronic pancreatitis. On the other hand, acute pancreatitis can be detected earlier by measuring plasma amylase levels, owing to the rapid release of this enzyme. The relative importance of these two laboratory tests is discussed.

[Occlusion of the pancreatic ducts with "Ethibloc" injections. Experimental study in the dog]. / Occlusion des canaux pancréatiques par injection d' "Ethibloc". Résultats d'une étude expérimentale chez le chien.

"Ethibloc" is a zeine-alcohol suspension which polymerizes in an aqueous medium after approximately 15 minutes. The product was injected in the pancreatic duct of 20 dogs. The procedure was free from complications, apart from the development of edematous pancreatitis without clinical manifestations. Healthy pancreases were transformed into fibrous organs within ten days or so, as is found in advanced stages of chronic pancreatitis. No effect occurred on the islets of Langerhans and a diabetes of the experimental type did'nt develop. "Ethibloc" would therefore appear to be of value in humans for the treatment of chronic pancreatitis, in order to reduce the period for transformation of the lesions without increasing the incidence of complications, and also for pancreatic fistulae of various origins, as a result of the rapid sclerosis of exocrine tissue that it provokes.