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PubMed1992

A PCR-mediated gene synthesis strategy involving the assembly of oligonucleotides representing only one of the strands.

A modification of PCR-mediated gene synthesis strategy is introduced. This modification enables the synthesis of a gene from oligonucleotides comprising only one of the two strands. Bridging oligonucleotides (approximately 20-mers in length) complementary to the junctions of template strand oligonucleotides and two outer primers are also needed for PCR. A two-step PCR containing a first step of 10 cycles, followed by a second step of 20 cycles, differing only in the annealing conditions was used. A single-step PCR combining the two different cycle conditions could also be used successfully. Optimal conditions for gene synthesis (and amplification) are described. Human and porcine colipase genes (297 and 309 bp, respectively) have been successfully synthesized, cloned into the Invitrogen TA cloning vector and sequenced. There was absolutely no error in the clones that were sequenced.

Animals↗
PubMed1988

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.

Acute Disease↗
PubMedDate not supplied

Review: the role of coenzymes in clinical enzymology.

Coenzymes participate in many of the enzyme analyses performed in the clinical laboratory. Supplementation of assay systems with optimal levels of coenzymes has recently been recommended as part of efforts to achieve interlaboratory standardization of enzyme measurements. Aspartate aminotransferase and alanine aminotransferase require pyridoxal phosphate for expression of enzyme activity. The role of this coenzyme in enzymatic transamination and the effects of its supplementation on the clinical estimation of these two enzymes is reviewed. Other coenzymes discussed are flavins, coenzymes for glutathione reductase, glucose oxidase, cholesterol oxidase and diaphorase, as well as thiamine pyrophosphate, coenzyme for transketolase. Catalase and peroxidase are used as examples of hemoproteins utilized in clinical measurements. Two peptide coenzymes, colipase and glutathione, are also considered. Measurement of apoenzyme stimulation upon supplementation with specific coenzymes is discussed as a valuable technique for quantitative coenzyme measurements or assessment of vitamin nutritional status.

Clinical Enzyme Tests↗
PubMed1986

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.

Acute Disease↗
PubMed1987

Turbidimetric measurement of lipase activity--problems and some solutions.

We optimized a commercial turbidimetric method for lipase (EC 3.1.1.3) activity (Boehringer Mannheim Diagnostics) and overcame some of its deficiencies. Increasing the bile salt concentration to 35 mmol/L and the colipase concentration to 6 mg/L and using a continuous recording of the reaction-rate curve greatly improved the reaction kinetics, eliminated false results from increases in absorbance, reduced the lag phase, and increased the analytical sensitivity and accuracy. Differentiation of pancreatitis from nonpancreatitis sera by adding NaCl, 140 mmol/L, to the assay mixture to observe the degree of enzyme activation has important limitations. Sera from patients with pancreatitis and only slight or modest increases in lipase behave like sera from healthy individuals or from patients with nonpancreatic disease. The assay shows no interference by lipoprotein lipase and carboxyl esterase. Results compare well by this optimized method and by an optimized "pH-Stat" titrimetric method.

Adult↗
PubMed1985

Lipid digestion and absorption.

The importance of nutrient lipids to the developing preterm and term infant has served to focus investigations upon the mechanisms of fat digestion and absorption. The unique physical-chemical properties of the human milk fat globule have been reviewed. Emphasis is placed on the newer understandings that have emerged concerning the intraluminal phase of triglycerides hydrolysis and solubilization of the lipolytic products. The mechanisms of action of the pancreatic lipase, colipase, and phospholipase A2 system, and the importance of intragastric lipolysis for initiating the lipid digestion are explored. Lastly, aspects of bile acid micelle formation and its role on nutrient lipid solubilization and absorption are examined in view of the postnatal adaptations that occur with weaning and the introduction of alternative nutrient sources to the older infant.

Bile Acids and Salts↗
PubMed1979

Fat necrosis.

Fat necrosis has been found to be associated with many forms of pancreatitis, carcinoma of the pancreas and pancreatic trauma. The causative agents seem to be pancreatic lipase and colipase, which presumably escape from the pancreas during the development of the disease. The precise mechanism by which these factors attack the adipose tissue, leading to the formation of foci of fat necrosis, is not known. The pathologic finding of fat necrosis is not restricted to the peritoneal-retroperitoneal region, where a direct contact with these factors is the most likely cause. In other patients, fat necrosis involves peripheral tissues, notably in subcutaneous adipose tissue throughout the body, in joints of the hand and foot and in bone marrow. This is associated with additional complications dependent upon the sites involved and is manifested as skin lesions, polyarthritis and osteolytic defects in patients who sometime suffer from a primary pancreatic disease.

Acute Disease↗
PubMed2000

Roles of molecular chaperones in pancreatic secretion and their involvement in intestinal absorption.

This review focuses on the contribution of molecular chaperones in the secretory process of digestive enzymes and their interaction with enterocytes. By using biochemistry and immunocytochemistry, we have shown that Grp94, Cpn10, Cpn60, and protein disulfide isomerase (PDI) are present all along the rough endoplasmic reticulum-Golgi-granule secretory pathway of the pancreatic acinar cells and are secreted into the acinar lumen. Two other molecular chaperones, Grp78 and the Hsp70, appear to be restricted to the rough endoplasmic reticulum and the trans-Golgi apparatus, respectively. We have found that chaperones can be associated with pancreatic enzymes along the secretory pathway. Indeed, double immunogold and immunocoprecipitation revealed an association between Cpn60 and the colipase-dependent lipase (CDL) and between Grp94 and the bile salt-dependent lipase (BSDL). These complexes are secreted into the acinar lumen and diverted to the duodenal lumen. These findings led us to investigate these enzyme-chaperone complexes in intestinal tissue. Grp94, Cpn60, and PDI are present on microvilli and on the endosomal compartment of enterocytes. Furthermore, we have shown that the Grp94-BSDL complexes are internalized by enterocytes through classical endocytosis. Upon dissociation of the BSDL-Grp94 complex in the late endosome, BSDL is transferred to the basolateral membrane. We propose that Grp94 interacts with specific receptors and/or could force the associated protein to adopt a specific conformation that allows its binding to corresponding membrane receptors and its internalization by enterocytes. These two hypotheses need not to be exclusive. The existence of such a pancreatic secretion-intestinal absorption link speaks in favor of a coordinated functional connection between these two entities, through molecular chaperones, in order to optimize intestinal activities.

Animals↗
PubMed2005

Kinetic properties of turkey pancreatic lipase: a comparative study with emulsified tributyrin and monomolecular dicaprin.

Using the classical emulsified system and the monomolecular film technique, we compared several interfacial properties of turkey pancreatic lipase (TPL) and human pancreatic lipase (HPL). TPL, like HPL, presented the interfacial activation phenomenon when vinyl ester was used as substrate. In the absence of colipase and bile salts, using tributyrin emulsion or monomolecular films of dicaprin at low surface pressure, TPL, unlike HPL, hydrolyzes pure tributyrin emulsion as well as dicaprin films maintained at low surface pressures. TPL was also able to hydrolyze triolein emulsion in the absence of any additive and despite the accumulation of long-chain free fatty acids at the interface. The difference of behaviors between TPL and HPL can be explained by the penetration power of each enzyme. The enzyme that presents the maximal pi(c) (TPL) interacts more efficiently with interfaces, and it is not denaturated at high interfacial energy. Turkey pancreatic lipase is more active on rac-dicaprin than HPL; a maximal ratio of 9 was found between the catalytic activities of the two lipases measured at their surface pressure optima (20 mN m(-1)). A kinetic study on the surface pressure dependency, stereospecificity, and regioselectivity of TPL was performed using enantiopure diglyceride (1,2-sn-dicaprin and 2,3-sn-dicaprin) and a prochiral isomer (1,3-dicaprin) that were spread as monomolecular films at the air-water interface. At low surface pressure (15 mN m(-1)), TPL acts preferentially on primary carboxylic ester groups of the diglyceride isomers (1,3-dicaprin), but at high surface pressure (23 mN m(-1)), this enzyme prefers both adjacent ester groups of the diglyceride isomers (1,2-sn-dicaprin and 2,3-sn-dicaprin). HPL prefers adjacent ester groups of the diglyceride isomers (1,2-sn-dicaprin and 2,3-sn-dicaprin). Furthermore, TPL was found to be markedly stereospecific for the sn-1 position of the 1,2-sn-enantiomer of dicaprin at low surface pressure (15 mN m(-1)), while at high surface pressure (23 mN m(-1)), this lipase presents a stereopreference for the sn-3 position of the 2,3-sn-enantiomer of dicaprin. HPL is stereospecific for the sn-1 position of the 1,2-sn-enantiomer of dicaprin both at 15 and 23 mN m(-1).

Animals↗
PubMed1991

Assessment of pancreatic function in screened infants with cystic fibrosis.

Previously we have reported that 37% of infants with cystic fibrosis diagnosed by neonatal screening with the dried blood spot immunoreactive trypsin assay have pancreatic sufficiency. However, 34 of the 78 infants had pancreatic function tests an average 2.3 years after diagnosis, thus it was possible that the percentage with neonatal pancreatic sufficiency was underestimated, due to the loss of pancreatic function with time in some infants. To assess this hypothesis we have assessed pancreatic function at the time of diagnosis in a further 20 infants since the completion of the previous study. Results of fecal fat determinations and/or pancreatic stimulation tests indicate that 10 (50%) of these infants have pancreatic sufficiency. Combining these results with those of the previous study, 31 of 64 patients (48%) have pancreatic sufficiency at this early age. We have also monitored the progression of pancreatic disease in the 39 patients with pancreatic sufficiency recognized to date. Eleven have developed pancreatic insufficiency and require enzyme replacement therapy. Five others have shown further improvement of colipase secretion with age. We conclude that the dried blood immunoreactive trypsin screening program for cystic fibrosis does recognize patients with pancreatic sufficiency, and at diagnosis nearly half the patients are in this category. To date, 28% of patients with pancreatic sufficiency have demonstrated a variable decline in pancreatic function with age.

Cystic Fibrosis↗
PubMed1998

Human pancreatic triglyceride lipase expressed in yeast cells: purification and characterization.

A cDNA clone encoding human pancreatic triglyceride lipase was cloned into a yeast expression vector so that the yeast PHO1 signal peptide replaced the native signal peptide. Pichia pastoris cells were transfected with the vector, and clones expressing human pancreatic triglyceride lipase were isolated. Recombinant human pancreatic lipase was expressed in broth cultures and was purified from the medium by DEAE blue Sepharose and hydroxyapatite chromatography. The highly purified lipase had specific activities for various triglyceride substrates identical to those of tissue-purified human pancreatic triglyceride lipase; it was inhibited by bile salts, required colipase for activity, and demonstrated interfacial activation. This expression system is suitable for the rapid, efficient production of human pancreatic triglyceride lipase in amounts adequate for biophysical studies.

Amino Acid Sequence↗
PubMed1995

Development and regulation of porcine pancreatic function.

A surgical and experimental procedure was developed to enable the collection of pure and inactivated pancreatic juice during the growth of the pig. Studies have shown that, during the suckling period, both the basal and the secretory responses to suckling are low, if present at all. After weaning, basal levels of the total exocrine secretion, total protein, amylase, and trypsin, respectively, increase slightly, while the postprandial levels of total protein, amylase, trypsin, lipase, colipase, and carboxylester lipase, respectively, increase markedly. The pancreatic juice enzyme composition changes qualitatively and the antibacterial activity of the pancreatic juice also significantly increases. Piglet age appeared to be of minor importance, since weaning at either 4 or 6 wk of age gave the same results. Secretin and CCK administered together in supraphysiological doses only significantly affect exocrine function from 3-4 wk of age. However, CCK may also affect the exocrine pancreas indirectly via reflexes initiated intraduodenally. Milk consumption in the suckling pig leads to a postprandial increase in glucose levels but not insulin. Milk appears to be able to regulate the exocrine pancreas to produce only the amount and type of enzymes required for digestion. Thus, milk components or digestive products may affect pancreas function regulation. Studies show that enterostatin, the procolipase activation peptide, may inhibit pancreatic secretion mediated indirectly through the GI tract. Pancreastatin, an endocrine peptide, inhibits both insulin secretion and protein and trypsin secretion to pancreatic juice. In hypoinsulinemic (alloxan+streptozotocin diabetes) pigs (15-20 kg), no postprandial pancreatic juice response is seen, although CCK 33 + secretin can stimulate pancreatic secretion. Hypoinsulinemic pigs have a reduced capacity for glucose tissue utilization, suggesting that tissue metabolism and exocrine pancreas secretion are related.

Animals↗
PubMed1987

Interfacial interactions between proteins and mammalian lipases.

The effects of proteins, both endogenous and exogenous, on the activity of lipases against water soluble and water insoluble substrates have been reviewed. The enzymes considered are pancreatic and gastric lipases, and lipoprotein, bile-salt-stimulated human milk and pancreatic carboxyl ester lipases. A brief account is given of the function of each enzyme and of the physical properties of the interacting proteins, which include albumins, lysozymes, globulins and immunoglobulins, myoglobin, transferrins, alpha-lactalbumin and melittin. With few exceptions (for example, the effect of colipase on pancreatic lipase), the interaction of proteins with lipases which act at the lipid-water interface of water insoluble substrates results in deactivation of enzymic activity. It seems that the amphiphilic nature of proteins allows them to aggregate at interfaces, thereby altering the nature of the interface and decreasing accessibility of the substrate to the enzyme. This discussion gives consideration to association of the proteins with the enzyme or the interface and to whether the interactions with specific binding sites or interfacial inactivation are responsible for the observations. However, the effect of proteins on lipases acting against water soluble substrates varies from protein to protein. Activation of enzyme-activity occurs if the interacting proteins are able to act as acyl transfer agents and thus introduce another catalytic hydrolysis pathway into the reaction mechanism. Inhibition may be caused by specific interactions between the protein and the enzyme or the substrate.

Animals↗
PubMed1980

Characterization of triacylglycerol lipase activity in human amniotic fluid.

A radiochemical assay was used to measure the triacylglycerol lipase activity found in normal human amniotic fluid at term. Enzyme activity was characterized in a partially purified extract of amniotic fluid and was found to be optimal at pH 8.0 +/- 0.2 in the presence of 5mM sodium taurocholate, with the use of emulsified tri-[3H]oleoyl glycerol as the substrate. The assay described made it possible to determine the lipase activity in as little as 25 microliters of a 12,000 x g supernatent of whole amniotic fluid as the source of enzyme. The lipase appeared to be distinct from another triacylglycerol lipase measurable in fetal membranes. In turn, it was shown that the amniotic fluid enzyme exhibited several catalytic properties which resembled those of pancreatic lipase. i.e., its substrate specificity, the bimodal effect of bile salt, and the influence of authentic pancreatic colipase on the catalytic process. The results suggest that the assay of triacylglycerol lipase activity may be clinically useful in the detection of enzyme abnormalities in human amniotic fluid.

Amniotic Fluid↗
PubMed1978

An improved large scale procedure for the purification of porcine pancreatic lipase.

A modified procedure for purifying porcine pancreatic lipase (triacyglycerol acyl-hydrodrolase, EC 3.1.1.3) is described. In comparison to the previous procedure reported by Verger, R., de Hass, G.H., Sarda, L and Desnuelle, P. (1969) Biochim. Biophys. Acta 188, 272--282) it is more rapid, more reproducible and results in a purer enzyme preparation. No colipase could be detected in the mixture of isoenzymes and, naturally, in the different separated lipases. In this process, butanol treatment is omitted. After pancreas powder extraction, a batch procedure was used for adsorption of DEAE-cellulose. Sephadex filtration (pH 8.0) was made in a largeer size column. Finally the isoenzymes were separated on CM-cellulose as in the Verger procedure, but under slightly modified conditions. Lipase LB was fully homogeneous as judged by end group determination, gel electrophoresis (in the presence of absence of sodium dodecyl sulfate) and sedimentation equilibrium.

Animals↗
PubMed1976

On the binding of bile salt to pancreatic lipase.

The binding of taurodeoxycholate to pancreatic lipase and a few other proteins has been studied with equilibrium dialysis and in gel filtration experiments. A three compartment dialysis cell has been used; with this cell, complete equilibration is not necessary for calculation of the binding even at bile salt concentrations above the critical micellar concentration. The results indicate that taurodeoxycholate does not bind to lipase below the critical micellar concentration, that the binding starts in the critical micellar concentration range of the bile salt and reaches around 12 mol taurodeoxycholate per mol of lipase at taurodeoxycholate concentrations well above the critical micellar concentration. Previous results indicating a binding of maximally 1-2 mol taurodeoxycholate/mol lipase were too low, depending on the experimental conditions in which complete equilibration was not obtained. The binding isotherm for taurodeoxycholate to lipase is similar to that for co-lipase; colipase and lipase in mixture bind as much taurodeoxycholate as the sum for the single proteins. Taurodeoxycholate binds to ribonuclease and chymotrypsinogen to a similar extent as to lipase.

Animals↗
PubMed1980

In vitro effect of adrenocorticotrophic hormone on the pH profile of tri- and diester lipase activities from rat brain.

The influence of adrenocorticotrophic hormone (ACTH) on the hydrolysis of tri- and dioleoylglycerol by a cerebral lipase was studied using a synaptosomal preparation from rat brain as source of enzyme. When ACTH was added to the lipolytic medium, it caused a marked shift of the optimum pH values of catalysis towards alkaline values. In the pH range 5.8-6.5, these shifts resulted in reaction rates 5- to 20-fold higher, depending on the experimental conditions, than those measured without hormone. The ACTH effect was dependent on the NH2-terminal sequence extending through the amino acid residues 15-18. The results suggest that the hormonal influence is specific and mediated through a colipase-like effect.

Adrenocorticotropic Hormone↗
PubMed1980

Effect of bile lipids on the adsorption and activity of pancreatic lipase on triacylglycerol emulsions.

Emulsions of natural triacylglycerols obtained with different shear forces were used to study lipase adsorption and lipolysis. The influence of the bile lipoprotein complex on these two processes was determined. Optimal lipase activity was observed to occur with a given phospholipid : triacylglycerol ratio. This ratio depended on the degree of triacylglycerol emulsification and was accompanied by maximal adsorption of the bile lipoprotein complex. These results support our previous model for pancreatic lipolysis under physiological conditions, according to which colipase controls lipase binding to the bile lipoprotein complex and the resulting association directs enzyme adsorption to the acylglycerol particle (Lairon, D., Nalbone, G., Lafont, H., Léonardi, J., Domingo, N., Hauton, J.C. and Verger, R. (1978) Biochemistry 17, 5263--5269).

Adsorption↗