Lipase Units Research Articles

Genetic determinants of gastrointestinal, hepatic and pancreatic involvement in adult patients with cystic fibrosis

This study assessed a) the potency of therapeutic doses of pancreatin to elicit feedback inhibition of enzyme secretion, b) putative underlying mechanisms, and c) the effect of exogenous enzymes on antroduodenal motility and gastric acid secretion. Methods: In 12 healthy male volunteers, we duodenally perfused pancreatin powder equivalent to 40,000 or 80,000 lipase units for 60 interdigestive min and, in parallel to a mixed liquid meal (2 kcallmin), for 120 postprandial min. Per 10,000 U lipase, the powder contained 8,000 U amylase and 600 U proteases. Loss of exogenous enzymes during duodenal passage was accounted for. Results: Both loads of pancreatin markedly reduced interdigestive and postprandial outputs of amylase, lipase, trypsin, chymotrypsin, and elastase. They did not interfere with interdigestive or postprandial CCK and PP release. gastric acid secretion and interdigestive motility apart from the low load reducing interdigestive IR-PP. Only the high load distinctly diminished postprandial contractile activity of antrum, duodenal bulb, and descending duodenum. Conclusions: Therapeutic duodenal loads of pancreatin elicit feedback inhibition of interdigestive and postprandial enzyme secretion. The mediatory effects of duodenopancreatic reflexes appear to outweigh those of CCK and vagal input. The cyclical pattern with secretory peaks is maintained with a reduction in nadir enzyme outputs. The unequivocal inhibition of fed antroduodenal motility with the high enzyme load appears to be independent of the feedback mechanism.

What to Do when a Patient with Exocrine Pancreatic Insufficiency Does Not Respond to Pancreatic Enzyme Substitution

In the majority of patients, pancreatin microsphere preparations containing 25,000–40,000 FIP units of lipase will lead to a clinical improvement and reduction of steatorrhea in patients with exocrine pancreatic insufficiency. In patients failing to respond, the following stepwise procedure is recommended: increasing enzyme dosage; checking the patient’s compliance; reevaluating the diagnosis; and as a last resort to decrease fat intake or adding H₂-blockers or proton pump inhibitors. Although this stepwise procedure is time-consuming, it is necessary to prevent in patients not responding to pancreatic enzyme substitution the development of late complications of exocrine pancreatic insufficiency.

Effect of Exogenous Lipase on Dough Lipids During Mixing of Wheat Flours

ABSTRACTIn control dough, endogenous wheat lipase was inactive, because the triacylglycerol (TAG), 1,2‐diacylglycerol (DAG1,2), and 1,3‐diacylglycerol (DAG1,3) fractions of nonpolar lipids were not affected by mixing. Conversely, the free fatty acid (FFA) and monoacylglycerol (MAG) fractions decreased, mainly due to the oxidation of polyunsaturated fatty acids (PUFA) catalyzed by wheat lipoxygenase. Addition of exogenous lipase to flour (15 lipase units [LU] per gram of dry matter) resulted in substantial modification of nonpolar lipids during dough mixing. Due to the 1,3 specificity of the lipase used in this experiment, the TAG and DAG1,3 fractions decreased, whereas the MAG and FFA fractions increased. The DAG1,2 fraction increased at the beginning of mixing and decreased after 40 min of mixing. Moreover, part of the PUFA released by lipase activity was oxidized by wheat lipoxygenase, resulting in major losses of PUFA. Conversely, the net content of the saturated and monounsaturated fatty acids (SMUFA) remained constant, because the free SMUFA content increased primarily at the expense of the esterified forms. For a constant mixing time of 20 min, increasing the amount of lipase added to dough (from 2.5 to 25 LU/g of dry matter) resulted in a linear decrease in the TAG fraction and a linear increase in the SMUFA content in the FFA fraction. At the same time, the PUFA content of the FFA fraction increased only for additions of lipase to flour of >5 LU/g of dry matter, due to partial oxidation by wheat lipoxygenase.

Ultrasound studies of the intestinal wall in patients with cystic fibrosis.

In 1994, first published reports described cystic fibrosis patients who experienced a then unknown complication-ileocecal and colonic stenoses with submucosal proliferation requiring surgical intervention. To investigate a suspected correlation between increased intestinal wall diameter and high doses of pancreatic enzymes, we carried out a prospective study in our CF-outpatient clinic. By ultrasound analysis we measured the intestinal wall diameter in 201 patients. One hundred ninety-three patients treated with pancreatic enzymes had pancreatic insufficiency. Eight patients showed normal pancreatic function, seven of them had never been treated with pancreatic enzymes. The control group included 12 healthy children. Measuring points were the distal ileum, cecum, ascending, and descending colon. Measurements were made by the longitudinal and cross sectional cut. The following aspects of the patients' history were recorded (a) current type of pancreatic enzyme medication; (b) total dosage per day (with reference to lipase units); (c) duration of therapy with standard-strength pancreatic enzyme (SSPE) preparations (< or = 10,000 lipase units per capsule) and HSPE preparations (> or = 20,000 lipase units per capsule); (d) gastrointestinal complication (distal intestinal obstruction syndrome, meconium ileus, abdominal surgery, intussusception), diabetes mellitus, and hepatobiliary complications. The intestinal wall diameter in patients receiving HSPE therapy was greater (with prominent submucosal layer) than that in patients receiving SSPE therapy or in patients with pancreatic sufficiency. Healthy subjects had the smallest intestinal wall diameter. There was no correlation between patient history and increased intestinal wall thickness. Ultrasound detects characteristic ileocecal wall lesions in the majority of cystic fibrosis patients on pancreatic enzymes. These lesions may lead to significantly increased ileocecal wall thickness, which is correlated but not restricted to HSPE.

High-dose pancreatic-enzyme supplements and fibrosing colonopathy in children with cystic fibrosis.

Fibrosing colonopathy has been reported in young children with cystic fibrosis, the majority of whom take high-strength pancreatic-enzyme supplements to control intestinal malabsorption. We conducted a case-control study in the United States to investigate the relation between dose and type of pancreatic-enzyme supplement and fibrosing colonopathy. Children with histopathologically confirmed cases of fibrosing colonopathy who required colectomy for colonic strictures from January 1, 1990, through December 31, 1994, were identified. Each of these patients was matched according to age at the time of surgery and medical center with up to four controls with cystic fibrosis who did not have fibrosing colonopathy. We studied 29 patients (mean age, 5.0 years) with fibrosing colonopathy (case patients) and 105 controls (mean age, 5.2 years). The mean dose of pancreatic-enzyme supplement was 50,046 units of lipase per kilogram of body weight per day for the case patients and 18,985 units per kilogram per day for the controls. A history of gastrointestinal complications attributed to cystic fibrosis and the use of histamine H2-receptor blockers, corticosteroids, or recombinant human DNase (dornase alfa) were associated with a higher incidence of fibrosing colonopathy. After adjustment for a history of such complications and the use of these medicines, the relative risk of fibrosing colonopathy that was associated with a dose of 24,001 to 50,000 units of lipase per kilogram per day, as compared with a dose of 0 to 24,000 units per kilogram per day, was 10.9 (95 percent confidence interval, 1.6 to 71.8), and that associated with a dose of more than 50,000 units per kilogram per day was 199.5 (95 percent confidence interval, 9.9 to 4026.0). The strength, coating, and manufacturer of the products used were not associated with the risk of fibrosing colonopathy. In young children with cystic fibrosis, we found a strong relation between high daily doses of pancreatic-enzyme supplements and the development of fibrosing colonopathy. Our findings support recommendations that the daily dose of pancreatic enzymes for most patients should remain below 10,000 units of lipase per kilogram.

Immobilization of lipases on porous polypropylene: Reduction in esterification efficiency at low loading

AbstractRhizomucor miehei, Humicola sp., Rhizopus niveus, and Candida antarctica B lipases were immobilized by physical adsorption onto a macroporous polypropylene support. In an esterification reaction, the enzyme efficiency, and therefore cost‐effectiveness, is greatly affected by enzyme loading, with an apparent suppression of efficiency at low lipase loadings for both R. miehei and Humicola sp. lipases. This results in the appearance of a pronounced maximum in the efficiency‐loading relationship at approximately 100,000 lipase units (LU)/g for R. miehei lipase (10% of its saturation loading) and at approximately 200,000 LU/g for Humicola sp. lipase (50% of its saturation loading). The other lipases studied do not show similar trends. At low loadings, only a small portion of the surface area is occupied and gives the lipase the opportunity to spread; it is hypothesized that the reduction in efficiency at low loadings is due to a distortion of the active molecular conformation caused by the lipase maximizing its contact with the support as a result of its high affinityfor the support surface. The relationship between efficiency and loading was different for each of the lipases studied, which may reflect both differences in the strength of the affinity of the lipase for the support and in the ease at which the molecular conformation of the lipase can be distorted.

Carboxyl Ester Lipase (Bile Salt-Stimulated Lipase), Colipase, Lipase, and Phospholipase A2 Levels in Pancreatic Enzyme Supplements

Background: Pancreatic lipolytic activity originates from lipase (LIP) and its cofactor colipase (COL), carboxyl ester lipase (CEL), and phospholipase A2 (PLA2). Yet there are few data on the levels of individual lipolytic enzymes in pancreatic enzyme supplements (PES). This study determines activity and immunoreactive mass in some commonly used PES and thus contributes to the understanding of the poor relationship between ‘lipase dose' and clinical improvements. Methods: Recommended doses of each PES were incubated at 37°C for 2 h in a 1-mM Tris-maleate buffer, pH 7.0, containing 150 mM NaCl and 1 mM CaCN. Aliquots for determinations of enzyme activities and for immunochemical mass were taken every half hour. For comparison a standard dose was defined as 10,000 declared lipase units. Results: No simple parallelism between LIP, COL, CEL, and/or PLA;. activities was seen. The LIP contents ranged from 135% to 301% of the standard dose. None of the PES were short of COL (227%-504%). The variation in CEL was twentyfold, and in PLA2 sevenfold. Less variations were seen in the mass composition. There was considerable variation in activity to mass ratios (particularly for CEL), declared lipase units per recommended dose (6000–160,000), and cost (0.36–3.52 SEK). Conclusions: PES differ considerably in their content of lipolytic enzymes. CEL activities were relatively low and COL and PLA2 activities high compared with normal duodenal content. The manufacturing procedure can be improved to increase the lipolytic activity in PES in a broader meaning. It seems to be most important to increase the amount of CEL. From these in vitro data we advocate a more careful decision in the choice of PES for each patient, depending on the total clinical picture. Money can be saved without disadvantage to the patient.

Chemiluminescence Triggered by Hydrolase Activity in a Horseradish Peroxidase/H2O2‐Coupled Assay*

ABSTRACT2‐Methyl‐1‐propenyl acetate (MPA) was hydrolyzed by wheat lipase, pancreatic lipase, hydrolases and lipopro‐tein lipase from serum in a reaction that produces 2‐methyl‐1‐propenol. The latter was subsequently oxidized by horseradish peroxidase (HRP)/H2O2/O2 yielding triplet acetone that emits visible light. The integrated light emission was proportional to the units of lipase or volume of serum present. When pancreatic lipase was assayed, light emission was observed in the presence of bile salt as surfactant and chlorophyll a as light sensitizer. The potential application of this reaction in determinations of hydrolase/Iipase activities is discussed.

Efficacy of a pancreatic enzyme formulation in the treatment of steatorrhea in patients with chronic pancreatitis

The effect of an acid-protected pancreatic enzyme preparation was evaluated in 10 patients with chronic pancreatitis and steatorrhea. (Twelve patients were enrolled but two withdrew because of poor compliance.) Fecal fat excretion and stool weight were measured, a p-aminobenzoic acid (PABA) test was performed, and gastrointestinal symptoms were assessed before and after 4 weeks of treatment with a standardized dose of pancreatic enzymes (two capsules three times daily, each capsule containing 13,000 FU [Italian Official Pharmacopeia] units of lipase). After treatment, we observed a statistically significant reduction in fecal fat excretion and stool weight as well as improved PABA test results and symptoms. This study confirmed the efficacy of this pancreatic enzyme preparation in the treatment of patients with pancreatogenic steatorrhea.

Functional molecular mass of rat hepatic lipase in liver, adrenal gland and ovary is different.

Lipoprotein lipase (LPL) is functionally active only as a dimer. It is also generally assumed that the highly homologous hepatic lipase functions as a dimer, but no clear evidence has been presented. A hepatic lipase-like activity, also indicated as L-type lipase, is present in adrenal and ovary tissues. This enzyme is thought to originate from the liver and to be identical to hepatic lipase. We determined the functional molecular mass of hepatic lipase in rat liver, adrenal gland and ovary by radiation inactivation, a method for determining the functional size of a protein without the need of prior purification. Samples were exposed to ionizing radiation at -135 degrees C. Hepatic lipase activity in liver homogenate showed a single exponential decay. The functional molecular mass was calculated to be 63 +/- 10 kDa. Hepatic lipase activity in adrenal homogenate was found to have a functional molecular mass of 117 +/- 16 kDa. The functional molecular masses of the lipases partially purified from rat liver perfusate, adrenal homogenate or ovarian homogenate showed the same pattern, a target mass for the liver enzyme of 56 +/- 6 kDa and a target mass of 117 +/- 14 kDa for the enzyme from adrenal gland or ovary. In Western blot analysis the mass of the structural units of hepatic lipase in liver was 57 kDa and in adrenal and ovary tissue 51 kDa. We conclude that the functional unit of hepatic lipase in the liver is a monomer. The enzyme in adrenal gland and ovary is different from the liver and the functional unit may be a dimer.

Pseudomonas sp. lipase‐catalyzed synthesis of geranyl esters by transesterification

AbstractPseudomonas sp. lipase was immobilized by adsorption onto five supports and tested for its ability to synthesize geranyl esters by transesterification using short‐chain triacylglycerols as acyl donors. Reaction mixtures were prepared in 2 mL ofn‐hexane, 0.1 M geraniol, 0.03M triacylglycerol, and 200 units of lipase, and incubated at 30°C and 200 rpm for 24 h. Overall, glass beads were the best support. Geranyl acetate and caproate performed best with Duolite (77.5 and 95.3%, respectively). Geranyl butyrate and caprylate performed best with polyvinylpyrrolidone, (80.2 and 95.5%, respectively). Values for nonimmobilized enzyme also were obtained. Immobilization improved yields, with geranyl caproate exhibiting best results.

Effect of the addition of pancreatic lipase on the ripening of dry-fermented sausages — Part 1. Microbial, physico-chemical and lipolytic changes

The effect of the addition of nine different amounts (3–500 units) of pancreatic lipase on the microbial and physico-chemical parameters and lipid fractions during the ripening of dry fermented sausages has been studied. No differences between conventional and lipase-added sausages were found for pH, dry matter and water activity. The addition of lipase caused a greater accumulation of products resulting from the triglyceride breakdown, mainly diglycerides and free fatty acids (FFA). The maximum rate of lipolysis was observed during the first week of the ripening process, specially in the fermentation phase. The greater the pancreatic lipase added, the higher lipolysis observed. At the end of the ripening, the levels of total FFA were clearly higher (1·5 to 5-fold) in all lipase-added batches than in the controls. This fact gave rise to the accumulation of a great amount of FFA, which can contribute either by themselves to the flavour of the sausage or can be available as substrates for further transformations which may generate other flavour compounds.

Effect of the addition of pancreatic lipase on the ripening of dry-fermented sausages — Part 2. Free fatty acids, short-chain fatty acids, carbonyls and sensory quality

The effect of the addition of nine different amounts ( 3–500 units) of pancreatic lipase on the composition of free fatty acids (FFA), short-chain fatty acids and carbonyls, as well as the sensory quality, has been studied on dry fermented sausages. The lipase produced a greater release of all fatty acids in relation to the control sausages. The greater the pancreatic lipase content, the higher the release of these fatty acids, the most important ones being myristic, palmitoleic and oleic acids. A lower release of linoleic acid was observed, probably because of its oxidative degradation. At the end of the ripening in all lipase-added batches, a clear increase of the carbonyl content was noticed in relation to conventional sausages. No consistent changes were observed in the short-chain fatty acid fraction. In the sensory evaluation, the highest significant differences ( P < 0·01) between control sausages and lipase-added batches were observed when 60 and 90 lipase units were used. According to the chemical and sensory analyses, it can be concluded that the addition of 60 and 90 units to sausages seems to be useful to enhanced the flavour of these products.

Production of optically active 3-phenylglycidic acid ester by the lipase from Serratia marcescens on a hollow-fiber membrane reactor

(±)- trans-3-(4-Methoxyphenyl)glycidic acid methyl ester [(±)-MPGM], a key intermediate in the synthesis of diltiazem hydrochloride, was efficiently hydrolyzed by lipase from Serratia marcescens Sr41 8000 immobilized on a hollow-fiber ultrafiltration membrane. The lipase was immobilized on a spongy layer of the shell side of the membrane by means of physical adsorption. Asymmetric hydrolysis was carried out semi-continuously at 22°C in a membrane reactor circulating both toluene solution containing (±)-MPGM in the shell loop and aqueous solution (pH 8.5) in the lumen loop. (+)-MPGM was hydrolyzed to (+)-(2 S, 3 R)-3-(4-methoxyphenyl)glycidic acid and methanol. p-Methoxyphenylacetaldehyde derived from the glycidic acid was accumulated in the toluene phase and inhibited the enzyme reaction. The inhibition, however, was suppressed by the addition of sodium hydrogen sulfite to the aqueous phase, because the aldehyde and sodium hydrogen sulfite formed an adduct and the adduct was transferred to the aqueous phase with the progress of enzyme reaction. The lipase immobilized on the hydrophilic membrane exhibited a high stability: the half-life of enzyme activity at 22°C was 127 h, which was about 30-fold that in the conventional emulsion reactor. The maximum value of the velocity constant ( k (+)) for the hydrolysis of (+)-MPGM was 0.25 h −1 under the condition of immobilization of 1.6 × 10 5 units of lipase per m 2. Since reaction and product separation were achieved simultaneously, crystalline (−)-MPGM with a high yield of 40–43% and optical purity of 100% enantiomeric excess [e.e.] was obtained through six runs by concentration of the toluene phase after the reaction.

Acidolysis of sardine oil by lipase to concentrate eicosapentaenoic and docosahexaenoic acids in glycerides

Lipoprotein lipase from Pseudomonas sp. was the best enzyme to concentrate eicosapentaenoic and docosahexaenoic acids (EPA and DHA) in sardine oil by acidolysis reaction, and acetone was more effective than n-hexane as a solvent for dissolving the reactants and concentrating the two fatty acids. The water concentration in the reaction mixture was a decisive factor governing the enrichment of EPA and DHA and the yield of glycerides. EPA and DHA were more concentrated, but the yield of glycerides decreased, when the water concentration was increased gradually. Thus, the concentration rates of both the fatty acids were low with 0.25% water, although a considerable amount of diglyceride was detectable in the reaction products. The effect of reaction temperature was very slight with the use of acetone; however, the ratio DHA/EPA increased when the temperature was lowered in the presence of n-hexane. When acidolysis was performed at 25°C for 1 h, using 10,000 units of lipase per g of the reactants, the total percentage of EPA and DHA reached 65% in the glycerides and the recoveries of the two acids were 87.4 and 81.3%, respectively, based on the contents in the original sardine oil. The relationship of the enzyme substrate specificity to the reaction results was also investigated.

Enteral pancreatic enzyme feedback inhibition of the exocrine secretion of the human transplanted pancreas.

The mechanism of regulation of negative feedback inhibition of the exocrine pancreas and its possible role in decreasing the exocrine secretion of the grafted human pancreas is unknown. To evaluate this we studied the effect of oral pancreatic enzymes on the stimulated transplanted pancreatic exocrine secretion in eight patients with allograft pancreaticocystostomies. After an 8-hr fast, all graft exocrine secretions via graft stent, fistula, and urinary anastomosis were collected for a 1-hr basal period. A standard 300-ml Lundh test meal was then ingested, and all exocrine secretions were collected in 30-min intervals for 3 hr. This test was repeated with 6 capsules of pancrelipase (24,000 units of lipase, 120,000 units of amylase, and 150,000 units of protease) given with the Lundh test meal. Stent, urine and fistula volume, amylase, and pH were measured for each collection period. The total 3-hr amylase secreted after the test meal and the test meal plus pancrelipase were compared. The period of peak amylase secretion after the test meal alone was compared with the same period after the test meal plus pancrelipase and the premeal basal period. The total amylase decreased 34% from 5550 +/- 1000 to 3680 +/- 740 IU/3 hr (P < .03) with pancrelipase. The peak amylase secretion decreased 63% from 1520 +/- 271 to 567 +/- 185 IU/30 min (P < .02) with the addition of pancrelipase to the test meal. Pancrelipase eliminated all meal-stimulated amylase secretion with the mean secretion 16% below the basal secretion of 674 +/- 117 IU/30 min. We conclude that pancreatic negative feedback inhibition significantly decreases meal-stimulated and basal exocrine secretion in the transplanted human pancreas.

Enzymic hydrolysis of animal fats in organic solvents at temperatures below their melting points

Lipase fromCandida rugosa catalyzed the hydrolysis of inedible beef tallow and pork lard (edible and inedible) in the presence of organic solvents at temperatures below the melting point of the fat. Reactions were carried out at 50% substrate with 180 lipase units per gram of fat in a two‐liter reactor. In the presence of isooctane (5‐10%) beef tallow yielded 94% hydrolysis in 24 hr both at 37° and 31°C. Edible pork lard yielded 97% hydrolysis under these conditions and at temperatures as low as 25°C, while inedible lard gave hydrolysis intermediate between the other two fats.

Synthesis of esters by lipase immobilized on poly (vinyl alcohol)-poly(ethyleneimine) copolymers in organic solvents.

The synthesis of lauric esters of mono-and polyhydric alcohols was studied in organic sol. vents using lipase from Candida cylindracea immobilized on poly (vinyl alcohol)-co-poly(ethyleneimine) gels. The conversion to n-hexyl laurate increased with increasing water content in the reaction system and levelled off above 3%. A similar result was obtained with wet-treated immobilized lipase. The synthesis of n-hexyl laurate in isooctane revealed the highest conversion and the highest rate of esterification among the organic solvents used. Fifty units of lipase was necessary to achieve the almost quantitative esterification in 22 h. Lauric esters of polyhydric alcohols, such as glycerol, ethylene glycol and 1, 3-butanediol, were similarly synthesized with a relatively high conversion in isooctane. Esters prepared were characterized by gel permeation chromatography.

Diagnosis of chronic pancreatitis by measurement of lactoferrin in duodenal juice.

Lactoferrin is a non-enzymatic secretory protein of human pancreas and is specifically increased in pancreatic juice of patients with chronic pancreatitis. Duodenal contents being easier to obtain than pure pancreatic juice, the possibility of using lactoferrin measurement in duodenal juice as a diagnosis test for chronic pancreatitis was explored. Forty-eight patients were studied. Duodenal juice was obtained devoid of salivary contamination by a special double lumen tube. Under these conditions lactoferrin secretion (concentration and output) is increased in patients with chronic pancreatitis. When expressed as the ratio of lactoferrin to lipase units, there was no overlap between chronic pancreatitis and other pancreatic disease or controls. The simplicity and the reproducibility of the technique on a material as readily available as duodenal juice confirms the diagnostic value of lactoferrin measurement in the assessment of patients with suspected pancreatic disease.

Pregastric Esterase in Milk Sham Fed to Adult Jersey Steers1,2

Pregastric esterase activity was detected in reconstituted nonfat milk sham fed from a nipple pail to two 4-yr-old rumen-fistulated steers. Lipolytic activity, determined in a medium containing 5% tri-n-butyrin, averaged 8.6±.4 lipase units. Further assays, in which activity was measured by free fatty acids released from a condensed milk substrate, averaged 166.9±9.2 μmol. These values are higher than those noted for young calves, indicating that secretion of pregastric esterase may persist in cattle beyond calfhood. Esterase activity in one of the steers fed whole milk until he was 2 yr of age showed no marked residual effect of earlier intake of milk fat.