Lipase Enzyme Activity Research Articles

Enzyme Activities of the Fruit Body of Ramaria botrytis DGUM 29001

The fruit body of Ramaria botrytis DGUM 29001 was used to determine enzyme activities of fruit body. The specific activity of laccase was the highest (6.5 unit/mg ⋅ protein) and that of α-amylase and xylanase was relatively high. However, little or no enzyme activity of β-glucosidase, CMCase, exo-β-1,4-glucanase, chitinase, lipase and protease was found.

Lipoprotein lipase and lipogenic enzyme activities in adipose tissue from rats fed different lipid sources.

This work was designed to study the effect of different lipid sources on the activities of lipoprotein lipase and lipogenic enzymes in adipose tissue from rats fed ad libitum or energy-controlled diets. Male Wistar rats were fed diets containing 40% of energy as fat (olive oil, sunflower oil, palm oil or beef tallow), for 4 wk. Under ad libitum feeding no differences were found among dietary fat groups in final body weight, adipose tissue weights and total body fat. Under energy-controlled feeding, despite isoenergetic intake, rats fed the beef tallow diet gained significantly less weight than rats fed the other three diets. Beef tallow fed rats showed the lowest values for adipose tissue weights and total body fat. When rats had free access to food no effect of dietary lipid source on lipogenic enzyme activities was found. In contrast, under energy-controlled feeding rats fed the beef tallow diet showed significantly higher activities of glucose-6-phosphate dehydrogenase and fatty acid synthase than rats fed the other three diets. Heparin-releasable lipoprotein lipase activity in perirenal and subcutaneous adipose tissues was not different among rats fed olive oil, safflower oil, palm oil or beef tallow. When comparing both adipose tissue anatomical locations, significantly higher activities were found in subcutaneous than in perirenal fat pad independently of dietary fat. In conclusion, under our experimental protocol, lipogenesis in rat adipose tissue does not seem to be affected by dietary fat type.

Influence of the porous texture of silica gels on the enzymatic activity of lipases in esterification reactions

Pseudomonas Cepacia lipases were encapsulated in hybrid silica-polyvinyl alcohol gels, which were dried either supercritically in order to form aerogels or by evaporation so as to obtain xerogels. In each case, the catalytic activity of the encapsulated enzymes was studied and compared to free enzyme biocatalysis. This study demonstrates that the activity of the enzyme is increased when the procedure used allows it to resist capillary stresses occurring during the drying of the gel. That is, esterification rates are higher when the gels are synthesized with a base catalyst, such as NaF, in the presence of polyvinyl alcohol and then dried supercritically.

Culture of porcine stromal-vascular cells in serum-free medium: differential action of various hormonal agents on adipose conversion.

We developed a strictly controlled serum-free culture system and tested the effects of adipogenic and antiadipogenic agents on the proliferation and(or) adipose conversion of porcine stromal-vascular cells. To avoid any interference with serum components, stromal-vascular cells were isolated, plated, and grown in absence of serum. In these culture conditions, a very limited growth phase and the absence of cell confluence were observed. However, when compared with continuous culture in serum-containing medium, the serum-free conditions were significantly more adipogenic as assessed by increased lipid content and increased enzymatic activities for lipoprotein lipase, glycerol 3-phosphate dehydrogenase, and malic enzyme. In serum-free medium, physiological concentrations of insulin or IGF-I were sufficient to significantly increase the percentage of lipid-containing cells, whereas triiodothyronine (T3) and GH had no effect. Insulin, IGF-I, and, more moderately, T3 also accelerated the lipid filling in the lipid-containing cells. In the presence of insulin, stimulation by T3 or hydrocortisone alone had no effect on glycerol 3-phosphate dehydrogenase activity, whereas their concomitant addition significantly increased it. Chronic exposure to tumor necrosis factor-alpha dose-dependently stimulated cell proliferation but clearly inhibited differentiation. Epidermal growth factor, another known antiadipogenic agent, also significantly increased the proliferation of stromal-vascular cells, but, surprisingly, this was not correlated with inhibition of adipocyte differentiation. Indeed, epidermal growth factor treatment did not decrease lipid filling and significantly improved lipoprotein lipase and malic enzyme activities. Taken together, the results obtained in these strictly controlled serum-free culture conditions point out functions for insulin, IGF-I, hydrocortisone, and T3 during early and(or) later steps of porcine adipose conversion. In addition, this study reports a positive activity of epidermal growth factor on porcine adipocyte differentiation that is in clear contrast with previous works performed with rodent cells.

Separation, Detection, and Functional Materials. Catalytic activity and enantioselectivity in organic solvent of lipase immobilized on ODS-sugar ester.

Three lipases were immobilized on ODS - Sugar, which was prepared by coating sucrose mono-stearate (sugar ester) on octadecyl silica gel (ODS-silica). The enantioselective esterification of (R)- or (S)-1-phenylethanol with aliphatic acid was studied in the presence of immobilized lipase (ODS - Sugar-Lipase) in organic solvents by varying the lipase origin, reaction media and substrate structures. The ODS - Sugar - Lipase (LPL-311) showed both high catalytic activity and enantioselectivity for the esterification of (R)-1- phenylethanol with long-chain aliphatic acid in dry iso-octane. On the other hand, the ODS- Sugar- Lipase (M) showed both a high catalytic activity and enantioselectivity for the esterification of (S)-ibuprofen with hexanol in dry iso-octane. The enzymatic activity of lipases in organic media was remarkably enhanced by immobilization with the sugar ester. The reaction rates of the immobilized lipases were increased by around 100200-fold compared to that of the native lipases. The activity of the immobilized lipases has been retained for at least one month in refrigerator. The ODS- Sugar- Lipase showed greater activity for ester synthesis than other systems, such as the PEG-grafted lipase and the powder dispersion system. We believe that the ODS - Sugar adsorbed enzyme system can be widely applicable to other enzymes whose substrates are lipophilic.

Modulator-mediated synthesis of active lipase of Pseudomonas sp. 109 by Escherichia coli cell-free coupled transcription/translation system

Catalytically active lipase was synthesized using Escherichia coli S30 extract from the signal-deleted lipL gene ( lipL) in the presence of its N-terminal hydrophobic fragment-truncated modulator (rLimL) that was purified from the overexpressing E. coli cells. The specific activity of the lipase thus synthesized was 125 times higher than that of the purified one from Pseudomonas sp. 109. No lipase activity was detected in the absence of rLimL, even though the lipase protein itself was synthesized. Active lipase was also produced in vitro by coexpression of rlipL and the modulator gene ( rlimL), although a much smaller amount of the lipase was formed. In the absence of rLimL, aggregates of the lipase were formed during its folding process. The addition of rLimL proportionally raised both lipase solubility and enzyme activity. An unstable but high activity peak of the lipase was found during its folding process.

Effect of sham feeding and acute suppression of acid secretion on human gastric lipase secretion.

Gastric lipase and gastric acid are secreted simultaneously. The aim of this study was to investigate whether the acid interferes with the lipase secretion. The secretion of human gastric lipase was studied during blockade of gastric acid secretion and modified sham feeding to estimate the impact of these conditions on both gastric lipase enzyme activity and immunoreactivity. Eight healthy volunteers were intubated with a nasogastric tube. We examined gastric aspirates for the amount and activity of lipase secretion during basal conditions, after blockade of acid secretion with a proton pump inhibitor (omeprazole iv. infusion), and in response to sham feeding (chewing gum) during the blockade. The amount of secreted gastric lipase was unaffected by blockade of acid secretion and increased significantly after sham feeding (169.9+/-35.7 microg/15 min to 348.1+/-79.2 microg/15 min; p < 0.01). Likewise, the output of enzyme activity increased after sham feeding (0.63+/-0.09 kU/15 min to 1.52+/-0.36 kU/15 min; p < 0.03). The concentration of enzyme activity remained unchanged by blockade of acid secretion, whereas the output of enzyme activity was decreased, probably because of reduced volume secretion or denaturation and conformational changes of the enzyme. Plasma concentrations of gastrin increased in response to blockade of acid secretion (basal 9.6+/-1.4 pmol/L to 13.3+/-2.9 pmol/L; p < 0.02). Gastric acid secretion is not a prerequisite for gastric lipase secretion. Lipase enzyme activity, though, is sensitive to anacidic conditions.

Effect of sham feeding and acute suppression of acid secretion on human gastric lipase secretion

Objective: Gastric lipase and gastric acid are secreted simultaneously. The aim of this study was to investigate whether the acid interferes with the lipase secretion. The secretion of human gastric lipase was studied during blockade of gastric acid secretion and modified sham feeding to estimate the impact of these conditions on both gastric lipase enzyme activity and immunoreactivity. Methods: Eight healthy volunteers were intubated with a nasogastric tube. We examined gastric aspirates for the amount and activity of lipase secretion during basal conditions, after blockade of acid secretion with a proton pump inhibitor (omeprazole i.v. infusion), and in response to sham feeding (chewing gum) during the blockade. Results: The amount of secreted gastric lipase was unaffected by blockade of acid secretion and increased significantly after sham feeding (169.9 ± 35.7 μg/15 min to 348.1 ± 79.2 μg/15 min; p < 0.01). Likewise, the output of enzyme activity increased after sham feeding (0.63 ± 0.09 kU/15 min to 1.52 ± 0.36 kU/15 min; p < 0.03). The concentration of enzyme activity remained unchanged by blockade of acid secretion, whereas the output of enzyme activity was decreased, probably because of reduced volume secretion or denaturation and conformational changes of the enzyme. Plasma concentrations of gastrin increased in response to blockade of acid secretion (basal 9.6 ± 1.4 pmol/L to 13.3 ± 2.9 pmol/L; p < 0.02). Conclusions: Gastric acid secretion is not a prerequisite for gastric lipase secretion. Lipase enzyme activity, though, is sensitive to anacidic conditions.

Enzymatic activities in the varieties of hazelnuts ( Corylus avellana L.) grown in Tarragona, Spain

Enzymatic activities of lipase, esterase, lipoxygenase (LOX), peroxidase (POD) and polyphenoloxidase (PPO) of the main local, Italian and Turkish varieties of hazelnuts grown in Tarragona, with and without irrigation, were determined. Enzymatic extract protein was estimated by calculating Δ Abs/min.mg of protein with spectrophotometry. Oleic (C 18:1) and linoleic (C 18:2) acid percentages were determined using high resolution gas chromatography (HRGC). Data were statistically analysed using General Linear Models and Duncan's multiple test. It is observed that variety is a determining factor for all enzymatic activities, especially lipase and peroxidase activity. Negret (Negret and Pauetet varieties), the most cultivated variety in Tarragona, has a high C 18.2 content and high enzymatic activities. Italian varieties have lower lipase, POD and PPO activities. All varieties show minimum activity of esterase and lipoxygenase.

A new mutation in the gene for lysosomal acid lipase leads to Wolman disease in an African kindred.

Cholesteryl ester storage disease (CESD) and Wolman disease (WD) are both autosomal recessive disorders associated with reduced activity and genetic defects of lysosomal acid lipase (LAL). The strikingly more severe course of WD is caused by genetic defects of LAL that leave no residual enzymatic activity. Mutations at the exon 8/intron 8 transition of the LAL gene have been identified in several CESD and WD patients and are responsible for the manifestation of the disease. We have determined the genetic defect in a 3-month-old boy of African origin affected by WD. No enzymatic activity of the lysosomal acid lipase was detectable in white blood cells and cultured fibroblasts. Analysis of his LAL cDNA and genomic DNA revealed that he was homozygous for a mutation at position -3 of the exon 8 splice donor site. A C-->T transition leads to a nonsense codon and to a premature termination of the LAL protein at amino acid 277. Due to this mutation, a shorter LAL mRNA species was also generated that lacked exon 8 and was deficient of the nonsense codon. As a consequence, the protein synthesis proceeded to the natural termination codon, but the enzyme generated had an internal deletion of 24 amino acids (254-277) and was also inactive. These findings, together with our previous observations when analyzing the mutations in WD and CESD patients lead to the conclusion that the more severe WD is due to mutations that absolutely abolish lysosomal acid lipase (LAL) enzyme activity and the cholesteryl ester storage disease phenotype is due to mutations that allow some residual LAL activity to be manifested.

Effects of weight reduction on the regulation of lipolysis in adipocytes of women with upper-body obesity.

1. The regulation of lipolysis was studied in 14 upper-body obese women aged 26-55 years. Isolated subcutaneous adipocytes from the abdominal region were examined before and after an 8 to 12-week-long weight reduction programme, during which the mean body mass index (kg/m2) of the group was reduced from 36.4 +/- 0.9 to 30.3 +/- 1.0. 2. Fat cell volume was reduced from 891 +/- 39 to 655 +/- 45 pl. The sensitivity to noradrenaline stimulation of lipolysis in vitro increased fivefold after weight reduction. A corresponding increase in sensitivity was found with the beta 2-selective adrenoceptor agonist terbutaline. However, the number of beta 2-adrenoceptor binding sites as assessed by radioligand binding with 125I-labelled cyanopindolol was not changed. No changes were observed when dobutamine (a beta 1-selective adrenoceptor agonist) and clonidine (an alpha 2-adrenoceptor agonist) were used. 3. The basal lipolysis rates decreased by about 50% after weight reduction and the maximum enzyme activity of hormone-sensitive lipase was also reduced by almost 50%. 4. Plasma concentrations of insulin, noradrenaline and total testosterone decreased and sex hormone-binding globulin increased after weight reduction. Calculated apparent free testosterone levels decreased by more than 40% after weight reduction. 5. In conclusion, weight reduction leads to increased efficiency of adipocyte lipolysis with decreased resting lipolysis rate but increased sensitivity to stimulation by catecholamines, which may be attributed to a decreased activity of hormone-sensitive lipase and an increased sensitivity of beta 2-adrenoceptors. Changes in circulating levels of catecholamines, insulin and testosterone may play a role in these modifications of adipocyte function.(ABSTRACT TRUNCATED AT 250 WORDS)

Heparan Sulfate Proteoglycans Are Primarily Responsible for the Maintenance of Enzyme Activity, Binding, and Degradation of Lipoprotein Lipase in Chinese Hamster Ovary Cells

Various aspects of lipoprotein lipase (LPL) metabolism, including cell surface binding, degradation, and enzymatic activity, were compared between Chinese hamster ovary (CHO) cells and two distinct proteoglycan-deficient CHO cell lines. The contribution of low density lipoprotein receptor-related protein in binding LPL was also analyzed by the use of a 39-kDa receptor-associated protein expressed as a glutathione S-transferase fusion protein (GST-RAP). Equilibrium binding data with 125I-LPL revealed the presence of a class of high affinity binding sites with a KD of 7.8 nM in CHO cells, whereas no high affinity binding was observed for proteoglycan-deficient cells. The high affinity binding of LPL in CHO cells appeared to be concentrated in cell surface projections and was not effectively inhibited by GST-RAP. Moreover, degradation of endogenous and exogenous LPL was significantly greater in control CHO cells than in proteoglycan-deficient cells. Degradation of LPL in CHO cells was not affected by GST-RAP, suggesting that proteoglycans and not low density lipoprotein receptor-related protein are responsible for the majority of binding and degradation of LPL in these cells. Our data also show that proteoglycan binding is not essential for the assembly of active LPL homodimers, although proteoglycan binding controls the distribution of LPL activity. Furthermore, LPL produced by CHO cells was more stable than LPL produced by proteoglycan-deficient cells.

Lipopolysaccharide regulation of lipoprotein lipase expression in murine macrophages.

The enzyme lipoprotein lipase is expressed in a number of cell types and plays a central role in lipid metabolism. Multiple factors regulate its expression in a tissue-specific manner. In murine macrophages, lipopolysaccharide inhibits lipoprotein lipase enzyme activity. The current work examines this process in the established J774 macrophage line and primary peritoneal macrophages from endotoxin-sensitive (C3HeB/Fej) and endotoxin-resistant (C3H/Hej) murine strains. Lipopolysaccharide inhibition of macrophage lipoprotein lipase occurred at the enzyme and mRNA levels in a time- and concentration-dependent manner. Cells from endotoxin-resistant animals maintained their expression of lipoprotein lipase following treatment with lipopolysaccharide. Results of gel retention assays showed that lipopolysaccharide treatment of the J774 macrophages altered the level of nuclear proteins recognizing and binding the lipoprotein lipase promoter DNA. Nuclear extracts from resting J774 cells contained proteins which bound specifically to the octamer motif and to the CAAT box within the lipoprotein lipase promoter. Exposure of the J774 cells to lipopolysaccharide for 16 h increased the level of protein-octamer DNA complexes. Similar responses were obtained in endotoxin-sensitive, but not endotoxin-resistant, primary macrophages following in vitro treatment with lipopolysaccharide. This finding suggests that transcriptional events may contribute to the lipopolysaccharide regulation of macrophage lipoprotein lipase expression.

Significance of elevated pancreatic enzymes in intracranial bleeding.

Hyperamylasemia of pancreatic origin has been noted in patients with severe head injury without abdominal trauma or evidence of pancreatitis. Thirty-eight patients with intracranial bleeding of various types were evaluated for elevated pancreatic amylase and lipase enzymes without associated pancreatitis. Twenty-five patients had elevated serum lipase; 17 of 25 also had elevated amylase without pancreatitis. Most lipase elevations occurred earlier than those of amylase. Six clinical variables--mannitol, ceftriaxone, nimodipine, steroids, Glasgow Coma Score, and total parenteral and enteral hyperalimentation--were evaluated to determine relationship to the enzyme elevations. A significant relationship exists between patients not treated with steroids and elevated lipase and amylase enzyme activities. Multivariate analysis revealed a significant interaction between lipase elevation and decreasing Glasgow Coma Score, indicative of increasing severity of intracranial bleeding. Proposed causes of enzyme elevations in intracranial bleeding include vagal stimulation, altered modulation of the central control of pancreatic enzyme release, and release of cholecystokinin from the brain. Physician awareness of the association of intracranial bleeding with the elevation of amylase and lipase without pancreatitis can save the patient needless cost and manipulation.

Regulation of bone marrow stromal cell differentiation by cytokines whose receptors share the gp130 protein.

The bone marrow stroma consists of a heterogeneous population of cells which participate in osteogenic, adipogenic, and hematopoietic events. The murine stromal cell line, BMS2, exhibits the adipocytic and osteoblastic phenotypes in vitro. BMS2 differentiation was examined in response to cytokines which share the gp130 signal transducing protein within their receptor complex. Four of the cytokines (interleukin 6, interleukin 11, leukemia inhibitory factor, and oncostatin M) inhibited hydrocortisone-induced adipocyte differentiation in a dose dependent manner based on lipid accumulation and lipoprotein lipase enzyme activity. Inhibition occurred only when the cytokines were present during the initial 24 h of the induction period; after 48 h their effects were diminished. Likewise, these cytokines increased alkaline phosphatase enzyme activity twofold in preadipocyte BMS2 cells. Both leukemia inhibitory factor and oncostatin M induced early active gene expression in resting preadipocyte BMS2 cells and decreased the steady state mRNA level of a unique osteoblastic gene marker, osteocalcin. A fifth cytokine whose receptor complex shares the gp130 protein, ciliary neurotrophic factor, did not significantly regulate stromal cell differentiation when added by itself. However, with the addition of a missing component of its receptor complex, ciliary neurotrophic factor receptor alpha protein, this cytokine also inhibited BMS2 adipogenesis. Together, these data indicate that the cytokines whose receptors share the gp130 protein can modulate stromal cell commitment to the adipocyte and osteoblast differentiation pathways.

Plasma disappearance of homologous and heterologous pancreatic enzymes in dogs.

Homologous and heterologous (human) pure pancreatic juice was infused into dogs to compare the behaviour of pancreatic enzymes in the circulation. This was achieved by estimating the magnitude of elevation and speed of disappearance of pancreatic enzymes in plasma. The half-lives of the plasma enzymes did not differ significantly between dogs and humans. However the half-lives of other enzymes (longest for amylase and shortest for trypsinogen) did differ. The magnitude of elevation expressed as a ratio of peak (5 min) to basal level was greatest for trypsinogen (28 times) and least for amylase (1.4 times). However, 289% and 410% of the initial lipase enzyme activity was recovered 5 min after infusion of dog and human pure pancreatic juice, respectively (44% when measured by immunoreactive human lipase activity). The excess recovery requires re-evaluation of diagnostic value of turbidimetric activity of plasma pancreatic lipase.

Absence of extracellular enzyme activity and cytotoxicity in cell-free culture filtrates of Haemophilus ducreyi

Ten isolates of Haemophilus ducreyi, including seven clinical isolates and three laboratory reference strains, were assessed for their ability to produce extracellular enzymes which might contribute to their pathogenicity. Protease, elastase, lecithinase, lipase or collagenase enzyme activity were not detected in culture filtrates from any of the isolates tested using either plate or spectrophotometrical assays. Furthermore, cell-free culture filtrates did not exhibit cytotoxic activity in vitro when tested using either an established tissue culture cell line (Vero) or a primary cell culture of human keratinocytes. These results suggest that extracellular products do not play a role in host invasion or production of tissue damage by H. ducreyi.

Expression of lipoprotein lipase in different human subcutaneous adipose tissue regions

Steady state expression of lipoprotein lipase was compared in abdominal and gluteal subcutaneous adipose tissue of nonobese men and women. In both regions enzyme activity and lipoprotein lipase mRNA levels were significantly higher in women than in men. In men the enzyme activity was higher in abdominal than in gluteal adipose tissue (P less than 0.01) whereas the opposite was observed in women (P less than 0.05). In both sexes, however, lipoprotein lipase mRNA levels were threefold higher in the abdominal as compared to the gluteal site, whether they were determined in isolated fat cells or in fat segments (P less than 0.01). This regional difference persisted when the mRNA values were expressed as a function of the mRNA concentration for beta-actin. There was a correlation between the two adipose tissue regions as regards the values for enzyme activity and mRNA level (r = 0.6-0.8). Northern blot analysis revealed two mRNA species of 3.5 and 3.7 kilobases, respectively. It is concluded that there are regional variations in the steady state expression of lipoprotein lipase in human subcutaneous adipose tissue. This involves site variations in gene expression as well as posttranslational modification of lipoprotein lipase enzyme activity and may contribute to the characteristic variations in adipose tissue mass and distribution between men and women.

Laboratory Abnormalities in Patients with Cancer

In this problem-oriented review of abnormalities associated with cancer, we have emphasized distinctive diagnostic points related to pathogenesis for each condition and outlined how the approach to management is determined by pathogenesis. For abnormalities of the complete blood count, it is important to distinguish between abnormalities directly related to marrow malignancy and abnormalities associated with extramarrow malignancy. Hemopoietic tumors consist of developmentally deficient blood cells produced by a clonal population of malignant stem cells. Tumors infiltrating marrow cause overcrowding in the limited marrow microenviroment. Extramarrow malignancies cause blood abnormalities, but the potential for normal marrow function is present. Abnormalities of blood cells secondary to therapy are usually clearly identified by consideration of clinical history. The initial differential diagnosis for hypercalcemia is malignancy. An aggressive diagnostic approach may be needed to identify the neoplasm, and therapy should incorporate measures to prevent renal failure. Hypoproteinemia and hyperproteinemia may be caused by neoplasia. Monoclonal gammopathies should be identified and may be associated with hyperviscosity syndrome. Hypoglycemia in the adult animal is most frequently caused by insulin-secreting tumors, but it has also been associated with hepatic and other tumors. Increased blood urea nitrogen, creatinine, lipase, amylase, and liver enzyme activities may also be caused by malignancy. Inadequate urine concentrating ability may be caused by hypercalcemia or malignancy-associated renal insufficiency. Hematuria in older animals is suggestive of urinary tract neoplasia. Exfoliated tumor cells may be identified in the urine sediment of these patients.

A New Microcalorimetric Method for the Determination of Lipase Activity and Substrate Concentration in Aqueous Medium

Abstract The lipase activity and the triglyceride concentration have been determined for the first time in a simple and direct way. The heat quantity involved in the main enzymatic hydrolysis reaction has been measured by means of batch microcalorimetry. The heat quantity measured was related linearly to the substrate concentration, and the heat change rate has been directly related to the enzymatic activity of lipase. No ancillary reactions were necessary. For the first time the lipolytic reaction has been directly studied in an aqueous medium, without organic solvents and/or emulsifying agents. The described calorimetric method has been applied to milk samples.