Determination Of Lipase Activity Research Articles

New Colorimetric Method for Lipases Activity Assay in Microbial Media

A simple, rapid and precise method has been developed for determination of lipase activity in microbial media. The method is based on using phenyl acetate as substrate for lipase and determination of liberated phenol by Folin Ciocalteu reagent. Reaction mixture containing substrate 2.4 ml of phenyl acetate 165 μM in Tris HCl buffer, 0.1 M and pH 7, with 1% (v/v) Triton X-100) and 0.1 ml lipase is incubated at 40?C during 10 minutes and the absorbance was measured at 750 nm. Linearity was observed in the concentration range 0-0.8 g/L lipase.

Nanoparticle modified QCM-based sensor for lipase activity determination

A highly sensitive lipase activity sensor was developed and tested. It is based on the application of SiO2 nanoparticle loaded olive oil as a lipase substrate, deposited on a QCM crystal. The heavier nanoparticles’ release during the substrate enzymatic degradation causes a QCM frequency response enhancement proportional to the nanoparticle/substrate mass ratio.

Inhibition of Propionibacterium acnes lipase by extracts of Indian medicinal plants

Lipases play an important role in pathogenesis of acne by hydrolysing sebum triglycerides and releasing irritating free fatty acids in the pilosebaceous follicles. Lipase is a strong chemotactic and proinflammatory antigen. Therefore, lipase has generated a high interest as a pharmacological target for antiacne drugs. The aim of this study was to identify inhibitory effects of plant extracts on the lipase activity of Propionibacterium acnes. Colorimetric microassay was used to determine lipase activity. Extracts from Terminalia chebula and Embelia ribes showed lower IC(50) value (1 μg mL(-1) ) for lipase inhibition as compared to Vitex negundo and Picrorhiza kurroa (19 and 47 μg mL(-1) , respectively). The active component responsible for lipase inhibition was isolated. This study reports for the first time the novel antilipase activity of chebulagic acid (IC(50) : 60 μmol L(-1) ) with minimum inhibitory concentration value of 12.5 μg mL(-1) against P. acnes. The inhibitory potential of plant extracts was further confirmed by plate assay. The organism was grown in the presence of subinhibitory concentrations of extracts from P. kurroa, V. negundo, T. chebula, E. ribes and antibiotics such as clindamycin and tetracycline. Extract from T. chebula showed significant inhibition of lipase activity and number of P. acnes.

Direct Measurement of Lipase Inhibition by Orlistat Using a Dissolution Linked In Vitro Assay.

PurposeTo develop a bio-assay that would be able to directly test gastrointestinal and/or dissolution samples to determine lipase activity and inhibition by Orlistat.MethodsEnzyme assays were performed with porcine pancreatic lipase and para-Nitrophenyl Palmitate (pNPP) in pH 8.0 reaction buffer at 37°C. Substrate hydrolysis was monitored by absorbance changes at 410 nm. The dissolution of two Orlistat formulations was tested with a USP II apparatus. Samples were HPLC analyzed to determine release profile in addition to being diluted and directly assayed for inhibitory effect.ResultsThe lipase-pNPP system demonstrates linearity and Michalis-Menten kinetics with a Km=2.7 ± 0.2 μM and Kcat = 0.019 s−1. Orlistat showed highly potent and time dependent inhibition with 5 ng/ml effecting 50% activity after 5 minutes in the Lipase-pNPP system. Dissolution studies showed a correlation of the drug release profile to the inhibitory effect of dissolution samples in the assay.ConclusionsThe lipase-pNPP method can be used as an in vitro assay to monitor orlistat inhibition from drug release or dissolution samples.

Establishment and evaluation of a new model for studying lipogenesis in grass carp (Ctenopharyngodon idella) preadipocytes

The objective of this study was to establish and evaluate a new model for studying lipogenesis in grass carp preadipocytes. The morphology characteristic from preadipocytes to mature adipocytes was observed with the microscopic morphology, and the proliferation kinetics of cells was tested by cell counting. In addition, the nature and differentiation degree of cells were evaluated using Oil Red O staining, lipase activity determination, and reverse transcription PCR (RT-PCR) assay. Morphologically, grass carp preadipocytes started to attach and grow on day 3, then they resembled fibroblasts, and most underwent attachment, proliferation, and growth arrest with subsequent accumulation of intracellular lipid droplets before becoming mature adipocytes. Glycerol-3-phosphate dehydrogenase (GPDH) activity was increased gradually during the progress of culture. Analysis of RT-PCR confirmed that peroxisome proliferator-activated receptor-γ expression patterns were consistent with my observations regarding GPDH activity. In summary, grass carp preadipocytes cultured with 10% FBS at 28°C in a humidified 5% CO2 atmosphere have high proliferation potential. Furthermore, the cells synthesize a range of markers that are consistent with this cell type. We conclude therefore that the grass carp preadipocytes described here have high capacity for lipogenesis and may, therefore, represent a unique tool for studying fish fat cell development and metabolism.

Adsorption of Bile Salts and Pancreatic Colipase and Lipase onto Digalactosyldiacylglycerol and Dipalmitoylphosphatidylcholine Monolayers

It is increasingly recognized that changes in the composition of the oil-water interface can markedly affect pancreatic lipase adsorption and function. To understand interfacial mechanisms determining lipase activity, we investigated the adsorption behavior of bile salts and pancreatic colipase and lipase onto digalactosyldiacylglycerol (DGDG) and dipalmitoylphosphatidylcholine (DPPC) monolayers at the air-water interface. The results from Langmuir trough and pendant drop experiments showed that a DGDG interface was more resistant to the adsorption of bile salts, colipase, and lipase compared to that of DPPC. Atomic force microscopy (AFM) images showed that the adsorption of bile salts into a DPPC monolayer decreased the size of the liquid condensed (LC) domains while there was no visible topographical change for DGDG systems. The results also showed that colipase and lipase adsorbed exclusively onto the mixed DPPC-bile salt regions and not the DPPC condensed phase. When the colipase and lipase were in excess, they fully covered the mixed DPPC-bile salt regions. However, the colipase and lipase coverage on the mixed DGDG-bile salt monolayer was incomplete and discontinuous. It was postulated that bile salts adsorbed into the DPPC monolayers filling the gaps between the lipid headgroups and spacing out the lipid molecules, making the lipid hydrocarbon tails more exposed to the surface. This created hydrophobic patches suitable for the binding of colipase and lipase. In contrast, bile salts adsorbed less easily into the DGDG monolayer because DGDG has a larger headgroup, which has strong intermolecular interactions and the ability to adopt different orientations at the interface. Thus, there are fewer hydrophobic patches that are of sufficient size to accommodate the colipase on the mixed DGDG-bile salt monolayer compared to the mixed DPPC-bile salt regions. The results from this work have reinforced the hypothesis that the interfacial molecular packing of lipids at the oil-water interface influences the adsorption of bile salts, colipase, and lipase, which in turn impacts the rate of lipolysis.

Oleate lipase activity in Gardnerella vaginalis and reconsideration of existing biotype schemes

BackgroundGardnerella vaginalis is a facultative gram positive organism that requires subculture every 1–2 days to maintain viability. It has been linked with bacterial vaginosis (BV), a syndrome that has been associated with increased risk for preterm delivery, pelvic inflammatory disease and HIV acquisition. About 10% of the G. vaginalis isolates have been reported to produce sialidase, but there have not been any studies relating sialidase production and biotype. Sialidase activity is dramatically increased in the vaginal fluid of women with BV and bacterial sialidases have been shown to increase the infectivity of HIV in vitro. There are 8 different biotypes of G. vaginalis. Biotypes 1–4 produce lipase and were reported to be associated with BV and the association of these biotypes with BV is under dispute. Other studies have demonstrated that G. vaginalis biotype 1 can stimulate HIV-1 production. Because of the discrepancies in the literature we compared the methods used to biotype G. vaginalis and investigated the relationship of biotype and sialidase production.ResultsA new medium for maintenance of Gardnerella vaginalis which allows survival for longer than one week is described. Some isolates only grew well under anaerobic conditions. Sialidase producing isolates were observed in 5 of the 6 biotypes tested. Using 4-methylumbelliferyl-oleate to determine lipase activity, instead of egg yolk agar, resulted in erroneous biotypes and does not provide reliable results.ConclusionPrevious studies associating G. vaginalis biotype with bacterial vaginosis were methodologically flawed, suggesting there is not an association of G. vaginalis biotypes and bacterial vaginosis. Sialidase activity was observed in 5 of the 8 biotypes.

A potential high-throughput method for the determination of lipase activity by potentiometric flow injection titrations

Potentiometric FIA titrations were performed to determine enzyme activities of lipase type B from Candida antarctica, CAL-B. Two substrates, triacetin and tributyrin were hydrolyzed in phosphate buffer solutions, and the concentration change of the base component of the buffer was titrated in a carrier solution containing hydrochloric acid and potassium chloride. The system was calibrated with butyric acid and acetic acid, respectively. FIA titration peaks were evaluated with respect to peak height and peak area. Butyric acid and acetic acid could be titrated in the buffer solution from 3 × 10 −3 mol L −1 to 0.1 mol L −1. The detection limit of enzyme activity was determined to be 0.07 U mL −1 (15 min reaction time) and the minimum activity was calculated to be 0.035 units corresponding to 35 nmol min −1. The specific activities of lipase B for the hydrolysis of tributyrin and triacetin were determined as 16 ± 2 U mg −1 and 2 ± 0.2 U mg −1 (per mg commercial lipase preparation), respectively.

Quantitative Determination of Lipase Activity by Liquid Chromatography-Mass Spectrometry

We developed a novel in vitro lipase assay based on the quantitation of fatty acids by liquid chromatography-mass spectrometry. Oleic acids enzymatically released from triolein substrates were isolated from the reaction mixture by reverse-phase chromatography, ionized in negative mode electrospray mass spectrometry and quantitated with the aid of [(13)C]-oleic acid internal standard. The enzymatic activity was measured by monitoring oleic acid productions at multiple time points. This method overcomes the substrate and pH limitations of conventional techniques and thus serves as a generic lipase activity assay.

Enzymatic activity of lipase in post-metamorphic phase bullfrogs

The knowledge of the digestive system of bullfrogs is an important step for the determination of their nutritional requirements throughout growth phases. With the objective of evaluating the enzymatic activity of lipase in the intestinal content of bullfrogs (Rana catesbeiana Shaw, 1802), 100 animals with median weight of 3.6 g were distributed in stalls under controlled temperature and photoperiod. The frogs, selected at the post-metamorphic phase, received commercial extruded diet ad libitum throughout the 87-day experiment. The collections of the intestinal content were performed by the desensitization of the frogs in ice and water at 0ºC and subsequent isolation of the small intestine. Determination of lipase activity was performed with a commercial enzymatic kit (Lipase-Bioclin, MG, Brazil), first measured in samples taken at day three (3.46 UI). During the initial phase the bullfrog possesses low lipase hydrolysis capacity was found, having a specific activity of 217 UI mg-1. In the subsequent period both lipase activity and specific lipase activity continuously increased. Lipase activity as a function of bullfrog weight fell after day twenty and reached 0.33 UI g-1, for frogs of medium weight (179 g). Feed for bullfrogs at the post-metamorphic phase weighing more than 10 g can have larger amounts of ingredients containg lipids, due to the increased digestive capacity of these frogs.

Methods for rapid screening and isolation of bacteria producing acidic lipase: feasibility studies and novel activity assay protocols

Acidic lipase finds its commercial values in medical applications and bioremediation of food wastes. In this work, approaches for rapid screening of lipase-producing bacteria were developed and the feasibility assessment of the screening methods was performed. From food waste samples, the proposed screening procedures allowed isolation of sixteen pure bacterial strains expressing higher lipase activity at acidic pH (pH 6.0) than at alkaline pH (pH 9.0). To enhance the accuracy of lipase activity determination under acidic conditions, a novel assay procedure was also developed by deactivating lipase activity by microwave treatment prior to back titration. This additional step could minimize interferences arising from residual lipase activity during conventional direct back-titration methods in measuring lipase activity at acidic pH. Using the four strategies proposed in this work, the best acidic-lipase-producing isolate was obtained by strategy C (SSC) and was identified as Aeromonas sp. C14, displaying an optimal lipase activity of 0.7 U/ml at an acidic pH of 6.0.

A thin-layer chromatography-densitometry assay for intracellular lipase activity in bivalves (Argopecten purpuratus)

This study provides a method for the determination of lipase activity in bivalves. Using this method, lipase activities were studied in extracts of digestive gland of Argopecten purpuratus fed a diet that mimics the food available in natural ecosystems over a short-term experiment (24–72 h). The scallops were acclimated to standard conditions seven days prior to initiating the enzymatic analysis. The diet consisted of a mixture of laboratory-cultured Isochrysis galbana and natural sediment. For the acclimation diet (control diet), a total particulate concentration of 3.9 mg L–1 was used with an organic content of 1.7 mg L–1 (42.5%), of which 28% were lipids. The experimental diet had a total particulate concentration of 20.8 mg L–1 with 26% of organic content, of which 20% were lipids. The effect of diet and experimental time on lipase activity was analyzed. The response of the intracellular lipases to changes in the food offered was abrupt and lower investment in enzymatic activity was observed with the experimental diet. No significant effects were observed either for the time or for the combined diet and time interaction.

Biosensors for the evaluation of lipase activity

In the review a comprehensive analysis is given of instrumental analytical approaches to control the activity of lipolytic enzymes and their substrates. A special attention is attached to the methods based on the biosensor technology. A number of electrochemical, optical and mechanical biosensors that were developed in several laboratories are analyzed in detail. In addition, perspectives are evaluated for the development of these biosensors and their practical application. It was concluded that biosensors may provide all practical demands which are in enzymology field at the express determination of lipase activity and triacylglycerol levels in biological objects.

Comparison of Lipase Activity in Peritoneal Fluid of Dogs with Different Pathologies – A Complementary Diagnostic Tool in Acute Pancreatitis?

A clinical diagnosis of acute pancreatitis is often difficult to obtain. Histopathology remains the gold standard, whereas clinical signs, diagnostic imaging and laboratory testing, even in combination, may be insufficient. In a prospective study, lipase activity in ascitic fluid of various aetiologies was determined in 44 dogs in order to investigate its performance in cases of acute pancreatitis. Data of simultaneously determined blood lipase activities were available in 27 dogs. Lipase activity was measured by a colorimetric assay. A complete peritoneal fluid analysis was performed. Dogs were divided into four groups, according to their final diagnosis: acute pancreatitis (A), abdominal trauma (B), abdominal neoplasia (C) and others (hepatic or cardiac diseases) (D). Dogs with acute pancreatitis had a significantly higher peritoneal lipase activity than those in other groups (P < or = 0.024), while no significant difference was found between the other groups (P > or = 0.734). Blood lipase activity as well as protein content and total cell count of the ascitic fluid did not show any significant difference between groups. Data show that determination of lipase activity in dogs that develop ascites may be useful in complementing the diagnosis of acute pancreatitis.

Determination of lipase activity using image analysis

The ability of lipases (triacylglycerol hydrolases, E.C. 3.1.1.3) to catalyze the hydrolysis of triacylglycerols has been known for many decades. However, since the early 1990s their importance in biotechnology has expanded signiWcantly, due to their ability to catalyze reactions in nonconventional media such as organic solvents and to recognize various diVerent esters as substrates [1]. For example, lipases have been used to catalyze the transesteriWcation of triacylglycerols and in the racemic resolution of various chiral esters. These special uses have driven a search for lipases able to catalyze novel reactions. Considering that typically such reactions are carried out under aggressive reactions conditions, such as the presence of organic solvents, it is necessary to Wnd lipases that have high stability and activity under these conditions [2]. The discovery of new enzymes through bioprospection [3], screening of metagenomic libraries [4], or molecular evolutionary approaches [5] demands a practical and sensitive screening method capable of manipulating a large number of samples of either whole cells or crude enzyme extracts. The development of such methods for screening lipases is hampered by the physicochemical properties of lipids. Methods that rely on titrimetric or radiometric measurements have been used, but methods in the Wrst group have low sensibility while those in the second group require expensive radiolabeled substrates. When a large number of samples are assayed, chromogenic or Xuorogenic substrates are used. These substrates are esters from which the enzy-

Determination of lipase activity in cheese using trivalerin as substrate

Abstract Lipolysis during cheese ripening is usually assessed by the accumulation of free fatty acids (FFA). An assay to determine total lipolytic activity present in a cheese during ripening was established. Finely grated cheese (1 g) was directly incubated with trivalerin (204 mg) as a substrate at 35 °C for 4 h. Free valeric acid was extracted and quantified by gas chromatography. The assay was linear with time up to 24 h and up to 0.11 Lipase Units g −1 cheese. The total amount of lipolytic activity determined with this assay was consistent with the total amount of FFA present in 5 types of cheese. In Idiazabal cheese samples manufactured with or without lipase added, the total lipolytic activity determined during ripening remained constant up to 6 months of ripening. In addition to trivalerin, other possible substrates investigated were triolein, triundecanoin and the endogenous butyric acid-containing triacylglycerols present in the cheese sample. Activities measured with these substrates were considerably lower than values obtained with trivalerin due to the high levels of oleic acid present in cheese, or to difficulties in mixing triundecanoin (solid at 35 °C) with the grated cheese sample. Endogenous triacylglycerols gave increasingly lower activity values as ripening time progressed due to substrate depletion.

Direct amperometric determination of lipase activity

Lipases, triacylglycerol hydrolases (EC 3.1.1.3) that cleave triacylglycerols at the oil/water interface, have extensive applications in the food, paper, pharmaceutical, cosmetic, detergent, leather, and textile industries [1–5]. Widespread practical use of these enzymes requires fast and reliable analytical routines to assess their activity. Typical protocols for lipase assay include use of a pH-stat and back titrations, application of a Langmuir–Blodget film balance, spectrophotometric determination of para-nitrophenyl palmitate (pNPP) hydrolysis, etc. (for critical review, see [4]). Though commonly used, the above-mentioned techniques have several drawbacks: the methods of pH-stat and monolayer film balance are relatively cumbersome, expensive, and difficult to adapt for portable hand-held devices, while the relatively simple method of back titration does not provide continuous kinetic data. At the same time, the continuous spectrophotometric pNPP assay is limited to the transparent solutions, and it and the pH-stat titration are unusable under acidic pH conditions. The turbidity problem can be resolved by chemical or physical pretreatment of the solution. For example, with regard to the pNPP-based procedure, Gupta et al. [6] proposed to overcome this problem by using the nonionic surfactant Triton X-100. The electrochemical analysis techniques are practically insensitive to the presence of solid or/and liquid lightscattering microparticles in the solutions and could be

Validation and diagnostic efficacy of a lipase assay using the substrate 1,2‐o‐dilauryl‐rac‐glycero glutaric acid‐(6′methyl resorufin)‐ester for the diagnosis of acute pancreatitis in dogs

Increased serum lipase activity has been used historically to support the diagnosis of acute pancreatitis, a common disease in dogs. Most of the lipase assays that are currently in use lack optimum sensitivity and specificity. The objectives of this study were to 1) validate the 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester (DGGR) assay for determination of lipase activity in canine serum and 2) compare results, reference intervals, sensitivity, and specificity of the DGGR assay with a standard 1,2-diglyceride (1,2 DiG) assay for diagnosing acute pancreatitis in dogs. Precision, linearity, and interference studies were performed for method validation on a Hitachi 911 analyzer. Lipase results from the DGGR and 1,2 DiG assays were compared by linear regression analysis. Sensitivity, specificity, and diagnostic efficacy were determined for both assays on a population of 30 dogs, 15 of which had acute pancreatitis based on history, clinical signs, and ultrasound findings. Within-run and within-day coefficients of variation (CVs) were low (<3%), with higher day-to-day CVs (< or =14 %). The assay was linear between 8 and 2792 U/L. No significant interference by hemolysis and lipemia was found. Poor correlation was found between the assays (r(s)=0.84). The lipase reference interval was 8-120 U/L for the DGGR assay and 30-699 U/L for the 1,2 DiG assay. Sensitivity and specificity for the diagnosis of pancreatitis were 93% and 53%, respectively, for the DGGR assay and 60% and 73% for the 1,2 DiG assay. Receiver operating characteristic curve analysis showed similar areas under the curve. On the basis of this study, the DGGR method is considered adequate for assaying serum lipase activity in dogs. The high sensitivity of the DGGR assay suggests it may be a useful screening test for canine pancreatitis.

Mutational Analysis of the “Regulatory Module” of Hormone-Sensitive Lipase

Hormone-sensitive lipase (HSL) is a rate-limiting enzyme in lipolysis that displays broad substrate specificity. HSL function is regulated by reversible phosphorylation that occurs within a 150 aa "regulatory module" of the protein. The current studies used mutational analysis to dissect the contribution of the "regulatory module" in HSL activity and substrate specificity. Deletion of the entire "regulatory module" or replacement of the "regulatory module" with the "lid" of lipoprotein lipase resulted in enzymatically inactive proteins. Deletion of sequentially longer stretches of the "regulatory module" resulted in a stepwise reduction in hydrolytic activity. Analysis of 7-19 amino acid deletional mutants that spanned the "regulatory module" showed that the N-terminal partial deletion mutants retained normal hydrolytic activity and activation by PKA. In contrast, the C-terminal partial deletion mutants displayed reduced hydrolytic activities, with preferential loss of activity against lipid-, as opposed to water-soluble, substrates. Single amino acid mutations of F650C, P651A, and F654D reduced activity against lipid-, but not water-soluble, substrates. The current results suggest that the length of the "regulatory module" and specific sequences within the C-terminal portion of the "regulatory module" of HSL (amino acids 644-683) are crucial for activity and appear to be responsible for determining lipase activity.

A colorimetric method for the determination of lipase activity in soil

A colorimetric method for the determination of lipase activity in soil has been developed. Using p-nitrophenyl butyrate as substrate, soil samples are incubated at 30 °C and pH 7.25 for 10 min. After cooling on ice and centrifugation, the released p-nitrophenol is determined at 400 nm. To allow for the adsorption of p-nitrophenol onto soil, a calibration curve is prepared in the presence of soil.