Assay Of Lipase Activity Research Articles

Use of naturally fluorescent triacylglycerols from Parinari glaberrimum to detect low lipase activities from Arabidopsis thaliana seedlings

The aim of this study was to design a convenient, specific, sensitive, and continuous lipase activity assay using natural long-chain triacylglycerols (TAGs). Oil was extracted from Parinari glaberrimum seed kernels and the purified TAGs were used as a substrate for detecting low levels of lipase activities. The purified TAGs are naturally fluorescent because more than half of the fatty acids from Parinari oil are known to contain 9,11,13,15-octadecatetraenoic acid (parinaric acid) in its esterified form. The presence of detergents (sodium taurodeoxycholate, CHAPS, Sulfobetaine SB12, Tween® 20, Brij® 35, Dobanol®, n-dodecylglucoside) above their critical micellar concentration dramatically increases the fluorescence of the parinaric acid released by various lipases. This increase in the fluorescence intensity is linear with time and proportional to the amount of lipase added.▪ This new method, performed under non-oxidative conditions, was applied successfully to detecting low lipase levels in crude protein extracts from plant seeds and could be scaled down to microtiterplate measurements. Quantities as low as 0.1 ng of pure pancreatic lipase could be detected under standard conditions (pH 8). Lipase activity can also be assayed in acidic media (pH 5) using human gastric lipase. This simple and continuous assay is compatible with a high sample throughput and might be applied to detecting true lipase activities in various biological samples.—Beisson, F., N. Ferté, J. Nari, G. Noat, V. Arondel, and R. Verger. Use of naturally fluorescent triacylglycerols from Parinari glaberrimum to detect low lipase activities from Arabidopsis thaliana seedlings.

Importance of the definition of catalytic properties for the commutability of an enzyme reference material: example of lipase

Assays for pancreatic lipase activity in serum need the use of calibrators, but there is a lack of a reference method and of a reference material. Under routine conditions, comparability of lipase catalytic activities was found to be poor when lipase was determined in 50 patients’ specimens by a turbidimetric (Boehringer) and a colorimetric (Sigma) assay. Mean values of the results differed by a ratio of 2.39. Catalytic properties of three materials, two commercial calibrators and a home-purified preparation of human pancreatic lipase (HPL), have been compared in titrimetry at constant pH. Optimal common conditions were defined for the titration of lipase activity in the three materials. When using these titres for each calibrator, comparability was greatly improved (ratio = 1.25). This result indicates that a significant part of between-method discrepancy was due to the lack of a reference method for the titration of lipase calibrators. The inter-method behavior of each material was compared to that of patients’ specimens. Using HPL as calibrator, the comparability of patients’ results was still dramatically improved (ratio = 1.01). This study shows the importance of verifying the similarity of catalytic properties of an enzyme reference material to those of the corresponding enzyme in human specimens, in order to ensure its commutability. The use of validated commutable calibrators, traceable to certified reference materials, is an opportunity to improve the inter-method comparability of results in clinical enzymology.

New fluorescent glycerolipids for a dual wavelength assay of lipase activity and stereoselectivity

A new type of fluorogenic alkyldiacyl glycerols was synthesized and used as fluorogenic substrates for the analysis of lipase activities and stereoselectivities. These compounds contain pyrene or perylene as a fluorophore and the trinitrophenyl-amino- (TNP) residue as a quencher. Both substituents are covalently bound to the ω-ends of the sn-2 and sn-1(3) acyl chains, respectively. Upon glycerolipid hydrolysis, the residues are separated from each other thus allowing determination of lipase activity from the continuous increase in fluorescence intensity which is caused by dequenching. Mixtures of enantiomeric fluorogenic alkyldiacyl glycerols, selectively labeled with pyrene or perylene as fluorophores, can be used for a dual-wavelength ‘stereoassay’ of lipases. Since absorption and emission maxima of both labels are clearly separated, hydrolysis of the respective enantiomeric substrates can be distinguished simultaneously, and the difference in their rates of hydrolysis can be taken as a parameter for the stereopreference of a lipase.

Hydrolysis of p-nitrophenyl palmitate in n-heptane by the Pseudomonas cepacia lipase: A simple test for the determination of lipase activity in organic media

The rate of p-nitrophenyl palmitate (pNPP) hydrolysis in n-heptane by a lipase preparation from Pseudomonas cepacia was studied. In organic media, the reaction rate could be easily measured by spectrophotometry after extraction of the liberated p-nitrophenol by an alkaline water phase. Optimal conditions for substrate hydrolysis were selected and proposed for the easy and fast assay of lipase activity in organic media. Agitation and the amount of added water showed little effect on reaction rates. A temperature of 37°C and substrate concentration of 50 m m were selected. The reaction rate in n-heptane was only 0.16% of that in water.

Periprandial systemic and regional lipase activity in normal humans.

An assay for plasma lipoprotein lipase activity was used without prior injection of heparin to study arteriovenous differences of lipases across skeletal muscle and adipose tissue of normal male volunteers. Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) activities and triglyceride?concentrations were measured in arterial plasma and in venous effluent plasma from forearm skeletal muscle and subcutaneous abdominal adipose tissue, in the postabsorptive state and after a mixed meal. Triglyceride clearance by the tissues was greater across adipose tissue than across muscle. There were no arteriovenous differences for HTGL activity. In the postabsorptive state skeletal muscle released LPL activity, but adipose tissue did not. Postprandially the arterial LPL and HTGL activities did not change. LPL activity in adipose tissue venous effluent rose, whereas that in muscle venous effluent decreased. These results show that the release of LPL from subcutaneous adipose and forearm tissues is regulated differently, reflecting in vivo differences in LPL regulation at the tissue level.

Enantiomeric perylene-glycerolipids as fluorogenic substrates for a dual wavelength assay of lipase activity and stereoselectivity.

A new type of fluorogenic alkyldiacyl glycerols was synthesized and used as fluorogenic substrates for the analysis of lipase activities and stereoselectivities. These compounds contain perylene as a fluorophore and the trinitrophenylamino (TNP) residue as a quencher. Both substituents are covalently bound to the omega-ends of the sn-2 and sn-1 (3) acyl chains, respectively. Upon glycerolipid hydrolysis, the residues are separated from each other thus allowing determination of lipase activity by the continuous increase in fluorescence intensity which is caused by dequenching. Using enantiomeric pairs of these compounds, we were able to analyze lipase stereoselectivity depending on the reaction medium. Mixtures of enantiomeric fluorogenic alkyldiacyl glycerols, selectively labelled with pyrene or perylene as fluorophores, can be used for a dual-wavelength "stereoassay" of lipases. Since absorption and emission maxima of both labels are clearly separated, hydrolysis of the respective enantiomeric substrates can be determined simultaneously, and the difference in the rates of hydrolysis can be taken as a parameter for the stereopreference of a lipase. Hydrolysis rates measured with perylene-substituted lipids are generally lower than those obtained with the pyrene analogs. Thus, with a mixture of perylene and pyrene-substituted lipids, we observe a higher apparent stereoselectivity of lipases since we measure a combination of stereo- and substrate selectivity. In the presence of albumin, all microbial lipases tested so far exhibit stereopreference for the sn-1 glycerol position. In our assay, the apparent stereoselectivities are highest if in the presence of albumin, the sn-1 position carries pyrene and the sn-3 position is substituted with perylene. The lipase stereoselectivity assay described here requires the simultaneous measurement of the fluorescence intensities at two different wavelengths in a single cuvette and can thus be carried out using existing and cheap instrumentation that was developed for the fluorimetric analysis of Ca+2 concentrations.

A new assay for lipase activity in organic solvents. Lipase-catalyzed synthesis of octyl-linolenate in a hexane microaqueous reaction system

Rhizopus arrhizus lipase (RAL) catalyzed the synthesis of the ester bond between linolenic acid and octanol in a hexane microaqueous system. Lipase activity was determined by measuring increases in absorbance of the hexane at 235 nm, as a function of reaction time, after addition of sufficient silica (0.1 g per 20 m m linolenic acid) to remove free fatty acids, but not the wax product (octyl-linolenate), followed by separation of the silica by centrifugation. The activity of the lipase freeze-dried to the bottom of a glass vial increased linearly as a function of enzyme concentration in the range 0–0.1 EU. RAL displayed maximum activity at pH 7.6 at both water activities of 0.33 and 0.75. Maximum synthetic activity was observed at a water activity of 0.75. The optimum reaction temperature was 40°C, both at pH 7.0 ( a w = 0.33) and pH 7.6 ( a w = 0.75). Preliminary kinetic analysis of the synthetic reaction provided an estimate of V max K m for linolenic acid of 0.00095 min −1 (K m = 15.2 m m, V max = 14.6 μ m min −1 at 50 m m octanol). In comparison, a V max K m of 0.0032 min −1 (K m = 12.6 m m, V max = 40.0 μ m min −1 at 50 m m octanol) was obtained using a fatty acid-copper salt method for the determination of free fatty acid concentration. No hydrolytic activity could be detected in this system.

Direct HPLC Monitoring of Lipase Activity in Reverse Micellar Media

Given the profusion of biotechnological applications of the nonaqueous use of lipases, we have evaluated the possibilities of exploiting the inherent advantages of high performance liquid chromatography (HPLC) for a simple, rapid assay of lipase activity in reverse micellar media, as a convenient alternative to previously reported spectroscopic methods, using both a model system and esterification reaction, and different commercial lipases. The results obtained after a screening for optimized chromatographic conditions in the reverse-phase mode indicate that a satisfactory resolution of the reaction components can be obtained following a straightforward protocol, which permits an accurate, reliable quantitation of the reaction progress, regardless of the enzyme used.

Two Automated Fully Enzymatic Assays for Lipase Activity in Serum Compared: Positive Interference from Post-Heparin Lipase Activity

The fully enzymatic Wako and Ektachem assays for pancreatic lipase in serum were found to yield precise (especially Ektachem) and consistent results that were significantly correlated (r = 0.995). Less good concordance was found with the turbidimetric aca method, results being on several occasions above normal by Wako or Ektachem but within the reference limit by the aca. Supplementation of aca packs with colipase generally increased lipase activities measured by the aca in these samples, thereby improving the correlation with Wako and Ektachem. In vitro addition experiments documented that both Wako and Ektachem were relatively insensitive to common chemical interferences. However, post-heparin lipase activities produced a positive interference in both assays, resulting in seemingly increased lipase activities that were suggestive of pancreatic disease. Substituting sodium glycocholate (6 mmol/L) for deoxycholic acid largely eliminated this positive interference in the Wako assay. Because Ektachem reagents cannot be modified in this way, we added sodium glycocholate (2-12 mmol/L) to sera; however, this failed to dissipate the in vivo effect of heparin on the Ektachem results.

High-performance liquid chromatographic assay of pancreatic lipase activity

High-performance liquid chromatographic assay of pancreatic lipase activity

A Spectrophotometric Lipase Activity Assay by Coupled Lipase-Lipoxygenase in Reverse Micelles

A new, continuous spectrophotometric method is described for determining lipase activity using a reverse micelle system, in which lipase (EC 3.1.1.3) and lipoxygenase (EC 1.13.11.12) are dissolved. The reverse micelle system consists of 2-ethyl hexyl sodium sulfosuccinate (AOT)-isooctane and water. Trilinolein is used as the lipase substrate; linoleate hydroperoxide is the end product of the oxidation catalyzed by lipoxygenase, which acts as an auxiliary coupled-enzyme of lipase. The method appears useful both for detailed kinetic studies of lipase and for serial analyses using sunflower oil, a cheaper substrate. This assay offers the typical advantages of the continuous direct photometric methods in that it is rapid, reproducible and sufficiently sensitive for measuring lipase activity even in some crude commercial preparations.

A novel spectrophotometric assay for lipase activity utilizing cis-parinaric acid.

A new spectrophotometric assay for determining the activity of acylglycerol hydrolases (lipases, E.C. 3.1.1.3) was developed and optimized for yeast lipase (Candida cylindracea). Studies with porcine pancreatic lipase were also conducted and the influence of various detergents and divalent cations on the assay was evaluated. The assay uses cis-parinaric acid (PnA), a naturally occurring fatty acid that has unique spectroscopic properties, and takes advantage of the reversible binding of fatty acids to bovine serum albumin (BSA). Free PnA has an ultraviolet absorption peak at 321.2 nm. When PnA is bound to BSA, however, the peak shifts to 324.2 nm. The assay mixture contains 6 microM PnA, 1 microM BSA, 75 microM triolein, and 0.3 mM taurocholate in a 50 mM tris-HCl buffer with 1 microM EDTA. The release of oleic acid from triolein is monitored over time by measuring the ratio of optical densities (OD) at 319.0 and 329.0 nm. Initially, there is maximum binding of PnA to BSA, and the OD ratio is approximately 1.0. Upon addition of lipase, PnA is displaced from the BSA by oleic acid released from triolein, and the OD ratio increases to a maximum of about 1.8. However, when calcium is present in the reaction mixture an insoluble calcium-PnA complex forms, resulting in a progressive decrease in OD at both 319.0 and 329.0 nm. The kinetic assay described here is simple, rapid, sensitive, reproducible, inexpensive, and it can be adapted to measure the activity of a variety of calcium-independent lipases. Under similar assay conditions, activities for Candida cylindracea lipase obtained with this assay are similar to those obtained with 14C-labelled triolein.

Measuring carboxypeptidase A activity with a centrifugal analyzer: analytical and clinical considerations.

This adaptation of a commercially available kit for automated measurement of carboxypeptidase A (CPA; EC 3.4.17.1) activity in serum with the Cobas Bio centrifugal analyzer extends the linear range to an activity concentration of 82 U/L. Results obtained by the described method correlated closely (r = 0.98) with those by the manual kit method. The reference interval for 150 apparently normal individuals was 0.12-0.91 U/L. Total CVs of the method ranged from 4.0% to 13.1%. Bilirubin and glucose decreased the CPA activity in serum by as much as 98% and 26%, respectively. Substantial CPA activity was found in pancreatic tissue, with little activity in intestinal tissue. CPA activity was not as widely distributed in extra-pancreatic tissues as were amylase and lipase activities. Peak activities of CPA, amylase, and lipase in the sera of patients with acute pancreatitis were significantly correlated (r = 0.45 to 0.78, P less than 0.05-0.01). The optimized diagnostic efficiency of CPA for acute pancreatitis was 0.85 at a cutoff value of 5 U/L. Amylase and lipase exhibited similar optimized efficiencies, and parallel testing did not significantly improve diagnostic accuracy. We conclude that automated analysis for CPA activity, even in the absence of interferences, does not add to the diagnostic information provided by the widely available assays for amylase and lipase activity.

A simple method for the separation of triacylglycerols from fatty acids released in lipase assays

A simple procedure for the partition of triacylglycerols from albumin-bound fatty acids is described. This procedure is based on the ability of fumed silicon dioxide to remove emulsified triacylglycerols from aqueous media. The method was developed for the assay of lipoprotein lipase activity but it may be used for the assay of other lipases.

An improved method for the colorimetric assay of lipase activity using an optically clear medium.

Lipase activity can be spectrophometrically measured in an optically clear medium using long chain fatty thioesters of 1-mercapto-2,3-propanediol or 2-mercaptoethanol as substrates. With hexamethylphosphoric triamide solutions of these thiosubstrates, the Michaëlis-Menten constants of lipase from Rhizopus arrhizus were determined. The effects of calcium chloride and of bovine serum albumin on the enzyme activity were established.

An in vitro method for assay of hormone or agonist-stimulated lipase activity

An assay for in vitro measurement of hormone or agonist-stimulated lipase activity, using exogenous prostaglandin synthetase as a trap for liberated arachidonic acid is described. Fifteen of twenty-six hormones and agonists previously tested in vivo , were tested in vitro and either stimulated or inhibited release of arachidonic acid parallel to their activity in vivo . Vasopressin stimulated the release of arachidonic acid in −5 linear, log-dose manner, from 10 −11 M to 10 −5 M.

Screening for Fungal Lipase Using Chromogenic Lipid Substrates

(1986). Screening for Fungal Lipase Using Chromogenic Lipid Substrates. Mycologia: Vol. 78, No. 2, pp. 298-300.

Lipid‐lipase interactions. 2. A new method for the assay of lipase activity

AbstractA simplified procedure for the measurement of activity was developed for lipases (EC 3.1.1.3) fromChromobacterium viscosum, Candida rugosa, Aspergillus niger andRhizopus arrhizus. It differs from existing procedures in that olive oil, refined with a bleaching clay, was used as the substrate, periodic sonication was applied to promote efficient emulsification, the hydrolysis time was extended to 1 hr, and no additive such as protective colloids and surfactants were used. The activity was determined arbitrarily for that amount of lipase required to bring about 24% hydrolysis/hr at room temperature Unlike currently used assay procedures, the present method gives results which are highly reproducible with a maximum standard deviation of 2.5% and, more commonly, less than 1.00%.

Hydrolysis of 4‐methylumbelliferyl butyrate: A convenient and sensitive fluorescent assay for lipase activity

AbstractA rapid, sensitive and convenient fluorescent assay was developed to screen for lipase activity. The non‐fluorescent substrate, 4‐methylumbelliferyl butyrate (4‐MUB), solubilized in either liposomal dispersions or bile salt/lecithin mixed micelles, is hydrolyzed to butyric acid and the highly fluorescent compound, 4‐methylumbelliferone (4‐MU). Assays are run at 37 C for 10 min, terminated, and the changes in fluorescence quantitated with a Turner III fluorometer. Both lingual and pancreatic lipases exhibit activity against this artificial substrate. The assay has several advantages: nmoles 4 MU/ml/hr are measured allowing the detection of very low lipolytic activity; multiple samples may be simultaneously assayed, and only a brief incubation period is required.

Subcellular localization and properties of lipase activities in human polymorphonuclear leukocytes

A fluorimetric assay for lipase activity has been optimized for measurement of the enzyme in human neutrophils. Activity was maximal at acid (4.5) and alkaline (9.5) pH, although there was also a neutral peak of activity at pH 6.5. Neutrophils were homogenised in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation. The gradient fractions were assayed for acid, neutral and alkaline lipase activity and for the principal organelle marker enzymes. Neutral lipase showed a unimodal distribution with an equilibrium density of 1.19 g δ cm −3, corresponding to the distribution of particulate leucine aminopeptidase. Acid and alkaline lipase activities showed very similar distribution profiles to each other with both soluble components and a broad peak of particulate activity. The broad modal density of 1.19–1.22 g · cm −3 suggests that acid and alkaline lipase activities could be localised to more than one population of cytoplasmic granule. Fractionation experiments with neutrophils homogenised in sucrose medium containing digitonin confirmed the localisation of neutral lipase and leucine aminopeptidase to the same cytoplasmic granule, and suggested that at least part of the acid lipase activity was localised to the specific granule. No lipase activity could be attributed to the alkaline phosphatase-containing granule. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and women in the third trimester of pregnancy. The specific activity of acid, neutral and alkaline lipase, and leucine aminopeptidase, in contrast to that of alkaline phosphatase, were similar in the three patient groups.