Lipase activity assay

Place 0.05 to 0.50 ml of 0.5 mM p-nitrophenol standard solution into ten individual 15- to 20-ml test tubes and dilute each to 5 ml with 0.1 M Tris-Cl buffer, pH 8.2. This yields a standard curve of 0.005 to 0.05 fimol p-nitrophenol/ml. 2. Measure A410 using 0.1 M Tris Cl buffer, pH 8.2, as a blank, and make a standard curve by plotting A410 versus the p-nitrophenol concentration in each tube. 3. For each lipase activity assay, place 2.5 ml of 0.1 M Tris-Cl buffer, pH 8.2, and 2.5 ml of 420 pM p-nitrophenyl laurate substrate solution into a 15- to 20-ml test tube. Prepare one extra tube for a reagent blank. 4. Add 1 ml water to the reagent blank. Lypolytic Enzymes... 

Table 1 Effect of soluble fractions on microsomal lipase activity. Assays were performed in the presence of 15 ig microsomal protein -... 

Recently Shihabi and Bishop (93) described a refinement in the preparation of a stable substrate and demonstrated the feasibility monitoring the reaction kinetically. This procedure has been evaluated by Lifton et. al. (9 ), who found that this method correlated well (r 0.914) with the copper soap-lipase method of Dirstine. They concluded that the method was rapid (less than 5 min. per sample), accurate, precise and linear over a clinically useful range. Its simplicity allows its application as an emergency procedure. Attempts to use this assay for urine lipase activity were unsuccessful. 

Shihabi, Z. R. and Bishop, C. Simplified turbidimetric assay for lipase activity. Clin. Chem. (1971), 17 1150-1153. 

The LPL catalytic assay measures the hydrolysis of a [14C[- or [3H]-triolein emulsion producing the 14C- or 3H -labeled free oleic acid [6]. The 14C- or 3H-labeled oleic acid is isolated by a selective extraction procedure and its radioactivity is determined by liquid scintillation counting [40]. Lipase activity is calculated as nanomoles of oleic acid released per minute per milliliter of postheparin plasma [41]. 

Two assays have been developed for measuring the relative concentration of lipoprotein lipase activator in milk. Anderson (1979) developed an immunoassay and Super et al (1976) used [23H]glycerol triolein and measured the liberated [23H]glycerol. 

The assays presented in this section deal with the measurement of enzyme activity, which is expected to be proportional to the amount of active enzyme present in a sample, food or otherwise, unit ci.i is an overview of the important considerations in performing activity assays unitc 1.2 illustrates how these considerations are applied to the assay of a representative food-relevant glycosyl hydrolase. Chapters C2, C3, and C4 present the first units on particular types of activity assays. In Chapter C2, two units present peptidase activity assays that use either synthetic substrates (UNITC2.1) or common, commercially available protein substrates (unit C2.2). unitC3.i presents three different assays for lipase activity. unitc4.i presents assays for diphenol oxidases, and unitc4.2 for lipoxygenase. 

Basic Protocol 1 Titrimetric Determination of Lipase Activity Basic Protocol 2 Colorimetric Assay of Lipase Activity Using the Copper Soap Method... 

Figure C3.1.1 Comparison of lipase activities using the titrimetric assay. The lipases used in the reaction mixture, all at 0.1 mg/ml, were from B. cepacia (diamonds), C. rugosa (triangles), and porcine pancreas (circles). Broken lines represent estimation of initial reaction rates.

Determine the number of units (U) of lipase activity, which is defined as the amount that produces 1 jimol of fatty acid per minute under the specified assay conditions. 

COLORIMETRIC ASSAY OF LIPASE ACTIVITY USING THE COPPER SOAP METHOD... 

Conceptually, assays for lipase activity using the colorimetric method (copper-soap procedure Basic Protocol 2) are similar to titrimetry in that liberated fatty acids are being measured however, the colorimetric method is more specific for fatty acids (Lowry and Tinsley, 1976). Quenched subsamples of emulsified acylglycerol/lipase reaction mixtures are combined with the biphasic mixture of cupric acetate/pyridine and benzene. Cupric salts of the fatty acids are formed (molar stoichiometry of fatty acid to Cu2+ of 4 2) and these soaps, which are blue in color, are partitioned into benzene to allow for quantification by measuring absorbance of the clear benzene phase at 715 nm. 

The greatest advantage of the spectrophotometric method is that it is direct and rapid, requires no sample workup, and allows for continuous assays of lipase activity compared to the multiple fixed-time-point analyses incumbent within Basic Protocols 1 and 2. The spectrophotometric method can also be done using very small volumes (as small as 1 ml) and is suitable for following the course of purification (such as in chromatographic fractions) or adaptable to 96-well plates (and subject to automation, if available). Thus, it is the method of choice for screening several samples or preparations for lipase (esterase) activity. 

In addition to assay features already mentioned, other factors may influence the choice of assay by the user. In terms of sensitivity of the assay, the threshold of detection of lipase activity, using the procedures as described in this unit, is on the order of 10 2 U for titrimetry, 10H U for colorimetry, and 10 4 U for spectrophotometry (where U is the amount of enzyme required to yield 1 imol product per minute). The smallest amounts (volumes) of materials, including enzyme, are required for the spectrophotometric method, and progressively more material is required for the colorimetric and titrimetric methods. Unless a flow cell adapter is available, the spectrophotometric method is not suitable for analysis of particulate (immobilized) enzyme preparations, whereas the other assay procedures are. 

Figure C3.1.3 Comparison of lipase activities using the copper soap colorimetric assay as affected by the degree of homogenization of olive oil substrate. The lipase used in the reaction mixture was from C. rugosa (at 0.40 mg/ml) in the presence mechanically homogenized (triangles) or poorly homogenized (shaken by hand diamonds) substrate emulsion. For comparison of linearity of reaction progress curves, the time course of C. rugosa lipase (at 0.20 mg/ml circles) is provided.

Contrasts several popular and less common methods for delecting and assaying lipase activity. Discusses advantages, disadvantages, and the unique requirements of each method for obtaining accurate estimations of lipase activity. 

Hydrolytic activities of free and immobilized lipase were assayed by the olive oil emulsion method according to the modification proposed by Soares et al. (11). One unit of enzyme activity was defined as the amount of enzyme that liberated 1 imol of free fatty acid/min under the assay conditions (37°C, pH 7.0,150 rpm). Analyses of hydrolytic activities carried out on the lipase loading solution and immobilized preparations were used to determine the activity-coupling yield (r %), which measures the recovered enzymatic activity according to Eq. 1 ... 

Because of the existence of the interfering catboxylester lipase activity (Table 2) in the pancreas, dissolved substrates are not adequate for the quantitative assay of... 

Application and Principle This procedure is used to determine the lipase activity in preparations derived from microbial sources and animal pancreatic tissues. The assay is based on the potentiometric measurement of the rate at which the preparations will catalyze the hydrolysis of tributyrin. 

Calculation From the Standard Test Dilution, plot the volume of 0.1 A sodium hydroxide titrated against time. Using only the points that fall on the straight-line segment of the curve, calculate the mean acidity released per min by the Assay Test Dilution. Taking into consideration dilution factors, calculate the lipase activity of the Standard Test Dilution, using the lipase activity of the USP Pancreatin Reference Standard stated on the label. 

Transcription ofT7 RNA polymerase is induced by addition of isopropyl-p-D-thiogalactoside. The polymerase then directs the transcription of the lipase operon. The lipase is correctly folded and secreted into the bacterial culture supernatant which can subsequently be used to assay lipase activities. 

EN92 Demanet, C., Goedhuys, W., Haentjens, M., Huyghens, L., Blaton, V. and Gorus, F. (1992). Two automated fully enzymatic assays for lipase activity in serum compared Positive interference from post-heparin lipase activity. Clin. Chem. 38, 288-292. 

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