3 Deec 51 Aca 15 Ef 0580

Eur. J. Lipid Sci. Technol.
2000, 133153 Frdric Beisson*, Ali Tiss*, Claude Rivire, Robert Verger
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Methods for lipase detection and assay: a critical review

1 2 2.1 2.1.1 2.1.2 2.1.2.1 2.1.2.2 2.1.2.3 2.1.3 2.1.4 2.2 2.2.1 2.2.1.1 2.2.1.2 2.2.2 2.2.2.1 2.2.2.1.1 2.2.2.1.2 2.2.2.1.3 2.2.2.1.4 2.2.2.1.5 2.2.2.2 2.2.2.2.1 2.2.2.2.2 2.2.2.2.3 2.2.3 2.2.3.1 2.2.3.2 2.2.3.3 2.2.4 2.2.5 2.2.6 3 3.1 3.2 3.2.1 3.2.2 3.3 3.4 4 4.1 4.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 Physico-chemical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 Disappearance of substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 Nephelometry and turbidimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 The Wilhelmy plate method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 Pure monomolecular films as substrates . . . . . . . . . . . . . . . . . . . . . . . . 135 Mixed monomolecular films as substrates . . . . . . . . . . . . . . . . . . . . . . . 136 Interfacial binding and film recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 Atomic force microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Infrared spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 Appearance of hydrolytic reaction products . . . . . . . . . . . . . . . . . . . . . . 138 Proton release as an indirect assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 Coloured indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 Titrimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 Analysis of the free fatty acids released. . . . . . . . . . . . . . . . . . . . . . . . . . 139 Glycerol derived carboxylic esters as substrates . . . . . . . . . . . . . . . . . . 139 Colourimetric assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 Fluorimetric assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 Chromatographic assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 Enzymatic assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 In situ detection by electron microscopy . . . . . . . . . . . . . . . . . . . . . . . . . 141 Synthetic carboxylic esters as substrates . . . . . . . . . . . . . . . . . . . . . . . . 141 Radioactive assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 Colourimetric assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 Fluorimetric assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Determination of the released alcohol or thiol . . . . . . . . . . . . . . . . . . . . . 143 Determination of the released glycerol from TAG . . . . . . . . . . . . . . . . . . 143 Determination of the released coloured or fluorescent alcohol from synthetic carboxylic esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Determination of the released thiol from synthetic thioesters . . . . . . . . . 144 Electric conductivity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 Acoustic wave conductance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 The oil-drop method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Immunological methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 ELISA for pure lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 Lipase immunodetection on physiological media . . . . . . . . . . . . . . . . . . 146 Lipase immunodetection in plasma or serum . . . . . . . . . . . . . . . . . . . . . 146 Lipase immunodetection in other physiological media . . . . . . . . . . . . . . 146 Immunocytolocalisation of lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 Immunoblot analysis of lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 Pure lipases or crude biological media . . . . . . . . . . . . . . . . . . . . . . . . . . 147 Level of lipase acitity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Abbreviations: AFM, atomic force microscopy; BSA, bovine serum albumin; ELISA, enzyme-linked immuno sorbent assay; FFA, free fatty acids; FTIR, Fourier transform infrared spectroscopy, HGL, human gastric lipase; HLL, Humicola lanuginosa lipase; HPL, human pancreatic lipase; IU, international units (moles of FFA released per min.); LPL, lipoprotein lipase; PPL, porcine pancreatic lipase; TAG, triacylglycerol.
Laboratoire de Lipolyse Enzymatique (UPR 9025 du CNRS), Institut de Biologie Structurale et Microbiologie, Marseille, France
Correspondence: Robert Verger, Laboratoire de Lipolyse Enzymatique (UPR 9025 du CNRS), Institut de Biologie Structurale et Microbiologie (IFR1 du CNRS et de lUniversit de la Mditerrane), 31 Chemin Joseph-Aiguier, 13402 Marseille Cedex 20, France. Phone: +33 4 91 16 40 93; Fax: +33 4 91 71 58 57; e-mail: verger@ibsm.cnrs-mrs.fr. * These authors have made equal contributions to the present review.
WILEY-VCH Verlag GmbH, 69451 Weinheim, 2000
0931-5985/2000/0202-0133 $17.50+.50/0
Review Article
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Eur. J. Lipid Sci. Technol. 2000, 133153 hydrocarbon chains of biologically usable TAGs. The lipases in the digestive tract therefore play a particularly important role in nutrition processes, in both humans and higher animals. Lipases are now being widely used as enantioselective catalysts in aqueous as well as in low-water media, and various synthetic molecules can serve as their substrates. In this review, we will deal only with lipase assays involving carboxylic ester hydrolysis and not with the methods designed for studying alcoholysis, acidolysis, ester synthesis, or inter- and transesterification reactions. The latter reactions are usually performed under low water activity conditions [2430]. Furthermore, the reader is referred to published articles concerning the stereoselective assays involving lipases [3140]. As can be seen from the literature [6, 41, 42], numerous methods are available for measuring the hydrolytic activity as well as for the detection of lipases. These methods can be classified as follows: 1. Titrimetry, 2. spectroscopy (photometry, fluorimetry, infra red), 3. chromatography, 4. radioactivity, 5. interfacial tensiometry, 6. turbidimetry, 7. conductimetry, 8. immunochemistry, 9. microscopy. It should be kept in mind, however, that the general triacylglycerol hydrolysis reaction catalysed by lipases can be written as follows:
1 Introduction
Lipids constitute a large part of the earths biomass, and lipolytic enzymes play an important role in the turnover of these water-insoluble compounds. Lipolytic enzymes are involved in the breakdown and thus in the mobilisation of lipids within the cells of individual organisms as well as in the transfer of lipids from one organism to another. One important aspect of lipolytic enzymes is the unique physico-chemical character of the reactions they catalyse at lipid-water interfaces, involving interfacial adsorption and subsequent catalysis sensu stricto. Most of the lipases are water-soluble enzymes acting on water-insoluble substrates (supersubstrates). The heterogeneous character of this catalysis makes it difficult to accurately quantitate both the amount of interface (specific surface) [1] as well as the interfacial parameters (such as the interfacial tension, surface viscosity, surface potential, etc.) responsible for the interfacial quality of the substrate [25], on which the lipolytic process greatly depends. The emulsification of the water-insoluble substrates, which requires the presence at the interface of surface active amphiphiles such as detergents, other lipids, proteins, etc., can therefore drastically influence lipase activity measurements: non-specific inhibition of lipases by proteins present at the oil/water interface is one well-known phenomenon of this kind [610]. Lipases were previously defined in kinetic terms, based on the interfacial activation phenomenon, i.e., on the increase in the activity which occurs when a partially watersoluble substrate becomes water-insoluble [11]. This process was not observed among esterases. The recently determined 3-D structures of lipases show an / hydrolase fold as well as a nucleophilic elbow where the catalytic serine is located [12, 13]. Some, but not all lipases show a lid controlling access to the active site. However, the above structural features, including the presence of a lid as well as the interfacial activation phenomenon, are not suitable criteria for classifying specific esterases as lipases. Since enzymes are usually named after the type of reaction they catalyse, lipases can be pragmatically redefined as carboxylesterases acting on long-chain acylglycerols: they are simply fat-splitting ferments [14]. Lipases are ubiquitous enzymes [6, 1517] which are found in animals, plants [18, 19], fungi [20] and bacteria [2123]. In the industrially developed countries, the edible lipids present in the human diet, which consist mainly of triacylglycerols (TAGs), from 100 to about 150 grams per day, i.e. 30% of each individuals daily caloric intake. TAG molecules cannot cross the intestinal barrier. A series of hydrolytic and absorption stages are therefore necessary to produce the chemical energy resources present in the
TAG
DAG FFA
MAG FFA
Glycerol FFA
TAG = triacylglycerols, DAG = diacylglycerols, MAG = monoacylglycerols, FFA = free fatty acids. As with all reactions catalysed by enzymes, activity measurements can be carried out using various physicochemical methods (by monitoring the disappearance of the substrate or the release of the product). Immunological methods are widely used to quantitate the presence of lipases in biological media, independently from their lipolytic activity. These two groups of methods will be described and discussed here. It is worth noting that lipases can also be quantitated by means of an active site titration method. For instance, Rotticci et al. [43] and Scholze et al. [44] developed a method which is based on the irreversible inhibition exerted by para-nitrophenyl phosphonate derivatives. However, since alkylphosphonates are not specific lipase inhibitors, this method should be restricted to the case of purified lipases.
Eur. J. Lipid Sci. Technol. 2000, 133153
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Fig. 1. Set up used with the Wilhelmy plate method to measure the catalytic activity of lipases on monomolecular films. A: Zero order trough for measuring lipase activity on medium chain substrates. B: Zero order trough for measuring lipase activity on long chain substrates in the presence of -CD in the subphase. (1): Teflon trough, (2): mobile Teflon barrier, (3): electromicrobalance, (4): platinum plate, (5): spread monolayer, (6): reaction compartment, (7): reservoir compartment, (8): surface channel, (9): -cyclodextrin, E: lipase in solution, E*: lipase absorbed to the lipid film.
2 Physico-chemical methods
2.1 Disappearance of substrate 2.1.1 Nephelometry and turbidimetry
In solid media: This method, which is comparable to those involving the use of coloured indicators in agar plates together with carboxylic esters as the lipase substrate (see chapter 2.2.1.1), consists basically of determining the diameter of the product diffusion area. The occurrence of lipolysis will give rise to a clarified zone on the agar plate [45]. This optical phenomenon is observable only if the released fatty acids are partly water-soluble. This clarification process results from the decrease in the size of the hydrolysed emulsified particles, which in turn causes a decrease in the diffused light. This type of technique can also be applied to monitor the hydrolysis of Tweens by some lipases [46]. The chemical structure of Tween esters is such, however, that they can be hydrolysed not only by lipases, but also by non-specific esterases. In liquid media: The method consists of monitoring the decrease with time in the absorbance of a TAG emulsion [4751]. This technique is sensitive to artefacts, however, since numerous factors present in blood serum for instance can interfere with the process [52, 53]. In addition, the results are not absolute lipase activity values, and to obtain reliable quantitative data, it is therefore necessary to perform a calibration curve using a pure lipase solution.
This calibration step had hampered a wide diffusion of this sensitive method. A turbidimetric esterase assay was developed using a Tween 20 solution in the presence of CaCl2 and Lysobacter enzymogenes esterase as enzyme source [54]. The reaction was monitored by measuring the increase in the optical density occurring at 500 nm due to the hydrolytic release of the fatty acids from Tween 20 and their precipitation in the form of calcium salts. This turbidimetric assay was used to determine the specific activity of lipases from Chromobacterium viscosum (87 IU mg1) and Candida cylindracea (0.5 IU mg1). It should be pointed out that Tweens are not specific substrates for lipases, however.
2.1.2 The Wilhelmy plate method 2.1.2.1 Pure monomolecular films as substrates
Among the interfacial tensiometry methods, the monomolecular film technique at the air-water interface has been extensively developed and used by our group [5564] (Fig. 1) as well as in the group of Brockman [6567]. This technique consists basically of taking a Teflon trough filled with an aqueous solution. A thin platinum plate dipped in the surface of the aqueous phase is attached to
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an electromicrobalance in order to measure the surface pressure (), which is directly related to the interfacial tension () by the relationship = o , where o is the reference value of the interfacial tension with a clean air-water interface. This equipment is commercially available at several companies: KSV (Helsinki, Finland), Krss GmbH (Hamburg, Germany) and Kibron Inc. (Helsinki, Finland). A monomolecular film of lipids can be spread at the surface of the aqueous phase from a lipid solution in a volatile solvent poorly water-soluble, e.g. chloroform. The lipid solution is applied in the form of small droplets which are allowed to evaporate. The area occupied by the lipid film can be limited and adjusted with a Teflon barrier that sweeps the surface of the aqueous phase. The surface pressure can therefore be maintained automatically constant, using the displacement of the Teflon barrier that is controlled and regulated depending on the output from the electromicrobalance. If a lipase solution is injected into the aqueous phase below the lipid film, the surface pressure will decrease, due to the solubilisation of the lipolytic reaction products. Since the barrier moves in order to maintain the surface pressure constant, the kinetics of the reaction can be monitored by recording the movement of the barrier versus time. With this technique, it is possible to measure and control some important interfacial parameters such as the surface pressure (the interfacial free energy) and the molecular area of the substrate, as well as the surface excess of the water-soluble lipases. One prerequisite of the monolayer technique, however, is that the water insoluble monomolecular film of substrate should generate watersoluble products during the reaction process. This is why synthetic medium-acyl chain lipids were originally mainly used as substrates with lipolytic enzymes [66, 68, 69]. We recently developed an alternative method using a non surface-active agent, -cyclodextrin, dissolved in the aqueous subphase in order to trap the long-chain lipolytic products generated by the lipolysis of monomolecular films of long chain neutral acylglycerols [61] or phospholipids [63]. The monomolecular film method is suitable for studying enzymatic reactions on lipid films spread at the air/water interface. This method is highly sensitive, and very low lipid amounts are required to perform reliable kinetic measurements. However, in terms of the amounts of lipase used, the monomolecular film technique requires as much lipase as the pH-stat method (from 0.1 to 1 g per assay). Nevertheless, the question remains to be answered whether the behaviour of a lipid film at the air-water interface is actually representative of what is occurring either at an oil-water interface or in a complex biomembrane.
2.1.2.2 Mixed monomolecular films as substrates

Most studies on the kinetics of lipolytic enzyme activities have been carried out in vitro with pure lipids as substrates. Virtually all biological interfaces are composed of complex mixtures of lipids and proteins. The monolayer technique is ideally suited for studying the mode of action of lipolytic enzymes at interfaces using controlled mixtures of lipids. Two methods of forming mixed lipid monolayers exist at the air-water interface: either by spreading a mixture of water-insoluble lipids from a volatile organic solvent, or by injecting a micellar detergent solution into the aqueous subphase covered with preformed insoluble lipid monolayers. A new application of the zero-order trough was proposed by Pironi and Verger [70] for studying the hydrolysis of mixed monomolecular films at a constant surface density and a constant lipid composition as shown schematically in Fig. 1A. A Teflon barrier was placed transversally over the small channel of the zero-order trough in order to block the surface communication between the reservoir and the reaction compartment. The surface pressure was first determined by placing the platinum plate in the reaction compartment, where the mixed film was spread at the required pressure. The surface pressure was then measured after switching the platinum plate to the reservoir compartment, where the pure substrate film was subsequently spread. The surface pressure of the reservoir was equalised with that of the reaction compartment by moving the mobile barrier. The barrier between the two compartments was then removed in order to allow the surfaces to communicate, and the enzyme was injected into the reaction compartment and the kinetics were recorded as described [55].
2.1.2.3 Interfacial binding and film recovery

Using the monomolecular film technique, several investigators have reported that an optimum occurs in the velocity-surface pressure profile. The exact value of the optimum varies considerably with the particular enzyme/substrate combination used. Qualitative interpretations have been given to explain this phenomenon. The first hypothesis, which was proposed by Hughes [71] and supported by later workers [72, 73], was that the packingdependent orientation of the substrate may be one of the factors on which the regulation of lipolysis depends. Using radiolabelled enzymes, Verger et al. [56] and Pattus et al. [74] subsequently established that the maxima observed in the velocity-surface pressure profile disappear when they are correlated with the interfacial excess of enzyme. Indeed, the main difference between the monolayer and the bulk system lies in the interfacial area to volume ratios, which differ from each other by several orders
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of magnitude. In the monolayer system, this ratio is usually about 1 cm1, depending upon the depth of the trough, whereas in the bulk system, it can be as high as 105 cm1, depending upon the amount of lipid used and the state of lipid dispersion. Consequently, under bulk conditions, the adsorption of nearly all the enzyme occurs at the interface, whereas with a monolayer, only one enzyme molecule out of hundred may be at the interface [69]. Owing to this situation, a small but unknown amount of enzyme, which is responsible for the observed hydrolysis rate, is adsorbed on the monolayer. In order to circumvent this limitation, different methods were proposed for recovering and measuring the quantity of enzymes adsorbed at the interface [62, 67, 7577]. After performing velocity measurements, Momsen and Brockman [77] transferred the monolayer to a piece of hydrophobic paper and the adsorbed enzyme was then assayed titrimetrically. After correcting for blank rate and subphase carry-over, the amount of adsorbed enzyme were calculated from the net velocity and the specific enzyme activity. In assays performed with radioactive enzymes [75], the film was aspirated by inserting the end of a bent glass capillary into the liquid meniscus emerging above the ridge of the Teflon compartment walls. As radiolabelled enzyme molecules dissolved in the subphase were unavoidably aspirated with the film constituents, the results had to be corrected by counting the radioactivity in the same volume of aspirated subphase. The difference between the two values, which actually reflected the excess radioactivity existing at the interface, was attributed to the enzyme molecules having bound to the film. Since it is possible with the monolayer technique to measure the reaction rate expressed in mol cm2 min1 and the interfacial excess of enzyme in mg cm2, it is easy to obtain an enzymatic specific activity value, which can be expressed as usual in mol min1 mg1 (IU mg1). By combining a sandwich ELISA technique with the monomolecular film technique, it was possible to measure the enzymatic activity of human gastric lipase (HGL) on 1,2-didecanoyl-sn-glycerol (dicaprin) monolayers as well as to determine the corresponding interfacial excess of the enzyme [62]. The HGL turnover number increased steadily with the lipid packing. The specific activities determined on dicaprin films spread at 35 mN m1 were found to be in the same range of the values measured under optimal bulk assay conditions, using tributyrin emulsion as the substrate (i.e. 1000 IU per mg of enzyme). At a given lipase concentration in the water subphase, the interfacial binding of HGL to the non hydrolysable egg yolk phosphatidylcholine monolayers was found to be ten times lower than in the case of dicaprin monolayers [62].
However, we have to keep in mind that the surface-bound enzyme molecules include not only those enzyme molecules which are directly involved in the catalysis, but also an unknown amount of protein which is present close (adsorbed) to the monolayer. These enzyme molecules were not necessarily involved in the enzymatic hydrolysis of the film.
2.1.3 Atomic force microscopy

In order to monitor the kinetics of the hydrolysis of phospholipid bilayers by phospholipase A2, Nielsen et al. [78] have performed experiments using atomic force microscopy (AFM) in a liquid medium. Following these studies, the enzymatic hydrolysis of mixed bilayers of acylglycerols/phospholipids by Humicola lanuginosa lipase (HLL), was also investigated using AFM. Mica supported lipid bilayers are hydrolysed by HLL, and as the products dissolve in the buffer, regions of the bilayer with deep defects were detected by the AFM tip. Real time images of the hydrolysis occurring in the bilayer were thus obtained and analysed using purpose built software. These images showed the occurrence of increasingly large nanoscale indentations at the interface. In addition, the increase in the area of the holes in the lipid bilayer was recorded as a function of time and the specific activity of the enzyme was thus estimated, assuming one molecule of lipase to be acting in each hole. The results were used to model
Fig. 2. Principle of the pH-stat method. The enzyme is injected into the thermostated reaction vessel containing the emulsified substrate. The lipase activity is measured by recording the amount of titrant (NaOH) added to maintain the pH at a constant endpoint value during the reaction.
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Eur. J. Lipid Sci. Technol. 2000, 133153 with time by adding titrated NaOH in order to maintain the pH at a constant end point value. The pH-stat equipment is commercially available at Radiometer (Copenhagen, Denmark) or Metrohm Ltd. (Herisau, Switzerland). In the particular case of the pancreatic lipase/colipase complex, the most common routine assay is that performed in a thermostated (37 C) reaction vessel containing 0.5 ml of tributyrin present in 15 ml of a buffered (pH 8) aqueous solution, in the absence of any surfactant or emulsifier other than bile salts [86, 87]. Usually, tributyrin is simply emulsified in situ by the efficient mechanical stirring in the pH-stat vessel. The effect of the periodic sonication in the pH-stat method has been reported, however. Sonication of olive oil emulsified with gum arabic has been shown to improve the sensitivity of the assay [88]. Periodic sonication has also been used to promote efficient emulsification of olive oil in the absence of any surfactant in order to improve the reproducibility of the assay [89]. However, sonication procedures are difficult to reproduce experimentally and chemical degradation of the substrate cannot be excluded. The pH-stat method was used to detect a plant lipase activity in seedling rape homogenates, in the presence of deoxycholate, using triolein emulsified with gum arabic as the substrate [90]. The pH-stat technique has also been used to determine lipase activites in serum, plasma and duodenal juice [91, 92]. The pH-stat method is a quantitative method which is sensitive to within one mole of released fatty acid per min. When 0.1 M NaOH is used as titrant, it is not a reliable means of detecting activity levels lower than 0.1 mole per min. Apart from its low sensitivity, the main disadvantage encountered with the pH-stat method is the restricted range of pH values which can be investigated. More specifically, the detection of the protons released during the hydrolytic reaction catalysed by lipases requires a partial ionisation of the released fatty acids. The end point values of the pH of the reaction medium must therefore be roughly equal to, or preferably higher than the apparent pKa value of the released fatty acid. In fact, it has been reported by Benzonana and Desnuelle that the apparent pKa values of oleic acid could be as high as 7.5 under lipase assay conditions [93]. Furthermore, it is worth noticing that the ionic strength as well as the presence of calcium ions will tremendously decrease the measured values of the apparent pKa. It is generally believed that the positive effect of the presence of calcium ions on lipase assays is due to the water insolubility of the calcium soaps of long-chain fatty acids. This micro-precipitation phenomenon drives the chemical equilibrium by virtue of the mass action law. If at the selected pH value, the FFAs are not fully ionised, their continuous titration is either very inaccurate or im-
the kinetics of the enzyme reaction at the lipid-water interface. These data provide the first nanoscale picture of the kinetics of lipid degradation by lipases [79].
2.1.4 Infrared spectroscopy

A continuous assay for measuring lipase-catalysed hydrolysis of TAGs in reverse micelles using Fourier transform infrared spectroscopy (FTIR), was developed by Walde and Luisi [80]. Lipolysis can be monitored by recording the FTIR spectrum of the entire reaction mixture. Fatty acid esters and FFAs (peak maximum at 1751 cm1 and at 1715 cm1, respectively) can be quantitated on the basis of their molar extinction coefficients and Beers law. This method was applied to measuring the lipolysis of various substrates (trioctanoylglycerol, vegetable oils).
2.2 Appearance of hydrolytic reaction products 2.2.1 Proton release as an indirect assay 2.2.1.1 Coloured indicators
In solid media: When a lipase solution is placed on an agar plate containing a carboxylic ester as substrate, it is possible to monitor the drop in pH due to the fatty acids released by observing the change in colour of indicators previously incorporated together with the substrate into the agar gel [22, 45]. There exists a linear relationship between the diameter of the fatty acid diffusion spot and the logarithm of the enzyme concentration. This technique is very convenient for rapidly screening lipolytic microorganisms growing on agar plates. However, some positive false can result from the acidification of the medium, due to the generation of acidic metabolites other than FFAs, which are released by microbial lipases. In liquid media: Although it is a qualitative method, this technique provides a simple means of detecting lipase activity in chromatography column fractions at various lipase purification stages by observing the changes in colour of indicators mixed with the ester substrates [81]. An even simpler qualitative method is that based on detecting the characteristic strong butyric and caproic acid smell of lipolysed milk droplets used as a lipase substrate.
2.2.1.2 Titrimetry
The well-known pH-stat method (Fig. 2) is generally used as a reference lipase assay [82, 83]. This is a convenient technique for characterising lipase activity and specificity, as well the interfacial activation phenomenon [11, 84, 85]. As shown in Fig. 2 with the pH-stat method, lipase activity is measured on a mechanically stirred emulsion of natural or synthetic TAGs by neutralising the FFAs released
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possible to perform, even after introducing a correction factor. A back-titration has to be carried out, as in the case of HGL, which is active under acidic pH conditions. The HGL activity is therefore measured by incubating the enzyme solution with a TAG emulsion for several min at pH 3. The released non ionised FFAs are back-titrated at the end of the reaction by inducing a rapid shift of the pH from 3 to 9. In a subsequent control experiment (without any HGL), the amount of added titrant (NaOH) is then substracted from the assay [94, 95].
Rogel et al. [103] described a spectrophotometric lipase activity assay based on the displacement of parinaric acid previously bound reversibly to BSA, which is induced by the oleic acid released from triolein. The lipase activity is monitored by recording the changes in the ratio between the absorbances at 319 and 329 nm. This sensitive method is limited, however, by the fact that detergents and calcium ions interfere with the assay. A plate assay to detect bacterial lipase in a medium containing trioleoylglycerol and the fluorescent dye rhodamine B has been described [104]. Substrate hydrolysis causes the formation of orange fluorescent halos around bacterial colonies visible upon UV irradiation. The logarithm of lipase activity from cell-free culture supernatants is linearly correlated with the diameter of halos, thereby allowing quantitation of lipase activities ranging from 60 to 1800 mIU.
2.2.2 Analysis of the free fatty acids released 2.2.2.1 Glycerol derived carboxylic esters as substrates 2.2.2.1.1 Colourimetric assays
A continuous spectrophotometric assay based on the metachromatic properties of the cationic dye safranine has been described [96]. In this assay, the change in the net negative charge at the lipid /water interface during the lipolytic reaction is monitored by the absorbance change of safranine. Lipase activities as low as 50 mU could be detected using an olive oil emulsion as substrate. The sensitivity of this method is therefore about two fold higher than the pH-stat method. A colourimetric method, based on the formation in an organic phase of a copper soap of the fatty acid in the presence of a dye indicator (copper reagent), was first developed by Duncombe [97]. The copper complex was subsequently estimated spectrophotometrically at 440 nm. The sensitivity of the copper reagent and the efficacy of the fatty acid solvent extraction step have been improved by many workers for specific purposes [98, 99]. Nixon and Chan [100] described a procedure which resulted in a reference curve showing linearity between 10 and 130 nmole of FFAs. In addition, the presence of bovine serum albumin (BSA) and phospholipids was found not to interfere with the assay. In the above-described methods using copper soaps, TAGs have to be freed from endogeneous FFAs in order to obtain an acceptable background level. The use of rhodamine 6G to obtain a FFA complex which is extractable in hexane was described by Hirayama and Matsuda [101]. In the presence of fatty acids, a pink colour develops and its absorbance is read at 513 nm. The reference curve is linear between 20 and 200 nmol of FFAs [102]. However, the reproducibility of rhodamine 6G batches is difficult to control and a reference curve has to be performed with each new rhodamine 6G solution. Replacing hexane by heptane improved the shelf life of the rhodamine solution without entailing any loss of sensitivity (Dr. J. Nari, LLE-Marseille, personal communication).
2.2.2.1.2 Fluorimetric assays

Wilton [105] described an assay which involves the displacement of the highly fluorescent fatty acid probe, 11(dansylamino)undecanoic acid, in rat liver fatty acid-binding protein, which is induced by the long-chain FFAs released by lipases. Quantities as low as 20 pg of purified pig pancreatic lipase (PPL) could be detected with this method. However, since this highly sensitive assay is based on the same principle as that described above using BSA/parinaric acid [103], it is likely to be limited by the same factors. Moreover, the method may be not sufficiently reliable when used in the case of crude biological media containing membrane fractions or albumin. A commercially available kit (ADIFAB, ICN pharmaceuticals Inc.) which can be used to quantitate FFAs was developed, based on the properties of a 15 kDa mammalian intestinal fatty acid-binding protein conjugated to an acrylodan fluorophore. Without FFAs, the indicator emits fluorescence at 432 nm and 505 nm when binding occurs with FFAs. As mentioned by the manufacturer, the detection of the FFA binding is based on a movement of the acrylodan fluorophore which occurs relative to the nonpolar binding pocket of the protein. According to the manufacturer, this method can be used to detect concentrations of FFA as low as 1 nM. A method based on interactions between rhodamine B and the released FFAs has been described [106]. The reaction takes place in a gel containing a mixture of rhodamine B, TAGs and agarose deposited in microtiterplate wells. After adding the lipase solution, the released FFAs can be monitored by reading the fluorescence intensity (excitation 485 nm, emission 535 nm) every 10 min for 1 to 2 h. The amounts of released FFAs can be estimated
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Eur. J. Lipid Sci. Technol. 2000, 133153 matography or gas-liquid chromatography [110113]. The latter method requires that fatty acids are previously transformed into their methyl esters, however, so as to render them volatile. After thin-layer chromatography, a quantitative analysis of the released FFAs can be carried out using either densitometric or autoradiographic methods together with radiolabelled TAGs. The specificity and the sensitivity of these techniques are very satisfactory, since they can be used to detect fatty acid in quantities as small as a few pmoles [114]. However, these methods are rather time-consuming and they are not continuous. A high-performance liquid chromatographic (HPLC) assay for determining lipase activity was developed by Maurich et al. [115] using -naphtyllaurate as substrate. The specific activity of PPL was only 1.5 IU mg1, which is very low in comparison with the PPL activity measured with other methods and other substrates (around 10 000 IU mg1, using a tributyrin emulsion). With a view to determining PPL activity, Maurich et al. developed a highly sensitive HPLC method, using the palmitic and lauric esters of para-nitrophenol as substrates. These authors gave details as to the specific activity and reproducibility of lauric ester alone, because palmitic ester turned out to be a very poor substrate for PPL [116].
using the calibration curve obtained upon adding variable amounts of FFAs to the gel. Wolf et al. [107] used phosphatidylcholine containing parinaric acid [9 (cis), 11 (trans), 13 (trans), 15 (cis)-octadecatetraenoic acid], a naturally fluorescent fatty acid bearing four conjugated double bonds, to monitor phospholipase A2 activity. Upon hydrolysis, the binding to albumin of the free parinaric acid released was correlated with an increase in the fluorescence polarisation and in the total fluorescence intensity. This method prompted us to set up a lipase assay using parinaric acid-containing TAG purified from Parinari glaberrimum seed oil [108]. The purified TAGs are naturally fluorescent and more than half of the fatty acids from Parinari oil are known to contain parinaric acid in its esterified form [109]. Under the assay conditions used, the excitation and emission wavelengths of Parinari oil were 324 nm and 420 nm, respectively. 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 free 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 seedlings 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) HGL [108]. One drawback of this method, however, is the susceptibility of parinaric acid to oxidation by atmospheric oxygen. This drawback can be overcome by adding antioxidant agents to the buffers and by performing incubation steps under an argon or a nitrogen atmosphere. Furthermore, this method requires the presence of a selected detergent in order to solubilise the released parinaric acid into mixed micelles; and of course, this detergent must not inhibit lipase activity. We were not able to use this method to measure lipase activity in the stratum corneum of human skin (F. Beisson, Anal. Biochem., submitted), due to the highly fluorescent background of the adhesive tape strips used to collect the stratum corneum. This simple and continuous assay is compatible, however, with a high sample throughput and might be applicable to detecting true lipase activities in various biological samples as well as in directed evolution experiments.
2.2.2.1.4 Enzymatic assays

The light emitted by some reaction products of the luciferase from the marine luminous bacterium Beneckea harveyi in the presence of fatty acids can be utilised to detect levels as low as 1 pmole of myristic acid and 100 to 200 pmoles of palmitic or oleic acid [117]. One main drawback of this technique is the fact that oleic, linoleic, palmitoleic and linolenic acids inhibit the luciferase. Moreover, at pH 8, a 5-fold decrease in the luminescence is observed in comparison with that obtained at pH 6.5. Due to the action of some lipoxygenases, linoleic acid, in the presence of oxygen, generates a hydroperoxide derivative which can subsequently be revealed with thiocyanate as a red complex. The formation of this complex can either be quantatively monitored [118] or the oxygen consumption during the reaction catalysed by the lipoxygenase can be monitored by means of an oxygen electrode [119]. Some lipoxygenases do not act specifically on free linoleate leading to overestimation of the extent of hydrolysis. Furthermore, non-enzymatic co-oxidation may lead to erroneous results. Acyl-CoA synthetase can be used to catalyse the formation of acyl-CoA from Coenzyme A and FFAs. Using acyl CoA oxidase, acyl-CoA can then be converted into
2.2.2.1.3 Chromatographic assays

Various chromatographic techniques can be used to detect lipids as well as FFAs released from TAGs, namely florisil columns or silicic acid columns, thin-layer chro-
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Fig. 3. Principle of the resorufin ester assay. The absorption maximum of the released resorufin is at 572 nm at pH 6.8 and 583 nm in the alkaline range. In the case of incubations performed at pH < 6.8, the pH is adjusted to 6.8 by adding KOH or alcaline buffer before reading the absorbance at 572 nm.
enoyl CoA and hydrogen peroxide, which in turn is converted into red quinone dye by peroxidase in the presence of phenol and 4-aminoantipyrine. The reaction can be monitored by measuring the colour produced at 500 nm [120]. The great drawback of this method is that many of the enzymes present in a crude biological medium are likely to interfere with the enzymatic reactions during the assay.
then exposed to lead salts to form insoluble soaps, and finally processed for electron microscopy. Although the lead precipitates were difficult to detect, some sites of lipase activity were identified in the cells. Larger precipitates have been obtained by using Tweens instead of triolein, but the former are not specific lipase substrates [125, 126].
2.2.2.1.5 In situ detection by electron microscopy

Fatty acids released in animal tissues by lipases can be detected by electron microscopy [121]. This technique has been used for example to detect rat lingual lipase [122] and the lipase present in the pancreatic acinar cells of mice and rats [123, 124]. It has also been used to detect lipase activities in the outer epidermis: aldehyde-fixed tissues (100-m slices) were incubated with triolein and
2.2.2.2 Synthetic carboxylic esters as substrates 2.2.2.2.1 Radioactive assays

The methods involving the use of TAGs containing radiolabelled acyl chains [127, 128] are specific and very sensitive lipase assays. However, they cannot be monitored continuously and they need a time-consuming chromatographic step or an organic solvent fractionation step to isolate the released fatty acids.
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Tab. 1. Initial rates of hydrolyis of tributyrin emulsions and resorufin ester by various pure proteins. Tributyrin assay conditions: with all the proteins assayed except RGL, the buffer used was 1 mM Tris-HCl (pH 8), 150 mM NaCl, 10 mM CaCl2. With RGL, the buffer used was 50 mM acetate (pH 6), 150 mM NaCl, 2 mM NaTDC, 1.5 M BSA. Resorufin assay conditions (in the absence of Thesit): 10 l of a lipase sample were added to 90 l of KH2PO4 0.1 M (pH 6.8) and 7 l of resorufin ester stock solution in dioxane (1 mg ml1). With RGL, the buffer used was 20 mM KH2PO4 (pH 6.8), 150 mM NaCl, 0.05% Triton X100. Protein Fungal lipases Candida antarctica lipase B Candida rugosa lipase Fusarium solani cutinase Pseudomonas glumae lipase Rhizomucor miehei lipase Mammalian lipases Human pancreatic lipase + Colipase Lipoprotein lipase Rabbit gastric lipase Non enzymatic proteins Hemoglobin Bovine serum albumin Tributyrin [IU mg1) 184 1037 3180 3000 8240 8000 250 800 0 0 Resorufin ester (IU mg1) 0.2 214 32 401 450 1000 8 3 0.7 0 Tributyrin/Resorufin ester ratio 1022 4.8 99 7.5 18.3 9 31.2 267 0
A method based on the binding of radiolabelled 63Ni to FFAs extracted in organic solvents has been described [129]. The estimated quantity of 63Ni bound to oleic acid was found to be linear up to 100 nmol of fatty acid, and quantities as small as 1 nmol of FFA can be detected per assay. However, this method requires a time-consuming step, in which the heptane phase containing the FFAs has to be evaporated prior to adding the radiolabelled nickel solution. It also requires the use of completely fatty acid free glassware.
2.2.2.2.2 Colourimetric assays

When using as a lipase substrate a synthetic TAG containing fatty acyl chain labelled by a pyrene group in position, it is possible to determine the amount of labelled FFAs released, using spectrophotometric methods with a sensitivity of around one nmole. This technique is not straightforward to use, however, since it requires performing solvent extraction of lipid molecules and separating the labelled TAG from the released labelled products, both of which having a yellow colour [130]. Resorufin ester (1,2-O-dilauryl-rac-glycero-3-glutaric acid-resorufin ester) is a sensitive glycerol-derived substrate (Fig. 3), which is commercially available at Hoffmann-La Roche (previously Boehringer Mannheim), and can be used conveniently for lipase assays under specific experimental conditions in the presence of a nonionic
detergent named Thesit. This assay is widely used for the determination of lipase activity in serum and was also utilised in the case of a microbial [131] and a plant lipase [132]. However, we established at our laboratory that the hydrolytic activity measured in a plant homogenate with this resorufin ester was not in fact attributable to a true lipase activity. The catalytic activity on resorufin ester was not abolished by heating the extract for 5 min at 95 C, whereas the true lipase activity measured with radiolabelled triolein or TAGs containing parinaric acid (see chapter 2.2.2.1.2) decreased dramatically after heat treatment [108]. In addition, at our laboratory we have used resorufin ester, in absence of detergent (e.g. Thesit), in order to determine and compare the rates of hydrolysis of resorufin ester and tributyrin by various pure proteins (Tab. 1). Unlike tributyrin, resorufin ester was poorly hydrolysed by many lipases. Some lipases such as Candida rugosa, Pseudomonas glumae, Rhizomucor miehei lipases and human pancreatic lipase (HPL) can significantly hydrolyse resorufin ester, showing specific activities ranging from 200 to 1000 IU mg1. However, if one looks at the ratio between rates of hydrolysis of tributyrin and resorufin ester, it is striking that this ratio ranges from 5 to 1000 in the case of Candida rugosa and Candida antarctica, respectively. A low but significant resorufin ester hydrolysis rate was obtained with hemoglobin purified from pig liver. The data obtained using resorufin ester to mea-
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sure lipase activities should therefore be interpreted with great care.
2.2.2.2.3 Fluorimetric assays

The main advantages of fluorimetric assays is their sensitivity and the fact that they make it possible to continuously monitor the reaction kinetics. We will therefore review here only the continuous fluorescent assay described in the literature. Dansyl-phosphatidylethanolamine [133] and NBD-phospholipids [134] have been used as a substrate to detect the phospholipase activity of lipoprotein lipase. These fluorescent phospholipid molecules are not, however, typical and specific substrates for other lipases. Another important group of fluorogenic substrates is that consisting of pyrenic acylglycerol derivatives, which were originally used for phospholipase determination [135, 136]. Derivatives of this kind were used in lipase activity determinations in which the production of FFAs which occurs upon lipolysis causes a shift in the peak fluorescence intensity. The lipolytic activity can be quantitated in terms of the increase with time in the fluorescence intensity at a given wavelength. The use of a TAG containing fatty acyl chain in which a pyrene residue is linked to the position was first described by Ngre et al. [137]. However, this sensitive assay requires the pyrene-labelled FFA released to be isolated from the reaction medium. A quencher residue (trinitrophenylamine residue) was introduced by Duque et al. [138] as a means of decreasing the basal fluorescence of the TAG containing acyl-pyrene (1O-hexadecyl-2-pyrenedecanoyl-3-trinitrophenylaminododecanoyl-sn-glycerol and its enantiomer). This intramolecularly-quenched TAG containing acyl-pyrene can therefore be used in a continuous fluorescent assay [139]. Unfortunately, as can be seen from the data reported by Duque et al. [138], this kind of chemically-modified TAG is poorly hydrolysed by lipases, probably for steric reasons. We have confirmed that a poor rate of hydrolysis by human pancreatic lipase occurs with this kind of labelled TAG: 5 103 IU per mg, as compared with 3000 IU per mg on natural long chain TAG [108]. The overall sensitivity of a fluorescent assay using synthetic TAGs will obviously depend on two factors: first, on the sensitivity of the FFA detection, i.e. on the lowest amount of fluorescent released FFAs that can be detected, and secondly, on the specific activity of the lipase. The sensitivity of the quenched pyrene-TAG based assay is therefore offset by the very poor rate of lipase hydrolysis of the corresponding synthetic TAG, as compared with that of non chemically-modified TAGs (such as TAGs containing parinaric acid, see above, chapter 2.2.2.1.2).
2.2.3 Determination of the released alcohol or thiol 2.2.3.1 Determination of the released glycerol from TAG
Direct determination of free glycerol is not commonly performed, since all three acyl chains of a triacylglycerol molecule are rarely released by a single lipase, and therefore the initial hydrolysis rates cannot be determined. Three techniques have been described, however, in the literature for estimating the released free glycerol: G Periodic oxidation of free glycerol, which leads to the formation of formaldehyde that can then be assayed spectroscopically. G Bioluminescence using the luciferin-luciferase complex [140]. G Phosphorylation of glycerol into glycerol-3-phosphate followed by conversion of the latter product into dihydroxyacetone-phosphate, which leads to the production of hydrogen peroxide, which can be determined spectrophotometrically at 550 nm using peroxidase (Kit lipase-PS of Sigma) [141].
2.2.3.2 Determination of the released coloured or fluorescent alcohol from synthetic carboxylic esters
The hydrolysis of carboxylic esters of -naphthol, para-nitrophenol or 2,4-dinitrophenol leads to the release of alcohols that can be monitored continuously and quantitatively using a spectrophotometric method. The appearance of the yellow coloured para-nitrophenol (pKa 7.15) can be monitored by reading the absorbance at 405 nm [142145]. The formation of 2,4-dinitrophenol (pKa 3.96) can be monitored from the increase in the absorbance at 360 nm [146]. Since the molar extinction coefficient of a phenolic solution is generally very dependent on its ionisation state, dinitrophenyl esters are more convenient substrates than para-nitrophenyl esters when the pH of the assay is around 78. The red colour obtained with naphthol can be monitored after complexing the solution with a diazonium salt [147]. The use of acyl esters of 5-(4hydroxy-3,5-dimethoxyphenylmethylene)-2-thioxothiazoline-3,5-dimethoxyphenylmethylene)-2-thioxothiazoline3-acetic acid as chromogenic lipase substrates has also been described [148]. Upon hydrolysis, these substrates yield an intensely red colour which can be assayed at 505 nm. However, esters of this kind do not in fact seem to be specific substrates for determining true lipase activities. Berg et al. [149] have reported a colourimetric assay of HLL using para-nitrophenyl butyrate, which partitioned at the interface of small anionic unilamelar vesicles (SUVs) with a mean diameter of around 40 nm, such as palmitoleoyl-oleoyl-phosphatidylglycerol (POPG)-SUVs. The
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Eur. J. Lipid Sci. Technol. 2000, 133153 were first described by Jacks and Kircher [153]. Umbelliferone acyl esters have been found to be more sensitive and stable [154]. The use of methylumbelliferone esters as substrates to assay the lipase activities in human epidermal stratum corneum has been investigated at our laboratory. Methylumbelliferone oleate is likely to be a specific substrate for these epidermal lipase activities under given assay conditions, whereas methylumbelliferone heptanoate is not a specific substrate at all (Beisson et al., Anal. Biochem., submitted). Lauroyl pyrenemethanol has been used to assay gastric lipase, cellular lipases of hematopoietic cells and Rhizopus arrhizus lipase [155]. The use of monodecanoyl-fluorescein has also been tested as a means of assaying lipases [156]. The carboxylic esters containing fluorogenic secondary alcohols instead of glycerol are very sensitive and convenient substrates, but one should not forget that they are not, a priori, hydrolysed specifically by lipases. Moreover, they are often prone to high spontaneous hydrolysis.
hydrolysis rate measured was 100-fold higher with (POPG)-SUVs than with the monodispersed substrate or with the substrate partitioned into zwitterionic palmitoleoyl-oleoyl-phosphatidylcholine (POPC)-SUVs. The lipase acivity can then be measured using colourimetric methods. According to the authors, POPG-SUVs fulfil the criteria for a neutral diluent, since they provide an interface for the partitioning of both substrate and lipase and do not block the lipase active-site. In this well defined system, the primary rates and equilibrium constants for the interfacial catalysis by HLL have been established [149]. In our opinion, nitrophenol acyl esters should not be used as lipase substrates for several reasons: i. They are not at all specific lipase substrates, since they can be hydrolysed by non-specific esterases and proteases often present in biological samples. Moreover, BSA or the non catalytic C-terminal domain of PPL can hydrolyse para-nitrophenyl acetate at the same rate as purified PPL [150]. ii. The catalytic turn-over number of true lipases on paranitrophenol acyl esters is usually several orders of magnitude lower than that obtained with TAG. One should recall that phenol esters are esters of secondary alcohols, whereas the vast majority of all the known lipases act exclusively on primary ester bonds such as those present in the sn-1 and sn-3 positions of TAG. As an example, the specific activity ratios (tributyrin/para-nitrophenyl acetate) are 2320, 1000 and 1430 when using purified PPL, HGL and rabbit gastric lipase, respectively [151]. iii. The carbonyl function of these secondary esters is electronically activated (it bears a partial positive charge, due to the electronic delocalisation of the aromatic ring, which is enhanced by the electron attractive NO2 substituent). These esters are therefore liable to undergo non-enzymatic alkaline and acidic hydrolysis. If, however, long acyl chain para-nitrophenyl esters are dissolved in an inert micellar detergent, this disadvantage no longer applies. All the available methods of measuring lipase activity using nitrophenyl esters therefore need to be handled with great care, even when pure lipases are used. A series of para-nitrophenyl esters with variable acyl chains have been frequently used, however, as reported in the literature, to quickly assay the so-called chain length specificity of the microbial lipases used in many biotransformation processes [152]. The fluorophore of fluorogenic synthetic lipase substrates is sometimes located in the alcohol moiety. This is so in the case of 4-methylumbelliferone acyl esters, which
2.2.3.3 Determination of the released thiol from synthetic thioesters

There exist numerous thioesters which can be hydrolysed by lipases. This reaction causes the release of one or several sulfhydrile functions, which can subsequently be detected using the chromogenic Ellmans reagent (5, 5 dithiobis 2-nitro benzoate). These thioesters are, either poorly or non specifically hydrolysed by lipases, however. Two thioesters (2,3-dimercaptopropan-1-ol tributyroate and 3-mercaptopropan-1,2-diol tributyroate) have been used for lipase assays in serum samples pretreated with phenylmethylsulfonyl fluoride, a potent inhibitor of some serum esterases. Synthetic thioester TAG analogues can be synthesised that will allow rapid analysis of the stereopreference (sn-1/sn-3) of the investigated lipases [157159].
2.2.4 Electric conductivity

The electric conductivity of the medium increases during the lipase hydrolysis reaction due to the development of electric charges carried by the released FFAs [160, 161]. This technique has numerous drawbacks: the measurements are highly temperature-dependent, and the sensitivity is really satisfactory only when triacetin is used as substrate. However, it has turned out that triacetin is not a particularly suitable lipase substrate [14].
2.2.5 Acoustic wave conductance

A surface acoustic wave sensor system for assaying the activity of pancreatic lipase has been proposed [162]. The assay of this enzyme is based on the change in conductance of the solution caused by the release of a fatty acid,
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Fig. 4. Diagram of the experimental oil drop set-up. (1): Optical bench, (2): integrated sphere light source (halogen lamp), (3): thermostated cuvette, (4): syringe (Exmire type), (5): DC motor with a 500 count per revolution optical encoder, (6): telecentric gaging lens (Melles Griot, Rochester, NY, USA), (7): CCD camera 512 512, (8): personal computer, (9): video monitor. using triolein as a substrate. A linear relationship between frequency response and enzyme concentration is obtained. under similar conditions [166168]. The oil-drop methodology requires the oil to be carefully freed from any natural tensioactive compounds such as FFAs and di- and monoglycerides because of the amphipathic character of these contaminants, which might decrease the initial interfacial tension. Clean materials and equipment are also a strict requirement for the oil-drop methodology to give reliable results, as is also the case with the Wilhelmy plate method. At supra catalytic concentrations of lipases, the protein adsorption to the oil droplet could also affect the surface tension. In this case, hydrolysis kinetics should therefore be interpreted with care.
2.2.6 The oil-drop method

In 1987, Nury et al. [163] established at our laboratory that unique information can be gained by measuring the variations in the oil-water interfacial tension (o/w) as a function of time during lipase hydrolysis. These authors adapted the well-known hanging-drop method to study the rate of lipase hydrolysis of natural long chain TAGs. The interfacial tension is measured here by automatically analysing the oil drop profile on-line, using the LaplaceYoung equation. The accumulation of tensioactive hydrolysis products at the surface of an oil drop is responsible for the decrease in the interfacial tension, which in turn is correlated with changes with time in the drop profile [164] (Fig. 4). Fully automated oil drop tensiometers based on this principle are now commercially available at Interfacial Technology Concept SARL (France) and have been found to have many advantages [165]. As compared to the other interfacial techniques, the oildrop tensiometer presents the unique advantage of being able to monitor lipase activities on natural long-chain TAGs at a closely controlled oil-water interface. Furthermore, the surface behaviour (interfacial binding) of lipases and mutants at the oil-water interface can be studied
3 Immunological methods
We have decided not to develop the various spectroscopic methods which can be used to detect adsorbed lipases on model interfaces, such as infrared spectroscopy [169], circular dichroism [170], fluorescence microscopy [171] and ellipsometry [172]. The set of tests which go under the name of ELISA (enzyme-linked immuno sorbent assay) forms a highly sensitive and specific system for detecting and quantifying lipases [173175]. These immunological methods can be used to detect both the active and inactive forms of a given lipase. The immunological detection of lipases requires purifying the enzyme from a natural or recombinant
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Eur. J. Lipid Sci. Technol. 2000, 133153 and used the clinical diagnosis of pancreatitis, where they are of particular value due to their high specificity and sensitivity [183187]. Other immunoassays and commercial kits have been developed to quantitate immunoreactive LPL [188192] as well as hepatic lipase [188, 193] in human serum and carboxyl ester lipase [194]. ELISA using serum antibodies to Staphylococcus aureus lipase is a sensitive assay for serological diagnosis of staphylococcal infections [195197].
source as well as raising poly- or monoclonal antibodies using conventional methods. This assay essentially consists of first binding a polyclonal antibody to a solid support (such as a PVC microtiter plate), and then causing the antigen (lipase) to interact with the first antibody (the captor), before adding a second antibody (the detector, which is usually a monoclonal antibody) that recognises a different epitope on the antigen. The amount of antigen present is quantitated by estimating the amount of the second antibody labelled with either biotin [176] or a fluorescent probe [177, 178]. Biotinylation of lipases is not always straightforward and should be carefully checked, e.g. for the level of biotinylation, heterogeneity of protein material as well as for the effect on the overall charge and hydrophobicity. Even when the effects of biotinylation on activity are absent, the binding properties may be changed.
3.2.2 Lipase immunodetection in other physiological media

Some groups have developed ELISA for detecting and quantitating LPL in tissues or in cell culture lysates [177, 189, 190, 198, 199]. The availability of specific antibodies has led many authors to set up sensitive and specific ELISA tests for measuring the amounts of lipases in physiological media such as the duodenal contents, in which both HGL and HPL are present [176, 179, 200]. A radioimmunoassay has also been developed for determining the concentration of HPL in the urine [183] and in the duodenal contents [200]. An ELISA test has also been described which can be used to determine pancreatic insufficiency by quantitating the HPL levels in the stools [201] as well as providing an index of severity in patients suffering from cystic fibrosis [202].
3.1 ELISA for pure lipases

Several ELISAs have been specifically developed for quantitating the amounts of pure lipases, e.g. HPL [173, 179], HGL [62], lipoprotein lipase (LPL) [180182], and hepatic lipase [181]. At our laboratory, ELISA tests were developed and used by Aoubala et al. [62] to evaluate the surface excess of HGL at a lipid-water interface. This ELISA assay was adapted to the monomolecular film technique (see Chapter 2.1.2.3). HGL was biotinylated without any significant loss of its catalytic activity occurring and was further detected (detection limit 25 pg/well) by performing a sandwich ELISA using anti-HGL polyclonal antibodies as specific captors before revealing the biotin-labelled HGL, using a streptavidin-peroxidase conjugate as a tracer [62]. By combining the above sandwich ELISA technique with the monomolecular film technique, it was possible for the first time to measure the enzymatic activity of HGL on dicaprin monolayers as well as to determine the corresponding interfacial excess of the enzyme, and thus to calculate the specific activity of the lipase at the lipid-water interface [62]. A similar sandwich ELISA test was adapted at our laboratory by Labourdenne et al. [167] to determine the amount of HPL adsorbed at an oil/water interface, using the oildrop technique.
3.3 Immunocytolocalisation of lipases

Using various immunohistochemical techniques, many authors have mapped mammalian lipases such as gastric lipase [203], LPL [204206], hepatic lipase [207], hormono-sensitive lipase [208], and microsomal triacylglycerol hydrolase [209].
3.4 Immunoblot analysis of lipases

Immunoblot analysis consists of combining gel electrophoresis with an immunochemical method of detection. It therefore provides a valuable technique for determining the molecular mass of lipases, as well as the specificity of antibodies for a given lipase. Proteins are separated on a sodium dodecyl sulfate polyacrylamide gel, transferred to a supporting membrane, and washed and incubated with antibodies in the presence of blocking reagents that reduce non-specific binding. Western blotting analysis using monoclonal antibodies has been widely used to discriminate between sequential and conformational epitopes. However, Bezzine et al. have recently shown first that in HPL, the unfolding process might not be complete at 3% w/v SDS, and secondly, that the C-terminal domain of HPL may be denatured during treatment of the sample at 3% w/v SDS,
3.2 Lipase immunodetection on physiological media 3.2.1 Lipase immunodetection in plasma or serum
Several immunological assays for determining the pancreatic lipase concentration in sera have been developed
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and then become refolded within the separation gel at 0.1% w/v SDS [210].
4.2 Level of lipase activity

The pH-stat method is probably the most suitable method of measuring lipase activities greater than 0.1 IU. As mentioned above, however, this technique can be used only within a restricted pH range. When measuring lipase activities lower than 0.1 IU, the FFAs released have to be extracted and quantitated with methods involving the use of copper salts or rhodamine. If these methods are not sufficiently sensitive, radiolabelled substrates can be used, and the FFAs subsequently extracted. Lastly, fluorescent synthetic esters can be used to perform sensitive continuous or discontinuous tests, but great care needs to be taken with their substrate specificity. The method based on the use of a naturally fluorescent TAGs recently described by Beisson et al. provides a sensitive, specific and continuous assay. It is therefore a particularly promising method for measuring lipase activities in crude biological media [108].
4 Conclusions
In order to explore the relationships between the structure of the lipid bilayer and the catalytic activity of HLL, Vissing et al. [211] recently attempted to set up a universal lipase substrate consisting of monomyristoylglycerol (MMG) inserted into unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) in the proportion [DMPC/DMPG (70:30)]/[MMG](70:30). However, the HLL activity measured on this universal substrate was found to be around two orders of magnitude lower than that recorded on the same mixture of lipids spread at an air-water interface (M. Ivanova, LLE-Marseille, personal communication). The main conclusion to be drawn from the above review is that there exists no single universal method of lipase assay, but rather a whole range of different techniques. The choice of a particular method will depend on the users own specific requirements.
Acknowledgements
We are indebted to our colleagues at the Laboratoire de Lipolyse Enzymatique du CNRS, Dr. Alain de Caro, Dr. Vincent Arondel, Dr. Frdric Carrire and Dr. Abdelkarim Aboulsalham as well as Pr. Louis Sarda (Universit de Provence) for fruitful discussions. Our thanks are also due to our collegues Dr. Thomas Bjrnholm, Dr. Thomas Callissen, Dr. M. Ivanova, Dr. Liliane Dupuis, Dr. Richard Lehner and Isabelle Douchet for giving permission to mention their respective unpublished data, as well as to Sylvie Nevy for her contribution to the initial draft of this review. The assistance of Dr. Jessica Blanc in revising the English manuscript is acknowledged.
4.1 Pure lipases or crude biological media

As many non-specific esterases are often present in biological samples, we recommend the use of long-chain acylglycerols as substrates rather than other esters, in order to detect and assay a true lipase activity. If long-chain acylglycerols are not used as the substrate, when it is proposed to detect lipase activities in a crude sample and/or for the sake of convenience, great care should be taken in interpreting the results, since the activity measured might be due to enzymes other than lipases. Esters other than glycerides can be used in experiments involving purified lipases. Interfacial methods (monolayer and oil drop tensiometer techniques) can be used to perform detailed studies on the effects of various interfacial parameters on lipolysis. It is worth mentioning that monoacylglycerols, diacylglycerols and FFAs should be removed from the TAGs used as substrates in order to avoid detecting secondary activities, and even more importantly, to minimise any uncontrolled tensioactive effects occurring at the oilwater interface and due to partial glycerides and FFAs. It should be mentioned in addition that the choice of a synthetic or natural TAGs as the substrate is crucial, especially when dealing with lipases showing a high degree of selectivity for a given type of fatty acids (typoselectivity), which seems to occur in the case of some plant [18] or microbial [212] lipases. Since short acyl chains para-nitrophenyl esters can be hydrolysed by non enzymatic proteins, they should not be used to assay lipase activities, even with purified lipases.
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Eur. J. Lipid Sci. Technol.

Methods for lipase detection and assay: a critical review

WILEY-VCH Verlag GmbH, 69451 Weinheim, 2000

Eur. J. Lipid Sci. Technol. 2000, 133153

Methods for lipase detection and assay: a critical review

Eur. J. Lipid Sci. Technol. 2000, 133153

2.1.2.2 Mixed monomolecular films as substrates

2.1.2.3 Interfacial binding and film recovery

Eur. J. Lipid Sci. Technol. 2000, 133153

Methods for lipase detection and assay: a critical review

2.1.3 Atomic force microscopy

2.1.4 Infrared spectroscopy

Eur. J. Lipid Sci. Technol. 2000, 133153

Methods for lipase detection and assay: a critical review

2.2.2.1.2 Fluorimetric assays

2.2.2.1.4 Enzymatic assays

2.2.2.1.3 Chromatographic assays

Eur. J. Lipid Sci. Technol. 2000, 133153

Methods for lipase detection and assay: a critical review

2.2.2.1.5 In situ detection by electron microscopy

2.2.2.2 Synthetic carboxylic esters as substrates 2.2.2.2.1 Radioactive assays

Eur. J. Lipid Sci. Technol. 2000, 133153

2.2.2.2.2 Colourimetric assays

Eur. J. Lipid Sci. Technol. 2000, 133153

Methods for lipase detection and assay: a critical review

sure lipase activities should therefore be interpreted with great care.

2.2.2.2.3 Fluorimetric assays

2.2.3.3 Determination of the released thiol from synthetic thioesters

2.2.4 Electric conductivity

2.2.5 Acoustic wave conductance

Eur. J. Lipid Sci. Technol. 2000, 133153

Methods for lipase detection and assay: a critical review

2.2.6 The oil-drop method

3.2.2 Lipase immunodetection in other physiological media

3.1 ELISA for pure lipases

3.3 Immunocytolocalisation of lipases

3.4 Immunoblot analysis of lipases

Eur. J. Lipid Sci. Technol. 2000, 133153

Methods for lipase detection and assay: a critical review

4.2 Level of lipase activity

4.1 Pure lipases or crude biological media

Eur. J. Lipid Sci. Technol. 2000, 133153

Eur. J. Lipid Sci. Technol. 2000, 133153

Methods for lipase detection and assay: a critical review

Eur. J. Lipid Sci. Technol. 2000, 133153

Eur. J. Lipid Sci. Technol. 2000, 133153

Methods for lipase detection and assay: a critical review

Eur. J. Lipid Sci. Technol. 2000, 133153

Eur. J. Lipid Sci. Technol. 2000, 133153

Methods for lipase detection and assay: a critical review

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