FR2696193A1 - Measurement of phospholipase activity - based on effect on interfacial tension of oil drop with adsorbed phospholipid film - Google Patents

Measurement of phospholipase activity - based on effect on interfacial tension of oil drop with adsorbed phospholipid film Download PDF

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FR2696193A1
FR2696193A1 FR9211474A FR9211474A FR2696193A1 FR 2696193 A1 FR2696193 A1 FR 2696193A1 FR 9211474 A FR9211474 A FR 9211474A FR 9211474 A FR9211474 A FR 9211474A FR 2696193 A1 FR2696193 A1 FR 2696193A1
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oil
drop
phospholipids
process according
aqueous phase
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FR2696193B1 (en
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Verger Robert
Riviere Claude
Esposito Gerard
Cagna Alain
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INTERFACIAL TECHNOLOGY CONCEPT
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/918—Carboxylic ester hydrolases (3.1.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
    • G01N2405/04—Phospholipids, i.e. phosphoglycerides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
    • G01N2405/04—Phospholipids, i.e. phosphoglycerides
    • G01N2405/06—Glycophospholipids, e.g. phosphatidyl inositol

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Abstract

The hydrolytic activity of phospholipases on phospholipids is measured by: (a) adsorbing a phospholipid film on an oil drop; (b) immersing the oil drop in an aq. phase; (c) injecting the phospholipase into the aq. phase; and (d) measuring the change in interfacial tension of the oil drop. USE/ADVANTAGE - Pharmaceutical, medical and industrial applications. Phospholipase activity is measured with high sensitivity under conditions similar to those encountered in vivo.

Description

PROCEDE DE MESURE DE L'ACTIVISTE DES PHOSPHOLIPASES
La présente invention a trait au domaine de l'étude des enzymes phospholipases. Elle concerne plus spécialement un procédé de mesure de l'activité de ces phospholipases.METHOD FOR MEASURING THE ACTIVITY OF PHOSPHOLIPASES
The present invention relates to the field of the study of phospholipase enzymes. It relates more specifically to a method for measuring the activity of these phospholipases.

Les phospholipases sont des enzymes particulièrement importants d'un point de vue biologique puisqu'ils catalysent l'hydrolyse des phospholipides, constituants principaux des membranes cellulaires, générant à partir des phospholipides, des acides gras et des lysophospholipides. Deux d'entre eux, la phospholipase pancréatique, et la phospholipase intestinale jouent un rôle prépondérant dans les processus nutritionnels. Enfin on trouve certains de ces enzymes dans les venins de serpent. Phospholipases are particularly important enzymes from a biological point of view since they catalyze the hydrolysis of phospholipids, main constituents of cell membranes, generating from phospholipids, fatty acids and lysophospholipids. Two of them, pancreatic phospholipase, and intestinal phospholipase play a major role in nutritional processes. Finally, some of these enzymes are found in snake venoms.

Afin de mesurer l'activité hydrolytique de ces enzymes, diverses méthodes de dosage sont utilisées, consistant soit en la mesure de la disparition du substrat, soit en la mesure de l'apparition des produits de la réaction enzymatique. In order to measure the hydrolytic activity of these enzymes, various assay methods are used, consisting either in measuring the disappearance of the substrate, or in measuring the appearance of the products of the enzymatic reaction.

Parmi les méthodes chimiques, on peut citer la pH-métrie, permettant de déterminer à l'aide d'un pH-stat, les variations de pH, dues à la libération d'acides gras au cours de la réaction d'hydrolyse. La limitation principale de l'utilisation du pH-stat concerne la gamme de pH potentiellement utilisable. En effet, le pH du milieu réactionnel contenant les phospholipides doit être supérieur au pKa apparent de l'acide gras libéré. De plus cette méthode présente l'inconvénient d'être très peu sensible. Among the chemical methods, mention may be made of pH-metrics, making it possible to determine, using a pH-stat, the variations in pH, due to the release of fatty acids during the hydrolysis reaction. The main limitation in the use of pH-stat relates to the range of pH potentially usable. Indeed, the pH of the reaction medium containing the phospholipids must be greater than the apparent pKa of the fatty acid released. In addition, this method has the disadvantage of being very insensitive.

On connaît également une méthode immunologique, le test ELISA (Enzyme Linked Immuno Sorbent Assay) mettant en oeuvre des anti-corps spécifiques de la phospholipase. Ce test est hautement sensible et spécifique et permet de réaliser une mesure ponctuelle, à un temps donné de l'activité de l'enzyme. Cependant, il n'offre pas la possibilité d'étudier la cinétique de la réaction enzymatique. An immunological method is also known, the ELISA test (Enzyme Linked Immuno Sorbent Assay) using specific antibodies to phospholipase. This test is highly sensitive and specific and makes it possible to carry out a specific measurement, at a given time, of the activity of the enzyme. However, it does not offer the possibility of studying the kinetics of the enzymatic reaction.

Enfin, il existe une méthode biophysique, reposant sur la variation d'une tension interfaciale liquide/gaz. Finally, there is a biophysical method, based on the variation of a liquid / gas interfacial tension.

On utilise pour cela un milieu aqueux sur lequel repose une couche monomoléculaire de phospholipides, et on mesure la variation de la tension interfaciale à l'interface liquide/gaz, après injection de la phospholipase dans la sous-phase aqueuse. L'inconvénient de cette méthode réside dans le fait, qu'on ne peut mesurer l'activité des phospholipases que sur des phospholipides de synthèse à chaînes acyl courtes et non sur des substrats naturels à chaînes acyl longues, puisque les acides gras naturels ne sont pas solubles dans l'eau. De plus cette méthodologie ne permet pas de reproduire des conditions proches des conditions physiologiques de fonctionnement des enzymes, qui correspondent plutôt à une interface liquide/liquide qu'à une interface liquide/gaz.For this, an aqueous medium is used on which a monomolecular layer of phospholipids rests, and the variation of the interfacial tension at the liquid / gas interface is measured, after injection of the phospholipase into the aqueous sub-phase. The disadvantage of this method lies in the fact that the activity of phospholipases can only be measured on synthetic phospholipids with short acyl chains and not on natural substrates with long acyl chains, since natural fatty acids are not not soluble in water. In addition, this methodology does not make it possible to reproduce conditions close to the physiological operating conditions of enzymes, which correspond more to a liquid / liquid interface than to a liquid / gas interface.

Au vu du rôle capital des phospholipases en biologie cellulaire, et afin d'étudier leur fonctionnement dans un milieu naturel, la demanderesse s'est proposée de mettre au point une méthode de dosage des phospholipases offrant des conditions de réaction proches des conditions physiologiques de fonctionnement de ces enzymes. In view of the crucial role of phospholipases in cell biology, and in order to study their functioning in a natural environment, the applicant proposed to develop a method for assaying phospholipases offering reaction conditions close to the physiological operating conditions. of these enzymes.

L'article de S. NURY, G. PIERONI, C. RIVIERE, Y. The article by S. NURY, G. PIERONI, C. RIVIERE, Y.

GARGOURI, A. BOIS, R. VERGER, dans Chemistry and Physics of Lipids, 45 (1987) p.27-37 intitulé "lipase kinetics at the triacylglycerol-water interface using surface tension measurements" décrit une méthode analytique de mesure de l'activité lipasique, reproduisant les conditions in-vivo de l'interface huile/eau, fondée sur le principe de la variation de la tension interfaciale d'une goutte de substrat immergée dans une solution enzymatique.GARGOURI, A. BOIS, R. VERGER, in Chemistry and Physics of Lipids, 45 (1987) p.27-37 entitled "lipase kinetics at the triacylglycerol-water interface using surface tension measurements" describes an analytical method for measuring the lipase activity, reproducing the in-vivo conditions of the oil / water interface, based on the principle of the variation of the interfacial tension of a drop of substrate immersed in an enzymatic solution.

Cette méthode n'était cependant par applicable aux phospholipides. En effet de par leur hydrophobicité inférieure à celle des lipides, les phospholipides ne sont pas aptes à former une goutte au sein d'une solution aqueuse. Ils ont tendance à former des liposomes ou des phases lamellaires. However, this method was not applicable to phospholipids. Indeed, due to their hydrophobicity lower than that of lipids, phospholipids are not able to form a drop within an aqueous solution. They tend to form liposomes or lamellar phases.

L'invention a donc pour but de proposer une méthode de mesure de l'activité de phospholipases, de grande sensibilité, facile à mettre en oeuvre et présentant l'avantage de mesurer l'activité de ces enzymes dans des conditions proches des conditions naturelles de leur fonctionnement. The object of the invention is therefore to propose a method for measuring the activity of phospholipases, which is highly sensitive, easy to implement and has the advantage of measuring the activity of these enzymes under conditions close to the natural conditions of their functioning.

Pour atteindre ce but, l'invention propose un procédé de mesure de l'activité hydrolytique des phospholipases sur les phospholipides, du type consistant à mesurer une tension interfaciale d'une goutte d'un corps huileux, immergée dans une phase aqueuse caractérisé en ce que - on réalise une adsorption d'un film de phospholipides à la périphérie de ladite goutte, - on immerge ladite goutte au sein d'une phase aqueuse, - on injecte la phospholipase à doser dans la phase aqueuse - on mesure la variation de la tension interfaciale de ladite goutte, due à l'hydrolyse du film de phospholipides par la phospholipase. To achieve this object, the invention provides a method for measuring the hydrolytic activity of phospholipases on phospholipids, of the type consisting in measuring an interfacial tension of a drop of an oily body, immersed in an aqueous phase characterized in that - an adsorption of a phospholipid film is carried out at the periphery of said drop, - said drop is immersed in an aqueous phase, - the phospholipase to be dosed is injected into the aqueous phase - the variation in the interfacial tension of said drop, due to the hydrolysis of the film of phospholipids by phospholipase.

D'une façon générale le procédé selon l'invention peut être utilisé avec un nombre varié et non limitatif de phospholipases : on peut par exemple doser la phospholipase Al, A2, D, ou C. In general, the method according to the invention can be used with a varied and nonlimiting number of phospholipases: it is possible, for example, to measure the phospholipase Al, A2, D, or C.

Dans la description qui suit, on se réfère plus particulièrement à la phospholipasé A2. In the description which follows, reference is made more particularly to the phospholipase A2.

Parmi les phospholipides convenant à la mise en oeuvre de l'invention, on peut citer la phosphatidylcholine, la phosphatidylsérine, la phosphatidylethanolamine, le phosphatidylglycérol ou le phosphatidylinositol. Among the phospholipids which are suitable for implementing the invention, mention may be made of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol or phosphatidylinositol.

Conformément à l'invention, les phospholipides sont adsorbés en une couche monomoléculaire ou multimoléculaire à la surface de ladite goutte. According to the invention, the phospholipids are adsorbed in a monomolecular or multimolecular layer on the surface of said drop.

Enfin, à titre de corps huileux apte à former une goutte au sein d'un milieu aqueux on peut choisir ce dernier dans la famille des huiles végétales, notamment l'huile de soja, de tournesol ou l'huile d'olive, ou bien dans famille des huiles d'hydrocarbure, notamment l'huile de parafine, l'huile de silicone ou huile de vaseline. Finally, as an oily body capable of forming a drop within an aqueous medium, the latter can be chosen from the family of vegetable oils, in particular soybean, sunflower oil or olive oil, or else in the family of hydrocarbon oils, in particular paraffin oil, silicone oil or petrolatum oil.

Avantageusement la phase aqueuse est constituée d'une solution tampon de pH compris entre 4 et 9. Advantageously, the aqueous phase consists of a buffer solution of pH between 4 and 9.

L'invention sera mieux comprise à la lecture de la description de nombreux essais entrepris par la demanderesse, et en se référant aux dessins et graphiques annexés qui représentent - Figure 1 : la réalisation d'une goutte d'huile au sein d'une émulsion de phospholipides - Figure 2 : la formation par adsorption d'un film monomoléculaire de phospholipides sur la goutte d'huile - Figure 3 : un diagramme illustrant la variation de la tension interfaciale, le temps en abscisse étant exprimé en minutes et la tension interfaciale en millinewton/mètre. The invention will be better understood on reading the description of numerous tests undertaken by the applicant, and with reference to the attached drawings and graphics which represent - Figure 1: the production of a drop of oil within an emulsion of phospholipids - Figure 2: the formation by adsorption of a monomolecular film of phospholipids on the drop of oil - Figure 3: a diagram illustrating the variation of the interfacial tension, the time on the abscissa being expressed in minutes and the interfacial tension in millinewton / meter.

Les essais suivants mettent en exergue la variation de tension interfaciale d'une goutte d'huile.The following tests highlight the variation in interfacial tension of a drop of oil.

Premier essai a) la phospholipase utilisée dans le premier essai était
la phospholipase A2, purifiée à partir du venin de
Naja Mossambica. On a opéré avec une solution aqueuse
de phospholipase de concentration 33 nM.First test a) the phospholipase used in the first test was
phospholipase A2, purified from venom of
Naja Mossambica. We operated with an aqueous solution
phospholipase concentration 33 nM.

b) la source de phospholipides était la lécithine d'oeuf
purifiée se présentant sous la forme d'une émulsion
huile/eau, préparée à partir d'une solution mère
ayant la composition suivante
lecithine d'oeuf purifiée 1,2 g
huile de soja purifiée 20 g
glycérol 2,25 g
eau distillé qsp 100 ml,
et ayant été diluée 1.000 fois dans la solution
tampon décrite en d) c) la goutte servant de support aux phospholipides était
une goutte d'huile de soja purifiée.b) the source of phospholipids was egg lecithin
purified in the form of an emulsion
oil / water, prepared from a stock solution
having the following composition
purified egg lecithin 1.2 g
purified soybean oil 20 g
glycerol 2.25 g
distilled water qs 100 ml,
and having been diluted 1,000 times in the solution
buffer described in d) c) the drop serving as support for the phospholipids was
a drop of purified soybean oil.

d) la phase aqueuse est constituée d'une solution tampon
de pH 8.d) the aqueous phase consists of a buffer solution
pH 8.

e) l'essai proprement dit a consisté, comme on peut le
voir sur la Figure 1 à former une goutte d'huile (1)
à l'aide d'une seringue (2), reliée à un tube (3), au
sein de l'émulsion (4) de lécithine, contenue dans la
cuve (5).e) the actual test consisted, as can be
see on Figure 1 to form a drop of oil (1)
using a syringe (2), connected to a tube (3), at the
within the lecithin emulsion (4), contained in the
tank (5).

On a procédé à une préincubation pendant 15 minutes,
les molécules de lécithine ont alors migré vers la
goutte d'huile et se sont orientées en fonction de
leurs pôles hydrophile (6) et lipophile (7), afin de
former par adsorption un film monomoléculaire (8),
comme on peut le voir sur la Figure 2, sur ladite
goutte. We preincubated for 15 minutes,
the lecithin molecules then migrated to the
drop of oil and oriented according to
their hydrophilic (6) and lipophilic (7) poles, in order to
forming a monomolecular film (8) by adsorption,
as can be seen in Figure 2, on said
drop.

On a alors remplacé par périfusion l'émulsion dans
ladite cuve par la solution tampon à l'aide de deux
pompes péristaltiques synchrones (non représentées
sur la Figure), ayant des débits identiques de
l'ordre de 0,2 ml/min, l'une aspirant l'émulsion,
l'autre apportant la solution tampon. On a alors
procédé à une agitation pendant dix minutes avant
d'injecter à l'aide d'une deuxième seringue (9)
représentée en trait pointillé sur la Figure 1, la
solution de phospholipase (10) dans ladite cuve (5).The emulsion was then replaced by perifusion in
said tank by the buffer solution using two
synchronous peristaltic pumps (not shown
in Figure), having identical flow rates of
about 0.2 ml / min, one aspirating the emulsion,
the other bringing the buffer solution. We then have
stirred for ten minutes before
inject using a second syringe (9)
shown in dotted lines in Figure 1, the
phospholipase solution (10) in said tank (5).

Parallèlement à ceci on a enregistré, après l'étape
de périfusion les variations de forme et de
dimensions de la goutte à l'aide d'une caméra reliée
à une électronique de traitement d'images, permettant
de calculer la tension interfaciale d'après
l'équation de Laplace. Comme on peut le voir sur la
Figure 3, durant les dix premières minutes
d'agitation, précédant l'injection de phospholipase,
on a noté une valeur constante de la tension
interfaciale, indiquant une bonne stabilité du film
phospholipidique à l'interface huile-eau.Along with this we recorded, after the step
of perifusion the variations of form and
dimensions of the drop using a connected camera
an image processing electronics, allowing
to calculate the interfacial tension according to
Laplace's equation. As we can see on the
Figure 3, during the first ten minutes
agitation, preceding the phospholipase injection,
we noted a constant value of the voltage
interfacial, indicating good film stability
phospholipid at the oil-water interface.

Après l'injection de phospholipase A2 (PLA2), on
observe une diminution notable de la tension
interfaciale, qui traduit une hydrolyse du film de
lécithine présent à l'interface huile-eau.After the injection of phospholipase A2 (PLA2), one
observes a noticeable decrease in blood pressure
interfacial, which translates a hydrolysis of the film of
lecithin present at the oil-water interface.

Deuxième essai
Selon une variante de réalisation on a procédé à un deuxième essai, en opérant selon le même protocole que dans le premier essai, si ce n'est que l'expérience de périfusion n'a pas eu lieu. On a directement injecté la solution de phospholipase au sein de l'émulsion de lécithine. L'enzyme agit dans ce cas aussi bien sur les phospholipides présents dans l'émulsion que sur les phospholipides constituant la couche adsorbée à la surface de la goutte d'huile. En procèdant parallèlement à la mesure de la tension interfaciale, on a observé après injection de la phospholipase, une chute brutale de ladite tension interfaciale.Second trial
According to an alternative embodiment, a second test was carried out, operating according to the same protocol as in the first test, except that the perifusion experiment did not take place. The phospholipase solution was directly injected into the lecithin emulsion. In this case, the enzyme acts both on the phospholipids present in the emulsion and on the phospholipids constituting the layer adsorbed on the surface of the drop of oil. By proceeding in parallel with the measurement of the interfacial tension, a sudden drop in said interfacial tension was observed after injection of the phospholipase.

Ainsi, conformément à l'invention, la mesure de la variation de la tension interfaciale d'une goutte d'huile sur laquelle est fixée le substrat de l'enzyme permet d'étudier la cinétique de la réaction enzymatique. On peut citer de nombreuses applications notamment pharmaceutiques, médicales et industrielles.  Thus, in accordance with the invention, the measurement of the variation of the interfacial tension of a drop of oil on which the enzyme substrate is fixed makes it possible to study the kinetics of the enzymatic reaction. Mention may be made of numerous applications, in particular pharmaceutical, medical and industrial.

Claims (11)

REVENDICATIONS 1 - Procédé de mesure de l'activité hydrolytique des phospholipases sur les phospholipides, du type consistant à mesurer une tension interfaciale d'une goutte d'un corps huileux, immergée dans une phase aqueuse, caractérisé en ce que - on réalise une adsorption d'un film (8) de phospholipides à la périphérie de ladite goutte (1), - on immerge ladite goutte au sein d'une phase aqueuse, - on injecte la phospholipase à doser dans la phase aqueuse, - on mesure la variation de la tension interfaciale de ladite goutte, due à l'hydrolyse du film (8) de phospholipides par la phospholipase. 1 - Method for measuring the hydrolytic activity of phospholipases on phospholipids, of the type consisting in measuring an interfacial tension of a drop of an oily body, immersed in an aqueous phase, characterized in that - adsorption is carried out '' a film (8) of phospholipids at the periphery of said drop (1), - said drop is immersed in an aqueous phase, - the phospholipase to be dosed is injected into the aqueous phase, - the variation in the interfacial tension of said drop, due to the hydrolysis of the film (8) of phospholipids by phospholipase. 2 - Procédé selon la revendication 1, caractérisé en ce que l'adsorption du film de phospholipides est réalisée en formant ladite goutte au sein d'une cuve (5) contenant une émulsion huile/eau de phospholipides. 2 - Process according to claim 1, characterized in that the adsorption of the phospholipid film is carried out by forming said drop within a tank (5) containing an oil / water emulsion of phospholipids. 3 - Procédé selon les revendications 1 et 2, caractérisé en ce que l'immersion de ladite goutte au sein de la phase aqueuse est réalisée en aspirant la solution de phospholipides et en injectant de manière synchronisée ladite phase aqueuse. 3 - Method according to claims 1 and 2, characterized in that the immersion of said drop within the aqueous phase is carried out by aspirating the phospholipid solution and by synchronously injecting said aqueous phase. 4 - Procédé selon les revendications 1 à 3, caractérisé en ce que la phase aqueuse est constituée d'une solution tampon de pH compris entre 4 et 9. 4 - Process according to claims 1 to 3, characterized in that the aqueous phase consists of a buffer solution of pH between 4 and 9. 5 - Procédé selon les revendications 1 et 2, caractérisé en ce que la phase aqueuse est constituée par l'émulsion huile/eau de phospholipides et que la phospholipase est directement injectée dans l'émulsion de phospholipides.  5 - Process according to claims 1 and 2, characterized in that the aqueous phase consists of the oil / water emulsion of phospholipids and that the phospholipase is directly injected into the emulsion of phospholipids. 6 - Procédé selon les revendications 1 à 5, caractérisé en ce que la tension interfaciale est mesurée à l'aide d'une caméra reliée à une électronique de traitement d'images. 6 - Method according to claims 1 to 5, characterized in that the interfacial tension is measured using a camera connected to an electronic image processing. 7 - Procédé selon les revendications 1 à 6, caractérisé en ce que le corps huileux est choisi parmi le groupe des huiles végétales. 7 - Process according to claims 1 to 6, characterized in that the oily body is chosen from the group of vegetable oils. 8 - Procédé selon la revendication 7, caractérisé en ce que le corps huileux est choisi dans le groupe constitué par l'huile de soja, l'huile de tournesol, l'huile d'olive. 8 - Process according to claim 7, characterized in that the oily body is chosen from the group consisting of soybean oil, sunflower oil, olive oil. 9 - Procédé selon les revendications 1 à 6 caractérisé en ce que le corps huileux est choisi parmi le groupe des huiles d'hydrocarbure. 9 - Process according to claims 1 to 6 characterized in that the oily body is chosen from the group of hydrocarbon oils. 10 - Procédé selon la revendication 9, caractérisé en ce que l'huile d'hydrocarbure est choisi dans le groupe constitué par l'huile de parafine, l'huile de silicone et l'huile de vaseline. 10 - Process according to claim 9, characterized in that the hydrocarbon oil is chosen from the group consisting of paraffin oil, silicone oil and petroleum jelly oil. 11 - Procédé selon les revendications 1 à 10, caractérisé en ce que le phospholipide est choisi parmi le groupe constitué par la phosphatidylcholine, la phosphatidylserine, la phosphatidylethanolamine le phosphatidylglycérol et le phosphatidylinositol.  11 - Process according to claims 1 to 10, characterized in that the phospholipid is chosen from the group consisting of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol.
FR9211474A 1992-09-25 1992-09-25 Method for measuring the activity of phospholipases. Expired - Fee Related FR2696193B1 (en)

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FR2696193B1 FR2696193B1 (en) 1994-12-09

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4140579A (en) * 1977-04-04 1979-02-20 Moncla Bernard J Method of testing for phospholipases using a composition containing a uniform dispersion of a phospholipid
EP0021572A1 (en) * 1979-06-04 1981-01-07 American Hospital Supply Corporation A substrate for use in a turbidimetric assay for lipase and a method for making this substrate
EP0297387A2 (en) * 1987-07-01 1989-01-04 Abbott Laboratories An improved turbidimetric method for the determination of serum lipase

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4140579A (en) * 1977-04-04 1979-02-20 Moncla Bernard J Method of testing for phospholipases using a composition containing a uniform dispersion of a phospholipid
EP0021572A1 (en) * 1979-06-04 1981-01-07 American Hospital Supply Corporation A substrate for use in a turbidimetric assay for lipase and a method for making this substrate
EP0297387A2 (en) * 1987-07-01 1989-01-04 Abbott Laboratories An improved turbidimetric method for the determination of serum lipase

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 116, no. 13, 30 Mars 1992, Columbus, Ohio, US; abstract no. 123641, T. KONDO ET AL. 'Hydrolysis of phospholipid monolayers by phospholipase D at the oil/water interface under the control of the potential drop across the monolayer.' page 364 ;colonne 2 ; *
JOURNAL OF DAIRY SCIENCE vol. 59, no. 12, 1976, CHAPAIGN, ILLINOIS US pages 2024 - 2030 G. L. CHRISOPE ET AL. 'Combined action of lipase and microbial phospholipase C on a model fat globule emulsion and raw milk.' *

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