IL32910A - Agent and process for the determination of lipase - Google Patents

Description

New agent and process for the determination ot lipase n K3*V ny*3j7> arm T nm nsin The present invention is concerned with a new agent and process for the determination of lipase.

Lipases are enzymes which split fats and oils into glycerides and fatty acids. They are widely distributed in the animal organism. Particularly high concentrations are found in the liver and in the pancreatic secretion which digests the nutritional fats in the small intestine. In the case of diseases of the pancreas, lipases passes into the blood stream. Therefore, the determination of lipases in serum is an important diagnostic aid for the recognition of pancreatic diseases.

According to the known :processes, lipase is determined by first preparing an excess of a lipase substrate and the amount,of fatty acid liberated therefrom by the lipase is measured by titration with an alkali, using an indicator, or by extraction of the copper salt thereof. As indicators for the titrometric determination by the known method, there is used thymolphthalein or phenol-phthalein (see Deutsche Medizinische vVochenschrift , 90 , 1 170/1 965 ; and Analytical Biochemistry, 6 , 45 /1 63) .

As substrate, there is generally used an oil-in-water emulsion. The precise determination of lipase by this process involves, however, considerable difficulties which contraindicate an extensive use of the process in clinical laboratories. The substrate cannot be easily handled and, because of turbidity, a photometric measurement by transillumination is impossible. Furthermore, it is disadvantageous that the amount of acid to be determined is extremely small. For example, even in the case of the use of 1 ml. of serum and with an incubation period of minutes, the amount of acid liberated is, in normal cases, only 0.0005 mMole, corresponding to a utilisation of 0.005 ml. 0.1 N sodium hydroxide solution. Therefore, it is necessary to employ very expensive automatic micro-titration devices with which it is not possible to carry out serial investigations. Insofar as automatic micro-titration apparatus are available, in the case of the use of colour indicators for the titration, it is necessary to put up with very long incubation times, relatively large amounts of serum and imprecise results.

Because of these difficulties, in practice an esterase determination is carried out, i.e. there is used as substrate a soluble ester of small chain length and the constitution ¾f which is well defined but which, on the other hand, is hydrolysed not only by the lipase but also by other esterases contained in the serum (c.f. ' rztliches Labor, 3, 157/1967).. The use of such a soluble ester is permissible in the case of a lipase determination in enriched enzyme preparations but not in serum since the lipase concentration in serum is very small (normal range up to 0.1 U/ml.) and, in addition, many other esterases are present, some of them in high concentrations. Therefore, hitherto for the determination of the so-called "true lipase", it was necessary to use the above-described process with the indicated great d sadvantages.

We have now found that, in the case of the use of certain pH indicators, the mentioned difficulties in the lipase determination with the aid of an oil-in-water emulsion are substantially avoided.

Consequently, it is an object of the present invention to provide an agent for the determination of lipase in body fluids with aqueous fat or oil emulsions and pH colour indicators, which is characterised in that, as colour indicator, there is used m-cresol purple and/or _-xylenolsulphophthalein and/or dibromothymolsulphophthalein and or thymolsulphophthalein and/or a-naphtholph alein.

Furthermore, the present invention is concerned with a process for the determination of lipase in body fluids with aqueous fat or oil emulsions and pH colour indicators, which consists in that, to the sample to be investigated, there is added the agent described above, which will be described hereinafter in more detail, and the colour change taking place during the incubation period is determined.

It is pa»ticularly advantageous that, in the new process, there is only needed a very small amount of the sample to be determined (about 0.2 ml. in the case of a normal determination or 0.02 ml. for a micro-determination). Furthermore, the new agent and process offer the advantage that, in contradistinction to the known agents and the titrometric process, no expensive apparatus and no highl trained personnel are necessary and tliat the new process, in comparison with the known process in which, with the use of indicators, the amount of acid liberated by the lipase must be determined by titration, can be carried out considerably more quickly. Furthermore, a multiple pipetting, such as is necessary for. the known process, is avoided by -the use of the new agent. A further advantage is the high specificity and exactitude of the lipase determination with the agent according to the present invention.

The very good results and the simple manipulation of the new agent were not to have "been foreseen on the basis of the prior art. Since the substrate and indicator are subjected to the action of the lipase in a protein-containing solution, it was to have been assumed that a displacement would have taken place of the colour change region for the colour indicators to be used according to the present invention. Furthe more, it is known that the colour change region of the indicator is generally not only displaced in a protein-containing solution but usually also widened, so that exact pH readings are made difficult. Furthermore, such a widening of the colour change region is also brought about by the addition of emulsion-stabilising materials, such as gum arabic. Insofar as an additive is not completely colourless, such as is the case with gum arabic, the reading off of the colour is also made difficult. Because of these uncertainty factors, the good measurement results which, are obtained with the new agent according to the present invention are to be considered as being surprising.

In the following, the composition of the new agent according to the present invention is described in more detail: Amongst the above-mentioned colour indicators which are contained in the agent according to the present invention, m-cresol purple and _-xylenolsulphophthalein are preferred. The indicators show a sharp colour change, for example m-cresol purple from violet to yellow and JD-xylenolsulphophthalein from blue to yellow. In the preparation of the agent according to the present invention the pH of the solution is expediently adjusted in such a manner that the colour change even takes place by the addition of a minimum amount of acid. For example, in the case of the use of m-cresol purple, adjustment is made to a brownish-violet shade and, in the case of the use of p_-xylenolsulphophthalein, to a brownish-blue colour shade. The adjustment of the desired pH value is advantageously carried out by adding an alkaline solution of the indicator, for example a solution of m-cresol purple or of _-xylenol-sulphophthalein in dilute, for example 0.1 sodium hydroxide solution, to the emulsion and subsequently an acid, for example dilute hydrochloric acid or another dilute mineral acid, is added thereto until the desired colour shade is achieved. In principle, it is also possible to add the indicator in aqueous acid, for example as a suspension or solution, and subsequently, by the addition of alkali, for example of dilute sodium hydroxide solution, to adjust the desired colour shade. In general, however, because of the better solubility, the addition of the indicator in alkaline solution is preferred. If the agent is to be stored for a comparatively long period of time before the lipase determination is carried out, it is recommended to add the emulsion only a short time before use in order to increase the storage stability of the indicator. In this case, the emulsion is first prepared with a preferably neutral or only weakly alkaline or v/eakly acidic pH and, separately from this, the alkaline indicator solution is prepared. Both solutions are also very storage stable for comparatively long periods of time. The indicator is present in the emulsion ready for use in a concentration of about 0.001 to 1%, preferably of 0.05 to 0.2%, referred to tlie weight of the total emulsion.

The colour indicator in the agent according to the present invention preferably contains one of the above-mentioned compounds, i.e. m-cresol purple, jj-xylenolsulpho-phthalein, dibromothymolsulphophthalein, thymolsulpho-phthalein or a-naptholphthalein. However, the indicator can possibly also consist of a mixture of 2 or more of these compounds. In particular, in some cases it can be expedient to use, besides the main indicator, for example, m-cresol purple or p_-xylenolsulphophthalein, a further one of the mentioned indicators in a lesser concentration for the precise adjustment of the colour nuance desired for the colour change region of the agent according to the present invention. Such an adjustment can be necessary, for example, when, for the better possibility of comparison with a previously printed colour scale, the colour tone of the main indicator is to be displaced. Por example, by the addition of a small amount of σ-naphtholphthalein, which is blue in the alkaline region, to a compound used as the main component of the colour indicator, such as -cresol purple or _~xylenolsulphophthalein, the colour shade of the agent in the alkaline region can, as a whole be displaced towards blue.

Such colour displacements in the agent according to the present invention can possibly also be achieved by the addition of small amounts of colour indicators other than those mentioned above, for example by the addition of diphenol purple (red displacement, especially in the neutral region, in the case of the use of n-cresol purple as main component of the colour indicator) or, in some cases, also by the addition of small amounts of another coloured compound, such as carotin, with which a yellow colour shade is achieved. These compounds are added, for example, in a concentration of up to 0.05 ; referred to the emulsion.

As lipase substrate, the agent according to the present invention contains natural or synthetic esters of fatty acids, especially fatty acid glycerol esters.

In general, there are used triglycerides or possibly also diglycerides of fatty acids, such as palmitic, stearic, oleic, linoleic or lauric acids. Thus, natural oils and fats, for example also coconut fat, are suitable as lipase substrates for the agent according to the present invention. Olive oil is particularly preferred. It is recommended to remove from the oils or fats, before use thereof as lipase substrate, the free fatty acids, for example by chromatographic purification of the oil or fat over a basic adsorption agent, such as aluminium oxide. The oils and fats can possibly also be purified in some other manner or can also be hydrogenated. For example, hydrogenated rape seed oil is also suitable as a lipase substrate.

For increasing the storage stability of the emulsion, the agent according to the present invention preferably contains an emulsion-stabilising additive. This additive can consist of one or more compounds from the groups of substances described hereinafter.

As emulsion-stab ilising additives, the compounds which have proved to be especially useful are those with a protective colloidal action, especially gum arable.

Polyvinyl-pyrrolidone can possibly also be added as protective colloid. The emulsion stabilisers with a protective colloidal action, especially gum arabic, are present in the emulsion in a concentration of about 0.5 to 30%, preferably of 10 to 25%. Besides a protective colloidal action, gum arabic also exerts an activating influence upon the lipase.

Furthermore, the agent according to the present invention can also contain the usual emulsifiers for oil-or fat-in-water emulsions, for example polyethylene glycol fatty alcohol ethers, especially polyoxyet ylene derivatives or sorbitol anhydrides, such as polyoxyethylene sor itan oleates, palmitates and stearates, for example polyoxyethylene sorbitan monopalmitate , monooleate, monostearate, trioleate or tristearate; polyethylene glycol alkyl-phenol ethers; sulphonic acid salts or sulphuric acid esters, for example isopropyl-naphthalene-sulphonic acid salts, or cetyl-stearyl sulphuric acid ester, for the stabilisation of the emulsion. The concentration of these emulsifiers is, for example, up to 20%, preferably up to 1 „ .

As further additive which stabilises the emulsion and, at the same time, exerts an activating influence on the lipase to be measured in the case of a pancreatic lipase determination, there can be used salts of bile acids, for example alkali metal cholates, desoxycholates , tauro-cholates and glycocholates, including bile acid salts.

These salts are used in a concentration of up to about 2%, especially of up to 0.5'.

As further suitable additive, the emulsion can contain, for stabilisation, substances which hinder precipitation, for example, gelling agents based upon montmorillonite, especially alkaline earth metal or quaternary ammonium salts of montmorillonite-fatty acids. For example, there can be used, in particular, very finely-divided magnesium montmorillonite with a density of 2.k g./ cc. and a moisture content of about 6% or a dimethyl-dioctadecyl-ammonium montmorillonite with a density of 1.80 g./cc. and a moisture content of less than 3?' , such as are usually employed as emulsion stabilisers for oil-in-water systems, as additive for the emulsion used according to the present invention. These anti-precipitation agents are used in a concentration of up to about 3%> preferably up to %, Furthermore, the agent according to the present invention can, contain emulsion-stabilising substances which increase the viscosity of the emulsions at lower temperatures. For this purpose, there can be used, for example, gel-forming, high molecular weight compounds which are not split by esterases. Agar has proved to be especially useful as an additive for increasing the viscosity and thus for stabilising' the emulsion.

Furthermore, the agent according to the present invention can contain reducing compounds for the prevention of an autoxidation and demixing of the emulsion occasioned thereby, preferred reducing agents being mercaptoethanol, cysteine, a-tocopherol acetate and/or ascorbyl palmitate in concentrations of up to about "\ , preferably of up to 0.05%. Tocopherol acetate and ascorbyl palmitate have the advantage that they are soluble in fats and oils. - For stabilising the emulsion, the agent according to the present invention can also contain biostatic substances which prevent the action of microorganisras on the substrate used. For this purpose, there can be used, for example, organo-mercury compounds, such as _-chloro-mercu ibenzoate, phenyl mercury acetate, ethyl mercuric-thiobenz-3, -oxazole-1 -carboxylic acid or the sodium salt thereof, ethyl mercury thiosalicylate, ethyl mercury thio-phenol-sulphonic acids, ^-hydroxy-benzoic acid esters, especially the methyl, ethyl or isopropyl esters, hexamethyl-ene-tetramine , iodoacetates and/or azides. For this purpose there are preferably used sodium iodoacetate and sodium ethyl mercuric thiosalicylate. The biostatic substances are preferably present in a concentration of up to about ^%, especially in a concentration of up to about 0, %. * In the agent according to the present invention, as emulsion-stabilising additive, there is usually present a compound selected from the above-described classes of substances. Gum arabic is preferably used as the emulsion stabiliser. In a further preferred embodiment, biostatic substances, possibly together with gum arabic, are present^ as emulsion-stabilising additives. Generally, those emulsion stabilisers are especially suitable which are not changed by esterases, especially by esterases other than lipase, in the weakly alkaline region.

In the case of the preparation of the agent according to the present invention, in general, there is first prepared a solution of at least one emulsion-stabilising compound, for example of gum arabic, in water. In some cases, it is expedient to filter or to centrifuge this solution before the preparation of the emulsion in order to remove residues from the additives, for example from the gum arabic. Subsequently, by the addition of the substrate, for example of olive oil, the emulsion is formed by turbo-stirring. The other additives used for the agent, for example, further emulsion-stabilising substances, especially biostatic substances, can be added to the mixture before or after the addition of the substrate.

The indicator can be added most expedi ently in the form of a solution to the emulsion. There is thus obtained the emulsion ready for use. The indicator can also be stored separately from the emulsion in the form of a solution and only added to the emulsion before the lipase determination is to be carried out. This embodiment of the agent according to the invention is preferred when a long storage period is necessary.

It is especially suitable to place the oil-in-water emulsion, in an amount necessary for one lipase determination, in a suitable container, preferably in a synthetic resin cuvette with, in particular, a rectangular base. In this case, the necessary indicator solution can possibly be prepared separately from the emulsion. The amount of emulsion placed in the container is hereby such that it is sufficient for one lipase determination. For example, in this embodiment, 1 or 2 ml. of emulsion are placed in the cuvette. In the indicator solution, which is possibly separate from the emulsion, the concentration of the indicator is expediently so adjusted that an aliquot part, which is easy to measure, for example one drop, of the indicator solution is added to the previously prepared oil-in-water emulsion, for example in a synthetic resin cuvette, for a determination. An oil-in-water emulsion w ic contains the indicator can be kept for about h weeks, a shaking of the emulsion before use being recommended.

If the oil-in-water emulsion with the indicator according to the present invention is previously placed in a cuvette for one determination, then it has proved to be expedient to provide the content of the cuvette with a film-forming coating. By means of such a coating, it ■ is possible to prevent the content of the cuvette from solidifying at low temperatures and also to prevent a sealing off of the cuvette opening when the cuvette is stored upside down which upon opening, would force out the content by a slight pressure arising in the cuvette.

This coating is, according to the present invention, advantageously formed on the emulsion by covering the emulsion with a liquid which is scarcely miscible or immiscible with water, which is specifically lighter than the emulsion and which solidi ies some time after placing it on the emulsion. For the production of this coating, there are preferably used solutions of natural, synthetic or semi-synthetic polymers, for example of a polyethylene derivative, such as polystyrene, polyvinyl acetate, polyvinyl chloride or polymethacrylic acid esters, or of a cellulose derivative, for example, of a nitrated cellulose, such as collodium, or of cellulose acetate, or of epoxy lacquers or of two-component urethane lacquers or of urethane lacquers which harden under the influence of moisture, in readily evaporable organic solvents or solvent mixtures. For example, as solvents for this purpose there can be used ethers, for example, diethyl ether, lower alcohols, such as ethanol, lower esters, such as ethyl acetate, or low boiling point hydrocarbons, which can possibly also be substituted by, for example, chlorine, such as d chloromethane , or ketones, such as acetone or cyclohexanone .

A plaeticiser, such as camphor, can possibly also be added. The film-forming substances are preferably used in such a concentration that the coating formed over the emulsion is up to 3 mm. thick, preferably about 0.5 mm. thick.

After evaporation of the solvent , a t in membrane is formed on the emulsion which adheres securely to the walls of tiie cuvette. This membrane prevents the emulsion from escaping tiirough the opening of the cuvette when the cuvette is stored with the opening downwards. A membrane which has proved to be particularly useful is one obtained by coating the emulsion in a cuvette with about 0.05 ml. of a 2% solution of collodium.

The process for determining lipase with the agent according to the invention is carried out by combining the above-described agent with the lipase-containing sample to be investigated and the colour change of the indicator arising during the incubation period is determined. There is added thereto a small, measured volume, preferably 0.1 ml., of the body fluid to be measured, for example of serum or plasma, in a measured volume, for example 1 ml., of the emulsion ready for use. Hereafter, mixing up is carried out in the usual way and the colour change measured. The colour change can be measured by comparison of the colour shade of the mixture with the corresponding colour shade on a calibrated colour scale or on a calibrated coloured strip. For this purpose, for example, the calibration number on the colour scale or colour band is read off and noted which is present immediately after the addition of the sample to the emulsion. Subsequently, the reaction mixture is left to stand for a definite period of incubation, for example for one hour, preferably at a standard temperature, for example at ambient temperature or at 37°C., and then there is again read off on the calibrated colour scale or on the calibrated colour band the number related to the resulting colour shade. The difference gives the lipase content in the units forming the basis for the calibration of the colour scale or colour band. , A calibrated colour scale or calibrated colour band for use in the measurement can be produced, for example, in the manner described in French Patent Speci ication No.1 ,516,492. For the calibration of the scale or of the band, that period of time is used as a basis which is employed in the subsequent lipase determina ion for the incubation, for example 1 hour.

In another embodiment of the process according to the present invention, the colour change is determined by comparison of the samples under investigation with one another after an incubation time of, for example one hour. In the case of this method, the colour shades of the samples with a normal lipase content, which are substantially unchanged after the incubation period, are used as standard. This manner of carrying out the lipase determination according to the present invention is especially suitable for serial analyses since usually more than S'O, . of the blood sera investigated in clinics show a normal lipase content and thus can serve as standard. The few samples with a pathologically increased lipase content are thus recognised after the incubation period from the colour shade which differs from the almost unchanged colour of the majority of the samples investigated. In the case of the use of the indicators m-cresol purple or jo-xylenolsulphophthalein, the pathological samples show a pale brown to pale yellow colour. If a more precise determination is necessary, the samples recognised as being pathological can again be measured more accurately by comparison with a calibrated colour band or calibrated colour scale.

The following Examples are given for the purpose of illustrating the present invention:-Sxample 1. mg. ethyl mercury thiosalicylate and 0 g. gum arabic are added to 8Q ml. water in a mixing device operating at a high speed of rotation.

Subsequently, the mixture is adjusted to pH 10 by the addition of 2N aqueous sodium hydroxide solution. It is then centrifuged and the supernatant liquid is filtered through a cotton plug for the removal of impurities. The filtrate is thereafter mixed in the mixing device, at a high speed of rotation, with 120 ml. olive oil (purified over basic aluminium oxide), optionally with the addition of an emulsifier, for example, of a polyoxyethylene sorbitan oleate, palmitate or stearate.

Subsequently, the mixture is again mixed for minutes and then mixed with 600 mg. m-cresol purple dissolved in 60 ml. 0.1N sodium hydroxide solution.

By the addition of dilute acid, for example of dilute hydrochloric acid, the pH of the mixture is adjusted to 8.8 (colour: brownish violet).

By way of alteration of the above procedure, instead of 600 mg. m-cresol purple, there are used the following indicators : 600 mg. dibromothymolsulphophthalein (adjustment of the pH to 8.8, colour blue) 55Ο mg. thymolsulphophthalein (adjustment of the pH to 8.8, colour greenish blue) 600 mg. a-naphtholphthalein (adjustment of the pll to 9 j colour pale blue) .

The emulsion thus prepared is either used directly for the lipase determination or, advantageously, is mechanically filled in 1 ml. amounts into closable synthetic resin cuvettes. Expediently, the' emulsion in each synthetic resin cuvette is covered with about 0.05 ml. of a 2yi solution of collodium. After evaporation of the solvent, a thin membrane is formed on the emulsion. The cuvettes are subsequently closed. The content of one synthetic resin -cuvette is then used for one lipase determination.

Example 2.

An emulsion is prepared in a manner analogous to that described in Example 1 except that no indicator is added and the pH is adjusted to 7.0. Separately from the cuvettes, each of which contain 2 ml. of emulsion, there is prepared a solution of m-cresol purple in 0.055 sodium hydroxide solution, a 0.02N solution of sodium hydroxide and a 0.02N solution of hydrochloric acid. The 0.02 solution of sodium hydroxide or of hydrochloric acid can he used for the adjustment of the pK of the emulsion which might have changed after a comparatively long period of storage.

Example 3.

In a manner analogous to that described in Example 1 , an emulsion which is ready for use is prepared from the following components: 80 ml. water mg. sodium iodoacetate or 100 mg. of _-hydroxy- benzoic acid methyl, ethyl or isopropyl ester or of hexamethylene tetramine g. gum arabic 0.2 g. agar ml. olive oil 80 mg. _-xylenolsulphophthalein The colour of the emulsion is adjusted to a brov/nish-brue by the addition of dilute sodium hydroxide solution or of dilute hydrochloric acid.

According to a modification of the above Example, 8 mg. of carotin or of diphenyl purple are additionally added to the described mixture.

The agent thus prepared can be used for the detQr-mination of lipase in the manner described in Example 7 hereinafter.

Example h.

In a manner analogous to that described in iixample 3? there is prepared an emulsion but without an indicator and the pH is adjusted to 7.0. Separately from the emulsion, there is prepared a % solution of jD-xylenolculphophthalein in 0.055N sodium hydroxide solution, as well as a 0.02N solution of sodium hydroxide and a 0.02H solution of hydrochloric acid.

Example 5 . 23 g. gum arabic h mg. sodium ethyl-mercury thiosalicylate and mg. sodium azide are dissolved in 1 20 ml. water.

This solution is placed in a homogeniser . Subsequently, with the motor running, the e is allowed to flow in a solution of mg. a-tocopherol acetate and mg. ascorbyl palmitate in m. olive oil.

According to a modification of this procedure, to the described emulsion there are also added U mg. sodium desoxycholate or of sodium taurocholate .

An indicator solution, prepared by dissolving 1 g. m-cresol purple, optionally together with 100 mg. methylene blue, in 1 00 ml. 0.03N sodium hydroxide solution, is mixed with the above-described emulsion in a volume ratio of 1 : 1 0 so that, for example, for every 2 ml. of emulsion, there are present 0.2 ml. of indicator solution in the' mixture.

The reagent prepared in this manner is used in a manner analogous to that described in Examples 1 - k - Example 6. 3 g. gum arabic ml. sodium iodoacetate 1 g. magnesium montmorillonite or of dimethy 1-di- octadecyl-ammonium montmorillonite and 0.5 . a ar are dissolved or slurried in 100 ml. water. The solution is then homogenised in a mixer with 25 ml. purified triolein. 100 parts by volume of the emulsion thus prepared are mixed with one part by volume of a solution obtained from 1 g. m-cresol purple and 100 ml. 0.05H sodium hydroxide solution.

The reagent prepared in this manner is used in a manner analogous to that described in Examples 1 to h.

The following Examples are given for the purpose of illustrating the process according to the present invention: -Example 7. 0.1 mlN of serum is added to a cuvette containing 1 ml. of the emulsion prepared according to Example .

The mixture is then shaken up. Subsequently, the resultant colour shade is compared with a calibrated colour scale. The number related to the colour shade, for example 50, is read off. Thereafter, the sample is left to stand for one hour at ambient temperature and, by comparison of the resultant colour shade ?/ith the same one on the calibrated colour scale, there is obtained the number 350. The lipase content of the measured sample is thus 200 mt/ml.

The same result is obtained when using the emulsion ready for use described in Example 3.

In a repetition of the above-described measurements, the samples, after determination of the initial colour shade, are left to stand for 20 minutes at 37°G. The . lipase content hereby determined is the same as that obtained above.

In a serial investigation, 0.1 ml. amounts of serum are mixed with 1 ml. amounts ox" the emulsions prepared according to Examples 1 and 3, and thereafter left to stand for one hour. In the majority of the samples, the brownish-violet or brownish-blue colour shade is scarcely changed in comparison with the initial colour. The sera with a pathologically increased lipase content show a brown to yellow or a dirty green to yellow colour. The exact lipase content can be determined in these pathological sera in the manner described in Example 7· Example 9- 0.2 ml. of the indicator solution prepared in the manner described in Example 2 are added to 2 ml. of the emulsion also described in Example 2. Subsequently, they are mixed, the pH indicator normally showing the desired colour (brownish-violet ) . If the pH of the emulsion, for example as a result of a comparatively long period of storage, no longer has the desired ρΗ value, then a few drops of 0.02N sodium hydroxide solution or of hydrochloric acid are added thereto until the desired brownish-violet colour shade is reached.

In an analogous manner, there is prepared an emulsion ready to use with the indicator according to Example - The possibly necessary correction of the pH of an emulsion which has been stored for a coraparatively long period of time is carried out by the addition of 0.02N sodium hydroxide solution or of hydrochloric acid until a blue colour shade is reached.

To the emulsions ready for use, tnere are then added 0.2 ml. amounts of serum. After shaking, the lipas contents are measured in the manner described in Examples 7 and 8.

Claims (9)

1. What we claim is :- 1. Agent for the determination of lipase in body fluids comprising an aqueous emulsion of fat or oil and a pH colour indicator, wherein the colour indicator is m-cresol purple and/or p-xyl^olsul hophthalei and/or dibromothymol-sulphophthalein and/or thymolsulphophthalein and/or a-naphtholphthalein. 2. Agent according to claim 1 , wherein the colour indicator is m-cresol purple or £-xylenolsulphophthalein. 3. Agent according to claim 1 or 2, wherein the indicator is present in a concentration of about 0.001 to 1 , referred to the total weight of the emulsion. k* Agent according to claim 3, where n the indicator is present in* a concentration of 0.05 to 0.2;.., referred to the total weight of the emulsion. 5. . Agent according' to any of the preceding claims, -wherein at least one additional colour indicator and/or coloured compound is present. 6. Agent according to claim 5> wherein said additional colour indicator and/or coloured compound is present in an amount of up to 0.05/5, referred to the emulsion. 7. Agent according to any of the preceding claims, wherein the fat or oil used is a natural or synthetic fatty acid diglyceride or triglyceride. 8. Agent according to claim 7, wherein olive oil is used as the oil. 9. Agent according to any of the preceding claims, wherein at least one emulsion-stabilising additive is present . 10. , Agent according to claim 9, wherein the emulsion- stabilising additive used is one wit a protective colloidal action. 11. Agent accoz'ding to claim 10, wherein the additive with a protective colloidal action is gum araoic.

2. Agent according- to claim 9 or 10, wherein the additive with a protective colloidal action is present in a concentration of about 0.5 to 30^ .'· . 1

3. Agent according to claim 12, wherein the additive with a protective colloidal action is present in a concentration of 10 to 25 . Ί 2-4- . Agent according to any of the preceding claims, wherein at least one emulsif ier is present . 15. Agent according to claim 11+, wherein the enmlsifier is a polyethylene glycol fatty alcohol ether, a polyethylene glycol alkyl-phenol ether, a sulphonic acid salt ov a sulphuric acid ester. 16. Agent according to claim 11+ or 15» wherein the enmlsifier is present in a concentration of up to 20%. 17. Agent according to claim 16, wherein the emulsifier is present in a concentration of up to 1 /.' . 16. Agent according to any of the preceding claims, wherein there is present at least one additive which stabilises the emulsion and also activates the lipase: 19. Agent according to claim 18, wherein said additive is a salt of a bile acid. 20. Agent according to claim 18 or , wherein said additive is present in a concentration of up to about 2%. 21. Agent according to claim 20, wherein said additive is present in a concentration of up to 0.5;ΰ. 22. Agent according to any of the preceding claims, wherein an anti-precipitation substance is present. 23. Agent according to claim 22 , wherein the anti-precipitation agent is a gelling agent based on montmorill-onite . 21+. Agent according to claim 22 or 2.3? w e e n the anti-precipitation agent is present in a concentration of up to about 3i. 25. Agent according to claim 21+, wherein the anti-precipitation agent is present in a concentration of up to i> . 26 . Agent according to any of the preceding claims, wherein there is present at least one emulsion-stabilising substance which increases the viscosity of the emulsions at lower temperatures . 27. Agent? according to claim 26 , wherein said substance is agar. 28. Agent according to any of the preceding claims, wherein a reducing compound is present. 29. Agent according to claim 28, wherein the reducing compound is present in a concentration of up to 30. Agent according to claim 29, wherein the reducing compound is present in a concentration of up to 0.05, .' · 31. Agent according to any of the preceding claims , wherein at least one biostatic compound is present. 32. Agent according to claim 31 J wherein he biostatic compound is an organo-mercury compound, a jD-hydroxy-benzoic acid ester, hexamethylene tetramine, an iodoacetate or an azide . 33. Agent according to claim 31 or 32 , wherein the biostatic compound is present in a concentration of up to about 1 ;o . 3

4. Agent according to claim 33 , wherein the biostatic compound is present in a concentration of up to about 3

5. . Agent according to any of the preceding claims, whenever in a container in an amount sufficient for one lipase determination. 3

6. . Agent according to claim 35 , wherein said container is a synthetic resin cuvette , 37 · Agent according to claim 35 or 6 , wherein the content of the container is covered with a film- orming coating . 38. Agent according to claim 37 ? wherein the film-forming coating is applied in the form of a solution in a volent sol♦vent or mixture of solvents. 39. Agent according to claim 37 or 3<3 , wherein the film-forming coating comprises a natural, synthetic or semi-synthetic polymer. O . Agent according to claim 39 , wherein the polymer is a polymeric ethylene derivative, a cellulose derivative, an epoxy lacquer, a two-component urethane lacquer or a urethane lacquer which hardens under the influence of moisture. i+1. Agent according to claim ho , wherein the polymer is collodium. 42. Agent according to any of claims 37 - 4-1 , wherein the coating has a thickness of up to 3 mm. 43. Agent according to claim 1+2 , wherein the coating has a thieloiess of about 0.5 mm. 44. Agent according to claim 1 for the dete mina on of lipase in body fluids, substantially as hereinbefore described and exemplified. 5. Process for the determination of lipase with the use of an aqueous emulsion of fat or oil and a pll colour indicator, wherein a sample to be investigated is added to the agent according to any of claims 1 to 5 and the colour change occurring during the incubation period is determined. 6. Process according to claim 45, wherein the colour change is measured by comparison with a calibrated colour scale or with a calibrated colour strip. 4

7. Process according to claim 45, wherein the colour change is determined by comparison of several samples with one another. 4

8. Process according to any of claims 45 - 47, wherein the sample is mixed with the agent, the colour is then determined immediately and then, after a period of incubation at a standard temperature, the colour is again determined, the lipase content of the sample being assessed from the colour change which has occurred. 4

9. Process according to claim 45 for the determination of lipase, substantially as hereinbefore described and exemplified.