IE53472B1

IE53472B1 - Process and reagent for the determination of the activity of chymotrypsin or trypsin in faeces

Info

Publication number: IE53472B1
Application number: IE2146/82A
Authority: IE
Original assignee: Boehringer Mannheim Gmbh
Priority date: 1981-10-06
Filing date: 1982-09-02
Publication date: 1988-11-23
Also published as: ES516124A0; ATE12521T1; EP0076506A1; AU534001B2; CA1191772A; SU1367866A3; EP0076506B1; DK156225C; JPS58111699A; DK156225B; AU8835082A; DK434082A; ES8306389A1; JPS6036758B2; IE822146L; DD203777A5; HU187416B; DE3262892D1; DE3139719A1

Abstract

1. Process for the determination of the activity of chymotrypsin or trypsin in faeces by measurement of the velocity of the splitting of a suitable substrate by a faecal suspension in aqueous or aqueous-organic medium, characterised in that one suspends the faeces in the presence of a surface-active agent.

Description

The present invention is concerned with a process and a reagent for the determination of the activity of chymotrypsin and. trypsin in faeces.

In the case of a suspicion of chronic pancreatitis 5 and also of mucoviscidosis in neonates, the diagnosis of pancreatic insufficiency is an important parameter, the clinical-chemical detection of which has hitherto not been satisfactorily accomplished. The most dependable test for this purpose is generally regarded as being the pancreozymin secretin test. In this case, after stimulation by pancreozymin and secretin, the pancreatic secretion is collected by means of a probe and subsequently investigated for the following parameters: volume, bicarbonate concentration and the activities of amylase, lipase, trypsin and chymotrypsin. The chief disadvantage of these methods is the large amount of time and technical expense, as well as the great degree of discomfort for the patients.

Another method, not only as an investigating test but also for monitoring control in cases of chronic pancreatitis and of mucoviscidosis is the determination of chymotrypsin and trypsin in the faeces. In cases of chronic pancreatitis and of mucoviscidosis, the activity values of trypsin and chymotrypsin are lowered, the chymotrypsin values thereby having proved to be the better parameter. For the determination of chymotrypsin or trypsin in faeces, a faecal suspension is -3mixed with an aqueous methanolic solution of a specific substrate and the amino acid liberated in a given period of time is determined. The determination of the liberated amino acid can be carried out, for exanple, by tritration with an aqueous lye, especially an aqueous solution of sodium hydroxide (pH-stat process, Haverback et al.. Gastroenterology, 44, 588-597/1963; Ammann, Fortschritte in der Pankreasfunktionsdiagnostik, pub. Springer Verlag, Berlin, Heidelberg, New York, 1967) or the time is measured which leads to the lowering of the pH value of the solution by 0.1 unit (pH-drop process, cf. Robinson, Smith and Elliott, Clin. Chim. Acta., 62, 225-229/1975).

However, all known methods for the determination of the activity of chymotrypsin or trypsin in faeces possess the disadvantage of a high expenditure of apparatus and time. A rapid and simple determination of the activity by photometric means has hitherto not been possible: for a reasonably exact determination in the case of the use of a very small sample, the measurement values to be determined are too small and, in the case of a larger sample, the inherent colour and turbidity thereof due to suspension particles is too great. A photometric determination after centrifuging the faecal suspension is not possible because the enzyme is bound relatively firmly to the faecal particles and, after centrifuging, is to be found -4almost completely in the sediment or is only present incompletely and in a non-reproducible amount in solution.

Therefore, it is an object of the present invention to provide a process for the rapid, simple, exact and readily reproducible determination of the activity of chymotrypsin or trypsin in faeces.

Thus, according to the present invention, there is provided a process for the determination of the activity of chymotrypsin or trypsin in faeces which comprises measuring the rate of fission of an appropriate substrate by a faecal suspension in an aqueous or aqueous organic medium, the faeces being suspended in the presence of a surface-active agent.

The measurement of the rate of fission of the substrate can take place by one of the methods known from the literature, for example by titration of the liberated amino acid by means of a lye (pH-stat process).

We have found that when carrying out the process according to the present invention in the presence of a surface-active agent, in general more than 90% of the enzyme activity is solubilised, the rate of reaction of the substrate fission is considerably increased and the apparent Km value of the substrate is lowered (activation factor about 2 to 10), the problems whioh have previously arisen (especially binding of the 4 7 2 -5enzyme to the faecal particles and surprisingly high Km values for untreated faecal samples in comparison with crystalline enzyme in the case of particular substrates) thereby being overcome. In particular, it is also possible to measure the fission of the substrate by photometric means quickly, simply, exactly and with the use of only a small amount of substrate.

The results obtained with the process according to the present invention in the presence of a surface-active agent are especially surprising because, in the case of pure α-chymotrypsin from bovine pancreas, practically no difference can be ascertained when carrying out the determination with or without the use of a surfaceactive agent.

The surface-active agent used can, in principle, be any appropriate tenside, such as an anionic or anpholytic tenside and preferably a non-ionic tenside and especially a cationic tenside.

Anionic tensides which can be used include alkanesulphonates, olefin-sulphonates, for exanple cumenesulphonate, ester sulphonates, alkylarylsulphonates of the dodecylbenzenesulphonate type, alkylnaphthalenesulphonates, alkyl sulphates, for example sodium lauryl sulphate, ether sulphates and fatty alcohol sulphates and salts of fetty acids and of bile acids? anpholytic tensides are those with anion-active and cation-active hydrophilic groups, for example glycerol derivatives -6with a betaine structure, sulphobetaines and lecithins; non-ionic tensides include, for example, polyethers, especially alkylphenol polyglycol ethers and other ethoxylation products of fatty acids, fatty acid amides, fatty amines and fatty alcohols, for example ethoxylated lauryl alcohol, polymers of propylene and ethylene oxide, polyoxyethylene alkyl ethers and nonylphenyl ethers, polyoxyethylene sorbitan monooleate and laurate, addition products of propylene oxide/ethylenediamine/ ethylene oxide, amine oxides and fatty acid esters of polyalcohols, tallow alcohol polyglycol ethers; cationic tensides include, for example, straight-chained and cyclic ammonium compounds, for example N-cetyl-N-ethyl morpholine methosulphate, benzalkonium chlorides and other quaternary ammonium salts, amine salts, pyridinium salts and quaternary fatty amine polyglycol ethers.

The choice of the most appropriate tenside also depends upon the other reaction conditions, especially upon the nature of the enzyme and substrate, upon the nature and concentration of the salts and also upon the pH value of the medium.

Of the cationic detergents, which are especially preferred for the process according to the present invention, the strongest activation effect is displayed by quaternary ammonium compounds and preferably those of the general formula RjRjNCCH^Jj. wherein Rj is preferably an alkyl radical containing 3 to 14 carbon -7atoms and especially a lauryl or cetyl radical and R2 is preferably a lower alkyl radical containing up to 5 carbon atoms or an aralkyl radical or also a hydroxyalkyl radical and especially a benzyl or methyl radical? and alkylpyridinium salts with preferably 12 to 18 carbon atoms in the alkyl radical, for example lauryl pyridinium chloride, lauryl pyridinium disulphate and especially hexadecyl pyridinium chloride. A tenside which is especially preferred for the process according to the present invention is lauryl trimethyl ammonium chloride.

The concentration of the surface-active agent in the homogenising solution used for the suspension of the faeces is, in general, about 0.02 to 10% by weight and preferably 0.5 to 5% by weight. The concentration of the surface-active agent in the measurement solution (faecal suspension and substrate solution) should preferably be about 0.0005 to 0.5 and more preferably 0.01 to 0.3% by weight. The concentration of the faecal sample in the suspension is, for example, 0.2 to 2% when using Succ-Ala-Ala-Pro -Phe-pNA as substrate.

In addition to the surface-active agent, the homogenising solution used for the suspension of the faeces preferably also contains one or more watersoluble salts, for example alkali metal and/or alkaline earth metal chlorides or sulphates. A content of sodium 4 7 2 -8chloride of 100 to 1000 mmole/litre or a content of calcium chloride of 20 to 500 mmole/litre or a combination of both salts in the given concentration ranges has proved to be especially useful. However, organic salts can also be used, for example acetates or citrates, as well as salts of other cations. The salts are preferably present in a concentration which corresponds to an ionic strength of 20 to 1000 mval/litre.

Surprisingly, we have ascertained that by means of a homogenising solution containing a surface-active agent and salts (ionic strength), a superadditive increase of the enzyme activity (increase of the rate of reaction) takes place, i.e. the increase is greater than the sum of the values obtained in the presence of surface-active agent alone or in the presence of salts alone. Furthermore, only the combination of salt and detergent, in contradistinction to the individual components, gives a constantly high and representative portion of the particle-bound enzyme in solution.

As substrate, there can, in general, be used all appropriate substrates known for the determination of the activity of chymotrypsin or trypsin in faeces by previously known methods (cf. for exanple, Grossman, Proo. Soc. Biol. Med., 110. 41/1951; Del Mar et al., Anal. Biochem., 99., 315/1979; Hakaiama et al., J. Biol Chem., 254(10). 4027-4032/1979). Especially preferred for the determination of chymotrypsin, particularly -9 · with regard to water-solubility, stability, Rn value and velocity constants, is Ala-Ala-Phe-pNA and especially Succ-Ala-Ala-Pro-Phe-pNA and MeO-Succ-Arg-ProTyr-pNA; these substrates are particularly useful for photometric determinations. A substrate which is especially preferred for the determination of trypsin activity is, for exanple, ChronDzyme TRY (Trade Mark, carbobenzoxyvalyl-glycyl-arginine-p-nitroanilide acetate).

For the preparation of the reagent solution, the substrate is dissolved in water or in a mixture of water and an organic solvent. The organic solvent thereby serves as a solubiliser for the substrate and can be, for exanple, acetonitrile, dimethylsulphoxide, acetone or methanol. The reagent solution preferably also contains the same salts as are used for the homogenising solution; the concentration of these salts in the reagent solution is, in general, lower than the concentration in the homogenising solution; the total salt concentration is preferably about 10 to 500 mmole/ litre, for example 250 mmole/litre of sodium chloride and 20 mmole/litre of calcium chloride.

For carrying out the measurement, a definite amount of a suspension of a faecal sample in the homogenising solution or of the supernatant solution obtained by centrifuging until clear is added to a definite amount of the reagent solution (for example 100 /tl. of sanple suspension to 2 ml. of reagent 3 4 7 2 -10solution) and the rate of fission ia determined, for example titrimetrieally or photometrically. The amount of sample suspension (sample dilution) for the achievement of readily measurable values, for exanple readily measurable extinction increase/minute, thereby depends especially upon the enzyme activity to be expected. On the basis of the increase of sensitivity by means of the use of the detergent, the amount of sample used can be so small that the inherent absorption of the suspended solid bodies is low and thus a photometric measurement with the suspension only then becomes possible. The pH value of the homogenising solution is not especially criticalj in general, it is from 3 to 10 and preferably from 6 to 8. The pH value of the mixture of sample suspension and reagent solution is generally from 8 to 10 and preferably 9; these values are preferably adjusted via the pH value of the reagent solution. It can also be advantageous to add to the solutions, for the adjustment of the pH value, an appropriate buffer mixture, for example tris buffer, glycine buffer or glycylglycine buffer. In general, the buffer concentration is from 10 to 1000 mmole/litre and especially 50 to 200 mmole/litre.

If only very small amounts of chymotrypsin are present, such as occur, for example, when measuring devices are used which only permit the picking up of the amount of chymotrypsin present in a surface area, -11then it is expedient to employ an especially sensitive detection reaction. Such a sensitive reaction is described, for exanple, in J. Biol. Chem., 128. 537/ 1939 and is known as the Bratton/Marshall reaction.

It depends upon the addition of sodium nitrite and Ν-α-naphthylethylenediamine and the subsequent addition of acid. The acid used is preferably trichloroacetic acid or an acid of comparable strength.

The measurement can be carried out manually or with an automatic analysis apparatus. For the photometric determination of activity and especially for the automatic photometric measurement (determination of the extinction), it is preferable to centrifuge the sample suspension until clear before carrying out the measurement.

The device described in Federal Republic of Germany Patent Specification No. 31 39 702.6 entitled Vessel for handling pasty sample material has proved to be especially useful for carrying out the process according to the present invention. With the process according to the present invention, a simple and aesthetic sampling, sample measurement, working up and measurement of enzyme activity is possible, without discomfort either to the patient or to the laboratory personnel.

The present invention also provides a reagent for carrying out the process, which comprises a surfaceactive agent, an enzyme substrate and an aqueous salt 3 4 7 2 -12solution, the reagent having a pH value of from 7 to 11.

A preferred reagent according to the present invention contains the following componentss 0.02 to 10% by weight surface-active agent, 0.05 to 5 mmole/litre enzyme substrate, to 1000 mval/litre of a water-soluble salt and 10 to 1000 mmole/litre of a buffer (pH 7 to 11).

A more preferred reagent according to the present invention contains the following components! 0.5 to 5% by weight of surface-active agent, 0.2 to 2 mmole/litre of substrate, 100 to 1000 mmole/litre of sodium chloride and/or 20 to 500 mmole/litre of calcium chloride and 50 to 200 mmole/litre tris buffer (pH 8 to 10).

The following Examples are given for the purpose of illustrating the present invention; if not stated otherwise, the percentages are by weight; Example 1. a) Preparation of a homocrenisincr solution; sodium chloride 2.9 g. (500 mmole/1.) calcium chloride 1.1 g. (100 mmole/1.) 33% lauryl trimethyl ammonium chloride 2.0 g. (0,7%) distilled water ad 100 ml. b) Preparation of a reagent solution; Succ-Ala-Ala-Pro-Phe-pHA 29.5 mg. (0.5 mmole/1.) 1.46 g. (250 mmole/1.) sodium chloride 3 4 7 2 -13caloium chloride 222 mg. (20 mmole/1.) buffer tris/HCl (pH 9.0) ad 100 ml. (60 mmole/1.) c) Homogenisation of the sample; About 100 mg. of faecal sample are mixed with a 100 fold amount by weight of homogenisation solution and worked up in an appropriate apparatus to give a fine suspension. d) Carrying out of the measurement; Into a 1 cm. measurement cuvette there are pipetted 2 ml. of the reagent solution which is warmed to 25°C. and mixed with 100 p.1. of the sample homogenate. After brief mixing, the increase of the extinction/minute is determined at 405 nm. For calculating the enzyme activity/g. of faeces, the extinction increase/minute at 405 nm is multiplied by the factor 212.

Exanple 2.

The preparation of the homogenising solution and of the reagent solution is carried out as in Exanple 1, as well as the homogenisation of the sample. Subsequent to the homogenisation, the suspension is centrifuged until the solution is clear. An aliquot is taken from the centrifuge supernatant and the enzyme activity therein is determined either manually as in Exanple 1 or with the use of an automatic analysis device.

Example 3. a) Preparation of the homogenising solution; -14An aqueous solution is prepared with the following components: tris/HCl buffer (pH 9.0) 60 mmole/litre sodium chloride 250 mmole/litre calcium chloride 20 mmole/litre lauryl trimethyl ammonium chloride 0,7% b) Preparation of the reagent solution: The reagent solution is prepared in the manner described in Example 1. c) Homogenisation of the sample: This is carried out as described in Example 1. d) Carrying out of the measurement: This is also carried out as described in Example 1.

Example 4. (comparative) Example 4 corresponds to Example 3 with the sole difference that the homogenising solution does not contain detergent (lauryl trimethyl ammonium chloride).

The results of a comparative measurement of identical samples with a reagent according to Example 4 and one according to Example 3 are given in the following Table: sample No. without detergent (Example 4) mE/min. with detergent (Example 3) mE/min. 1 31 181 2 S 68 3 6 42 4 8 68 5 20 159 3 4 7» -15Example 5. a) Preparation of the homogenising aolutlon: This is carried out as described in Exanple 1. b) Preparation of the reagent solution: An aqueous solution is prepared containing the following components: Ala-Ala-Phe-2-nitroanilide 2 mmole/litre sodium chloride 250 mmole/litre calcium chloride 20 mmole/litre tris/HCl buffer (pH 9.0) 60 mmole/litre c) Homogenisation of the sample: 200 mg. of faecal sample are mixed with 10 ml. homogenising solution and worked up in an appropriate apparatus to give a fine suspension. d) Carrying out of the measurement: ml. of the reagent solution is pipetted into a 1 cm. cuvette, warmed to 25°C. and mixed with 200 jxl. of the sample homogenate. After briefly mixing, the increase of the extinction is measured at 405 nm. Example 6. (comparative) Example 6 corresponds to Example 5 with the sole difference that the homogenising solution does not contain detergent. The results obtained with an identical sample according to Examples 5 and 6 are as follows: without detergent (Example 6) mE/min. with detergent (Example 5) mE/min. 31 45 -16Bxample 7. a) Preparation of the homogenising solution; This is carried out as described in Example 1. b) Preparation of the reagent solution: An aqueous solution is prepared with the following components: 3-carbomethoxypropionyl-L-arginyl-L-prolylL-tyrosine p-nitroanilide hydrochloride 0.5 mmole/litre sodium chloride 250 mmole/litre calcium chloride 20 mmole/litre tris/HCl buffer (pH 9.0) 60 mmole/litre c) Homogenisation and measurement take place in the manner described in Example 1.

Example 8.(comparative) Example 8 corresponds to Example 7 with the sole difference that the homogenising solution does not contain any detergent. The results of the measurement of an identical sample according to Examples 7 and 8 are as follows: without detergent (Example 8) mS/min. with detergent (Example 7) mE/min. 19 148 Example 9.

Determination of trypsin. a) Preparation of the homogenising solution: This is carried out as described in Example 1. 3 4 7 2 -17b) Preparation of the reagent solution: An aqueous solution is prepared containing the following components: carbobenzoxy-Ii-valyl-L-glycyl-L-arginine jj-nitroanilide acetate mmole/litre 250 mmole/litre 20 mmole/litre 60 mmole/litre sodium chloride calcium chloride tris/HCl buffer (pH 9.0) c) Homogenisation and d) measurement take place in the manner described in Example 1.

Example 10. (comparative) Example 10 corresponds to Example 9 with the sole difference that the homogenisation solution does not contain any detergent. The results of the measurement with an identical sample according to Examples 9 and 10 15 are as follows: without detergent (Example 10) mB/min. with detergent (Example 9) mE/min. about 3 8 Example 11.

An aliquot of a faecal sample is measured by means of an appropriate device in such a manner that the sample is present in a chamber which is bounded on one side by a filter paper or the like. For this purpose, there can be used, for example, various commercially available devices 5347S -1810 for the determination, of blood in faeces., such as Hemo FEC (Trade Mark) or Feca-Nostic (Trade Mark), after exchange of the appropriate filter paper.

The filter paper is sprinkled with 50 «,1. of an aqueous solubilising solution consisting of: lauryl trimethyl ammonium chloride 50 g./litre (550) sodium chloride 500 mmole/litre calcium chloride 100 mmole/litre the chymotrypsin of a boundary surface thereby being solubilised and taken up by the filter paper. Subsequently, the filter paper is sprinkled with 50 ^.1. of a reagent solution of the following composition: Succ-Ala-Ala-Pro-Phe-jaNA H-α-naphthylethylenediamin e sodium nitrite tris/HCl buffer (pH 9.0) mmole/litre 5 g./litre 1 g./litre 100 mmole/litre After a reaction time of 5 minutes, a drop of trichloroacetic acid (3.2 mmole/litre) is applied to the paper. When chymotrypsin is present in the sample, an intense violet coloration is formed, the extent of which depends upon the amount of enzyme.

Example 12. (comparative) Example 11 is repeated but with the sole difference that the solution used for the solubilisation of the enzyme does not contain any detergent. No visible coloration develops.

Claims

1. CLAIMS :1. A process for the determination of the activity of chynotxypsin or trypsin in faeces viiich comprises measuring the rate of fission of an appropriate substrate by a faecal suspension in an aqueous or aqueous-organic medium, the faeces being suspended in the presence of a surfaceactive agent.

2. A process according to claim 1, wherein the amino acid acid liberated bv the fission of a substrate is measured.

3. A process according to claim 2, wherein the liberated amino acid is determined by titration with a base.

4. A process according to claim 1, wherein the rate of fission of the substrate is determined photometrically.

5. A process according to any of the preceding claims, wherein the measurement is carried out with a faecal suspension or with the supernatant obtained by centrifuging the suspension.

6. A process according to any of the preceding claims, wherein the determination is carried out manually or with an automatic analysis apparatus.

7. A process according to any of the preceding claims, wherein a cationic or non-ionic tenside is used as the surface-active agent.

8. A process according to claim 7, wherein the surface-active agent is a quaternary ammonium salt or an alkyl-pyridinium salt.

9. A process according to claim 8, wherein the quaternary ammonium salt is lauryl trimethyl ammonium chloride. 5 3 4 7 2 -2010. A process according to any of the preceding claims, wherein the concentration of the surface-active agent in the homogenising solution used for the suspension of the faeces is about 0.02 to 10% by 5 weight. 11. A process according to claim 10, wherein the concentration of the surface-active agent in the homogenising solution used for the suspension of the faeces is 0.5 to 5% by weight.

10. 12. A process according to any of the preceding claims, wherein the homogenising solution used for the suspension of the faeces contains, in addition to the surface-active agent, one or more water-soluble salts. 15 13. A process according to claim 12, wherein the watersoluble salt present in the homogenising solution is a sodium salt and/or a calcium salt.

11. 14. A process according to claim 12 or 13, wherein the ionic strength of the salts is from 20 to 1000 mval/ 20 litre.

12. 15. A process according to any of the preceding claims, wherein chymotrypsin is determined.

13. 16. A process according to claim 15, wherein the substrate used is Ala-Ala-Phe-pNA, Succ-Ala-Ala-Pro-Phe25 pNA or MeO-Succ-Arg-Pro-Tyr-joNA.

14. 17. A process according to any of claims 1 to 14, wherein trypsin is determined. -2118. A process according to claim 17, wherein the substrate used is carbobenzoxy-Val-Gly-Arg-jaNA acetate.

15. 19. A process according to claim 1 for the determination of the activity of chymotrypsin or trypsin in faeces, substantially as hereinbefore described and exemplified.

16. 20. A reagent for carrying out the process according to any of claims 1 to 19, comprising a surface-active agent, an enzyme substrate and an aqueous salt solution, said reagent having a pH value of from 7 to 11.

17. 21. a reagent according to claim 20, vhich contains the following components: 0.02 to 10% by weight surface-active agent, 0.05 to 5 mmole/litre enzyme substrate, 20 to 1000 mval/litre of a water-soluble salt and 10 to 1000 mmole/litre of a buffer (pH 7 to 11).

18. 22. A reagent according to claim 20, which contains the following components: 0.5 to 5% by weight of surface-active agent, 0.2 to 2 mmole/litre of substrate, 100 to 1000 mmole/litre of sodium chloride and/or 20 to 500 mmole/litre of calcium chloride and 50 to 200 mmole/litre tris buffer (pH 8 to 10).

19. 23. A reagent according to claim 20, substantially as hereinbefore described and exemplified.