DD203777A5 - PROCESS FOR DETERMINING THE ACTIVITY OF CHYMOTRYPSINE OR TRYPSINE IN THE CHAIR - Google Patents

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

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Method for determining the activity of chymotrypsin or trypsin in stool

Field of application of the invention

The invention relates to a novel method for determining the activity of chymotrypsin or trypsin in the stool, which contribute to the diagnosis of pancreatic insufficiency.

Well-known technical solutions

In suspected chronic pancreatitis as well as on cystic fibrosis in infants, the diagnosis of pancreatic insufficiency is an important parameter whose clinical chemical evidence has not yet been satisfactorily resolved. The most reliable method is generally considered to be the pancreozymin-secretin test. After stimulation by pancreozymin and secretin, the pancreatic secretions are collected by means of a probe and then tested for the following parameters. Volume, bicarbonate concentration activities of amylase, lipase, trypsin and chymotrypsin. The main disadvantage of this method is the high temporal and technical complexity and the great annoyance for the patient «

Another method, both as a screening test and for follow-up: chronic, pancreatitis and. cystic fibrosis, the determination of chymotrypsin or trypsin in stool. In the case of chronic pancreatitis and cystic fibrosis, the activity levels of trypsin and chymotrypsin are decreased, with chymotrypsin levels proving to be the better parameters. "To determine chymotrypsin or trypsin in stool, a stool suspension is treated with an aqueous methanolic solution of a specific substrate the amino acid released in the time unit determines »the determination of the released amino acid

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can ζ. Β. by titration by means of lye, in particular sodium hydroxide solution, (pH-stat method, Haverback et al., Gastroenterologe 44 (1963) 588-597; Ammann, Port steps in pancreatic function diagnostics, Springer Verlag, Berlin, Heidelberg, New York (1) 967)), or the time is measured which leads to a decrease in the pH of the solution by 0.1 unit (pH drop method, see Robinson, Smith, Elliott, Clin Chim, Acta 62 (1975) , 225-229) ·

However, all known methods for determining the activity of chymotrypsin or trypsin in the stool have the disadvantage of a high expenditure on equipment and time. A rapid and simple determination of the activity by photometric means has not been possible hitherto: for a reasonably accurate determination, the measured quantities to be determined are too small for very small sample use, and for higher sample use the inherent coloration and turbidity due to suspension particles is too strong Determination after centrifugation of the stool suspension is therefore not possible because the enzyme is relatively firmly bound to the stool particles and is almost completely in the sediment after centrifugation or is incomplete and not reproducible in solution.

Object of the invention

It is an object of the invention to avoid the disadvantages described.

reading the invention

The object of the invention is therefore to provide a method for rapid, simple, accurate and well reproducible determination of the activity of chymotrypsin or trypsin

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in the chair under changed process conditions ·

The invention relates to a method for determining the activity of chymotrypsin or trypsin in the stool by measuring the rate of cleavage of a suitable substrate by a stool suspension in aqueous or aqueous-organic medium, characterized in that one suspen the chair in the presence of a surfactant. diert.

The measurement of the rate of cleavage of the substrate can be carried out according to one of the methods known from the prior art, such as. By titration of the liberated amino acid by means of lye (pH-stat method) ° It has been found that in the inventive implementation of the method in the presence of a surfactant in .general more than 90 % of the _f ..Enzymaktivität solubilized, the reaction rate of substrate cleavage is significantly increased, and the apparent Km of the substrate is decreased (activation factor about 2 to 10), thereby alleviating the problems hitherto encountered (especially binding of the enzyme to the stool particles and surprisingly large Km values for untreated stool samples compared to crystalline enzyme in certain substrates). In particular, this makes it possible 'to measure the speed of cleavage of the substrate also by a photochemical means quickly, easily, accurately and with little use of the substrate. The results obtained by the method according to the invention in the presence of a surface active agent are also surprising in particular because with pure c *, chymotrypsin from bovine pancreas practically no difference was observed in carrying out the method of determination in or without the presence of a surface-active agent

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In principle, any suitable surfactant, such as anionic or ampholytic surfactants, and preferably nonionic surfactants, and in particular cationic surfactants, may be used as the surface-active agent.

Anionic surfactants are, for example, alkanesulfonates, olefinsulfonates, tin, example, cumene sulfonate, ester sulfonates, alkylarylsulfonates, in particular alkylbenzenesulfonates of the dodecylbenzenesulfonate type and alkylnaphthalenesulfonates, alkylsulfates, such as sodium lauryl sulfate, ether sulfates or fatty alcohol sulfates, salts of fatty acids and cholic acids; ampholytic surfactants are those having anionic and cationic hydrophilic groups, such as betaine-based glycerol derivatives, sulfobetaines, and lecithins; Nonionic surfactants are, for example, polyethers, in particular alkylphenol polyglycol ethers and other products of atoxylation of fatty acids, fatty acid amides, fatty amines and fatty alcohols, for example ethoxylated lauryl alcohol, polymers of propylene and ethylene oxide, polyoxyethylen len alkyl ether or nonylphenyl ether, polyoxyethylene sorbitan mono oleate or laureate, addition products of propylene oxide / Äthylendiamiη / ethylene oxide, amine oxides and fatty acid esters of polyalcohols, tallow-alcohol polyglycol ethers cationic surfactants are, for example, straight-chain and cyclic ammonium compounds, eg. As U-cetyl-N-ethyl-morpholine metosulfate, benzalkonium chlorides and other quaternary ammonium salts, amine salts, pyridinium salts or quaternary fatty amine polyglycol ether.

The choice of the most suitable surfactant is also dependent on the other reaction conditions, in particular on the type of enzyme and substrate, on the type and concentration of the salts, but also on the pH of the medium.

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Of the cationic detergents which are most suitable for the process according to the invention, the strongest activating effect is quaternary ammonium compounds, preferably of the formula H ₁ RpH (CHo) PJ where IL is preferably an alkyl radical having 8 to 14 carbon atoms, and especially lauryl and cetyl and Sp is preferably a lower alkyl radical having 1 to 5 carbon atoms or an aralkyl radical, or else a hydroxyalkyl radical, and in particular methyl or benzyl, and alkyl pyridinium salts having preferably 12 to 18 carbon atoms in the alkyl radical, such as Bo laurylpyridinium chloride, laurylpyridinium disulfate, and in particular hexadecylpyridinium chloride. A particularly suitable surfactant for the process according to the invention is lauryltrimethylammonium chloride.

As a substrate, in general, any of the prior art, for,., The determination, the. Activity. chymotrypsin or trypsin in the stool can be used as a suitably known substrate according to previous methods (see, for example, Grossman, Proc Soc., Biol. Med., VK) (1962); 41; Del Mar et al., Anal. Biochem. 9j3 (1979) 316; Nakajama et al., J. Biol. Chem. 254 (10), 1979, 4027-4032). Particularly suitable for chymotrypsin determination, especially with regard to water solubility, stability, Km value and rate constant, proved to be the Ala-Ala-Phe-pilA and in particular the Succ-Ala-Ala-Pro-Phe-plA and MeO -Succ-Arg-Pro-Tyr-pNA; In particular, they are very well suited for a photometric determination. A substrate which is well suited for the determination of the trypsin activity is, for example, B. Chromozyme TRY ^ (carbobenzoxyvalyl-glycyl-arginine-p-nitroanilide-acetate) <,

To prepare the reagent solution, the substrate is dissolved in water or a mixture of water and an organic solvent.

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It serves as a solubilizer for the substrate, and is ζ. The acetone solution also preferably contains the same salts as those used for the homologation solution, the concentration of these salts in the reagent solution being generally lower than the concentration in the homogenizing solution the total salt concentration is preferably about 10 to 500 mmol / l, for example 250 mmol / l sodium chloride and 20 mmol / l calcium chlorido

To carry out the measurement, a certain amount of a suspension of the stool sample in the homogenizing solution or the centrifugation solution obtained by centrifugation until clarification is added to a certain amount of the reagent solution (eg 100 μl sample suspension to 2 ml reagent solution) and the Speed of cleavage z. B. titrimetrisch or photometrically determined · The amount of sample suspension (sample dilution) is aimed to achieve good measurable values, eg. B. well measurable Extinktionsanstiege / minute, especially after the expected enzyme activity. Due to the sensitivity increase by use of the detergent, the amount of sample can be chosen so small that the intrinsic absorption of the suspended plague is low and thus a photometric measurement with the suspension is made possible. The pH of the homogenizing solution is not particularly critical; it is generally between 3 and 10, and more particularly between 6 and 8. The pH of the mixture of sample suspension and reagent solution is generally between 8 and 10, and preferably at pH = 9; These values are preferably adjusted via the pH of the reagent solution. It may also be advantageous to add to the solutions for adjusting the pH a suitable buffer, such as B. iris buffer, glycine buffer or

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GIycylglyein buffer. The buffer concentration is generally between 10 and 1000 mmol / l, in particular 50 to 200 mmol / l.

If there are very small amounts of chymotrypsin, as occur, for example, when dosing devices are used which allow only the amount of chymotrypsin present in an interface to be detected, it is expedient to use a particularly sensitive detection reaction. Such a sensitive reaction is described in J. Biol. Chem. 128 (1939) 537, and known as the Bratton / Marshall reaction. It is based on the addition of iTatriumnitrit and IT-Caphthylethylenediamine and 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 analyzer. For the photometric determination of the activity, and in particular for the automatic photometric measurement (determination of the extinction), it is expedient to centrifuge the sample suspension until clarification before the measurement.

The device described in the German patent application P.31 39 702.6 under the name "vessel for handling pasty sample material" of the Applicant has proved to be particularly useful for carrying out the process according to the invention. With the method according to the invention, a simple and aesthetic sampling, dosing, processing and measurement is possible, which means that neither patient nor laboratory personnel are loaded «

The following examples illustrate the invention without limiting it to them, '''iarni otherwise stated,

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percentages are by weight. example 1

a) Preparation of the homogenizing solution:

IaCl ί 2.9 g (= 500 mmol / l)

CaCl ₂ : 1.1 g (= 100 mmol / l)

33% ig Lauryl trimethyl ammonium chloride: 2.0 g (= 0.7 %) H ₂ O dest * -: ad 100 ml

b) Preparation of the reagent solution! Succ-Ala-Ala-Pro-Phe-pNA 29.5 mg (0.5 mmol / l)

IaCl 1.46 g (250 mmol / l)

CaCl ₂ "222 mg (20 mmol / l)

Buffer Tris / HCl, pH 9.0, 60 mmol / l ad 100 ml

c) Homogenization of the sample:

About 100 mg of stool sample are mixed with 100 times the amount by weight of HomogenisierIosung and in a suitable. Device processed into a fine suspension.

d) Carrying out the measurement:

In a 1 cm Meßkiivette 2 ml of the reagent solution are pipetted, heated to 25 ⁰ C and mixed with 100 ul of the sample homogenate. After a short mixing, the extinction increase / min. determined at 405 nm. To calculate the enzyme activity / g stool, the increase in absorbance / min. at 405 nm with the factor 212 to multiply.

Example 2

The preparation of the homogenizing solution and the reagent solution

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as in Example 1, as well as the homogenization of the sample »Following homogenization, the suspension is centrifuged until the solution is clarified. An aliquot was taken from the centrifugation stand and the enzyme activity was determined either manually as in Example 1 or using an automatic analyzer.

Example 3

a) Preparation of the homogenizing solution: An aqueous solution with the following constituents is prepared! Tris / HCl buffer, pH 9.0 60 mmol / l

UaCl 250 mmol / 1

CaCl ₂ 2 0 mmol / l

Lauryltrimethylammonium chloride 0.7 %

b) Preparation of the reagent solution:

The preparation of the reagent solution is carried out according to Example 1

c) Homogenization of the sample: carried out according to Example

d) carrying out the measurement: carried out in accordance with Example 4 (comparative example )

Example 4 corresponds to Example 3 with the only difference that the Eomogenisierlösung no detergent (Lauryltrimethyl-ammoniumchlcrid) contains The results of a comparative measurement of identical Froben with a reagent according to Example 3 and Example 4 are compared in the following <>

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Without detergent (Example 4) mU / min. 01/26/1983 31 AP G 01 N / 243 765 8th 61 362 12 Sample Erβ 6 With detergent (Example 3) mU / min. 1 8th 181 2 20 68 3 42 4 68 VJL 159 Example 5

a) The preparation of the homogenizing solution is carried out according to Example 1. ,

b) Preparation of the reagent solution: An aqueous solution is prepared having the following constituents: Ala-Ala-Phe-p-nitroanilide 2 mmol / l of IaCl 25 0 mmol / l

CaCl ₂ Tris / HCl buffer, pH 9.0

20 mmol / l 60 mmol / l

c) Homogenization of the sample: 200 ml of stool sample are mixed with 10 ml of homogenizing solution and processed with a suitable device into a fine suspension

d) Carrying out the measurement:

In a 1 cm Klivette 1 ml of the reagent solution are pipetted, heated to 25 ⁰ C and mixed with 200 ul sample homogenate. After a short mixing, the increase in absorbance at 405 nm is determined.

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Example 6 (Comparative Example)

Example 6 corresponds to Example 5 with the only difference that the homogenizing solution contains no detergent.

The result of an identical sample according to Example 5 and Example 6 is compared in the following:

Without detergent With detergent

(Example 6) (Example 5)

mE / min, mB / inin

31 45

Example 7

a) Preparation of Homogenisierlösimg carried out according to Example-1.

b) Preparation of the reagent solution: An aqueous solution is prepared having the following constituents:

3-carbomethoxypropionyl-L-arginyl-L-

prolyl-L-tyrosine p-nitroanoid hydrochloride 0.5 mmol / l

NaCl 250 mmol / 1

CaCl ₂ 20 mmol / l

Tris / HCl buffer, pH 9.0 6 0 mmol / l

c) homogenization and measurement are carried out according to Example 1 » Example 8 (Comparative Example )

Example 8 corresponds to Example 7 with the only difference that the homogenizing solution contains no detergent. The result of measuring an identical sample in accordance with

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Game 7 and Example 8 are compared below:

Without detergent Mt Detergens

(Example 8) (Example 7)

mE / min mS / min

19 148

Example 9 Determination of trypsin

a) The preparation of the homogenizing solution is carried out according to Example 1

b) Preparation of the reagent solution: An aqueous solution with the following constituents is prepared: carbobenzoxy-L-.valy1-L-glycy1-L-arginine-p-nitroanilide acetate 5 mmol / l

IaCl 250 mmol / l

CaCl ₂ . 20 mmol / l

Tris / HCl buffer, pH 9.0 60 mmol / l

c) homogenization and d) measurement is carried out in accordance with Example 10 (comparative example )

Example 10 corresponds to Example 9 with the only difference that the homogenizing solution contains no detergent. The result of the measurement with an identical sample according to Example 9 and Example 10 is compared in the following:

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Without detergent With detergent

(Example 10) (Example 9)

mE / min "mE / min

about 3 8

Example 11

An aliquot of the stool specimen is metered by a suitable device such that the specimen is in a space delimited on one side by a filter paper or the like. For this purpose, z. B * Various commercial devices for the determination of elut in the stool such as Hemo FEC ^ or Peca-Hostic ^ after replacement of the appropriate filter paper suitable.

The filter paper is filled with 50 μl of an aqueous solubilization solution consisting of:

Lauryltrimethylammonium chloride 5 0 g / l (5 %) HaCl 500 mmol / l CaOl ₂ 100 mmol / l

the chymotrypsin is solubilized at an interface and taken up by the filter paper. The filter paper is then drizzled with 50 μl of a reagent solution of the following composition:

Succ-Ala-Ala-Pro-Phe-pKA 2 mmol / l l-soaphthylethylenediamine 5 g / l sodium nitrate 1 g / l buffer I _r is / HCl pH 9.0 100 aunol / l

After a reaction time of 5 minutes, one drop of trichloroacetic acid (3.2 mmol / l) is added to the paper

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in the presence of chymotrypsin in the sample, an intense purple color, the extent of which depends on the amount of enzyme, was formed

Example 12 (comparative example )

Example 11 is repeated with the only difference that the solution used to solubilize the enzyme contains no detergent. There is no visible coloration

Claims (10)

65 6 01/26/1983 AP G 01 11/243 765 61 362 12 invention claim -1. A method for determining the activity of chymotrypsin or trypsin in stool by measuring the rate of cleavage of a suitable substrate through a stool suspension in an aqueous or aqueous-organic medium, characterized by suspending the stool in the presence of a surfactant. 2. The method according to item 1, characterized in that one determines amino acid released in a Substratspaltiing. 3 · Method according to item 2, characterized in that the amino acid released is determined by titration with a base. « 4. Method according to item 1, characterized in that the rate of cleavage of the substrate is determined photometrically « 5. The method according to any one of items 1 to 4, characterized in that one carries out the measurement with the stool suspension or with the centrifugation supernatant obtained by centrifugation of the suspension » 6 «method according to one of the points 1 to 5, characterized in that one carries out the determination manually or with an automatic analyzer. 7. The method according to any one of items 1 to 6, characterized in that one uses as a surface-active agent, a cationic or nonionic surfactant. 243765 6 01/26/1983 AP G 01 N / 243 765 61 362 12 8 »Procedure after. Item 7, characterized in that the surface active agent used is a quaternary ammonium salt or an alkylpyridinium salt, and in particular lauryltrimethyl-AEHnonium chloride. Method according to one of the items 1 to 8, characterized in that the concentration of the surfactant in the homogenizing solution used for the suspension of the stool is about 0.02 to about 10% by weight, preferably 0.5 to 5% by weight. , is « 10. Method according to one of the items 1 to 3, characterized in that the homogenizing solution used for the suspension of the stool contains, in addition to the surface-active agent, one or more water-soluble salts. 11. The method according to item 10, characterized in that the Homogenisi erlösung sodium or / and calcium salt contained 12o Method according to one of the items 10 or 11, characterized in that the ionic strength is 20 to 1000 meq / 1 · 13 «Method according to any one of items 1 to 12, characterized in that chymotrypsin is determined« Process according to item 13, characterized in that Ala-Ala-Phe-pHA, Succ-Ala-Ala-Pro-Phe-pEA or MeO-Succ-Arg-Pro-Tyr-pHA is used as the substrate « Method according to one of the items 1 to 12, characterized in that trypsin is determined. 37 6 5 01/26/1983 AP G 01 N / 243 61 362 16. The method according to item 15, characterized in that the substrate used is carbenzene-oxy-Val-Gly-Arg-pNA-acetate. Reagent for carrying out the process according to items 16 to 16, characterized by a content of surface-active agent, enzyme substrate, water-soluble salt, water and a pH of 7 to 11 Reagent according to item 17, characterized in that it contains from 0.02 to 10% by weight of surfactant, 0.05 to 5 mmol / l of Snzyme substrate, 20 to 1000 meq / l of water soluble salt and 10 to 1000 mmol / l Buffer, pH 7 to 11, contain Reagent according to item 18, characterized in that it contains 0.5 to 5% by weight of surfactant, 0.2 to 2 mmol / l of substrate, 100 to 1000 mmol / l of ITaGl or / and 20 to 500 mmol / 1 CaCl ₂ and 50 to 200 mmol / l Tris buffer, pH 8 to 10.