CA1191772A - Process and reagent for determining the activity of chymotrypsin and trypsin in faeces - Google Patents
Process and reagent for determining the activity of chymotrypsin and trypsin in faecesInfo
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- CA1191772A CA1191772A CA000410589A CA410589A CA1191772A CA 1191772 A CA1191772 A CA 1191772A CA 000410589 A CA000410589 A CA 000410589A CA 410589 A CA410589 A CA 410589A CA 1191772 A CA1191772 A CA 1191772A
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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
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
-
- 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
- C12Q2337/00—N-linked chromogens for determinations of peptidases and proteinases
- C12Q2337/10—Anilides
- C12Q2337/12—Para-Nitroanilides p-NA
-
- 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)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/976—Trypsin; Chymotrypsin
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- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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- Engineering & Computer Science (AREA)
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- Biotechnology (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
ABSTRACT
The present invention provides a process for the determination of the activity of chymotrypsin or trypsin in faeces by measurement of the rate of fission of an appropriate substrate by a faecal suspension in an aqueous or aqueous-organic medium, wherein a faecal sample is suspended in the presence of a surface-active agent.
The present invention also provides a reagent for carrying out this process, comprising a surface-active agent, an enzyme substrate and an aqueous salt solution, said reagent having a pH value of from 7 to 11.
The present invention provides a process for the determination of the activity of chymotrypsin or trypsin in faeces by measurement of the rate of fission of an appropriate substrate by a faecal suspension in an aqueous or aqueous-organic medium, wherein a faecal sample is suspended in the presence of a surface-active agent.
The present invention also provides a reagent for carrying out this process, comprising a surface-active agent, an enzyme substrate and an aqueous salt solution, said reagent having a pH value of from 7 to 11.
Description
:~19:~L'77;~
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 and also of mucoviscidosis in neonates, the diagnosis of pancreatic insufficiency is an important para~eter, the clinical-chemical detection of which haq 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 subse-quently inve~tigated for the following parameters:
volume, bicarbonate concentration and the act~vities 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 ~5 chymotrypsin values thereby having proved to be the better parameter. For the determination of chymo-trypsin or trypsin in faeces, a faecal suspension is '7'7Z
mixed with an aqueous methanolic solution of a ~speciEic 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 example, by tritration with an aqueous lye~ especially an aqueous solution oE sodium hydroxide (pH-stat proces~q, Haverback et al., Gastroenterology, 44, 588-5g7/l963, Ammann, Fortschritte in der Pankreasfunktionsdiagnostik, pub. Springer Verla~, Berlin, Heidelberg, ~ew 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., _, 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 centri-fuging the faecal suspension is not possible because the enzyme is bound relatively firmly to the faecal particles and, after centrifuging, is to be found '7'7~
almost 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 by measurement of the rate of fission of an appropriate substrate by a faecal sample in an aqueous or aqueous-organic medium, wherein a faecal sample is 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 (p~I-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 9~O 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 which have previously arisen (especially binding of the '77~
enzyme 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, în the case of pure a-chymotrypsin from bovine pancreas, practically no dif erence can be ascertained when carrying out the determina-tion with or without the use of a surface-active agent.
The surface-active agent used can, in principle, be any appropriate tenside, such as an anionic or ampholytic tenside and preferably a non-ionic tenside and especially a cationic tenside.
Anionic tensides which can be used include alkane-sulphonates, ole~in-sulphonates, for example cumene-sulphonate, ester sulphonates, alkylarylsulphonates of the dodecylbenzenesulphonate type, alkylnaphthalene-sulphonates, alkyl sulphates, for example sodium lauryl sulphate, ether sulphates and fatty alcohol sulphates and salts of fetty acids and of bile acids, ampholytic tensides are those with anion-active and cation-active hydrophilic groups, for example glycerol derivatives `7'~
with a betaine stxucture, sulphobe~aines and lecithins non-ionic tensides includeO for example, polyethers, especially alkylphenol polyglycol ethers and other ethoxylation products of fatty acids, fatty acid amides, fatty amines and fatty alcohols, for exarnple 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/ethylenediarnine/
ethylene oxide, arnine oxides and fatty acid esters of polyalcohols, tallow alcohol polyglycol ethers, cationic tensides include, for example, straight-chained and cyclic ammonium compounds, for example ~-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 enzyrne 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 ammoniurn compounds and preferably those of the general formula RlR2~(CH3)2. wherein Rl is preferably an alkyl radical containing ~ to 1~ carbon _7_ atoms 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 hydroxy-alkyl radical and especially a ben~yl or methyl radical, and alkylpyridinium salts with preferably 12 to 18 carbon atoms in the alkyl radical, for example lauryl pyridinium chloride, lauryl pyridinium disulp~ate 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 about lOo/o 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 concen-tration of the faecal sample in the suspension is, for example, 0.2 to 2~ when using Succ-Ala-Ala~Pro-Phe-p~A
as substrate.
In addition to the surface-active agent, the homogenising solution used for the suspension of the faeces preferably also contains one or mcre water-soluble salts, for example alkali metal and/or alkaline earth metal chlorides or sulphates. A content of sodium`
chloride of 100 to 1000 mmole/litre or a content of calcium chloride of 20 to 500 mmole/litre or a combin-ation of both salts in the given concentration ranges has proved to be especially useful. However, organic salts can al50 be used, for example acetates or citrates, as well as salts of other cations. The salts are prefer-ably 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 deterge~t, in contradistinction to the individual com-ponents, gives a constantly high and representative portion of the particle-bound enzyme in solution.
As substrate, there can, in general, be used all ap2ropriate substrates known for the determination of the activity of chymotrypsin or trypsin in faeces by previously known methods (cf. for example, Grossman, Proc. Soc. Biol. Med., 110, 41/1951, Del Mar et al., Anal. Biochem., 99, 315/1979, Nakajama et al., J. Biol.
Chem., 254(10), 4027-4032/1979). Especially preferred for the determination of chymotrypsin~ particularly '7~
g .
with regard to water-solubility~ stability~ Km value and velocity constant~, is Ala-Ala-Phe-p~A and espec-ially Succ-~la-Ala-Pro-Phe-p~A and MeO-Succ-Arg-Pro-Tyr-pNA, these substrates are particularly useful for photometric determinations. ~ substrate which is especially pre-ferred for the determination of trypsin activity is, for example, Chromozyme TR ~ carbobenzoxy-valyl-glycyl-arginine-p-nitroanilide acetate).
For the preparation of the reagent solution, the substrate is dissolved in water or in a mixtura of water and an organic solvent. The organic solvent thereby serves as a solubiliser for the substrate and can be, for example, acetonitrile, dimethylsulphoxide, acetone or methanol. The reagent solution preferably also contains the same salts as are used for the homogenis-ing solution, the concentration of these salts in the reagent solution is, in general, lower than the con-centration 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 centrifug:ing until clear is added to a definite amount of the reagent solution (for example 100 ~1. of sample suspension to 2 ml. of reagent .~ 10 solution) and the rate of fission is determined, for example titrimetrically or photometrically. The amount of sample suspension (sample dilution) for the achievement of readily measurable value~, for example 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 u~ed can be so small that the inherent absorption of the suspended solid bodies is low and thus a photo-metric measurement with the suspension only then becomes possible. The pH value of the homogenising solution is not especially critical, in general, it is from 3 to 10 and preferably from 5 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, '7'~
then it is expedient to employ an especially sensitive detection reaction. Such a sensitive reaction is described, for example, in J. Biol~ Chem., 128, 537/
1939 and is known as the Bratton/Marshall reaction.
It depends upon the addition of sodium nitrite and ~-a-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 photo-metric 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 measure-ment.
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 surface-active agent, an enzyme substrate and an aqueous salt solution, the reagent having a pH value of from 7 to 11.
A preferred reagent according -to the present invention contains the follow~ng components:
0.02 to 10% by weight surface-active agent, 0.05 to 5 mmole/litre enzyme substrate, 20 to 1000 m~al/litre of a water-soluble salt and 10 to 1000 mmole/litre of a buffer ~pH 7 to 11).
A ~ore 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 ~p~ 8 to 10).
The following Examples ar~ given for the purpose of illustrating the present invention, if not stated otherwise, the percentages are by weight:
Example 1.
a) Preparation of a homoaenisinq solution:
sodium chloride 2.9 g. ~500 mmole/l.) calcium chloride 1.1 g. ~100 mmole/l.) 33% lauryl trimethyl ammonium chloride 2.0 g. (0,7%) distilled water ad 100 ml.
b) Preparation of a reaqent solution:
Succ-Ala-Ala-Pro-Phe-pNA 29.5 mg. ~0.5 mmole/l.) sodium chloride 1.46 g, (250 mmole/l.
calcium chloride 222 mg. (20 r~ole/l.) buffer tris/HCl (pH 9.0) ad 100 ml.
(60 mmole/l.) c) ~omoqenisation 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) Carryin~ out of the measurement:
Into a 1 cm. measurement cuvette there are pipetted 2 ml. of the reagent solution which is warmed to 25C. and mixed with 100 ~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.
~.
The preparation of the homogenising solution and of the reagent solution is carried out as in Example 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 Example 1 or with the use of an automatic analysis device.
E~ mple 3.
a~ Pre~aration oE the homoqenisinq solution:
~917'~'~
An aqueous solution is prepared with the following components:
tris/EICl 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 reaqent solution:
The reagent solution is prepared in the manner described in Example 1.
c) Homo~1enisation of the sample:
This is carried out as described in Example 1.
d) Carryinq out of the measurement:
This i~ also carried out as described in Example 1.
Exam~le 4. (comparative) Example 4 corresponds to Example 3 with the sole difference that the homogenising solution does not con-tain detergent (lauryl trimethyl a~monium 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 with detergent (Example 4) (Example 3) . _ mE/min. mE/min.
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 and also of mucoviscidosis in neonates, the diagnosis of pancreatic insufficiency is an important para~eter, the clinical-chemical detection of which haq 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 subse-quently inve~tigated for the following parameters:
volume, bicarbonate concentration and the act~vities 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 ~5 chymotrypsin values thereby having proved to be the better parameter. For the determination of chymo-trypsin or trypsin in faeces, a faecal suspension is '7'7Z
mixed with an aqueous methanolic solution of a ~speciEic 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 example, by tritration with an aqueous lye~ especially an aqueous solution oE sodium hydroxide (pH-stat proces~q, Haverback et al., Gastroenterology, 44, 588-5g7/l963, Ammann, Fortschritte in der Pankreasfunktionsdiagnostik, pub. Springer Verla~, Berlin, Heidelberg, ~ew 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., _, 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 centri-fuging the faecal suspension is not possible because the enzyme is bound relatively firmly to the faecal particles and, after centrifuging, is to be found '7'7~
almost 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 by measurement of the rate of fission of an appropriate substrate by a faecal sample in an aqueous or aqueous-organic medium, wherein a faecal sample is 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 (p~I-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 9~O 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 which have previously arisen (especially binding of the '77~
enzyme 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, în the case of pure a-chymotrypsin from bovine pancreas, practically no dif erence can be ascertained when carrying out the determina-tion with or without the use of a surface-active agent.
The surface-active agent used can, in principle, be any appropriate tenside, such as an anionic or ampholytic tenside and preferably a non-ionic tenside and especially a cationic tenside.
Anionic tensides which can be used include alkane-sulphonates, ole~in-sulphonates, for example cumene-sulphonate, ester sulphonates, alkylarylsulphonates of the dodecylbenzenesulphonate type, alkylnaphthalene-sulphonates, alkyl sulphates, for example sodium lauryl sulphate, ether sulphates and fatty alcohol sulphates and salts of fetty acids and of bile acids, ampholytic tensides are those with anion-active and cation-active hydrophilic groups, for example glycerol derivatives `7'~
with a betaine stxucture, sulphobe~aines and lecithins non-ionic tensides includeO for example, polyethers, especially alkylphenol polyglycol ethers and other ethoxylation products of fatty acids, fatty acid amides, fatty amines and fatty alcohols, for exarnple 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/ethylenediarnine/
ethylene oxide, arnine oxides and fatty acid esters of polyalcohols, tallow alcohol polyglycol ethers, cationic tensides include, for example, straight-chained and cyclic ammonium compounds, for example ~-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 enzyrne 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 ammoniurn compounds and preferably those of the general formula RlR2~(CH3)2. wherein Rl is preferably an alkyl radical containing ~ to 1~ carbon _7_ atoms 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 hydroxy-alkyl radical and especially a ben~yl or methyl radical, and alkylpyridinium salts with preferably 12 to 18 carbon atoms in the alkyl radical, for example lauryl pyridinium chloride, lauryl pyridinium disulp~ate 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 about lOo/o 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 concen-tration of the faecal sample in the suspension is, for example, 0.2 to 2~ when using Succ-Ala-Ala~Pro-Phe-p~A
as substrate.
In addition to the surface-active agent, the homogenising solution used for the suspension of the faeces preferably also contains one or mcre water-soluble salts, for example alkali metal and/or alkaline earth metal chlorides or sulphates. A content of sodium`
chloride of 100 to 1000 mmole/litre or a content of calcium chloride of 20 to 500 mmole/litre or a combin-ation of both salts in the given concentration ranges has proved to be especially useful. However, organic salts can al50 be used, for example acetates or citrates, as well as salts of other cations. The salts are prefer-ably 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 deterge~t, in contradistinction to the individual com-ponents, gives a constantly high and representative portion of the particle-bound enzyme in solution.
As substrate, there can, in general, be used all ap2ropriate substrates known for the determination of the activity of chymotrypsin or trypsin in faeces by previously known methods (cf. for example, Grossman, Proc. Soc. Biol. Med., 110, 41/1951, Del Mar et al., Anal. Biochem., 99, 315/1979, Nakajama et al., J. Biol.
Chem., 254(10), 4027-4032/1979). Especially preferred for the determination of chymotrypsin~ particularly '7~
g .
with regard to water-solubility~ stability~ Km value and velocity constant~, is Ala-Ala-Phe-p~A and espec-ially Succ-~la-Ala-Pro-Phe-p~A and MeO-Succ-Arg-Pro-Tyr-pNA, these substrates are particularly useful for photometric determinations. ~ substrate which is especially pre-ferred for the determination of trypsin activity is, for example, Chromozyme TR ~ carbobenzoxy-valyl-glycyl-arginine-p-nitroanilide acetate).
For the preparation of the reagent solution, the substrate is dissolved in water or in a mixtura of water and an organic solvent. The organic solvent thereby serves as a solubiliser for the substrate and can be, for example, acetonitrile, dimethylsulphoxide, acetone or methanol. The reagent solution preferably also contains the same salts as are used for the homogenis-ing solution, the concentration of these salts in the reagent solution is, in general, lower than the con-centration 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 centrifug:ing until clear is added to a definite amount of the reagent solution (for example 100 ~1. of sample suspension to 2 ml. of reagent .~ 10 solution) and the rate of fission is determined, for example titrimetrically or photometrically. The amount of sample suspension (sample dilution) for the achievement of readily measurable value~, for example 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 u~ed can be so small that the inherent absorption of the suspended solid bodies is low and thus a photo-metric measurement with the suspension only then becomes possible. The pH value of the homogenising solution is not especially critical, in general, it is from 3 to 10 and preferably from 5 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, '7'~
then it is expedient to employ an especially sensitive detection reaction. Such a sensitive reaction is described, for example, in J. Biol~ Chem., 128, 537/
1939 and is known as the Bratton/Marshall reaction.
It depends upon the addition of sodium nitrite and ~-a-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 photo-metric 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 measure-ment.
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 surface-active agent, an enzyme substrate and an aqueous salt solution, the reagent having a pH value of from 7 to 11.
A preferred reagent according -to the present invention contains the follow~ng components:
0.02 to 10% by weight surface-active agent, 0.05 to 5 mmole/litre enzyme substrate, 20 to 1000 m~al/litre of a water-soluble salt and 10 to 1000 mmole/litre of a buffer ~pH 7 to 11).
A ~ore 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 ~p~ 8 to 10).
The following Examples ar~ given for the purpose of illustrating the present invention, if not stated otherwise, the percentages are by weight:
Example 1.
a) Preparation of a homoaenisinq solution:
sodium chloride 2.9 g. ~500 mmole/l.) calcium chloride 1.1 g. ~100 mmole/l.) 33% lauryl trimethyl ammonium chloride 2.0 g. (0,7%) distilled water ad 100 ml.
b) Preparation of a reaqent solution:
Succ-Ala-Ala-Pro-Phe-pNA 29.5 mg. ~0.5 mmole/l.) sodium chloride 1.46 g, (250 mmole/l.
calcium chloride 222 mg. (20 r~ole/l.) buffer tris/HCl (pH 9.0) ad 100 ml.
(60 mmole/l.) c) ~omoqenisation 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) Carryin~ out of the measurement:
Into a 1 cm. measurement cuvette there are pipetted 2 ml. of the reagent solution which is warmed to 25C. and mixed with 100 ~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.
~.
The preparation of the homogenising solution and of the reagent solution is carried out as in Example 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 Example 1 or with the use of an automatic analysis device.
E~ mple 3.
a~ Pre~aration oE the homoqenisinq solution:
~917'~'~
An aqueous solution is prepared with the following components:
tris/EICl 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 reaqent solution:
The reagent solution is prepared in the manner described in Example 1.
c) Homo~1enisation of the sample:
This is carried out as described in Example 1.
d) Carryinq out of the measurement:
This i~ also carried out as described in Example 1.
Exam~le 4. (comparative) Example 4 corresponds to Example 3 with the sole difference that the homogenising solution does not con-tain detergent (lauryl trimethyl a~monium 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 with detergent (Example 4) (Example 3) . _ mE/min. mE/min.
2 6 628 S 20 lS9 7~;Z
Exa_ple 5.
a) Preparation of the homoqenisinq solut n:
This is carried out as described in Example 1.
b) Preparation of the reaqent solution:
An aqueous solution is prepared containing the ~ following components:
~la-Ala-Phe-~-nitroanilide2 mmole/litre sodium chloride250 mmole/litre calcium chloride20 mmole/litre tris/HCl buffer (pH 9.0)60 mmole/litre c) Homoqenisation 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 suspen~ion.
d) Carr~inq out of the measurement:
1 ml. of the reagent solution is pipetted into a 1 cm. cuvette, warmed to 25 C. and mixed with 200 ~ .
of the sample homogenate. After briefly mixing, the increase of the extinction is measured at 405 nm.
EXamD1e 6. (comparative) Example 6 corresponds to Example 5 with the sole difference that the homogenising solution does not con-tain detergent. The results obtained with an identical sample according to Examples 5 and 6 are as follows:
,.._. ._ .
without detergent with detergent (Example 6) (Example 5) mE/min. mE/min.
_ -_ _ '7;~
Example 7.
a) Preparation of the homoqenisinq solution:
This is carried out as described in Example 1.
b) Preparation of the reaqent solution:
An aqueous solution is prepared with the following components:
Exa_ple 5.
a) Preparation of the homoqenisinq solut n:
This is carried out as described in Example 1.
b) Preparation of the reaqent solution:
An aqueous solution is prepared containing the ~ following components:
~la-Ala-Phe-~-nitroanilide2 mmole/litre sodium chloride250 mmole/litre calcium chloride20 mmole/litre tris/HCl buffer (pH 9.0)60 mmole/litre c) Homoqenisation 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 suspen~ion.
d) Carr~inq out of the measurement:
1 ml. of the reagent solution is pipetted into a 1 cm. cuvette, warmed to 25 C. and mixed with 200 ~ .
of the sample homogenate. After briefly mixing, the increase of the extinction is measured at 405 nm.
EXamD1e 6. (comparative) Example 6 corresponds to Example 5 with the sole difference that the homogenising solution does not con-tain detergent. The results obtained with an identical sample according to Examples 5 and 6 are as follows:
,.._. ._ .
without detergent with detergent (Example 6) (Example 5) mE/min. mE/min.
_ -_ _ '7;~
Example 7.
a) Preparation of the homoqenisinq solution:
This is carried out as described in Example 1.
b) Preparation of the reaqent solution:
An aqueous solution is prepared with the following components:
3-carbomethoxyproplonyl-L-arginyl-L-prolyl-L-tyrosine ~-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 ta]ce 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 con-tain any detergent. The results of the measurement of an identical sample according to Examples 7 and 8 are as follows:
without detergentwith detergent (Example 8) (Example 7) mE/min. mE/min.
, _ _ _ ~ e~
Determination of tr~psin.
a) Preparation of the homoqenisinq solution:
Thls is carried out as described in Example 1.
1~9~"~7;~
b) Preparation of the reaqent solution:
An aqueous solution i~ prepared containing the following components:
carbobenzoxy-L--valyl-L-glycyl-L-arginine ~-nitroanilide acetate5 mmole/litre sodium chloride 250 mmole/litre calcium chloride 20 mmole/litre tris/HCl buffer (pH 9.0)60 mmole/litre 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 are as follows:
~b~ t ~et~r3en= I with detergent (Example 10) ~Example 9) mE/min. mE/min.
A~ t ~ ..,... - ...._.....
Example 11 An aliquot of a faecal sample is measured by means of an appropriate device in such a manner that the sarnple i~ 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 cornmercially available devices <31'~'îJ'~
for the determination of blood in faeces, such as Hemo FEC ~ or Feca-~ostic~ after exchange of the appropriate filter paper.
The filter paper is sprinkled with 50 ~1. of an a~ueous solubilising solution consisting of:
lauryl trimethyl ammonium chloride 50 g./litre (5,') sodium chloride 500 mmole/litre calcium chloride 100 rnmole/litre the chymotrypsin of a boundary surface thereby being solubilised and taken up by the filter paper. Subse-quently, the filter paper is sprinkled with 50 ~1. of a reagent solution of the following composition:
Succ-Ala-Ala-Pro-Phe-~A 2 mmole/litre ~-a-naphthylethylenediamine 5 g./litre sodium nitrite 1 g./litre tris/HCl buffer (pH 9.0) 100 mmole/litre After a reaction time of 5 minutes, a drop of trichloro-acetic acid 13.2 mmole/l~tre) is applied to the paper.
When chymotrypsin is present in the sample, an intense violet coloration is formed, the extent of whlch depends upon the amount of enzyme.
Exam~le 12. (comparative) Example 11 is repeated but with the sole difference that the solution used for the solubilisation of the ~5 enzyme does not contain any deterqent. No visible coloration develops~
Example 8.(comparative) Example 8 corresponds to Example 7 with the sole difference that the homogenising solution does not con-tain any detergent. The results of the measurement of an identical sample according to Examples 7 and 8 are as follows:
without detergentwith detergent (Example 8) (Example 7) mE/min. mE/min.
, _ _ _ ~ e~
Determination of tr~psin.
a) Preparation of the homoqenisinq solution:
Thls is carried out as described in Example 1.
1~9~"~7;~
b) Preparation of the reaqent solution:
An aqueous solution i~ prepared containing the following components:
carbobenzoxy-L--valyl-L-glycyl-L-arginine ~-nitroanilide acetate5 mmole/litre sodium chloride 250 mmole/litre calcium chloride 20 mmole/litre tris/HCl buffer (pH 9.0)60 mmole/litre 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 are as follows:
~b~ t ~et~r3en= I with detergent (Example 10) ~Example 9) mE/min. mE/min.
A~ t ~ ..,... - ...._.....
Example 11 An aliquot of a faecal sample is measured by means of an appropriate device in such a manner that the sarnple i~ 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 cornmercially available devices <31'~'îJ'~
for the determination of blood in faeces, such as Hemo FEC ~ or Feca-~ostic~ after exchange of the appropriate filter paper.
The filter paper is sprinkled with 50 ~1. of an a~ueous solubilising solution consisting of:
lauryl trimethyl ammonium chloride 50 g./litre (5,') sodium chloride 500 mmole/litre calcium chloride 100 rnmole/litre the chymotrypsin of a boundary surface thereby being solubilised and taken up by the filter paper. Subse-quently, the filter paper is sprinkled with 50 ~1. of a reagent solution of the following composition:
Succ-Ala-Ala-Pro-Phe-~A 2 mmole/litre ~-a-naphthylethylenediamine 5 g./litre sodium nitrite 1 g./litre tris/HCl buffer (pH 9.0) 100 mmole/litre After a reaction time of 5 minutes, a drop of trichloro-acetic acid 13.2 mmole/l~tre) is applied to the paper.
When chymotrypsin is present in the sample, an intense violet coloration is formed, the extent of whlch depends upon the amount of enzyme.
Exam~le 12. (comparative) Example 11 is repeated but with the sole difference that the solution used for the solubilisation of the ~5 enzyme does not contain any deterqent. No visible coloration develops~
Claims (22)
1. A process for the determination of the activity of chymotrypsin or trypsin in faeces by measurement of the rate of fission of an appropriate substrate by a faecal suspension in an aqueous or aqueous-organic medium, wherein a faecal sample is suspended in the presence of a surface-active agent.
2. A process according to claim 1, wherein the amino acid formed by 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 claim 1, 2 or 3, wherein the measurement is carried out with a faecal suspension.
6. A process according to claim 1, 2 or 3, wherein the determination is carried out manually.
7. A process according to claim 1, 2 or 3, wherein the determination is carried out with an automatic analysis apparatus.
8. A process according to claim 1, wherein a cationic or non-ionic tenside is used as the surface-active agent.
9. A process according to claim 8, wherein the surface-active agent is a quaternary ammonium salt or an alkyl-pyridinium salt.
10.. A process according to claim 9, wherein the quaternary ammonium salt is lauryl trimethyl ammonium chloride.
11. A process according to claim 1, wherein the concentration of the surface-active agent in the homogenising solution used for the suspension of the faecal sample is about 0.02 to about 10%
by weight.
by weight.
12. A process according to claim 11, wherein the concentration of the surface-active agent in the homogenising solution used for the suspension of the faecal sample is 0.5 to 5% by weight.
13. A process according to claim 1, wherein the homogenising solution used for the suspension of the faecal sample contains, in addition to the surface-active agent, at least one water-soluble salt.
14. A process according to claim 13, wherein the water-soluble salt present in the homogenising solution is at least one of a sodium salt and a calcium salt.
15. A process according to claim 13 or 14, wherein the ionic strength of the at least one salt is from 20 to 1000 mval/litre.
16. A process according to claim 1, wherein chymotrypsin is determined.
17. A process according to claim 16, wherein the substrate is Ala-Ala-Phe-?NA, Succ-Ala-Ala-Pro-Phe-?NA or MeO-Succ-Arg-Pro-Tyr-?NA.
18. A process according to claim 1, wherein trypsin is determined.
19. A process according to claim 18, wherein the substrate is carbobenzoxy-Val-Gly-Arg-?NA acetate.
20. A reagent for the determination of the activity of chymotrypsin or trypsin in faeces com-prising a surface-active agent, an enzyme substrate and an aqueous salt solution, said reagent having a pH value of from 7 to 11.
21. A reagent according to claim 20, which contains the following components:
0.02 to 10% by weight surface-active agent, 0.05 to 5 mmol/litre enzyme substrate, 20 to 1000 mval/litre of a water-soluble salt and 10 to 1000 mmole/litre of a buffer effective to establish a pH of 7 to 11.
0.02 to 10% by weight surface-active agent, 0.05 to 5 mmol/litre enzyme substrate, 20 to 1000 mval/litre of a water-soluble salt and 10 to 1000 mmole/litre of a buffer effective to establish a pH of 7 to 11.
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 effective to establish a pH of 8 to 10.
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 effective to establish a pH of 8 to 10.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3139719.0 | 1981-10-06 | ||
| DE19813139719 DE3139719A1 (en) | 1981-10-06 | 1981-10-06 | METHOD FOR DETERMINING THE ACTIVITY OF CHYMOTRYPSIN OR TRYPSIN IN THE CHAIR |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1191772A true CA1191772A (en) | 1985-08-13 |
Family
ID=6143516
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000410589A Expired CA1191772A (en) | 1981-10-06 | 1982-09-01 | Process and reagent for determining the activity of chymotrypsin and trypsin in faeces |
Country Status (12)
| Country | Link |
|---|---|
| EP (1) | EP0076506B1 (en) |
| JP (1) | JPS6036758B2 (en) |
| AT (1) | ATE12521T1 (en) |
| AU (1) | AU534001B2 (en) |
| CA (1) | CA1191772A (en) |
| DD (1) | DD203777A5 (en) |
| DE (2) | DE3139719A1 (en) |
| DK (1) | DK156225C (en) |
| ES (1) | ES516124A0 (en) |
| HU (1) | HU187416B (en) |
| IE (1) | IE53472B1 (en) |
| SU (1) | SU1367866A3 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6601148B2 (en) | 2015-10-27 | 2019-11-06 | スズキ株式会社 | Engine lubrication structure and motorcycle |
| JP7355140B2 (en) * | 2022-02-28 | 2023-10-03 | 住友ベークライト株式会社 | Reagents for detecting or measuring serine proteases |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SE380258B (en) * | 1972-05-02 | 1975-11-03 | Bofors Ab | NEW DIAGNOSTIC OPERATING SUBSIDIES WITH HIGH SPECIFICITY FOR TRYPSIN AND OTHER PEPTIDYL-PEPTIDE HYDROLASES OF THE PEPTIDYL TYPE |
| DE2829531A1 (en) * | 1978-07-05 | 1980-01-24 | Heuck Claus Christian Dr Rer N | METHOD FOR QUANTITATIVE DETERMINATION OF A SERUM PROTEIN IN BLUE SERUM AND PLASMA SAMPLES |
-
1981
- 1981-10-06 DE DE19813139719 patent/DE3139719A1/en not_active Withdrawn
-
1982
- 1982-09-01 CA CA000410589A patent/CA1191772A/en not_active Expired
- 1982-09-02 IE IE2146/82A patent/IE53472B1/en not_active IP Right Cessation
- 1982-09-13 AU AU88350/82A patent/AU534001B2/en not_active Ceased
- 1982-09-20 SU SU823493078A patent/SU1367866A3/en active
- 1982-09-30 ES ES516124A patent/ES516124A0/en active Granted
- 1982-09-30 DK DK434082A patent/DK156225C/en not_active IP Right Cessation
- 1982-10-04 DD DD82243765A patent/DD203777A5/en not_active IP Right Cessation
- 1982-10-04 DE DE8282109156T patent/DE3262892D1/en not_active Expired
- 1982-10-04 JP JP57174430A patent/JPS6036758B2/en not_active Expired
- 1982-10-04 AT AT82109156T patent/ATE12521T1/en not_active IP Right Cessation
- 1982-10-04 EP EP82109156A patent/EP0076506B1/en not_active Expired
- 1982-10-05 HU HU823190A patent/HU187416B/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| EP0076506A1 (en) | 1983-04-13 |
| ES8306389A1 (en) | 1983-06-01 |
| SU1367866A3 (en) | 1988-01-15 |
| JPS58111699A (en) | 1983-07-02 |
| IE822146L (en) | 1983-04-06 |
| AU534001B2 (en) | 1983-12-22 |
| AU8835082A (en) | 1983-05-05 |
| EP0076506B1 (en) | 1985-04-03 |
| DK156225C (en) | 1989-12-04 |
| DE3139719A1 (en) | 1983-04-21 |
| JPS6036758B2 (en) | 1985-08-22 |
| DD203777A5 (en) | 1983-11-02 |
| DK156225B (en) | 1989-07-10 |
| IE53472B1 (en) | 1988-11-23 |
| DE3262892D1 (en) | 1985-05-09 |
| ES516124A0 (en) | 1983-06-01 |
| HU187416B (en) | 1986-01-28 |
| ATE12521T1 (en) | 1985-04-15 |
| DK434082A (en) | 1983-04-07 |
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