Method and test composition for determination of enzyme activity

Abstract: Disclosed is a method for determination of an activity of γ-glutamyl transpeptidase, leucine aminopeptidase, alanine aminopeptidase, cystine aminopeptidase, X factor as a coagulation factor, thrombin, plasmin of plasminogen series, kallikrein, chymotrypsin, alkali phosphatase, N-acetyl glucosaminase and amylase, by allowing a particular substrate to act on the enzyme to thereby form an enhancer; oxidizing a chromogen by an oxidase in the presence of the enhancer and oxygen to form a pigment; and determining the pigment. Also disclosed is a test composition for carrying out the determination.

Description: The present invention relates to a method and test composition for determination of an enzyme activity, and more particularly to a method for determination of an activity of an enzyme contained in a living body. The method comprises adding an appropriate substrate for an enzyme to a living body sample to thereby form a reaction product, which is capable of accelerating a rate of a reaction in which a chromogen is oxidised by an oxidase in the presence of oxygen; oxidising the chromogen by the oxidase in the presence of that reaction product and oxygen to form a pigment; and quantitatively determining the pigment in a conventional manner. The present invention also pertains to a test composition suitable for carrying out such determination. The enzymes to which the present invention is applicable include, for example, γ-glutamyl transpeptidase (γ-GTP), leucine aminopeptidase (LAP), alanine aminopeptidase (AAP), cystine aminopeptidase (CAP), blood coagulation factor X, thrombin, plasmin, kallikrein, chymotrypsin, alkali phosphatase, N-acetyl glucosaminase and amylase. γ-GTP, LAP and alkali phosphatase reflect troubles of biliary and hepatic organs. AAP and N-acetyl glucosaminase are an indicator for renal disorder. CAP is an indicator for movement in the uterus of a pregnant woman. Coagulation factor X, thrombin and plasmin are involved in the coagulation of blood. Kallikrein is an indicator for primary aldosteronism and hypertension. Chymotrypsin is an indicator for chronic pancreatitis. Amylase is an indicator for pancreatic disorder. Until now the determination of the activity of γ-GTP, LAP and AAP has been carried out by adding a substrate for the enzyme to a sample containing the enzyme, further adding a necessary enzyme for deriving an analysable substance from the substrate, if necessary, and measuring the rate of formation of a resulting measurable compound. For example, the activity of γ-GTP or LAP is quantitatively determined by decomposing an appropriate substrate for γ-GTP or LAP by the enzyme, thereby forming an aniline derivative, allowing the aniline derivative to react with a chromogen to thereby form a pigment, and quantitatively determining the rate of formation of the pigment. When the amount of the enzyme is small, a chromogen capable of forming a pigment having a high molecular extinction coefficient is used, but such a conventional method has a limit in determination of an activity of a trace amount of enzyme. As prior art mention may be made of FR-A-2567907 and US-A-4681841. Each discloses an enzyme assay procedure in which, in a first enzymatic reaction an aniline derivative is liberated from a synthetic substrate by the enzyme under assay. That aniline derivative is then oxidatively condensed with a coupler by the action of a second enzyme, viz. an oxidase, resulting in the formation of a pigment. By contrast, the present invention utilises a substrate or synthetic substrate which liberates an aniline derivative that merely catalyses the enzymatic oxidation of a chromogen with an oxidase to form a pigment, without the formation of a condensation product with the chromogen. In other words, the aniline derivative merely acts as an activator and enhances the activity of the oxidase, thus increasing the rate at which the pigment is produced, that rate being proportional to the activity of the first enzyme. In accordance with the present invention, therefore, a method for the chromogenic determination of the activity of an enzyme in a sample is provided, which comprises reacting the sample with a substrate for that enzyme and capable of reacting with the enzyme to produce an aniline derivative in an amount proportional to the activity of the enzyme, enzymatically oxidising a chromogen by reaction with an oxidase in the presence of the aniline derivative produced in said sample by the reaction of the said enzyme with the said substrate so as to form a pigment in an amount proportional to the amount of the aniline derivative produced in said sample by said enzyme, and quantitatively determining the rate at which the pigment is formed, wherein there is used as the said substrate, a substrate which upon reaction with the said enzyme produces or releases as the said aniline derivative an aniline derivative of the formula (I): wherein R₁, R₂, R₃ and R₄ may be the same or different and represent hydrogen, halogen, alkyl, sulfo or hydroxyl, R₅ represents hydroxyl, amino or substituted amino, and the substituent represents alkyl, sulfoalkyl or hydroxyalkyl, that aniline derivative serving to increase the rate of oxidation of the chromogen by the said oxidase, the increase of rate being proportional to the activity of the enzyme and to the amount of aniline derivative produced in the sample by the reaction of said enzyme with said substrate and wherein there is used as the chromogen a compound oxidisable by said oxidase in the presence of said aniline derivative to form a pigment which does not react with that aniline derivative to form a condensation product, the enzyme activity being determined from the increase in the rate of formation of the pigment consequent upon the production of said aniline derivative by the said enzyme. In the method of the present invention, the aniline derivative produced or released by the enzymatic reaction increases the rate at which the chromogen is oxidised by the action of the oxidase in the presence of oxygen. Thus by quantitatively determining the rate at which the pigment is formed in the presence of the aniline derivative the activity of the enzyme can be quantitatively determined. This is based on the finding that a linear relation exists between the amount of the aniline derivative and the rate of formation of the pigment. Even if the amount of the enzyme contained in a sample is very small, so that the rate of formation of the aniline derivative is slow, the derivative will gradually accumulate in the reaction medium and the rate of formation of the pigment will gradually increase accordingly. By measuring the rate of formation of the pigment the activity of the enzyme contained in a sample can be quantitatively determined. The enzyme activity of the sample is proportional to the absorbance of the coloured solution containing the pigment, and thus it is advantageous to obtain a calibration curve on the basis of the relation between the enzyme activity and the absorbance. Quantitative determination of the thus formed pigment can be made according to any one of the known procedures. The determination is simply carried out by measuring the absorbency of the reaction solution coloured by formation of a pigment at the maximum absorption wavelength of the pigment. As indicated, in the present invention, a specific substrate is used which reacts with the enzyme to produce or release an aniline derivative of formula (I). Typical aniline derivatives of formula (I) are 3,5-dibromo-4-hydroxyaniline (DBHA), 3,5-dichloro-4-hydroxyaniline (DCHA), p-N,N-disulfopropylaminoaniline (SPA), 3,5-diiodo-4-hydroxyaniline (DIHA), 3,3-diaminostilben-4,4'disulfonic acid (DSDA), p-phenylenediamine (PPD), 4-aminoaniline-3-sulfonic acid (DAS), 2-methyl-3,5-dibromo-4-hydroxyaniline (DMBHA), 2,6-dimethyl-3,5-dichloro-4-hydroxyaniline (DMDBHA), 4-N,N-disulfopropylamino-3,5-dibromoaniline (SDBA), 4-(N-ethyl-N-hydroxyethylamino)-3,5-dibromoaniline (EHDBA), 4-N,N-diethylamino-3,4-dihydroxyaniline (DEDHA) and N,N-disulfopropylaniline (DSPA). When a substrate capable of directly forming an aniline derivative of formula I is not available as a substrate for the enzyme, a conjugate substrate can be used, that is to say a conjugate formed between a known substrate for the enzyme and an aniline derivative of formula (I), that conjugate releasing that derivative when reacted upon by the enzyme. For example, aniline derivatives such as DBHA, DCHA, have a hydroxyl group by means of which they may be linked to a known substrate for the enzyme through an appropriate amino acid or peptide. Typical enzyme and substrate or substrate conjugate are combinations given below: Enzyme Substrate or Substrate Conjugate γ-GTP γ-glutamyl-DBHA LAP L-leucyl-DBHA AAP L-alanyl-DBHA Cystine aminopeptidase S-Bz-Cys-DBHA Blood Coagulation Factor X Bz-Ileu-Glu-Gly-Arg-DBHA Thrombin D-Phe-Pip-Arg-DBHA Plasmin (from the plasminogen series) D-Val-Leu-Lys-DBHA Kallikrein Z-Pro-Phe-Arg-DBHA Chymotrypsin Bz-Tyr-DBHA Alkali phosphatase 2,6-dibromo-4-amino-phenyl phosphate N-acetyl glucosaminase p-aminophenyl-N-acetyl-glucosamine Amylase 2,6-diiodo-4-aminophenyl-G7 (or G5) Any oxidase which can oxidise the chromogen in the presence of oxygen and the aniline derivative to form a pigment, can be used. Preferred oxidases are bilirubin oxidase (BLOD EC 1.3.3.5), monophenol monooxygenase (MPO, EC 1.14.18.1), ascorbate oxidase (AOD, EC 1.10.3.3), catechol oxidase (CAO, EC 1.10.3.1), laccase (EC 1.10.3.2), o-aminophenol oxidase (APO, EC 1.10.3.4), 3-hydroxyanthranilate oxidase (HAO, EC 1.10.3.5) and phenol monooxygenase (PMO, EC1,14,13,7). Any chromogen which can be oxidised by an oxidase to develop a colour and which does not form a condensation product with the aniline derivative of formula (I) can be used. In order to obtain a higher sensitivity in the determination system, a chromogen with a higher molecular extinction coefficient is preferred. Typical and preferred chromogens are, for example, the compounds P-1 to P-14 represented by the formulae shown in Table 1. The maximum absorption of these compounds is shown in the following Table 2 Chromogen No. Maximum Absorption (nm) P-1 630 P-2 630 P-3 755 P-4 630 P-5 630 P-6 630 P-7 666 P-8 655 P-9 668 P-10 670 P-11 435 P-12 650 P-13 520 P-14 590 Chromogens P-1, P-2, P-4, P-5 and P-6 are disclosed in EP-A-206316. They are chromogens known as an intermediate for dye synthesis, and are synthesised by a condensation reaction of Michler's hydrol with a naphthalene derivate or by a reduction reaction of a commercially available pigment. Chromogens P-3 and P-10 are disclosed in EP-A-124287. Chromogens P-7, P-8, P-9 and P-13, and the preparation thereof are disclosed in EP-B-38205. Chromogens P-11, P-12 and P-14 are commercially available and can be purchased from Aldrich Co. In the quantitative determination of enzyme activity according to this invention, a substrate capable of forming or releasing the aniline derivative upon reaction with the enzyme, a chromogen, an oxidase, a buffer reagent and a surfactant, etc. are added to a sample to carry out the enzyme reaction. In the enzymatic reaction, the respective reagents are used at the following concentrations: Buffer reagent 10 mM- 1M Oxidase 0.001 - 1,000 U/ml Chromogen 0.01 - 10 mg/L Substrate 0.1 - 100 mg/ml Surfactant 1 - 10 mg/ml The reaction is usually carried out at a temperature of 20 - 40°C and at a pH of 5 - 9. Suitable buffers for use in the present invention include, for example, Good's buffer, phosphate, borate, acetate and tris-hydrochloride. In another aspect of the present invention, test compositions are provided for determination of enzyme activity by the method of this invention, those compositions containing the enzyme substrate, the chromogen and the oxidase, and preferably the buffer and/or a surfactant. Also in accordance with this invention diagnostic devices are provided comprising a porous substrate, e.g. a sheet, film or stick of porous material, e.g. filter paper etc. impregnated with a test composition as above described, and which produce a colour response when contacted with a sample, e.g. a urine sample, containing the enzyme, the intensity of that colour response being proportional to the activity of the enzyme in the sample and comparable with that of a blank control to give an evaluation of the enzymatic activity of the samples. Certain specific embodiments of the invention are illustrated by the following representative examples. Example 1 DIPSO (Good's buffer solution, made by Dojin Kagaku Kenkyusho) (pH 7.5) 0.1M Dispanol M-32A (made by Nihon Yushi K.K.) 5 mg/ml Chromogen P-1 0.2 mg/ml BLOD (Bilirubin oxidase) 0.02 U/ml Gly-Gly 3 mg/ml MgCl₂ 1 mg/ml γ-glutamyl-DBHA (G-DBHA) 2 mg/ml To 3.0 ml quantities of the foregoing reagent solution were added 0.02 ml quantities of aqueous γ-GTP solution at concentrations of 2.5, 5, 7.5 and 10 mU/ml respectively. Each mixture was then allowed to stand at 37°C for 30 minutes and changes in the absorbency of the reaction solutions at 630 nm were measured. The reactions involved are as follows: The amount of DBHA in the reaction solution gradually increases with a consequent increase in rate of formation of the pigment. By determining the amount of pigment formed, the activity of γ-GTP in the reaction solution can be determined. As a control, the same procedure was repeated with a reagent of the same composition as described above, except that 1.5 mg/ml of N-ethyl-N-(3-methylphenyl)-N'-succinyl ethylenediamine (EMSE) was used as a chromogen in place of P-1, and that 0.4 U/ml of BLOD was used. The absorbency of the reaction solution at 710 nm was measured. The reactions proceed as follows: In these reactions the rate of oxidation of the chromogen is not accelerated and the rate of formation of pigment is much slower. Moreover it is clear from the test results given in Table 3 that the absorbency of the pigment solution is proportional to the concentration of the γ-GTP in the sample. γ-GTP (mU/ml) Aniline Derivative Absorbency Chromogen P-1 EMSE 2.5 DBHA 0.32 0.03 5.0 DBHA 0.67 0.06 7.5 DBHA 1.02 0.09 10.0 DBHA 1.35 0.12 Example 2 Reagent solution A DIPSO Buffer solution (pH7.5) 0.1 M Dispanol M-32A 5 mg/ml Chromogen P-2 0.2 mg/ml AOD (ascorbic acid oxidase) 50 U/ml MgCl₂ 1 mg/ml L-leucyl DSPA 2 mg/ml To 3.0 ml of reagent solution A were added 0.02 ml of an aqueous solution containing 20 mU/ml LAP and the mixture then allowed to stand for 30 minutes. The absorbency of the reaction solution was then measured at 630 nm. As a control, the same procedure was repeated except that EMSE was used in place of P-2, and the absorbency of the reaction solution was measured at 745 nm. When using the chromogen P-2 the sensitivity of reagent solution A was 5.2 times greater than the control Example 3 Reagent solution B Phosphate buffer solution (pH 7.2) 0.2 M Triton X-100 5 mg/ml Compound P-1 1 mg/ml AOD 200 U/ml γ-glutamyl-DBHA 10 mg/ml Gly-Gly 20 mg/ml A Wattman No. 41 filter paper, thickness of 0.21 mm, was dipped in solution B and then dried in vacuo . (Triton and Wattman are Registered Trade Marks). Reagent solution C A reagent solution C was prepared having the same composition as solution B, except that 4 mg/ml of EMSE was used in place of Compound P-1, and that 500 U/ml of AOD was used. A test paper was impregnated with solution C and dried as above. A series of aqueous test solutions were prepared containing 0, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10, 20 or 50 mU/ml of γ-GTP respectively and 100 µl of each test solution was dropped onto each of the test papers B and C to investigate the minimum concentration of γ-GTP that could be detected. At 20 minutes, test paper C could detect γ-GTP down to 5.0 mU/ml, whereas test paper B could detect down to 0.1 mU/ml. At 3 minutes, test paper C could detect γ-GTP down to 50 mU/ml, whereas test paper B could detect down to 1 mU/ml. When urine of a normal male and urine of a male patient with renal disease were tested on test papers B and C, it was found that the differentiation between the two urine samples required 20 minutes for test paper C, but only 5 minutes with test paper B. Example 4 Similar pairs of test papers were prepared using the same solutions and procedures as in Example 3, except that each of oxidases shown in Table 3 was used in place of the AOD used in the test papers of Example 3. Urine from a patient with renal disease was dropped onto each pair of test papers and the colour development on each pair of test papers was compared on a chromatoscanner. The sensitivity ratios for each pair of test papers are shown in Table 4. Oxidase Concentration U/ml Sensitivity ratio MPO 0.2 16.5 CPO 1.2 78.1 Laccase 0.05 4.3 APO 0.02 6.9 HAO 2.5 9.2 PMO 0.08 11.6 Example 5 Example 1 was repeated except that the enzymes and substrates given in Table 5 were used to compare the sensitivity ratios of the reagent solutions according to the invention (A) and the controls (B) Enzyme Substrate Sensitivity ratio A/B CAP S-Bz-Cys-DBHA 10.2 X factor Bz-Ile-Glu-Gly-Arg-DBHA 14.5 Thrombin D-Phe-Pip-Arg-DBHA 9.8 Plasmin D-Val-Leu-Lys-DBHA 13.5 Kallikrein Z-Pro-Phe-Arg-DBHA 12.6 Example 6 The activities of the enzymes given in Table 7 were determined in the manner described in Example 2, except that the substrates given in the Table were used in place of DSPA, and the sensitivity ratios were obtained in the same way. Enzyme Substrate Sensitivity ratio A/B Alkali phosphatase 2,6-dichloro-4-aminophenylphosphate 10.6 N-Acetylglucosaminase p-aminophenyl-N-acetyl-glucosamine 4.2 Amylase 2,6-diiodo-4-amino-phenyl-G7 (or G5) 2.8 Example 7 Example 2 was repeated except that DIHA, DSDA, PPD, DAS, DMBHA, DMDBHA, SDBA, EHDBA and DEDHA were used separately in place of DSPA. The sensitivity ratios, A/B, were found to be 46.1, 3.4, 6.8, 4.4, 16.6, 20.8, 5.0, 11.4 and 8.9, respectively.

Claims: A method for the chromogenic determination of the activity of an enzyme in a sample, which comprises reacting the sample with a substrate for that enzyme capable of reacting with the enzyme to form an aniline derivative in an amount proportional to the activity of the enzyme, enzymatically oxidising a chromogen by reaction with an oxidase in the presence of the aniline derivative produced in said sample by the reaction of the said enzyme with the said substrate so as to form a pigment in an amount proportional to the amount of aniline derivative produced in said sample by said enzyme, and quantitatively determining the rate at which the pigment is formed, characterised in that there is used as the said substrate, a substrate which upon reaction with the said enzyme produces or releases as the said reaction product an aniline derivative of the formula (I): wherein R₁, R₂, R₃ and R₄ may be the same or different and represent hydrogen, halogen, alkyl, sulfo or hydroxyl, R₅ represents hydroxyl, amino or substituted amino, and the substituent represents alkyl, sulfoalkyl or hydroxyalkyl, that aniline derivative serving to increase the rate of oxidation of the chromogen by the said oxidase, that rate of increase being proportional to the activity of the enzyme and to the amount of aniline derivative produced in the sample by the reaction of said enzyme with the said substrate, and wherein there is used as the chromogen, a compound oxidisable by said oxidase in the presence of said aniline derivative to form a pigment which does not react with that aniline derivative to form a condensation product, the enzyme activity being determined from the increase in the rate of formation of the pigment consequent upon the production of said aniline derivative by the said enzyme. A method according to claim 1, wherein the enzyme is γ-glutamyl transpeptidase, leucine aminopeptidase, alanine aminopeptidase, cystine aminopeptidase, blood coagulation factor X, thrombin, plasmin, kallikrein, chymotrypsin, alkali phosphatase, N-acetyl glucosaminase or amylase. A method according to claim 1 or 2, wherein the oxidase is bilirubin oxidase, monophenol monooxygenase, ascorbate oxidase, catechol oxidase, laccase, o-aminophenol oxidase or 3-hydroxyanthranilate oxidase or phenol monooxygenase. A method according to any one of claims 1 to 3, wherein the chromogen is any one of compounds P-1 to P-14 as follows: A method according to any one of claims 1 to 4, wherein the substrate comprises a conjugate formed between an aniline derivative of formula (I), and a known substrate for the enzyme, the reaction between the enzyme and the substrate conjugate serving to decompose that conjugate and to release that aniline derivative into the sample. A method according to claim 5, wherein the substrate comprises a conjugate formed between an amino acid or peptide, being a substrate for the enzyme whose activity is to be determined, and an aniline derivative of formula (I). A method according to any one of claims 1 to 4, wherein the substrate comprises a compound which itself is converted to an aniline derivative of formula (I) by reaction with the said enzyme. A reagent composition for use in the method of claim 1, that composition comprising a substrate for the enzyme whose activity is to be determined, and being a compound which upon reaction with that enzyme produces an aniline derivative of formula (I), as defined in claim 1, a chromogen oxidisable by an oxidase in the presence of that aniline derivative to form a pigment and an oxidase capable of oxidising the chromogen to form a pigment in the presence of said aniline derivative characterized in that the chromogen and the aniline derivative do not react to form a condensate. A reagent composition according to claim 8, wherein the chromogen is a compound as defined in claim 4. A reagent composition according to claim 8 or 9, wherein the oxidase is as defined in claim 3. A reagent composition according to claim 8, 9 or 10, wherein the enzyme substrate is as defined in claim 5, 6 or 7. A diagnostic device for use in the determination of enzymatic activity by the method of claim 1, comprising a porous substrate material impregnated with a composition as claimed in any one of claims 8 to 11 and which device produces a colour response when contacted with a sample containing the enzyme, the intensity of that colour response being proportional to the activity of the enzyme in the sample.

Inventors: MIIKE AKIRA, TATANO TOSHIO, MIIKE, AKIRA, TATANO, TOSHIO
Applicants: KYOWA MEDEX CO LTD, KYOWA MEDEX CO. LTD.
Application year: 1988
Main classification: C12Q1
IPC codes: C12Q1, G01N21