Test system for measuring enzymatic activity of angiotensin converting enzyme (ACE)

Abstract

In order to provide a method for the quantitative measurement of the enzymatic activity of angiotensin converting enzyme (ACE), which is as sensitive as possible, is substantially insensitive to spectral interference, and enables automation on conventional analyzers for conventional clinical use, according to the invention, a method is provided for the quantitative measurement of the enzymatic activity of ACE. A method for quantitative measurement of the enzymatic activity of angiotensin converting enzyme (ACE) in a sample is proposed in which a reaction mixture is produced by contacting the ACE contained in the sample with a first reagent set, with a second reagent set, and with aminopyrine, the first reagent group comprises hydroxybenzoyl tripeptide and an oxidation substrate of a weak oxidant and / or oxidoreductase, and the second reagent group comprises hippuricase and optional oxidoreductase.

Description

Technical Field

[0001] The present invention relates to an analytical or diagnostic test system for quantitatively determining the enzymatic activity of angiotensin-converting enzyme (ACE) in a sample. In particular, the present invention relates to a method for quantitatively determining the enzymatic activity, and a test system composed of a plurality of reagents, by which the method can be implemented. Background Art

[0002] The angiotensin-converting enzyme (synonyms: ACE, kininase II, peptidyl dipeptidase a) studied in the present invention is a zinc metalloprotease that cleaves the prohormone angiotensin I into the vasoconstrictor hormone angiotensin II. Angiotensin II is an effector in the renin-angiotensin-aldosterone system, which has a vasoconstrictive effect and, in this way, causes an increase in blood pressure and extracellular volume.

[0003] Angiotensin-converting enzyme (ACE) exists in a membrane-bound form on the luminal surface of endothelial cells, mainly in the lungs, followed by the brain, intestine, kidney, adrenal gland, and testis. In the nervous system, the ACE concentration in the choroid plexus is relatively high. In addition to the membrane-bound form, ACE also exists in a free soluble form in plasma and other body fluids (such as amniotic fluid, cerebrospinal fluid, etc.). The soluble form of ACE is formed by the hydrolysis of the C-terminal membrane anchor protein.

[0004] Determining the activity of ACE in blood can be used for the diagnosis and process monitoring of granulomatous lung diseases, such as tuberculosis and sarcoidosis. In addition, ACE diagnosis is also combined with the diagnostic studies of leprosy, Gaucher's disease, and various preeclampsia.

[0005] The determination of the ACE content in serum is usually carried out using one of the following two test systems.

[0006] In the FAPGG method, the peptide FAPGG is used as a substrate, which is hydrolyzed and degraded by the patient's own ACE in the sample, resulting in a decrease in absorbance in the ultraviolet light range. However, since the measurement wavelength is in the ultraviolet light range, the measurement based on the FAPGG method is easily affected by spectral interference from endogenous bilirubin, hemoglobin, or lipids.

[0007] In contrast, in the so-called "hippuricase method", the synthetic substrate p-hydroxybenzoyl-glycyl-L-histidyl-L-leucine is degraded by ACE in the sample into p-benzoic acid under the action of hippuricase provided by the reagent. The generated p-hydroxybenzoic acid is converted into a dye through the Trinder reaction, and its absorbance is measured at 505 nm.

[0008] Measuring the photometric signal in the visible light range enables the determination of ACE that is slightly more sensitive and less sensitive to spectral interference compared to the FAPGG method. However, this test system typically consists of five independent, partially lyophilized reagent components and is automated on many conventional analyzers used for routine clinical applications. Summary of the Invention

[0009] Against this background, the object of the inventors of the present application is to provide a method for quantitatively measuring the enzyme activity of ACE that is as sensitive as possible, is substantially insensitive to spectral interference, and can be automated on conventional analyzers used in routine clinical practice.

[0010] Therefore, according to the present invention, there is provided a method for quantitatively measuring the enzyme activity of angiotensin-converting enzyme (ACE) in a sample, wherein a reaction mixture is produced by contacting the ACE contained in the sample with the following substances:

[0011] a) A first reagent group, wherein the first reagent group contains the following reagent components:

[0012] a1) A hydroxybenzoyl tripeptide of general formula (I) as an ACE-substrate

[0013]

[0014] Wherein, in general formula (I),

[0015] -R 1 is an alkoxy group, a halogen atom or an unsubstituted amino group; 1-4 is an alkoxy group, a halogen atom or an unsubstituted amino group;

[0016] -R 2 and R 3 are each independently an alkoxy group, a halogen atom or H; 1-4 is an alkoxy group, a halogen atom or H;

[0017] -A is selected from the amino acid groups His, Gly or Ala, and

[0018] -B is selected from the amino acid groups Leu, Gly or Phe,

[0019] a2) An oxidizing agent having a standard potential E° in the range of 0.1 V to 0.5 V measured relative to the standard hydrogen electrode at 25 °C and an effective concentration of 1 mol / l, and / or

[0020] An oxidation substrate of an oxidoreductase that oxidizes the oxidation substrate and reduces oxygen to hydrogen peroxide; and

[0021] b) A second reagent group, wherein the second reagent group contains the following reagent components:

[0022] b1) An enzyme of the hippuricase type, and optionally

[0023] b2) An oxidoreductase that oxidizes an oxidation substrate optionally included in the first reagent group as reagent component a2) and reduces oxygen to hydrogen peroxide; and

[0024] c) Aminopyrine of general formula (II),

[0025]

[0026] wherein, in general formula (II), R1 and R2 are each independently of the other C 1-4 alkyl or H.

[0027] The inventors have found that by using a relatively weak oxidizing agent in the first reagent group - the oxidizing agent having a standard potential E° in the range of 0.1 V to 0.5 V measured relative to the standard hydrogen electrode at 25 °C and an effective concentration of 1 mol / l, or by dividing the reagents used in ACE diagnostics into two separate reagent groups as defined above, a stable test system suitable for clinical routine applications is obtained, which test system provides extremely high sensitivity and is hardly affected by interference from other substances contained in the sample to be measured.

[0028] The first reagent group used according to the invention contains, in addition to the ACE-substrate hydroxybenzoyl tripeptide, a relatively weak oxidizing agent or an oxidation substrate of an oxidoreductase, and the second reagent group proposed according to the invention contains hippuricase; and in embodiments where the first reagent contains an oxidation substrate, it further additionally contains an oxidoreductase that oxidizes the oxidation substrate contained in the first reagent group and simultaneously reduces the oxygen contained in the reaction mixture to hydrogen peroxide.

[0029] In a specific embodiment of the invention, the first reagent contains both a relatively weak oxidizing agent and an oxidation substrate for the oxidoreductase contained in the second reagent.

[0030] In the reaction mixture formed by the first reagent group and the second reagent group, the hydroxybenzoyl tripeptide derived from the first reagent group is first converted by ACE contained in the sample into 4-hydroxyhippuric acid by cleaving two terminal amino acids, and this 4-hydroxyhippuric acid is then converted by the hippuricase contained in the second reagent group into 4-hydroxybenzoic acid by cleaving the amino acid glycine.

[0031] The 4-hydroxybenzoic acid in-situ generated by the above reaction condenses with the aminopyrine contained in the reaction mixture in the presence of the hydrogen peroxide in-situ formed in the reaction mixture to form a quinoneimine, which quinoneimine has a maximum absorption peak in the visible light range.

[0032] In certain embodiments, the conversion of the above 4-hydroxybenzoic acid and aminopyrine to form a quinoneimine is optionally carried out in the presence of peroxidase, thereby enhancing the rate of the condensation reaction and thus increasing the signal intensity. The peroxidase can be added to the reaction mixture as a component of the first reagent set or as a component of the second reagent set or as a separate, isolated component as needed.

[0033] The above reaction can be schematically represented as follows:

[0034]

[0035] The advantage of the method according to the invention is that, due to the reaction chemistry principle established by the inventors, the reagent components of the first reagent set can be premixed in a common reagent container, and the reagent components of the second reagent set can also be premixed in a common reagent container, and no reaction occurs between the reagent components even during a long storage period, which can avoid affecting the measurement results of the enzyme activity of ACE in the sample. Thus, the number of reagent containers to be handled in the diagnosis can be significantly reduced.

[0036] One of the obstacles to be overcome for this is that the ACE-substrate hydroxybenzoyl tripeptide is gradually converted by hippuricase during a long storage period, which correspondingly leads to distorted measurement results. Another problem to be solved is that if the oxidant required for the color reaction is a strong oxidant, the oxidant will destroy the enzyme activity during a long storage period when mixed with hippuricase, and a strong oxidant is especially required when a rapid oxidation reaction is needed.

[0037] The division of the ACE-substrate hydroxybenzoyl tripeptide and hippuricase into two reagent sets as proposed according to the invention prevents the gradual conversion of hydroxybenzoyl tripeptide by hippuricase during a long storage period.

[0038] Furthermore, by the arrangement of the reagents proposed according to the invention, or rather, by dividing the reagents into two reagent sets, it is prevented that the oxidant required for the color reaction will destroy the enzyme used. This is achieved in the following ways: either a relatively weak oxidant is used in the first reagent set, or hydrogen peroxide is in-situ generated as an oxidant immediately before photometric measurement by a suitable oxidase / substrate pair. For the in-situ generated oxidant, the first reagent set contains the substrate for the oxidase, and the oxidase is located in the second reagent set.

[0039] The high storage stability of the premixed reagent sets used according to the invention is in the range of 12 to 24 months, where the high storage stability can be understood as that when stored at a temperature in the range of 2 to 8 °C for a period of 12 to 24 months from the date of manufacture, the maximum deviation of the sensitivity of the test system according to the invention from the sensitivity of the test system measured immediately after manufacture is 5%.

[0040] In order to keep the number of reagent containers to be processed during the implementation of the method according to the invention as small as possible, in a specific embodiment of the test system according to the invention, either the two reagent components a1) and a2) of the first reagent group are respectively placed in a common reagent container, or the two reagent components b1) and b2) of the second reagent group are respectively placed in a common reagent container. In a specific embodiment, the reagent components of the first reagent group are placed in a common first reagent container, while the reagent components of the second reagent group are placed in a common second reagent container.

[0041] The additional reagent aminopyrine can either be placed in a separate third reagent container ("ready-to-use" three-component system), or can be a component of the reagent mixture in the reagent container of the first or second reagent group ("ready-to-use" two-component system).

[0042] In principle, the reagent components of the first reagent group and the second reagent group can contact the ACE contained in the sample in any order to generate a reaction mixture. In an embodiment of the invention, the (preferably premixed) reagent components of the first reagent group are first added to the reaction mixture, and then the (preferably premixed) reagent components of the second reagent group are added. For this order, particularly high sensitivity has been found. In an alternative embodiment of the invention, the (preferably premixed) reagent components of the second reagent group are first added to the reaction mixture, and then the (preferably premixed) reagent components of the first reagent group are added. Very good sensitivity has also been found for this order, although the sensitivity is slightly lower than that of the previous order.

[0043] In the system described by the above chemical reaction formula, 4-hydroxybenzoyl-glycine-histidine-leucine is used as the substrate for ACE contained in the sample. In certain embodiments of the invention, the hydroxybenzoyl tripeptide of the reagent component a1) of the first reagent group is selected from:

[0044] -N-(4-hydroxybenzoyl)-glycyl-histidyl-leucine,

[0045] -N-(4-hydroxy-3-methoxybenzoyl)-glycyl-glycyl-glycine,

[0046] -N-(4-hydroxy-3-methoxybenzoyl)-glycyl-histidyl-leucine,

[0047] -N-(3-chloro-4-hydroxybenzoyl)-glycyl-glycyl-glycine,

[0048] -N-(3,5-dimethoxy-4-hydroxybenzoyl)-glycyl-histidyl-leucine,

[0049] -N-(3,5-dibromo-4-hydroxybenzoyl)-glycyl-glycyl-glycine,

[0050] -N-(3,5-dichloro-4-hydroxybenzoyl)-glycyl-histidyl-leucine,

[0051] -N-(3-hydroxy-2,4,6-triiodobenzoyl)-glycyl-histidyl-leucine,

[0052] -N-(4-hydroxy-3-methoxybenzoyl)-glycyl-alanyl-phenylalanine,

[0053] -N-(3-chloro-4-hydroxybenzoyl)-glycyl-alanyl-phenylalanine,

[0054] -N-(3,5-dimethoxy-4-hydroxybenzoyl)-glycyl-alanyl-phenylalanine.

[0055] In some embodiments of the present invention, in order to generate hydrogen peroxide in situ, in the presence of oxygen in the reaction mixture, glucose already contained in the sample or added to the reaction mixture is converted by glucose oxidase into gluconic acid and hydrogen peroxide. In alternative specific embodiments of the present invention, the oxidoreductase of reagent component b2) and its oxidation substrate of reagent component a2) are selected from the following combinations:

[0056] -cholesterol oxidase / cholesteryl chloride

[0057] -choline oxidase / choline chloride

[0058] -lactate oxidase / lactic acid.

[0059] In these cases, the corresponding oxidation substrate can either be naturally contained in the sample or be added to the reaction mixture purposefully, or both.

[0060] In the test system schematically described above, the aminopyrine set for condensation reaction with 4-hydroxybenzoic acid is 4-aminoantipyrine.

[0061] In some embodiments of the method according to the present invention, the method comprises a relatively weak oxidant as reagent component a2) in the first reagent group. The term "weak oxidant" can be understood herein as an oxidant having a standard potential E° in the range of 0.1 V to 0.5 V measured at 25 °C and with an effective concentration of 1 mol / l relative to the standard hydrogen electrode.

[0062] In certain embodiments of the method according to the present invention, the relatively weak oxidant optionally comprised as reagent component a2) in the first reagent group is selected from:

[0063] - Copper(II) sulfate tetraammine

[0064] - Molybdate containing divalent metal ions

[0065] - Potassium hexacyanoferrate(III)

[0066] - Hexachlororhodate(III)

[0067] - Manganese

[0068] - Oxalic acid

[0069] - Sulfate, thiosulfate and nitrite.

[0070] Typically, the sample analyzed using the method according to the invention is a liquid sample containing an amount of ACE to be determined. In most cases, the sample is a body fluid containing soluble ACE, and depending on the specific application, the body fluid can be selected from blood, serum, plasma or cerebrospinal fluid. However, in special embodiments, the liquid sample can also be a liquid sample obtained from other biological materials, in which initially membrane-bound ACE has been mobilized.

[0071] At the end of the method according to the invention, the absorbance is usually measured in the visible wavelength range. Depending on the different aminopyrine used in the method according to the invention, the suitable range for absorbance measurement is also different. Ideally, in order to achieve the highest possible sensitivity and accuracy, the measurement should be carried out in the range of 5 - 50 nm near the maximum absorbance of the quinoneimine produced in the reaction. In certain embodiments, the absorbance is measured in the wavelength range of 480 - 530 nm.

[0072] It has been demonstrated that the method according to the invention has extremely high sensitivity, so that even a very small amount of sample is sufficient to measure the enzyme activity of ACE. In certain embodiments of the invention, the sample volume is in the range of 0.1 μl - 100 μl. In a preferred embodiment, the sample volume is less than 20 μl; in a particularly preferred embodiment, the sample volume < 10 μl, < 5 μl or even < 1 μl.

[0073] The method according to the invention can determine the enzyme activity extremely accurately within a relatively wide range. In certain embodiments, the enzyme activity determined using this method is in the range of 0.1 U / L - 250 U / L. In a preferred embodiment, the enzyme activity range < 20 U / L, in a more preferred embodiment, the enzyme activity < 10 U / L, < 5 U / L or even < 1 U / L.

[0074] The method for determining the enzymatic activity of ACE according to the present invention can be carried out in a patient sample for pharmacological analysis or for diagnosis or progression monitoring. Preferably, the analysis using the method according to the present invention is directed to patients having at least one of the following clinical manifestations or symptoms or having at least one of the following physiological conditions:

[0075] Hypertension, chronic heart failure, neurosarcoidosis, leprosy, Gaucher's disease, tuberculosis, preeclampsia, chronic beryllium poisoning, proliferative retinopathy, HIV infection, pregnancy, chronic fatigue syndrome, cancer, Alzheimer's disease, kidney disease, fibrosis, COVID-19.

[0076] To carry out the method according to the present invention, the present invention also provides an analytical or diagnostic test system, wherein the test system is characterized in that it comprises a first reagent group and a second reagent group, wherein the first reagent group comprises at least the following reagent components:

[0077] a1) A hydroxybenzoyl tripeptide of general formula (I) as an ACE-substrate,

[0078]

[0079] wherein, in general formula (I),

[0080] -R 1 is an alkoxy group, a halogen atom or an unsubstituted amino group; 1-4 -R

[0081] and R 2 and R 3 are each independently an alkoxy group, a halogen atom or H; 1-4 -A is selected from the amino acid groups His, Gly or Ala, and

[0082] -B is selected from the amino acid groups Leu, Gly or Phe,

[0083]

[0084] and

[0085] a2) An oxidizing agent having a standard potential E° in the range of 0.1 V to 0.5 V measured relative to the standard hydrogen electrode at 25 °C and an effective concentration of 1 mol / l, and / or

[0086] An oxidation substrate of an oxidoreductase that oxidizes the oxidation substrate and reduces oxygen to hydrogen peroxide;

[0087] And the second reagent group comprises at least the following reagent components:

[0088] b1) An enzyme of the hippuricase type, and optionally

[0089] b2) An oxidoreductase that oxidizes an oxidation substrate optionally included as reagent component a2) in the first reagent group and reduces oxygen to hydrogen peroxide;

[0090] Wherein, the first reagent group, or the second reagent group, or a reagent additionally provided in the test system contains aminopyrine of general formula (II)

[0091]

[0092] Wherein, in general formula (II), R1 and R2 are each independently of the other C 1-4 alkyl or H.

[0093] Herein, the term "reagent group" can be understood as: a reagent group always contains at least two liquid or solid chemical substances participating in the chemical reaction according to the method of the present invention as reagent components, wherein the individual reagent components of the reagent group can either exist separately in different reagent containers or can exist together at least partially in the form of a reagent mixture in the same reagent container, provided that the reagent components of the first reagent group will never be mixed with the reagent components of the second reagent group.

[0094] In addition to the reagent components of the first reagent group and the second reagent group, the test system of the present invention further contains the above-defined aminopyrine, which is either in a separate reagent container or as a component of reagent group 1 or as a component of reagent group 2.

[0095] In addition to the reagent components of the first reagent group and the second reagent group, the test system of the present invention further contains the above peroxidase, which is either in a separate reagent container or as a component of reagent group 1 or as a component of reagent group 2.

[0096] In a specific embodiment of the test system according to the present invention, the reagent components of the first reagent group are present in a common reagent container, or the reagent components of the second reagent group are present in a common reagent container. In a specific embodiment, the reagent components of the first reagent group are present in a common first reagent container, while the reagent components of the second reagent group are present in a common second reagent container.

[0097] The additional reagent aminopyrine can either be placed in a separate third reagent container or as a component of the reagent mixture in the reagent containers of the first or second reagent group.

[0098] In a particularly advantageous embodiment of the test system according to the invention, the two reagent components a1) and a2) of the first reagent group are present in a premixed manner as components of a first liquid reagent mixture in a first reagent container, and / or the two reagent components b1) and b2) of the second reagent group are present in a premixed manner as components of a second liquid reagent mixture in a second reagent container.

[0099] In a specific embodiment of the invention, the reagent components are provided as components of a liquid reagent mixture, and this specific embodiment is characterized in that the reagent mixture contains a buffer, and the buffers are independently selected from PIPES, MES, MOPS, HEPES, BIS-TRIS-propane, TRIS, AMPSO, borate, TABS and TAPS, wherein HEPES is preferred in the case of the first liquid reagent mixture, and PIPES is preferred in the case of the second liquid reagent mixture.

[0100] In some embodiments of the invention, it is also possible to further enhance the signal intensity by adding monovalent metal ions (such as K + , Na + , NH4 + ) and / or divalent metal ions (such as Mg 2+ , Ca 2+ , Ba 2+ , Cu 2+ , Fe2+, Zn 2+ ) and / or by adding mild detergents (such as Triton, Tergitol, CTaB, SDS, Thesit and Tween), wherein nonionic surfactants such as Triton and Tergitol are preferred.

[0101] A specific embodiment of the invention having a premixed liquid reagent mixture of the first reagent group and / or the second reagent group is characterized in that the pH value of the reagent mixture of the first reagent group is preferably in the range of 6.0 to 9.0 and particularly preferably in the range of 7.5 to 9.0, and / or the pH value of the second reagent mixture of the second reagent group is preferably in the range of 6.0 to 9.0 and particularly preferably in the range of 6.5 to 8.5.

[0102] In certain embodiments of the invention, the components of the first reagent group are mixed and used in the following proportions for each component:

[0103]

[0104] In certain embodiments of the invention, the components of the second reagent group are mixed and used in the following proportions for each component:

[0105]

[0106] In certain embodiments, the analytical or diagnostic test system of the present invention includes at least one control solution and / or at least one calibration solution, wherein the control solution and / or calibration solution preferably contains human plasma and / or sodium azide as matrix components, and optionally additionally contains 6-aminocaproic acid and / or calcium chloride.

[0107] In certain embodiments, the first and / or second liquid reagent mixture contains a preservative combination, which contains at least gentamicin sulfate and optionally one of amphotericin and onium 46.

[0108] For the purpose of the original disclosure, it should be noted that all features that are obvious to those skilled in the art from this specification and the claims, even if they are specifically described only in combination with certain other features, can be combined alone or in any arrangement with other features or groups of features disclosed herein, unless such combination is explicitly excluded or chemical, physicochemical or pharmacological conditions make such combination impossible or meaningless. For the sake of brevity and readability of the specification, a comprehensive and explicit description of all possible feature combinations is omitted herein.

[0109] Furthermore, it should be noted that those skilled in the art should understand that the following examples are only used to exemplarily illustrate the possible embodiments of the present invention reproduced as examples. Therefore, those skilled in the art will easily understand that all other embodiments having the features or combinations of features according to the present invention mentioned in the claims are also within the protection scope of the present invention. For the sake of brevity and readability of the specification, a comprehensive and explicit description of all conceivable embodiments is omitted herein. Detailed Description

[0110] In one embodiment of the present invention, the components of the first reagent group are used in a mixed form, and the proportions of the respective components are as follows:

[0111]

[0112] In one embodiment of the present invention, the components of the second reagent group are used in a mixed form, and the proportions of the respective components are as follows:

[0113]

[0114] In one embodiment of the present invention, the usage of the first reagent group and the second reagent group is as follows:

[0115]

[0116] The method according to the present invention is compared with an in-house test according to an alternative FAPGG method and a conventional hippuricase test employed by competitors, and the following results are obtained:

[0117]

Claims

1. A method for quantitatively measuring the enzymatic activity of angiotensin converting enzyme (ACE) in a sample, wherein, A reaction mixture is produced by contacting the ACE contained in a sample with the following substances: a) A first reagent group, wherein the first reagent group contains the following reagent components: a1) A hydroxybenzoyl tripeptide of general formula (I) as an ACE-substrate Wherein, in general formula (I), -R 1 is C 1-4 an alkoxy group, a halogen atom or an unsubstituted amino group; -R 2 and R 3 are each independently C 1-4 alkoxy, a halogen atom or H; - A is selected from the amino acid groups His, Gly or Ala, and - B is selected from the amino acid groups Leu, Gly or Phe, a2) An oxidizing agent having a standard potential E° in the range of 0.1 V to 0.5 V measured relative to the standard hydrogen electrode at 25 °C and an effective concentration of 1 mol / l, and / or An oxidation substrate of an oxidoreductase, which oxidizes the oxidation substrate and reduces oxygen to hydrogen peroxide; and b) A second reagent group, wherein the second reagent group contains the following reagent components: b1) An enzyme of the hippuricase type, and optionally b2) An oxidoreductase that oxidizes the oxidation substrate optionally contained in the first reagent group as reagent component a2) and reduces oxygen to hydrogen peroxide; and c) Aminopyrine of general formula (II), Among them, in general formula (II), R1 and R2 are each independently of the other C 1-4 alkyl or H.

2. The method according to claim 1, wherein The hydroxybenzoyl tripeptide of reagent component a1) is selected from - N-(4-Hydroxybenzoyl)-glycyl-histidyl-leucine, - N-(4-Hydroxy-3-methoxybenzoyl)-glycyl-glycyl-glycine, - N-(4-Hydroxy-3-methoxybenzoyl)-glycyl-histidyl-leucine, - N-(3-Chloro-4-hydroxybenzoyl)-glycyl-glycyl-glycine, - N-(3,5-Dimethoxy-4-hydroxybenzoyl)-glycyl-histidyl-leucine, - N-(3,5-Dibromo-4-hydroxybenzoyl)-glycyl-glycyl-glycine, - N-(3,5-Dichloro-4-hydroxybenzoyl)-glycyl-histidyl-leucine, - N-(3-Hydroxy-2,4,6-triiodobenzoyl)-glycyl-histidyl-leucine, - N-(4-Hydroxy-3-methoxybenzoyl)-glycyl-alanyl-phenylalanine, - N-(3-Chloro-4-hydroxybenzoyl)-glycyl-alanyl-phenylalanine, - N-(3,5-Dimethoxy-4-hydroxybenzoyl)-glycyl-alanyl-phenylalanine.

3. The method according to any one of claims 1 and 2, characterized in that, The oxidoreductase of reagent component b2) and its oxidation substrate of reagent component a2) are selected from the following combinations: - Glucose oxidase / glucose - Cholesterol oxidase / cholesteryl chloride - Choline oxidase / choline chloride - Lactate oxidase / lactic acid.

4. The method according to any one of claims 1 to 3, characterized in that The aminopyrine is 4-aminoantipyrine.

5. The method according to any one of claims 1 to 4, characterized in that, The oxidizing agent optionally contained in the first reagent group as reagent component a2) is selected from: - Tetraamminecopper(II) sulfate - Molybdate containing divalent metal ions - Potassium hexacyanoferrate(III) - Potassium hexachlororhodate(III) - Manganese - Oxalic acid - Sulfates, thiosulfates and nitrites.

6. The method according to any one of claims 1 to 4, characterized in that The sample for measuring the enzyme activity of ACE therein is a liquid sample, and the liquid sample is selected from blood, serum, plasma or cerebrospinal fluid.

7. The method according to any one of claims 1 to 5, characterized in that The absorbance of the reaction mixture is measured in the wavelength range of 480 to 530 nm.

8. The method according to any one of claims 1 to 6, characterized in that The amount of the ACE-containing sample used in generating the reaction mixture ranges from 0.1 μl to 100 μl and is preferably <20 μl, <10 μl, <5 μl or even <1 μl, and / or the enzymatic activity of ACE in the sample ranges from 0.1 U / L to 250 U / L and is preferably <20 U / L, <10 U / L, <5 U / L or even <1 U / L.

9. The method according to any one of claims 1 to 8, characterized in that For the purpose of pharmacological analysis or for diagnosis or progress monitoring, the enzymatic activity of ACE is measured in samples from patients with at least one of the following clinical manifestations or symptoms or with at least one of the following physiological conditions: hypertension, chronic heart failure, neurosarcoidosis, leprosy, Gaucher's disease, tuberculosis, preeclampsia, chronic beryllium poisoning, proliferative retinopathy, HIV infection, pregnancy, chronic fatigue syndrome, cancer, Alzheimer's disease, kidney disease, fibrosis, COVID-19.

10. An analytical or diagnostic test system for quantitatively measuring the enzymatic activity of angiotensin converting enzyme (ACE) in a sample, characterized in that, The test system comprises a first reagent group and a second reagent group, wherein the first reagent group at least comprises the following reagent components: a1) a hydroxybenzoyl tripeptide of general formula (I) as an ACE-substrate, wherein, in general formula (I), -R 1 is C 1-4 an alkoxy group, a halogen atom or an unsubstituted amino group; -R 2 and R 3 are each independently C 1-4 alkoxy, a halogen atom or H; -A is selected from the amino acid groups His, Gly or Ala, and -B is selected from the amino acid groups Leu, Gly or Phe, and a2) an oxidizing agent having a standard potential E° in the range of 0.1 V to 0.5 V measured relative to the standard hydrogen electrode at 25 °C and an effective concentration of 1 mol / l, and / or an oxidation substrate of an oxidoreductase, which oxidizes the oxidation substrate and reduces oxygen to hydrogen peroxide; and the second reagent group at least comprises the following reagent components: b1) an enzyme of the hippuricase type, and b2) an oxidoreductase, which oxidizes the oxidation substrate optionally comprised in the first reagent group as reagent component a2) and reduces oxygen to hydrogen peroxide; wherein the aminopyrine of general formula (II) is comprised in the first reagent group or the second reagent group or a reagent additionally provided in the test system Among them, in general formula (II), R1 and R2 are each independently C 1-4 alkyl or H.

11. The analysis or diagnostic test system according to claim 10, characterized in that, The two reagent components a1) and a2) of the first reagent group are present in a first reagent container in a premixed manner as components of a first liquid reagent mixture, and / or the two reagent components b1) and b2) of the second reagent group are present in a second reagent container in a premixed manner as components of a second liquid reagent mixture.

12. The analysis or diagnostic test system according to claim 11, characterized in that, The first liquid reagent mixture and the second liquid reagent mixture comprise a buffer, which is independently selected from PIPES, MES, MOPS, HEPES, BIS-TRIS-propane and TAPS, wherein HEPES is preferably used in the case of the first liquid reagent mixture, and PIPES is preferably used in the case of the second liquid reagent mixture.

13. The analysis or diagnostic test system according to any one of claims 11 and 12, characterized in that The pH value of the first liquid reagent mixture ranges from 8.0 to 8.5, and / or the pH value of the second liquid reagent mixture ranges from 7.0 to 9.

0.

14. The analysis or diagnostic test system according to any one of claims 10 to 13, characterized in that, The analysis or diagnostic test system comprises at least one control solution and / or at least one calibration solution, wherein the control solution and / or the calibration solution preferably comprise human plasma and / or sodium azide as matrix components, and optionally additionally comprise 6-aminocaproic acid and / or calcium chloride.

15. The analysis or diagnostic test system according to any one of claims 10 to 14, characterized in that The first liquid reagent mixture and / or the second liquid reagent mixture comprise a preservative combination, the preservative combination comprising at least gentamicin sulfate and optionally comprising one of amphotericin and onium 46.

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