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

Abstract

In order to provide a method for quantitatively determining the enzymatic activity of angiotensin-converting enzyme (ACE) that is as sensitive as possible, is hardly affected by spectral interferences, and can be practically automated on standard analytical equipment for routine clinical use, the present invention proposes a method for quantitatively determining the enzymatic activity of angiotensin-converting enzyme (ACE) in a sample, which comprises contacting the ACE contained in the sample with a first reagent set comprising a hydroxybenzoyl tripeptide and a weak oxidizing agent and / or an oxidation substrate for an oxidoreductase, contacting this with a second reagent set comprising hippuricase and optionally an oxidoreductase, and contacting this with aminopyrine to prepare a reaction mixture.

Description

[Technical Field]

[0001] The present invention relates to an analytical or diagnostic test system for quantitatively measuring the enzymatic activity of angiotensin-converting enzyme (ACE) in a sample. In particular, the present invention relates to a method for quantitatively measuring the enzymatic activity and a test system comprising various reagents for carrying out the method. [Background technology]

[0002] Angiotensin-converting enzyme (synonyms: ACE, kininase II, peptidyl-dipeptidase A), the subject of 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 and exerts vasoconstriction, resulting in an increase in blood pressure and extracellular volume.

[0003] ACE is present in a membrane-bound form on the luminal surface of endothelial cells, primarily in the lungs, with smaller amounts present in the brain, intestine, kidneys, adrenal glands, and testes. In the nervous system, high concentrations of ACE are found in the choroid plexus. In addition to the membrane-bound form, ACE also exists in a free, soluble form in plasma and other body fluids (e.g., amniotic fluid, spinal fluid). The soluble form of ACE is generated by proteolysis of the C-terminal membrane anchor.

[0004] ACE activity in the blood is measured for purposes including the diagnosis and monitoring of granulomatous lung diseases such as tuberculosis and sarcoidosis. ACE diagnostics are also used in connection with the diagnostic investigation of leprosy and Gaucher disease, and various forms of preeclampsia. Summary of the Invention

[0005] ACE content in serum is usually measured using one of two test systems:

[0006] The FAPGG method uses the peptide FAPGG as a substrate. This peptide is hydrolyzed by the patient's own ACE in the sample, resulting in a decrease in absorbance in the UV region. However, due to the measurement wavelength in the UV region, tests based on the FAPGG method are highly susceptible to spectral interference from endogenous bilirubin and hemoglobin or lipids.

[0007] In contrast, in the so-called "Hippuricase method," the synthetic substrate p-hydroxybenzoylglycyl-L-histidyl-L-leucine is decomposed to p-benzoic acid by the Hippuricase enzyme provided by the reagent and ACE from the sample. The resulting p-hydroxybenzoic acid is converted to a dye by the Trinder reaction, and its absorbance is measured at 505 nm.

[0008] Measurement of the photometric signal in the visible light (VIS) region allows for ACE measurements that are somewhat more sensitive and less susceptible to spectral interferences than the FAPGG method. However, this test system typically consists of five separate reagent components, some of which are lyophilized, making it difficult to automate practically for routine clinical use on many common analyzers.

[0009] Against this background, the inventors of the present application set themselves the task of providing a method for the quantitative determination of ACE enzymatic activity that is as sensitive as possible, is largely unaffected by spectral interferences, and can be practically automated for routine clinical use on conventional analytical equipment. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention provides a) a first reagent set comprising the following reagent components: a1) Hydroxybenzoyl tripeptidyls of general formula (I) as ACE substrates [ka] [In the general formula (I), R 1 is C 1-4an alkoxy group, a halogen atom, or an unsubstituted amino group; R 2 and R 3 are independent of each other, C 1-4 an alkoxy group, a halogen atom, or H; A is selected from the amino acid group His, Gly, or Ala; 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 to 0.5 V measured against a standard hydrogen electrode at 25°C and an effective concentration of 1 mol / L, and / or an oxidizing substrate of an oxidoreductase that oxidizes the oxidizing substrate and reduces oxygen to hydrogen peroxide; and b) a second reagent set comprising the following reagent components: b1) enzymes of the hippuricase type, and Optionally, b2) an oxidoreductase that oxidizes the oxidizable substrate optionally included in the first set of reagents as reagent component a2) and reduces oxygen to hydrogen peroxide; and c) Aminopyrine of general formula (II) [ka] [In the general formula (II), R1 and R2 each independently represent C 1-4 is an alkyl group or H. The present invention discloses a method for quantitatively measuring the enzymatic activity of angiotensin-converting enzyme (ACE) in a sample, which comprises contacting ACE contained in the sample with a reaction mixture to prepare a reaction mixture.

[0011] The inventors have discovered that this results in the use of a relatively weak oxidizing agent in the first reagent set, having a standard potential E° in the range of 0.1 to 0.5 V measured against a standard hydrogen electrode at 25°C and an effective concentration of 1 mol / L, or by dividing the reagents used in the ACE diagnostic method into two separate reagent sets as defined above, a stable test system is obtained which is suitable for routine clinical use, provides very high sensitivity and can be performed with little interference from other substances contained in the sample being tested.

[0012] The first reagent set used in the present invention contains, in addition to the ACE substrate hydroxybenzoyl tripeptide, either a relatively weak oxidizing agent or an oxidation substrate for oxidoreductase. The second reagent set proposed in the present invention contains the enzyme hippuricase, and in embodiments in which the first reagent contains an oxidation substrate, it further contains an oxidoreductase that oxidizes the oxidation substrate contained in the first reagent set, thereby reducing oxygen contained in the reaction mixture to hydrogen peroxide.

[0013] In particular embodiments of the invention, the first reagent comprises both a relatively weak oxidizing agent and an oxidizable substrate for the oxidoreductase contained in the second reagent.

[0014] In the reaction mixture formed from the first and second reagent sets, the hydroxybenzoyl tripeptide derived from the first reagent set is first converted to 4-hydroxyhippuric acid by ACE contained in the sample through cleavage of the two terminal amino acids, and then converted to 4-hydroxybenzoic acid by hippuricase contained in the second reagent set through cleavage of the amino acid glycine.

[0015] The 4-hydroxybenzoic acid produced in situ by the above reaction condenses with aminopyrine contained in the reaction mixture in the presence of hydrogen peroxide produced in situ in the reaction mixture to form a quinoneimine with an absorption maximum in the visible light (VIS) region.

[0016] In certain embodiments, the conversion of 4-hydroxybenzoic acid to quinoneimine with aminopyrine is optionally carried out in the presence of peroxidase, which increases the reaction rate of the condensation reaction and therefore the signal intensity. The peroxidase can be added to the reaction mixture as a component of the first set of reagents, as a component of the second set of reagents, or as a separate component, as needed.

[0017] The above reaction can be represented schematically as follows:

[0018] [ka]

[0019] The method of the present invention has the advantage that, thanks to the reaction chemistry established by the inventors, the reagent components of the first reagent set can be premixed in standard reagent containers, and the reagent components of the second reagent set can also be premixed in standard reagent containers, without the reagent components reacting with each other even during long-term storage. If the reagent components react with each other during long-term storage, this could affect the results of measuring ACE enzyme activity in the sample. This significantly reduces the number of reagent containers that need to be handled for diagnosis.

[0020] One problem to be overcome was that the ACE substrate, hydroxybenzoyl tripeptide, is slowly converted by the enzyme hippuricase during long-term storage, leading to inaccurate measurements. Another problem to be solved was that the oxidizing agent required for the color reaction, which is a strong oxidizing agent especially when a fast oxidation reaction is desired, impairs the activity of the enzyme when mixed with hippuricase during long-term storage.

[0021] As proposed according to the present invention, by dividing the ACE substrate hydroxybenzoyl tripeptide and the enzyme hippuricase into two reagent sets, the gradual conversion of hydroxybenzoyl tripeptide by the enzyme hippuricase during long-term storage is prevented.

[0022] Furthermore, the proposed reagent composition or division of the reagents into two reagent sets prevents the oxidizing agent required for the color reaction from destroying the enzyme used. This can be achieved by using a relatively weak oxidizing agent in the first reagent set or by generating hydrogen peroxide in situ as an oxidizing agent using an appropriate oxidase / substrate combination immediately before spectrophotometric measurement. When generating an oxidizing agent in situ, the first reagent set contains a substrate for the oxidase, and the oxidase is contained in the second reagent set.

[0023] The high storage stability of the premixed reagent sets used in the present invention is in the range of 12 to 24 months, where high storage stability is understood to mean that the sensitivity of the test system of the present invention varies by a maximum of 5% from the sensitivity of the test system immediately after manufacture when stored at a temperature of 2 to 8°C for 12 to 24 months from manufacture.

[0024] In order to minimize the number of reagent containers that need to be handled when carrying out the method of the present invention, in specific embodiments of the test system of the present invention, the two reagent components a1) and a2) of the first reagent set are each provided in a common reagent container, or the two reagent components b1) and b2) of the second reagent set are each provided in a common reagent container. In particular embodiments, the reagent components of the first reagent set are present in a common first reagent container, and the reagent components of the second reagent set are present in a common second reagent container.

[0025] The additional reagent aminopyrine can be provided in a separate third reagent container (a "ready-to-use" three-component system) or can be part of the reagent mixture in the reagent container of the first or second reagent set (a "ready-to-use" two-component system).

[0026] The reagent components of the first and second reagent sets can be contacted with the ACE contained in the sample in essentially any order to form a reaction mixture. In one embodiment of the present invention, the (preferably premixed) reagent components of the first reagent set are added to the reaction mixture first, followed by the (preferably premixed) reagent components of the second reagent set. This order has been found to provide particularly high sensitivity. In an alternative embodiment of the present invention, the (preferably premixed) reagent components of the second reagent set are added to the reaction mixture first, followed by the (preferably premixed) reagent components of the first reagent set. This order also provides very good sensitivity, although slightly lower than the first procedure described above.

[0027] In the system represented by the above chemical formula, 4-hydroxybenzoyl-glycine-histidine-leucine serves as a substrate for ACE contained in the sample. In a specific embodiment of the present invention, the hydroxybenzoyl tripeptide of the reagent component a1) of the first reagent set is N-(4-hydroxybenzoyl)glycylhistidylleucine, N-(4-hydroxy-3-methoxybenzoyl)glycylglycylglycine, N-(4-hydroxy-3-methoxybenzoyl)glycylhistidylleucine, N-(3-chloro-4-hydroxybenzoyl)glycylglycylglycine, N-(3,5-dimethoxy-4-hydroxybenzoyl)glycylhistidylleucine, N-(3,5-dibromo-4-hydroxybenzoyl)glycylglycylglycine, N-(3,5-dichloro-4-hydroxybenzoyl)glycylhistidylleucine, N-(3-hydroxy-2,4,6-triiodobenzoyl)glycylhistidylleucine, N-(4-hydroxy-3-methoxybenzoyl)glycylalanylphenylalanine, N-(3-chloro-4-hydroxybenzoyl)glycylalanylphenylalanine, N-(3,5-dimethoxy-4-hydroxybenzoyl)glycylalanylphenylalanine is selected from.

[0028] In some embodiments of the present invention, glucose already present in the sample or glucose added to the reaction mixture is converted to gluconic acid and hydrogen peroxide by glucose oxidase in the presence of oxygen present in the reaction mixture, in order to generate hydrogen peroxide in situ. In an alternative particular embodiment of the present invention, the oxidoreductase of reagent component b2) and its oxidized substrate of reagent component a2) are Cholesterol oxidase / cholesterol chloride Choline oxidase / choline chloride Lactate oxidase / lactic acid is selected from a combination of:

[0029] Again, the respective oxidizable substrate may already be naturally present in the sample, may be specifically added to the reaction mixture, or both.

[0030] In the test system shown schematically above, the aminopyrine intended for condensation reaction with 4-hydroxybenzoic acid is 4-aminoantipyrine.

[0031] In some embodiments of the method according to the present invention, the first reagent set comprises as reagent component a2) a relatively weak oxidizing agent, where the term "weak oxidizing agent" refers to an oxidizing agent having a standard potential E° in the range of 0.1 to 0.5 V measured against a standard hydrogen electrode at 25°C and an effective concentration of 1 mol / L.

[0032] In a particular embodiment of the method according to the invention, the relatively weak oxidizing agent optionally included in the first set of reagents as reagent component a2) is Tetraaminecopper(II) sulfate Molybdates containing divalent metal ions Potassium hexacyanoferrate(III) Hexachlororhodate(III) ·manganese Oxalic acid Sulfates, thiosulfates, and nitrites is selected from.

[0033] Typically, the sample analyzed using the method of the present invention is a liquid sample containing the amount of ACE to be measured. In most cases, this is a body fluid containing soluble ACE, which can be selected from blood, serum, plasma, or cerebrospinal fluid depending on the specific application. However, in a special embodiment, it can also be a liquid sample obtained from other biological materials in which the ACE originally bound to the membrane has been released.

[0034] At the end of the process according to the present invention, absorbance is typically measured in the visible wavelength range. The wavelength range suitable for absorbance measurement varies depending on the type of aminopyrine used in the process according to the present invention. Ideally, to obtain the highest possible sensitivity and accuracy, measurements are performed in the 5-50 nm range near the absorption maximum of the quinoneimine produced during the reaction. In a specific embodiment, the wavelength at which absorbance is measured is in the 480-530 nm range.

[0035] The sensitivity of the method of the present invention is extremely high, and it has been shown that even very small amounts of sample are sufficient to measure the enzymatic activity of ACE. In certain embodiments of the present invention, the amount of sample used ranges from 0.1 to 100 μL. In preferred embodiments, less than 20 μL of sample is used, and in particularly preferred embodiments, the amount of sample used is less than 10 μL, less than 5 μL, or even less than 1 μL.

[0036] The method of the present invention allows for highly accurate measurement of enzyme activity over a relatively wide range. In certain embodiments, the range of enzyme activity measured by the method is 0.1 to 250 U / L. In preferred embodiments, the range of enzyme activity is less than 20 U / L, and in preferred embodiments, the enzyme activity is less than 10 U / L, less than 5 U / L, or even less than 1 U / L.

[0037] The method of the invention for measuring ACE enzyme activity can be carried out in patient samples for pharmacological analytical purposes or for diagnostic or follow-up purposes. Preferably, the analysis using the method of the invention is carried out in patients with at least one of the following clinical features or symptoms or at least one of the following physiological conditions: Hypertension, chronic heart failure, neurosarcoidosis, leprosy, Gaucher disease, tuberculosis, preeclampsia, chronic beryllium disease, proliferative retinopathy, HIV infection, pregnancy, chronic fatigue syndrome, cancer, Alzheimer's disease, nephropathy, fibrosis, COVID-19.

[0038] For the purpose of carrying out the method according to the invention, the invention also provides a first reagent set and a second reagent set; The first reagent set includes at least the following reagent components: a1) Hydroxybenzoyl tripeptides of general formula (I) as ACE substrates [ka] [In the 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 independent of each other, C 1-4 an alkoxy group, a halogen atom, or H; A is selected from the amino acid group His, Gly, or Ala; 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 to 0.5 V measured against a standard hydrogen electrode at 25°C and an effective concentration of 1 mol / L, and / or an oxidizing substrate of an oxidoreductase that oxidizes the oxidizing substrate and reduces oxygen to hydrogen peroxide; The second reagent set includes at least the following reagent components: b1) enzymes of the hippuricase type, and Optionally, b2) an oxidoreductase that oxidizes the oxidation substrate optionally included in the first set of reagents as reagent component a2) and reduces oxygen to hydrogen peroxide; Either the first reagent set or the second reagent set, or a reagent additionally provided in the test system, comprises an aminopyrine of general formula (II): [ka] [In the general formula (II), R1 and R2 each independently represent C 1-4 is an alkyl group or H. The present invention provides an analytical or diagnostic test system, characterized in that:

[0039] The term "reagent set" is understood here to mean that a reagent set always comprises at least two liquid or solid chemicals as reagent components that are involved in the chemical reactions of the method of the invention, the individual reagent components of a reagent set being present separately in different reagent containers or together in one and the same reagent container at least partially in the form of a reagent mixture, provided that the reagent components of a first reagent set are never present mixed with the reagent components of a second reagent set.

[0040] In addition to the reagent components of the first reagent set and the reagent components of the second reagent set, the test system of the present invention comprises aminopyrine as defined above in a separate reagent container or as a component of Reagent Set 1 or as a component of Reagent Set 2.

[0041] In addition to the reagent components of the first reagent set and the reagent components of the second reagent set, the test system of the present invention includes the peroxidase described above in a separate reagent container or as a component of Reagent Set 1 or as a component of Reagent Set 2.

[0042] In certain embodiments of the testing system of the present invention, the reagent components of the first reagent set are contained in a common reagent container, or the reagent components of the second reagent set are contained in a common reagent container, and in particular embodiments, the reagent components of the first reagent set are provided in a common first reagent container and the reagent components of the second reagent set are provided in a common second reagent container.

[0043] The additional reagent, aminopyrine, can be provided in a separate third reagent container or can be part of the reagent mixture in the reagent container of the first or second reagent set.

[0044] In a particularly advantageous embodiment of the test system according to the invention, the two reagent components a1) and a2) of the first reagent set are premixed in a first reagent container as components of a first liquid reagent mixture, and / or the two reagent components b1) and b2) of the second reagent set are premixed in a second reagent container as components of a second liquid reagent mixture.

[0045] A particular embodiment of the invention in which the reagent components are provided as components of a liquid reagent mixture is characterized in that the reagent mixture comprises, independently of one another, buffers selected from PIPES, MES, MOPS, HEPES, bis-TRIS-propane, TRIS, AMPSO, borate, TABS and TAPS, with TRIS, TAPS and AMPSO being preferred for reagent mixtures of the first reagent set and PIPES and MOPS being preferred for reagent mixtures of the second reagent set.

[0046] In some embodiments of the present invention, K + , Na + , N.H. 4+ Monovalent metal ions such as Mg 2+ , Ca 2+ , Ba 2+ , Cu 2+ , Fe 2+ , Zn 2+ Signal intensity can be further enhanced by adding divalent metal ions such as HCl, ...

[0047] A particular embodiment of the invention using premixed liquid reagent mixtures of the first and / or second reagent sets is characterized in that the pH of the reagent mixture of the first reagent set is preferably in the range of 6.0 to 9.0, particularly preferably in the range of 7.5 to 9.0, and / or the pH of the second reagent mixture of the second reagent set is preferably in the range of 6.0 to 9.0, particularly preferably in the range of 6.5 to 8.5.

[0048] In a particular embodiment of the invention, the components of the first set of reagents are used in a mixture with the following proportions of each component:

[0049] [Table 1]

[0050] In a particular embodiment of the invention, the components of the second set of reagents are used in a mixture of the following proportions of each component:

[0051] [Table 2]

[0052] In certain embodiments, the analytical or diagnostic test system of the present invention comprises at least one control solution and / or at least one calibrator solution, said control solution and / or calibrator solution comprising human plasma and / or sodium azide as matrix components, and optionally further comprising 6-aminohexanoic acid and / or calcium chloride.

[0053] In certain embodiments, the first liquid reagent mixture and / or the second liquid reagent mixture comprises a preservative combination comprising at least gentamicin sulfate and, optionally, one of amphotericin and onium 46.

[0054] It should be noted that for the purposes of the original disclosure, all features that are apparent to a person skilled in the art from the present specification and claims, even if specifically described only in connection with certain further features, can be combined individually or in any combination with other features or feature groups disclosed herein, unless expressly excluded or unless chemical, physicochemical or pharmacological circumstances make such a combination impossible or insignificant. For the sake of brevity and readability, a comprehensive and explicit listing of all possible feature combinations is omitted here.

[0055] Furthermore, it should be noted that it is obvious to those skilled in the art that the following embodiments are merely illustrative of possible embodiments of the present invention by way of example. Therefore, those skilled in the art will easily understand that all other embodiments having the inventive features or combinations of features set forth in the claims also fall within the protection scope of the present invention. For the sake of brevity and readability, a comprehensive and explicit presentation of all possible embodiments is omitted here. [Example]

[0056] In one embodiment of the present invention, the components of the first reagent set were used in a mixture of the following proportions of each component:

[0057] [Table 3]

[0058] In one embodiment of the present invention, the components of the second reagent set were used in a mixture of the following proportions of each component:

[0059] [Table 4]

[0060] In one embodiment of the present invention, the first and second reagent sets were used as follows.

[0061] [Table 5]

[0062] When the method of the present invention was compared with in-house testing using an alternative FAPGG method and with conventional hippuricase testing performed by a competitor, the following results were obtained:

[0063] [Table 6]

Claims

1. a) a first reagent set comprising the following reagent components: a1) Hydroxybenzoyl tripeptidyls of general formula (I) as ACE substrates 【Chemistry 1】 (I) [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 １－４ an alkoxy group, a halogen atom, or H; A is selected from the amino acid groups His, Gly, or Ala; 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 to 0.5 V measured against a standard hydrogen electrode at 25°C and an effective concentration of 1 mol / L, and / or an oxidizing substrate of an oxidoreductase that oxidizes the oxidizing substrate and reduces oxygen to hydrogen peroxide; and b) a second reagent set comprising the following reagent components: b1) enzymes of the hippuricase type, and Optionally, b2) an oxidoreductase that oxidizes the oxidizable substrate optionally included in the first reagent set as reagent component a2) and reduces oxygen to hydrogen peroxide; and c) Aminopyrine of general formula (II) 【Chemistry 2】 (II) [In the general formula (II), R1 and R2 each independently represent C １－４ is an alkyl group or H. A method for quantitatively measuring the enzymatic activity of angiotensin-converting enzyme (ACE) in a sample, comprising contacting ACE contained in the sample with a reaction mixture to prepare a reaction mixture.

2. The hydroxybenzoyl tripeptidyl of reagent component a1) is N-(4-hydroxybenzoyl)glycylhistidylleucine, N-(4-hydroxy-3-methoxybenzoyl)glycylglycylglycine, N-(4-hydroxy-3-methoxybenzoyl)glycylhistidylleucine, N-(3-chloro-4-hydroxybenzoyl)glycylglycylglycine, N-(3,5-dimethoxy-4-hydroxybenzoyl)glycylhistidylleucine, N-(3,5-dibromo-4-hydroxybenzoyl)glycylglycylglycine, N-(3,5-dichloro-4-hydroxybenzoyl)glycylhistidylleucine, N-(3-hydroxy-2,4,6-triiodobenzoyl)glycylhistidylleucine, N-(4-hydroxy-3-methoxybenzoyl)glycylalanylphenylalanine, N-(3-chloro-4-hydroxybenzoyl)glycylalanylphenylalanine, N-(3,5-dimethoxy-4-hydroxybenzoyl)glycylalanylphenylalanine 2. The method of claim 1, wherein the compound is selected from the group consisting of:

3. The oxidoreductase in reagent component b2) and its oxidized substrate in reagent component a2) Glucose oxidase / glucose Cholesterol oxidase / cholesterol chloride Choline oxidase / choline chloride Lactate oxidase / lactic acid 3. The method according to claim 1 or 2, characterized in that the combination is selected from the following:

4. The method according to any one of claims 1 to 3, wherein the aminopyrine is 4-aminoantipyrine.

5. The oxidizing agent optionally included in the first reagent set as reagent component a2) is Tetraamine copper (II) sulfate Molybdate salts containing divalent metal ions Potassium hexacyanoferrate(III) Hexachlororhodate(III) ·manganese Oxalic acid Sulfates, thiosulfates, and nitrites The method according to any one of claims 1 to 4, characterized in that the compound is selected from the group consisting of:

6. The method according to any one of claims 1 to 4, wherein the sample for measuring the enzymatic activity of ACE is a liquid sample selected from blood, serum, plasma, or cerebrospinal fluid.

7. 6. The method according to claim 1, wherein the absorbance of the reaction mixture is measured at a wavelength in the range of 480 to 530 nm.

8. the amount of ACE-containing sample used in preparing the reaction mixture is in the range of 0.1 to 100 μL, preferably less than 20 μL, less than 10 μL, less than 5 μL, or less than 1 μL; and / or The enzyme activity of ACE in the sample is in the range of 0.1 to 250 U / L, preferably less than 20 U / L, less than 10 U / L, less than 5 U / L, or less than 1 U / L. The method according to any one of claims 1 to 6, characterized in that

9. 9. The method according to any one of claims 1 to 8, characterized in that the enzymatic activity of ACE is measured in a sample from a patient suffering from at least one of the following conditions: hypertension, chronic heart failure, neurosarcoidosis, leprosy, Gaucher disease, tuberculosis, preeclampsia, chronic beryllium disease, proliferative retinopathy, HIV infection, pregnancy, chronic fatigue syndrome, cancer, Alzheimer's disease, nephropathy, fibrosis, clinical picture or symptom of COVID-19, or at least one of these physiological conditions, for the purposes of pharmacological analysis or for diagnostic or follow-up purposes.

10. A first reagent set and a second reagent set are provided, The first reagent set includes at least the following reagent components: a1) Hydroxybenzoyl tripeptides of general formula (I) as ACE substrates 【Transformation 3】 (I) [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 １－４ an alkoxy group, a halogen atom, or H; A is selected from the amino acid groups His, Gly, or Ala; 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 to 0.5 V measured against a standard hydrogen electrode at 25°C and an effective concentration of 1 mol / L, and / or an oxidizing substrate of an oxidoreductase that oxidizes the oxidizing substrate and reduces oxygen to hydrogen peroxide; The second reagent set includes at least the following reagent components: b1) enzymes of the hippuricase type, and b2) an oxidoreductase that oxidizes the oxidation substrate optionally included in the first reagent set as reagent component a2) and reduces oxygen to hydrogen peroxide; Either the first reagent set or the second reagent set, or a reagent additionally provided in the test system, comprises an aminopyrine of general formula (II): 【Chemistry 4】 (II) [In the general formula (II), R1 and R2 each independently represent C １－４ is an alkyl group or H.

1. An analytical or diagnostic test system for the quantitative determination of enzymatic activity of angiotensin converting enzyme (ACE) in a sample, comprising:

11. the two reagent components a1) and a2) of the first reagent set are present as components of a first liquid reagent mixture premixed in a first reagent container; and / or The two reagent components b1) and b2) of the second reagent set are present as components of a second liquid reagent mixture premixed in a second reagent container.

11. An analytical or diagnostic test system according to claim 10.

12. 12. The analytical or diagnostic test system of claim 11, wherein the first and second liquid reagent mixtures each independently comprise a buffer selected from PIPES, MES, MOPS, HEPES, bis-TRIS-propane, and TAPS, with HEPES being preferred for the first reagent mixture and PIPES being preferred for the second reagent mixture.

13. 13. An analytical or diagnostic test system according to claim 11 or 12, characterized in that the pH value of the first reagent mixture is in the range of 8.0 to 8.5 and / or the pH value of the second reagent mixture is in the range of 7.0 to 9.

0.

14. 14. An analytical or diagnostic test system according to any one of claims 10 to 13, characterised in that it comprises at least one control liquid and / or at least one calibrator liquid, said control liquid and / or said calibrator liquid comprising as matrix components preferably human plasma and / or sodium azide, and optionally further comprising 6-aminohexanoic acid and / or calcium chloride.

15. 15. An analytical 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 comprises a preservative combination comprising at least gentamicin sulfate and optionally one of amphotericin and onium 46.

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