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

Abstract

In order to provide a method which is as sensitive as possible for quantitative measurement of the enzyme activity of ACE, which method is by contrast largely insensitive to spectral interferences and can be practicably automated in currently used analysers for routine clinical operation, according to the invention a method is proposed for quantitative measurement of the enzyme activity of angiotensin-converting enzyme (ACE) in a sample, in which method a reaction mixture is produced by bringing the ACE contained in the sample into contact with a first batch of reagents, which contains a hydroxybenzoyl tripeptidyl and a weak oxidant and / or an oxidation substrate of an oxidoreductase, and into contact with a second batch of reagents, which contains a hippuricase and optionally an oxidoreductase, and into contact with an aminopyrine.

Description

[0001] Test system for measuring the enzyme activity of angiotensin-converting enzyme (ACE)

[0002] The present invention relates to an analytical or diagnostic test system for the quantitative measurement of angiotensin-converting enzyme (ACE) activity in a sample. In particular, the present invention relates to a method for quantitatively measuring enzyme activity and a test system consisting of various reagents with which the method can be carried out.

[0003] The angiotensin-converting enzyme (synonyms: ACE, kininase II, peptidyl dipeptidase A), which is the subject of the present invention, is a zinc metalloprotease that cleaves the prohormone angiotensin I into the vasoconstrictor hormone angiotensin II. Angiotensin II, in turn, is an effector in the renin-angiotensin-aldosterone system that exerts a vasoconstrictor effect, thus leading to an increase in blood pressure and extracellular volume.

[0004] ACE is present in a membrane-bound form on the luminal surface of endothelial cells—primarily in the lungs and, to a lesser extent, 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 occurs in a free, soluble form in blood plasma and other body fluids (e.g., amniotic fluid, spinal fluid, etc.). The soluble form of ACE is formed by proteolysis of the C-terminal membrane anchor.

[0005] Determination of ACE activity in the blood is used, among other things, for the diagnosis and monitoring of granulomatous lung diseases such as tuberculosis and sarcoidosis. ACE testing is also used in conjunction with the diagnostic evaluation of leprosy and Gaucher disease, as well as various forms of preeclampsia.

[0006] The determination of ACE levels in blood serum is typically performed using one of the following two test systems. The FAPGG method uses the peptide FAPGG as a substrate, which is hydrolytically degraded by the patient's own ACE in the sample, resulting in a decrease in absorbance in the UV range. However, the measurement wavelength in the UV range makes assays based on the FAPGG method quite susceptible to spectral interference from endogenous bilirubin, hemoglobin, or lipids.

[0007] In comparison, in the so-called "hippuricase method," the synthetic substrate p-hydroxybenzoyl-glycyl-L-histidyl-L-leucine is degraded from the sample by ACE in conjunction with the enzyme hippuricase, which is provided by the reagent, to p-benzoic acid. The resulting p-hydroxybenzoic acid is converted into a dye via a trinder reaction, whose absorption is measured at 505 nm.

[0008] Measuring the photometric signal in the VIS range allows for a somewhat more sensitive ACE determination and less sensitive to spectral interference than the FAPGG method. However, the test system typically consists of five separate, partially lyophilized reagent components and is practically impossible to automate on many common analyzers for routine clinical use.

[0009] Against this background, the inventors of the present application have set themselves the task of providing a method for the quantitative measurement of the enzyme activity of ACE that is as sensitive as possible, which is largely insensitive to spectral interference and can be practically automated on the common analyzers for clinical routine operation.

[0010] According to the invention, a method for quantitatively measuring the enzyme activity of the angiotensin-converting enzyme (ACE) in a sample is proposed, in which a reaction mixture is produced by bringing ACE contained in the sample a) into contact with a first set of reagents, wherein the first set of reagents comprises the following reagent components: a1) a hydroxybenzoyl-tripeptidyl of the general formula (I) as ACE substrate where in the general formula (I)

[0011] R 1 a Ci-4 alkoxy group, a halogen atom or an unsubstituted amino group,

[0012] R 2 and R 3 each independently represents a Ci-4 alkoxy group, a halogen atom or H,

[0013] A is selected from the amino acid groups His, Gly or Ala and

[0014] B is selected from the amino acid groups Leu, Gly or Phe, a2) an oxidizing agent with a standard potential E° measured at 25°C and an effective concentration of 1 mol / l against a standard hydrogen electrode in the range of 0.1 to 0.5 V and / or an oxidation substrate of an oxidoreductase which oxidizes the oxidation substrate and reduces oxygen to hydrogen peroxide, and b) bringing into contact with a second reagent set, wherein the second reagent set comprises the following reagent components: b1) an enzyme of the hippuricase type and optionally b2) an oxidoreductase which oxidizes the oxidation substrate optionally contained in the first reagent set as reagent component a2) and reduces oxygen to hydrogen peroxide, and c) with an aminopyrine of the general formula (II) wherein in the general formula (II) R1 and R2 are each independently a Ci-4 alkyl group or H.

[0015] The inventors have discovered that by using a relatively weak oxidizing agent in the first set of reagents, the standard potential E° of which, measured at 25°C and an effective concentration of 1 mol / l against a standard hydrogen electrode, is in the range of 0.1 to 0.5 V, or by dividing the reagents used in ACE diagnostics between the two separate reagent sets defined above, a stable test system suitable for clinical routine operation is obtained that provides a very high sensitivity and can be carried out largely unaffected by interference from other substances contained in the sample to be examined.

[0016] The first set of reagents used according to the invention contains, in addition to the ACE substrate hydroxybenzoyl tripeptidyl, either a relatively weak oxidizing agent or an oxidation substrate of an oxidoreductase, and the second set of reagents proposed according to the invention contains the enzyme hippuricase, and in the embodiments in which the first reagent contains an oxidation substrate, additionally the oxidoreductase, which oxidizes the oxidation substrate contained in the first set of reagents and thereby reduces oxygen contained in the reaction mixture to hydrogen peroxide.

[0017] In specific embodiments of the invention, the first reagent contains both the relatively weak oxidizing agent and an oxidation substrate for an oxidoreductase contained in the second reagent.

[0018] In the reaction mixture formed from the first set of reagents and the second set of reagents, the hydroxybenzoyl tripeptidyl originating from the first set of reagents is first converted by the ACE contained in the sample to 4-hydroxyhippuric acid by cleavage of the two terminal amino acids, which is then converted to 4-hydroxybenzoic acid by the hippuricase contained in the second set of reagents by cleavage of the amino acid glycine.

[0019] The 4-hydroxybenzoic acid produced in situ by the reaction described above condenses with the aminopyrine contained in the reaction mixture in the presence of the hydrogen peroxide formed in situ in the reaction mixture to form a quinone imine with an absorption maximum in the VIS range.

[0020] In certain embodiments, the above-described reaction of 4-hydroxybenzoic acid with the aminopyrine to form a quinone imine optionally takes place in the presence of a peroxidase, which increases the reaction rate of the condensation reaction and thus the signal intensity. The peroxidase can be added to the reaction mixture either as a component of the first set of reagents or as a component of the second set of reagents, or as an individual, separate component, as needed.

[0021] The reactions described above can be represented schematically as follows: + His-Leu

[0022] 4-Hydroxyhippuryl-His-Leu 4-Hydroxyhippuricacid + Gly

[0023] 4-Hydroxyhippuricacid 4-Hydroxybenzoicacid

[0024] POD

[0025] + H2O2 + 4-AA quinone imines

[0026] 505 nm

[0027] 4-Hydroxybenzoic acid

[0028] glucose Gluconic acid

[0029] The method according to the 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 a common reagent container, and the reagent components of the second reagent set can also be premixed in a common reagent container, without the reagent components reacting with each other, even during extended storage periods, which could influence the result of the measurement of the enzyme activity of ACE in a sample. This significantly reduces the number of reagent containers required for diagnostics.

[0030] One of the obstacles that had to be overcome was the fact that the ACE substrate hydroxybenzoyl tripeptidyl is gradually converted by the enzyme hippuricase during extended storage times, which would accordingly distort the measurement results. Another problem that had to be solved was that the oxidizing agent required for the color reaction, when mixed with hippuricase for extended storage times, would destroy the enzyme's activity if the oxidizing agent was a strong oxidant, which is particularly necessary when a rapid oxidation reaction is desired.

[0031] The division of the ACE substrate hydroxybenzoyl-tripeptidyl and the enzyme hippuricase into two sets of reagents proposed according to the invention prevents the hydroxybenzoyl-tripeptidyl from being gradually converted by the enzyme hippuricase during longer storage times.

[0032] Furthermore, the combination of reagents proposed by the invention, or rather the division of the reagents into two sets of reagents, prevents the oxidizing agent required for the color reaction from destroying the enzymes used. This is achieved either by using a relatively weak oxidizing agent in the first set of reagents or by generating the hydrogen peroxide as the oxidizing agent in situ by a suitable oxidase / substrate pair immediately before the photometric measurement. For the in situ generated oxidizing agent, the first set of reagents contains a substrate for an oxidase, which is located in the second set of reagents.

[0033] The high storage stability of the premixed reagent sets used according to the invention is in the range of 12 to 24 months, whereby high storage stability is understood here to mean that when stored at a temperature in the range of 2 to 8°C over a period of 12 to 24 months from production, the sensitivity of the test system according to the invention deviates by a maximum of 5% from the sensitivity of the test system immediately after production.

[0034] In order to keep the number of reagent containers that need to be handled when carrying out the method according to the invention as low 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 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 specific 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.

[0035] The additional reagent aminopyrine can either be provided in a separate third reagent container (“ready-to-use” 3-component system) or be part of a reagent mixture in a reagent container of the first or second reagent set (“ready-to-use” 2-component system).

[0036] The reagent components of the first reagent set and the second reagent set can, in principle, be brought into contact with the ACE contained in the sample in any order to generate the reaction mixture. In one embodiment of the 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. A particularly high sensitivity has been observed for this order. In an alternative embodiment of the 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.A very good sensitivity was also found for this sequence, although somewhat lower than in the case of the first mentioned sequence.

[0037] In the system described by the chemical equations presented above, 4-hydroxybenzoyl-glycine-histidine-leucine serves as the substrate for the ACE contained in the sample. In certain embodiments of the invention, the hydroxybenzoyl-tripeptidyl of reagent component a1) of the first reagent set is selected from:

[0038] ■ N-(4-hydroxy-benzoyl)-glycyl-histidy-leucine,

[0039] ■ N-(4-hydroxy-3-methoxybenzoyl)-glycyl-glycyl-glycine,

[0040] ■ N-(4-hydroxy-3-methoxybenzoyl)-glycyl-histidyl-leucine,

[0041] ■ N-(3-chloro-4-hydroxybenzoyl)-glycyl-glycyl-glycine,

[0042] ■ N-(3,5-dimethoxy-4-hydroxy-benzoyl)-glycyl-histidyl-leucine,

[0043] ■ N-(3,5-dibromo-4-hydroxy-benzoyl)-glycyl-glycyl-glycine,

[0044] ■ N-(3,5-dichloro-4-hydroxy-benzoyl)-glycyl-histidyl-leucine,

[0045] ■ N-(3-hydroxy-2,4,6-triiodo-benzoyl)-glycyl-histidyl-leucine,

[0046] ■ N-(4-hydroxy-3-methoxybenzoyl)-glycyl-alanyl-phenylalane in,

[0047] ■ N-(3-chloro-4-hydroxybenzoyl)-glycyl-alanyl-phenylalanine,

[0048] ■ N-(3,5-dimethoxy-4-hydroxybenzoyl)-glycyl-alanyl-phenylalanine.

[0049] In some embodiments of the invention, for the in situ generation of hydrogen peroxide, glucose already present in the sample or glucose added to the reaction mixture is converted by glucose oxidase in the presence of oxygen present in the reaction mixture to gluconic acid and hydrogen peroxide. In alternative specific embodiments of the invention, the oxido-reductase of reagent component b2) and its oxidation substrate of reagent component a2) are selected from the following combinations:

[0050] ■ Cholesterol oxidase / cholesterol

[0051] ■ Choline oxidase / choline

[0052] ■ Lactate oxidase / lactate

[0053] In these cases, the respective oxidation substrate can either already be naturally present in the sample or it can be deliberately added to the reaction mixture, or both.

[0054] In the test system schematically illustrated above, the aminopyrine intended for the condensation reaction with 4-hydroxybenzoic acid is 4-aminoantipyrine. In some embodiments of the method according to the invention, this contains a relatively weak oxidizing agent as reagent component a2) in the first reagent set. The term "weak oxidizing agent" is understood here to mean those oxidizing agents that, measured at 25°C and an effective concentration of 1 mol / l against a standard hydrogen electrode, have a standard potential E° in the range of 0.1 to 0.5 V.

[0055] In certain embodiments of the process according to the invention, the relatively weak oxidizing agent optionally contained in the first reagent set as reagent component a2) is selected from:

[0056] - Tetraamine copper(II) sulfate

[0057] - Molybdates with divalent metal ions

[0058] - Potassium hexacyanoferrate(III)

[0059] - Hexachlororhodate(lll)

[0060] - Manganese,

[0061] - oxalic acid,

[0062] - Sulfates, thiosulfates and nitrites.

[0063] Typically, the sample analyzed using the method according to the invention is a liquid sample containing a determined amount of ACE. In most cases, this will be a body fluid containing soluble ACE, which, depending on the specific application, can be selected from blood, blood serum, blood plasma, or cerebrospinal fluid. In special embodiments, however, it can also be a liquid sample obtained from another biological material, in which membrane-bound ACE was originally mobilized.

[0064] At the end of the process according to the invention, the absorption is typically measured in the visible wavelength range. Depending on which aminopyrine is used in the process according to the invention, the range for absorption measurement varies. Ideally, for the highest possible sensitivity and accuracy, the measurement is carried out in a range of 5-50 nm around the absorption maximum of the quinone imine formed during the reaction. In certain embodiments, the wavelength at which the absorption is measured is in the range of 480-530 nm.

[0065] The sensitivity of the method according to the invention has been shown to be extremely high, so that even very small amounts of sample are sufficient for measuring ACE enzyme activity. In certain embodiments of the present invention, the amount of sample used is in the range of 0.1–100 μl. In preferred embodiments, less than 20 μl of sample is used, and in particularly preferred embodiments, the amount of sample used is <10 μl, <5 μl, or even <1 μl.

[0066] The method according to the invention allows enzyme activities to be determined extremely accurately over a relatively wide range. In certain embodiments, the range of enzyme activity determined by the method is 0.1–250 U / L. In preferred embodiments, the range of enzyme activity is <20 U / L, and in preferred embodiments, the enzyme activity is <10 U / L, <5 U / L, or even <1 U / L.

[0067] The method according to the invention for determining the enzyme activity of ACE can be carried out in a patient sample for the purposes of pharmacological analysis or for the purposes of diagnosis or follow-up. Preferably, the analysis using the method according to the invention is carried out in a patient who has at least one of the following clinical pictures or symptoms or at least one of the following physiological conditions:

[0068] Hypertension, chronic heart failure, neurosarcosides, leprosy, Gaucher's disease, tuberculosis, preeclampsia, chronic berylliosis, proliferative retinopathy, HIV infection, pregnancy, chronic fatigue syndrome, cancer, Alzheimer's disease, nephropathy, fibrosis, COVID-19.

[0069] For the purposes of carrying out the method according to the invention, the present invention also proposes an analytical or diagnostic test system, which test system is characterized in that the test system comprises a first set of reagents and a second set of reagents, wherein the first set of reagents comprises at least the following reagent components: a1) a hydroxybenzoyl-tripeptidyl of the general formula (I) as ACE substrate where in the general formula (I)

[0070] R 1 a Ci-4 alkoxy group, a halogen atom or an unsubstituted

[0071] amino group, R 2 and R 3 each independently represents a Ci-4 alkoxy group, a halogen atom or H,

[0072] A is selected from the amino acid groups His, Gly or Ala and

[0073] B is selected from the amino acid groups Leu, Gly or Phe, and a2) an oxidizing agent with a standard potential E° measured at 25°C and an effective concentration of 1 mol / l against a standard hydrogen electrode in the range of 0.1 to 0.5 V and / or an oxidation substrate of an oxidoreductase which oxidizes the oxidation substrate and reduces oxygen to hydrogen peroxide, and the second reagent set comprises at least the following reagent components: b1) an enzyme of the hippuricase type and optionally b2) an oxidoreductase which oxidizes the oxidation substrate optionally contained in the first reagent set as reagent component a2) and reduces oxygen to hydrogen peroxide, wherein either the first reagent set or the second reagent set or a reagent additionally provided in the test system comprises an aminopyrine of the general formula (II) wherein in the general formula (II) R1 and R2 are each independently a Ci-4 alkyl group or H.

[0074] The term "reagent set" is to be understood here as meaning that a reagent set always contains at least two liquid or solid chemical substances involved in the chemical reaction of the process according to the invention as reagent components, wherein the individual reagent components of a reagent set can either be present separately in different reagent containers or can be present at least partially in the form of a reagent mixture together in one and the same reagent container, with the proviso that the reagent components of the first reagent set are never present in a mixture with reagent components of the second reagent set.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 the aminopyrine defined above either in a separate reagent container or as a component of reagent set 1 or as a component of reagent set 2.

[0075] 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 the above-mentioned peroxidase either in a separate reagent container or as a component of reagent set 1 or as a component of reagent set 2.

[0076] In a specific embodiment of the test system according to the invention, the reagent components of the first reagent set are present in a common reagent container, or the reagent components of the second reagent set are present in a common reagent container. In specific 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.

[0077] The additional reagent aminopyrine can either be provided in a separate third reagent container or be part of a reagent mixture in a reagent container of the first or second reagent set.

[0078] 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 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.

[0079] Special embodiments of the invention, in which the reagent components are provided as components of liquid reagent mixtures, are characterized in that the reagent mixtures contain a buffer which is each independently selected from PIPES, MES, MOPS, HEPES, BIS-TRIS-propane, TRIS, AMPSO, borate, TABS and TAPS, wherein in the case of the reagent mixture of the first reagent set, TRIS, TAPS and AMPSO are preferred and in the case of the reagent mixture of the second reagent set, PIPES and MOPS are preferred. In some embodiments of the invention, the signal intensity can be further increased by the addition of monovalent metal ions, such as K + , N / a + , NH4 + , and / or of divalent metal ions, such as Mg 2+ , Ca 2+ , Ba 2+ , Cu 2+ , Fe 2+ , Zn 2+and / or by the addition of mild detergents such as Triton, Tergitol, CT AB, SDS, Thesit and Tween, with non-ionic surfactants such as Triton and Tergitol being preferred.

[0080] Particular embodiments of the invention with premixed liquid reagent mixtures of the first and / or second reagent set are characterized in that the pH of the reagent mixture of the first reagent set is preferably in the range from 6.0 to 9.0 and particularly preferably in the range from 7.5 to 9.0 and / or the pH of the second reagent mixture of the second reagent set is preferably in the range from 6.0 to 9.0 and particularly preferably in the range from 6.5 to 8.5.

[0081] In certain embodiments of the present invention, the components of the first set of reagents are used in a mixture with the following proportions of the individual components:

[0082] In certain embodiments of the present invention, the components of the second set of reagents are used in a mixture with the following proportions of the individual components: In certain embodiments, the analytical or diagnostic test system of the invention comprises at least one control liquid and / or at least one calibrator liquid, wherein the control liquid and / or calibrator liquid preferably contains human plasma and / or sodium azide as matrix components and optionally additionally 6-aminohexanoic acid and / or calcium chloride.

[0083] In certain embodiments, the first and / or second liquid reagent mixture contains a combination of preservatives comprising at least gentamycin sulfate and optionally one of amphotericin and onium 46.

[0084] For the purposes of the original disclosure, it is pointed out that all features as they become apparent to a person skilled in the art from the present description and the claims, even if they were specifically described only in conjunction with certain other features, can be combined both individually and in any combination with other features or groups of features disclosed herein, unless this has been expressly excluded or chemical, physicochemical, or pharmacological conditions make such combinations impossible or pointless. A comprehensive, explicit presentation of all conceivable combinations of features is omitted here solely for the sake of brevity and readability of the description.

[0085] Furthermore, it should be understood by those skilled in the art that the following exemplary embodiments merely serve to illustrate the possible embodiments of the present invention presented as exemplary embodiments. Those skilled in the art will therefore readily understand that, in addition, all other embodiments that have the inventive features or combinations of features recited in the claims are also within the scope of protection of the invention. A comprehensive, explicit presentation of all conceivable embodiments is omitted here solely for the sake of brevity and readability of the description.

[0086] Examples of implementation

[0087] In one embodiment of the present invention, the components of the first set of reagents were used in a mixture with the following proportions of the individual components:

[0088] In one embodiment of the present invention, the components of the second set of reagents were used in a mixture with the following proportions of the individual components:

[0089] In one embodiment of the present invention, the first set of reagents and the second set of reagents were applied as follows: When comparing the method according to the invention with an in-house test using the alternative FAPGG method and with a conventional hippuricase test from a competitor, the following results were obtained:

Claims

Patent claims Method for the quantitative measurement of the enzyme activity of the angiotensin-converting enzyme (ACE) in a sample, in which a reaction mixture is produced by bringing ACE contained in the sample a) into contact with a first set of reagents, wherein the first set of reagents comprises the following reagent components: a1) a hydroxybenzoyl-tripeptidyl of the general formula (I) as ACE substrate where in the general formula (I) R 1 a Ci-4 alkoxy group, a halogen atom or an unsubstituted amino group, R 2 and R 3 each independently represents a Ci-4 alkoxy group, 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 with a standard potential E° measured at 25°C and an effective concentration of 1 mol / l against a standard hydrogen electrode in the range of 0.1 to 0.5 V and / or an oxidation substrate of an oxidoreductase which oxidizes the oxidation substrate and reduces oxygen to hydrogen peroxide, and b) bringing into contact with a second reagent set, wherein the second reagent set comprises the following reagent components: b1) an enzyme of the hippuricase type and optionally b2) an oxidoreductase which oxidizes the oxidation substrate optionally contained in the first reagent set as reagent component a2) and reduces oxygen to hydrogen peroxide, and with an aminopyrine of the general formula (II) in contact, wherein in the general formula (II) R1 and R2 are each independently a Ci-4 alkyl group or H. Process according to claim 1, characterized in that the hydroxybenzoyl tripeptiyl of the reagent component a1) is selected from: - N-(4-hydroxy-benzoyl)-glycyl-histidy-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-hydroxy-benzoyl)-glycyl-histidyl-leucine, - N-(3,5-dibromo-4-hydroxy-benzoyl)-glycyl-glycyl-glycine, - N-(3,5-dichloro-4-hydroxy-benzsyl)-glycyl-histidyl-leucine, - N-(3-hydroxy-2,4,6-triiodo-benzoyl)-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. Process according to 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 / cholesterol chloride - Choline oxidase / choline chloride - Lactate oxidase / lactate method according to one of claims 1 to 3, characterized in that the aminopyrine is 4-aminoantipyrine. Method according to one of claims 1 to 4, characterized in that the oxidizing agent optionally contained in the first reagent set as reagent component a2) is selected from: - Tetraamine copper(II) sulfate - Molybdates with divalent metal ions - Potassium hexacyanoferrate(III) - Hexachlororhodate(lll) - Manganese, - oxalic acid, - Sulfates, thiosulfates and nitrites.

6. Method according to one of claims 1 to 4, characterized in that the sample in which the enzyme activity of ACE is measured is a liquid sample selected from blood, blood serum, blood plasma or cerebrospinal fluid.

7. Method according to one of claims 1 to 5, characterized in that the absorption of the reaction mixture is measured at a wavelength in the range of 480 to 530 nm.

8. Method according to one of claims 1 to 6, characterized in that the amount of ACE-containing sample used in the production of the reaction mixture is in the range of 0.1 to 100 pl and preferably < 20 pl, < 10 pl, < 5 pl or even < 1 pl, and / or that the enzyme activity of ACE in the sample is in the range of 0.1 to 250 U / L and preferably < 20 U / L, < 10 U / L, < 5 U / L or even < 1 U / L.

9. Method according to one of claims 1 to 8, characterized in that the enzyme activity of ACE is measured for the purposes of pharmacological analysis or for the purposes of diagnosis or follow-up in a sample from a patient who has at least one of the following clinical pictures or symptoms or at least one of the following physiological conditions: hypertension, chronic heart failure, neurosarcosides, leprosy, Gaucher's disease, tuberculosis, preeclampsia, chronic berylliosis, proliferative retinopathy, HIV infection, pregnancy, chronic fatigue syndrome, cancer, Alzheimer's disease, nephropathy, fibrosis, COVID-19.

10. Analytical or diagnostic test system for the quantitative measurement of the enzyme activity of the angiotensin-converting enzyme (ACE) in a sample, characterized in that the test system comprises a first set of reagents and a second set of reagents, wherein the first set of reagents comprises at least the following reagent components: a1) a hydroxybenzoyl-tripeptidyl of the general formula (I) as ACE substrate where in the general formula (I) R 1 a Ci-4 alkoxy group, a halogen atom or an unsubstituted amino group, R 2 and R 3 each independently represents a Ci-4 alkoxy group, 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 with a standard potential E° measured at 25°C and an effective concentration of 1 mol / l against a standard hydrogen electrode in the range of 0.1 to 0.5 V and / or an oxidation substrate of an oxidoreductase which oxidizes the oxidation substrate and reduces oxygen to hydrogen peroxide, and the second reagent set comprises at least the following reagent components: b1) an enzyme of the hippuricase type and b2) an oxidoreductase which oxidizes the oxidation substrate optionally contained in the first reagent set as reagent component a2) and reduces oxygen to hydrogen peroxide, wherein either the first reagent set or the second reagent set or a reagent additionally provided in the test system comprises an aminopyrine of the general formula (II) contains, wherein in the general formula (II) R1 and R2 are each independently a Ci-4 alkyl group or H. Analytical or diagnostic test system according to claim 10, characterized in that 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.Analytical or diagnostic test system according to claim 11, characterized in that the first and second liquid reagent mixtures contain a buffer which is each independently selected from PIPES, MES, MOPS, HEPES, BISTRIS-propane, and TAPS, wherein HEPES is preferred in the case of the first reagent mixture and PIPES is preferred in the case of the second reagent mixture. Analytical or diagnostic test system according to one of claims 11 and 12, characterized in that the pH of the first reagent mixture is in the range from 8.0 to 8.5 and / or the pH of the second reagent mixture is in the range from 7.0 to 9.0.Analytical or diagnostic test system according to one of claims 10 to 13, characterized in that it contains at least one control liquid and / or at least one calibrator liquid, wherein the control liquid and / or calibrator liquid preferably contains human plasma and / or sodium azide as matrix components and optionally additionally 6-aminohexanoic acid and / or calcium chloride. Analytical or diagnostic test system according to one of claims 10 to 14, characterized in that the first and / or second liquid reagent mixture contains a combination of preservatives comprising at least gentamycin sulfate and optionally one of amphotericin and onium 46.

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