A method of preparing an incubation medium for histochemical analysis of diamine oxidase in a native tissue sample and a kit comprising said incubation medium

Abstract

The present invention relates to a method of preparing an incubation medium for histochemical analysis of diamine oxidase in a native tissue sample. In a first step, phenazine methosulfate, preferably 0.5 to 1.0 mg phenazine methosulfate; an aqueous tetrazolium salt solution at a concentration of 0.01 to 0.2 wt. % and in a molar ratio of tetrazolium salt to phenazine methosulfate of 2:1 to 1 :2, preferably 1.5 to 2.5 mL 0.1 wt. % nitro blue tetrazolium chloride solution; an aqueous substrate solution comprising at least one D-amino acid at a concentration of 0.4 to 1.2 M and in a molar ratio of D-amino acid to phenazine methosulfate of 10:1 to 230:1, preferably 0.1 to 0.3 mL substrate solution comprising 1 M D-alanine and 0.5 M D-phenylalanine; and optionally, an aqueous cyanide salt solution at a concentration of 0.01 to 0.2 M and in a molar ratio of cyanide salt to phenazine methosulfate of 40:1 to 1 :4, preferably 0.1 to 0.3 mL 0.1 M potassium cyanide solution are mixed. In a second step, the incubation medium is illuminated for 1 to 10 min. to form an incubation medium for histochemical analysis of diamine oxidase in the native tissue sample.

Description

[0001] A method of preparing an incubation medium for histochemical analysis of diamine oxidase in a native tissue sample and a kit comprising said incubation medium

[0002] Field of technology

[0003] The present invention relates to a method of preparing an incubation medium for histochemical analysis of diamine oxidase in a native tissue sample, and to a kit comprising said incubation medium.

[0004] Prior art

[0005] Histamine is a biogenic amine, one of the neurotransmitters naturally occurring in the human body. Its abundance is associated with a number of biochemical processes associated with allergic, inflammatory and other reactions. Its increased amount negatively affects the organism and manifests itself in a very rich symptomatology, grouped under the name of histamine intolerance. The enzyme diamine oxidase is responsible for the regulation and degradation of histamine. Diamine oxidase (DAO; D- amino acid oxidase; D-amino acid:oxygen reductase, deaminating, 1.4.3.3) is a copper- comprising, flavoprotein enzyme found mainly in the kidney, liver and intestine.

[0006] The total circulating DAO in the blood can be examined immunochemically using ELISA methods from a serum sample. The presence of DAO can be investigated immunohistochemically using IHC (immunohistochemical) methods on a fixed tissue sample, i. e. with an inactivated (non-native) enzyme, e. g. using a mouse monoclonal antibody ABP1 (F-9), KAO by Santa Cruz. Further, it must be emphasized that various processes are constantly occurring in the human body in which, under ideal circumstances, the amount of histamine is regulated by the DAO enzyme, and therefore the total DAO value does not give us information about the amount and functionality of enteral DAO. At the same time, however, even examination of an immunohistochemical enteral sample (with enzyme inactivation caused by tissue fixation) cannot assess enzyme activity, only the presence of the molecule.

[0007] Enteral DAO has an important role in the degradation of exogenously ingested histamine. In the absence or lack of a functional molecule (or when the enzyme is blocked by drugs or other foods, i. e. in the case of a non-functional DAO molecule), exogenous histamine crosses over into the blood, thereby increasing the level of circulating histamine. The role of circulating DAO is precisely to degrade this elevated level, and of course the enzyme is consumed, resulting in reduced serum levels. Therefore, it is not possible to diagnose histamine intolerance by indirect examination of serum DAO levels without absolute exclusion of allergic or inflammatory reactions in the body.

[0008] Diamine oxidase catalyses the oxidation of D-amino acids to iminoacids, which further yield ammonia and ketoacids, with the reaction producing hydrogen peroxide. The hydrogen acceptor can be, for example, a tetrazolium salt or phenazine methosulfate (PMS) instead of oxygen, thus providing the possibility of their histochemical representation.

[0009] Diamine oxidase is not very tightly bound in the cells, so it passes into the incubation environment. Sufficient activity usually survives a 15-minute fixation of the blocks in a buffered formol solution, and in addition to the improvement in cytological image, there is also a partial immobilization of the enzyme. Histochemical evidence can be performed on non-fixed (i. e. native) cryostat sections or on frozen sections obtained from blocks fixed for 15 min. in a buffered formol solution and washed for 10 min. in a buffer.

[0010] The publications of Lojda et al., entitled "Fundamentals of histochemical detection of enzymes : (preparation of solutions and buffers)" of 1970, 1971 and 1978 disclose a method for a histochemical analysis of D-diamine oxidase in a rat kidney tissue sample, using an incubation medium comprising 2 mL distilled water; 0.8 mg phenazine methosulphate; 2 mL aqueous nitro blue tetrazolium salt solution prepared by mixing 0.4 wt. % tetrazolium salt solution in distilled water and 0.1 M phosphate buffer at pH = 7.6 in a volume ratio of 1 :1 ; and 0.2 mL aqueous substrate solution comprising 1 M D-alanine, 1 M D-phenylalanine, 1 M L-lactic acid, 1 M a-L-hydroxybutyrate and 1 M a-L- hydroxyvalerate. The tissue sample was incubated in the dark at 37 °C for 5 to 30 min., followed by rinsing in saline, fixation with formol for 10 min., rinsing in water, and mounting in an aqueous medium. Sites with D-diamine oxidase activity are stained blue; localization at the cellular level is good. As a control, incubation of parallel sections in media in which water replaces the substrate or quinacrine is added at a concentration of 1 mM. The incubation medium can be supplemented with an a-keto acid-binding agent in the form of a cyanide to a final concentration of max. 0.01 M, but the same medium without substrate is used as a control, since the controls with cyanide are more potent than the controls without cyanide. Unfortunately, the inventor named in this application has not been able to reproduce the procedure using the above incubation medium. It was not possible to prepare a 1 M phenylalanine solution due to insufficient dissolution, incubation did not take place in the dark, nor in less than 20 min.

[0011] Allen, Beard et al., J. Exp. Zool., 160:329-344 (1965), entitled " The localization of a- hydroxy acid oxidase in renal microbodies" discloses a method for a histochemical analysis of D-diamine oxidase by determination of the ketoacids formed, using an incubation medium comprising 0.05 M Sorensen phosphate buffer having pH = 8.0; 0.1 M D-alanine; and 20p M flavin adenine dinucleotide. After incubation at 37 °C, the reaction was stopped by the addition of half volume of 15% trichloroacetic acid and the corresponding ketoacid was subsequently determined. A disadvantage of the above method is the absence of a colorimetric determination.

[0012] Gould et al., Analytical Biochemistry 67, 515-519 (1975) entitled "A new stain for copperprotein complexes: its use with crustacean hemocyanins" discloses an electrophoretic method of staining a D-diamine oxidase-copper complex, using a staining solution prepared by mixing 18 mL 0.06M KCN having pH = 8; 10 mL 0.05 M Tris buffer having pH = 8; and 0.5 g polyvinylpyrrolidone; wherein within one hour of use, 10 mL aqueous p-nitro blue tetrazolium chloride (NBT) solution at a concentration of 5 mg / mL is further added to the solution; and just prior to use, 0.6 mL aqueous phenazine methosulfate (PMS) solution at a concentration of 3 mg / mL is further added to the solution; so that the resulting ratio of the components is 5:9:5:0.3 (buffer:KCN:NBT:PMS). The prepared staining solution is then poured onto the sample gel and electrophoresis is carried out in the dark for 30-40 min. The bands are stained pink, and over time and after repeated rinsing, this colour darkens. The disadvantage of the above method is the impossibility of its application to a native tissue sample.

[0013] Worsfold et al., Analytical biochemistry 79, 152-156 (1977) entitled "Enzyme detection using phenazine methosulphate and tetrazolium salts: interference by oxygen", Shebela et al., Plant Physiol. Biochem. 39 (2001) 831-839 entitled "Light microscopic localization of aminoaldehyde dehydrogenase activity in plant tissues using nitroblue tetrazolium- based staining method", and Shebela et al., Inflamm. res. 51 , Supplement 1 (2002) S95- S96, entitled "Metabolism of polyamine oxidation products in plants: localisation of pea aminoaldehyde dehydrogenase activity" disclose methods for a detection of other enzymes using phenazine methosulphate and tetrazolium salts. The Chinese patent CN 105699640 B describes a kit for detecting the barrier function of the intestinal wall, with a staining solution for detecting D-diamine oxidase comprising, as one of many possibilities, phenazine methosulfate as an electron carrier and nitro blue tetrazolium chloride as a dye. The analytical method is not described in detail herein.

[0014] The European patent EP1948819 B1 describes a method for a determination of D- diamine oxidase in a tissue sample by reacting it with an amount of diamine (e. g. histamine, putrescine, spermidine, cadaverine) and then determining the remaining, unreacted diamine by derivatizing it (e. g. by acylation with NHS-biotin). A disadvantage of the above method is indirect determination of DAO using the consumed substrate.

[0015] The manual for Thermofischer Scientific's nitro blue tetrazolium chloride solution for glucose oxidase detection (available November 29, 2023 at https: / / www.thermofisher.cn / document-connect / document- connect.htmL?url=https: / / assets.thermofisher.cn / TFS- Assets%2FLSG%2Fmanuals%2FMAN001 1192_NBT_UG.pdf) discloses mixing of: a substrate solution comprising 41.7 mM (7.5 g / L) a-D-glucose and 326 pM (0.1 g / L) phenazine methosulfate (PMS) in 0.1 M Na2HPO4 at pH 6.9; and a stock nitro blue tetrazolium chloride solution (NBT) comprising 0.5 g NBT in 10 mL 70% N,N- dimethylformamide in water to a final NBT concentration of 2.45 mM (0.5 g / L). The manual cautions about the photosensitivity of PMS, which may reduce the tetrazolium salt during storage, and recommends preparing the solution only just prior to analysis.

[0016] Halaka et al., The Journal of Biological Chemistry Vol. 257, No. 3, pp. 1458-1461 , 1982, entitled "Properties of 5-methylphenazinium methyl sulfate" reports that phenazine methosulfate absorbs in an aqueous solution at 388 nm and that at pH = 7.4 in the presence of oxygen, its slow photodecomposition to pyocyanine (1-hydroxy-5- methylphenazinium cation) occurs, absorbing at 312 nm (see Fig. 6 of said publication). Experiments were carried out on the reduction of PMS by an NADH reagent, depending on the presence of oxygen and light. The reduction of phenazine methosulfate is a two- step process with two one-electron transfers and with a distinguishable semi-quinonoid form, which, according to Zaugg, The Journal of Biological Chemistry Vol. 289, No. 11 , pp. 3964-3970, 1964, entitled "Spectroscopic characteristics and some chemical properties of N-methylphenazinium methyl sulfate (phenazine methosulfate) and pyocyanine at the semiquinoid oxidation level", is coloured green in an aqueous solution and absorbs at 440 nm.

[0017] Feinstein et al., Analytical Biochemistry, 1973, Vol. 56, No. 2, pp. 353-360, entitled "Detection of oxidases on polyacrylamide gels" discloses detection of oxidases by electron transfer from reduced flavin oxidase to phenazine methosulfate, and subsequently to a nitro blue tetrazolium salt.

[0018] Horiike et al., Acta Histochemica et Cytochemica, 1985, Vol. 18, No. 5, pp. 539-550, entitled "Histochemical staining of cells comprising flavo-enzyme D-amino acid oxidase based on its enzymatic activity: application of a coupled peroxidation method" discloses a histochemical method for the analysis of D-amino acid oxidase by peroxidase.

[0019] The European patent application EP 0606296 A1 (corresponding to the international patent application WO 93 / 06487 A1 ) describes a reagent for the detection of phenazinebased enzymes.

[0020] The Korean patent application KR 20180004027 A discloses in one of many possible combinations a kit for analysing enzymatic activity by electron transfer to phenazine methosulfate, and subsequently to a nitroblue tetrazolium salt.

[0021] A need therefore arises in the prior art, and it is an object of the present invention to provide a reproducible method of preparing an incubation medium for histochemical analysis of diamine oxidase in a native tissue sample, as well as a kit comprising said incubation medium.

[0022] Summarv of the invention

[0023] Said object is achieved in a first aspect of the present invention by a method of preparing an incubation medium for histochemical analysis of diamine oxidase in a native tissue sample. In a first step of this method, phenazine methosulfate (PMS), preferably 0.5 to 1 .0 mg phenazine methosulfate, is mixed with an aqueous tetrazolium salt solution at a concentration of 0.01 to 0.2 wt. % and in a molar ratio of tetrazolium salt to phenazine methosulfate of 2:1 to 1 :2, preferably 1.5 to 2.5 mL 0.1 wt. % nitro blue tetrazolium chloride solution; an aqueous substrate solution comprising at least one D-amino acid at a concentration of 0.4 to 1.2 M and in a molar ratio of D-amino acid to phenazine methosulfate of 10:1 to 230:1 , preferably 0.1 to 0.3 mL substrate solution comprising 1 M D-alanine and 0.5 M D-phenylalanine; and optionally, an aqueous cyanide salt solution at a concentration of 0.01 to 0.2 M and in a molar ratio of cyanide salt to phenazine methosulfate of 40:1 to 1 :4, preferably 0.1 to 0.3 mL 0.1 M potassium cyanide solution.

[0024] Phenazine methosulfate serves as a hydrogen carrier from the hydrogen peroxide formed. Since the reduced phenazine methosulphate is unstable, the tetrazolium salt (yellow in solution) is further reduced by this hydrogen to formazan in the form of a detectable, dark blue to purple precipitate. The cyanide anion is a strong nucleophile and serves to liberate copper from the enzyme, leading to better reproducibility and a faster analytical reaction, but this reaction will also take place in the absence of the cyanide anion. D-alanine and D-phenylalanine serve as substrates for the enzymatic reaction of diamine oxidase.

[0025] In a second step of the method, the prepared incubation medium is illuminated for 1 to 10 min., preferably 5 min. at room temperature, to form an incubation medium suitable for histochemical analysis of diamine oxidase in a native tissue sample. Illumination activates the incubation medium and causes it to tint slightly blue. Although the mechanism of this activation in relation to diamine oxidase analysis has not yet been fully elucidated, phenazine methosulphate is known to be photolabile, to reduce the tetrazolium salt (see Thermofischer Scientific manual above), to absorb at 388 nm, and to undergo a gradual photodecomposition to pyocyanine (1-hydroxy-5-methylphenazine cation) absorbing at 310 nm in the presence of oxygen at pH = 7.4 (see Fig. 6 in Halaka et al. and the Zaugg publication cited above).

[0026] The aqueous tetrazolium salt solution is preferably prepared by mixing 0.4 wt. % tetrazolium salt solution in distilled water (with initial dissolution of the salt using a small amount of N,N-dimethylformamide) and 0.1 M phosphate buffer (at pH = 7 to 8, e. g. pH = 7.6). The phosphate buffer may be the Sorensen phosphate buffer comprising 0.15 M Na2HPO4.12H2O and 0.15 M KH2PO4.

[0027] The aqueous cyanide salt solution is preferably prepared by acidification with 1 M HCI to pH = 7 to 8 (e. g. pH = 7.4).

[0028] The incubation medium is preferably prepared by mixing 0.8 mg phenazine methosulfate; 2.0 mL 0.1 wt. % aqueous nitro blue tetrazolium chloride solution; 0.2 mL aqueous substrate solution comprising 1 M D-alanine and 0.5 M D-phenylalanine; and 0.2 mL 0.1 M aqueous potassium cyanide solution. The incubation medium is preferably prepared by mixing 1 mL distilled water; 0.8 mg phenazine methosulfate; 1.0 mL 0.2 wt. % aqueous nitro blue tetrazolium chloride solution; 0.2 mL aqueous substrate solution comprising 1 M D-alanine and 0.5 M D- phenylalanine; and 0.2 mL 0.1 M aqueous potassium cyanide solution.

[0029] The aqueous substrate solution preferably further comprises at least one L-hydroxy acid or derivative thereof at a concentration of 0.8 to 1.2 M, preferably L-lactic acid and / or methyl-R-3-hydroxybutyrate and / or ethyl-2-hydroxyvalerate at a concentration of 0.8 to 1.2 M, more preferably 1 M.

[0030] In a preferred embodiment, the above method is further used to perform histochemical analysis of diamine oxidase in a native tissue sample. In a first step, a frozen, sectioned native (i. e. non-fixed) tissue sample is provided. In a second step, incubation medium is prepared according to the method described above. In a third step, the sample is incubated with the incubation medium with simultaneous illumination for 20 to 40 min., preferably 30 min. at 37 °C. The illumination during incubation ensures the reproducibility of the analytical method. In a fourth step, the sample is rinsed, fixed and mounted in an aqueous medium to form a microscopic slide for histochemical analysis of diamine oxidase, wherein sites of diamine oxidase activity are highlighted by staining (dark blue, the background being blue, purple to pink) and the histochemical analysis of diamine oxidase in the native tissue sample is performed.

[0031] Preferably, transport and storage of the sample prior to the analysis is in a petroleum ether at approximately -18 °C or on ice or dry ice.

[0032] Preferably, the sample is a sample of duodenal tissue cut on a cryostat microtome. The sections may be about 5 pm thick, cut on a cryostat microtome at about -25 to -28 °C.

[0033] Preferably, visible radiation, e. g. white, violet, blue, green, yellow, orange or red LED light, or UV light, is used as illumination in the method herein, and the incubation medium storage container, as well as the incubation container for incubation, is transparent.

[0034] Preferably, after incubation, the sample is rinsed with saline for 5 to 15 min. (e. g. 10 min.), fixed (e. g. with 10% neutral formol) for 5 to 15 min. (e. g. 10 min.), washed with water for 5 to 15 min. (e. g. 10 min.), and rinsed with distilled water for 1 to 5 min., optionally dried, and mounted in an aqueous medium to form a microscopic slide for histochemical analysis of diamine oxidase, with sites of diamine oxidase activity highlighted by staining.

[0035] Said object is achieved in a second aspect of the present invention by a kit for performing the above method, said kit being used for histochemical analysis of diamine oxidase in a native tissue sample, said kit comprising: phenazine methosulfate; an aqueous tetrazolium salt solution, preferably nitro blue tetrazolium chloride, at a concentration of 0.01 to 0.2 wt. %; an aqueous substrate solution comprising at least one D-amino acid, preferably D-alanine and D-phenylalanine, at a concentration of 0.4 to 1.2 M; and optionally, an aqueous cyanide salt solution, preferably potassium cyanide, at a concentration of 0.01 to 0.2 M. Distilled water may further be present in the kit. The aqueous cyanide salt solution is an optional component which improves reproducibility and increases the speed of the analytical reaction.

[0036] Preferably, said kit comprises the aqueous nitro blue tetrazolium salt solution at a concentration of 0.1 wt. %; the aqueous substrate solution comprising D-alanine at a concentration of 1 M and D-phenylalanine at a concentration of 0.5 M; and the aqueous potassium cyanide solution at a concentration of 0.1 M.

[0037] Preferably, said kit comprises distilled water; the aqueous nitro blue tetrazolium salt solution at a concentration of 0.2 wt. %; the aqueous substrate solution comprising D- alanine at a concentration of 1 M and D-phenylalanine at a concentration of 0.5 M; and the aqueous potassium cyanide solution at a concentration of 0.1 M.

[0038] Preferably, the aqueous substrate solution further comprises at least one L-hydroxy acid or derivative thereof at a concentration of 0.8 to 1.2 M, preferably L-lactic acid and / or methyl-R-3-hydroxybutyrate and / or ethyl-2-hydroxyvalerate at a concentration of 0.8 to 1.2 M, more preferably 1 M.

[0039] Preferably, the aqueous tetrazolium salt solution comprises 0.4 wt. % tetrazolium salt solution in distilled water (with initial dissolution of the salt using a small amount of N,N- dimethylformamide) and 0.1 M phosphate buffer (at pH = 7 to 8, e. g. pH = 7.6) in a volume ratio of 1 :3. The phosphate buffer may be the Sorensen phosphate buffer comprising 0.15 M Na2HPC>4.12H2O and 0.15 M KH2PO4.

[0040] Preferably, the aqueous cyanide salt solution also contains 1 M HCI, which acidifies the solution to pH = 7 to 8 (e. g. pH = 7.4). The individual components in the kit are stored in separate containers, and mixing occurs prior to the histochemical analysis. Stock solutions (tetrazolium salt, phosphate buffer, substrate, cyanide salt) without the use of preservative stabilisers will last 6-8 months when stored in a refrigerator at approximately 8 °C.

[0041] Brief description of the

[0042] Figures 1 and 2 show microscopic preparations of a native sample of duodenal tissue for histochemical analysis of diamine oxidase, wherein sites in intestinal villi with diamine oxidase activity are highlighted by staining dark blue, the background being blue, purple to pink. In Fig. 1 , the dark blue-coloured microgranules occur focally in enterocytes and massively in the lamina propria mucosae, indicating DAO activity within normal limits. In Fig. 2, the reaction is absent, indicating histamine intolerance.

[0043] Example 1

[0044] In a first step, the incubation medium for histochemical analysis of diamine oxidase in a native tissue sample is prepared by mixing 0.8 mg phenazine methosulfate (2.611 pmol); 2.0 mL 0.1 wt. % aqueous nitro blue tetrazolium chloride solution (2.446 pmol); and 0.2 mL aqueous substrate solution comprising 1 M D-alanine (200 pmol) and 0.5 M D- phenylalanine (100 pmol).

[0045] The 0.1 wt. % aqueous nitro blue tetrazolium chloride solution is prepared by mixing, in a 1 :3 volume ratio, 0.4 wt. % stock nitro blue tetrazolium chloride solution in distilled water (after initially dissolving NBT-CI with a small amount of N,N-dimethylformamide); and 0.1 M Sorensen phosphate buffer (at pH = 7.6), comprising 0.15 M Na2HPC>4.12H2O and 0.15 M KH2PO4.

[0046] The aqueous substrate solution may further comprise L-lactic acid, methyl-R-3- hydroxybutyrate and ethyl-2-hydroxyvalerate, each at a concentration of 1 M.

[0047] Said components may be included in a kit for histochemical analysis of diamine oxidase in a native tissue sample.

[0048] In a second step, the incubation medium is illuminated for 5 min. at room temperature to form an incubation medium suitable for histochemical analysis of diamine oxidase in a native tissue sample (see Example 5). Example 2

[0049] In a first step, the incubation medium for histochemical analysis of diamine oxidase in a native tissue sample is prepared by mixing 0.8 mg phenazine methosulfate (2.611 pmol); 2.0 mL 0.1 wt. % aqueous nitro blue tetrazolium chloride solution (2.446 pmol); 0.2 mL aqueous substrate solution comprising 1 M D-alanine (200 pmol) and 0.5 M D- phenylalanine (100 pmol); and 0.2 mL 0.1 M aqueous potassium cyanide solution (20 pmol).

[0050] The 0.1 wt. % aqueous nitro blue tetrazolium chloride solution is prepared by mixing, in a 1 :3 volume ratio, 0.4 wt. % stock nitro blue tetrazolium chloride solution in distilled water (after initially dissolving NBT-CI with a small amount of N,N-dimethylformamide); and 0.1 M Sorensen phosphate buffer (at pH = 7.6), comprising 0.15 M Na2HPC>4.12H2O and 0.15 M KH2PO4.

[0051] The 0.1 M aqueous potassium cyanide solution is prepared by acidification with 1 M HCI to pH = 7.4.

[0052] The aqueous substrate solution may further comprise L-lactic acid, methyl-R-3- hydroxybutyrate and ethyl-2-hydroxyvalerate, each at a concentration of 1 M.

[0053] Said components may be included in a kit for histochemical analysis of diamine oxidase in a native tissue sample.

[0054] In a second step, the incubation medium is illuminated for 5 min. at room temperature to form an incubation medium suitable for histochemical analysis of diamine oxidase in a native tissue sample (see Example 5).

[0055] Example 3

[0056] In a first step, the incubation medium for histochemical analysis of diamine oxidase in a native tissue sample is prepared by mixing 1 mL distilled water; 0.8 mg phenazine methosulfate (2.611 pmol); 1.0 mL 0.2 wt. % aqueous nitro blue tetrazolium chloride solution (2.446 pmol); and 0.2 mL aqueous substrate solution comprising 1 M D-alanine (200 pmol) and 0.5 M D-phenylalanine (100 pmol).

[0057] The 0.2 wt. % aqueous nitro blue tetrazolium chloride solution is prepared by mixing, in a 1 :1 volume ratio, 0.4 wt. % stock nitro blue tetrazolium chloride solution in distilled water (after initially dissolving NBT-CI with a small amount of N,N-dimethylformamide); and 0.1 M Sorensen phosphate buffer (at pH = 7.6), comprising 0.15 M Na2HPC>4.12H2O and 0.15 M KH2PO4.

[0058] The aqueous substrate solution may further comprise L-lactic acid, methyl-R-3- hydroxybutyrate and ethyl-2-hydroxyvalerate, each at a concentration of 1 M.

[0059] Said components may be included in a kit for histochemical analysis of diamine oxidase in a native tissue sample.

[0060] In a second step, the incubation medium is illuminated for 5 min. at room temperature to form an incubation medium suitable for histochemical analysis of diamine oxidase in a native tissue sample (see Example 5).

[0061] Example 4

[0062] In a first step, the incubation medium for histochemical analysis of diamine oxidase in a native tissue sample is prepared by mixing 1 mL distilled water; 0.8 mg phenazine methosulfate (2.611 pmol); 1.0 mL 0.2 wt. % aqueous nitro blue tetrazolium chloride solution (2.446 pmol); 0.2 mL aqueous substrate solution comprising 1 M D-alanine (200 pmol) and 0.5 M D-phenylalanine (100 pmol); and 0.2 mL 0.1 M aqueous potassium cyanide solution (20 pmol).

[0063] The 0.2 wt. % aqueous nitro blue tetrazolium chloride solution is prepared by mixing, in a 1 :1 volume ratio, 0.4 wt. % stock nitro blue tetrazolium chloride solution in distilled water (after initially dissolving NBT-CI with a small amount of N,N-dimethylformamide); and 0.1 M Sorensen phosphate buffer (at pH = 7.6), comprising 0.15 M Na2HPC>4.12H2O and 0.15 M KH2PO4.

[0064] The 0.1 M aqueous potassium cyanide solution is prepared by acidification with 1 M HCI to pH = 7.4.

[0065] The aqueous substrate solution may further comprise L-lactic acid, methyl-R-3- hydroxybutyrate and ethyl-2-hydroxyvalerate, each at a concentration of 1 M.

[0066] Said components may be included in a kit for histochemical analysis of diamine oxidase in a native tissue sample.

[0067] In a second step, the incubation medium is illuminated for 5 min. at room temperature to form an incubation medium suitable for histochemical analysis of diamine oxidase in a native tissue sample (see Example 5). Example 5

[0068] In a first step of a method for performing histochemical analysis of diamine oxidase in a native tissue sample, a frozen, sectioned, native (i. e. non-fixed) duodenal tissue sample is provided. Transport and storage of the sample prior to analysis is in petroleum ether at approximately -18 °C or on ice or dry ice. Sample sections may be approximately 5 pm thick, cut in a cryostat microtome at approximately -25 to -28 °C.

[0069] In a second step, the incubation medium is prepared according to any one of Examples 1 to 4.

[0070] In a third step, the sample is incubated with the above incubation medium with simultaneous illumination with white LED light for 30 min. at 37 °C.

[0071] In a fourth step, the sample is rinsed with saline for 10 min., fixed with 10% neutral formol for 10 min., washed with water for 10 min., rinsed with distilled water for 3 min., optionally dried, and finally mounted in an aqueous medium to form a microscopic slide for histochemical analysis of diamine oxidase. Sites with diamine oxidase activity (e. g. intestinal villi) are highlighted by staining in dark blue (see Figs. 1 and 2).

[0072] Industrial applicability

[0073] The method and kit described above can be used in laboratories for histochemical analysis of diamine oxidase in native tissue samples.

Claims

PATENT CLAIMS1. A method of preparing an incubation medium for histochemical analysis of diamine oxidase in a native tissue sample, characterised in that it comprises the steps of: a. mixing: i. phenazine methosulfate, preferably 0.5 to 1.0 mg phenazine methosulfate; ii. an aqueous tetrazolium salt solution at a concentration of 0.01 to 0.2 wt. % and in a molar ratio of tetrazolium salt to phenazine methosulphate of 2:1 to 1 :2, preferably 1.5 to 2.5 mL 0.1 wt. % nitro blue tetrazolium chloride solution; and iii. an aqueous substrate solution comprising at least one D-amino acid at a concentration of 0.4 to 1 .2 M and in a molar ratio of D-amino acid to phenazine methosulfate of 10:1 to 230:1 , preferably 0.1 to 0.3 mL substrate solution comprising 1 M D-alanine and 0.5 M D- phenylalanine; b. Illumination of the incubation medium for 1 to 10 min. to form an incubation medium for histochemical analysis of diamine oxidase in a native tissue sample.

2. The method according to claim 1 , characterised in that an aqueous cyanide salt solution at a concentration of 0.01 to 0.2 M and in a molar ratio of cyanide salt to phenazine methosulfate of 40:1 to 1 :4, preferably 0.1 to 0.3 mL 0.1 M potassium cyanide solution, is mixed in step a. of claim 1 .

3. The method according to claim 1 or 2, characterised in that the aqueous tetrazolium salt solution is prepared by mixing 0.4 wt. % tetrazolium salt solution in distilled water and 0.1 M phosphate buffer in a volume ratio of 1 :3.

4. The method according to any one of the preceding claims, characterised in that 0.8 mg phenazine methosulfate; 2.0 mL 0.1 wt. % aqueous nitro blue tetrazolium chloride solution; 0.2 mL aqueous substrate solution comprising 1 M D-alanine and 0.5 M D- phenylalanine; and 0.2 mL 0.1 M aqueous potassium cyanide solution are mixed.

5. The method according to any one of the preceding claims, characterised in that it further comprises the steps of: a. providing a frozen, sectioned, native tissue sample; b. incubation of the sample with the incubation medium prepared according to any of the preceding claims 1 to 4 with simultaneous illumination for 20 to 40 min.; and c. rinsing, fixing and mounting the sample in an aqueous medium to form a microscopic slide for histochemical analysis of diamine oxidase, wherein sites of diamine oxidase activity are highlighted by staining to perform the histochemical analysis of diamine oxidase in the native tissue sample.

6. The method according to claim 5, characterised in that the sample is a sample of duodenal tissue cut on a cryostat microtome.

7. The method according to any one of claims 5 to 6, characterised in that after incubation in step c., the sample is rinsed with saline for 5 to 15 min., fixed with formol for 5 to 15 min., washed with water for 5 to 15 min., rinsed with distilled water for 1 to 5 min., optionally dried, and mounted in an aqueous medium.

8. A kit for performing the method according to any one of the preceding claims 5 to 7, characterised in that it comprises: a. phenazine methosulfate; b. an aqueous tetrazolium salt solution, preferably nitro blue tetrazolium chloride, at a concentration of 0.01 to 0.2 wt. %; and c. an aqueous substrate solution comprising at least one D-amino acid, preferably D-alanine and D-phenylalanine, at a concentration of 0.4 to 1 ,2 M.

9. The kit according to claim 8, characterised in that it further comprises an aqueous cyanide salt solution, preferably potassium cyanide, at a concentration of 0.01 to 0.2 M.

10. The kit according to claim 8 or 9, characterised in that it comprises the aqueous nitro blue tetrazolium salt solution at a concentration of 0.1 wt. %; the aqueous substratesolution comprising D-alanine at a concentration of 1 M and D-phenylalanine at a concentration of 0.5 M; and the aqueous potassium cyanide solution at a concentration of 0.1 M.

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