IN-VITRO DIAGNOSIS OF HISTAMINE INTOLERANCE SYNDROME
20230109016 · 2023-04-06
Assignee
Inventors
Cpc classification
International classification
Abstract
A method of diagnosis of histamine intolerance in a person suspected of suffering from histamine intolerance syndrome. Isotope-labeled histamine metabolites, namely imidazole acetic acid and methylimidazole acetic acid are identified and measured in serum, plasma, urine and other bodily fluids following derivatization with a hydrazinoquinoline derivatization agent using the LC-MS/MS technique. The method and analytical technique is very sensitive and allows a safe use of an isotope-labeled oral histamine load as well as time-dependent measurements of the total activity of secreted and membrane-associated DAO enzymes for the sake of a differential diagnosis of histamine intolerance syndrome compared to food allergy, food hypersensitivity or food intolerance.
Claims
1-14. (canceled)
15. A method of diagnosis of histamine intolerance syndrome in a subject suspected of suffering from insufficient DAO enzyme activity comprising the steps of: administering orally a preparation, solution or suspension containing a known amount of stable isotopic labeled histamine; obtaining a sample of bodily fluid selected from blood, serum, plasma, urine, or saliva after a predefined period of time, optionally followed by removal of proteins; chemical derivatization of histamine metabolites using a derivatization agent which reacts with imidazole acetic acid and N-methyl-imidazole acetic acid compounds; measuring derivatized isotopic labeled histamine metabolites contained in said sample of bodily fluid using mass spectrometry which comprises an LC-MS/MS technique; and calculating the subjects' DAO activity and/or the subjects' histamine degradation capacity on basis of the amount of isotopic labeled imidazole acetic acid compounds and methyl imidazole acetic acid contained in said sample of bodily fluid.
16. The method of claim 15, wherein the stable isotopic labeled histamine is selected from histamine .sup.15N-labeled at positions at N1 and/or N3 or histamine .sup.13C-labeled at positions C2, C4, C5, C6, or C7 or histamine deuterium labeled at anyone or more hydrogen position, or histamine having a combination of stable isotope labels.
17. The method of claim 15, further comprising an immunological determination of secreted human diamine oxidase in serum or plasma.
18. The method of claim 15, wherein the derivatization agent is selected from hydrazinoquinoline, 2-hydrazinoquinoline, substituted hydrazinoquinoline, 7-chloro-4-hydrazinoquinoline, dialkyl-hydrazinoquinoline, 3,8-diethyl-2-hydrazinoquinoline, 6-fluoro-4-hydrazinoquinoline.
19. The method of claim 18, wherein the carboxyl group of the imidazole acetic acid or a derivative thereof is first activated prior to derivatization.
20. A method of diagnosis of histamine intolerance syndrome in a subject suspected of suffering from insufficient DAO enzyme activity comprising the steps of: — obtaining a sample of plasma or serum from a said subject suspected of suffering from histamine intolerance; adding to said sample a predefined amount of stable isotope-labeled DAO substrate as defined in claim 16 to produce a defined concentration of isotope-labeled DAO substrate in said sample; incubation of said sample for a predefined period under physiological conditions to obtain degradation of the said isotope-labeled substrate by the activity of DAO enzyme contained in said sample of plasma or serum; determining by mass spectrometry which comprises an LC-MS/MS technique the concentration of isotope-labeled metabolites of histamine following derivatization, to determine the activity of DAO and a histamine conversion rate per unit time for diagnosis of histamine intolerance syndrome.
21. The method of claim 20, which comprises a spiking of the sample with isotope-labeled histamine to achieve a spiked-diamine concentration of from 200 to 500 nmol/liter-1 serum.
22. The method of claim 20, which further calculates the activity of soluble human DAO enzyme in a said sample at given physiological conditions from the amounts of DAO substrates converted per unit of time and determining the histamine degradation capacity or half-life of histamine and/or DAO substrate in said patient, to diagnose a histamine intolerance when the patient's diamine oxidase activity is below 3 enzyme units (U) per milliliter serum or plasma or below 10 enzyme units (U) per milliliter as measured by the immunoassay and/or the half-life of carbon-13 histamine in serum is above a threshold found in a sample of a healthy subject.
23. The method of claim 20, comprising an immunological determination of soluble human diamine oxidase in serum or plasma and a determination of the enzyme activity of DAO in serum or plasma.
24. A method for diagnosis of histamine intolerance syndrome, comprising the use of a kit for a defined histamine oral load in the form of a solution, dispersion, or tablet, which contains a defined amount of stable isotope-labeled histamine selected from histamine .sup.15N-labeled at positions N1 and/or N3 or histamine .sup.13C-labeled at positions C2, C4, C5, C6, or C7 or histamine deuterium labeled at one or more hydrogen positions; and a derivatization agent for derivatization of imidazole acetic acid and N-methyl imidazole acetic acid selected from the group comprising derivatized or substituted hydrazinoquinolines, hydrazinoquinoline, 2-hydrazinoquinoline, 4- hydrazinoquinoline; 7-chloro-4-hydrazinoquinoline, dialkyl-hydrazinoquinoline, 3,8-diethyl-2- hydrazinoquinoline, 6-fluoro-4-hydrazinoquinoline and suitable buffers and solutions for sample preparation.
25. The method of claim 24, in which the kit for diagnosis of histamine intolerance syndrome comprises a histamine spiking solution, which contains a defined amount of stable isotope-labeled histamine selected from histamine .sup.15N-labeled at positions N1 and/or N3 or histamine .sup.13C-labeled at positions C2, C4, C5, C6, or C7 or histamine deuterium labeled at anyone or more hydrogen position, or histamine having a combination of stable isotope labels; a derivatization agent for derivatization of imidazole acetic acid and N-methyl imidazole acetic acid selected from the group comprising derivatized or substituted hydrazinoquinolines, hydrazinoquinoline, 2-hydrazinoquinoline, 7-chloro-4- hydrazinoquinoline, dialkyl-hydrazinoquinoline, 3,8-diethyl-2-hydrazinoquinoline, 6-fluoro-4-hydrazinoquinoline; and suitable buffers and solutions for sample preparation.
26. The method of claim 24, in which the kit for diagnosis of histamine intolerance syndrome comprises an internal standard solution of imidazole acetic acid and methyl imidazole acetic acid additionally isotopic labeled at least at one more position compared to the expected isotopic labeled metabolites.
27. The method of claim 24, in which the kit for diagnosis of histamine intolerance syndrome comprises two to six matrix standards with varying concentrations of a mixture of the expected isotopic labeled imidazole acetic acid and methyl imidazole acetic acid.
28. The method of claim 24, in which the kit for diagnosis of histamine intolerance syndrome further comprises an enzyme solution of catalase, peroxidase, and/or alcohol dehydrogenase.
Description
DETAILED DESCRIPTION OF THE INVENTION
[0055] Histamine is present in many fermented foods and beverages and high levels of ingested histamine can trigger pseudoallergic reactions, asthmatic wheezing, swelling of eyelids, eye turgor and respiratory mucosa, flushing, skin reddening, rushes, urticaria, pruritus. High levels of ingested histamine may also lead to a lowering of the blood pressure, contraction of the smooth muscles in the intestine and respiratory tract, and therefore to gastrointestinal problems, abdominal pain, constipation, diarrhoea, migraine, headache, arrhythmia, oedema and sleep disturbances.
[0056] Histamine intolerance syndrome is observed in cases of increased availability of histamine or impaired histamine degradation or both. An increased availability of histamine may be due to an overproduction of endogenous histamine under conditions of allergies, mastocytosis, bacterial infections, gastrointestinal bleedings and/or due to increased ingestion of histidine or histamine by a consumption of fermented food, often in combination with a consumption of alcohol. Alcohol and histamine metabolic pathways have the common enzymes aldehyde dehydrogenase and aldehyde oxygenase and a consumption of alcohol will therefore enhance the physiological effects of histamine (Zimatkin S M et al, Alcohol- histamine interactions, Alcohol Alcohol 1999, 34(2):151-7). Histamine degradation may be inhibited or compromised by a lack of co-factors such as vitamin C, vitamin B6, copper or manganese ions or the presence of competing biogenic diamines. It is further known that the activity of DAO in serum is inhibited by numerous drugs, including muscle relaxants (pancuronium, alcuronium, D-tubocurarine), narcotics (thiopental), analgetics (morphine, pethidine, non-steroidal and anti-inflammatory drugs (acetylsalicylic acid, metamizole), local anaesthetics (prilocaine), antihypotonics (dobutamine), antihypertensive drugs (verapamil, alprenolol, dihydralazin), antiarrhythmics (propafenone), diuretics (amiloride), drugs influencing gut motility (metoclopramide), antibiotics (cefuroxime, cefotiam, isoniazid, pentamidin, clavulanic acid, chloroquine), mucolytics (acetylcysteine, ambroxol), broncholytics (aminophylline), H2-receptor antagonists (cimetidine), cytostatics (cyclophosphamide), antidepressants (amitriptyline). DAO levels in humans further increase sharply within 30 minutes of heparin administration while the presence of heparin in experiments with cell culture has no impact on the DAO level or its activity.
[0057] There are two primary pathways for histamine inactivation:—(i) by methylation of its imidazole ring which is catalysed by histamine-N-methyltransferase (NMT). The resulting N-methylhistamine is physiologically not active. And, (ii) by oxidative deamination of the primary amino group of histamine catalysed by diamine oxidase (DAO). NMT in the cytosol controls the transmission of the histaminergic signals in the nervous system and DAO in serum or tissue-bound (e.g. intestinal mucosa, kidney) degrades the dietary histamine when passing into the circulation and thereafter.
[0058] The symptoms of histamine intolerance are only observed when histamine levels reach in serum a pathological level of 9 nmol (about 1 ng/mL serum) or above. The histamine levels in serum are however not directly dependent on the amount of DAO enzyme in the blood or its activity. As foods and beverages may contain histamine in amounts of up to 50 mg per 100 grams, their consumption corresponds to a spiking of the physiological histamine clearance capacity. The spiking of a plasma or serum sample with isotopic labeled histamine (“diamine-spiking”) challenges the histamine clearance. A measured DAO activity of 3 enzyme units in serum would then correspond to a clearance of 3 000 nmol histamine per minute. Pathological histamine concentrations of up to 5 to 10 000 nmol.Math.litre-1 serum have been determined in patients with anaphylactoid reactions. Thus, a measured DAO activity in serum of 3 enzyme units as determined by a spiking experiment would corresponds to a half-life of less than 2 minutes to 4 hours for highly increased histamine levels, e.g. as would be obtained after the normal consumption of non-toxic food and beverages high in histamine. Thus, a DAO activity in serum of 3 enzyme units or more excludes any symptoms of histamine intolerance.
[0059] The measured DAO activity following a spiking of a plasma or serum sample with a histamine challenge should not be confused with a measurement of the statutory DAO in serum as disclosed by prior art methods. The metabolic pathways of histamine and alcohol are linked because the primary metabolite of ethanol degradation, acetaldehyde, and the primary metabolites of histamine degradation, methylimidazole acetaldehyde and imidazole acetaldehyde, compete for the same aldehyde dehydrogenase and aldehyde oxidase. Alcohol and acetaldehyde further induce a release of histamine from the storage granules in peripheral mast cells so that a consumption of alcohol can provoke food-induced histaminosis which leads to gastric and intestinal damages and bronchial asthma in many persons. An increasing epidemiological problem. Histamine levels in common alcoholic beverages range from 3 to 120 micrograms/L in white wine, 20 to 300 micrograms/L in beers, from 15 to 700 micrograms/L in champagnes and from 60 to 3800 micrograms/L in red wine.
[0060] If the measured half-life of “spiked” (dietary) histamine in serum is below a threshold (120 seconds to 4 hours, depending on circumstances) the patient will likely not suffer from increased histamine levels and this will also apply to a normal consumption of foods rich in histamine. On the other hand, we now know that a measurement of the statutory DAO or its activity in serum allows no reliable differentiation between histamine intolerance and e.g. a food allergy. A conventional determination of a diamine/histamine conversion rate in serum may therefore be not enough to diagnose histamine intolerance or the aetiology of histamine intolerance and why patients are suffering from an unphysiological histamine boost over an extended period of time (several hours or overnight).
[0061] The patient's total capacity for ingested histamine clearance (tissue associated DAO and secreted serum DAO) must be known for differential diagnosis of histamine intolerance from food hypersensitivity, food intolerance and food allergy and this non-compromised (not inhibited by alcohol or drugs) total dietary histamine clearance requires a measurement of histamine metabolites in bodily fluids. This is because the amount and activity of tissue-associated DAO of a patient also contributes to the total dietary histamine clearance capacity. In the prior art, the serum DAO activity was never measured having regard to the total clearance of ingested histamine which involves tissue-associated DAO. The prior art methods merely determined which histamine clearance is achieved in the serum by an addition external diamine (histamine or putrescine). In other words, the measurements were done with the expectation that the soluble DAO activity in serum would coincide with the total amount of DAO enzyme in the body. That model did not consider the various histamine inactivation pathways nor the multiple forms of DAO.
[0062] The present invention considers that the metabolic pathways may change by an excess of dietary histamine, that histamine degradation in serum can be compromised by the presence of drugs or alcohol and that serum DAO activity is dependent on multiple factors, including pH because histamine may also be a substrate of monoamine oxidases (MAO) at higher pH above 8.0. Prior art assays even recommend using a Tris-HCl buffer (tris-(hydroxymethyl)-aminomethane) having a pH of 8.0 to 8.5 for a determining the diamine/histamine conversion ratio and rate as such a buffer solution has “particularly positive effects on the DAO activity” (see U.S. Pat. No. 8,003,343, column 5). The conventional assays for diagnosis of histamine intolerance never examined or quantified the “inactivated” metabolites of histamine and could not differentiate between monoamine oxidase (MAO) activity and diamine oxidase (DAO) activity nor detect any inhibition, e.g. by external drugs or a lack of co-factors.
[0063] The present application considers that the DAO Michaelis constants, the Km values of histamine and putrescine (1,4-diamine butane) are about 2.8 micromole and 20 micromole, respectively, as measured using isolated recombinant human kidney DAO expressed in Drosophila cells (see Elmore B O et al., Human kidney diamine oxidase: heterologous expression, purification, and characterization, J Biol Inorg Chem 2002, 7(6):565-579). Thus, the substrate concentrations for histamine and putrescine at which the DAO reaction rate is half of the maximum reaction rate differ by a factor of 7. Putrescine can therefore not be used in methods of diagnosis of histamine intolerance syndrome. Human serum DAO is further partly inhibited by 100 micromolar histamine in the assay and a histamine concentration of 500 micromoles inhibits human DAO activity by about 60% (see U.S. Pat. No. 8,003,343). The present disclosure therefore considers for as well as the diamine/histamine conversion ratio and a diamine/histamine conversion rate per unit time diagnosis of histamine intolerance in accordance with the Michaelis-Menten model for catalysts in biological system that the DAO enzyme (E) must first combine with the diamine substrate (S) to form an enzyme-substrate (ES) complex. This enzyme-substrate (ES) complex can then proceed with the oxidative degradation of the diamine/histamine and dissociate again. Thus, the degradation rate of histamine is given by the equation
in which Vmax is the degradation rate when the DAO enzyme is fully saturated with substrate and Km, the Michaelis-Menten constant, is the substrate concentration at which the reaction rate is half maximal and [S] is the concentration of the substrate.
[0064] The prior art works with an excess of histamine or diamine in the assay and ignores the different Km values for histamine and putrescine so that no more than a maximal DAO activity at an artificial “pH and enzyme environment” is determined which is further compromised by competing metabolic pathways and varying amounts of histamine, diamine, DAO and/or MAO in the sample. Prior art solutions for measuring the DAO enzyme activity work with histamine or diamine in an amount of about 80 to 100 micromoles per litre, claiming a range from 5 to 400 micromoles, which is well above physiological levels. The usual limit for pathological histamine values in serum is 9 nanomoles per litre. Histamine concentrations in serum of patients with an anaphylactoid reactions are below 10 000 nanomoles (nmol) per litre (L), say less than 10 micromolar, with histamine disappearing from serum at 37° C. with an apparent half-life of 2 minutes to 4 hours. Thus, a theoretical maximal DAO activity is determined or a wrong activity but not the actual DAO enzyme activity in serum. On the other hand, the present method allows an administration or oral administration of typical dietary or physiological concentrations of histamine due to the high sensitivity of the LC-MS/MS detection method. The resulting serum levels of isotopic labeled imidazole acetic acid and methyl imidazole acetic acid, as well as of other histamine metabolites take account of the activity of tissue-associated DAO, e.g. in the mucosal membrane and in the kidney, in addition to serum DAO.
[0065] The kinetic studies on the deaminating oxidation of .sup.14C-putrescine at concentrations from 1 micromole to 5 millimole confirm the existence of two enzymes in serum: DAO with high affinity which is sensitive to alpha-aminoguanidine, and MAO with a lower affinity for the substrate which is selegiline-sensitive. The present disclosure refers to a method of diagnosing histamine intolerance when the diamine oxidase activity in serum is below 3 enzyme units (U) per millilitre serum, if determined in vitro at RT (25° C.). The disclosure further comprises a method of diagnosis wherein the actual diamine oxidase activity in serum is 50% of the normalized diamine oxidase activity in serum as determined by an immunoassay for human DAO, indicating an inhibition of the enzyme DAO in serum by the presence of drugs or other biogenic amines.
[0066] The serum DAO concentration and the measured serum DAO activity by REA are already used biomarkers for histamine intolerance and associated symptoms. Both, serum DAO and the measured DAO activity in serum complement each other as the DAO activity in serum depends on the absence of inhibitors (drugs, alcohol) and cofactors such copper ions, manganese ions and vitamin C or vitamin B6. By further quantitating the cofactors in blood, the clinical laboratory will allow a treatment of the disorder in respect of the co-factors which have to be supplemented or which drug should be better replaced in order not to inhibit dietary histamine degradation. A differential diagnosis of histamine intolerance syndrome vis-à-vis food intolerance, food hypersensitivity or food allergy is not possible.
[0067] Histamine intolerance (HIT) occurs when the combined activities of serum DAO and tissue-associated DAOs in the intestinal mucosal membrane, kidney, lungs are not enough to degrade a dietary histamine spike in addition to the histamine produced in the body. The overall histamine degradation capacity in the body depends likewise on the absence of DAO inhibitors and the presence of co-factors. The large sequence identity between secreted DAO and tissue-associated DAOs suggests that the inhibitors and co-factors affect most similar the overall histamine degradation capacity in the body as can be determined for the degradation capacity of secreted serum DAO alone. The specific enzyme activity of DAO in a patient's sample is therefore a reference measure of the overall histamine degradation ability in the body.
[0068] The specific enzyme activity is the amount of product formed in each amount of time under given conditions per milligram of enzyme. The specific activity is equal to the rate of reaction multiplied by the volume of reaction divided by the mass of total protein. The SI unit is the katal, 1 katal=1 mol s−1, but this is an excessively large unit. A more commonly used value is the (one) enzyme unit (U) which is 1 micromole min-1. (One enzyme unit (U) corresponds to 16.67 nanokatals). The specific enzyme activity is therefore a measure of the processivity at a specific (usually saturating) substrate concentration. Where the molecular weight and the precise amount of the enzyme is not known, as in the present case, the measured turnover of isotope-labeled histamine in serum and the separately measured specific activity of serum DAO allow calculation of how much DAO enzyme, secreted or tissue-associated, is effectively present in the body and its total activity for degradation of dietary histamine taken up with the foods. The turnover number can be visualized as the number of times each enzyme molecule conducts its catalytic cycle per second. The method of the invention therefore allows a reliable differentiation between histamine intolerance syndrome and food intolerance or food allergy. The histamine intolerance syndrome goes in line with an insufficient degradation of a spike of dietary histamine for a lack of tissue-associated DAO in the intestinal mucosal membrane and to a minor degree for a lack of serum DAO. The symptoms of food allergy or food hypersensitivity are caused by a spike of endogenous histamine.
[0069] Gas chromatography-mass spectrometry (GC-MS) has been an analytical platform for detecting polar ketones, aldehydes, carboxylic acids because of its high resolution and sensitivity. The derivatization methods for GC analysis comprise a tandem oximation-silylation procedure. The oximation of aldehydes and ketones is achieved by a reaction between the carbonyl group and methoxyamine. The reaction with silylation agents replaces the active hydrogen in alcohols, carboxylic acids, amines, and thiols. Both reactions for a GC-based analysis are incompatibility with water. The necessary dehydration by solvent extraction, evaporation, or lyophilization makes the sample preparation for GC-MS analysis time-consuming and the entire process is therefore too inefficient for a use in a clinical laboratory. Thus, a method for liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed which is more compatible with bodily fluids. A direct LC-MS/MS analysis of histamine and its degradation/inactivation products of histamine is difficult due to their poor chromatographic performance and low ionization efficiency. The reverse phase LC column are not well suited for retaining and separating these hydrophilic metabolites, leading to poor or no signals in the mass detector. The hydrophilic interaction liquid chromatography (HILIC) offers improved chromatographic separation of polar compounds. On the other hand, a chemical derivatization leads to a better chromatographic performance and detectability of the histamine degradation products: imidazole acetic acid and N-methylimidazole acetic acid. Esterification and amidation are typical derivatization reactions for carboxylic acids. The functional groups 2-picolylamine and 2-hydrazinopyridine give improved ionization efficiency of carboxylic acids (Higashi T et al, Simple and practical derivatization procedure for enhanced detection of carboxylic acids in liquid chromatography-electrospray ionization-tandem mass spectrometry. J Pharm Biomed Anal 2010, 52: 809-818) Other derivatization reagents for carboxylic acids include 2-chloro-1-methylpyridinium (Cartwright A J et al., Derivatisation of carboxylic acid groups in pharmaceuticals for enhanced detection using liquid chromatography with electrospray ionisation tandem mass spectrometry. Rapid Commun Mass Spectrom 2005, 19:1058-1062), triethylamine (Barry S J et al., Derivatisation for liquid chromatography-/electrospray mass spectrometry: synthesis of pyridinium compounds and their amine and carboxylic acid derivatives. Rapid Commun Mass Spectrom 2003, 17:603-620), and heptadecafluoroundecylamine (Hayama T et al., Fluorous derivatization combined with liquid chromatography/tandem mass spectrometry: a method for the selective and sensitive determination of sialic acids in biological samples. Rapid Commun Mass Spectrom 2010, 24, 2868-2874). Derivatives of phosphonium bromide, hydroxylamine and hydrazines such as 2,4-dinitrophenylhydrazine and dansyl hydrazine lead to derivatives which can be more easily ionized than the parent compounds. A most preferred hydrazine derivatization agent are hydrazinoquinolines, in particular, 2-hydrazinoquinoline, as they allow a derivatization of the oxidized histamine metabolites in urine, serum, plasma and tissue extracts in one hour. Compared to derivatives of 2-hydrazinopyridine, 2-picolylamine and dansyl hydrazine, hydrazinoquinolines derivatives achieve better chromatographic performance in reversed phase LC system and higher ionization efficiency. The use of 2-hydrazinoquinoline as derivatization agent of substituted and non-substituted imidazole acetic acid for simultaneous LC-MS analysis provides therefore a new platform for determining the total histamine degradation rate in the body.
[0070] The isotopic labeled histamine and N-methyl-histamine may be labeled at positions shown in formulae I. Preferred internal standards of the expected histamine metabolites may be labeled additionally, e.g. at least at one more position, preferably at three or four positions with the corresponding heavy isotope.
[0071] As shown in
[0072] The present application discloses that the degradation products of histamine-imidazole-4-acetic acid and methyl imidazole-4- acetic acid—can be jointly derivatized—in-situ like—by a one-pot derivatization and thereafter be separated on a reverse phase column. Common derivatization reactions include silylation, esterification, acetylation, and alkylation. A suitable derivatization agent is 2-hydrazinoquinoline (2-HQ). Using 2-HQ as derivatization agent the LC-MS/MS method can be optimized for the resulting 2-HQ derivatives shown in formulae IV and V.
[0073] The combination of the LC-MS/MS technique, the use of an isotopic labeled histamine for an oral histamine load and/or for spiking and the high analytical sensitivity for the histamine degradation products after a derivatization of imidazole acetic acid and N-methylimidazole acetic acid, this combination allows a differential diagnosis of histamine intolerance syndrome compared to anaphylactoid and allergic reactions because the inactivation of isotopic labeled dietary histamine is determined. The alternative clinical laboratory method may comprise the steps: (i) taking an aliquot of plasma or serum from a patient suspected of suffering from histamine intolerance syndrome; (ii) optionally, determining the concentration of human diamine oxidase (hDAO—EC 1.4.3.22) in said aliquot using an immunoassay for human diamine oxidase (DAO); (iii) mixing (spiking) an aliquot of plasma or serum with a predefined amount of DAO substrate comprising isotopic labeled histamine of formula I at a concentration range of from 10 to 10 000 nanomoles/L, preferably from 100 to 500 nanomoles/L and incubation of the histamine spiked sample at physiological conditions (pH 7.2 to 7.8, preferably 7.3 to 7.6; 36-38° C.) for a predefined period from 2 minutes to 24 hours, followed by a chemical derivatization reaction; (iv) transferring said aliquot to a mass spectrometry device, preferably a device comprising a combination of liquid chromatography and tandem mass spectrometry and (v) determining by mass spectrometry in said aliquot the concentrations of histamine metabolites, and optionally of histamine, to obtain a histamine conversion ratio and a histamine conversion rate; to diagnose a histamine intolerance when the patient's DAO activity in said sample is below a threshold level. This threshold level may be set at 3 enzyme units (U) per milliliter serum at physiological conditions of the blood and/or normalized 10 enzyme units (U) per milliliter. or a half-life of dietary histamine levels of above a certain threshold for healthy subjects (2 minutes to 24 hours, depending on concentrations).
[0074] A person skilled in the art will also contemplate to directly determine the histamine concentration in the plasma sample to determine whether it is in a pathological range.
EXAMPLES
Example 1— Representative Histamine Catabolites and their Determination by LC-MS/MS
[0075] For determination and quantitation of representative histamine catabolites 500 μl serum sample and control were transferred into 1.5 ml reaction vials and incubated at 37 degrees Celsius with 100 μl aqueous buffer containing 500 nM (92 ng/mL) isotopic labeled histamine and 1 U/100 μl catalase/peroxidase and aldehyde dehydrogenase for predefined periods (10′ ,20′ ,30′ ,60′ , 2 h, 4 h, 6 h, 24 h).
[0076] 10 μl sample/control were placed in 0.5 ml reaction vial, mixed with 80 μl acetonitrile, and followed by centrifugation at 9000×g for 10 minutes. 85 μl supernatant was transferred into a closed reaction vial. The carboxyl groups were activated by 5 μl 200 mM diphenyl-2-pyridylphosphine/2,2-dipyridyldisulfide in acetonitrile. 10 μl 2-hydrazinoquinoline dissolved in acetonitrile was added to a final concentration of 400 mM. The derivatization sample was incubated at 60 degrees Celsius for 1.5 hours, the reaction stopped by cooling and addition of 100 μl H.sub.2O. The derivatized sample was centrifuged at 9000×g for 10 minutes and transferred into autosampler vials. 5 μl sample were injected into the LC-MS/MS system.
[0077] The baseline separation was achieved on a reversed phase column (Waters ACQUITY UPLC BEH C18 Column, particle 1.7 μm, 2.1 mm×50 mm—other columns such as C18, C8, C4, PFP and others with varying size and particle sized were also tested). The derivatized sample was separated using two mobile phases. Mobile phase A: 2% acetonitrile in water. Mobile phase B: 98% acetonitrile. The mobile phases were adjusted at pH 8.2 with 1 mM ammonium acetate, applying a flow rate of 400 μl/min. The column temperature was 40 degrees Celsius (range from 30-50 degrees Celsius). The gradient comprised a loading step of mobile phase A for 1 minute (range 0.1-2 mins); a slope to 20% mobile phase B within 0.1 minutes, (range 0-50% B in 0.1 to 1 mins); a separation slope from 20% B to 60% B in 3 mins; a washout phase with 100% mobile phase B for 1.5 mins (range 95-100% B for 0.5 to 10 mins), a first re-equilibration phase from 100% B to 100% A in 0.1 mins (range time 0.1 to 1 mins) and a second re-equilibration phase with 100% A for 2 mins (range time 0.1 to 10 mins). A skilled person will of course adapt the gradient to the specific LC-system and column. An exemplary gradient (Flowrate: 400 μl/mi; column temperature: 40° C.; solvent A: 2% acetonitrile with 1 mM ammonium acetate, pH 8.2; solvent B: 98% acetonitrile with 1 mM ammonium acetate, pH 8.2) is given in Table I below:
TABLE-US-00001 TABLE 1 time [min] mobile phase B [%] 0 0 1 0 1.1 20 4.1 60 5 100 6.5 100 6.6 0 8.6 0
[0078] The exemplary fragments of Table 2 were used as qualifiers/quantifiers, originated from imidazole-4-acetic acid and methyl imidazole acetic acid. A person skilled in the art can chose alternative fragments (labelled or unlabelled) for unambiguous detection by MS/MS.
##STR00004##
TABLE-US-00002 TABLE 2 Monoisotopic Monoisotopic mass unlabeled mass 13C Ion [M + H]+ [M + H]+ Precursor 268.119 273.155 Fragment (1) 81.045 85.059 Fragment (2) 109.04 114.058 Fragment (3) 160.087 160.087
##STR00005##
[0079]
##STR00006##
TABLE-US-00003 TABLE 3 Monoisotopic Monoisotopic mass unlabeled mass 13C Ion [M + H]+ [M + H]+ Precursor 282.128 282.134 Fragment (1) 95.06 99.075 Fragment (2) 123.055 128.073 Fragment (3) 160.087 160.087
##STR00007##
[0080] Representative chromatograms of 2-HQ-imidazole-4-acetic acid and 2-HQ-methylimidazole-4-acetic acid from spiked and analysed serum and plasma samples are shown in
[0081] With respect to 2-HQ-Imidazole-4-acetic acid the described method has a sensitivity of 0.65 ng/mL (average blank signal+3 standard deviations) and an average accuracy of 101.26% (±7.25%) as calculated over a calibration range of 1-100 ng/mL (increments 1, 2, 5, 10, 20, 50, 100 ng/mL, triple injections); see
Example 2—Determination of the Histamine Metabolites Using SPE Preconcentration
[0082] The sample was treated as described in example 1. 100 μl sample was transferred to new reaction vial, mixed with 300 μl ice-cold MeOH, and centrifuged at minimum 9000 g at 4° C. for 10 minutes. The supernatant was transferred to a new vial and mixed with SPE loading buffer, containing 25 mM TRIS acetate buffer pH 9.5 in a ratio of 1:3. The mixed-mode-strong anion exchange SPE cartridge was conditioned with 1 ml MeOH, followed by an equilibration using 1 mL of loading buffer. The sample was loaded onto the equilibrated SPE cartridge, washed with 1 mL loading buffer, 1 mL aqua bidest and 1 mL methanol. The sample was eluted with 2×250 μl methanol containing 5% formic acid, dried in a rotary evaporator under nitrogen and reconstituted in 25 μL reconstitution solution, comprising 1:3 water/acetonitrile. 25 μl activation mix was prepared as described, added to the sample, and 50 μL derivatization agent (400 mM 2-hydrazinoquinoline dissolved in acetonitrile) added. The sample was sample further processed as described in example 1 and the derivatized metabolites determined and quantified.
Example 3 Histamine Provocation and Measuring Total Histamine Degradation
[0083] The subject is provoked using an oral provocation solution comprising isotopic labeled histamine at a concentration of 0.001-0.5 mg/kg, preferably between 0.01 mg/kg and 0.1 mg/kg. Thereafter, urine and serum sample are collected at regular intervals, preferably for 1-4 h. The samples are then processed and analysed as stated in example 1.
Synopsis
[0084] A method of differential diagnosis of histamine intolerance syndrome in a human subject suspected of suffering from histamine intolerance syndrome compared to food allergy, food hypersensitivity or food intolerance. The patient is first subjected to an oral load comprising a defined amount of histamine and stable-isotope labelled histamine. The oral histamine load is preferably typical or equivalent to that of a histamine containing food or beverage. The as well as the diamine/histamine conversion ratio and a diamine/histamine conversion rate per unit time method comprises the steps of (i) obtaining a sample of bodily fluid from said patient after a defined period of time, (ii) derivatization of imidazole acetic acid and methylimidazole acetic acid using 2-hydrazinoquinoline (2HQ) and (iii) determining the amount of isotope-labeled 2-HQ-imidazole acetic acid and 2HQ-methylimidazole acetic acid, to determine the degradation rate of dietary histamine and for differential diagnosis of histamine intolerance syndrome when slow compared to the histamine degradation rate of a healthy individual. The extreme sensitivity of the described method allows a use of a low oral histamine load and monitoring of the histamine degradation via its imidazole metabolites in plasma or serum. The described method allows a measurement of the combined activities of tissue-associated and secreted serum DAO for differential diagnosis of histamine intolerance.
[0085] Alternatively, histamine intolerance syndrome can also be diagnosed fully as well as the diamine/histamine conversion ratio and a diamine/histamine conversion rate per unit time. This method comprises the steps of: —
[0086] adding to plasma or serum a predefined amount of isotope-labeled DAO substrate, e.g. .sup.13C-labeled histamine,
[0087] incubation of said sample at physiological conditions for a predefined period from 1 minute to 10 minutes to obtain oxidative degradation of histamine by the activity of the human DAO enzyme contained in said sample;
[0088] determining by LC-MS/MS technique the concentration of isotope-labeled imidazole acetic acid or methyl imidazole acetic acid and, optionally, other isotope-labeled diamine metabolites, to determine a histamine degradation rate and/or the histamine half-life in serum or plasma.