WO2018002173A1

WO2018002173A1 - Method of detecting copd by mass spectrometry

Info

Publication number: WO2018002173A1
Application number: PCT/EP2017/066053
Authority: WO
Inventors: Lukas BREGY; Diego GARCIA-GÓMEZ; Malcolm KOHLER; Yvonne NUSSBAUMER-OCHSNER; Pablo Martinez-Lozano Sinues; Yannick SUTER; Renato Zenobi
Original assignee: Eth Zurich; Universität Zürich
Priority date: 2016-06-29
Filing date: 2017-06-28
Publication date: 2018-01-04

Abstract

The present invention provides a method for determining whether a patient suffers from COPD, said method comprising the step of: a. providing at a sample comprising, or consisting of, exhaled breath of said patient to an ionization chamber of a mass spectrometer; b. simultaneously determining the presence, absence or relative concentration of at least two metabolites in said sample using said mass spectrometer; c. determining if the presence, absence or relative concentration of said metabolites is indicative of COPD by comparing the determined presence, absence or relative concentration of at least two metabolites to predetermined reference values, characterized in that the metabolites are chosen from the group of metabolites of amino acids, metabolites emanating from of an oxidative or carbonyl stress process, or from combinations of said groups, optionally together with pyridine.

Description

TITLE METHOD OF DETECTING COPD BY MASS SPECTROMETRY

TECHNICAL FIELD

The present invention relates to a method for determining whether an individual suffers from COPD (chronic obstructive pulmonary disorder), and to a computer-controlled medical diagnosis system for determining whether a individual suffers from COPD and in particular if the individual suffering from COPD additionally suffers from acute exacerbations of COPD (AECOPD).

PRIOR ART

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. Diagnosis is currently based on fixed airflow limitation in spirometry, respiratory symptoms and an appropriate history of exposure to risk factors. However, extensive training on how to perform and interpret spirometry has to be done to guarantee adequate disease assessment by medical personnel. While spirometry can help identifying the disease at a stage at which breathing symptoms arise, it is inherently unsuited for use in the diagnosis of the early stages of COPD, where breathing symptoms might be absent. Also, because of the breathing symptoms can potentially be caused by other diseases of the respiratory tract, such as asthma, the results from spirometry suffer from a lack of specificity and are therefore usually complemented by correlation to other symptoms indicative of COPD, such as dyspnea, chronic cough or sputum production, or to a history of exposure to risk factors. Based on the aforementioned specificity shortcomings of spirometry, a more reliable diagnostic instrument is desirable.

Biochemical markers such as proteins have recently been used as alternative means for diagnosis of COPD. However, the use of proteins such as interleukins, heat shock proteins, caspase-cleaved cytokeratin-18 (ccCK-18) and others requires taking samples of tissue, blood or urine, and a subsequent sample preparation that is both time-consuming and technically demanding for target proteins to be analyzed quantitatively, in most cases, through immunoassays.

The exhaled air or breath of a patient, on the other hand, can easily be sampled and contains certain amounts of metabolites. These metabolites are either produced locally in the respiratory tract itself or systemically and then released across the blood-gas barrier during gas-exchange, i.e. breathing. As such, these metabolites may be useful biomarkers of the internal biochemical processes underlying COPD and serve as fast and non-invasive biomarkers in the diagnosis and monitoring of COPD, and of AECOPD in particular.

WO2010/031788A1 discloses a method for the diagnosis of COPD, in which volatile organic carbon (VOC) compounds are detected in exhaled breath of a patient. The exhaled air is collected in a bag and then lead over an absorption medium capable of trapping VOCs. The trapped VOCs are subsequently released through heating the absorption medium and lead into a gas chromatography capillary column and detected by a mass spectrometer equipped with a time-of-flight detector (TOF-MS). A problem with such methodology and/or set-up is that the heating of the absorption medium and of the gas chromatography column can in some cases result in fragmentation of the compounds comprised in the exhaled breath, thereby making it impossible to determine some of the more thermally fragile compounds. The VOCs used as biomarkers for COPD are described such as isoprene, saturated an unsubstituted branched alkanes such as C 16H34; 4,7,- dimethyl undecane; 2,6-dimethyl heptane; 4-methyl octane and linear C i6H₃₄. WO2010/031788A1 thus discloses a method in which VOCs are determined in stored/adsorbed breath samples using GC-MS.

US 9 121 844 Bl discloses a method for analyzing vapors emanating from the human body, such as for example breath or skin vapors, by combining a SESI ionizer with a mass spectrometer for the purpose of classifying humans with respect to ion mass and signal intensity of the volatile or semi-volatile species comprised in said vapors. The disclosed method is in essence improved vis-a-vis the prior art in the sense that the data collected is made more reliable by bringing both the sample flow and the background flow to the same humidity before being sampled into the SESI ionizer. While it is disclosed that the data pair of ion mass and signal intensity can be used to recognize the state of health of a person, there is however no disclosure to be found which could be used to relate a particular medical condition to the disclosed to ion mass and/or signal intensity.

It is thus desirable to provide a method that allows determining almost instantly if a person suffers from COPD or AECOPD on the basis of alternative biomarkers with high and specificity and sensitivity.

SUMMARY OF THE INVENTION

The present invention provides for a method and computer-controlled medical diagnosis system that allows determining easily and non-invasively if an individual suffers from COPD and AECOPD by analysis of the exhaled breath of an individual, using multiple, newly-found biomarkers comprised in the exhaled breath. The method and computer- controlled medical diagnosis system of the present invention has the advantage of being more reliable than traditional spirometry analysis and less complex than other biomarker- based methods that require blood sampling and subsequent preparation/purification of blood samples before biomarker analysis. The newly-found biomarkers that can be used in the method and the computer-controlled medical diagnosis system of the present invention have been identified by correlating features comprised in the exhaled breath of control individuals and COPD patients, and measured by SESI-MS, to the FEV1 (% predicted). FEV1 (forced expiratory volume in 1 second) represents the proportion of a person's vital capacity that they are able to expire in the first second of forced expiration. In total, this methodology has yielded a plurality of compounds which can be used to classify for COPD / non-COPD status. Without wishing to be held to a particular theory, the applicants have classified the newly-found biomarkers with respect to certain types of metabolites of biomolecules such as for example fatty acids. It is understood that the newly-found biomarkers are, in and of themselves as compounds, reliable indicators in exhaled breath that allow classifying for COPD/ non- COPD status, irrespective of their assumed or true metabolic or catabolic origin. Thus, any teaching relative to a group of metabolites or individual metabolites should be considered to apply to the individual compounds themselves as enumerated within the group of metabolites, unless indicated otherwise.

Accordingly, it is an object of the present invention to provide a method for determining whether a patient suffers from a disease of the lower respiratory tract such as COPD or AECOPD, said method comprising the step of:

a. providing at a sample comprising, or consisting of, exhaled breath of said patient to an ionization chamber of a mass spectrometer;

b. simultaneously determining the presence, absence or relative concentration of at least two metabolites in said sample using said mass spectrometer;

c. determining if the presence, absence or relative concentration of said metabolites is indicative of COPD or AECOPD by comparing the determined presence, absence or relative concentration of at least two metabolites to predetermined reference values, characterized in that the metabolites are chosen from the group of metabolites of amino acids, in particular of proteinogenic amino acids, from the group of metabolites emanating from of an oxidative or carbonyl stress process such as ω-oxidation products of linear aliphatic fatty acids, from the group of metabolites emanating from nitrosative stress such as nitrophenol derivatives or from combinations of said groups, optionally together with pyridine and/or uric acid and/or fucose and/or 4-aminobenezene-l ,2-diol.

It is an alternative object of the present invention to provide a method for determining whether a patient suffers from a disease of the lower respiratory tract such as COPD or AECOPD, said method comprising the step of:

b. simultaneously determining the presence, absence or relative concentration of at least two compounds in said sample using said mass spectrometer;

c. determining if the presence, absence or relative concentration of said compounds is indicative of COPD or AECOPD by comparing the determined presence, absence or relative concentration of at least two compounds to predetermined reference values, characterized in that the compounds are chosen from dehydroalanine, 2-oxoglutaric acid semialdehyde, aspartic acid semialdehyde, ω-hydroxyalkanoic acids (C_XH2_X0₃) such as 5- hydroxypentanoic acid, 6-hydroxyhexanoic acid, 7-hydroxyheptanoic acid, 8- hydroxyoctanoic acid, 9-hydroxynonanoic acid, 10-hydroxydecanoic acid, 11- hydroxyundecanoic acid, 12-hydroxydodecanoic acid, 13-hydroxytridecanoic acid, 14- hydroxytetradecanoic acid, 15-hydroxypentadecanoic acid, ω-oxoalkanoic acids such as 5- oxopentanoic acid, 6-oxohexanoic acid, 7-oxoheptanoic acid, 8-oxooctanoic acid, 9- oxononanoic acid, 10-oxodecanoic acid, 1 1-oxoundecanoic acid, 12-oxododecanoic acid, 13-oxotridecanoic acid, 14-oxotetradecanoic acid, 15-oxopentadecanoic acid, alkanedioic acids such as pentanedioic acid, hexanedioic acid, heptanedioic acid, octanedioic acid, nonanedioic acid, decanedioic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid, niti tyrosine, dimethylnitrophenol, ethylnitrophenol, 2-methyl-3,5-dinitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol, 2- nitrohydroquinone, 4-nitrocatechol, 2-methyl-4-nitrophenol, 2-methyl-6-nitrophenol and 4- nitrophenol, pyridine, uric acid, fucose, 4-aminobenezene-l ,2-diol.

It is further an object to provide a computer-controlled medical diagnosis system for determining whether an individual suffers from a disease of the lower respiratory tract such as COPD or AECOPD, comprising

a. a computer and a mass spectrometer, wherein the mass spectrometer comprises i. an ionization chamber and an inlet fluidly connected to the ionization chamber for providing a sample to said ionization chamber, and

ii. a mass analysis and detector device capable of detecting metabolites and a data line connecting the mass analysis and detector device to a computer for providing data to the computer

b. wherein the computer is configured for

i. receiving data provided by the mass analysis and detector device to the computer using the data line and,

ii. processing said data such as to simultaneously determine the presence, absence or relative concentration of at least two metabolites in said sample on the basis of the data provided to said computer;

iii. comparing the determined presence, absence or relative concentration of at least two metabolites in said sample to predetermined reference values for said at least two metabolites, characterized in that the at least two metabolites are chosen from the group of metabolites of amino acids, from the group of metabolites emanating from of an oxidative or carbonyl stress process such as ω-oxidation products of linear fatty acids, from the group of metabolites emanating from nitrosative stress such as nitrophenol derivatives, or from combinations of the metabolites of said groups optionally together with pyridine and/or uric acid and/or fucose and/or 4-aminobenezene-l,2-diol.

Further embodiments of the invention are laid down in the dependent claims. BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,

Fig. 1 shows a schematic representation of a computer-controlled medical diagnosis system comprising a mouthpiece (2), through which a test subject (1) can exhale breath into the heated PTFE tube (3), where the pressure in the heated PTFE tube (3) is controlled using a digital manometer (4). The heated PTFE tube (3) is fluidly connected to the ionization chamber (9) of the ionization source (5) such as to allow the flow of heated breath (10) to enter the ionization chamber (9), where a nano electrospray nozzle (8) generates a nano electrospray plume (12) which ionizes the heated breath

(13) and redirects it towards the interface (1 1) of the mass spectrometer (6). Excess breath is removed from the ionization chamber via an excess breath outlet (14). The measurement and data analysis computer is connected to the mass spectrometer (6).

Fig. 2a, 2b shows a mass spectrum of the breath of a healthy control (top, 2a) and a mass spectrum of the breath of a COPD patient (bottom, 2b). In both mass spectra (2a, 2b), the section corresponding of C5N5H, C₈H₂2N₆02 and Ci₇H₃₂03 is shown enlarged in the respective, annotated boxes. As can be seen, the peak corresponding to C5N5H displays a roughly two-fold increase in intensity in the COPD patients when compared to healthy controls whereas the peaks corresponding to C8H22N6O2 and Ci₇H₃20₃ display a relative decrease in intensity in in the COPD patients when compared to healthy controls.

Fig. 3a - 3f shows the receiver operating characteristic (ROC) according to the present invention and from multiple scientific publications, which are described in more detail below.

DESCRIPTION OF PREFERRED EMBODIMENTS The present invention provides a method for determining whether an individual suffers from a disease of the lower respiratory tract such as COPD or AECOPD, said method comprising the step of:

a. providing a sample comprising, or consisting of, exhaled breath of said individual to an ionization chamber of a mass spectrometer;

c. determining if the presence, absence or relative concentration of said metabolites is indicative of COPD by comparing the determined presence, absence or relative concentration of said at least two metabolites to predetermined reference values, characterized in that the metabolites are chosen from the group of metabolites of amino acids, from the group of metabolites emanating from of an oxidative or carbonyl stress process such as ω-oxidation products of linear fatty acids, from the group of metabolites emanating from nitrosative stress such as nitrophenol derivatives, or from combinations of the metabolites from said groups, optionally together with pyridine and/or uric acid and/or fucose and/or 4-aminobenezene-l,2-diol. It is understood that the at least two metabolites of which the presence, absence or relative concentration is determined can be chosen from the same group of metabolites (e.g. solely from co-oxidation products of linear fatty acids) or from different groups of metabolites. In the case where one or more metabolites of the at least two metabolites of which the presence, absence or relative concentration is determined is chosen from pyridine and/or uric acid and/or fucose and/or 4- aminobenezene-l ,2-diol, the remaining metabolites of which the presence, absence or relative concentration is determined can likewise be chosen from the same group of metabolites (e.g. solely from co-oxidation products of linear fatty acids) or from different groups of metabolites.

In a preferred embodiment, the present invention provides a method for determining whether an individual suffers from a disease of the lower respiratory tract such as COPD or AECOPD, said method comprising the step of:

b. simultaneously determining the presence, absence or relative concentration of pyridine and/or uric acid and/or fucose and/or 4-aminobenezene-l,2-diol, and at least two metabolites in said sample using said mass spectrometer;

c. determining if the presence, absence or relative concentration of pyridine and/or uric acid and/or fucose and/or 4-aminobenezene-l,2-diol and said at least two metabolites is indicative of COPD by comparing the determined presence, absence or relative concentration of pyridine and/or uric acid and/or fucose and/or 4-aminobenezene-l,2-diol and said at least two metabolites to predetermined reference values, characterized in that the metabolites are chosen from the group of metabolites of amino acids, from the group of metabolites emanating from of an oxidative or carbonyl stress process such as co-oxidation products of linear fatty acids, from the group of metabolites emanating from nitrosative stress such as nitrophenol derivatives, or from combinations of the metabolites of said groups.

In general, the sample can consist either of as-is exhaled breath of the individual for which the determination of whether or not he or she suffers from COPD or AECOPD is to be carried out, or it may comprise exhaled breath in combination with other inert substances. While it is possible that the exhaled breath is comprised in an analytical matrix such as for example an inert gaseous matrix, it is however preferred that the sample consists of neat exhaled breath, i.e. without the admixture of another substance, since it is possible that for example the matrix, even though it should be inert, can provoke a chemical change in the metabolites of the exhaled breath.

In a preferred embodiment of the method of the present invention, the sample is formed by at least a part of an exhaled breath flow of the individual. This is achieved by redirecting at least part of a breath flow being exhaled directly into the ionization chamber of the mass spectrometer. In order to redirect the breath flow into the ionization chamber, a mouth piece or face mask that is fluidly connected to an inlet of the ionization chamber of the mass spectrometer can be used. For instance, the mouth piece connected to the inlet can be sealed by the lips of the individual whereas the face mask can be fitted to the face of the patient in an air sealing manner. In preferred embodiment, the flow of exhaled breath can be controlled by for example monitoring the pressure in a connection means fluidly connecting the mouthpiece or face mask to the inlet, such as a PTFE tube, using a manometer. Further, the temperature and humidity of the flow of exhaled breath may be controlled, for example by heating the exhaled breath to a predetermined temperature and adjusting the relative humidity of the exhaled breath, in order to provide for the same conditions in each sample analysis. In a yet another preferred embodiment of the method of the present invention, the sample is formed by redirecting at least part of the final 50% by volume, or final 25% by volume, of the exhaled breath flow of said patient into the ionization chamber of the mass spectrometry device. The reason for this is that in context of the present invention, the sample should contain the part of the exhaled breath which best reflects the composition of the breath at the blood-air barrier, i.e. the alveolar breath. The alveolar breath is released towards the later part of a single exhalation, and thus it is preferable that the sample be formed by the final 50% by volume, or final 25% by volume, of the exhaled breath flow. This may be for example achieved by selectively redirecting the breath through the use of a valve system that allows splitting the exhaled breath into the corresponding final volume fractions. Alternatively the patient may be directed to only exhale the latter part of his exhaled breath into a mouthpiece fluidly connected to the ionization chamber. Preferably the individual fasts overnight to minimize interferences coming from the mouth and obtain a repeatable breath pattern. In an alternative embodiment of the method of the present invention, the sample comprises, or consists of, exhaled breath condensate (EBC). Using a sample of exhaled breath condensate can be useful in the case where the patient, for either medical or practical reasons, is not able to be brought into the proximity of the mass spectrometer. A person skilled in the art will know how to collect exhaled breath concentrate, since multiple publications relating to the analysis of EBC exist.

In an alternative embodiment of the method of the present invention, the metabolites of amino acids are in particular metabolites of nonpolar amino acids such as glycine, alanine, phenylalanine, proline valine, isoleucine and leucine, metabolites polar amino acids such as lysine, ornithine, glutamic acid, aspartic acid, serine or cysteine. The amino acids diffuse across the blood-air barrier and are exhaled with the breath. Therefore, the presence, absence or concentration of these amino acids and their corresponding metabolites can be detected with suitable methods such as mass spectrometry and can be assigned to a medical condition, in particular COPD.

Metabolites of amino acids that have been identified in the context of the present invention as being indicative for COPD are, without wishing to be limited, dehydroalanine, 2- oxoglutaric acid semialdehyde, and aspartic acid semialdehyde.

In an alternative embodiment of the method of the present invention, the group of metabolites of an oxidative or carbonyl stress process consists of ω-oxidation products of linear fatty acids. For instance, ω-oxidation products of linear fatty acids are co- hydroxyalkanoic acids (Ο_χΗ_2χθ3), co-oxoalkanoic acids (C_xH2_X-20₃) or alkanedioic acids (C_xH2x_-20 ). In a preferred embodiment, the aforementioned metabolites of linear fatty acids are metabolites of C5-C15 linear fatty acids. Examples of ω-hydroxyalkanoic acids (C_xH_2x03) are 5-hydroxypentanoic acid, 6-hydroxyhexanoic acid, 7-hydroxyheptanoic acid, 8-hydroxyoctanoic acid, 9-hydroxynonanoic acid, 10-hydroxydecanoic acid, 11- hydroxyundecanoic acid, 12-hydroxydodecanoic acid, 13-hydroxytridecanoic acid, 14- hydroxytetradecanoic acid, 15-hydroxypentadecanoic acid. Examples of suitzable co- oxoalkanoic acids are 5-oxopentanoic acid, 6-oxohexanoic acid, 7-oxoheptanoic acid, 8- oxooctanoic acid, 9-oxononanoic acid, 10-oxodecaiioic acid, 11-oxoundecanoic acid, 12- oxododecanoic acid, 13-oxotridecanoic acid, 14-oxotetradecanoic acid, 15- oxopentadecanoic acid. Examples of alkanedioic acids are pentanedioic acid, hexanedioic acid, heptanedioic acid, octanedioic acid, nonanedioic acid, decanedioic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid. In addition to being metabolites indicative of COPD, the co- oxidation products of linear fatty acids allow further to discriminate between COPD patients and patients suffering from acute exacerbation of COPD (AECOPD). A patient suffering from AECOPD will exhibit a decreased relative concentration of co-oxidation products of linear aliphatic fatty acids audi as co-hydroxyalkanoic acids (C_xH_2x0₃), co- oxoalkanoic acids (C_xH_2x-203) or alkanedioic acids (C_xH₂x-₂04) when compared to healthy individuals. Thus, a decreased relative concentration of co-hydroxyalkanoic acids (C_xH₂x0₃), co-oxoalkanoic acids (C_xH_2x.₂03) or alkanedioic acids (C_xH_2x-204) is not only indicative of COPD but in particular is further indicative of AECOPD.

In an alternative embodiment of the method of the present invention, the group of metabolites emanating from nitrosative stress includes nitrophenol derivatives such as for example nitrotyrosine and nitrophenol derivatives such as nitrohydroquinones, dinitrophenols, nitrocatechols and alkylated derivatives thereof. In particular, dimethylnitrophenol, ethylnitrophenol, 2-methyl-3,5-dinitrophenol, 2,4-dinitrophenol, 2,5- dinitrophenol, 2-nitrohydroquinone, 4-nitrocatechol, 2-methyl-4-nitrophenol, 2-methyl-6- nitrophenol and 4-nitrophenol have been found to be indicative of COPD but in particular are further indicative of AECOPD.

In an alternative embodiment of the method of the present invention, the metabolites of an oxidative or carbonyl stress process are derivatives of fatty acids, saturated or unsaturated, such as medium-chain fatty acids, long-chain fatty acids or very long-chain fatty acids, which are preferably hydroxylated, carbonylated or carboxylated derivatives thereof. A class of compounds that show particular promise as indicators of COPD are hydroxy- or keto-derivatives of fatty acids, especially of long- or medium-chain fatty acids, such as the above-mentioned ω-oxidation products of linear C5-C15 fatty acids. For instance, it has been found that hydroxylated, carbonylated or carboxylated derivatives of saturated fatty acids are particularly useful.

Metabolites emanating from of an oxidative or carbonyl stress process that have been identified in the context of the present invention as being indicative of COPD are, without wishing be limited to the following substances, hydroxyisobutyric acid, acetohydroxybutanoic acid, hydroxyundecanoic acid, oxo-tetradecenoic acid, hexadecatrienoic acid, oxo-heptadecanoic acid, hydroxyalkanaoic acid, 4-2-aminophenyl- 2,4-dioxobutanoic acid, oxoalkenoic acid, dodecanedioic acid, oxotetradecandioic acid, oxoalkanoic acid, 4-oxobutainoic acid, 2-aminomalonic acid semi-aldehyde, 4-amino-2 butenoic acid, 2-oxoglutaric acid semialdehyde, 2-amino-4-oxobutanoic acid, benzoic acid, 3-(2,3-dihydroxyphenyl) propanoic acid, alkenal, oxoalkenoic acid, alkandioic acid. Thus, in general terms, metabolites emanating from an oxidative or carbonyl stress process include the above-mentioned oxidation products of aliphatic fatty acids, preferably of aliphatic linear fatty acids have been found to be indicative of COPD.

In an alternative embodiment of the method of the present invention, the presence, absence or relative concentration of more than at least two metabolites can be determined. In order to increase both specificity and sensitivity of the method according to the present invention, it is preferable to determine at least 3, 4, 5, 6, 7, 8 , 9, 10, or more metabolites simultaneously in said sample, optionally in addition to pyridine and/or uric acid and/or fucose and/or 4-aminobenezene-l,2-diol. It is understood that the at least two metabolites can either be solely chosen from one of the groups or from any combination of groups, i.e. the group of metabolites of amino acids, the group of metabolites emanating from of an oxidative or carbonyl stress process such as co-oxidation products of linear fatty acids, the group of metabolites emanating from nitrosative stress or from combinations of the metabolites of said groups, optionally together with pyridine and/or uric acid and/or fucose and/or 4-aminobenezene-l,2-diol. In some cases, the detected relative concentration of a metabolite may increase or decrease in the case of COPD. For example, pyridine is present in higher amounts in the breath of COPD patients when compared to healthy controls, whereas derivatives of saturated fatty acids such as oxo-heptadecanoic acid are present in lower amounts in the breath of COPD patients, when compared to healthy controls. This is also true for co-oxidation products of linear fatty acids, which are present in lower amounts in the breath of COPD and AECOPD patients, when compared to healthy controls.

The present invention further provides a computer-controlled medical diagnosis system for determining whether an individual suffers from a disease of the lower respiratory tract such as COPD or AECOPD, comprising a computer and a mass spectrometer, wherein the mass spectrometer comprises i. an ionization chamber and an inlet fluidly connected to the ionization chamber for providing a sample to said ionization chamber, and ii. a mass analysis and detector device capable of detecting metabolites and a data line connecting the mass analysis and detector device to a computer for providing data to the computer, wherein the computer is configured for i. receiving data provided by the mass analysis and detector device to the computer using the data line and, ii. processing said data such as to simultaneously determine the presence, absence or relative concentration of at least two metabolites in said sample on the basis of the data provided to said computer; iii. comparing the determined presence, absence or relative concentration of at least two metabolites in said sample to predetermined reference values for said at least two metabolites, characterized in that the at least two metabolites are chosen from the group of metabolites of amino acids, from the group of metabolites emanating from of an oxidative or carbonyl stress process such co-oxidation products of linear fatty acids, from the group of metabolites emanating from nitrosative stress such as nitrophenol derivatives, or from combinations of the metabolites of said groups optionally together with pyridine and/or uric acid and/or fucose and/or 4-aminobenezene-l,2-diol. The mass spectrometer comprises ionization chamber and an inlet fluidly connected to the ionization chamber for providing a sample to said ionization chamber, and a mass analysis and detector device capable of detecting metabolites and a data line connecting the mass analysis and detector device to a computer for providing data to the computer. The sample can be a flow of exhaled breath, which is preferably adjusted to a given temperature, humidity and/or pressure.

In a preferred embodiment of the computer-controlled medical diagnosis system of the present invention, the computer is configured for i. receiving data provided by the mass analysis and detector device to the computer using the data line and, ii. processing said data such as to simultaneously determine the presence, absence or relative concentration of at least two metabolites in said sample on the basis of the data provided to said computer; iii. comparing the determined presence, absence or relative concentration of at least two metabolites in said sample to predetermined reference values for said at least two metabolites. For the purpose of the present invention, the computer can be configured for example by providing it with an appropriate software package that enables a user to set the masses, or ranges of masses, that correspond to the at least two metabolites that are to be qualitatively and quantitatively detected. Once the masses or range of masses are set, the software package allows the computer to process the data received from the detector such as to simultaneously determine the presence, absence or relative concentration of at least two metabolites in said sample on the basis of the data provided to said computer. The determined presence, absence or relative concentration can then be compared to predetermined reference values for said at least two metabolites by either displaying the comparison result in the form of a table or graph, or a score for example. The predetermined reference values for the presence, absence or relative concentration of at least two metabolites can be collected by determining the presence, absence or relative concentration of the at least two metabolites of interest in a population of control individuals not suffering from COPD or AECOPD and computing a range or threshold value for the at least two metabolites that allows to separate a healthy individual from an individual suffering from COPD or AECOPD with sufficient significance.

EXAMPLES

The experimental setup consists of a high-resolution mass spectrometer modified to allow for the admission of breath sample through a heated tube made from an inert material. Exhaled breath is mixed with a nano electrospray plume (water and 0.2% formic acid) whereby some compounds can be detected in real time. The mass spectrometer can be operated in positive or negative ion mode. The subjects are instructed to provide a deep exhalation through a disposable mouth piece, while keeping the pressure through the sampling tube at 20 mbar (monitored by digital manometer), thereby ensuring that each subject breathed at the approximate same flow rate of 1.8 1/min. This process is repeated several times per subject, with typically 6 replicate measurements, taking less than 6 min. The setup is shown in Figure 1.

Figure 3a shows the result classification based on a statistical evaluation of all detected compounds according to the present invention, a so-called receiver operating characteristic (ROC) curve. The compounds are enumerated in Table 1 , below. The data was collected from 22 patients with COPD and 14 healthy controls. The two groups were balanced concerning age, gender, smoking state, pack-years and body mass index. The area under curve (AUC) is 0.92.

Figure 3 b shows the receiver operating characteristic (ROC) curve based on the findings described in Thorn J, Tilling B, Lisspers K, Jorgensen L, Stenling A, Stratelis G. Improved prediction of COPD in at-risk patients using lung function pre-screening in primary care: a real-life study and cost-effectiveness analysis. Prim Care Respir J. 2012;21(2): 159-166. doi: 10.4104/pcrj.201 1.00104. Thorn et al. attempted to discriminate between COPD patients and healthy controls through the ratio between FEVi/FEV₆. The area under curve (AUC) is 0.84.

Figure 3c shows the receiver operating characteristic (ROC) curve based on the findings described in Stanley AJ, Hasan I, Crockett AJ, van Schayck OCP, Zwar NA. Validation of the COPD Diagnostic Questionnaire in an Australian general practice cohort: a cross- sectional study. Prim Care Respir J. 2014;23(l):92-97. doi: 10.4104/pcrj.2014.00015 Stanley et al. attempted to discriminate between COPD patients and healthy controls through a COPD diagnostic questionnaire. The area under curve (AUC) is 0.71. Figure 3d shows the receiver operating characteristic (ROC) curve based on the findings described in Martinez FJ, Raczek AE, Seifer FD, et al. Development and initial validation of a self-scored COPD Population Screener Questionnaire (COPD-PS). COPD.

2008;5(2):85-95. doi: 10.1080/15412550801940721 Martinez et al. attempted to discriminate between COPD patients and healthy controls through a score derived from a population screener questionnaire. The area under curve (AUC) is 0.81.

Figure 3e shows the receiver operating characteristic (ROC) curve based on the findings described in Kon SSC, Canavan JL, Jones SE, et al. Minimum clinically important difference for the COPD Assessment Test: a prospective analysis. Lancet Respir Med. 2014;2(3): 195-203. doi : 10.1016/S2213 -2600( 14)70001-3 Kon et al. attempted to discriminate between COPD patients and healthy controls through a score derived from a "St George's respiratory questionnaire". The area under curve (AUC) is 0.65.

Figure 3f shows the receiver operating characteristic (ROC) curve based on the findings described in Kon SSC, Canavan JL, Jones SE, et al. Minimum clinically important difference for the COPD Assessment Test: a prospective analysis. Lancet Respir Med. 2014;2(3): 195-203. doi: 10.1016/S2213-2600( 14)70001 -3 Kon et al. attempted to discriminate between COPD patients and healthy controls through a score derived from a chronic respiratory questionnaire. The area under curve (AUC) is 0.70.

Thus, as can be seen from the presented data, when using the method according to the present invention based on real-time mass spectrometry, the area under curve (AUC) is 0.92, which is the highest value amongst Figures 3a, 3b, 3c, 3d, 3e and 3f. This indicates an increased sensitivity and specificity for the method according to the present invention, when compared with methods relying on traditional forced expiratory volume

measurements or diagnostic questionnaires. LIST OF REFERENCE SIGNS test subject

mouthpiece

heated PTFE tube

digital manometer

ionization source

mass spectrometer

measurement and data

analysis computer

nano electrospray nozzle

ionization chamber

flow of heated breath

mass spectrometer interface

nano electrospray plume

floe of ionized heated breath

excess breath outlet

breath inlet

Claims

1. A method for determining whether an individual suffers from a disease of the lower respiratory tract such as COPD or AECOPD, said method comprising the step of: a. providing a sample comprising, or consisting of, exhaled breath of said individual to an ionization chamber of a mass spectrometer;

b. simultaneously determining the presence, absence or relative concentration of at least two metabolites in said sample using said mass spectrometer; c. determining if the presence, absence or relative concentration of said metabolites is indicative of COPD by comparing the determined presence, absence or relative concentration of said at least two metabolites to predetermined reference values,

characterized in that the metabolites are chosen from the group of metabolites emanating from of an oxidative or carbonyl stress process such as co-oxidation products of linear fatty acids, from the group of metabolites of amino acids, from the group of metabolites emanating from nitrosative stress such as nitrophenol derivatives or from combinations of the metabolites of said groups, optionally together with pyridine and/or uric acid and/or fucose and/or 4-aminobenezene-l,2- diol.

2. The method according to claim 1, wherein the sample is provided by redirecting at least part of a breath flow of said individual being exhaled into the ionization chamber of the mass spectrometer.

3. The method according to claim 1 or 2, wherein the sample is formed by redirecting at least part of the final 50% by volume, or final 25% by volume, of the exhaled breath flow of said individual into the ionization chamber of the mass spectrometry device.

4. The method according to any preceding claim, wherein the sample comprises, or consists of, exhaled breath condensate (EBC).

5. The method according to any preceding claim, wherein the group of co-oxidation products of linear fatty acids comprises co-hydroxyalkanoic acids (C_xH_2x0₃), co- oxoalkanoic acids (C_xH_2x-20₃) or alkanedioic acids (C_xH_2x-20₄) and/or the group of metabolites emanating from nitrosative stress such as nitrophenol derivatives includes nitrohydroquinones, dinitrophenols, nitrocatechols and alkylated derivatives thereof.

6. The method according to any preceding claim, wherein the metabolites of amino acids are in particular metabolites of nonpolar amino acids such as glycine, alanine, phenylalanine, proline valine, isoleucine and leucine, metabolites of polar amino acids such as lysine, ornithine, glutamic acid, aspartic acid, serine or cysteine.

7. The method according to claim 6, wherein the metabolites of amino acids are chosen from dehydroalanine, 2-oxoglutaric acid semialdehyde, aspartic acid semialdehyde.

8. The method according to any preceding claim, wherein the ionization chamber is configured to produce ions using an electrospray, and in particular is configured to produce ions using secondary electrospray ionization (SESI), and/or wherein the mass spectrometer is preferably a time-of-flight mass spectrometer.

9. The method according to any preceding claim, wherein the metabolites emanating from of an oxidative or carbonyl stress process are derivatives of fatty acids, saturated or unsaturated, such as medium-chain fatty acids, long-chain fatty acids or very long-chain fatty acids, which are preferably hydroxylated, carbonylated or carboxylated derivatives thereof.

10. The method according to claim 9, wherein the metabolites emanating from of an oxidative or carbonyl stress process are chosen from 2-hydroxyisobutyric acid, acetohydroxybutanoic acid, hydroxyundecanoic acid, oxo-tetradecenoic acid, hexadecatrienoic acid, oxo-heptadecanoic acid.

1 1. The method according to any preceding claim, wherein the presence, absence or relative concentration of at least 3, 4, 5, 6, 7, 8 , 9, 10 or more metabolites are simultaneously determined in said sample.

12. A computer-controlled medical diagnosis system for determining whether an individual suffers from a disease of the lower respiratory tract such as COPD or AECOPD, comprising

a. a computer and a mass spectrometer, wherein the mass spectrometer comprises

i. an ionization chamber and an inlet fluidly connected to the ionization chamber for providing a sample to said ionization chamber, and

wherein the computer is configured for

iii. comparing the determined presence, absence or relative concentration of at least two metabolites in said sample to predetermined reference values for said at least two metabolites, characterized in that the at least two metabolites are chosen from the group of metabolites emanating from of an oxidative or carbonyl stress process such as ω-oxidation products of linear fatty acids, from the group of metabolites of amino acids, from the group of metabolites emanating from nitrosative stress such as nitrophenol derivatives or from combinations of the metabolites of said groups, wherein preferably the metabolites of amino acids are in particular metabolites of nonpolar amino acids such as glycine, alanine, phenylalanine, proline valine, isoleucine and leucine, metabolites of polar amino acids such as lysine, ornithine, glutamic acid, aspartic acid, serine or cysteine and/or wherein preferably the metabolites emanating from of an oxidative or carbonyl stress process are derivatives of fatty acids, saturated or unsaturated, such as medium-chain fatty acids, long-chain fatty acids or very long-chain fatty acids, which are preferably hydroxylated, carbonylated or carboxylated derivatives thereof and/or wherein preferably the group of ω-oxidation products of linear fatty acids comprises o- hydroxyalkanoic acids (C_xH_2x0₃), co-oxoalkanoic acids (C_xH_2x-20₃) or alkanedioic acids (C_xH_2x.₂0₄) and/or wherein preferably the group of metabolites emanating from nitrosative stress such as nitrophenol derivatives includes nitrohydroquinones, dinitrophenols, nitrocatechols and alkylated derivatives thereof.

13. The computer-controlled medical diagnosis system of claim 12, wherein the ionization chamber is configured to produce ions using an electrospray, and in particular is configured to produce ions using secondary electrospray ionization (SESI), and/or wherein the mass spectrometer is preferably a time-of-flight mass spectrometer.

14. The computer-controlled medical diagnosis system of claim 12 or 13, wherein the inlet is fluidly connected to the ionization chamber for providing a sample to said ionization chamber on one side, and on the other side is connected to a mouthpiece.

15. The computer-controlled medical diagnosis system of any of claims 12 to 14, wherein the computer is configured for

ii. processing said data such as to simultaneously determine the presence, absence or relative concentration of at least 3, 4, 5, 6, 7, 8 , 9, 10 or more metabolites in said sample on the basis of the data provided to said computer;

iii. comparing the determined presence, absence or relative concentration of said at least 3, 4, 5, 6, 7, 8 , 9, 10 or more metabolites in said sample to predetermined reference values for said of at least 3, 4, 5, 6, 7, 8 , 9, 10 or more metabolites.

16. Use of a computer-controlled medical diagnosis system according to any of claims 12 to 15 for determining whether an individual suffers from a disease of the lower respiratory tract such as COPD or AECOPD.