Pure non-invasive method for identification of organ diseases or impaired organ function by investigation of marker substances in exhaled air stimulated by inhaled marker substances

Abstract

The disclosure relates to a method for providing original data that can be used for subsequently determining the function of an organ of a living organism or for subsequently diagnosing a disease or a severity of a disease of an organ of a living organism. This method is characterized by several steps, one of which is administering a marker substance to a living organism by inhalation, wherein the marker substance has a vapor pressure above 0.01 mmHg at 37° C. In other method steps, the concentration of this marker substance in exhaled air is determined at at least two different time points. Then, a difference in marker substance concentration is calculated.

Claims

1. A method for determining the liver function of a human patient, the method comprising the following steps: a) administering a volatile organic compound to a human patient by inhalation, wherein the volatile organic compound has a vapor pressure above 0.01 mmHg at 37° C., wherein the volatile organic compound is at least one substance chosen from the group consisting of octane, furan-2-ylmethanethiol, 1,2-diazine, 1,3-diazine, 1,4-diazine, a terpene, 2-pentanone, hexane and 4-hydroxynonenal, b) collecting exhaled air from the human patient, c) determining a concentration of the volatile organic compound in exhaled air which is exhaled by the human patient at a first time point, wherein the first time point is a time point after inhalation of the volatile organic compound, d) determining the concentration of the volatile organic compound in the exhaled air which is exhaled by the human patient at a plurality of further time points after the first time point, so as to obtain a plurality of data points relating to the concentration of the volatile organic compound at different time points, wherein a difference between the first time point and the latest of the further time points lies in a range of 30 seconds to 70 minutes, e) determining a time-resolved metabolization dynamics of the volatile organic compound on the basis of differences between the concentration of the volatile organic compound determined at the first time point and the concentration of the volatile organic compound determined at each of the further time points, and f) determining the liver function based on the time-resolved metabolization dynamics determined in step d).

2. The method of claim 1, wherein the method also comprises the step of reporting the liver function.

3. The method of claim 1, wherein the method comprises determining the health status of the human patient with respect to and based on the determined liver function.

4. The method of claim 3, wherein the method also comprises the step of reporting the health status of the human patient.

5. The method of claim 1, wherein the terpene is at least one of the group consisting of limonene, α-pinene, β-pinene and γ-pinene.

6. A method for diagnosing a disease or for diagnosing a severity of a disease of the liver of a human patient, the method comprising the following steps: a) administering a volatile organic compound to a human patient by inhalation, wherein the volatile organic compound has a vapor pressure above 0.01 mmHg at 37° C., wherein the volatile organic compound is at least one substance chosen from the group consisting of octane, furan-2-ylmethanethiol, 1,2-diazine, 1,3-diazine, 1,4-diazine, a terpene, 2-pentanone, hexane and 4-hydroxynonenal, b) collecting exhaled air from the human patient, c) determining a concentration of the volatile organic compound in exhaled air which is exhaled by the human patient at a first time point, wherein the first time point is a time point after inhalation of the volatile organic compound, b) determining the concentration of the volatile organic compound in the exhaled air which is exhaled by the human patient at a plurality of further time points after the first time point, so as to obtain a plurality of data points relating to the concentration of the volatile organic compound at different time points, wherein a difference between the first time point and the latest of the further time points lies in a range of 30 seconds to 70 minutes, e) determining a time-resolved metabolization dynamics of the volatile organic compound on the basis of differences between the concentration of the volatile organic compound determined at the first time point and the concentration of the volatile organic compound determined at each of the further time points, and f) making a diagnosis based on the time-resolved metabolization dynamics determined in step d).

7. The method of claim 6, wherein the terpene is at least one of the group consisting of limonene, α-pinene, β-pinene and γ-pinene.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Aspects of the instant invention will now be explained with respect to exemplary embodiments and accompanying Figures.

(2) FIG. 1A shows the results of proton-transfer reaction mass spectrometry (PTR-MS) with respect to compounds having an m/z value of 137 of exhaled breath of individuals having divers health conditions or nutritional states;

(3) FIG. 1B shows the results of PTR-MS of exhaled breath with respect to compounds having an m/z value of 137 of exhaled breath of the same individuals as in FIG. 1A in dependence on the liver power (expressed as LiMAx value) that has been determined for these individuals independently on the PTR-MS measurement;

(4) FIG. 2A shows the results of proton-transfer reaction mass spectrometry (PTR-MS) with respect to compounds having an m/z value of 61 of exhaled breath of individuals having divers health conditions or nutritional states;

(5) FIG. 2B shows the results of PTR-MS of exhaled breath with respect to compounds having an m/z value of 61 of exhaled breath of the same individuals as in FIG. 2A in dependence on the liver power (expressed as LiMAx value) that has been determined for these individuals independently on the PTR-MS measurement;

(6) FIG. 3A shows the results of proton-transfer reaction mass spectrometry (PTR-MS) with respect to compounds having an m/z value of 109 of exhaled breath of individuals having divers health conditions or nutritional states;

(7) FIG. 3B shows the results of PTR-MS of exhaled breath with respect to compounds having an m/z value of 109 of exhaled breath of the same individuals as in FIG. 3A in dependence on the liver power (expressed as LiMAx value) that has been determined for these individuals independently on the PTR-MS measurement;

(8) FIG. 4A shows the results of proton-transfer reaction mass spectrometry (PTR-MS) with respect to cysteine having an m/z value of 122 of exhaled breath of individuals having divers health conditions or nutritional states;

(9) FIG. 4B shows the results of PTR-MS of exhaled breath with respect to cysteine having an m/z value of 122 of exhaled breath of the same individuals as in FIG. 4A in dependence on the liver power (expressed as LiMAx value) that has been determined for these individuals independently on the PTR-MS measurement;

(10) FIG. 5A shows the results of proton-transfer reaction mass spectrometry (PTR-MS) with respect to selenocysteine having an m/z value of 169 of exhaled breath of individuals having divers health conditions or nutritional states;

(11) FIG. 5B shows the results of PTR-MS of exhaled breath with respect to selenocysteine having an m/z value of 169 of exhaled breath of the same individuals as in FIG. 5A in dependence on the liver power (expressed as LiMAx value) that has been determined for these individuals independently on the PTR-MS measurement;

(12) FIG. 6A shows the results of proton-transfer reaction mass spectrometry (PTR-MS) with respect to compounds having an m/z value of 115 of exhaled breath of individuals having divers health conditions or nutritional states;

(13) FIG. 6B shows the results of PTR-MS of exhaled breath with respect to compounds having an m/z value of 115 of exhaled breath of the same individuals as in FIG. 6A in dependence on the liver power (expressed as LiMAx value) that has been determined for these individuals independently on the PTR-MS measurement;

(14) FIG. 7A shows the results of proton-transfer reaction mass spectrometry (PTR-MS) with respect to compounds having an m/z value of 129 of exhaled breath of individuals having divers health conditions or nutritional states;

(15) FIG. 7B shows the results of PTR-MS of exhaled breath with respect to compounds having an m/z value of 129 of exhaled breath of the same individuals as in FIG. 7A in dependence on the liver power (expressed as LiMAx value) that has been determined for these individuals independently on the PTR-MS measurement;

(16) FIG. 8A shows the results of proton-transfer reaction mass spectrometry (PTR-MS) with respect to compounds having an m/z value of 81 of exhaled breath of individuals having divers health conditions or nutritional states;

(17) FIG. 8B shows the results of PTR-MS of exhaled breath with respect to compounds having an m/z value of 81 of exhaled breath of the same individuals as in FIG. 8A in dependence on the liver power (expressed as LiMAx value) that has been determined for these individuals independently on the PTR-MS measurement;

(18) FIG. 9A shows the results of proton-transfer reaction mass spectrometry (PTR-MS) with respect to compounds having an m/z value of 87 of exhaled breath of individuals having divers health conditions or nutritional states;

(19) FIG. 9B shows the results of PTR-MS of exhaled breath with respect to compounds having an m/z value of 87 of exhaled breath of the same individuals as in FIG. 9A in dependence on the liver power (expressed as LiMAx value) that has been determined for these individuals independently on the PTR-MS measurement;

(20) FIG. 10A shows the results of proton-transfer reaction mass spectrometry (PTR-MS) with respect to 4-hydroxynonenal having an m/z value of 157 of exhaled breath of individuals having divers health conditions or nutritional states; and

(21) FIG. 10B shows the results of PTR-MS of exhaled breath with respect to 4-hydroxynonenal having an m/z value of 157 of exhaled breath of the same individuals as in FIG. 10A in dependence on the liver power (expressed as LiMAx value) that has been determined for these individuals independently on the PTR-MS measurement.

DETAILED DESCRIPTION

Exemplary Embodiments

(22) Suited marker substances were identified by re-evaluating the experimental data of the dissertation “Analysis of breath allows for non-invasive identification and quantification of diseases and metabolic dysfunction” of Suha Adel Al-Ani that is freely available under the following internet address:

(23) www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000100227

(24) Further details of the concrete experimental work that has been done to obtain the data explained in the following can be found in chapter 4 of this dissertation. This dissertation, in particular chapter 4 regarding the experimental work, chapter 3 regarding details of DOB kinetics, and the graphically depicted results of chapter 5, is hereby incorporated by reference.

(25) Briefly, the exhaled breath of healthy individuals belonging to two groups of different nutritional states (namely based on a normal diet on the one hand and based on a vegan diet on the other hand) as well as of patients suffering from a liver disease has been measured by proton-transfer reaction mass spectrometry (PTR-MS) for quantitatively identifying different volatile organic compounds (VOCs) in the measured exhaled breath.

(26) FIG. 1A shows the results of according PTR-MS measurements regarding limonene and different pinenes as marker substances that are typically inhaled from the surrounding to identify liver diseases or impaired liver function. The concentration of limonene, α-pinene, β-pinene and γ-pinene (all having an m/z ratio of 137 including an additional H.sup.+; their mass without a proton is 136 au) is almost identical for all three groups of individuals tested (taking into account the error bars).

(27) In addition, the liver power of the same individuals was tested by determining the LiMAx value on the basis of a breath test after .sup.13C-methacetin administration. Thereby, the LiMAx value was calculated according to the following formula:

(28) $\mathrm{LiMAx}=\frac{{\mathrm{DOB}}_{\max }.Math.R_{\mathrm{PDB}}.Math.P_{{\mathrm{CO}}_2}.Math.M}{\mathrm{BW}},$
wherein
the unit of the LiMAx value is (m/kg)/h, DOB.sub.max denotes the maximum value of the DOB (delta over baseline) kinetics, R.sub.PDB is the Pee Dee Belemnite standard and is 0.011237, P.sub.CO2 denotes the CO.sub.2 production rate that is to be calculated by (300 mmol/h)*BSA, wherein BSA means body surface area; it is indicated in m.sup.2 and is calculated according to the Du Bois formula: BSA=0.007184*W.sup.0.425*H.sup.0.725, wherein W is the weight in kg and H is the height in cm of the respective individual; M is the molar mass of .sup.13C-methacetin (166.19 g/mol), BW is the body weight of the individual in kg.

(29) FIG. 1B shows the results of PTR-MS measurements in dependence on an according determination of the LiMAx value. It can be seen from FIG. 1B that the concentration of limonene, α-pinene, β-pinene and γ-pinene is significantly increased in the expiratory air of patients that have a strongly impaired liver function represented by a LiMAx value of below 176 (both bars on the left) as compared to patients with only slightly impaired liver function represented by a LiMAx value of 176 to 351 (both bars in the middle) or individuals with normal liver function represented by a LiMAx value of above 351 (both bars on the right) that were erroneously grouped as patients suffering from a liver disease but that have in fact no decreased liver function. Thereby, the LiMAx value has been determined by two independent devices, namely by a modified non-dispersive isotope-selective infrared spectrometer of Fischer Analysen Instrumente GmbH (black bars, abbreviated by FANci or FANci2-db16) or by a Flow-through Fast Liver Investigation Packet available from Humedics GmbH (grey bars, abbreviated by FLIP).

(30) The modified non-dispersive isotope-selective infrared spectrometer FANci2-db16 has a frequency of approximately 1/min. It was used to draw and analyze breath samples. This spectrometer measures the .sup.13CO.sub.2 to .sup.12CO.sub.2 ratio. As a light source, a black body radiator is used. Two detection chambers are filled with .sup.13CO.sub.2 or .sup.12CO.sub.2, respectively, wherein a microphone is present in each detection chamber. Between the light source and the detection chamber there is a chopper to modulate the IR radiation. A measuring chamber is filled with the gas to be tested. The molecules in the detection chambers absorb the modulated IR radiation and convert it to thermal energy. The so-modulated density fluctuations cause sound waves, and each is measured with a microphone.

(31) The disadvantage of the device is that it is very sensitive to vibrations and to temperature changes. Also, the breath cannot be measured when flowing so that it is instead kept stationary in an aluminum bag. In standard mode, the breath is collected in a bag and the bag is connected to the device, then it pumps the exhaled air into the measuring chamber. During exhaling air in to the bag, it is important to make sure that only the alveolar air is used. The air that does not reach the alveoli has the CO.sub.2 content of the inspired air. It would distort the measured values. With this measuring method an accuracy of ±2 DOB can be achieved according to the manufacturer, but it does not provide absolute values for exhaled CO.sub.2 volumes.

(32) The FLIP device can measure the .sup.13CO.sub.2 to .sup.12CO.sub.2 ratio in exhaled breath. The ultra-sensitive laser spectroscopy system of the FLIP device can quickly and reliably determine the capacity of the liver function. The FLIP/LiMAx system greatly improves the surgical intervention planning. The laser based FLIP device detects a metabolic product (.sup.13CO.sub.2) of the enzymatic conversion of the drug methacetin in the liver in the exhaled air. .sup.13CO.sub.2 is stable, non-radioactive and detected by the unique sensors in the device even at extremely low concentrations (100 ppb) in every single breath.

(33) The FLIP device measures in real-time a continuous flow of air. It has been developed in cooperation with medical professionals and is adapted to various clinical situations. The FLIP has unified the mobility, the usability and the practicality. It has been used successfully in various intensive care units, emergency rooms, operating theatres and outpatient stations.

(34) The data shown in FIGS. 1A and 1B was interpreted in the above-mentioned dissertation such that the according substances were held to be no good biomarkers, since the amount of inhaled marker substance was typically not known, and thus the reference was missing.

(35) However, it turned out that this statement is a misinterpretation of the data. In contrast to this statement, limonene is a well suited marker substance within the context of aspects of the present invention. Limonene has a low vapor pressure and a pleasant odor.

(36) After inhalation of air with marker substance vapor (marker substance gas) the exhaled marker substance concentration is measured. This provides useful information on the lung status, when the concentration of the marker substance gas is known, because this provides direct information on the exchange rate of the lung.

(37) After some time after the inhalation, the concentration of the marker substance in the exhaled air is measured again. A reduction in concentration (in particular the course of concentration over time) of the marker substance directly reflects the metabolization of the marker substance and thus its decomposition within the living organism.

(38) The marker substances referred to in FIGS. 1A and 1B are clearly decomposed slower in patients with impaired liver function. It was reported that limonene is metabolized by enzymes of the Cytochrome P450 family. [Mizayawa, M. et al. The American Society for Pharmacology and Experimental Therapeutics, Vol. 30, No. 5, (2002), 602-607] Thus, the higher concentration of the marker substance in the exhaled air in case of patients suffering from liver disease provides direct qualitative or quantitative information on the impaired liver function. Hence, measurement of exhaled marker substance concentration provides a fast test for severe liver diseases or impaired liver function.

(39) Mizayawa et al. also reported that limonene is known to have chemopreventive activity, and is metabolized in human liver cells by CYP 2C9 and CYP 2C19 to carveol and perillyl alcohol. Other enzymes like CYP 2C8, 2C18, and 3A4 could also play a role in this metabolization. Michael N. Gold reported in Environmental Health Perspectives, Vol. 105, Supplement 4, 1997, pages 977-979 that “Monoterpenes such as limonene and perillyl alcohol have been shown to prevent mammary, liver, lung, and other cancers”.

(40) The use of limonene as marker substances to detect liver diseases or an impaired liver function is thus connected with the additional effect of preventing mammary, liver, lung and other cancers. Moreover, the liver metabolization product of limonene, perillyl alcohol, also prevents liver and other cancers.

(41) In summary, by using limonene as marker substance, a natural product is used as marker substance to detect liver diseases or an impaired liver function. Limonene itself has a preventive influence on liver and other cancers. Moreover, limonene can be easily inhaled, thus allowing its use in a purely non-invasive method. Limonene is also comfortable for the patients, because of its pleasant odor. Furthermore, it provides direct information on the lung function by detecting the blood concentration change upon inhalation of the marker substance.

(42) In contrast to prior art methods, in this exemplary embodiment limonene is administered as marker substance by inhalation. Afterwards, its metabolization via enzymes of the Cytochrome P450 enzyme family is followed by detecting the level of the administered marker substance in the exhaled air (not of the metabolized product).

(43) Besides limonene and/or the pinenes referred to in the first exemplary embodiment, other marker substances can be used for the described methods. These marker substances are generally characterized by two properties that need to be fulfilled. First property: Comparison of the marker substance concentrations of the three groups of vegan persons, volunteers, and patients (cf. FIG. 1A) shows no significant difference (within the error bars, i.e. taking the error bars into account). Second property: Comparison of the marker substance concentrations of three different liver function groups represented by three different ranges of the LiMAx value (cf. FIG. 1B) shows differences (within the error bars, i.e. taking the error bars into account).

(44) By applying these criteria to experimental data that has been previously obtained and already analyzed under a different point of view, more suited marker substances for determining the function of an organ of a living organism or for subsequently diagnosing a disease or a severity of a disease of an organ of a living organism were identified. The according data is shown in FIGS. 2A to 10B.

(45) Thereby, all FIGS. indicate the m/z ratio of the substances identified in exhaled breath by considering an additional proton applied to the substances during PTR-MS for ionization purposes.

(46) The results shown in FIGS. 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A, and 10A has been obtained in the same way as in case of FIG. 1A. The results shown in FIGS. 2B, 3B, 4B, 5B, 6B, 7B, 8B, 9B, and 10B has been obtained in the same way as in case of FIG. 1B.

(47) FIGS. 2A and 2B indicate that carbonyl sulfide, dimethylsilane, acetic acid and/or propanol (having an m/z ratio of 61) are suited markers for determining the function of an organ of a living organism or for subsequently diagnosing a disease or a severity of a disease of an organ of a living organism.

(48) FIGS. 3A and 3B indicate that bis(methylthio)methane and/or 3-mercaptopropane-1,2,-diol (having an m/z ratio of 109) are suited markers for determining the function of an organ of a living organism or for subsequently diagnosing a disease or a severity of a disease of an organ of a living organism.

(49) FIGS. 4A and 4B indicate that cysteine (having an m/z ratio of 122) is a suited marker for determining the function of an organ of a living organism or for subsequently diagnosing a disease or a severity of a disease of an organ of a living organism.

(50) FIGS. 5A and 5B indicate that selenocysteine (having an m/z ratio of 169) is a suited marker for determining the function of an organ of a living organism or for subsequently diagnosing a disease or a severity of a disease of an organ of a living organism.

(51) FIGS. 6A and 6B indicate that octane and/or furan-2-ylmethanethiol (having an m/z ratio of 115) are suited markers for determining the function of an organ of a living organism or for subsequently diagnosing a disease or a severity of a disease of an organ of a living organism.

(52) FIGS. 7A and 7B indicate that nonane and/or naphthalene (having an m/z ratio of 129) are suited markers for determining the function of an organ of a living organism or for subsequently diagnosing a disease or a severity of a disease of an organ of a living organism.

(53) FIGS. 8A and 8B indicate that 1,2-diazine (pyridazine), 1,3-diazine (pyrimidine) and/or 1,4-diazine (pyrazine) (having an m/z ratio of 81) are suited markers for determining the function of an organ of a living organism or for subsequently diagnosing a disease or a severity of a disease of an organ of a living organism.

(54) FIGS. 9A and 9B indicate that 2-pentanone and/or hexane (having an m/z ratio of 87) are suited markers for determining the function of an organ of a living organism or for subsequently diagnosing a disease or a severity of a disease of an organ of a living organism.

(55) FIGS. 10A and 10B indicate that 4-hydroxynonenal (having an m/z ratio of 157) is a suited marker for determining the function of an organ of a living organism or for subsequently diagnosing a disease or a severity of a disease of an organ of a living organism.